RU2830496C1 - Vectors based on recombinant herpes virus of turkeys expressing antigens of avian pathogens, and versions thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology and virology. Disclosed is a recombinant turkey herpes virus (HVT) genome encoding and expressing an antigenic polypeptide of a pathogenic agent, wherein said genome contains one or more nucleotide sequences coding one or more heterologous antigens inserted into intergenic loci UL 35/UL 36 in a unique long (UL) region of the HVT genome. Disclosed is a recombinant turkey herpes virus (HVT) genome encoding and expressing an antigenic polypeptide of a pathogenic agent, wherein said genome contains one or more nucleotide sequences coding one or more heterologous antigens inserted into intergenic loci UL 35/UL 36 in a unique long region of the HVT genome and one or more nucleotide sequences coding one or more heterologous antigens inserted into the UL55/gene 3 site of the unique long region (UL) of the HVT genome. Disclosed are: recovered DNA coding recombinant HVT gene; immunogenic composition; vaccine composition; method of poultry vaccination for treating or preventing Marek's disease and one or more poultry diseases caused by one or more poultry pathogens, and a method for inducing an immune response in an animal bird to a Marek disease virus and one or more avian pathogens.

EFFECT: invention enables to obtain vaccines for providing immunity to several pathogens simultaneously.

60 cl, 33 dwg, 30 tbl, 29 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC 119(e) to U.S. Provisional Application No. 62/898,651, filed September 11, 2019, which is incorporated herein by reference in its entirety.

AREA OF TECHNOLOGY

The present invention relates to recombinant virus-based vectors for insertion and expression of foreign genes for use in safe immunizations for protection against various pathogens. It also relates to polyvalent compositions or vaccines containing one or more recombinant virus-based vectors for protection against various pathogens. The present invention relates to methods for obtaining and using said recombinant virus-based vectors.

LEVEL OF TECHNOLOGY

Marek's disease, a highly contagious lymphoproliferative disorder, is one of the most common avian infections that primarily affects young chickens. Marek's disease is caused by the Marek disease virus. Marek disease virus (MDV), a herpesvirus, is a member of the genus Mardivirus, which contains three serotypes (species): MDV-1 (herpesvirus Gallid 2), MDV-2 (herpesvirus Gallid 3), and MDV-3 (herpesvirus Meleagrid 1, turkey herpesvirus (HVT)). MDV-1 is the most virulent of the three serotypes, causing widespread disease in unvaccinated poultry. Birds infected with MDV-1 exhibit neurological, visceral, and cutaneous clinical signs such as paralysis of the lower limbs, wings, and neck; eye lesions and visual impairment, weight loss, cancers in many organs such as the thymus, heart, lungs, gonads, muscles, and feather follicles. Morbidity in affected birds is 10-50%, and mortality can be as high as 100%. Although Marek's disease can affect birds of any age, acute Marek's disease causes the death of large numbers of unvaccinated birds at an early age of four to eight weeks. Marek's disease is spread by direct or indirect contact with the dander of infected chickens, and the virus is inhaled. MDV-2 and MDV-3 are avirulent virus strains and have been used in the preparation of a vaccine against the related and virulent MDV-1.

In addition to Marek's disease, there are several pathogens that affect poultry and pose a threat to the poultry industry. Producers must rely on the immunity provided by vaccines to protect flocks from viral, bacterial and other pathogens. Live, killed and recombinant vaccines have been used to vaccinate birds. Live vaccines have the advantage of providing strong and long-lasting immunity, but they require careful handling as they can cause mild to severe reactions. On the other hand, killed vaccines are more stable and safer than live vaccines, but they produce a weaker immune response, requiring multiple administrations. Both live and killed vaccines have proven to be safe and effective, but there remains a need to develop and continually improve polyvalent vaccines to provide protection against more than one pathogen with a single vaccination.

Recombinant vector vaccines have been developed to provide immunity to multiple pathogens simultaneously. These vaccines are made by removing some nonessential gene regions from the host genome of a non-pathogenic organism and replacing them with one or more genes encoding antigens that are responsible for producing an immune response against the pathogenic organism. The newly created vector is then used to infect the host, where it will replicate and express the antigens of the virulent organism(s), inducing an immune response. Recombinant vector vaccines combine the advantages of live and killed vaccines. Recombinant vectors, similar to a live vaccine, provide longer-lasting immunity while causing a milder response after vaccination than killed vaccines. In addition, both the vector and the inserted gene(s) can provide immunity that protects birds against two or more diseases.

Marek's disease viruses are among the most effective polyvalent vaccine vectors for immunization against poultry diseases because they provide lifetime protection after just one vaccination. In addition, these viruses are restricted to avian hosts, so there is no risk of infection to other animals or people working in poultry farms. Among the Marek's disease viruses, turkey herpes virus (HVT) has been used more widely both as a live vaccine and as a recombinant vaccine vector against the more virulent MDV-1. HVT was first isolated from turkeys in 1969-1970 and was soon found to be protective against MDV, after which it was approved for use as a vaccine in 1971. HVT has similar antigenic properties to Marek's disease virus (MDV-1), but is not pathogenic to chickens. In addition, HVT is not sensitive to maternal antibodies against MDV or HVT, so live HVT vaccine has been used to effectively vaccinate against MDV-1 in ovo or early pre-hatch. In addition, the HVT genome has been used as a vaccine vector to contain foreign DNA sequences of other avian pathogens.

BRIEF SUMMARY OF THE PRESENT INVENTION

The present invention provides recombinant virus-based vectors for insertion and expression of foreign genes for use in safe immunizations for protecting birds from various pathogens. The present invention also provides polyvalent compositions or vaccines containing one or more recombinant HVT virus-based vectors for protection against various pathogens. In addition, the present invention also provides methods for producing and using recombinant virus-based vectors alone or in combination with other vaccines or pharmaceutical compositions.

In one aspect, the present invention provides a recombinant turkey herpes virus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL 35/UL 36 intergenic loci in the unique long (UL) region of the HVT genome.

In one aspect, the present invention provides a recombinant turkey herpes virus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL 35/UL 36 intergenic loci in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL55/gene 3 site of the unique long region (UL) of the HVT genome.

In one or more embodiments, the present invention provides a recombinant HVT, wherein one or more heterologous antigens are characterized by a protective effect against avian pathogens or pathogens selected from the group consisting of: infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV).

In one or more embodiments, the present invention provides a recombinant HVT, wherein one or more heterologous antigens are selected from the group consisting of VP2, VP3, or VP4 proteins of infectious bursal disease virus (IBDV); VP1 or VP2 proteins of chicken anemia virus (CAV); a chimeric F/HN protein or F, NP, P, M, HN, or L proteins of Newcastle disease virus (NDV); S1, S2, or M proteins of infectious bronchitis virus (IBV); gB, gC, gD, gE, gH, gI, or gL proteins of infectious laryngotracheitis virus (ILTV); and any of the HA, NA, NP, or M proteins of avian influenza virus (AIV).

In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens have a protective effect against IBDV. In one embodiment, the recombinant HVT of the present invention provides that one or more heterologous antigens are the VP2 protein of IBDV. In one embodiment, the recombinant HVT of the present invention provides that the VP2 protein is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence comprising SEQ ID NO. 5 or SEQ ID NO. 10. In one embodiment, the recombinant HVT of the present invention provides that the VP2 protein is encoded by a nucleotide sequence comprising either SEQ ID NO. 5 or SEQ ID NO. 10.

In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens have a protective effect against Newcastle disease virus (NDV). In one embodiment, the recombinant HVT of the present invention provides that one or more heterologous antigens are NDV F protein. In one embodiment, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence comprising SEQ ID NO. 3. In one embodiment, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence comprising SEQ ID NO. 3.

In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens have a protective effect against NDV and IBDV. In one or more embodiments, the recombinant HVT of the present invention provides that at least one heterologous antigen is the F protein of NDV and the VP2 protein of IBDV.

In one or more embodiments, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence comprising SEQ ID NO. 3 and the VP2 protein of IBDV is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence comprising SEQ ID NO. 5 or SEQ ID NO. 10.

In one or more embodiments, the recombinant HVT of the present invention provides the F protein from NDV encoded by a nucleotide sequence comprising SEQ ID NO. 3, and the VP2 protein from IBDV is encoded by a nucleotide sequence comprising SEQ ID NO. 5 or SEQ ID NO. 10.

In one or more embodiments, the recombinant HVT of the present invention comprises a genome comprising one or more expression cassettes comprising one or more nucleotide sequences that encode one or more heterologous antigens. In one embodiment, the recombinant HVT comprises a recombinant HVT-based genome, an expression cassette that comprises a nucleotide sequence encoding promoters that are operably linked to one or more nucleotides that encode antigens to be expressed. In one embodiment, the antigen to be expressed comprises the F protein from NDV. In one embodiment, the antigen to be expressed comprises the VP2 protein from IBDV. In one embodiment, the antigens to be expressed comprise both the F protein from NDV and the VP2 protein from IBDV.

In one embodiment, the recombinant HVT of the present invention provides one or more promoters selected from the group consisting of: the human cytomegalovirus (hCMV) immediate early promoter; the guinea pig CMV immediate early promoter; the mouse CMV immediate early promoter; the Rec promoter; the chicken β-actin promoter; the SV40 promoter; the pseudorabies virus glycoprotein X promoters; the herpes simplex virus-1 alpha 4 promoter; the Marek's disease virus glycoprotein gA, gC, gB, gE or gI promoters; the infectious laryngotracheitis virus glycoprotein gB, gE, gI, gD promoters; and the bovine herpes virus 1/1 VP8 promoter. In one embodiment, the recombinant HVT comprises the human CMV promoter. In one embodiment, the recombinant HVT comprises the mouse CMV promoter. In one embodiment, the recombinant HVT comprises the hCMV and mCMV promoter.

In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a polyadenylation (poly A) signal sequence. In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a poly A signal sequence and is selected from the poly A BGH sequence (SEQ ID NO. 6) or the poly A SV40 sequence (SEQ ID NO. 12). In one embodiment, the poly A signal sequence is the poly A BGH signal sequence. In one embodiment, the poly A signal sequence is the poly A SV40 signal sequence.

In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding the VP2 protein from IBDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence, wherein the entire VP2-based expression cassette is inserted into the non-coding region of the HVT genome. In one embodiment, the CMV promoter comprises the hCMV promoter (SEQ ID NO. 1). In one embodiment, the nucleotide sequence encoding the VP2 protein of IBDV is selected from SEQ ID NO. 5 or SEQ ID NO. 10. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO. 5. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO. 10. In one embodiment, the polyadenylation signal sequence comprises SEQ ID NO. 6. In one embodiment, the polyadenylation signal sequence comprises SEQ ID NO. 12. In one embodiment, the promoter, the nucleotide sequence encoding the VP2 protein, and the poly A signal sequence comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55/gene 3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35/36 site in the genome. In one embodiment, the expression cassette comprises, in order, SEQ ID NO. 1, SEQ ID NO. 5, or SEQ ID NO. 10 and SEQ ID NO. 6 inserted into the HVT genome at the UL55/gene 3 site.

In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding an F protein from NDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence, wherein the entire portion of the NDV F protein-based cassette is inserted into a non-coding region in the HVT genome. In one embodiment, the CMV promoter comprises the mCMV promoter (SEQ ID NO.2). In one embodiment, the nucleotide sequence encodes an F protein from NDV that comprises SEQ ID NO.3. In one embodiment, the polyadenylation signal sequence is encoded by a nucleotide sequence comprising SEQ ID NO. 12. In one embodiment, the promoter, the nucleotide sequence encoding the F protein, and the poly A signal sequence comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55/gene 3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35/36 site in the genome. In one embodiment, the expression cassette comprises, in order, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 12 inserted into the HVT genome at the UL55/gene 3 site.

In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding the VP2 protein from IBDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence, all comprising a VP2 expression cassette inserted into a non-coding region in the HVT genome. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein from NDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence as part of an F-based expression cassette from NDV inserted into the same insertion site as the VP2-based cassette. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein from NDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence as part of an F-based expression cassette from NDV inserted at a different site than the VP2-based cassette.

In one embodiment, the recombinant HVT of the present invention provides that the VP2-based expression cassette comprises, in order, a nucleotide sequence encoding the hCMV promoter (SEQ ID NO. 1), a nucleotide sequence encoding VP2 from IBDV (selected from SEQ ID NO. 5 or SEQ ID NO. 10), and a nucleotide sequence encoding the BGH polyadenylation signal sequence (SEQ ID NO. 6) inserted into the HVT genome in the UL35/36 non-coding region, and, in order, a nucleotide sequence encoding the mCMV promoter (SEQ ID NO. 2), a nucleotide sequence encoding the F protein from NDV (SEQ ID NO. 3), and a nucleotide sequence encoding the SV40 polyadenylation signal sequence (SEQ ID NO. 12) inserted into the HVT genome in the UL55/gene non-coding region 3. In one aspect, the recombinant HVT of the present invention comprises a promoter operably linked to a nucleotide sequence encoding an infectious laryngotracheitis virus antigen, further comprising a nucleotide sequence encoding a polyadenylation signal sequence. In one embodiment, the ILT antigen comprises one or more antigens selected from the group consisting of: gB, gC, gD, gE, gH, gI, gL, or chimeric proteins of one or more ILT antigens of infectious laryngotracheitis virus (ILTV). In one embodiment, the recombinant HVT of the present invention further comprises a nucleotide sequence encoding one or more antigens selected from the group consisting of infectious bursal disease virus, chicken anemia virus, Newcastle disease virus, infectious bronchitis virus, and avian influenza virus. In one embodiment, the recombinant HVT of the present invention further comprises a promoter operably linked to a nucleotide sequence encoding antigens selected from the group consisting of: the VP1, VP2, VP3 or VP4 antigen of infectious bursal disease virus (IBDV); the VP1 or VP2 proteins of chicken anemia virus (CAV); the F/HN chimeric protein or the F, NP, P, M, HN or L proteins of Newcastle disease virus (NDV); the S1, S2 or M proteins of infectious bronchitis virus (IBV); and any of the HA, NA, NP or M proteins of avian influenza virus (AIV).

In one embodiment, the recombinant HVT of the present invention comprises one or more ILT antigens as part of an expression cassette comprising a promoter operably linked to a nucleotide encoding an ILT antigen and further comprising a nucleotide sequence encoding a polyadenylation signal sequence. In one embodiment, the recombinant HVT of the present invention comprises a second and a third expression cassette, each comprising nucleotide sequences encoding a promoter operably linked to a nucleotide sequence encoding avian antigens selected from the group consisting of the VP1, VP2, VP3, or VP4 antigen of infectious bursal disease virus (IBDV); the VP1 or VP2 proteins of chicken anemia virus (CAV); the F/HN chimeric protein or the F, NP, P, M, HN, or L proteins; the S1, S2, or M proteins of infectious bronchitis virus (IBV); and any of the HA, NA, NP or M proteins of the avian influenza virus (AIV), and further comprises a nucleotide sequence encoding a polyadenylation signal sequence.

In one or more aspects, the present invention provides a recombinant DNA encoding a recombinant HVT genome of the present invention.

In one or more aspects, the present invention provides an immunogenic composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient or adjuvant.

In one or more aspects, the present invention provides a vaccine composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient or adjuvant.

In one embodiment, the vaccine of the present invention further comprises an additional Marek's disease virus (MDV) selected from the group consisting of: the naturally attenuated MDV-1 Rispens strain (CVI-988); or the Gallid 3 herpes virus strain SB-1. In one embodiment, the vaccine of the present invention provides that the additional MDV comprises a recombinant genome. In one embodiment, the vaccine of the present invention provides that the additional genome of the recombinant MDV comprises one or more nucleotide sequences encoding one or more heterologous antigens that are characterized by a protective effect against one or more avian pathogens.

In one embodiment, the vaccine of the present invention is provided for vaccinating a bird against one or more diseases caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention is provided for use in protecting a bird from clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention is provided for use in protecting a bird from clinical symptoms caused by Marek's disease virus and clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention comprises one or more avian pathogens selected from the group consisting of: infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). In one embodiment, the vaccine of the present invention provides that the one or more avian pathogens comprise Newcastle disease virus. In one embodiment, the vaccine of the present invention provides that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the vaccine of the present invention provides that one or more avian pathogens include Newcastle disease virus and infectious bursal disease virus.

In one or more embodiments, the vaccine of the present invention is provided for use in vaccinating birds, wherein the vaccine is administered by at least one of vaccine administration by spraying, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration, or a combination thereof. In one embodiment, the vaccine of the present invention provides that the vaccine is administered by in ovo administration. In one embodiment, the vaccine of the present invention provides that the in ovo administration occurs into an egg with a developing embryo in the period between about 16 to 22 days of development. In one or more embodiments, the vaccine of the present invention provides that the in ovo administration occurs into an egg with a developing embryo in the period of about 18 days of development. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises in ovo administration followed by spray administration. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises spray administration.

In one aspect, the present invention provides a method of vaccinating a bird for the treatment or prevention of Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising the step of administering an effective amount of a vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that one or more avian pathogens comprise infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV).

In one aspect of the present invention, there is provided a method for inducing an immune response in an avian animal to Marek's disease virus and one or more avian pathogens, comprising the step of administering to the avian an effective amount of an immunogenic or vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). In one or more embodiments, the method of the present invention provides that the administration is carried out by spraying, in ovo administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration. In one embodiment, the method provides for in ovo administration. In one embodiment, the method provides that the in ovo administration occurs into an egg with a developing embryo in the period between about 16 to 22 days of development. In one or more embodiments, the method provides that the in ovo administration occurs into an egg with a developing embryo in the period of about 18 days of development. In one or more embodiments, the method provides that the route of administration includes in ovo administration followed by spray administration. In one embodiment, the method provides that the route of administration includes spray administration. In one or more embodiments, the method provides that the bird is selected from the group consisting of a chicken, a turkey, a goose, a duck, a pheasant, an ostrich, a pigeon, and a quail. In one embodiment, the method provides that the bird is a chicken.

In one aspect of the present invention, there is provided a vaccine composition comprising a recombinant HVT of the present invention, which comprises a nucleotide sequence encoding a Newcastle disease virus F protein, further comprising a composition comprising an attenuated infectious bursal disease virus and an antibody that specifically binds to the infectious bursal disease virus. In one or more embodiments, the composition comprising IBDV is an attenuated strain I2512 BD and comprises a Bursaplex™ vaccine. In one or more embodiments, the composition comprising IBDV is an attenuated strain V877 IBD and comprises a Magniplex™ vaccine.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows a PCR reaction demonstrating proper insertion of the gfp gene into the UL55/gene 3 site of the HVT genome.

Figure 2 shows a PCR reaction demonstrating the gfp gene integration site within the UL35/36 integration site of the HVT genome.

Fig. 3A, 3B, 3C show PCR reactions demonstrating proper integration of the VP2 gene into the HVT genome for HVT IBD 1.

Figures 4A and 4B show images of transfected/infected JBJ-1 cells stained for IBDV VP2 (panel A) and HVT infection (panel B) for HVT IBD5.

Figure 5 shows an image of the PCR reactions performed to confirm the proper orientation of the VP2 insert into the UL35/36 integration site of the HVT genome for HVT IBD 5.

Figure 6 shows a Western blot of infected cell lysate using a monoclonal antibody against IBDVR63, showing a protein band of approximately 50 kDa for HVT IBD 6a.

Figures 7A and 7B show PCR reactions demonstrating proper integration of the VP2 gene into the UL35/36 site in the HVT genome for HVT IBD 6a.

Figure 8 shows an image of PCR reactions demonstrating proper integration of the VP2 gene into the UL55/gene 3 site in the HVT genome for HVT IBD 9.

Figure 9 shows an image of PCR reactions demonstrating proper integration of the VP2 gene into the UL55/gene 3 site in the HVT genome for HVT IBD 30.

Fig. 10A and 10B show Western blot images of transfected/infected cell lysates using anti-IBDVR63 monoclonal antibody for HVT IBD 31.

Figure 11 shows images of PCR reactions demonstrating proper integration of the VP2 gene into the UL35/36 integration site in the HVT genome for HVT IBD 31.

Figure 12 shows images of PCR reactions demonstrating proper integration of the VP2 gene into the IL55/gene 3 integration site in the HVT genome for HVT IBD 34.

Fig. 13 shows a graphical representation of IBDV serologic responses of HVT-IBD 1, 5, 9, and 15.

Fig. 14 shows a graphical representation of IBDV serologic responses to HVT-IBD 6a, 30, 31.

Figures 15A and 15B show images of PCR reactions demonstrating the proper orientation of the NDVF insert for HVT ND#38.

Figures 16A and 16B show images of PCR reactions demonstrating the proper orientation of the NDVF insert for HVT ND#39.

Fig. 17A, 17B, 17C show images of PCR reactions demonstrating the proper orientation of the NDV F insert for HVT ND#40.

Figure 18 shows an image of several PCR reactions demonstrating the proper orientation of the NDVF insert for NDV 42 HVT.

Figure 19 shows an image of PCR reactions demonstrating the proper orientation of the NDVF insert for NDV 45 HVT.

Fig. 20A, 20B, 20C show an image of PCR reactions demonstrating the proper orientation of the NDVF insert for NDV 46 HVT.

Fig. 21 shows a linear representation of the HVT genome indicating the insertion site A, UL35 (HVT043)-UL36 (HVT044).

Fig. 22 shows a linear representation of the HVT genome indicating the insertion site B, UL55 (HVT065)-gene 3 (HVT066).

Fig. 23 shows an image of the Faragher IBDV VP2 F52/70 strain.

Fig. 24 shows an image of the synthesis of plasmid pHVT-IBD #30.

Fig. 25 shows an image of the synthesis of plasmid pHVT-ND No. 42.

Fig. 26 shows a circular map image of the pSiteA #30 transfer plasmid.

Fig. 27 shows a circular map image of the pSiteB #42 transfer plasmid.

Fig. 28 shows the production of intermediate recombinant HVT-ND #42.

Fig. 29 shows the production of HVT-IBD #30-ND #42.

Fig. 30 shows an image of the characterization of the HVT-IBD #30-ND #42 construct based on PCR and restriction endonuclease digestion of site A.

Fig. 31 shows an image of the characterization of the HVT-IBD #30-ND #42 construct based on PCR and restriction endonuclease digestion of site B.

Fig. 32 shows a Western blot image of the expression of the target protein HVT-IBD #30-ND #42 IBD VP2.

Fig. 33 shows a Western blot image of the expression of the target protein HVT-IBD #30-ND #42 NDV F.

BRIEF DESCRIPTION OF SEQUENCES

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

The following detailed description is provided to assist those skilled in the art. Those skilled in the art can make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention.

The present invention relates to vaccines for use in birds based on live recombinant avian herpes viruses, namely, in particular, Marek's disease virus (MDV) and more particularly based on the HVT virus (turkey herpes virus), into which one or more nucleotide sequences have been inserted encoding and expressing an antigenic polypeptide of a pathogenic agent under conditions that ensure immunization leading to effective protection of the vaccinated animal from said pathogenic agent or agents. Marek's disease (MD) is a common lymphoproliferative disease of chickens caused by the Marek's disease virus (MDV), which can lead to significant losses in poultry farming. Currently, MD is controlled in poultry by vaccines using serotype 3 MDV, which is related to the turkey herpes virus (HVT). By introducing genes from poultry viruses other than MDV into the HVT genome at specific genetic positions, the present inventors were able to develop novel recombinant virus-based vaccines that provide simultaneous protection of poultry against MD and one or more additional diseases through the administration of a single viral vaccine.

The present invention provides recombinant virus-based vectors for insertion and expression of foreign genes for use in safe immunizations for protecting birds from various pathogens. The present invention also provides polyvalent compositions or vaccines containing one or more recombinant HVT virus-based vectors for protection against various pathogens. In addition, the present invention also provides methods for producing and using recombinant virus-based vectors alone or in combination with other vaccines or pharmaceutical compositions.

In one aspect, the present invention provides a recombinant turkey herpes virus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL 35/UL 36 intergenic loci in the unique long (UL) region of the HVT genome.

In one aspect, the present invention provides a recombinant turkey herpes virus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL 35/UL 36 intergenic loci in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL55/gene 3 site of the unique long region (UL) of the HVT genome.

In one or more embodiments, the present invention provides a recombinant HVT, wherein one or more heterologous antigens are characterized by a protective effect against avian pathogens or pathogens selected from the group consisting of: infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV).

In one or more embodiments, the present invention provides a recombinant HVT, wherein one or more heterologous antigens are selected from the group consisting of VP2, VP3, or VP4 proteins of infectious bursal disease virus (IBDV); VP1 or VP2 proteins of chicken anemia virus (CAV); a chimeric F/HN protein or F, NP, P, M, HN, or L proteins of Newcastle disease virus (NDV); S1, S2, or M proteins of infectious bronchitis virus (IBV); gB, gC, gD, gE, gH, gI, or gL proteins of infectious laryngotracheitis virus (ILTV); and any of the HA, NA, NP, or M proteins of avian influenza virus (AIV).

In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens have a protective effect against IBDV. In one embodiment, the recombinant HVT of the present invention provides that one or more heterologous antigens are the VP2 protein of IBDV. In one embodiment, the recombinant HVT of the present invention provides that the VP2 protein is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence comprising SEQ ID NO. 5 or SEQ ID NO. 10. In one embodiment, the recombinant HVT of the present invention provides that the VP2 protein is encoded by a nucleotide sequence comprising either SEQ ID NO. 5 or SEQ ID NO. 10.

In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens have a protective effect against Newcastle disease virus (NDV). In one embodiment, the recombinant HVT of the present invention provides that one or more heterologous antigens are NDV F protein. In one embodiment, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence comprising SEQ ID NO. 3. In one embodiment, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence comprising SEQ ID NO. 3.

In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens have a protective effect against NDV and IBDV. In one or more embodiments, the recombinant HVT of the present invention provides that at least one heterologous antigen is the F protein of NDV and the VP2 protein of IBDV.

In one or more embodiments, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence comprising SEQ ID NO. 3 and the VP2 protein of IBDV is encoded by a nucleotide sequence that is at least 80% identical to a nucleotide sequence comprising SEQ ID NO. 5 or SEQ ID NO. 10.

In one or more embodiments, the recombinant HVT of the present invention provides the F protein from NDV encoded by a nucleotide sequence comprising SEQ ID NO. 3, and the VP2 protein from IBDV is encoded by a nucleotide sequence comprising SEQ ID NO. 5 or SEQ ID NO. 10.

In one or more embodiments, the recombinant HVT of the present invention comprises a genome comprising one or more expression cassettes comprising one or more nucleotide sequences that encode one or more heterologous antigens. In one embodiment, the recombinant HVT comprises a recombinant HVT-based genome, an expression cassette that comprises a nucleotide sequence encoding promoters that are operably linked to one or more nucleotides that encode antigens to be expressed. In one embodiment, the antigen to be expressed comprises the F protein from NDV. In one embodiment, the antigen to be expressed comprises the VP2 protein from IBDV. In one embodiment, the antigens to be expressed comprise both the F protein from NDV and the VP2 protein from IBDV.

In one embodiment, the recombinant HVT of the present invention provides one or more promoters selected from the group consisting of: the human cytomegalovirus (hCMV) immediate early promoter; the guinea pig CMV immediate early promoter; the mouse CMV immediate early promoter; the Rec promoter; the chicken p-actin promoter; the SV40 promoter; the pseudorabies virus glycoprotein X promoters; the herpes simplex virus-1 alpha 4 promoter; the Marek's disease virus glycoprotein gA, gC, gB, gE or gI promoters; the infectious laryngotracheitis virus glycoprotein gB, gE, gI, gD promoters; and the bovine herpes virus 1/1 VP8 promoter. In one embodiment, the recombinant HVT comprises the human CMV promoter. In one embodiment, the recombinant HVT comprises the mouse CMV promoter. In one embodiment, the recombinant HVT comprises the hCMV and mCMV promoter.

In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a polyadenylation (poly A) signal. In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a poly A signal and is selected from the poly A BGH sequence (SEQ ID NO. 6) or the poly A SV40 sequence (SEQ ID NO. 12). In one embodiment, the poly A signal sequence is the poly A BGH signal sequence. In one embodiment, the poly A signal sequence is the poly A SV40 signal sequence.

In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding the VP2 protein from IBDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence, wherein the entire VP2-based expression cassette is inserted into the non-coding region of the HVT genome. In one embodiment, the CMV promoter comprises the hCMV promoter (SEQ ID NO. 1). In one embodiment, the nucleotide sequence encoding the VP2 protein of IBDV is selected from SEQ ID NO. 5 or SEQ ID NO. 10. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO. 5. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO. 10. In one embodiment, the polyadenylation signal sequence comprises SEQ ID NO. 6. In one embodiment, the polyadenylation signal sequence comprises SEQ ID NO. 12. In one embodiment, the promoter, the nucleotide sequence encoding the VP2 protein, and the poly A signal sequence comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55/gene 3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35/36 site in the genome. In one embodiment, the expression cassette comprises, in order, SEQ ID NO.1, SEQ ID NO.5, or SEQ ID NO. 10 and SEQ ID NO.6, inserted into the HVT genome at the UL55/gene 3 site.

In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding an F protein from NDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence, wherein the entire portion of the NDV F protein-based cassette is inserted into a non-coding region in the HVT genome. In one embodiment, the CMV promoter comprises the mCMV promoter (SEQ ID NO.2). In one embodiment, the nucleotide sequence encodes an F protein from NDV that comprises SEQ ID NO.3. In one embodiment, the polyadenylation signal sequence is encoded by a nucleotide sequence comprising SEQ ID NO. 12. In one embodiment, the promoter, the nucleotide sequence encoding the F protein, and the poly A signal sequence comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55/gene 3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35/36 site in the genome. In one embodiment, the expression cassette comprises, in order, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 12 inserted into the HVT genome at the UL55/gene 3 site.

In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding the VP2 protein from IBDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence, all comprising a VP2 expression cassette inserted into a non-coding region in the HVT genome. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein from NDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence as part of an F-based expression cassette from NDV inserted into the same insertion site as the VP2-based cassette. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein from NDV, further comprising a nucleotide sequence encoding a polyadenylation signal sequence as part of an F-based expression cassette from NDV inserted at a different site than the VP2-based cassette.

In one embodiment, the recombinant HVT of the present invention provides that the VP2-based expression cassette comprises, in order, a nucleotide sequence encoding the hCMV promoter (SEQ ID NO. 1), a nucleotide sequence encoding VP2 from IBDV (selected from SEQ ID NO. 5 or SEQ ID NO. 10), and a nucleotide sequence encoding the BGH polyadenylation signal sequence (SEQ ID NO. 6) inserted into the HVT genome in the UL35/36 non-coding region, and, in order, a nucleotide sequence encoding the mCMV promoter (SEQ ID NO. 2), a nucleotide sequence encoding the F protein from NDV (SEQ ID NO. 3), and a nucleotide sequence encoding the SV40 polyadenylation signal sequence (SEQ ID NO. 12) inserted into the HVT genome in the UL55/gene non-coding region 3. In one embodiment, the recombinant HVT of the present invention further comprises a nucleotide sequence encoding one or more antigens selected from the group consisting of infectious bursal disease virus, chicken anemia virus, Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus and avian influenza virus. In one embodiment, the recombinant HVT of the present invention further provides a promoter operably linked to a nucleotide sequence encoding antigens selected from the group consisting of: the VP1, VP2, VP3 or VP4 antigen of infectious bursal disease virus (IBDV); the VP1 or VP2 proteins of chicken anemia virus (CAV); the F/HN chimeric protein or the F, NP, P, M, HN or L proteins of Newcastle disease virus (NDV); the S1, S2 or M proteins of infectious bronchitis virus (IBV); and any of the HA, NA, NP or M proteins of avian influenza virus (AIV).

In one embodiment, the recombinant HVT of the present invention comprises one or more ILT antigens as part of an expression cassette comprising a promoter operably linked to a nucleotide encoding an ILT antigen and further comprising a nucleotide sequence encoding a polyadenylation signal sequence. In one embodiment, the recombinant HVT of the present invention comprises a second and a third expression cassette, each comprising nucleotide sequences encoding a promoter operably linked to a nucleotide sequence encoding avian antigens selected from the group consisting of the VP1, VP2, VP3, or VP4 antigen of infectious bursal disease virus (IBDV); the VP1 or VP2 proteins of chicken anemia virus (CAV); the F/HN chimeric protein or the F, NP, P, M, HN, or L proteins; the S1, S2, or M proteins of infectious bronchitis virus (IBV); and any of the HA, NA, NP or M proteins of the avian influenza virus (AIV), and further comprises a nucleotide sequence encoding a polyadenylation signal sequence.

In one or more aspects, the present invention provides a recombinant DNA encoding a recombinant HVT genome of the present invention.

In one or more aspects, the present invention provides an immunogenic composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient or adjuvant.

In one or more aspects, the present invention provides a vaccine composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient or adjuvant.

In one embodiment, the vaccine of the present invention further comprises an additional Marek's disease virus (MDV) selected from the group consisting of: the naturally attenuated MDV-1 Rispens strain (CVI-988); or the Gallid 3 herpes virus strain SB-1. In one embodiment, the vaccine of the present invention provides that the additional MDV comprises a recombinant genome. In one embodiment, the vaccine of the present invention provides that the additional genome of the recombinant MDV comprises one or more nucleotide sequences encoding one or more heterologous antigens that are characterized by a protective effect against one or more avian pathogens.

In one embodiment, the vaccine of the present invention is provided for vaccinating a bird against one or more diseases caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention is provided for use in protecting a bird from clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention is provided for use in protecting a bird from clinical symptoms caused by Marek's disease virus and clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention comprises one or more avian pathogens selected from the group consisting of: infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). In one embodiment, the vaccine of the present invention provides that the one or more avian pathogens comprise Newcastle disease virus. In one embodiment, the vaccine of the present invention provides that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the vaccine of the present invention provides that one or more avian pathogens include Newcastle disease virus and infectious bursal disease virus.

In one or more embodiments, the vaccine of the present invention is provided for use in vaccinating birds, wherein the vaccine is administered by at least one of vaccine administration by spraying, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration, or a combination thereof. In one embodiment, the vaccine of the present invention provides that the vaccine is administered by in ovo administration. In one embodiment, the vaccine of the present invention provides that the in ovo administration occurs into an egg with a developing embryo in the period between about 16 to 22 days of development. In one or more embodiments, the vaccine of the present invention provides that the in ovo administration occurs into an egg with a developing embryo in the period of about 18 days of development. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises in ovo administration followed by spray administration. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises spray administration.

In one aspect, the present invention provides a method of vaccinating a bird for the treatment or prevention of Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising the step of administering an effective amount of a vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that one or more avian pathogens comprise infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV).

In one aspect of the present invention, there is provided a method for inducing an immune response in an avian animal to Marek's disease virus and one or more avian pathogens, comprising the step of administering to the avian an effective amount of an immunogenic or vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens comprise infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). In one or more embodiments, the method of the present invention provides that the administration is carried out by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration. In one embodiment, the method provides in ovo administration. In one embodiment, the method provides that the in ovo administration occurs into an egg with a developing embryo in the period between about 16 to 22 days of development. In one or more embodiments, the method provides that the in ovo administration occurs into an egg with a developing embryo in the period of about 18 days of development. In one or more embodiments, the method provides that the route of administration includes in ovo administration followed by spray administration. In one embodiment, the method provides that the route of administration includes spray administration. In one or more embodiments, the method provides that the bird is selected from the group consisting of a chicken, a turkey, a goose, a duck, a pheasant, an ostrich, a pigeon, and a quail. In one embodiment, the method provides that the bird is a chicken.

General methodologies:

It should be understood that the present invention is not limited to the particular methodology, protocols, reagents, etc. described herein and, thus, may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise indicated, scientific and technical terms used in connection with the present invention described herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization, and the techniques described herein are well known and commonly used in the art.

For recombinant DNA, oligonucleotide synthesis, tissue culture, and transfection, standard techniques are used that are well known to those skilled in the art. Enzymatic reactions and purification procedures are performed according to the manufacturer's descriptions or as commonly done in the art or as described herein. The above techniques and procedures are generally performed according to conventional methods well known in the art and as described in, but without limitation, the various general and more specific references cited and discussed herein, see, for example, Sambrook et al. MOLECULAR CLONING: LAB. MANUAL (3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y., 2001), and Ausubel et al. Current Protocols in Molecular Biology (New York: Greene Publishing Association JWiley Interscience), Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. 1. Freshney, ed.1987); Introduction to Cell and Tissue Culture (1. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and S. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (Y. T. DeVita et al., eds., J. B. Lippincott Company, 1993).

Except as provided in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term “about.”

All patents and other publications cited are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methodologies described in such publications that may be applied in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application.

Definitions

Before describing the present invention in detail, several terms used in the context of the present invention will be defined. In addition to these terms, other terms are defined elsewhere in the description as needed. Unless otherwise expressly stated herein, terms from the art used in the present description shall have the meanings recognized in the art.

It should be noted that in the present invention, such terms as “comprises,” “comprised,” “comprising,” “contained,” “consisting,” “consisted,” “consisting essentially of,” “includes,” “included,” etc. are defined in accordance with the standard practice of the patent law of the United States of America and international practice.

The term "about" is used herein to indicate that the value includes the standard deviation of the error of the device or method used to determine the value. The use of the term "or" in the claims is used to mean "and/or" unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, although the present invention supports a definition that refers only to alternatives and to "and/or". When not used in combination with a closed wording in the claims or otherwise specifically stated, the singular forms mean "one or more". Thus, the term "conveyed by a transgene", for example, covers one or more transgenes.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those that are encoded by the genetic code as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogues contain modified R-groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the overall chemical structure of the amino acid, but that function similarly to the naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes. Macromolecular structures, such as polypeptide structures, may be described in terms of different levels of organization. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to the locally ordered three-dimensional structures within a polypeptide. These structures are commonly known as domains, such as enzymatic domains, extracellular domains, transmembrane domains, pore domains, or cytoplasmic tail domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide. Exemplary domains include domains with enzymatic activity. The domain may consist of regions of lesser organization, such as β-sheet and α-helix regions. "Tertiary structure" refers to the overall three-dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three-dimensional structure formed by the non-covalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

As used herein, the term "antibody" refers to a polypeptide comprising a framework region of an immunoglobulin gene or fragments thereof that specifically binds to and recognizes an antigen.

Commonly recognized immunoglobulin genes may include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as many immunoglobulin variable region genes. Light chains may be classified as either kappa or lambda. Heavy chains may be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgY, IgG, IgM, IgA, IgD, and IgE, respectively.

An exemplary structural unit of an immunoglobulin (antibody) may comprise a tetramer, each tetramer consisting of two identical pairs of polypeptide chains, each pair containing one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain and variable heavy chain refer to these light and heavy chains. Antibodies exist, for example, as intact immunoglobulins or as several well-characterized fragments produced by cleavage with various peptidases. While various antibody fragments are defined in terms of cleavage of an intact antibody, one skilled in the art will appreciate that such fragments may be synthesized de novo, either chemically or using recombinant DNA methodologies. Thus, the term "antibody" as used herein also includes antibody fragments either produced by modification of whole antibodies, or synthesized de novo using recombinant DNA methodologies, or identified using other methods known in the art.

Many methods known in the art can be used to produce antibodies, such as recombinant, monoclonal or polyclonal antibodies. Genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell and used to produce a recombinant monoclonal antibody. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be used. Random combinations of heavy and light chain gene products create a large pool of antibodies with different antigen specificities. Methods for producing single-chain antibodies or recombinant antibodies are known in the art and can be adapted to produce antibodies to the polypeptides of the present invention. Phage display technology can also be used to identify antibodies and heteromeric fragments that specifically bind to selected antigens. Antibodies can also be bispecific, i.e., capable of recognizing two different antigens, or be heteroconjugates, such as two covalently linked antibodies or immunotoxins.

As used herein, the term "antigen" refers to a viral protein or polypeptide, such as a viral polypeptide, as well as viral particles. In some embodiments, an antigen according to the present invention may also be a viral nucleic acid. An antigen is a molecule that is recognized by the immune system and is capable of inducing an immune response in the host organism. An antigen may be a whole, attenuated, killed or living organism or a subunit or part of an organism. It may also be a part or fragment of DNA, a polypeptide, an epitope, a hapten or any combination thereof that can induce an immune response.

As used herein, the term "poultry" includes poultry such as members of the order Galliformes. More specifically, the class of birds primarily of economic and/or agronomic interest such as chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons and quails, etc.

As used herein, the term "biological sample" or "specimen" may include blood and blood parts, including but not limited to serum, plasma, platelets, or red blood cells; sputum, cloacal swabs, mucosa, tissue, cultured cells, including primary cultures, explants, and transformed cells; biological fluids, feces, and urine. A biological sample may also include tissue sections, such as biopsy and autopsy specimens, and frozen sections taken for histological purposes. A biological sample may be obtained from a eukaryotic organism, such as a bird, including but not limited to a bird of the order Galliformes, such as chickens, quail, and turkeys. Any tissue suitable for use in accordance with the present invention may be used, such as skin, brain, spinal cord, adrenal glands, pectoral muscles, lung, heart, liver, crop, stomach, ventricle, duodenum, small intestine, large intestine, cloaca, kidney, bursa of Fabricius, spleen, pancreas, bone marrow, lumbosacral spinal cord, or blood.

The term "conservative amino acid substitution" means any amino acid substitution of a specified amino acid residue, wherein the substituting residue is so chemically similar to the specified residue that it does not result in a substantial reduction in the function of the polypeptide (e.g., enzymatic activity). Conservative amino acid substitutions are widely known in the art, and examples thereof are described, for example, in U.S. Patent Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, a conservative amino acid substitution is any substitution that occurs in one of the following six groups:

• 1. Small aliphatic, virtually non-polar residues: Ala, Gly, Pro, Ser and Thr;

• 2. Large aliphatic, non-polar residues: He, Leu and Val; Met;

• 3. Polar, negatively charged residues and their amides: Asp and Glu;

• 4. Amides of polar negatively charged residues: Asn and Gin; His;

• 5. Polar, positively charged residues: Arg and Lys; His; and

• 6. Large aromatic residues: Trp and Tyr; Phe.

In a preferred embodiment, the conservative amino acid substitution is any of the following pairs listed as pairs of native residues (conservative substitutions): Ala (Ser); Arg (Lys); Asn (Gin; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gin); He (Leu; Val); Leu (Ile; Val); Lys (Arg; Gin; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (He; Leu).

The phrase "functional effects" in the context of assays for testing compounds that modulate the activity of a virus as described herein includes the determination of a parameter that is directly or indirectly affected by such virus, such as a phenotypic or chemical effect. "Functional effects" may include in vitro, in vivo, and ex vivo activity and may be measured by any method known to those skilled in the art, such as changes in spectroscopic characteristics, shape, chromatographic properties, or solubility properties of the protein, measurement of inducible markers, or transcriptional activation of the protein; measurement of binding activity or binding assays, i.e., binding to antibodies; measurement of changes in ligand or substrate binding activity, measurement of viral replication, measurement of cell surface marker expression, measurement of changes in protein levels, measurement of RNA stability, identification of downstream or reporter gene expression by, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, and/or inducible markers.

The term "gene" refers to components that include viral DNA or RNA, cDNA, viral intron and exon DNA, artificial polynucleotide of viral DNA or other DNA that encodes a viral peptide, viral polypeptide, viral protein or viral RNA transcript molecule, and genetic elements that may flank the coding sequence that are involved in the regulation of expression, such as promoter regions, 5' leader regions, 3' untranslated region, which may exist as native genes or transgenes in the viral genome. A gene or fragment thereof may be subjected to polynucleotide sequencing methods that determine the order of nucleotides that make up the gene.

The term "turkey herpesvirus (HVT)" is defined as a nonpathogenic virus of domestic turkeys and is classified as the third serotype in the Marek disease virus group of antigenically and genetically related lymphotropic avian herpesviruses.

The term "heterologous," when used in reference to portions of a nucleic acid, indicates that the nucleic acid contains two or more sequences that are not found in the same ratio to each other in nature. For example, a nucleic acid is typically produced recombinantly by having two or more sequences from unrelated genes arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein contains two or more subsequences that are not found in the same ratio to each other in nature (e.g., a fusion protein). Heterologous may also refer to a viral sequence, such as a gene or transgene, or portion thereof, inserted into a viral genome in which it is not normally found, or to a gene introduced into an organism in which it is not normally found.

The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or processed in any way to cause the cell to produce a substance, such as the cell to express a gene, DNA or RNA sequence, protein, or enzyme. A host cell is intended to include any single cell or cell culture that may be or has been a recipient for vectors or for the incorporation of exogenous nucleic acid molecules, polynucleotides, and/or proteins. It is also intended to include the progeny of a single cell. The progeny may not necessarily be completely identical (in morphology or in genomic or total DNA composition) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may be prokaryotic or eukaryotic.

As used herein, the term "host", "subject", "patient" or "organism" may include animals, in particular birds, especially poultry. For veterinary applications, birds may be of the order Galliformes, which includes chickens, quail, turkeys, etc. The term "living host" refers to a host as defined above or to another organism that is alive. The term may also refer to the entire host or organism, and not just a portion (e.g., the brain or other organ) that has been surgically removed from a living host. These terms also include humans in all stages of development, including embryonic and fetal stages.

The terms “identical” or “percent identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum match over a comparison window or a designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below or by manual alignment and visual inspection (see, for example, the NCBI website at ncbi.nlm.nih.gov/BLAST/ or similar). Such sequences are then said to be "substantially identical". This definition also refers to or applies to the complement of a particular sequence. The definition may also include sequences that contain deletions, additions, and/or substitutions.

In sequence comparison, one sequence typically serves as a reference sequence to which other sequences are compared. Using a sequence comparison algorithm, the reference and comparison sequences can be entered into a computer and the parameters of the sequence algorithm program are selected as desired. Percent sequence identity for the comparison sequences relative to the reference sequence is then generated based on the selected parameters. An example of an algorithm that may be suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (Nuc Acids Res 25:3389-3402, 1977) and Altschul et al, (JMol Biol 215:403-410, 1990), respectively. BLAST and BLAST 2.0 are well known in the art and can be used to determine the percent sequence identity of any nucleic acids or proteins, such as those described herein.

As used herein, the terms "immunogenic composition" or "pharmaceutical composition" or "vaccine" are intended to encompass a composition comprising an antigen suitable for administration to a subject, such as an avian subject. Said composition is typically intended to induce an immune response in the subject. The immune response may include a T cell response, a B cell response, or both a T cell and a B cell response. The composition may function to sensitize the subject by presenting the antigen in association with MHC molecules on the cell surface. In addition, antigen-specific T lymphocytes or antibodies may be produced to provide subsequent protection to the immunized host. An "immunogenic composition" may comprise a live, attenuated, or killed/inactivated vaccine comprising a whole microorganism or an immunogenic portion thereof that induces either a cell-mediated (T-cell) immune response or an antibody-mediated (B-cell) immune response, or both, and can protect an animal from one or more symptoms associated with infection with the microorganism or can protect an animal from mortality due to infection with the microorganism. Typically, the "immunogenic composition" is sterile and preferably free of impurities that may induce an undesirable response in a subject (e.g., the compound(s) in the immunogenic composition are of pharmaceutical grade). Immunogenic compositions can be designed for administration to subjects in need thereof via a variety of different routes of administration, including in ovo, oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intrathecal, intramuscular, subcutaneous, inhalation, etc.

The term "immunogenic" protein or peptide as used herein includes polypeptides that are immunologically active in the sense that, upon administration to a host, they are capable of inducing a humoral and/or cellular immune response directed against the protein. Preferably, the protein fragment has the same immunological activity as the full-length protein. Thus, the protein fragment according to the present invention comprises or essentially consists of or consists of at least one epitope or antigenic determinant. The term "immunogenic" protein or polypeptide as used herein includes the full-length protein sequence, its analogs or immunogenic fragments thereof. By "immunogenic fragment" is meant a protein fragment that comprises one or more epitopes and thus induces an immunological response as described above.

The term "immunogenic protein or peptide" further contemplates deletions, additions and substitutions in the sequence so long as the polypeptide functions to produce an immunological response as defined herein. The term "conservative variation" means the replacement of an amino acid residue with another biologically similar residue or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue is unchanged or is another biologically similar residue. Particularly preferred substitutions in this regard will generally be conservative in nature, i.e., those substitutions that occur within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) the uncharged polar amino acids glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, etc.; or similar conservative substitution of an amino acid for a structurally related amino acid that will not significantly affect biological activity. Thus, proteins containing substantially the same amino acid sequence as a reference molecule but characterized by minor amino acid substitutions that do not significantly affect the immunogenicity of the protein fall within the definition of a reference polypeptide. All polypeptides produced by these modifications are included in this document. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies induced by the substituted polypeptide also react immunologically with the unsubstituted polypeptide.

The term "immunologically effective amount" as used herein refers to an amount of an antigen or vaccine sufficient to induce an immune response, either a cellular (T cell) or a humoral (B cell or antibody) response, as measured by standard assays known to those skilled in the art. For example, in relation to the present invention, the term "immunologically effective amount" is the minimum protective dose (titer). The effectiveness of an antigen as an immunogen can be measured either by proliferation assays or by cytolytic assays, such as chromium release assays, to measure the ability of T cells to lyse their specific target cell, or by measuring the levels of B cell activity by measuring the levels of circulating antibodies specific to the antigen in the blood serum, or by other assays that are known and used by those skilled in the art. In addition, the level of protection of the immune response can be measured by exposing the immunized host to the antigen that has been injected. For example, if the antigen to which an immune response is desired is a virus or a tumor cell, the level of protection induced by an "immunologically effective amount" of the antigen is measured by determining the percentage survival or percentage mortality after exposure to the virus or tumor cells in animals.

A person skilled in the art can determine what amount of the vaccine according to the present invention is immunologically effective, for example, by monitoring the immunological response after vaccination or after exposure to an infection, for example, by re-isolating the pathogen or by observing clinical signs of disease or serological parameters in targets and comparing them with the responses observed in sham-vaccinated animals. The dosing regimen for using the vaccine of the present invention to a target organism can be presented as single or multiple doses that can be administered simultaneously or sequentially, in a manner compatible with the formulation of the vaccine, and in such an amount that will be immunologically effective.

The terms "inhibitors", "activators" and "modulators" of viral nucleic acid and polypeptide sequences are used to refer to activating, inhibiting or modulating molecules identified using in vitro and in vivo analyses of viral nucleic acid and polypeptide sequences. Inhibitors are compounds that can bind to, partially or completely block the activity of, reduce, prevent, delay the activation of, inactivate, desensitize or suppress the activity or expression of the virus. Activators refer to compounds that enhance, open, activate, facilitate, enhance the activation of, sensitize, agonize or potentiate the activity of the virus. Inhibitors, activators, or modulators also include genetically modified versions of the virus as described herein, such as variants with altered activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, small chemical molecules, and the like. Assays for inhibitors and activators include, for example, expressing the virus of the present invention in vitro, in cells or cell membranes, using putative modulator compounds, and then determining the functional effects on activity as described herein.

Test samples or assays containing the virus of the present invention that have been treated with a potential activator, inhibitor or modulator can be compared to a control sample that does not contain the inhibitor, activator or modulator to determine the degree of inhibition. The control samples to which the test sample or assay is compared can be assigned a relative protein activity value of 100%. Inhibition of the virus is achieved when the activity value of the test sample relative to the control sample is less than about 80%, including about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% and about 0%.

Intergenic loci, as described herein, are defined as the region of DNA sequence located between genes, including untranslated regions, 5' and 3' flanking regions, introns, etc. Intergenic regions are the portion of non-coding DNA that may contain gene control elements such as promoters and enhancers. The term "isolated" means a substance that has been substantially separated from or enriched relative to other substances with which it occurs in nature. Isolated substances are typically characterized by a purity of at least about 80%, at least 90%, at least 98%, or at least about 99% by weight.

A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include ^32P , fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin or haptens and proteins that can be made detectable, such as by incorporating a radioactive label into a peptide or using antibodies specifically reactive with the peptide for detection.

As used herein, the term "Marek's disease virus" or "MDV" refers to any alphaherpesvirus of the genus Mardivirus, which includes the herpesvirus of turkeys (HVT) as described herein. In a particular embodiment, the present invention relates to Marek's disease virus, its genetic components, genes and proteins produced by them. As used herein, such a virus may include the genetic components of the virus, i.e., the genome and its transcripts, the proteins encoded by the genome (including structural and non-structural proteins), and functional or non-functional viral particles. Polynucleotide and polypeptide sequences encoding such viruses are well known in the art and can be readily detected by one of skill in the art.

The terms "mutant" and "mutation" mean any detectable change in genetic material, such as DNA, or any process, mechanism, or result of such a change. They include gene mutations in which the structure (e.g., DNA sequence) of a gene is changed, any gene or DNA resulting from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" may also be used to refer to a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.

As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded polymer of deoxyribonucleotide bases or ribonucleotide bases read from the 5' end to the 3' end. A "nucleic acid" may also optionally contain unnatural or altered nucleotide bases that ensure proper reading by the polymerase and do not reduce expression of the polypeptide encoded by the nucleic acid. The term "nucleotide sequence" or "nucleic acid sequence" refers to both the sense and antisense strands of the nucleic acid, either as separate single strands or as a duplex. The term "ribonucleic acid" (RNA) includes RNAi (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro RNA), tRNA (transfer RNA charged with or freed from the appropriate acylated amino acid) and cRNA (complementary RNA). The terms "nucleic acid segment", "nucleotide sequence segment" or more generally "segment" will be understood by those skilled in the art as a functional term that includes genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences and smaller engineered nucleotide sequences that express or can be adapted to express proteins, polypeptides or peptides. This document uses the nomenclature required by 37 CFR §1.822 and set forth in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

The term "operably linked" is used herein to refer to an arrangement of flanking sequences, wherein the flanking sequences so described are configured or assembled to perform their normal function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting replication, transcription, and/or translation of the coding sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. The flanking sequence need not be adjacent to the coding sequence as long as it functions properly. Thus, for example, there may be intervening untranslated but transcribed sequences between a promoter sequence and a coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is physiologically compatible for administration to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, an excipient, a stabilizer, an adjuvant, a preservative, a diluent, an aqueous or non-aqueous vehicle, and other additives. Additionally, the term refers to an element of an immunogenic composition or vaccine that is generally approved by a regulatory agency of the federal, state, or other regulatory agency, or listed in the United States Pharmacopeia or other recognized pharmacopeia for use in both humans and non-human animals. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol, and the like. If necessary, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can also be formulated as a suppository, with conventional binders and carriers such as triglycerides. The oral formulation may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should be appropriate to the route of administration.

As used herein, the term "poultry" refers to domestic or commercial birds kept for the production of eggs, as well as their meat and feathers. In some embodiments, poultry may include birds of the Galliformes order, which includes chickens, quails, and turkeys, and may also include geese, ducks, swans, guinea fowl, pigeons, and the like.

The polynucleotides described herein may be complementary to all or part of a viral gene sequence, including a promoter, intron, coding sequence, exon, 5' untranslated region, and 3' untranslated region.

A defined nucleic acid sequence may also include "splice variants". Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. Splice variants are products of alternative splicing of a gene. Following transcription, the original nucleic acid transcript may be spliced such that different (alternative) nucleic acid splice products encode different polypeptides. The mechanisms by which splice variants are produced vary, but include alternative splicing of exons. Alternative polypeptides produced from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of the splicing reaction, including recombinant forms of the splice products, are included within this definition.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and a polymer of non-naturally occurring amino acids.

The terms "multivalent vaccine", "combination or combo vaccine" and "polyvalent vaccine" are used interchangeably to refer to a vaccine containing more than one antigen. A multivalent vaccine may contain two, three, four or more antigens. A multivalent vaccine may contain recombinant virus vectors, active, attenuated or killed wild-type viruses, or a mixture of recombinant virus vectors and wild-type viruses in active, attenuated or killed forms.

The term "promoters" as used herein refers to DNA sequences that determine the location where transcription of a gene begins by RNA polymerase. Promoters are typically located upstream of the transcription initiation site. A promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand nucleotides from the transcription start site. Promoters determine the direction of transcription and specify which strand of DNA will be transcribed. A promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate expression of a gene component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Typical examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the RSV-LTR promoter, the CMV IE promoter, the human CMV promoter; the mouse CMV promoter; the Rec promoter; the chicken β-actin promoter; the guinea pig CMV promoter, the pseudorabies virus promoter; the glycoprotein X promoter, the herpes simplex virus 1 promoter; the Marek's disease virus promoter; and the SV40 promoter.

As used herein, the terms "prophylactically treat" or "prophylactically treat" refer to the total or partial prevention of a disease or a symptom thereof and/or may be therapeutic in the sense of a partial or total cure of a disease and/or an adverse effect associated with the disease.

The term "recombinant," when used in reference to, for example, a cell or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by altering a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise expressed abnormally, underexpressed, or not expressed at all. In some embodiments, recombinant sequences may also include nucleic acids, proteins, or recombinant genomes, such as viral genomes. Recombinant viral vectors as described herein may contain transgenes that are operably linked to a heterologous promoter to effect transcription of the transgene.

The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target sequence, usually in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions may be sequence dependent and will vary under different circumstances. Longer sequences hybridize specifically at higher temperatures. Stringent conditions can be achieved by adding destabilizing agents such as formamide.

Suitable stringent conditions to promote DNA hybridization are well known to those skilled in the art and may include, for example, 6X sodium chloride/sodium citrate (SSC) at a temperature of about 45°C followed by a wash with 2X SSC at 50°C. The salt concentration in the wash step may be selected from a low stringency of about 2X SSC at 50°C to a high stringency of about 0.2X SSC at 50°C. The temperature in the wash step may be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. The temperature and/or salt conditions may be varied as appropriate to obtain optimal results. According to the present invention, a nucleic acid can exhibit at least about 80% to about 100% sequence identity with one or more nucleic acid molecules as described herein, such as at least about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity.

Nucleic acids that do not hybridize to each other under stringent conditions are still essentially identical if the polypeptides they encode are essentially identical. This occurs, for example, when a copy of a nucleic acid is made using the maximum codon degeneracy allowed by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

The term "therapeutically effective amount", "effective amount" or "therapeutically effective dose" as used herein refers to a dose that produces the effect for which it is administered. Such a dose or amount may also refer to the amount of the agent embodiment administered that will alleviate to some extent one or more symptoms of the disease, i.e., the infection being treated, and/or the amount that will prevent to some extent one or more symptoms of the disease, i.e., the infection, that the treated host has or is at risk of developing. The exact dose will depend on the purpose of treatment, and one skilled in the art will be able to determine such a dose using techniques known in the art.

As used herein, the term "transgene" refers to a segment of DNA containing a heterologous coding sequence or other genetic material for introduction from one organism to another. For example, in some embodiments, a transgene of the present invention may contain a sequence encoding an antigen, such as a viral gene, or a sequence encoding a viral protein.

As used herein, the terms "treating," "treating," and "treating" are defined as affecting a disease, disorder, or condition with an agent to reduce or improve the pharmacological and/or physiological effects of the disease, disorder, or condition and/or its symptoms. The term "treating," as used herein, encompasses any treatment of a disease in a subject or host (e.g., an animal of veterinary interest), and includes: (a) reducing the risk of developing the disease in a subject that is known to be predisposed to the disease but that has not yet been diagnosed as infected with the disease, (b) preventing the development of the disease, and (c) alleviating the disease, i.e., by inducing regression of the disease and/or alleviating one or more symptoms of the disease. The term "treating" is also intended to include delivery of an inhibitory agent to provide a pharmacological effect even in the absence of the disease or condition. For example, the term "treatment" includes delivery of a disease-inhibiting agent or pathogen that provides enhanced or desired effects in a subject (e.g., reduction in pathogen load, reduction in disease symptoms, etc.).

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for animal subjects, each unit containing a predetermined quantity of a compound (e.g., an antiviral compound as described herein) calculated in sufficient quantity to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of unit dosage forms depend on the particular compound employed, the route and frequency of administration, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The terms "vaccine" or "vaccine composition" as used interchangeably herein refer to pharmaceutical compositions comprising at least one immunogenic composition of the present invention that induces an immune response in a subject. The vaccine or vaccine composition can protect the subject from disease or possible death and may or may not contain one or more additional components that enhance the immunological activity of the active component. A composition of the present invention that induces a protective immune response comprises a recombinant HVT virus comprising one or more genes encoding a heterologous antigen inserted into the HVT genome at the UL 35/36 intergenic region. In some embodiments, a composition of the present invention comprises a recombinant HVT virus comprising one or more genes encoding a heterologous antigen inserted into the HVT genome at UL 35/36 and one or more genes encoding an antigen inserted into the HVT genome at UL55. In some embodiments, the genes encoding the antigen are antigens derived from poultry pathogens such as Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, avian influenza virus, infectious laryngotracheitis virus and/or chicken anemia virus. In some embodiments, the recombinant HVT is combined with another recombinant Marek's disease virus vaccine that elicits a protective immune response in poultry. The vaccine or vaccine composition of the present invention may further comprise additional components typical of vaccines or vaccine compositions, including, for example, an adjuvant or an immunomodulator. The vaccine may comprise one or more of the elements described above.

The vaccine of the present invention may further comprise a suitable pharmaceutical carrier. The term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and agents delaying absorption with respect to hosts. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier if the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions may also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition may be formulated with conventional binders and carriers such as triglycerides, depending on the route of administration. Certain formulations may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The composition should be appropriate to the route of administration. A suitable carrier is obvious to those skilled in the art and depends largely on the route of administration. Additional components that may be present in the present invention are adjuvants, preservatives, surface-active agents, chemical stabilizers, suspending or dispersing agents. Typically, stabilizers, adjuvants and preservatives are optimized to determine the best composition for effectiveness in the target subject.

A "variant" of a peptide refers herein to a peptide whose amino acid sequence differs from the amino acid sequence of a "parent" vaccine peptide due to the addition, deletion and/or substitution of one or more amino acid residues in the parent peptide sequence, and retains at least one desired activity of the parent vaccine peptide. For example, a variant may comprise at least one, such as from about one to about ten, and preferably from about two to about five substitutions in one or more amino acid sequences of a peptide used as part of a vaccine of the present invention. Typically, a variant will comprise an amino acid sequence having at least 50% amino acid sequence identity to the parent amino acid sequences, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95% sequence identity. Identity or homology to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to residues in the parent peptide, after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage sequence identity. No N-terminal, C-terminal or internal extensions, deletions or insertions in the peptide sequence should be considered as affecting the sequence identity or homology. The variant retains the ability to induce an immune response and is preferably characterized by a desired activity that is superior to that of the parent peptide.

Peptide variants may be fully functional or may lack one or more functions. Fully functional variants typically contain only conservative variations or variations in non-critical residues or in non-critical regions. Functional variants may also contain substitutions of similar amino acids that result in little or no change in function. Alternatively, such substitutions may have some positive or negative effect on function. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Moreover, polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification methods well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme, covalent attachment of nucleotide or nucleotide derivative, covalent attachment of lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination. Such modifications are well known to those skilled in the art and have been described in great detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, have been described in most of the major publications, such as Proteins-Structure and Molecular Properties (2nd ed., T. E. Creighton, W. H. Freeman & Co., NY, 1993). There are many detailed reviews on this subject, such as Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983); Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

Accordingly, the peptides of the present invention also encompass derivatives or analogs in which the substituted amino acid residue is not encoded by the genetic code. Likewise, additions and substitutions in the amino acid sequence, as well as the variations and modifications just described, may equally apply to the amino acid sequence of the antigen and/or its epitope or peptides and are thus encompassed by the present invention.

A "variant" of a nucleic acid refers herein to a molecule that differs in sequence from a "parent" nucleic acid. Divergence of a polynucleotide sequence may result from mutational changes such as deletions, substitutions, or additions of one or more nucleotides. Each of these changes may occur alone or in combination, one or more times in a particular sequence.

Just as a polypeptide may comprise conservative amino acid(s) substitution(s), its polynucleotide may comprise conservative codon(s). A codon substitution is considered conservative if, when expressed, it results in a conservative amino acid substitution as described above. A degenerate codon substitution that does not result in an amino acid substitution is also useful in the polynucleotides of the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide useful in an embodiment of the present invention may be mutated by a degenerate codon substitution to approximate the codon usage frequency exhibited by the expression host cell to be transformed therefrom, or otherwise improve its expression.

As used herein, the term "vector" refers to a construct that is capable of delivering and preferentially expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. Vectors, as described herein, contain expression control sequences, which means a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is "operably linked" to the nucleic acid sequence to be transcribed. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As used herein, the term "viral proteins" or "viral polypeptides" refers to a protein encoded by a virus described herein, including structural and non-structural proteins. Such proteins may include naturally occurring or non-naturally occurring MDV, NDV and/or IBDV viral proteins, including the VP2, F and/or HN, NP, P, M or L proteins. Such proteins may also include naturally occurring or non-naturally occurring ILTV viral proteins such as gB, gC, gD, gE, gH, gI or gL, infectious bronchitis virus (IBV) S1, S2 or M proteins, chicken anemia virus (CAV) VP1 or VP2 proteins and/or any of the HA, NA, NP or M proteins of avian influenza virus (AIV).

According to the present invention, the recombinant viral vectors described herein can provide protection to poultry against two or more different viral pathogens by administering recombinant viral vectors that express genes from such viral pathogens. In some embodiments, the recombinant viral vectors of the present invention can be administered to poultry in an immunogenic composition as described herein. Genes from any viral pathogen suitable for use with a recombinant viral vector as described herein can be used. For example, in some embodiments, a recombinant viral vector can express genes from Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), chicken anemia virus (CAV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV), or the like.

According to the present invention, a transgene that provides protection or resistance to a particular virus or viruses can be inserted into the viral genome at a particular location. For example, in some embodiments, a transgene described herein can be inserted into the viral genome in the intergenic region flanked by HVT UL 35/UL 36 in a unique long region of the genome. In another embodiment of the present invention, a transgene described herein comprises one or more heterologous genes inserted into the viral genome in the intergenic region flanked by HVT UL 35/UL 36 of the HVT genome, in addition to the second usable site, wherein the one or more heterologous genes are inserted into the UL55 site in the HVT genome. In other embodiments, more than one transgene can be inserted into one or both of these regions.

In some embodiments, a recombinant virus vector may express multiple genes from a single virus species or may express genes from more than one virus species to provide resistance to multiple viruses. For example, in one embodiment, the present invention provides a recombinant virus vector comprising an HVT genome and at least one transgene from another viral pathogen, thereby providing protection to a bird, such as poultry, from Marek's disease and at least one other viral disease. For example, in one embodiment, a recombinant virus vector according to the present invention may provide protection to poultry from MDV and NDV, or may provide protection from MDV and IBDV, or may provide protection from MDV, NDV and IBDV.

Viral antigens for expression in poultry using the recombinant viral vector of the present invention may be encoded by a viral gene, such as the viral gene described herein. One skilled in the art will appreciate in this regard that it may not be necessary to include the entirety of a particular viral gene to obtain the desired viral resistance. Rather, a portion of such a gene may be used. It may be desirable to select a particular portion of the desired gene that is specific for any particular target virus or viruses. Optimization of the desired viral protein or sequence encoding such a protein, regardless of the length of the protein, can be readily accomplished using methodologies known in the art that are suitable for use in the present invention. One skilled in the art will appreciate that modifications can be made to the viral gene or genes or proteins encoded by them to enhance the activity of the viral protein when administered to a subject. Modifications made to the viral genes or proteins can enhance or reduce the host response to a specific virus.

In certain embodiments, a recombinant Marek's disease virus or a recombinant viral vector of the present invention may comprise a transgene encoding an IBDV viral protein or gene product, such as the IBDV VP2 protein or gene product. In another embodiment, such a recombinant virus or viral vector may comprise a transgene encoding an NDV viral protein or gene product, such as the NDV F or HN protein or gene product. In another embodiment, such a recombinant virus or viral vector may comprise a transgene encoding an avian influenza virus (AIV) viral protein or gene product, such as the AIV HA or N protein or gene product. In another embodiment, such a recombinant virus or viral vector may comprise a transgene encoding an infectious bronchitis virus (IBV) viral protein or gene product, such as the IBV S1 or S2 protein or gene product. The transgene of the present invention may comprise more than one gene, including a gene-based fusion protein or gene product, such as a fusion protein, chimera, or NDV F-HN gene product. In some embodiments, the entire coding sequence of such a gene may be used to produce a full-length or fully functional protein or polypeptide. Alternatively, a portion or fragment of a viral protein or polypeptide may be sufficient to provide protection against or resistance to a particular virus or viruses.

In certain embodiments, the recombinant Marek's disease virus or recombinant virus vector of the present invention may comprise a transgene encoding an immunomodulator, such as a cytokine protein or gene product. According to the present invention, the cytokine may be an interleukin (IL), including but not limited to IL2, IL6, IL7, IL8, IL12, IL18, etc. Such a transgene encoding a cytokine may be inserted into one or both genomic sites as described herein. In some embodiments, the transgene encoding a cytokine may be inserted into one site described herein and the transgene encoding a viral protein may be inserted into the other site. Other immunomodulators such as interferons, chemokines, glucans, granulocyte colony stimulating factors, oligodeoxynucleotides may also be used in accordance with the present invention.

Isolation of viral genes or proteins

In embodiments of the present invention, the viral gene described herein can be isolated using nucleic acid probes and/or oligonucleotides under stringent hybridization conditions, PCR or microarrays, DNA library screening, or any other methods known in the art. It will be readily apparent to one skilled in the art how to isolate viral genes or proteins for use in the present invention. Alternatively, expression libraries can be used to clone the virus, its polymorphic variants, orthologs, or alleles by immunologically detecting homologs using antiserum or purified antibodies directed against another species of virus or portions thereof.

Methods for creating and screening cDNA libraries are well known in the art. For example, to create a cDNA library for cloning viral genes expressed by the genome, mRNA can be reverse transcribed into cDNA using reverse transcriptase. The cDNA can then be ligated into a vector, such as a recombinant vector, and introduced into a host cell or organism for propagation, screening, and cloning.

For a genomic library, DNA may be extracted from the desired tissue and may be digested using biological enzymes or may be mechanically digested. The resulting DNA fragments may then be isolated from unwanted DNA fragments and constructed into an appropriate vector, which may then be packaged in vitro. Recombinant vectors may be analyzed by any method known in the art.

Methods such as polymerase chain reaction (PCR and RT-PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences directly from mRNA, from genomic libraries, or from cDNA libraries. Degenerate oligonucleotides can be designed to amplify homologues using the sequences provided herein. Restriction endonuclease sites can be included in the primers. Polymerase chain reaction or other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences that encode proteins to be expressed to produce nucleic acids that can be used as probes to detect the presence of a disease to be targeted, such as MDV, NDV and/or IBDV mRNA encoding, in biological samples, for nucleic acid sequencing, or for other purposes. Genes amplified by PCR can be purified from agarose and cloned into an appropriate vector.

Expression of viral genes can also be analyzed by techniques known in the art, such as reverse transcription and mRNA amplification, isolation of total RNA or polyA RNA, Northern blotting, dot blotting, in situ hybridization, RNase protection, high-density polynucleotide array technology, etc.

Nucleic acids encoding a viral genome or protein can be used via high-density oligonucleotide array technology (e.g., GeneChip™) to identify viral genes, orthologs, alleles, variants, and polymorphic variants thereof in the present invention. The selected gene can be cloned into an intermediate vector prior to transformation into prokaryotic or eukaryotic cells for replication and/or expression. These intermediate vectors can be prokaryotic vectors, such as plasmids or shuttle vectors.

Modification of nucleic acids

Any number of methods well known to those skilled in the art may be used to isolate and manipulate a DNA molecule. For example, polymerase chain reaction (PCR) technology may be used to amplify a particular original DNA molecule and/or to produce variants of the original DNA molecule. DNA molecules or fragments thereof may also be produced by any of the techniques known in the art, including direct synthesis of a fragment by chemical means. In this manner, all or part of a nucleic acid may be synthesized as described herein.

As used herein, the term "complementary nucleic acids" refers to two nucleic acid molecules that are capable of specifically hybridizing with each other, wherein the two molecules can form an antiparallel double-stranded nucleic acid structure. In this regard, a nucleic acid molecule is said to be complementary to another nucleic acid molecule if they exhibit complete complementarity. Two molecules are said to be "minimally complementary" if they can hybridize to each other with sufficient stability to allow them to remain annealed from each other under at least ordinary low stringency conditions. Similarly, molecules are said to be complementary if they can hybridize to each other with sufficient stability to allow them to remain annealed from each other under ordinary high stringency conditions. Ordinary stringency conditions are described by Sambrook, et al. (1989) and Haymes et al. (1985).

Deviations from perfect complementarity are permissible as long as the ability of the molecules to form a double-stranded structure is retained. Thus, for a nucleic acid molecule or fragment of a nucleic acid molecule to act as a primer or probe, such a molecule or fragment must be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the specified conditions of solvent and salt concentration used.

As used herein, the terms "sequence identity", "sequence similarity" or "homology" are used to describe the sequence relationships between two or more nucleotide sequences. The percentage of "sequence identity" between two sequences is determined by comparing two optimally aligned sequences over a specified number of nucleotides, wherein a portion of the sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence. Two sequences are said to be identical if the nucleotides at each position are the same. A nucleotide sequence observed in the 5' to 3' direction is said to be the "complement" or complementary to a second nucleotide sequence observed in the 3' to 5' direction if the first nucleotide sequence exhibits complete complementarity with the second or reference sequence. As used herein, nucleic acid sequence molecules are said to exhibit "perfect complementarity" when every nucleotide of one of the sequences, read in the 5' to 3' direction, is complementary to every nucleotide of the other sequence, read in the 3' to 5' direction. A nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit a sequence that is identical to the reverse sequence of the complement of the reference nucleotide sequence.

Recombinant vectors and host cells

A recombinant DNA vector may be, for example, a linear or circular plasmid. A vector system may be a single vector or plasmid, or two or more vectors or plasmids that together contain a common DNA for introduction into the genome of a host cell. A recombinant vector described herein may be an expression vector, for example, to provide for the production of a desired protein in a host cell, such as a bacterial cell. The nucleic acid molecules described herein, or complements or fragments thereof, may be inserted into a vector under the control of a suitable promoter that functions in one or more microbial hosts to direct the expression of the associated coding sequence or other DNA sequence. Many vectors are available and known in the art for this purpose, and the choice of a suitable vector depends on the size of the nucleic acid to be inserted into the vector and the host cell to be transformed with the vector. Each vector may contain different components depending on its function (e.g., DNA amplification or DNA expression) and the particular host cell with which it is compatible. Components of a bacterial transformation vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selectable marker genes, and an inducible promoter that allows expression of exogenous DNA.

As used herein, the term "recombinant Marek's disease virus" or "recombinant HVT" or "recombinant virus" refers to an infectious virus or viral particle that has been genetically modified by the incorporation into the viral genome of one or more heterologous nucleic acid sequences, i.e., DNA encoding a viral gene or a fragment or portion thereof that is not identical to the nucleic acid sequence of the gene naturally present in the virus. When a cell is infected with the recombinant Marek's disease virus, the recombinant virus expresses the heterologous gene in the form of a heterologous polypeptide.

The term "recombinant virus vector" or "viral vector" as used herein refers to a recombinant construct that is inserted into a virus for introduction into a host cell. Such a vector according to the present invention can be obtained from any strain of HVT. If necessary, viral genes or protein encoding sequences can be included in such a recombinant virus vector as described herein for introduction into a chicken or other poultry for protection against one or more viral diseases.

As used herein, the term "insertion site" refers to a region of the viral genome into which transgenic or exogenous DNA is inserted. The insertion sites of the present invention may be intergenic regions. The intergenic region of the present invention may be flanked by HVT UL35 and HVT UL36 in a unique long region of the genome. In some embodiments of the present invention, one or more heterologous nucleotides encoding antigens may also be inserted into regions defined by the UL55 locus of the HVT genome. In some embodiments, the insertion sites of the present invention may include all or part of the flanking gene on either side of the intergenic region. Insertion of one or more transgenes into one of these regions produces a recombinant virus vector that can then be administered to a chicken or other poultry to protect against one or more diseases.

As used herein, the term "operably linked" when used with respect to a regulatory sequence and a nucleotide sequence means that the regulatory sequence causes the regulated expression of the associated structural nucleotide sequence. The terms "regulatory sequences," "regulatory elements," or "control elements" refer to nucleotide sequences located upstream (5' sequences), within, or downstream (3' sequences) of a structural nucleotide sequence. Such sequences influence the timing and level or amount of transcription, RNA processing or stability, or translation of the associated structural nucleotide sequence. Regulatory sequences may include, but are not limited to, promoters, leader sequences, introns, enhancers, stem-loops, repressor binding sequences, and polyadenylation recognition sequences, including, but not limited to, the bovine growth hormone polyA signal sequence, the simian virus 40 (SV40) polyA signal sequence, the Autographa californica nuclear nojm3ppo3a (AcNPV) polyA 1629 ORF signal sequence, and the herpes simplex virus (HSV) thymidine kinase (TK) polyA signal sequence. One skilled in the art will appreciate that various combinations of promoters and/or regulatory elements may be used to increase or decrease expression of a transgene as described herein.

Promoters that function in different species are also well known in the art. Promoters useful for expressing polypeptides include promoters that are inducible, viral, synthetic, or constitutive, and/or promoters that are tissue-specific, temporally regulated, spatially regulated, and spatially and temporally regulated. For example, promoters useful in accordance with the present invention may include, but are not limited to, the immediate early (IE) cytomegalovirus (CMV) promoter, the guinea pig CMV promoter, the SV40 promoter, pseudorabies virus promoters such as the glycoprotein X promoter, the herpes simplex virus 1 promoter such as the alpha-4 promoter, Marek's disease virus promoters including any MDV isolate or strain such as MDV-1, MDV-2 and HVT, for example, a promoter controlling the expression of glycoproteins such as gC, gB, gE or gI, infectious laryngotracheitis virus promoters such as the glycoprotein gB, gE, gI, gD promoters, or any other suitable promoters. One skilled in the art will know how to identify a promoter useful in accordance with the present invention.

According to the present invention, a recombinant Marek's disease virus or a recombinant virus vector described herein may comprise one or more transgenes operably linked to one or more promoters for expressing one or more viral proteins or peptides, or fragments or portions thereof. In some embodiments, one transgene may be operably linked to one promoter, or multiple transgenes may be operably linked to one promoter. In other embodiments, more than one transgene may be present in a recombinant vector, wherein a first transgene is operably linked to a first promoter and a second transgene is operably linked to a second promoter.

Construction and selection of vectors

Those skilled in the art are familiar with the construction of vectors containing one or more of the components described herein, useful for inserting genes or transgenes or portions thereof into a target site, and can employ standard recombinant DNA techniques. The vector or recombinant DNA construct may contain a selectable marker that confers a selectable phenotype on the cell. Selectable markers may also be used to select cells containing exogenous nucleic acids encoding polypeptides or proteins as described herein. Such a marker may encode, for example, biocide resistance or antibiotic resistance (e.g., kanamycin, G418, bleomycin, hygromycin, etc.). Selectable markers are well known to those skilled in the art and may include any markers suitable for use in accordance with the present invention.

The recombinant vector or construct may also include a screenable marker that can be used to monitor expression, but which may not result in cell death. Suitable markers for screening may include, for example, the β-glucuronidase or uidA (GUS) gene, one or more different fluorescent protein genes such as green fluorescent protein (GFP), red fluorescent protein (RFP), or any of a large family of proteins that fluoresce at characteristic wavelengths, a gene that encodes an enzyme for which various chromogenic substrates are known, a luciferase gene, a xylE gene that encodes a catechol dioxygenase that converts chromogenic catechols, a β-amylase gene, a tyrosinase gene that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn are condensed into melanin, or an α-galactosidase that catalyzes the chromogenic substrate α-galactose.

Expression of proteins in host cells

To obtain high level expression of the cloned viral gene as described herein, the nucleic acid can be subcloned into an expression vector that contains a strong promoter to direct transcription and a transcription/translation terminator. For the encoded proteins, a ribosome binding site for translation initiation can also be included. Suitable promoters for use in expression vectors are well known in the art, such as a bacterial promoter, a viral promoter, and the like. Expression systems for protein expression are available in several prokaryotic and eukaryotic species known in the art. Commercial kits for such expression systems are also readily available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

The selection of a suitable promoter for direct expression of a heterologous nucleic acid will depend on the particular application. Such a promoter may be located at a distance from the heterologous transcription initiation site similar to the distance in its natural environment, although one skilled in the art will appreciate that some variation in this distance may be permitted without loss of promoter function.

In addition to the promoter, the expression vector typically contains a transcription or expression cassette, which contains all the elements necessary for expression of the nucleic acid in the host cell. Any standard vectors known in the art that can be used for expression in eukaryotic or prokaryotic cells can be used to transfer the genetic information into the cell. Thus, a typical expression cassette contains a promoter operably linked to a nucleic acid sequence encoding the selected nucleic acid and appropriate signal sequences necessary for efficient processing, such as ribosome binding, polyadenylation, and translation termination sites. Additional elements may include enhancers and, in the case of genomic DNA as a structural gene, introns with functional splice donor and acceptor sites.

In addition to a promoter sequence, such as the promoter described herein, the expression cassette may also contain a transcription termination region downstream of the structural gene to ensure efficient transcription termination. The termination region may be from the same gene as the promoter sequence, or it may be from another gene. Markers such as fluorescent proteins, green or red fluorescent protein, β-gal, CAT, etc., may be included in the vectors as markers for vector transduction. Epitope tags or sequence tags may also be added to the recombinant proteins to provide convenient isolation methods.

Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40-based vectors, papillomavirus-based vectors, retroviral vectors, and Epstein-Barr virus-derived vectors. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, pDSVE baculovirus, and any other vector that provides for the expression of proteins under the control of the CMV promoter, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or other promoters known in the art that can be effective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can also be regulated using inducible promoters. With inducible promoters, expression levels are linked to the concentration of inducing agents such as tetracycline or ecdysone by incorporating response elements for these agents into the promoter. High expression levels can be obtained from inducible promoters in the presence of the inducing agent. Some expression systems contain markers such as thymidine kinase and dihydrofolate reductase that allow gene amplification.

The expression vector may also contain a replicon that functions in E. coli, an antibiotic resistance gene for selection of bacteria containing the recombinant plasmids, and unique restriction sites in non-essential regions of the plasmid to allow insertion of eukaryotic sequences. Any antibiotic resistance gene suitable for use in the present invention may be used.

Standard transfection techniques known in the art can be used to obtain bacterial, mammalian, yeast or insect cell lines that express significant amounts of the protein. Such cell lines can then be purified using standard techniques known in the art, and prokaryotic and/or eukaryotic cells can be transformed according to any method known in the art to introduce cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into the host cell. Such methods can include, but are not limited to, plasmid or viral vectors, calcium phosphate transfection, protoplast fusion, electroporation, biolistics, liposomes, microinjection or any methods available in the art.

After introducing the expression vector or transgene into the host cell, the cell can then be cultured under conditions optimal for expression of the desired protein, which can be isolated using standard techniques known in the art. Viral pathogens or viral proteins, such as those described herein, can then be purified for use in diagnostic assays, for the production of antibodies and immunogenic compositions, and for the identification of antiviral compounds. Naturally occurring proteins can be purified from biological samples, such as a tissue sample from a bird infected with a virus, as described herein, while recombinant proteins can be purified using any suitable methods or expression systems known in the art.

A number of procedures for purifying recombinant proteins are available in the art. For example, proteins characterized by certain molecular adhesion properties can be reversibly fused to another protein. In addition, a particular protein can be selectively adsorbed onto a purification column and then isolated from the column in relatively pure form using appropriate ligands or substrates. The fusion protein can then be removed by enzymatic activity. The protein can also be purified using affinity columns. The recombinant protein can be purified from any suitable source.

Protein purification from recombinant bacteria

Recombinant proteins can be expressed in large quantities by bacteria, for example, using an inducible or constitutive promoter. Promoter induction using IPTG is an example of an inducible promoter system. Bacteria can be grown from fresh or frozen culture according to standard procedures known in the art.

Proteins expressed in bacteria may form insoluble aggregates called inclusion bodies. Suitable protocols for purifying protein inclusion bodies are known in the art. Lysing the bacteria to isolate the expressed proteins may be accomplished using any of the methods known in the art, which may include the introduction of chemical buffers, sonication, mechanical disruption, etc. Inclusion bodies may also be solubilized, and the lysed cell suspension may be centrifuged to remove unwanted cellular debris. Inclusion body proteins may be renatured by dilution or dialysis with an appropriate buffer.

Recombinant proteins can also be obtained from the periplasm of bacteria. After lysis of bacterial cells, the periplasmic fraction of bacteria can be isolated by any methods known in the art. Recombinant proteins present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art.

Proteins can be separated using any of the techniques known in the art, such as solubility-based fractionation or size-based differential filtration, in which the protein is isolated based on molecular weight using filtration through membranes of different pore sizes. Column chromatography can be used to separate a protein from other proteins based on size, net surface charge, hydrophobicity, or affinity for ligands or substrates. Additionally, antibodies raised against the protein of interest can be conjugated to the column and the proteins immunopurified. All of these methods are well known to those skilled in the art. It will be apparent to those skilled in the art that the chromatographic techniques can be applied at any scale and using any suitable commercial equipment. Antibody Production

Methods for producing polyclonal and monoclonal antibodies that specifically react with viral proteins, viral particles and/or nucleic acids are known in the art. Such methods may include producing antibodies by selecting antibodies from recombinant antibody libraries in phage or other vectors, as well as producing polyclonal and monoclonal antibodies by immunizing rabbits or mice.

A number of antigens or antigenic regions comprising a viral protein or portions thereof, viral particles and/or nucleic acids can be used to produce antibodies that specifically react with a desired viral pathogen. For example, a recombinant viral protein or antigenic fragment thereof can be isolated using any of the methods described herein or known in the art. Recombinant proteins can be expressed in prokaryotic or eukaryotic cells and purified as described herein. Monoclonal and/or polyclonal antibodies can be produced using naturally occurring (pure or mixed) or recombinant proteins using methods known in the art. Synthetic peptides derived from a viral sequence can also be used to produce antibodies and can be conjugated to a carrier protein and administered to an animal capable of producing antibodies (e.g., a rabbit).

Methods for producing polyclonal antibodies are known to those skilled in the art. For example, an inbred strain of mice or rabbits can be immunized with a protein using a standard adjuvant, such as the adjuvant described herein, using a standard immunization protocol known in the art. When sufficiently high titers of antibodies to the protein are obtained, antiserum can be prepared and enriched to obtain antibodies that react with the protein.

Monoclonal antibodies can also be produced by various methods known in the art. For example, spleen cells from an animal immunized with a desired antigen can be immortalized, typically by fusion with a myeloma cell or by transformation with Epstein-Barr virus (EBV), oncogenes or retroviruses, or other methods well known in the art. The immortalized cells can then be screened for the production of antibodies with the desired specificity and affinity for the antigen. The yield of monoclonal antibodies produced by such cells can be increased by various techniques known in the art, for example by injection into the peritoneal cavity of a vertebrate host.

Monoclonal antibodies and polyclonal sera can be collected and titrated against the desired antigen or protein in an immunoassay, such as a solid-phase immunoassay with the protein immobilized on a solid support. Antibodies specific only for a particular viral protein can also be obtained by removing other cross-reacting proteins. In this way, antibodies can be obtained that bind only to the selected protein.

When specific antibodies against a desired viral antigen, such as a protein, virus and/or nucleic acid, become available, the desired antigen can be detected using various immunoassay methods. The antibody can also be used therapeutically.

A protein, either associated with or distal to a viral particle, as described herein, can be detected and/or quantified using any of a number of well-known immunoassays for binding. Viral particles can be detected based on an epitope defined by viral proteins presented in the viral particle and/or an epitope defined by a viral protein that is distal to the viral particle (e.g., such as may be present in an infected cell). Immunoassays can utilize an antibody that specifically binds to a selected protein or antigen. The antibody can be produced by any of a number of methods well known to those skilled in the art. Immunoassays can also utilize a labeling agent to specifically bind a complex formed by an antibody and an antigen for detection purposes. The labeling agent itself can be one of the moieties comprising the antibody/antigen complex. Thus, the labeling agent may be a labeled viral protein nucleic acid or a labeled antiviral antibody. Alternatively, the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody/antigen complex. The secondary antibody may be specific for antibodies of the species from which the first antibody is derived. The labeling agent may be modified with a detectable moiety, such as biotin, to which another molecule, such as streptavidin, can specifically bind. Various detectable moieties are well known to those skilled in the art.

Immunoassays for detecting viral protein, virus and/or nucleic acid in samples are well known in the art. Such assays may be either competitive or non-competitive, and quantitative or non-quantitative. Non-competitive immunoassays are assays in which antigen can be directly detected and, in some cases, the amount of antigen is directly measured. In competitive assays, viral antigen present in a sample is detected indirectly by a detectable signal associated with a known added (exogenous) viral antigen displaced from an antibody to an antiviral antigen by viral antigen present in the sample. Thus, such assays can also be adapted to indirectly measure the amount of viral antigen present in a sample. Competitive binding immunoassays can also be used to determine cross-reactivity, in which any cross-reacting antibodies can be removed from the pooled antiserum. Additional types of assays, including but not limited to Western blotting or liposome-based immunoassays, may also be used in accordance with the present invention.

It will be appreciated by those skilled in the art that it is often desirable to minimize non-specific binding in immunoassays. In particular, when the assay involves an antigen or antibody immobilized on a solid substrate, it is desirable to minimize the extent of non-specific binding to the substrate. Means for reducing such non-specific binding are well known to those skilled in the art.

The assay described herein may include a label or detectable group that does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group may be any material characterized by a detectable physical or chemical property. Such detectable labels are known in the art, and in general, any label used in such methods can be used in the present invention. Thus, the term "label" as used herein may be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention may include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive labels (e.g., ^3H , ^125I , ^35S , ^14C , or ^32P ), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and/or any others known in the art and used in ELISA), and colorimetric labels such as colloidal gold, colored glass, or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

The label according to the present invention can be linked directly or indirectly to the desired component of the assay according to methods well known in the art. As described above, a wide range of labels can be used, with the choice of label depending, among other things, on sensitivity, ease of conjugation with the compound, stability requirements or available equipment.

Non-radioactive labels can be applied by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently linked to the molecule. The ligand can then be linked to another molecule (e.g., streptavidin), which can be detected either by itself or covalently linked to a signaling system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Ligands and their corresponding targets can be used in any suitable combination with antibodies that recognize the viral antigen or secondary antibodies that recognize the antiviral antigen. Molecules can also be conjugated directly to signal-generating compounds, such as by conjugation to an enzyme or fluorophore. Enzymes of interest for use as labels can be hydrolases, such as phosphatases, esterases, and glycosidases, or oxidotases, such as peroxidases. Fluorescent compounds may include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds may include luciferin, 2,3-dihydrophthalazinediones such as luminol, or other compounds known in the art.

Methods for detecting labels are well known to those skilled in the art and will depend on the type of label used. For example, autoradiographic analysis can be used to detect a radioactive label, and fluorochromes can be used to detect a fluorescent label. Fluorescence can be detected visually, for example, by electronic detectors such as charge-coupled devices (CCDs) or photomultipliers, etc. Similarly, enzymatic labels can be detected by introducing appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels can be detected by observing the color associated with a particular label. In some embodiments, assay formats may not require the use of a labeled component, but can be detected by simple visual inspection.

Pharmaceutical/immunogenic compositions and their administration

In some aspects, recombinant vectors comprising one or more transgenes expressing one or more viral proteins or peptides or fragments thereof as described herein can be used as pharmaceutical compositions or immunogenic compositions for administration to a subject, such as a chicken or other poultry, to provide protection against one or more viruses. For example, an immunogenic composition described herein comprises a recombinant vector with one or more transgenes described herein inserted into a viral genome, such as in an intergenic region flanked by the UL 35/UL 36 intergenic loci in a unique long (UL) region of the HVT genome. In one aspect, the present invention provides a recombinant turkey herpes virus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL 35/UL 36 intergenic loci in the unique long region of the HVT genome and one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL55 site of the unique long region (UL) of the HVT genome.

In other aspects, proteins or peptides and immunogenic fragments thereof and/or polynucleotides, as well as antiviral antibodies and/or T cells, can be included in pharmaceutical compositions or immunogenic compositions (e.g., vaccines). In another embodiment, an immunogenic composition of the present invention can contain at least a third transgene, a fourth transgene, etc., which can encode additional viral proteins. Thus, an immunogenic composition can be administered to a subject, such as poultry, that provides protection against any desired number of viruses. A whole virus vaccine (live and attenuated, or replication incompetent, or killed) or subunit vaccines, such as structural or non-structural viral proteins or immunogenic fragments thereof, can be used to treat or prevent viral infections by inducing an immune response in a subject. Alternatively, the pharmaceutical composition may comprise an antigen-presenting cell transfected with a viral polynucleotide such that the antigen-presenting cell expresses the viral peptide.

Immunogenic compositions according to the present invention can be designed to generate antibody immunity and/or cellular immunity in a subject. Such compositions can comprise one or more such compounds together with a non-naturally occurring pharmaceutically acceptable carrier. In other embodiments, an immunogenic composition according to the present invention can include more than one adjuvant or pharmaceutically acceptable carrier, of which at least one is non-naturally occurring. A pharmaceutically acceptable carrier or adjuvant can be any substance that enhances the immune response in a subject to an exogenous antigen, including but not limited to adjuvants, liposomes, biodegradable microspheres. A pharmaceutically acceptable carrier or adjuvant can comprise a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, or a stimulator of immune responses, such as proteins derived from Bortadella pertussis or Mycobacterium tuberculosis. Commercially available adjuvants may include, for example, Freund's incomplete and complete adjuvant, Merck 65 adjuvant, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; CpG oligonucleotides, calcium, iron or zinc salts; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and monophosphoryl lipid A. One skilled in the art will be able to identify suitable pharmaceutically acceptable carriers for use in the present invention.

Pharmaceutical or immunogenic compositions and/or vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other antigens may be present either as part of a fusion polypeptide or as a separate compound in a composition or vaccine of the present invention. In some embodiments, the polypeptides used in the present invention may be conjugated to other macromolecules. Pharmaceutical or immunogenic compositions and vaccines may generally be used for prophylactic and/or therapeutic purposes. For example, in accordance with the present invention, a composition described herein may be administered to a subject, such as a bird, prior to infection with or exposure to a virus, to provide protection against infection with one or more viruses or the development of symptoms of an infection. In other embodiments, such a composition may be administered to a subject, such as a bird, after infection with or exposure to one or more viruses, to provide treatment of the viruses in the subject, such as by reducing or eliminating the infection in the subject.

Nucleic acid vaccines encoding a genome, structural or non-structural protein, or fragment thereof of a virus described herein may also be used to induce an immune response to treat or prevent a viral infection. Numerous gene delivery techniques are well known in the art. Suitable nucleic acid expression systems may contain the necessary DNA sequences for expression in a subject (such as a suitable promoter and termination signal sequence). In some embodiments, the DNA described herein may be administered using a viral expression system (e.g., Marek's disease virus or HVT), which may involve the use of a non-pathogenic virus capable of replication.

The pharmaceutical or immunogenic compositions may be supplied in single-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be sealed to maintain the sterility of the composition until use. Typically, the compositions described herein may be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, such a composition may be stored in a lyophilized state, requiring only the addition of a sterile liquid vehicle immediately prior to use.

As described herein, the immunogenic composition can be combined with a pharmaceutically acceptable carrier. The choice of a suitable carrier can be determined in part by the particular composition being administered (e.g., a nucleic acid, protein, modulator compounds, or a transduced cell), as well as the particular route used to administer the composition. Accordingly, a wide variety of suitable formulations of pharmaceutical or immunogenic compositions are available that can be used in the present invention. Administration can be by any convenient route, such as injection, oral administration, inhalation, transdermal application, or rectal administration. The injection of a recombinant vector or immunogenic composition as described herein can be provided to a subject, such as a poultry animal, as a single administration or dose, or can be administered more than once, such as in repeated doses.

Formulations suitable for parenteral administration, such as, for example, intra-articular (into joints), intravenous, intramuscular, intradermal, intraperitoneal, in ovo and subcutaneous administration, include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that make the formulation isotonic with the blood of the intended subject, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. In the practice of the present invention, the compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.

Such compositions may also contain buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that make the formulation isotonic, hypotonic, or slightly hypertonic with respect to the subject's blood, suspending agents, thickening agents, and/or preservatives. Alternatively, the compositions of the present invention may be formulated as a lyophilisate. The compounds may also be encapsulated in liposomes using methods known in the art.

Injectable solutions and suspensions can be prepared from sterile powders, granules and tablets as described herein. Cells transduced with nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above. An injection as described herein can contain a suspension of one or more killed, inactivated, attenuated or otherwise non-virulent viral cultures, a purified or unpurified solution of viral protein or nucleic acid as described herein. An injection solution can also contain a pharmaceutically acceptable carrier as described herein.

Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of packaged viral protein or nucleic acid, suspended in diluents such as water, saline or PEG 400; (b) capsules or tablets, each containing a predetermined amount of the active ingredient as a liquid, solid, granules or gelatin; (c) a suspension in an appropriate liquid; or (d) suitable emulsions. Tablet forms may include lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other auxiliary substances, colorants, fillers, binders, diluents, buffering agents, wetting agents, preservatives, flavorings, dyes, disintegrants and pharmaceutically compatible carriers. Lozenges may contain the active ingredient in a flavoring agent, such as sucrose, as well as lozenges containing the active ingredient in an inert base such as gelatin and glycerol or emulsions of sucrose and acacia, gels, etc., containing, in addition to the active ingredient, carriers known from the prior art.

The selected compound, alone or in combination with other suitable components, can be prepared as aerosol formulations for administration by inhalation. Aerosol formulations can be placed in suitable propellants under pressure, such as dichlorodifluoromethane, propane, nitrogen, etc.

The dose administered to a subject in the context of the present invention should be sufficient to provide a positive therapeutic response in the subject over time. The dose will be determined by the potency of the particular vector used and the condition of the subject, as well as the body weight and/or surface area of the patient being treated. The size of the dose may also be determined by the presence, nature, and extent of any adverse side effects that accompany the administration of the particular vector or transduced cell type in the particular patient. For vector-containing compositions as described herein, the effective amount of vector administered may be determined in part based on circulating plasma levels of the vector, vector toxicity, the health of the subject, and the production of antibodies to the vector.

With respect to administration, the compounds and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the inhibitor, vector or transduced cell type, as well as the side effects of the inhibitor, vector or cell type at various concentrations, in relation to the weight and general health of the subject. Administration can be carried out in single, multiple or divided doses.

Immunological determination of polypeptides and nucleic acids

Immunoassays can be used to detect viral proteins, viral particles, and/or nucleic acids. Such assays can be useful for therapeutic and/or diagnostic applications, such as those described herein. Immunoassays are well known in the art and can be used to qualitatively or quantitatively analyze proteins, viral particles, and/or nucleic acids.

Analysis of viral proteins and antibodies to viral antigens

In one embodiment of the present invention, the presence of a virus as described herein, a viral nucleic acid, or a viral protein in a sample can be determined by an immunoassay. Enzyme-mediated immunoassays such as immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), capture assays, microagglutination tests, and immunoblotting assays (e.g., Western blotting) can be readily adapted to detect a virus or viral proteins. An ELISA method can be effective for detecting a virus or viral protein as described herein. Such an ELISA can, for example, include the steps of: (1) binding an antiviral antibody or antigen to a substrate; (2) contacting the bound receptor with a biological sample containing the virus, viral antigen, viral protein, or antibodies to the virus; (3) contacting the biological sample with an antibody linked to a detectable moiety (e.g., a horseradish peroxidase enzyme or an alkaline phosphatase enzyme); (4) contacting the biological sample with a substrate for the enzyme; (5) contacting the biological sample with a detection reagent, such as a staining reagent; (6) observing a detectable result. In some embodiments, a biological sample suitable for use in such an ELISA can be blood or other fluids. In another embodiment, the ELISA described herein can detect a virus or a viral protein in a tissue sample. Such methods can be readily modified by those skilled in the art to detect the presence of an antiviral antibody in a sample or a specific viral protein, as well as the virus itself. In some embodiments, the ELISA of the present invention can detect the presence of an antiviral antibody.

ELISA assays as described herein may comprise the use of a nitrocellulose strip impregnated with a viral protein as described herein. The nitrocellulose strip may provide a visual result upon contact with a test sample containing antiviral nucleoprotein antibodies. Such a test may identify a subject who already has antibodies against the viral protein, and thus the subject may have immunity to the virus. Administration of an immunogenic composition for preventing a viral infection, such as described herein, may be unnecessary to such a subject, and therefore, identification of subjects who already have immunogenic antibodies may prevent unnecessary administration of an immunogenic compound to such a subject. In this regard, an embodiment of the present invention may comprise identifying a subject who lacks antiviral antibodies using an assay described herein, such as an ELISA assay, and then administering an immunogenic composition described herein to the subject to prevent a viral infection. In another embodiment, a nitrocellulose strip for use in the ELISA of the present invention may be impregnated with an antibody, such as an antiviral antibody, and may produce a visual result upon contact with a test sample containing a viral protein. Such a test may identify a subject infected with a virus described herein.

Another immunological technique that may be useful for detecting a virus is a competitive inhibition assay. This assay uses monoclonal antibodies (MABs) that react with a specific virus. A biological fluid (e.g., blood) from a subject may be contacted with a first antibody bound to a substrate, and a labeled monoclonal antibody is contacted with the first antibody-virus complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control.

As will be understood by one skilled in the art, the biological sample for use in the above assays may be taken directly from the subject or may be in a partially purified form. An antibody specific for a particular virus will react by binding to the virus as a primary reaction. A secondary reaction with an antibody bound or labeled with a detectable fragment may also be added thereafter to enhance the detection of the primary reaction. Typically, in the secondary reaction, the antibody or other ligand that specifically or non-specifically reacts with another binding site (epitope) of the virus is selected for its ability to react with several sites of the antibody-virus complex. For example, several antibody molecules in the secondary reaction may react with each complex formed as a result of the primary reaction, which makes the primary reaction more detectable.

The detectable fragment may provide visual detection of sediment or color change, visual detection by microscopy or automatic detection by spectrometry, radiometric measurements, etc. Examples of detectable fragments include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change). The detection methods and fragments used may be selected, for example, from any disclosed herein or available from the prior art.

Detection of the presence of viral nucleic acid

In some embodiments, a viral infection described herein can be detected based on the level of a certain RNA or DNA in a biological sample. Primers from a certain virus or viral pathogen can be used to detect, diagnose and determine the presence of a virus. Any suitable primer can be used to detect genomic DNA or any sequence thereof, an open reading frame or a gene or a selected protein using any suitable methods known in the art. A suitable nucleic acid sequence can be used as single-stranded or double-stranded probes or primers to detect viral mRNA or cDNA derived therefrom that may be present in a biological sample. The viral polynucleotides described herein can also be used to produce additional copies of the polynucleotides, to produce antisense oligonucleotides or as triple-stranded oligonucleotides. For example, two oligonucleotide primers can be used in a PCR-based assay to amplify a portion of viral cDNA obtained from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) the viral polynucleotide. Such primers may be of any length sufficient to hybridize and allow amplification of viral nucleic acid as described herein, including at least or about 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, or 50 nucleotides; or from about 12 to about 50 nucleotides in length, from 15 to 30 nucleotides in length, from 15 to 25 nucleotides in length, or from 20 to 30 nucleotides in length. DNA primers suitable for use in the present invention may be any of the primers described herein, such as those set forth in SEQ ID NOs: 40-157.

The amplified nucleotide, such as cDNA, can then be isolated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to the viral polynucleotide can be used in a hybridization assay to detect the presence of the viral polynucleotide in a biological sample.

Nucleic acid probes or primers specific for a virus as described herein can be generated using the polynucleotide sequences disclosed herein. The probes are preferably at least about 12, 15, 16, 18, 20, 22, 24 or 25 nucleotide fragments or another polynucleotide sequence encoding a viral nucleic acid or polypeptide. The length of the nucleic acid probes can be less than about 200 bp, 150 bp, 100 bp, 75 bp, 50 bp, 60 bp, 40 bp, 30 bp, 25 bp, 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.25 kb, 0.1 kb, or 0.05 kb. The probes can be prepared, for example, by chemical synthesis, PCR amplification, preparation from longer polynucleotides using restriction enzymes, or other methods well known in the art. The polynucleotides described herein can also be used in methods or assays involving the use of solid substrates, such as arrays. Such a chip may contain one or more different polynucleotides, which can be immobilized on the chips using methods known in the art.

In some embodiments, a polynucleotide of the present invention may be labeled with a detectable label. Detectable labels may include, but are not limited to, radioactive labels, fluorochromes including fluorescein isothiocyanate (FITC), rhodamine, Texas red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM), or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); radioactive labels such as ^32P , ^35S and ^3H , etc. In some embodiments, the detectable label may comprise multiple steps (e.g., biotin-avidin, hapten-antibody to hapten, etc.).

In accordance with the present invention, any suitable qualitative or quantitative methods known in the art for detecting specific viral nucleic acids (e.g., RNA or DNA) can be used. Viral nucleic acid as described herein can be detected, for example, by in situ hybridization in tissue sections using methods that detect single base pair differences between hybridizing nucleic acids, by reverse transcriptase PCR or in Northern blots containing mRNA poly A, or other methods well known in the art. Methods that detect single base pair mismatches can be used to detect viral polynucleotides in blood or samples obtained from blood.

The viral nucleic acid sequence may be present in a biological sample obtained from an infected individual in relatively low quantities, and thus amplification techniques known in the art (e.g., PCR) can be used to amplify the sequence prior to hybridization assays.

Nucleic acid probes can be prepared using a viral genome as described herein. Such a probe can contain at least about 8 nucleotides or more and can be produced synthetically or by excision from recombinant polynucleotides. The probe described herein can hybridize with a viral nucleic acid and, thus, such a probe can be useful for detecting a particular virus in a biological sample. The probes described herein can also be useful for identifying infected subjects, as well as for further characterizing viral genomes. A probe for detecting viral polynucleotides (natural or derived) can be characterized by a certain length or contain a sequence that allows detection of unique viral sequences by hybridization. Although about 6-8 nucleotides can be useful, longer sequences can be preferred, for example, sequences of about 10-12 nucleotides or about 20 nucleotides or more. One skilled in the art will know how to make and use a probe as described herein.

Nucleic acid probes can be prepared using conventional methods, including but not limited to automated oligonucleotide synthesis methods. A sequence useful for preparing such a probe can include the complement of any unique portion of a viral genome, such as a portion of a viral genome that distinguishes a particular virus from other viruses that may be present in a sample. A probe described herein can be fully complementary to a target sequence of interest, or it can contain one or more mismatches. A probe used in accordance with the present invention that contains one or more mismatches will still hybridize to a target sequence of interest. For use of such probes as a diagnostic, the biological sample to be analyzed can be processed prior to analysis, if necessary, to extract nucleic acids contained therein. The resulting nucleic acids from the sample can be subjected to gel electrophoresis or other size-based separation techniques. The probe may be labeled with a detectable label as described herein. Suitable labels and methods for labeling probes are known in the art and may include any of the labels described herein or other labels useful in the present invention.

The probe may be fully complementary to the viral genome or a portion thereof (e.g., all or a portion of a viral protein coding sequence as described herein). High stringency conditions may be desirable to prevent or at least minimize false positive results. Hybridization stringency may be determined by several factors during hybridization and washing, including temperature, ionic strength, length of time, and concentration of reagents. The probe or nucleic acid from the sample may be present in solution for such assays or may be attached to a support (e.g., a solid or semi-solid support). Examples of supports that can be used include, but are not limited to, nitrocellulose (e.g., membrane or well form), polyvinyl chloride (e.g., sheets or microtiter wells), polystyrene latex (e.g., beads or microtiter plates, polyvinylidene fluoride, diazotized paper, nylon membranes, activated beads, and protein A beads).

In one embodiment, a nucleic acid probe or sample can be presented on a chip for detection. Chips can be created, for example, by applying polynucleotide probes to a support (e.g., glass, nitrocellulose, etc.) in a two-dimensional array or chip. The probes can be linked to the support either by covalent bonds or by non-specific interactions, such as hydrophobic interactions. Polynucleotide samples can be labeled with a detectable label (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double-stranded polynucleotides containing labeled sample polynucleotides bound to probe polynucleotides can be detected after removal of the unbound portion of the sample. Techniques for constructing chips and methods for using these chips are known in the art. Chips can be used to analyze a single sample for the presence of two or more target nucleic acid regions. In this case, probes for each of the target regions, as well as controls (both positive and negative) can be presented on a single chip. Thus, the chips facilitate fast and convenient analysis.

Diagnostic tests and kits

The present invention further provides diagnostic reagents and kits comprising one or more such reagents for use in various diagnostic assays, including, for example, immunoassays such as ELISA and sandwich immunoassays, as well as nucleic acid assays, such as PCR-based assays. In a related embodiment, the assay can be carried out in a flow test or strip format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. Such kits can preferably comprise at least a first peptide or a first antibody or antigen-binding fragment of the present invention, a functional fragment thereof, or a mixture thereof, or a first pair of oligonucleotides, as well as means for generating a signal. In some embodiments, the kit can comprise an immunogenic composition, such as a recombinant virus, as described herein. Reagents and other compounds, such as a pharmaceutically acceptable carrier, can be included in the kit. The immunogenic composition provided in such a kit may be in solution, such as in a pre-measured dose or amount, or may be a dry composition, such as in a dried or lyophilized form suitable for rehydration or resuspension. The components of the kit may be pre-attached to a solid support or may be applied to the surface of a solid support upon use of the kit. The signal generating means may be pre-associated with the antibody or nucleic acid of the present invention or may require combination with one or more components, such as buffers, nucleic acids, antibody-enzyme conjugates, enzyme substrates, etc., prior to use.

The kits may also contain additional reagents, such as blocking reagents to reduce non-specific binding to the solid phase surface, washing reagents, enzyme substrates, enzymes, etc. The solid phase surface may be in the form of microtiter plates, microspheres, or other materials suitable for immobilizing nucleic acids, proteins, peptides, or polypeptides. An enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is one such component of the signal generating means. Such enzymes are well known in the art. If a radioactive label, chromogenic, fluorogenic or other type of detectable label or detection means is included in the kit, the labeling means may be provided either in the same container as the diagnostic or therapeutic composition itself, or alternatively may be placed in a second separate container into which the second composition may be placed and appropriately aliquoted. Alternatively, the detection reagent and label may be prepared in a single container.

All patents and other publications cited are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methodologies described in such publications that may be applied in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application.

The present invention is further illustrated and supported by the following examples. However, these examples should in no way be considered as further limitations on the scope of the present invention. On the contrary, it will be easy for a person skilled in the art to understand that there are other embodiments, modifications and equivalents of the present invention without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLES

Example 1

Construction of HVT-gfp-based plasmids

Construction of a transfer tazmid based on HVT-green fluorescent protein (gfp)-B

Transfer plasmid HVT-gfp-B (SEQ ID NO. 18) was chemically synthesized by GeneArt, Thermo Fisher. 2.5 μg of plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. About 4-6 h later, the transfected cells were infected with HVT at moi 0.006 (1.5 × 10 ⁴ PFU/2.5 × 10 ⁶ cells). Three days later, the cells were passaged 1:15 in T75 with freshly prepared CEF (1 × 10 ⁷ cells/T75). Then three days later, the cells were seeded at a ratio of 1:50 in 24-well plates. Cells from wells containing green fluorescent protein foci were plated in 96-well plates at 1:200, 1:500, and 1:1000 dilutions with freshly prepared cells (6 x ¹⁰⁴ cells/well). Wells containing single green foci were purified in 3 rounds by limiting dilution method using 96-well plates. Purified virus was expanded using CEF cells and recovered from frozen stock solution. It was designated as “HVT-gfp-B”.

PCR analysis of the three purified clones using primers just outside the UL55-gene 3 integration site (upper primer: SEQ ID NO. 49; lower primer: 5'-SEQ ID NO. 50) resulted in a 1.893-kb band, as expected. HVT resulted in a 0.15-kb band, as expected. See Fig. 1.

Construction of modified HVT-gfp-A transfer plasmid

The modified HVT-gfp-A transfer plasmid (SEQ ID NO. 16) was generated by site-directed mutagenesis using two pairs of primers (upper primer pairs to obtain SbfI upstream of the gfp gene: SEQ ID NO. 40 and SEQ ID NO. 41; lower primers to obtain SbfI downstream of the gfp gene: - SEQ ID NO. 42 and SEQ ID NO. 43 for the original HVT-gfp-A transfer plasmid (SEQ ID NO. 17), which was chemically synthesized by GeneArt, ThermoFisher. 0.01 μg of the modified HVT-gfp-A transfer plasmid was co-transfected with 2.5 μg of HVT DNA using 7.5 μl of PEI (polyethyleneimine) into secondary CEF cells in a 6-well plate. Green foci fluorescent protein became visible at passage 1. After three rounds of limiting dilution purification, clone 1 of HVT-gfp-A was further expanded and recovered from frozen stock.

PCR analysis of the purified clone (left lane) using primers just outside the UL35-UL36 integration site (upper primer: SEQ ID NO. 44; lower primer: SEQ ID NO. 45) resulted in a band of 1.922 kb, as expected (Fig. 4). Modified transfer plasmid HVT-gfp-A DNA was used as a control (right lane). See Fig. 2.

Example 2

HVT-IBD Contouring

Design of HVT-IBD #1

Transfer plasmid HVT-IBD#1 (SEQ ID NO. 20) was chemically synthesized by GeneArt, ThermoFisher. 2.5 μg of plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. About 4-6 h later, transfected cells were infected with HVT at moi of 0.055. Three days later, cells were passaged 1:7.5 in T75 with freshly prepared CEF (1 x 10 ⁷ cells/T75). Cells were then seeded in 10 96-well plates and three days later duplicate plates were created. One set of plates was fixed and stained with chicken antiserum to IBDV. Two wells containing foci staining positive for IBD were identified. The corresponding wells containing positive staining foci were purified in three rounds by limiting dilution method using 96-well plates.

Purified virus was expanded using CEF cells and recovered from frozen stock and designated as "HVT-IBD#1".

PCR analysis of the different clones using primers just outside the UL55-gene 3 integration site (upper primer: SEQ ID NO. 46; lower primer: SEQ ID NO. 47, panel A) resulted in a 2.414 kb band, while the parental vector PCR band was 1.922 kb. Proper integration was further confirmed using primers flanking the downstream insert (upper primer SEQ ID NO. 48, which is located in the coding region of IBDV VP2; lower primer SEQ ID NO. 49, located downstream of the transfer plasmid, panel B). As expected, a PCR band of 1.118 kb was obtained. Proper integration of the upstream integration site was accomplished using primers flanking the upstream insert (upstream primer SEQ ID NO. 50, which is located upstream of the transfer plasmid; downstream primer SEQ ID NO. 51, which is within the IBDV VP2 coding region, panel C). As expected, a PCR band of 1.428 kb was obtained. See Fig. 3A, B, and C.

Design of HVT-IBD #5

Transfer plasmid HVT-IBD#5 (SEQ ID NO. 21) was chemically synthesized by Gene Art, ThermoFisher. JBJ-1 cells (chicken fibroblast cell line) in a 6-well plate were transfected with 2.5 μg of plasmid using LTX transfection reagent (mvitrogen). Transfected cells were infected with HVT at an moi of 0.05 approximately 5 hours after transfection. Transfected/infected cells were amplified by serial passage (1:4-1:10) and a portion were then plated in 96-well plates at limiting dilutions. Expression of IBDV VP2 antigen was assessed by staining live cell monolayers with antibody without fixation. See Fig. 4A and 4B. Stained foci were collected by trypsinization of cells using cloning cylinders placed around the positive foci. This "live staining" followed by passage of the cloning cylinder was repeated four times to obtain pure VP2 positive cultures. The cultures were amplified by serial passages on JBJ-1 cells and before final amplification on primary CEF cells in roller bottles. The collected CEF cells were used to prepare a frozen cell solution and designated "HVT-IBD#5".

PCR analysis of clone #7 was performed using 2 primer sets to confirm integration of the insert at both insertion sites. In PCR A, the upstream primer (SEQ ID NO.52) binds to the IBDV VP2 coding region while the downstream primer (SEQ ID NO.53) binds downstream of the UL35-UL36 integration site. This primer set resulted in a PCR band of 1.244 kb, as expected. In PCR B, the upstream primer (SEQ ID NO.54) binds upstream of the UL35-UL36 insertion site and the downstream primer (SEQ ID NO.55) binds within the human CMV promoter of the insert and resulted in a PCR band of 0.926 kb, as expected. See Fig. 5.

Design of HVT-IBD #6a

Transfer plasmid HVT-IBD #6a (SEQ ID NO.22) was chemically synthesized by BioBasic Inc. 0.1 μg and 0.01 μg of linearized transfer plasmid (by EcoR1 and HindIII digestion) were cotransfected with 2.5 μg of HVT-gfp-A, which was digested with Sbfl in secondary CEF cells using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Four days after transfection, four non-green foci were observed when 0.01 μg of transfer plasmid was transfected and three non-green foci were observed when 0.1 μg of transfer plasmid was transfected, whereas no foci were observed when HVT-gfp-A digested with Sbf1 only. The two non-green foci were purified three times by limiting dilution in a 96-well plate. The purified virus was expanded using CEF cells and recovered from frozen stock. It was designated as “HVT-IBD#6a”.

Infected cell lysate was prepared and Western blotted using anti-IBDV R63 monoclonal antibody. A protein band of approximately 50 kDa was visible in all lanes except the band containing the HVT-gfp-A vector lysate. See Fig. 6.

PCR analysis of clones using 2 sets of primers to confirm proper integration. The first set of primers targeted the upstream integration site: Upstream primer 5' SEQ ID NO. 56, located upstream of the UL35-UL36 integration site; Downstream primer SEQ ID NO. 57, which is located within the Rec promoter. This set of primers resulted in a PCR band of 0.911 kb, as expected. The second set of primers targeted the downstream integration site: Upstream primer 5' SEQ ID NO. 58, which is located within the coding region of IBDV VP2; Downstream primer 5' SEQ ID NO. 59, which is located downstream of the UL35-UL36 insertion site. A PCR band of 1.244 kb was obtained, as expected. See Fig. 7A and 7B.

Design of HVT-IBD #9

Transfer plasmid HVT-IBD#9 (SEQ ID NO.23) was chemically synthesized by GeneArt, Thermo Fisher. 2.5 μg of plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. About 4-6 h later, the transfected cells were infected with HVT-gfp-B at moi 0.075. Three days later, the cells were passaged 1:10 in T75 with freshly prepared CEF (1× ¹⁰⁷ cells/T75) 3 times. The cells were then seeded in 10 96-well plates and 90 non-green foci were obtained. Three of them stained positively with chicken antiserum to IBDV. Two clones were purified in 3 rounds by limiting dilution method using 96-well plates. The purified virus was expanded using CEF cells and recovered from frozen stock and designated as "HVT-IBD #9".

PCR analysis of different clones using primers just outside the UL55-gene 3 integration site (upper primer: SEQ ID NO. 60; lower primer: SEQ ID NO. 61, panel A) resulted in a 2.536 kb band, while the PCR band of the original HVT-gfp-B vector was 1.922 kb. Proper integration was further confirmed using primers flanking the upstream insertion junction (upper primer SEQ ID NO. 62, which is located upstream of the UL55-gene 3 insertion site; lower primer SEQ ID NO. 63, which is located within the coding region of IBDV VP2. As expected, a PCR band of 1.482 kb was obtained. Proper integration of the downstream site was accomplished using primers flanking the downstream insertion junction (upper primer SEQ ID NO. 64, which is located within the coding region of IBDV VP2; lower primer SEQ ID NO. 65, which is located downstream of the UL55-gene 3 insertion site. As expected, a PCR band of 1.166 kb was obtained. See Fig. 8A and B.

Transfer plasmid HVT-IBD#30 (SEQ ID NO.24) was chemically synthesized by GeneArt, ThermoFisher. Secondary CEF cells were co-transfected with 1 μg plasmid and 2.5 μg HVT using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Three days later, cells were passaged 1:12 with freshly prepared CEF cells. Foci expressing IBD VP2 were visualized by staining unfixed cultures with chicken polyclonal IBDV antiserum and labeling these foci using a fluorescence microscope. A total of 16 positive foci were passaged with freshly prepared CEF cells by trypsinization using cloning cylinders to separate foci from those not expressing VP2. Four of these cultures were cloned three times using the same procedure before amplification on primary CEF cells in roller bottles. The frozen cell stock was set aside and designated "HVT-IBD #30".

PCR analysis of four different clones using primers for the upstream region of the UL55-gene 3 integration site (upper primer: SEQ ID NO.66; lower primer: SEQ ID NO.67, panel A) resulted in a 1,673 kb band. Proper integration was further confirmed using primers surrounding the 3′ junction of the insert (upper primer SEQ ID NO.68, which is located in the coding region of IBDV VP2; lower primer SEQ ID NO.69, which is located downstream of the UL55-gene 3 insertion site, panel B). As expected, a PCR band of 1,082 kb was obtained. Proper integration of the downstream site was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO. 70; lower primer SEQ ID NO. 71 (panel C). As expected, a PCR band of 2.558 kb was obtained. See Fig. 9A-C.

Coplasting HVT-IBD No. 31

Transfer plasmid HVT-IBD #31 (SEQ ID NO. 25) was chemically synthesized by GeneArt, ThermoFisher. 0.1 μg and 0.01 μg of linearized transfer plasmid (by EcoR1 and HindIII digestion) were cotransfected with 2.5 μg of HVT-gfp-A digested with Sbf1 in secondary CEF cells using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Four days after transfection, 1 non-green focus was observed, while no foci were observed with HVT-gfp-A digested with Sbf1 alone. After passage, 2 non-green foci were purified 3 times by limiting dilution using a 96-well plate. Purified virus was expanded using CEF cells and recovered from frozen stock and designated as "HVT-IBD #31".

Infected cell lysates were prepared and Western blotted using monoclonal antibody to IBDV R63. A protein band of approximately 50 kDa was visible in all lanes with chicken antiserum to IBDV as a probe only, but not with mAb to IBDV R63. See Fig. 10A and B.

PCR analysis of clones using 2 sets of primers to confirm proper integration. The first set of primers targeted the upstream integration site: Upstream primer SEQ ID NO. 72, which is located upstream of the UL35-UL36 integration site; Downstream primer SEQ ID NO. 73, which is located within the chicken beta-actin promoter. This set of primers resulted in a PCR band of 0.835 kb, as expected. The second set of primers targeted the downstream integration site: Upstream primer SEQ ID NO. 76, which is located within the coding region of IBDV VP2; Downstream primer SEQ ID NO. 77, which is located downstream of the UL35-UL36 insertion site. A PCR band of 1.248 kb was obtained, as expected. See Fig. 11A and B.

Design of HVT-IBD #34

Transfer plasmid HVT-IBD #34 (SEQ ID NO. 28) was chemically synthesized by GeneArt, ThermoFisher. 2.5 μg of plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. About 4-6 hours later, transfected cells were infected with HVT-gfp-B at moi 0.05. Three days later, cells were passaged 1:15 in T75 with freshly prepared CEF (1× ¹⁰⁷ cells/T75). Infected cells were seeded in 10×96-well plates, grown for 3 days, then subcultured in 96-well plates in duplicate. One replicate was fixed and stained with chicken anti-IBDV antiserum, and 3 wells containing foci that stained positive for IBDV were identified. The corresponding wells from the live cell plate for the replicate were purified by 3 rounds of limiting dilution cloning using 96-well plates. One of the purified viruses was expanded using CEF cells and recovered from frozen stock. It was designated "HVT-IBD#34".

PCR analysis of three different clones using primers targeting the upstream region of the 3-UL55 gene integration site (upper primer: SEQ ID NO.78; lower primer: SEQ ID NO.79, which is located in the chicken beta-actin promoter, SEQ ID NO:79, panel A) yielded a 0.815 kb band, as expected. Proper integration was further confirmed using primers surrounding the downstream insertion junction (upper primer: SEQ ID NO.80, which is located in the coding region of IBDV VP2; lower primer: SEQ ID NO.81, which is located downstream of the 3-UL55 gene insertion site, panel B). A PCR band of 1.296 kb was obtained, as expected. The correct construction was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO.82; lower primer SEQ ID NO.83 (panel C). As expected, a PCR band of 3.001 kb was obtained. See Fig. 12A-C.

Design of HVT-ND #38

Transfer plasmid HVT-IBD#38 (SEQ ID NO.29) was chemically synthesized by BioBasic, Inc. Transfer plasmid HVT-ND#38 digested with HindIII and ApoI was cotransfected with Sbf1 digested HVT-gfp-A DNA using PEI (polyethyleneimine, 7.5 μl) in a 6-well plate with secondary CEF cells. Six days after transfection, transfected cells were seeded in a 96-well plate and live-stained with chicken anti-NDV serum. Seven wells containing positive foci were purified 3-fold by limiting dilution. One of the purified viruses was expanded using CEF cells and recovered from frozen stock. It was designated as "HVT-ND#38".

PCR analysis of 5 different clones using primers for the upstream region of the UL35-UL36 integration site (upper primer: SEQ ID NO. 84; lower primer: SEQ ID NO. 85, which is located within the coding region of NDV F, panel A) resulted in a 2.122 kb band. Proper integration was further confirmed using primers surrounding the 3′ junction insert (upper primer SEQ ID NO. 86, which is located within the coding region of NDV F; lower primer SEQ ID NO. 87, which is located downstream of the UL35-UL36 insertion site, panel B). As expected, a PCR band of 1.127 kb was obtained. The correct construction was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO. 88; lower primer SEQ ID NO. 89 (panel C). As expected, a PCR band of 3.657 kb was obtained. See Fig. 15A and B.

Design of HVT-ND #39

Transfer plasmid HVT-IBD #39 (SEQ ID NO. 30) was chemically synthesized by BioBasic, Inc. Secondary CEF cells were cotransfected with 0.01 μg of plasmid and 2.5 μg of HVT using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Six days later, cells were passaged at a ratio of 1:24 with freshly prepared CEF cells. Three days after passage, foci expressing NDV F protein were visualized by staining unfixed cultures with polyclonal chicken antiserum specific for NDV, and these foci were labeled using a fluorescence microscope. A total of 4 positive foci were passaged with freshly prepared CEF cells by trypsinization using cloning cylinders to separate foci from those not expressing the F protein. Four of these cultures were cloned three times by the same procedure before amplification on primary CEF cells in roller bottles. A frozen stock solution of cells (clone 2 in the figures) was set aside and designated “HVT-ND#39”.

PCR analysis of three different clones using primers for the upstream region of the UL35-UL36 integration site (upper primer: SEQ ID NO. 90; lower primer: SEQ ID NO. 92, which is located in the chicken beta-actin promoter, SEQ ID NO. 79, panel A) resulted in a 0.835 kb band. Proper integration was further confirmed using primers surrounding the downstream junction insert (upper primer SEQ ID NO. 92, which is located in the coding region of poly A; lower primer SEQ ID NO. 93, which is located downstream of the UL35-UL36 insertion site, panel B). As expected, a PCR band of 0.856 kb was obtained. The correct construction was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO. 94; lower primer SEQ ID NO. 95 (panel C). As expected, a PCR band of 3.449 kb was obtained. See Fig. 16A and B.

Design of HVT-ND #40

Transfer plasmid HVT-IBD#40 (SEQ ID NO.31) was chemically synthesized by BioBasic, Inc. Transfer plasmid HVT-ND#40 digested with HindIII was cotransfected with Sbf1 digested HVT-gfp-A DNA using PEI (polyethyleneimine, 7.5 μl) in a 6-well plate with secondary CEF cells. Seven days after transfection, transfected cells were seeded in 24-well plates and live-stained with chicken anti-NDV antiserum. Four wells containing positive foci were purified 3-fold by limiting dilution. One of the purified viruses was expanded using CEF cells and recovered from frozen stock. It was designated as "HVT-ND#40".

PCR analysis of 4 different clones using primers for the upstream region of the UL35-UL36 integration site (upper primer: SEQ ID NO.96; lower primer: SEQ ID NO.97, which is located in the chicken beta-actin promoter, SEQ ID NO.79, panel A) resulted in a 0.835 kb band. Proper integration was further confirmed using primers surrounding the downstream insertion junction (upper primer SEQ ID NO.98, which is located in the coding region of NDV F; lower primer SEQ ID NO.99, which is located downstream of the UL35-UL36 insertion site, panel B). As expected, a PCR band of 0.856 kb was obtained. The correct construction was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO. 100; lower primer SEQ ID NO. 101 (panel C). As expected, a PCR band of 3.449 kb was obtained. See Fig. 17A-C.

Design of HVT-ND #42

The original transfer plasmid HVT-ND#42 (SEQ ID NO. 33) was chemically synthesized by BioBasic, Inc. The cloning plasmid was chemically synthesized by DNA2.0.PCR amplification of the NDV F gene expression cassette of the transfer plasmid HVT-ND#42 using the following primers: up primer, SEQ ID NO. 102; down primer, 5' of SEQ ID NO. 103. The amplified PCR fragment was cloned into the AscI and NheI sites of UL55/gene 3 to obtain the final transfer plasmid HVT ND#42 (SEQ ID NO. 35).

Transfer plasmid was transfected into HVT-infected CEF cells using Lipofectamine LTX in a 6-well plate. Three days post-transfection, transfected/infected cells were plated in duplicate in a 6-well plate and then in a 96-well plate to screen for ND-expressing foci by staining with NDV antiserum. Wells corresponding to ND-expressing foci were purified 3-fold by limiting dilution. One of the purified viruses was expanded using CEF cells and recovered from frozen stock. It was designated as “HVT-ND#42”.

PCR analysis of one final clone using primers outside the expression cassette (Primer Set 1: Upstream Primer SEQ ID NO. 104; Downstream Primer SEQ ID NO. 105) resulted in a 3.597 kb band, as expected. Four primer sets for the upstream UL55-gene 3 integration region, with all upstream primers located upstream and outside the expression cassette and all downstream primers located within the ND F coding region. Primer Set 2: Upstream Primer: SEQ ID NO. 106, Downstream Primer: SEQ ID NO. 107, which resulted in a 2.243 kb band; Primer Set 3: Upstream Primer: SEQ ID NO. 108; Downstream Primer: SEQ ID NO. 109, which resulted in a PCR band of 2.356 kb. Primer Set 4: Upstream primer: SEQ ID NO. 110; Downstream primer: SEQ ID NO. 111, which resulted in a PCR band of 2.424 kb. Primer Set 5: Upstream primer: SEQ ID NO. 112; Downstream primer: SEQ ID NO. 113, which resulted in a PCR band of 2.170 kb. Proper integration was further confirmed using primers surrounding the downstream insert (Primer Set 6: Upstream primer SEQ ID NO. 114, which is located in the coding sequence of the NDV F gene; Downstream primer SEQ ID NO. 115, which is located downstream of the UL55-gene 3 insertion site, panel C). As expected, a PCR band of 0.971 kb was obtained. See Fig. 18.

Transfer plasmid HVT-IBD #44 (SEQ ID NO.36) was chemically synthesized by BioBasic, Inc. Transfer plasmid #44 was digested with restriction enzymes EcoRI and HindIII to release the insert from the plasmid sequences, and the resulting digested DNA (10 ng) was used along with 2.5 μg of HVT-gfpB DNA to cotransfect secondary cells with PEI (polyethyleneimine). Four days after transfection, transfected cells were passaged at a 1:6 ratio with freshly prepared secondary cells and live-stained with chicken polyclonal antiserum to NDV to identify NDV-expressing foci three or four days after passage. Three positively stained foci were collected by trypsinization using cloning cylinders. The collected cells were serially diluted and plated on freshly prepared secondary CEF cells. This process was repeated every three to four days until NDV staining showed homogeneity, and then four sequential clonings were performed. The cloned culture was then amplified and prepared from frozen stock. The frozen stock was designated “HVT-ND #44.”

PCR analysis of 1 final clone using primers targeting the upstream region of the UL55-gene 3 integration site (upper primer: SEQ ID NO. 116, which is located upstream of UL55; lower primer: SEQ ID NO. 117, which is located in the chicken beta-actin promoter, SEQ ID NO. 79, panel A) resulted in a 0.71 kb band; a similarly localized pair of primers: upper primer: SEQ ID NO. 118, lower primer: SEQ ID NO. 119 (panel B, which resulted in a 0.965 kb PCR band). Proper integration was further confirmed using primers flanking the downstream insertion junction (upper primer SEQ ID NO. 120, which is located within the coding sequence of the NDV F gene; lower primer SEQ ID NO. 121, which is located downstream of the UL55-gene 3 insertion site, panel C). As expected, a PCR band of 0.971 kb was obtained. Proper construction was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO. 122; lower primer SEQ ID NO. 123 (panel D). As expected, a PCR band of 3.438 kb was obtained.

Design of HVT-ND #45

Transfer plasmid HVT-IBD#45 (SEQ ID) was chemically synthesized by BioBasic, Inc. This plasmid was transfected into CEF cells infected with HVT-GFP-B using Lipofectamine LTX in a 6-well plate. Three days after transfection, transfected/infected cells were plated in a 96-well plate for screening of GFP negative foci. Wells containing GFP negative foci were purified 3-fold by limiting dilution. Purified viruses were stained with IFA chicken anti-NDV serum to confirm NDV F gene expression. One of the purified viruses was expanded using CEF cells and recovered from frozen stock. It was designated as “HVT-ND#45”.

PCR analysis of 1 final clone using primers outside the expression cassette (Primer Set 1: Upper Primer SEQ ID NO. 124; Lower Primer SEQ ID NO. 125) resulted in a 2,830 kb band, as expected. Two primer sets for the upstream integration region of the 3-UL55 gene, with both upper primers located upstream and outside the expression cassette and both lower primers located within the ND F coding region. Primer Set 2: Upper Primer: SEQ ID NO. 126; Lower Primer: SEQ ID NO. 127, which resulted in a 1,635 kb band; Primer Set 3: Upper Primer SEQ ID NO. 128; Lower Primer: SEQ ID NO. 129, which resulted in a PCR band of 1.588 kb. Proper integration was further confirmed using 2 primer sets flanking the downstream insertion junction: Primer Set 4: Upstream primer SEQ ID NO. 130, which is located within the coding sequence of the NDV F gene; Downstream primer SEQ ID NO. 131, which is located downstream of the 3-UL55 gene insertion site. As expected, a PCR band of 0.993 kb was obtained. Primer Set 5: Upstream primer: SEQ ID NO. 132; Downstream primer: SEQ ID NO. 133, resulted in a PCR band of 1.137 kb, as expected. See Fig. 19.

Design of HVT-ND #46

Transfer plasmid HVT-ND#46 (SEQ ID NO.38) was chemically synthesized by BioBasic, Inc. This plasmid was transfected along with HVT-GFP-B viral DNA into CEF cells using PEI (polyethyleneimine, 7.5 μl) in a 6-well plate. Four days after transfection, the transfected cells were seeded in a 96-well plate for screening of GFP negative foci. Wells containing GFP negative foci were purified 3-fold by limiting dilution. Purified viruses were stained with IFA chicken antiserum to NDV to confirm NDV F gene expression. One of the purified viruses was expanded using CEF cells and recovered from frozen stock. It was designated as "HVT-ND#46".

PCR analysis of 1 final clone using primers outside the expression cassette (upper primer SEQ ID NO. 134; lower primer SEQ ID NO. 135) resulted in a 3.597 kb band, as expected. One set of primers for the upstream integration region of the 3-UL55 gene, with the upper primers located upstream and outside the expression cassette (SEQ ID NO. 136) and the lower primers located in the mouse CMV promoter (upper primer SEQ ID NO. 137), resulted in a 1.107 kb PCR band, as expected. Proper integration was further confirmed using 4 sets of primers surrounding the downstream junction insert: P1: upper primer SEQ ID NO. 138, which is located in the coding sequence of the NDV F gene; the lower primer SEQ ID NO. 139, which is located downstream and outside the expression cassette. As expected, a PCR band of 1.003 kb was obtained. P2: upper primer: SEQ ID NO. 140, lower primer: SEQ ID NO. 141, as expected, a PCR band of 1.147 kb was obtained. P3: upper primer: SEQ ID NO. 142, lower primer: SEQ ID NO. 143, as expected, a PCR band of 1.019 kb was obtained. P4: upper primer: SEQ ID NO. 144, lower primer: SEQ ID NO. 145, as expected, a PCR band of 1.018 kb was obtained. See Fig. 20 A-C.

Design of HVT-ND #48

Linearized transfer plasmid for HVT-ND#48 (SEQ ID NO.39) was cotransfected with HVT-gfp-B DNA using PEI (polyethyleneimine, 7.5 μl) in a 6-well plate with secondary CEF cells. Four days after transfection, transfected cells were seeded in a 96-well plate and live-stained with chicken anti-NDV serum. Four non-green foci were detected and 2 were positive with chicken anti-NDV serum. See Fig. 33A and B. Two clones were purified 3-fold by limiting dilution. Purified virus was expanded using CEF cells and prepared from frozen stock. It was designated as "HVT-ND#48".

Transfer plasmid HVT-IBD#48 was chemically synthesized by BioBasic, Inc. The transfer plasmid was digested with EcoRI and HindIII to release the insert from the plasmid sequences, and the resulting digested DNA (10 ng) was used along with 2.5 μg of HVT-gfp-B DNA to cotransfect secondary cells with PEI (polyethyleneimine). Three days after transfection, transfected cells were passaged at a 1:6 ratio with freshly prepared secondary cells and live-stained with chicken polyclonal NDV antiserum to identify NDV-expressing foci four days after passage. Three positively stained foci were collected by trypsinization using cloning cylinders. The collected cells were serially diluted and plated on freshly prepared secondary CEF cells. This process was repeated every three or four days until NDV staining showed homogeneity, and then four sequential clonings were performed. The cloned culture was then amplified from a six-well plate into a 75 ^cm2 flask, then into a 225 ^cm2 flask before a final amplification in an 850 ^cm2 spinner flask using primary CEF cells. The final culture was harvested and designated “HVT-ND #48.”

PCR analysis of 1 final clone using primers targeting the upstream region of the 3-UL55 gene integration site (upper primer: SEQ ID NO. 146; lower primer: SEQ ID NO. 147, which is located in the chicken beta-actin promoter, panel A) yielded a 0.815 kb band as expected; proper integration was further confirmed using primers flanking the downstream insertion junction (upper primer: SEQ ID NO. 148, which is located in the NDV F coding region; lower primer: SEQ ID NO. 149, located downstream of the 3-UL55 gene insertion site, panel B). A PCR band of 1.003 kb was obtained as expected. Other similarly located primers were: upper primer: SEQ ID NO. 150; lower primer: SEQ ID NO. 151 (panel C), which resulted in a PCR band of 1.147 kb, as expected. The proper construction was further confirmed using primers outside the expression cassette (upper primer SEQ ID NO. 152; lower primer SEQ ID NO. 153 (panel D). A PCR band of 3.430 kb was obtained, as expected.

Example 3

In vivo HVT-IBD Efficacy Test against IBDV

#9, #34 in SPF birds against IBDV

Two HVT-IBD recombinants, HVT-IBD#9,#34 were tested for their in vivo efficacy against virulent IBDV (USDA STD strain) challenge in SPF birds. A commercial vaccine, Vaxxitek (Merial), was used as a positive control in this study. 1500 PFU of each recombinant virus was inoculated in ovo into El8. The reciprocal titer of each vaccine virus was also determined for each recombinant after vaccination. While 100% of HVT-IBD#9 expresses the IBDV VP2 antigen, only 96% of HVT-IBD#34 were found to express this antigen. IBDV STD challenge was performed on day 28 according to USDA guidelines. All birds were necropsied 5 days post challenge. 100% protection against HVT-IBD #9 and 90% protection against HVT-IBD #34 were observed, while the positive control Vaxxitek provided 97% protection.

Example 4

In vivo HVT-IBD Efficacy Test against IBDV

#1, #5, #6a, #9, #30, #34 SPF Birds

Six recombinants HVT-IBD, HVT-IBD#1, #5, #6a, #9, #30, #34 were tested for their in vivo efficacy against virulent IBDV (USDA STD strain) challenge in SPF birds. A positive control, commercial vaccine Vaxxitek (Merial), was used in this study. 1500 PFU of each recombinant virus was inoculated in ovo at E18. The reverse titer of each vaccine virus was also determined for each recombinant after vaccination. All recombinants were found to have 100% expression of IBDV VP2 antigen. IBDV STD challenge was performed on day 28 according to USDA guidelines. All birds were necropsied 5 days post challenge. 100% protection against HVT-IBD #9 and 96% protection against HVT-IBD #1, #30, #34, and 92% protection against HVT-IBD #6a were observed, while the positive control Vaxxitek provided 92% protection.

Example 5:

Serologic responses to IBDV HVT-IBD

No. 1, No. 5, No. 9, No. 15 commercial broiler birds

Serological responses to IBDV antigen were measured using a commercial ProFlok ND plus ELISA kit. 1500 PFU (0.2 ml) of each recombinant (HVT-IBD #1, #5, #9, #15) were injected subcutaneously (SC) into 1 day old chickens. Serum samples were collected at days 12, 19, 26, 33, 39, 47 and 54 and can be found in Table 3 below. The percentage of positive samples for each construct over time is shown in Fig. 13.

Example 6:

Serologic responses to IBDV HVT-IBD

No. 6a, No. 30, No. 31 commercial broiler birds

Serological responses to IBDV antigen were measured using the commercial ProFlok IBD plus ELISA kit. 1500 PFU (0.2 ml) of each recombinant (HVT-IBD #6a, #30, #31) were injected subcutaneously (SC) into 1 day-old chickens. Serum samples were collected at days 12, 19, 26, 33, 39, 47, and 54 and are presented in Table 4 below. The percentage of positive samples for each construct over time is shown in Fig. 14.

Example 7:

In vivo efficacy test of HVT-ND

#38, #39, #44, #48 for SPF birds

Four recombinants HVT-IBD, HVT-IBD #38, #39, #44, #48 were tested for their in vivo efficacy against virulent NDV (Texas GB strain provided by USDA) challenge in SPF birds. A commercial vaccine Vectormune ND (Ceva) was used as a positive control in this study. 1500 PFU of each recombinant virus were inoculated in ovo at E18. The reverse titer of the vaccine virus was also determined for each recombinant after vaccination. While 100% of HVT-ND #38 and #48 expressed NDV F antigen, only 95-96% of HVT-IBD #39 and #44 were found to express this antigen. NDV Texas GB challenge was performed on day 28 according to USDA guidelines. All birds were monitored 2 weeks post-challenge. 90% protection was observed for HVT-ND #38 and #48, 50% protection for HVT-ND #39, and 60% protection for HVT-ND #44, while the positive control Vectormune ND provided 90% protection. See Table 5 below.

The antibody response to the various HVT-ND vaccine candidates was analyzed using the ProFlok ND plus kit (Zoetis LLC). All titers were included without using the cutoff value (345) recommended by the kit. The percentage of birds with a positive ND titer is shown in Table 6 below.

Example 8:

In vivo anti-NDV efficacy test HVT-ND

#40, #42, #45, #46 for SPF birds

Four recombinants HVT-IBD, HVT-IBD #40, #42, #45, #46 were tested for their in vivo efficacy against virulent NDV (Texas GB strain provided by USDA) challenge in SPF birds. A commercial vaccine Vectormune ND (Ceva) was used as a positive control in this study. 1500 PFU of each recombinant virus were inoculated in ovo at E18. The reverse titer of the vaccine virus was determined for each recombinant after vaccination. While 100% of HVT-ND #42, #45, #46 express NDV F antigen, only 94-99% of HVT-IBD #40 were found to express this antigen. NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were monitored 2 weeks post-challenge. 95% protection was observed for HVT-ND #42 and #45, 80% protection was observed for HVT-ND #46, 55% protection was observed for HVT-ND #40, while the positive control Vectormune ND provided 90% protection. See Table 7 below.

The antibody response to the various HVT-ND vaccine candidates was analyzed using the ProFlok ND plus kit (Zoetis LLC). All titers were included without using the cutoff value (345) recommended by the kit. The percentage of birds with positive ND titers is shown in Table 8 below.

Example 9

In vivo anti-MDV efficacy test HVT-ND

#38, #42, #45 for SPF birds

Three recombinants HVT-IBD, HVT-IBD #38, #42, #45 were tested for their in vivo efficacy against virulent MDV (GA22) challenge in SPF birds. A positive control, the commercial vaccine Vectormune ND (Ceva), was used in this study. 1500 PFU of each recombinant virus were inoculated in ovo at E18. The reverse titer of the vaccine virus was determined for each recombinant after vaccination. MDV GA22 challenge was performed on day 5 according to USDA guidelines. All birds were observed 54 days post challenge. 69% protection was observed against HVT-ND #42 and #45, and 46% protection against HVT-ND #38, while the positive control Vectormune ND provided 62% protection. See Table 9 below.

Example 10

In vivo anti-ND efficacy test HVT-ND

No. 38, No. 42, No. 45 for broiler birds

Three recombinants HVT-IBD, HVT-IBD #38, #42, #45 were tested for their in vivo efficacy against virulent NDV (Texas GB strain provided by USDA) challenge in broiler birds. A positive control, a commercial vaccine Vectormune ND (Ceva), was used in this study. 4000 PFU of each recombinant virus were inoculated in ovo at E18. The reverse titer of the vaccine virus was determined for each recombinant after vaccination. NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were observed 2 weeks after challenge. 100% protection was observed against HVT-ND #42 and #45, and 37% protection against HVT-ND #38, while the positive control Vectormune ND provided 50% protection. The Vectormune ND reciprocal titer was 0.

Antibody responses to various HVT-ND vaccine candidates were analyzed using the ProFlok ND plus kit (Zoetis LLC). All titers were included without using the cutoff value (345) recommended by the kit.

Example 11

In vivo anti-NDV efficacy test HVT-ND

#38, #42, #45 in SPF birds when infected on day 20

Three recombinants HVT-IBD, HVT-IBD #38, #42, #45 were tested for their in vivo efficacy against virulent NDV (Texas GB strain provided by USDA) challenge in SPF birds at day 20 challenge. A commercial Vectormune ND vaccine (Ceva) was used as a positive control in this study. 2000 PFU of each recombinant virus were inoculated in ovo into El8. Reverse titer of vaccine virus was determined for each recombinant after vaccination. NDV Texas GB challenge was performed at day 20 according to USDA guidelines. All birds were observed 2 weeks post challenge. 87.5% protection was observed with HVT-ND#42, 70% protection with HVT-ND#45, 65% protection with HVT-ND#38, while the positive control Vectormune ND provided 87.5% protection. See Table 11 below.

Example 12

In vivo anti-NDV efficacy test HVT-ND

(No. 42, MSV+5) in SPF birds when infected on days 17, 18 and 19

Three recombinants HVT-ND, HVT-ND(#42, MSV+5) were tested for their in vivo efficacy against virulent NDV (Texas GB strain) challenge in SPF birds at day 17, 18 and 19 challenges. All birds were observed 2 weeks post challenge. 100% (40/40), 88% (35/40), 98% (39/40) protection were observed at day 17, 18 and 19 NDV challenges respectively for in ovo vaccination. 75% (30/40), 88% (35/40), 93% (37/40) protection were observed at day of hatch subcutaneous vaccination. See Table 12 below.

Example 13

In vivo anti-NDV efficacy test HVT-ND

(No. 42, MSV+5) in SPF birds when infected on day 16 and 19

Three recombinants HVT-ND, HVT-ND(#42, MSV+5) were tested for their in vivo efficacy against virulent NDV (Texas GB strain) challenge in SPF birds at day 16 and day 19 challenge. All birds were monitored 2 weeks post challenge. 85% (37/40) and 93% (37/40) protection were observed at day 16 and day 19 for NDV challenge for in ovo vaccination. 70% (28/40) and 95% (38/40) protection were observed for subcutaneous vaccination at day of hatch. See Table 13 below.

Example 14

HVT-ND Immunity Duration Test

(No. 42, MSV+5) in SPF birds at infection on day 63

Three recombinants HVT-ND, HVT-ND(#42, MSV+5) were tested for duration of immunity against virulent NDV (Texas GB strain) challenge in SPF birds challenged on day 63. All birds were monitored 2 weeks post challenge. 100% (30/30) protection was observed with both in ovo and subcutaneous vaccination on day of hatch. See Table 14 below.

Example 15

Immunogenicity test of ND HVT-ND (No. 42, MSV+5) in SPF birds

Three recombinants HVT-ND, HVT-ND(#42, MSV+5) were tested for immunogenicity against challenge with virulent NDV (Texas GB strain, provided by USDA) on day 28 in SPF birds. All birds were observed 2 weeks after challenge. 100% (30/30) protection was observed with both in ovo and subcutaneous vaccination on day of hatch. See Table 15 below.

Example 16

Immunogenicity test of MD HVT-ND (No. 42, MSV+5) in SPF birds

Three recombinants HVT-ND, HVT-ND(#42, MSV+5) were tested for immunogenicity against virulent MDV (GA22 strain) challenge on day 5 in SPF birds. All birds were followed up at 54 days post challenge. 100% (30/30) protection was observed with both in ovo and subcutaneous vaccination on day of hatch. See Table 16 below.

Example 17

In vitro growth experiment

In vitro growth experiment was performed for HVT-ND #38, #42, #45. Roller bottles of 490 ^cm2 were seeded with 5× ¹⁰⁸ primary CEF cells. HVT-ND #38, #42, #45 were inoculated into each roller bottle at three different MOI: 0.001, 0.003, 0.008. Infected cells were collected 48 h after infection and titrated on CEF cells. Both HVT-ND #42 and #45 grew satisfactorily and were characterized by titer of 2.86× ¹⁰⁶ and 2.97× ¹⁰⁶ PFU/ml, respectively. HVT-ND #38 had a titer of 1.67 x 10 ⁶ PFU/ml. See Table 17 below.

Example 18

Design of HVT-IBD-ND #42-#30 LP C2

Creation of transfer plasmid -№42:

The parent transfer plasmid HVT-ND#42 was chemically synthesized by BioBasic, Inc. The cloning plasmid UL55/gene 3 was chemically synthesized by DNA2.0 as described above. PCR amplification of the NDV F gene expression cassette of transfer plasmid HVT-ND#42 was carried out using the following primers: upper primer SEQ ID NO. 154; lower primer SEQ ID NO. 155.

The amplified PCR product was cloned into the AscI and NheI sites of UL55/gene 3 to obtain final transfer plasmid #42. This plasmid was used for transfection/infection to produce HVT-ND #42.

Creation of transfer plasmid -№30:

The original transfer plasmid HVT-IBD#30 was chemically synthesized by BioBasic, Inc. The cloning plasmid was chemically synthesized by DNA2.0. PCR amplification of the IBD gene expression cassette of plasmid #30 was performed using the following primers: upper primer SEQ ID NO. 156, lower primer SEQ ID NO. 157. The amplified PCR product was cloned into the AgeI and KpnI sites of UL35/36 to obtain the final transfer plasmid #30. This plasmid was used for transfection/infection to produce HVT-IBD-ND#42-#30 LP C2.

Construction of HVT-ND #42:

Co-infection/transfection: CEF cells were seeded in a 6-well plate, the next day they were infected with HVT working seed (140 µl) + transfected with plasmid -#42 (linearized by SpeI + SbfI digestion) using Lipofectamine™ LTX reagent (ThermoFisher). Transfected cells were harvested on day 2 post-transfection. Positive foci were screened in a 6-well plate by IFA with chicken polyclonal antibody to NDV (live spot, dilution ~1:250), then further purified once (live spot) in a 96-well plate by limiting dilution to obtain single clones. The purified clone was passaged twice in a 6-well plate in duplicate, and the clone purity was confirmed by IFA (by fixation and staining). The harvest from the 6-well plate was used to construct HVT-IBD-ND #42-#30.

Design of HVT-IBD-ND #42-#30:

Co-infection/transfection: CEF cells were seeded in a 6-well plate, the next day they were infected with HVT-ND #42 + transfected with -#30 plasmid (linearized by SbfI digestion) using Lipofectamine™ LTX reagent (ThermoFisher). Transfected cells were harvested on day 3 post-transfection. Positive foci were screened in a 6-well plate by IFA with chicken polyclonal antibody to IBD (live spot, dilution ~1:250), then further purified once (live spot) by limiting dilution to obtain single clones. Two purified clones were selected and passaged in a 6-well plate in duplicate, and the purity of the clones was confirmed by IFA (by fixation and staining). Clones were propagated sequentially in a T-75 flask, T-150 flask, T-225 flask, and 850-mL roller flask. Recombinant virus was collected and aliquoted into 1-mL/flask, frozen at -80°C overnight, and then transferred to a reservoir containing LN.

Example 19:

In vivo efficacy test against NDV HVT-IBD-ND

#42-#30, #42-#32, #104 for SPF birds

Seven recombinants HVT-IBD-ND, HVT-IBD-ND #42-#30 (3 clones), #42-#32 (2 clones), #104 (2 clones) were tested for their in vivo efficacy against virulent NDV challenge in SPF birds. NDV Texas GB challenge was performed on day 28. Approximately 1500 PFU of each recombinant virus was inoculated in ovo at E18. All birds were observed 2 weeks post challenge. See Table 18 below.

Example 20:

In vivo anti-IBD efficacy test HVT-IBD-ND

#42-#30, #42-#32, #104 for SPF birds

Seven recombinants HVT-IBD-ND, HVT-IBD-ND #42-#30 (3 clones), #42-#32 (2 clones), #104 (2 clones) were tested for their in vivo efficacy against virulent IBDV challenge in SPF birds on day 14 and day 21, respectively. About 2000 PFU of each recombinant virus were inoculated in ovo into El8. All birds were necropsied 5 days post challenge. See Table 19 below.

Example 21

In vivo anti-MDV efficacy test HVT-IBD-ND #42 - #30

(4 clones)

at SPF birds

Three HVT-IBD-ND recombinants #42-#30 (4 clones) were tested for their in vivo efficacy against virulent MDV (GA22) challenge in SPF birds. Approximately 1500 PFU of each recombinant virus were inoculated in ovo at E18. MDV GA22 challenge was initiated on day 5. All birds were observed 54 days post challenge. See Table 20 below.

Example 22

In vivo efficacy test against wIBD HVT-IBD-ND

№42-№30, №42-№32, №04 for SPF birds

Three HVT-IBD-ND recombinants, #42-#30 (2 clones), #42-#32 (2 clones), #104 were tested for their in vivo efficacy against highly virulent IBDV challenge in SPF birds. Approximately 1500 PFU of each recombinant virus were inoculated in ovo at E18. vvIBDV challenge was performed on day 14 and day 21. All birds were observed 10 days post challenge. Histological examination of the bursa of Fabricius was performed for each bird at the end of the study. See Table 21 below.

Example 23

Test for duration of immunity against IBD when used

HVT-IBD-ND (No. 42-No. 30, X + 5) in SPF birds at infection on day 63

Recombinant HVT-IBD-ND, #42-#30 (MSV+5) was tested for duration of immunity against challenge with virulent classical IBDV in SPF birds challenged on day 63. All birds were observed four days post-challenge and necropsied. See Table 22.

Example 24

Immunogenicity test of ND HVT-IBD-ND (No. 42-No. 30, MSV+5) in SPF birds

Recombinant HVT-IBD-ND, #42-#30 (MSV+5) was tested for immunogenicity against challenge with virulent NDV (Texas GB strain) on day 28 in SPF birds. All birds were observed 2 weeks after challenge. See Table 23.

Example 25

IBD HVT-IBD-ND (No. 42-No. 30, MSV+5) Immunogenicity Test in SPF Birds

Recombinant HVT-IBD-ND, #42-#30 (MSV+5) was tested for immunogenicity against challenge with virulent IBDV on day 34 in SPF birds. All birds were observed 4 days post-challenge and necropsied for bursa of Fabricius lesions. See Table 24 below.

Example 26

Immunogenicity test of MD HVT-IBD-ND (No. 42-No. 30, MSV+5) in SPF birds

Three HVT-IBD-ND recombinants, #42-#30 (MSV+5), were tested for immunogenicity against challenge with virulent MDV (GA22 strain) on day 5 in SPF birds. All birds were followed for 54 days post-challenge. See Table 25 below.

Example 27

ND Immunogenicity Test HVT-IBD-ND

(No. 42-No. 30, MSV+5) in SPF birds against infection with the EU strain

Recombinant HVT-IBD-ND, #42-#30 (MSV+5) was tested for immunogenicity against challenge with virulent NDV Europe (Herts Weybridge 33/56) on day 21 in SPF birds. All birds were observed 2 weeks post-challenge. See Table 26 below.

Example 28 HVT-ND Compatibility with Bursaplex

Efficacy against IBDV

BURSAPLEX™ (Zoetis, US 5871748, incorporated herein by reference) is an infectious bursal disease (IBD) vaccine that contains a vaccine conjugate consisting of a live attenuated IBD strain 2512 and a neutralizing antibody, BDA, linked to the virus. Bursaplex generates active immunity against IBD in poultry, particularly chickens. At E18, eggs were injected in ovo with either control or test vaccine (HVT-ND to Bursaplex at a 1:1 ratio) and transferred to a designated incubator as specified by Biometrics, along with uninjected eggs. On the day of hatch, T04 birds were vaccinated subcutaneously. Blood samples were collected on days 5, 12, 19, 26, and 33 for IBDV serology. On day 34, selected birds were challenged with classical virulent IBDV and on day 38, all birds were necropsied for the presence of bursa of Fabricius lesions. No chicks in the negative group T01 developed clearly visible lesions, while 100 percent of the chicks in the control challenge group T02 developed clearly visible lesions. Both T03 (HVT-ND + Bursaplex, in ovo) and T04 (HVT-ND + Bursaplex, SC) were 100% protective. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Bursaplex are compatible when administered together and remain effective against IBDV challenge when administered either in ovo or subcutaneously.

Efficacy against NDV

At El8, eggs were injected in ovo with either control or test vaccine (HVT-ND with Bursaplex at a 1:1 ratio) and transferred to a designated incubator as specified by Biometrics, along with uninjected eggs. On the day of hatch, T04 birds were vaccinated subcutaneously. Blood samples were collected on days 6, 13, 20, and 27 for NDV serology. On day 28, select birds were challenged with velogenic NDV and on day 42, all surviving birds were euthanized. No chicks in the T01 negative group developed clinical signs, while 100 percent of chicks in the T02 challenged control group developed clinical signs of Newcastle disease, including mortality. Both T03 (HVT-ND + Bursaplex, in ovo) and T04 (HVT-ND + Bursaplex, SC) were 92.5% and 95% protective, respectively. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Bursaplex are compatible when administered together and remain effective against NDV challenge when administered either in ovo or subcutaneously.

Example 29 Compatibility of HVT-ND with Magniplex Efficacy against IBD

MAGNIPLEX™ (Zoetis) is an infectious bursa disease (IBD) vaccine that contains a vaccine conjugate consisting of a live attenuated IBD strain V877 and a neutralizing antibody, BDA, linked to the virus. Bursaplex generates active immunity against IBD in poultry, specifically chickens. At E18, eggs were injected in ovo with either control or test vaccine (pre-clearance HVT-ND series with Magniplex at a 1:1 ratio) and transferred to a designated incubator as specified by Biometrics, along with uninjected eggs. On the day of hatch, T04 birds were vaccinated subcutaneously. Blood samples were collected on days 5, 12, 19, 26, and 33 for IBDV serology. On day 34, selected birds were challenged with classical virulent IBDV and on day 38, all birds were necropsied for the presence of bursa of Fabricius lesions. No chicks in the negative group T01 developed clearly visible lesions, while 100 percent of the chicks in the control challenge group T02 developed clearly visible lesions. Both T03 (HVT-ND + Magniplex, in ovo) and T04 (HVT-ND + Magniplex, SC) were 100% protective. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Magniplex are compatible when administered together and remain effective against IBDV challenge when administered either in ovo or subcutaneously.

Efficacy against ND

At E18, eggs were injected in ovo with either control or test vaccine (HVT-ND with Magniplex at a 1:1 ratio) and transferred to a designated incubator as specified by Biometrics, along with uninjected eggs. On the day of hatch, T04 birds were vaccinated subcutaneously. Blood samples were collected on days 6, 13, 20, and 27 for NDV serology. On day 28, select birds were challenged with velogenic NDV and on day 42, all surviving birds were euthanized. No chicks in the T01 negative group developed clinical signs, while 100 percent of chicks in the T02 challenged control group developed clinical signs of Newcastle disease, including mortality. Both T03 (HVT-ND + Magniplex, in ovo) and T04 (HVT-ND + Magniplex, SC) were 92.5% and 95% protective, respectively. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Magniplex are compatible when administered together and remain effective against NDV challenge when administered either in ovo or subcutaneously.

--->

List of sequences

<110> Zoetis

Rong, Sing

Luo, Yugang

Brown, Tyler

<120> VECTORS BASED ON RECOMBINANT TURKEY HERPES VIRUS,

EXPRESSING ANTIGENS OF BIRD PATHOGENS,

AND OPTIONS FOR THEIR APPLICATION

<130> ZP000168

<140> 62/898651

<141> September 4, 2020

<160> 158

<170> PatentIn version 3.5

<210> 1

<211> 687

<212> DNA

<213> Artificial sequence

<220>

<223> Human CMV (hCMV) promoter DNA sequence

<400> 1

ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 60

cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 120

caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 180

gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 240

tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 300

ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 360

attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 420

gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 480

ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 540

aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag 600

agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc 660

tggctagcgt ttaaacttaa gcttacc 687

<210> 2

<211> 1391

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse CMV (mCMV) promoter DNA sequence

<400> 2

aactccgccc gttttatgac tagaaccaat agtttttaat gccaaatgca ctgaaatccc 60

ctaatttgca aagccaaacg ccccctatgt gagtaatacg gggacttttt acccaatttc 120

ccaagcggaa agccccctaa tacactcata tggcatatga atcagcacgg tcatgcactc 180

taatggcggc ccatagggac tttccacata gggggcgttc accatttccc agcatagggg 240

tggtgactca atggccttta cccaagtaca ttgggtcaat gggaggtaag ccaatgggtt 300

tttcccatta ctggcaagca cactgagtca aatgggactt tccactgggt tttgcccaag 360

tacattgggt caatgggagg tgagccaatg ggaaaaaccc attgctgcca agtacactga 420

ctcaataggg actttccaat gggtttttcc attgttggca agcatataag gtcaatgtgg 480

gtgagtcaat agggactttc cattgtattc tgcccagtac ataaggtcaa tagggggtga 540

atcaacagga aagtcccatt ggagccaagt acactgcgtc aatagggact ttccattggg 600

ttttgcccag tacataaggt caatagggga tgagtcaatg ggaaaaaccc attggagcca 660

agtacactga ctcaataggg actttccatt gggttttgcc cagtacataa ggtcaatagg 720

gggtgagtca acaggaaagt cccattggag ccaagtacat tgagtcaata gggactttcc 780

aatgggtttt gcccagtaca taaggtcaat gggaggtaag ccaatgggtt tttcccatta 840

ctggcacgta tactgagtca ttagggactt tccaatgggt tttgcccagt acataaggtc 900

aataggggtg aatcaacagg aaagtcccat tggagccaag tacactgagt caatagggac 960

tttccattgg gttttgccca gtacaaaagg tcaatagggg gtgagtcaat gggtttttcc 1020

cattattggc acgtacataa ggtcaatagg ggtgagtcat tgggtttttc cagccaattt 1080

aattaaaacg ccatgtactt tcccaccatt gacgtcaatg ggctattgaa actaatgcaa 1140

cgtgaccttt aaacggtact ttcccatagc tgattaatgg gaaagtaccg ttctcgagcc 1200

aatacacgtc aatgggaagt gaaagggcag ccaaaacgta acaccgcccc ggttttcccc 1260

tggaaattcc atattggcac gcattctatt ggctgagctg cgttctacgt gggtataaga 1320

ggcgcgacca gcgtcggtac cgtcgcagtc ttcggtctga ccaccgtaga acgcagagct 1380

cctcgctgca g 1391

<210> 3

<211> 1662

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of NDV F protein strain D26-76

<400> 3

atgggctcca gatcttctac caggatccca gtacctctga tgctgaccgt ccgaatcatg 60

ttggcactga gttgcgtctg tccgaccagc tcccttgatg gcaggcctct tgcagctgca 120

gggattgtgg taacaggaga caaagcagtc aacatataca cctcatctca gacagggtca 180

atcataatca agttactccc aaatatgccc aaggataaag aggcgtgtgc aaaagcccca 240

ttggaagcat acaacaggac attgactact ttgctcaccc cccttggtga ttctatccgt 300

aggatacaag agtctgtgac cacatccgga ggagggaaac agggacgtct tataggcgcc 360

attatcggtg gtgtagctct cggggttgca accgctgcac agataacagc agcctcggct 420

ctgatacaag ccaatcaaaa tgctgccaac atcctccggc tcaaagagag cattgctgca 480

accaatgagg ctgtgcacga ggtcactgac ggattatcac aactagcagt ggcagttggg 540

aagatgcagc aatttgttaa tgaccagttt aataaaacag ctcaggaatt ggactgtata 600

aaaattacac agcaggttgg tgtagaactc aacctgtacc taactgaatt gactacagta 660

ttcgggccac aaatcacttc ccctgcctta actcagctga ctatccaggc gctttacaat 720

ctagctggtg ggaatatgga ttacttgttg actaagttag gtgtaggaaa caaccaactc 780

agctcattaa ttggtagtgg cctgattacc ggcaacccta tcctgtacga ctcacagact 840

caactcttgg gtatacaggt caccctaccc tcagtcggga atctaaataa tatgcgtgcc 900

acctacctgg aaaccttgtc tgtaagtaca accaaaggat ttgcctcagc acttgtccca 960

aaagtagtga cacaggttgg ttccgtgata gaagagcttg acacctcgta ctgtatcgag 1020

accgatttgg acctatattg tacaagaata gtgacattcc ctatgtctcc tggtatttat 1080

tcctgtttga gtggcaatac atctgcttgc atgtattcaa agactgaagg cgcactcact 1140

acgccgtata tgaccctcaa aggctcagtt attgccaact gtaagatgac aacatgtaga 1200

tgtgcagacc ccccggggtat catatcgcag aattatggag aagctgtgtc tctaatagat 1260

aggcaatcat gcaatatctt atccttagac gggataactt tgaggctcag tggggaattt 1320

gatgcaactt atcaaaagaa tatctcaata caagattctc aagtaatagt tacaggcaat 1380

cttgacatct cgactgagct tgggaatgtc aacaactcga taagtaatgc tttggataag 1440

ttagaggaaa gcaacagcaa actagacaag gtcaatgtta aactgaccag cacatccgct 1500

cttattacct atatcgtttt aactgtcata tctcttgtat gtggtatact tagcctggtt 1560

ctagcatgct acctgatgta caagcaaaag gcgcaacaga agaccttgtt gtggcttggg 1620

aataataccc tagaccagat gagggccact acaaaaatgt ag 1662

<210> 4

<211> 553

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence of DNA protein of NDV F strain D26-76

<400> 4

Met Gly Ser Arg Ser Ser Thr Arg Ile Pro Val Pro Leu Met Leu Thr

1 5 10 15

Val Arg Ile Met Leu Ala Leu Ser Cys Val Cys Pro Thr Ser Ser Leu

20 25 30

Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys

35 40 45

Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Ile Lys

50 55 60

Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro

65 70 75 80

Leu Glu Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly

85 90 95

Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly

100 105 110

Lys Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly

115 120 125

Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ser Ala Leu Ile Gln Ala

130 135 140

Asn Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala

145 150 155 160

Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala

165 170 175

Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys

180 185 190

Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Thr Gln Gln Val Gly Val

195 200 205

Glu Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gly Pro Gln

210 215 220

Ile Thr Ser Pro Ala Leu Thr Gln Leu Thr Ile Gln Ala Leu Tyr Asn

225 230 235 240

Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Val Gly

245 250 255

Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn

260 265 270

Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr

275 280 285

Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu

290 295 300

Thr Leu Ser Val Ser Thr Thr Lys Gly Phe Ala Ser Ala Leu Val Pro

305 310 315 320

Lys Val Val Thr Gln Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser

325 330 335

Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr

340 345 350

Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser

355 360 365

Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met

370 375 380

Thr Leu Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg

385 390 395 400

Cys Ala Asp Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val

405 410 415

Ser Leu Ile Asp Arg Gln Ser Cys Asn Ile Leu Ser Leu Asp Gly Ile

420 425 430

Thr Leu Arg Leu Ser Gly Glu Phe Asp Ala Thr Tyr Gln Lys Asn Ile

435 440 445

Ser Ile Gln Asp Ser Gln Val Ile Val Thr Gly Asn Leu Asp Ile Ser

450 455 460

Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asp Lys

465 470 475 480

Leu Glu Glu Ser Asn Ser Lys Leu Asp Lys Val Asn Val Lys Leu Thr

485 490 495

Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Val Ile Ser Leu

500 505 510

Val Cys Gly Ile Leu Ser Leu Val Leu Ala Cys Tyr Leu Met Tyr Lys

515 520 525

Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu

530 535 540

Asp Gln Met Arg Ala Thr Thr Lys Met

545 550

<210> 5

<211> 1362

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of vIBDV (F52/70) VP2

<400> 5

atgaccaatc tccaggacca gacccagcag attgtgcctt ttattaggag tctcttgatg 60

cctacaaccg gccccgccag catcccggac gacacactgg aaaaacatac actgagaagc 120

gagacatcta catacaattt gaccgtgggc gataccggct ccgggcttat cgtgttcttc 180

ccaggttttc ccggatctat cgtaggagcg cactacaccc tccaaagtaa cggcaattac 240

aaattcgacc agatgctcct gacagcccag aaccttcctg cttcttacaa ttactgtaga 300

cttgtgtcca ggtccctgac tgtgcggagt agcacgcttc caggaggcgt atacgccctg 360

aacggaacta taaacgccgt caccttccag ggctccttgt ccgaacttac cgacgtgtcc 420

tacaatggcc tcatgagcgc aacggccaac ataaacgata agatcggcaa tgttcttgtg 480

ggcgaggggg ttacagtcct ttctctgcca accagttatg atctgggata cgtgcggctt 540

ggcgatccca ttcccgctat cggtctcgac cctaaaatgg tggctacttg cgactcatct 600

gaccgcccaa gggtctatac aattactgca gccgatgact atcagttttc cagccaatac 660

cagccagggg gtgtgacaat cacacttttc agcgccaata ttgacgctat cacatccctc 720

tcaatcggag gtgagcttgt gttccagact tctgttcagg gcttggtatt gggcgccact 780

atttacttga tcgggttcga cgggaccgca gtgatcactc gggcagtggc tgcggataac 840

ggactcactg ccggaactga caaccttatg ccttttaatc tggtcatccc cactaacgag 900

atcacccagc ctattacctc cataaagctc gaaattgtga ccagcaagag cggagggcag 960

gcaggcgacc aaatgagttg gtctgcaagc gggtccctcg ccgtgaccat ccacggtggc 1020

aactatcctg gggcgctcag acccgtcacc ctggtagcct acgaaagggt tgccacaggc 1080

tcagttgtca cggtggctgg agtaagcaat ttcgagctca tcccgaatcc tgagctcgct 1140

aaaaatcttg tgaccgagta tggaaggttc gaccctggcg caatgaatta cacaaagctg 1200

attctgtccg aacgggatag gctgggtatc aagacagttt ggcccacgcg cgaatacaca 1260

gatttcaggg agtactttat ggaggtcgca gatttgaata gcccacttaa gatcgctgga 1320

gcatttggct ttaaggatat tatccgcgca atcagaaggt ag 1362

<210> 6

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Bovine growth hormone Poly A DNA sequence

<400> 6

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggt 225

<210> 7

<211> 580

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for flanking HVT UL55

<400> 7

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg 580

<210> 8

<211> 678

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence flanking HVT gene 3

<400> 8

gtttaatgtt agtttattca atgcattggt tgcaaatatt cattacttct ccaatcccag 60

gtcattcttt agcgagatga tgttatgaca ttgctgtgaa aattactaca ggatatattt 120

ttaagatgca ggagtaacaa tgtgcatagt aggcgtagtt atcgcagacg tgcaacgctt 180

cgcatttgag ttaccgaagt gcccaacagt gctgcggtta tggtttatgc gcacagaatc 240

catgcatgtc ctaattgaac catccgattt ttcttttaat cgcgatcgtt gtttgggcaa 300

ctgcgttatt tcagatctaa aaaatttacc ctttatgacc atcacatctc tctggctcat 360

accccgcttg gataagatat catgtagatt ccgccctaag aaatgcaaac taacattatt 420

gtcggttcca tatacacttc catcttgtcc ttcgaaaata acaaactcgc gcaatagacc 480

gtccgtacat gcatggccga tgtgtgtcaa catcattggt ctgctagatc ccgatgggac 540

gaatcgtaca gtcgtcgctc cagcattggc aaaaatcccc agataccctc catgcggcaa 600

atctaaattg cgaccccgaa gagactgcac caaagtctta tcgacgcacg ctgatttttt 660

tgaacagcgg gagcccat 678

<210> 9

<211> 561

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the Pec promoter

<400> 9

agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 60

gttacataac ttacggtaaa tggcccgccg gctgaccgcc caacgacccc cgcccattga 120

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 180

gggtggagta tttacggtaa actgcccatt ggcagtacat caagtgtatc atatgccaag 240

tacgccccct attgacgtca atgacggtaa atggatgcag tattttgtgc agcgatgggg 300

gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 360

gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 420

ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 480

tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 540

ctgactgacc gcgtctagag g 561

<210> 10

<211> 1362

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence vvIBDV VP2

<400> 10

atgacaaatc ttcaggatca gacccagcag atagttccct ttattaggtc ccttctgatg 60

ccaaccacag ggcccgctag cattccggac gataccctgg agaaacacac tttgcggagt 120

gagacaagca cttacaatct gacggtggga gataccggct caggtcttat cgtgtttttt 180

cccggctttc ctggatccat tgttggcgcg cattacacgc tgcagagcaa cggcaactat 240

aaattcgatc agatgctcct gacggctcag aatctccccg ccagttacaa ttactgccgc 300

cttgtaagta ggtccttgac tgttagaagc tcaacgctgc caggcggagt atatgccctg 360

aatggaacca ttaatgctgt aacattccaa ggatcactgt ccgagctcac cgatgtgtct 420

tacaatggat tgatgtctgc cacggctaac attaacgaca agatcgggaa tgtgctcgtg 480

ggcgagggag tgaccgtttt gagcctgccg acaagctacg acctcggcta cgtaaggctc 540

ggggatccaa tccccgcgat cggcttggat cccaaaatgg ttgctacgtg cgacagcagc 600

gatagaccca gggtctatac catcaccgct gccgatgatt accagtttag ctcccagtac 660

caggcgggag gggtcacgat cacccttttt agcgccaaca tcgacgccat aacctcactt 720

tctatagggg gcgagttggt ttttcagacc agtgtccagg ggctcatcct cggtgcgaca 780

atctatctga tcggctttga cggaacagct gtcatcacga gggccgtagc tgcagataat 840

ggcctgactg ctgggacaga taatctgatg ccgttcaaca tagtgatccc caccagtgag 900

attacgcaac ccatcacgag catcaaactg gagatcgtga cgtcaaaatc cggcggtcag 960

gcaggtgacc agatgtcttg gtccgcaagc ggaagtttgg ccgtgacaat tcacgggggg 1020

aattaccccg gcgcactcag gcccgtgacc ctcgtcgcct acgaaagagt tgcaacggga 1080

agtgtagtga cagtcgctgg agtgagtaac ttcgaactca tccctaatcc cgagctcgcc 1140

aaaaatctcg tcacggagta tgggaggttt gatcccggcg ccatgaacta cacaaaactg 1200

atattgtccg aaagggatag gttgggcatt aaaaccgtgt ggcctactag ggaatacacc 1260

gatttccgcg aatattttat ggaggtcgcg gatctgaact ctcccctgaa gatagcaggc 1320

gcttttgggt tcaaggatat tatccgggcg ttgcggcggt ag 1362

<210> 11

<211> 197

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the SV40 promoter

<400> 11

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 60

ctccgcccag ttccgcccat tctccgcccc atcgctgact aatttttttt atttatgcag 120

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 180

gcctaggctt ttgcaaa 197

<210> 12

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the polyA sequence of SV40

<400> 12

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 60

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 120

tt 122

<210> 13

<211> 531

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for flanking HVT UL35

<400> 13

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat a 531

<210> 14

<211> 700

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence to flank HVT UL36

<400> 14

gtacataatt cttatttatc tttaatccat gaggagcatt tttattttaa aaatgtcagc 60

cgccagccct ataaccctag atcgcaactg atccctagtc tgcgttattt gtcttgcaat 120

cttttcgcac gcctttgtga gtgcatacaa tgcccccctg ctcgcttttc tgaaatcgcg 180

tcgggtcatt aatgtgtcgg ctatcacaat gcgagatgta ctcgacatgt ccgtgtctgt 240

actattggga ttgtaaatag tcgaccgcga atcatcagag tcggaatctg taaaggatac 300

agattccgac tctgagcgct tatgaatggg atccactcgg acgttgttga acttccgttc 360

ggattctgct tcagtcaaca ccggcccccg atagctacta aggttggggg gtttgtgggt 420

tgtttgtgaa actgctttgc ggtgtgcatt accacggggg gtgtggggaa gtatctgttt 480

ccacgatgcg ataacgttcg gtggcggagg gggcgattca ttctctagtg tacgcgtttc 540

aacttcagga acgtgattat ttctttcagg acactctttc caatttcctt cttccttcac 600

ttcgggtaca ggtatattct taatgtttac atacatgtcg tctgctcgtc tcaactgcgg 660

ggttatgatg ggtggtggtg acagtctctc cgaatgatcg 700

<210> 15

<211> 1278

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the chicken beta-actin promoter

<400> 15

tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 60

ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 120

cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg 180

gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg 240

cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgtt gccttcgccc 300

cgtgccccgc tccgcgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 360

ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa 420

tgacggctcg tttcttttct gtggctgcgt gaaagcctta aagggctccg ggagggccct 480

ttgtgcgggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc 540

gtgcggcccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg 600

ctccgcgtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc gggggggctg 660

cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg 720

cgcggcggtc gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc 780

cggcttcggg tgcggggctc cgtgcggggc gtggcgcggg gctcgccgtg ccgggcgggg 840

ggtggcggca ggtggggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg 900

gggaggggcg cggcggcccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca 960

ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctggcg 1020

gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcgggcg aagcggtgcg 1080

gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc 1140

ttctccatct ccagcctcgg ggctgccgca gggggacggc tgccttcggg ggggacgggg 1200

cagggcgggg ttcggcttct ggcgtgtgac cggcggggtt tatatcttcc cttctctgtt 1260

cctccgcagc cagccatg 1278

<210> 16

<211> 2882

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of modified transfer plasmid for

HVT-gfp-A #14*

<400> 16

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat attgacattg 540

attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 600

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 660

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 720

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 780

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 840

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 900

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 960

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 1020

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 1080

taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac 1140

tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg 1200

tttaaactta agcttacctg caggccacca tggtgagcaa gggcgccgag ctgttcaccg 1260

gcatcgtgcc catcctgatc gagctgaatg gcgatgtgaa tggccacaag ttcagcgtga 1320

gcggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 1380

ccggcaagct gcctgtgccc tggcccaccc tggtgaccac cctgagctac ggcgtgcagt 1440

gcttctcacg ctaccccgat cacatgaagc agcacgactt cttcaagagc gccatgcctg 1500

agggctacat ccaggagcgc accatcttct tcgaggatga cggcaactac aagtcgcgcg 1560

ccgaggtgaa gttcgagggc gataccctgg tgaatcgcat cgagctgacc ggcaccgatt 1620

tcaagggagga tggcaacatc ctgggcaata agatggagta caactacaac gcccacaatg 1680

tgtacatcat gaccgacaag gccaagaatg gcatcaaggt gaacttcaag atccgccaca 1740

acatcgagga tggcagcgtg cagctggccg accactacca gcagaatacc cccatcggcg 1800

atggccctgt gctgctgccc gataaccact acctgtccac cgagcgcc ctgtccaagg 1860

accccaacga gaagcgcgat cacatgatct acttcggctt cgtgaccgcc gccgccatca 1920

cccacggcat ggatgagctg tacaagtgac ctgcaggtgt gccttctagt tgccagccat 1980

ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 2040

tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 2100

ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 2160

gggatgcggt gggctctatg gtgtacataa ttcttattta tctttaatcc atgaggagca 2220

tttttatttt aaaaatgtca gccgccagcc ctataaccct agatcgcaac tgatccctag 2280

tctgcgttat ttgtcttgca atcttttcgc acgcctttgt gagtgcatac aatgcccccc 2340

tgctcgcttt tctgaaatcg cgtcgggtca ttaatgtgtc ggctatcaca atgcgagatg 2400

tactcgacat gtccgtgtct gtactattgg gattgtaaat agtcgaccgc gaatcatcag 2460

agtcggaatc tgtaaaggat acagattccg actctgagcg cttatgaatg ggatccactc 2520

ggacgttgtt gaacttccgt tcggattctg cttcagtcaa caccggcccc cgatagctac 2580

taaggttggg gggtttgtgg gttgtttgtg aaactgcttt gcggtgtgca ttaccacggg 2640

gggtgtgggg aagtatctgt ttccacgatg cgataacgtt cggtggcgga gggggcgatt 2700

cattctctag tgtacgcgtt tcaacttcag gaacgtgatt atttctttca ggacactctt 2760

tccaatttcc ttcttccttc acttcgggta caggtatatt cttaatgttt acatacatgt 2820

cgtctgctcg tctcaactgc ggggttatga tgggtggtgg tgacagtctc tccgaatgat 2880

cg 2882

<210> 17

<211> 2869

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the original transfer plasmid for HVT-gfp-A #14

<400> 17

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat attgacattg 540

attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 600

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 660

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 720

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 780

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 840

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 900

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 960

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 1020

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 1080

taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac 1140

tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg 1200

tttaaactta agcttaccgc caccatggtg agcaagggcg ccgagctgtt caccggcatc 1260

gtgcccatcc tgatcgagct gaatggcgat gtgaatggcc acaagttcag cgtgagcggc 1320

gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 1380

aagctgcctg tgccctggcc caccctggtg accaccctga gctacggcgt gcagtgcttc 1440

tcacgctacc ccgatcacat gaagcagcac gacttcttca agagcgccat gcctgaggc 1500

tacatccagg agcgcaccat cttcttcgag gatgacggca actacaagtc gcgcgccgag 1560

gtgaagttcg agggcgatac cctggtgaat cgcatcgagc tgaccggcac cgatttcaag 1620

gaggatggca acatcctggg caataagatg gagtacaact acaacgccca caatgtgtac 1680

atcatgaccg acaaggccaa gaatggcatc aaggtgaact tcaagatccg ccacaacatc 1740

gaggatggca gcgtgcagct ggccgaccac taccagcaga atacccccat cggcgatggc 1800

cctgtgctgc tgcccgataa ccactacctg tccacccaga gcgccctgtc caaggacccc 1860

aacgagaagc gcgatcacat gatctacttc ggcttcgtga ccgccgccgc catcaccac 1920

ggcatggatg agctgtacaa gtgatgtgcc ttctagttgc cagccatctg ttgtttgccc 1980

ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2040

tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 2100

gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 2160

ctctatggtg tacataattc ttatttatct ttaatccatg aggagcattt ttattttaaa 2220

aatgtcagcc gccagcccta taaccctaga tcgcaactga tccctagtct gcgttatttg 2280

tcttgcaatc ttttcgcacg cctttgtgag tgcatacaat gcccccctgc tcgcttttct 2340

gaaatcgcgt cgggtcatta atgtgtcggc tatcacaatg cgagatgtac tcgacatgtc 2400

cgtgtctgta ctattgggat tgtaaatagt cgaccgcgaa tcatcagagt cggaatctgt 2460

aaaggataca gattccgact ctgagcgctt atgaatggga tccactcgga cgttgttgaa 2520

cttccgttcg gattctgctt cagtcaacac cggcccccga tagctactaa ggttgggggg 2580

tttgtgggtt gtttgtgaaa ctgctttgcg gtgtgcatta ccacgggggg tgtggggaag 2640

tatctgtttc cacgatgcga taacgttcgg tggcggaggg ggcgattcat tctctagtgt 2700

acgcgtttca acttcaggaa cgtgattatt tctttcagga cactctttcc aatttccttc 2760

ttccttcact tcgggtacag gtatattctt aatgtttaca tacatgtcgt ctgctcgtct 2820

caactgcggg gttatgatgg gtggtggtga cagtctctcc gaatgatcg 2869

<210> 18

<211> 2895

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-gfp-B #13

<400> 18

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg ttgacattga ttattgacta 600

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 660

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 720

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 780

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 840

gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 900

tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 960

tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 1020

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 1080

actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 1140

ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc 1200

ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1260

gcttaccgcc accatggtga gcaagggcgc cgagctgttc accggcatcg tgcccatcct 1320

gatcgagctg aatggcgatg tgaatggcca caagttcagc gtgagcggcg agggcgaggg 1380

cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcctgt 1440

gccctggccc accctggtga ccaccctgag ctacggcgtg cagtgcttct cacgctaccc 1500

cgatcacatg aagcagcacg acttcttcaa gagcgccatg cctgagggct acatccagga 1560

gcgcaccatc ttcttcgagg atgacggcaa ctacaagtcg cgcgccgagg tgaagttcga 1620

gggcgatacc ctggtgaatc gcatcgagct gaccggcacc gatttcaagg aggatggcaa 1680

catcctgggc aataagatgg agtacaacta caacgcccac aatgtgtaca tcatgaccga 1740

caaggccaag aatggcatca aggtgaactt caagatccgc cacaacatcg aggatggcag 1800

cgtgcagctg gccgaccact accagcagaa tacccccatc ggcgatggcc ctgtgctgct 1860

gcccgataac cactacctgt ccacccagag cgccctgtcc aaggacccca acgagaagcg 1920

cgatcacatg atctacttcg gcttcgtgac cgccgccgcc atcaccaccacg gcatggatga 1980

gctgtacaag tgatgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 2040

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 2100

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 2160

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggttt 2220

taatgttagt ttattcaatg cattggttgc aaatattcat tacttctcca atcccaggtc 2280

attctttagc gagatgatgt tatgacattg ctgtgaaaat tactacagga tatattttta 2340

agatgcagga gtaacaatgt gcatagtagg cgtagttatc gcagacgtgc aacgcttcgc 2400

atttgagtta ccgaagtgcc caacagtgct gcggttatgg tttatgcgca cagaatccat 2460

gcatgtccta attgaaccat ccgatttttc ttttaatcgc gatcgttgtt tgggcaactg 2520

cgttatttca gatctaaaaa atttaccctt tatgaccatc acatctctct ggctcatacc 2580

ccgcttggat aagatatcat gtagattccg ccctaagaaa tgcaaactaa cattattgtc 2640

ggttccatat acacttccat cttgtccttc gaaaataaca aactcgcgca atagaccgtc 2700

cgtacatgca tggccgatgt gtgtcaacat cattggtctg ctagatcccg atgggacgaa 2760

tcgtacagtc gtcgctccag cattggcaaa aatccccaga taccctccat gcggcaaatc 2820

taaattgcga ccccgaagag actgcaccaa agtcttatcg acgcacgctg atttttttga 2880

acagcggggag cccat 2895

<210> 19

<211> 2908

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-gfp-B #13

<400> 19

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg ttgacattga ttattgacta 600

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 660

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 720

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 780

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 840

gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 900

tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 960

tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 1020

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 1080

actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 1140

ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc 1200

ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1260

gcttacctgc aggccaccat ggtgagcaag ggcgccgagc tgttcaccgg catcgtgccc 1320

atcctgatcg agctgaatgg cgatgtgaat ggccacaagt tcagcgtgag cggcgagggc 1380

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 1440

cctgtgccct ggcccaccct ggtgaccacc ctgagctacg gcgtgcagtg cttctcacgc 1500

taccccgatc acatgaagca gcacgacttc ttcaagagcg ccatgcctga gggctacatc 1560

caggagcgca ccatcttctt cgaggatgac ggcaactaca agtcgcgcgc cgaggtgaag 1620

ttcgaggcg ataccctggt gaatcgcatc gagctgaccg gcaccgattt caaggaggat 1680

ggcaacatcc tgggcaataa gatggagtac aactacaacg cccacaatgt gtacatcatg 1740

accgacaagg ccaagaatgg catcaaggtg aacttcaaga tccgccacaa catcgaggat 1800

ggcagcgtgc agctggccga ccactaccag cagaataccc ccatcggcga tggccctgtg 1860

ctgctgcccg ataaccacta cctgtccacc cagagcgccc tgtccaagga ccccaacgag 1920

aagcgcgatc acatgatcta cttcggcttc gtgaccgccg ccgccatcac ccacggcatg 1980

gatgagctgt acaagtgacc tgcaggtgtg ccttctagtt gccagccatc tgttgtttgc 2040

ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 2100

aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 2160

gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg 2220

ggctctatgg ttttaatgtt agtttattca atgcattggt tgcaaatatt cattacttct 2280

ccaatcccag gtcattcttt agcgagatga tgttatgaca ttgctgtgaa aattactaca 2340

ggatatattt ttaagatgca ggagtaacaa tgtgcatagt aggcgtagtt atcgcagacg 2400

tgcaacgctt cgcatttgag ttaccgaagt gcccaacagt gctgcggtta tggtttatgc 2460

gcacagaatc catgcatgtc ctaattgaac catccgattt ttcttttaat cgcgatcgtt 2520

gtttgggcaa ctgcgttatt tcagatctaa aaaatttacc ctttatgacc atcacatctc 2580

tctggctcat accccgcttg gataagatat catgtagatt ccgccctaag aaatgcaaac 2640

taacattatt gtcggttcca tatacacttc catcttgtcc ttcgaaaata acaaactcgc 2700

gcaatagacc gtccgtacat gcatggccga tgtgtgtcaa catcattggt ctgctagatc 2760

ccgatgggac gaatcgtaca gtcgtcgctc cagcattggc aaaaatcccc agataccctc 2820

catgcggcaa atctaaattg cgaccccgaa gagactgcac caaagtctta tcgacgcacg 2880

ctgatttttt tgaacagcgg gagcccat 2908

<210> 20

<211> 3538

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #1

<400> 20

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg ttgacattga ttattgacta 600

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 660

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 720

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 780

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 840

gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 900

tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 960

tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 1020

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 1080

actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 1140

ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc 1200

ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1260

gcttaccgcc accatgacaa atcttcagga tcagacccag cagatagttc cctttattag 1320

gtcccttctg atgccaacca cagggcccgc tagcattccg gacgataccc tggagaaaca 1380

cactttgcgg agtgagacaa gcacttacaa tctgacggtg ggagataccg gctcaggtct 1440

tatcgtgttt tttcccggct ttcctggatc cattgttggc gcgcattaca cgctgcagag 1500

caacggcaac tataaattcg atcagatgct cctgacggct cagaatctcc ccgccagtta 1560

caattactgc cgccttgtaa gtaggtcctt gactgttaga agctcaacgc tgccaggcgg 1620

agtatatgcc ctgaatggaa ccattaatgc tgtaacattc caaggatcac tgtccgagct 1680

caccgatgtg tcttacaatg gattgatgtc tgccacggct aacattaacg acaagatcgg 1740

gaatgtgctc gtgggcgagg gagtgaccgt tttgagcctg ccgacaagct acgacctcgg 1800

ctacgtaagg ctcggggatc caatccccgc gatcggcttg gatcccaaaa tggttgctac 1860

gtgcgacagc agcgatagac cccaggtcta taccatcacc gctgccgatg attaccagtt 1920

tagctcccag taccaggcgg gaggggtcac gatcaccctt tttagcgcca acatcgacgc 1980

cataacctca ctttctatag ggggcgagtt ggtttttcag accagtgtcc aggggctcat 2040

cctcggtgcg acaatctatc tgatcggctt tgacggaaca gctgtcatca cgagggccgt 2100

agctgcagat aatggcctga ctgctgggac agataatctg atgccgttca acatagtgat 2160

ccccaccagt gagattacgc aacccatcac gagcatcaaa ctggagatcg tgacgtcaaa 2220

atccggcggt caggcaggtg accagatgtc ttggtccgca agcggaagtt tggccgtgac 2280

aattcacggg gggaattacc ccggcgcact caggcccgtg accctcgtcg cctacgaaag 2340

agttgcaacg ggaagtgtag tgacagtcgc tggagtgagt aacttcgaac tcatccctaa 2400

tcccgagctc gccaaaaatc tcgtcacgga gtatgggagg tttgatcccg gcgccatgaa 2460

ctacacaaaa ctgatattgt ccgaaaggga taggttgggc attaaaaccg tgtggcctac 2520

tagggaatac accgatttcc gcgaatattt tatggaggtc gcggatctga actctcccct 2580

gaagatagca ggcgcttttg ggttcaagga tattatccgg gcgttgcggc ggtagtgtgc 2640

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 2700

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 2760

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 2820

acaatagcag gcatgctggg gatgcggtgg gctctatggt gtttaatgtt agtttattca 2880

atgcattggt tgcaaatatt cattacttct ccaatcccag gtcattcttt agcgagatga 2940

tgttatgaca ttgctgtgaa aattactaca ggatatattt ttaagatgca ggagtaacaa 3000

tgtgcatagt aggcgtagtt atcgcagacg tgcaacgctt cgcatttgag ttaccgaagt 3060

gcccaacagt gctgcggtta tggtttatgc gcacagaatc catgcatgtc ctaattgaac 3120

catccgattt ttcttttaat cgcgatcgtt gtttgggcaa ctgcgttatt tcagatctaa 3180

aaaatttacc ctttatgacc atcacatctc tctggctcat accccgcttg gataagatat 3240

catgtagatt ccgccctaag aaatgcaaac taacattatt gtcggttcca tatacacttc 3300

catcttgtcc ttcgaaaata acaaactcgc gcaatagacc gtccgtacat gcatggccga 3360

tgtgtgtcaa catcattggt ctgctagatc ccgatgggac gaatcgtaca gtcgtcgctc 3420

cagcattggc aaaaatcccc agataccctc catgcggcaa atctaaattg cgaccccgaa 3480

gagactgcac caaagtctta tcgacgcacg ctgatttttt tgaacagcgg gagcccat 3538

<210> 21

<211> 3511

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #5

<400> 21

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat attgacattg 540

attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 600

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 660

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 720

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 780

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 840

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 900

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 960

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 1020

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 1080

taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac 1140

tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg 1200

tttaaactta agcttaccgc caccatgaca aatcttcagg atcagaccca gcagatagtt 1260

ccctttatta ggtcccttct gatgccaacc acagggcccg ctagcattcc ggacgatacc 1320

ctggagaaac acactttgcg gagtgagaca agcacttaca atctgacggt gggagatacc 1380

ggctcaggtc ttatcgtgtt ttttcccggc tttcctggat ccattgttgg cgcgcattac 1440

acgctgcaga gcaacggcaa ctataaattc gatcagatgc tcctgacggc tcagaatctc 1500

cccgccagtt acaattactg ccgccttgta agtaggtcct tgactgttag aagctcaacg 1560

ctgccaggcg gagtatatgc cctgaatgga accattaatg ctgtaacatt ccaaggatca 1620

ctgtccgagc tcaccgatgt gtcttacaat ggattgatgt ctgccacggc taacattaac 1680

gacaagatcg ggaatgtgct cgtgggcgag ggagtgaccg ttttgagcct gccgacaagc 1740

tacgacctcg gctacgtaag gctcggggat ccaatccccg cgatcggctt ggatcccaaa 1800

atggttgcta cgtgcgacag cagcgataga cccagggtct ataccatcac cgctgccgat 1860

gattaccagt ttagctccca gtaccaggcg ggaggggtca cgatcaccct ttttagcgcc 1920

aacatcgacg ccataacctc actttctata gggggcgagt tggtttttca gaccagtgtc 1980

caggggctca tcctcggtgc gacaatctat ctgatcggct ttgacggaac agctgtcatc 2040

acgagggccg tagctgcaga taatggcctg actgctggga cagataatct gatgccgttc 2100

aacatagtga tccccaccag tgagattacg caacccatca cgagcatcaa actggagatc 2160

gtgacgtcaa aatccggcgg tcaggcaggt gaccagatgt cttggtccgc aagcggaagt 2220

ttggccgtga caattcacgg ggggaattac cccggcgcac tcaggcccgt gaccctcgtc 2280

gcctacgaaa gagttgcaac gggaagtgta gtgacagtcg ctggagtgag taacttcgaa 2340

ctcatcccta atcccgagct cgccaaaaat ctcgtcacgg agtatgggag gtttgatccc 2400

ggcgccatga actacacaaa actgatattg tccgaaaggg ataggttggg cattaaaacc 2460

gtgtggccta ctagggaata caccgatttc cgcgaatatt ttatggaggt cgcggatctg 2520

aactctcccc tgaagatagc aggcgctttt gggttcaagg atattatccg ggcgttgcgg 2580

cggtagtgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2640

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2700

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 2760

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg tgtacataat 2820

tcttatttat ctttaatcca tgaggagcat ttttatttta aaaatgtcag ccgccagccc 2880

tataacccta gatcgcaact gatccctagt ctgcgttatt tgtcttgcaa tcttttcgca 2940

cgcctttgtg agtgcataca atgcccccct gctcgctttt ctgaaatcgc gtcgggtcat 3000

taatgtgtcg gctatcacaa tgcgagatgt actcgacatg tccgtgtctg tactattggg 3060

attgtaaata gtcgaccgcg aatcatcaga gtcggaatct gtaaaggata cagattccga 3120

ctctgagcgc ttatgaatgg gatccactcg gacgttgttg aacttccgtt cggattctgc 3180

ttcagtcaac accggccccc gatagctact aaggttgggg ggtttgtggg ttgtttgtga 3240

aactgctttg cggtgtgcat taccacgggg ggtgtgggga agtatctgtt tccacgatgc 3300

gataacgttc ggtggcggag ggggcgattc attctctagt gtacgcgttt caacttcagg 3360

aacgtgatta tttctttcag gacactcttt ccaatttcct tcttccttca cttcgggtac 3420

aggtatattc ttaatgttta catacatgtc gtctgctcgt ctcaactgcg gggttatgat 3480

gggtggtggt gacagtctct ccgaatgatc g 3511

<210> 22

<211> 3385

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #1

<400> 22

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat aagttattaa 540

tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 600

cttacggtaa atggcccgcc ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 660

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 720

atttacggta aactgcccat tggcagtaca tcaagtgtat catatgccaa gtacgccccc 780

tattgacgtc aatgacggta aatggatgca gtattttgtg cagcgatggg ggcggggggg 840

gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 900

aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 960

gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 1020

ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 1080

cgcgtctaga gggccaccat gacaaatctt caggatcaga cccagcagat agttcccttt 1140

attaggtccc ttctgatgcc aaccacaggg cccgctagca ttccggacga taccctggag 1200

aaacacactt tgcggagtga gacaagcact tacaatctga cggtgggaga taccggctca 1260

ggtcttatcg tgttttttcc cggctttcct ggatccattg ttggcgcgca ttacacgctg 1320

cagagcaacg gcaactataa attcgatcag atgctcctga cggctcagaa tctccccgcc 1380

agttacaatt actgccgcct tgtaagtagg tccttgactg ttagaagctc aacgctgcca 1440

ggcggagtat atgccctgaa tggaaccatt aatgctgtaa cattccaagg atcactgtcc 1500

gagctcaccg atgtgtctta caatggattg atgtctgcca cggctaacat taacgacaag 1560

atcgggaatg tgctcgtggg cgagggagtg accgttttga gcctgccgac aagctacgac 1620

ctcggctacg taaggctcgg ggatccaatc cccgcgatcg gcttggatcc caaaatggtt 1680

gctacgtgcg acagcagcga tagacccagg gtctatacca tcaccgctgc cgatgattac 1740

cagtttagct cccagtacca ggcgggaggg gtcacgatca ccctttttag cgccaacatc 1800

gacgccataa cctcactttc tatagggggc gagttggttt ttcagaccag tgtccagggg 1860

ctcatcctcg gtgcgacaat ctatctgatc ggctttgacg gaacagctgt catcacgagg 1920

gccgtagctg cagataatgg cctgactgct gggacagata atctgatgcc gttcaacata 1980

gtgatcccca ccagtgagat tacgcaaccc atcacgagca tcaaactgga gatcgtgacg 2040

tcaaaatccg gcggtcaggc aggtgaccag atgtcttggt ccgcaagcgg aagtttggcc 2100

gtgacaattc acggggggaa ttaccccggc gcactcaggc ccgtgaccct cgtcgcctac 2160

gaaagagttg caacgggaag tgtagtgaca gtcgctggag tgagtaactt cgaactcatc 2220

cctaatcccg agctcgccaa aaatctcgtc acggagtatg ggaggtttga tcccggcgcc 2280

atgaactaca caaaactgat attgtccgaa agggataggt tgggcattaa aaccgtgtgg 2340

cctactaggg aatacaccga tttccgcgaa tattttatgg aggtcgcgga tctgaactct 2400

cccctgaaga tagcaggcgc ttttgggttc aaggatatta tccgggcgtt gcggcggtag 2460

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 2520

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 2580

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 2640

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggtgtaca taattcttat 2700

ttatctttaa tccatgagga gcatttttat tttaaaaatg tcagccgcca gccctataac 2760

cctagatcgc aactgatccc tagtctgcgt tatttgtctt gcaatctttt cgcacgcctt 2820

tgtgagtgca tacaatgccc ccctgctcgc ttttctgaaa tcgcgtcggg tcattaatgt 2880

gtcggctatc acaatgcgag atgtactcga catgtccgtg tctgtactat tgggattgta 2940

aatagtcgac cgcgaatcat cagagtcgga atctgtaaag gatacagatt ccgactctga 3000

gcgcttatga atgggatcca ctcggacgtt gttgaacttc cgttcggatt ctgcttcagt 3060

caacaccggc ccccgatagc tactaaggtt ggggggtttg tgggttgttt gtgaaactgc 3120

tttgcggtgt gcattaccac ggggggtgtg gggaagtatc tgtttccacg atgcgataac 3180

gttcggtggc ggagggggcg attcattctc tagtgtacgc gtttcaactt caggaacgtg 3240

attatttctt tcaggacact ctttccaatt tccttcttcc ttcacttcgg gtacaggtat 3300

attcttaatg tttacataca tgtcgtctgc tcgtctcaac tgcggggtta tgatgggtgg 3360

tggtgacagt ctctccgaat gatcg 3385

<210> 23

<211> 3538

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #9

<400> 23

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg ttgacattga ttattgacta 600

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 660

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 720

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 780

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 840

gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 900

tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 960

tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 1020

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 1080

actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 1140

ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc 1200

ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1260

gcttaccgcc accatgacca atctccagga ccagacccag cagattgtgc cttttattag 1320

gagtctcttg atgcctacaa ccggccccgc cagcatcccg gacgacacac tggaaaaaca 1380

tacactgaga agcgagacat ctacatacaa tttgaccgtg ggcgataccg gctccgggct 1440

tatcgtgttc ttcccaggtt ttcccggatc tatcgtagga gcgcactaca ccctccaaag 1500

taacggcaat tacaaattcg accagatgct cctgacagcc cagaaccttc ctgcttctta 1560

caattactgt agacttgtgt ccaggtccct gactgtgcgg agtagcacgc ttccaggagg 1620

cgtatacgcc ctgaacggaa ctataaacgc cgtcaccttc cagggctcct tgtccgaact 1680

taccgacgtg tcctacaatg gcctcatgag cgcaacggcc aacataaacg ataagatcgg 1740

caatgttctt gtgggcgagg gggttacagt cctttctctg ccaaccagtt atgatctggg 1800

atacgtgcgg cttggcgatc ccattcccgc tatcggtctc gaccctaaaa tggtggctac 1860

ttgcgactca tctgaccgcc caagggtcta tacaattact gcagccgatg actatcagtt 1920

ttccagccaa taccagccag ggggtgtgac aatcacactt ttcagcgcca atattgacgc 1980

tatcacatcc ctctcaatcg gaggtgagct tgtgttccag acttctgttc agggcttggt 2040

attgggcgcc actatttact tgatcgggtt cgacgggacc gcagtgatca ctcgggcagt 2100

ggctgcggat aacggactca ctgccggaac tgacaacctt atgcctttta atctggtcat 2160

ccccactaac gagatcaccc agcctattac ctccataaag ctcgaaattg tgaccagcaa 2220

gagcggaggg caggcaggcg accaaatgag ttggtctgca agcgggtccc tcgccgtgac 2280

catccacggt ggcaactatc ctggggcgct cagacccgtc accctggtag cctacgaaag 2340

ggttgccaca ggctcagttg tcacggtggc tggagtaagc aatttcgagc tcatcccgaa 2400

tcctgagctc gctaaaaatc ttgtgaccga gtatggaagg ttcgaccctg gcgcaatgaa 2460

ttacacaaag ctgattctgt ccgaacggga taggctgggt atcaagacag tttggcccac 2520

gcgcgaatac acagatttca gggagtactt tatggaggtc gcagatttga atagcccact 2580

taagatcgct ggagcatttg gctttaagga tattatccgc gcaatcagaa ggtagtgtgc 2640

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 2700

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 2760

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 2820

acaatagcag gcatgctggg gatgcggtgg gctctatggt gtttaatgtt agtttattca 2880

atgcattggt tgcaaatatt cattacttct ccaatcccag gtcattcttt agcgagatga 2940

tgttatgaca ttgctgtgaa aattactaca ggatatattt ttaagatgca ggagtaacaa 3000

tgtgcatagt aggcgtagtt atcgcagacg tgcaacgctt cgcatttgag ttaccgaagt 3060

gcccaacagt gctgcggtta tggtttatgc gcacagaatc catgcatgtc ctaattgaac 3120

catccgattt ttcttttaat cgcgatcgtt gtttgggcaa ctgcgttatt tcagatctaa 3180

aaaatttacc ctttatgacc atcacatctc tctggctcat accccgcttg gataagatat 3240

catgtagatt ccgccctaag aaatgcaaac taacattatt gtcggttcca tatacacttc 3300

catcttgtcc ttcgaaaata acaaactcgc gcaatagacc gtccgtacat gcatggccga 3360

tgtgtgtcaa catcattggt ctgctagatc ccgatgggac gaatcgtaca gtcgtcgctc 3420

cagcattggc aaaaatcccc agataccctc catgcggcaa atctaaattg cgaccccgaa 3480

gagactgcac caaagtctta tcgacgcacg ctgatttttt tgaacagcgg gagcccat 3538

<210> 24

<211> 3511

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #30

<400> 24

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat attgacattg 540

attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 600

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 660

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 720

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 780

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 840

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 900

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 960

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 1020

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 1080

taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac 1140

tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg 1200

tttaaactta agcttaccgc caccatgacc aatctccagg accagaccca gcagattgtg 1260

ccttttatta ggagtctctt gatgcctaca accggccccg ccagcatccc ggacgacaca 1320

ctggaaaaac atacactgag aagcgagaca tctacataca atttgaccgt gggcgatacc 1380

ggctccgggc ttatcgtgtt cttcccaggt tttcccggat ctatcgtagg agcgcactac 1440

accctccaaa gtaacggcaa ttacaaattc gaccagatgc tcctgacagc ccagaacctt 1500

cctgcttctt acaattactg tagacttgtg tccaggtccc tgactgtgcg gagtagcacg 1560

cttccaggag gcgtatacgc cctgaacgga actataaacg ccgtcacctt ccagggctcc 1620

ttgtccgaac ttaccgacgt gtcctacaat ggcctcatga gcgcaacggc caacataaac 1680

gataagatcg gcaatgttct tgtgggcgag ggggttacag tcctttctct gccaaccagt 1740

tatgatctgg gatacgtgcg gcttggcgat cccattcccg ctatcggtct cgaccctaaa 1800

atggtggcta cttgcgactc atctgaccgc ccaagggtct atacaattac tgcagccgat 1860

gactatcagt tttccagcca ataccagcca gggggtgtga caatcacact tttcagcgcc 1920

aatattgacg ctatcacatc cctctcaatc ggaggtgagc ttgtgttcca gacttctgtt 1980

cagggcttgg tattgggcgc cactatttac ttgatcgggt tcgacgggac cgcagtgatc 2040

actcgggcag tggctgcgga taacggactc actgccggaa ctgacaacct tatgcctttt 2100

aatctggtca tccccactaa cgagatcacc cagcctatta cctccataaa gctcgaaatt 2160

gtgaccagca agagcggagg gcaggcaggc gaccaaatga gttggtctgc aagcgggtcc 2220

ctcgccgtga ccatccacgg tggcaactat cctggggcgc tcagacccgt caccctggta 2280

gcctacgaaa gggttgccac aggctcagtt gtcacggtgg ctggagtaag caatttcgag 2340

ctcatcccga atcctgagct cgctaaaaat cttgtgaccg agtatggaag gttcgaccct 2400

ggcgcaatga attacacaaa gctgattctg tccgaacggg ataggctggg tatcaagaca 2460

gtttggccca cgcgcgaata cacagatttc agggagtact ttatggaggt cgcagatttg 2520

aatagcccac ttaagatcgc tggagcattt ggctttaagg atattatccg cgcaatcaga 2580

aggtagtgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 2640

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 2700

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 2760

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg tgtacataat 2820

tcttatttat ctttaatcca tgaggagcat ttttatttta aaaatgtcag ccgccagccc 2880

tataacccta gatcgcaact gatccctagt ctgcgttatt tgtcttgcaa tcttttcgca 2940

cgcctttgtg agtgcataca atgcccccct gctcgctttt ctgaaatcgc gtcgggtcat 3000

taatgtgtcg gctatcacaa tgcgagatgt actcgacatg tccgtgtctg tactattggg 3060

attgtaaata gtcgaccgcg aatcatcaga gtcggaatct gtaaaggata cagattccga 3120

ctctgagcgc ttatgaatgg gatccactcg gacgttgttg aacttccgtt cggattctgc 3180

ttcagtcaac accggccccc gatagctact aaggttgggg ggtttgtggg ttgtttgtga 3240

aactgctttg cggtgtgcat taccacgggg ggtgtgggga agtatctgtt tccacgatgc 3300

gataacgttc ggtggcggag ggggcgattc attctctagt gtacgcgttt caacttcagg 3360

aacgtgatta tttctttcag gacactcttt ccaatttcct tcttccttca cttcgggtac 3420

aggtatattc ttaatgttta catacatgtc gtctgctcgt ctcaactgcg gggttatgat 3480

gggtggtggt gacagtctct ccgaatgatc g 3511

<210> 25

<211> 4102

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #31

<400> 25

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat atcgaggtga 540

gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt 600

tatttatttt ttaattattt tgtgcagcga tggggggcggg gggggggggg gcgcgcgcca 660

ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc 720

aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc 780

tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgt tgccttcgcc ccgtgccccg 840

ctccgcgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 900

agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctc 960

gtttcttttc tgtggctgcg tgaaagcctt aaagggctcc gggagggccc tttgtgcggg 1020

ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg ggggagcgccg cgtgcggccc 1080

gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcgtg 1140

tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cgggggggct gcgaggggaa 1200

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcggcggt 1260

cgggctgtaa cccccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg 1320

gtgcggggct ccgtgcgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc 1380

aggtggggggt gccggggcggg gcggggccgc ctcggggccgg ggagggctcg ggggaggggc 1440

gcggcggccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1500

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctggc ggagccgaaa 1560

tctgggaggc gccgccgcac cccctctagc gggcgcgggc gaagcggtgc ggcgccggca 1620

ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccatc 1680

tccagcctcg gggctgccgc agggggacgg ctgccttcgg gggggacggg gcagggcggg 1740

gttcggcttc tggcgtgtga ccggcggggt ttatatcttc ccttctctgt tcctccgcag 1800

ccagccatgg ccaccatgac caatctccag gaccagaccc agcagattgt gccttttatt 1860

aggagtctct tgatgcctac aaccggcccc gccagcatcc cggacgacac actggaaaaa 1920

catacactga gaagcgagac atctacatac aatttgaccg tgggcgatac cggctccggg 1980

cttatcgtgt tcttcccagg ttttcccgga tctatcgtag gagcgcacta caccctccaa 2040

agtaacggca attacaaatt cgaccagatg ctcctgacag cccagaacct tcctgcttct 2100

tacaattact gtagacttgt gtccaggtcc ctgactgtgc ggagtagcac gcttccagga 2160

ggcgtatacg ccctgaacgg aactataaac gccgtcacct tccagggctc cttgtccgaa 2220

cttaccgacg tgtcctacaa tggcctcatg agcgcaacgg ccaacataaa cgataagatc 2280

ggcaatgttc ttgtgggcga gggggttaca gtcctttctc tgccaaccag ttatgatctg 2340

ggatacgtgc ggcttggcga tcccattccc gctatcggtc tcgaccctaa aatggtggct 2400

acttgcgact catctgaccg cccaagggtc tatacaatta ctgcagccga tgactatcag 2460

ttttccagcc aataccagcc agggggtgtg acaatcacac ttttcagcgc caatattgac 2520

gctatcacat ccctctcaat cggaggtgag cttgtgttcc agacttctgt tcagggcttg 2580

gtattgggcg ccactattta cttgatcggg ttcgacggga ccgcagtgat cactcgggca 2640

gtggctgcgg ataacggact cactgccgga actgacaacc ttatgccttt taatctggtc 2700

atccccacta acgagatcac ccagcctatt acctccataa agctcgaaat tgtgaccagc 2760

aagagcggag ggcaggcagg cgaccaaatg agttggtctg caagcgggtc cctcgccgtg 2820

accatccacg gtggcaacta tcctggggcg ctcagacccg tcaccctggt agcctacgaa 2880

agggttgcca caggctcagt tgtcacggtg gctggagtaa gcaatttcga gctcatcccg 2940

aatcctgagc tcgctaaaaa tcttgtgacc gagtatggaa ggttcgaccc tggcgcaatg 3000

aattacacaa agctgattct gtccgaacgg gataggctgg gtatcaagac agtttggccc 3060

acgcgcgaat acacagattt cagggagtac tttatggagg tcgcagattt gaatagccca 3120

cttaagatcg ctggagcatt tggctttaag gatattatcc gcgcaatcag aaggtagtgt 3180

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 3240

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 3300

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 3360

agacaatagc aggcatgctg gggatgcggt gggctctatg gtgtacataa ttcttattta 3420

tctttaatcc atgaggagca tttttatttt aaaaatgtca gccgccagcc ctataaccct 3480

agatcgcaac tgatccctag tctgcgttat ttgtcttgca atcttttcgc acgcctttgt 3540

gagtgcatac aatgcccccc tgctcgcttt tctgaaatcg cgtcgggtca ttaatgtgtc 3600

ggctatcaca atgcgagatg tactcgacat gtccgtgtct gtactattgg gattgtaaat 3660

agtcgaccgc gaatcatcag agtcggaatc tgtaaaggat acagattccg actctgagcg 3720

cttatgaatg ggatccactc ggacgttgtt gaacttccgt tcggattctg cttcagtcaa 3780

caccggcccc cgatagctac taaggttggg gggtttgtgg gttgtttgtg aaactgcttt 3840

gcggtgtgca ttaccacggg gggtgtgggg aagtatctgt ttccacgatg cgataacgtt 3900

cggtggcgga gggggcgatt cattctctag tgtacgcgtt tcaacttcag gaacgtgatt 3960

atttctttca ggacactctt tccaatttcc ttcttccttc acttcgggta caggtatatt 4020

cttaatgttt acatacatgt cgtctgctcg tctcaactgc ggggttatga tgggtggtgg 4080

tgacagtctc tccgaatgat cg 4102

<210> 26

<211> 3394

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #32

<400> 26

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat aactctcagt 540

acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag 600

gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat 660

tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 720

tatacgcgta tctgagggga ctagggtgtg tttaggcgaa aagcggggct tcggttgtac 780

gcggttagga gtcccctcag gatatagtag tttcgctttt gcatagggag ggggaaatgt 840

agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct 900

tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg 960

ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattccgca 1020

ttgcagagat attgtattta agtgcctagc tcgatacaat aaacgccatt tgaccattca 1080

ccacattggt gtgcacctcc agccaccatg accaatctcc aggaccagac ccagcagatt 1140

gtgcctttta ttaggagtct cttgatgcct acaaccggcc ccgccagcat cccggacgac 1200

acactggaaa aacatacact gagaagcgag acatctacat acaatttgac cgtgggcgat 1260

accggctccg ggcttatcgt gttcttccca ggttttcccg gatctatcgt aggagcgcac 1320

tacaccctcc aaagtaacgg caattacaaa ttcgaccaga tgctcctgac agcccagaac 1380

cttcctgctt cttacaatta ctgtagactt gtgtccaggt ccctgactgt gcggagtagc 1440

acgcttccag gaggcgtata cgccctgaac ggaactataa acgccgtcac cttccagggc 1500

tccttgtccg aacttaccga cgtgtcctac aatggcctca tgagcgcaac ggccaacata 1560

aacgataaga tcggcaatgt tcttgtgggc gagggggtta cagtcctttc tctgccaacc 1620

agttatgatc tgggatacgt gcggcttggc gatcccattc ccgctatcgg tctcgaccct 1680

aaaatggtgg ctacttgcga ctcatctgac cgcccaaggg tctatacaat tactgcagcc 1740

gatgactatc agttttccag ccaataccag ccagggggtg tgacaatcac acttttcagc 1800

gccaatattg acgctatcac atccctctca atcggaggtg agcttgtgtt ccagacttct 1860

gttcagggct tggtattggg cgccactatt tacttgatcg ggttcgacgg gaccgcagtg 1920

atcactcggg cagtggctgc ggataacgga ctcactgccg gaactgacaa ccttatgcct 1980

tttaatctgg tcatccccac taacgagatc acccagccta ttacctccat aaagctcgaa 2040

attgtgacca gcaagagcgg agggcaggca ggcgaccaaa tgagttggtc tgcaagcggg 2100

tccctcgccg tgaccatcca cggtggcaac tatcctgggg cgctcagacc cgtcaccctg 2160

gtagcctacg aaagggttgc cacaggctca gttgtcacgg tggctggagt aagcaatttc 2220

gagctcatcc cgaatcctga gctcgctaaa aatcttgtga ccgagtatgg aaggttcgac 2280

cctggcgcaa tgaattacac aaagctgatt ctgtccgaac gggataggct gggtatcaag 2340

acagtttggc ccacgcgcga atacacagat ttcagggagt actttatgga ggtcgcagat 2400

ttgaatagcc cacttaagat cgctggagca tttggcttta aggatattat ccgcgcaatc 2460

agaaggtagt gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 2520

cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 2580

gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 2640

ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggtgtacat 2700

aattcttatt tatctttaat ccatgaggag catttttatt ttaaaaatgt cagccgccag 2760

ccctataacc ctagatcgca actgatccct agtctgcgtt atttgtcttg caatcttttc 2820

gcacgccttt gtgagtgcat acaatgcccc cctgctcgct tttctgaaat cgcgtcgggt 2880

cattaatgtg tcggctatca caatgcgaga tgtactcgac atgtccgtgt ctgtactatt 2940

gggattgtaa atagtcgacc gcgaatcatc agagtcggaa tctgtaaagg atacagattc 3000

cgactctgag cgcttatgaa tgggatccac tcggacgttg ttgaacttcc gttcggattc 3060

tgcttcagtc aacaccggcc cccgatagct actaaggttg gggggtttgt gggttgtttg 3120

tgaaactgct ttgcggtgtg cattaccacg gggggtgtgg ggaagtatct gtttccacga 3180

tgcgataacg ttcggtggcg gagggggcga ttcattctct agtgtacgcg tttcaacttc 3240

aggaacgtga ttatttcttt caggacactc tttccaattt ccttcttcct tcacttcggg 3300

tacaggtata ttcttaatgt ttacatacat gtcgtctgct cgtctcaact gcggggttat 3360

gatgggtggt ggtgacagtc tctccgaatg atcg 3394

<210> 27

<211> 3243

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #33

<400> 27

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat agcgcagcac 540

catggcctga aataacctct gaaagaggaa cttggttagc taccttctga ggcggaaaga 600

accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 660

gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 720

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 780

ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 840

gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc 900

agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc gccaccatga 960

ccaatctcca ggaccagacc cagcagattg tgccttttat taggagtctc ttgatgccta 1020

caaccggccc cgccagcatc ccggacgaca cactggaaaa acatacactg agaagcgaga 1080

catctacata caatttgacc gtgggcgata ccggctccgg gcttatcgtg ttcttcccag 1140

gttttcccgg atctatcgta ggagcgcact acaccctcca aagtaacggc aattacaaat 1200

tcgaccagat gctcctgaca gcccagaacc ttcctgcttc ttacaattac tgtagacttg 1260

tgtccaggtc cctgactgtg cggagtagca cgcttccagg aggcgtatac gccctgaacg 1320

gaactataaa cgccgtcacc ttccagggct ccttgtccga acttaccgac gtgtcctaca 1380

atggcctcat gagcgcaacg gccaacataa acgataagat cggcaatgtt cttgtgggcg 1440

aggggggttac agtcctttct ctgccaacca gttatgatct gggatacgtg cggcttggcg 1500

atcccattcc cgctatcggt ctcgacccta aaatggtggc tacttgcgac tcatctgacc 1560

gcccaagggt ctatacaatt actgcagccg atgactatca gttttccagc caataccagc 1620

caggggggtgt gacaatcaca cttttcagcg ccaatattga cgctatcaca tccctctcaa 1680

tcggaggtga gcttgtgttc cagacttctg ttcagggctt ggtattgggc gccactattt 1740

acttgatcgg gttcgacggg accgcagtga tcactcgggc agtggctgcg gataacggac 1800

tcactgccgg aactgacaac cttatgcctt ttaatctggt catccccact aacgagatca 1860

cccagcctat tacctccata aagctcgaaa ttgtgaccag caagagcgga gggcaggcag 1920

gcgaccaaat gagttggtct gcaagcgggt ccctcgccgt gaccatccac ggtggcaact 1980

atcctggggc gctcagaccc gtcaccctgg tagcctacga aagggttgcc acaggctcag 2040

ttgtcacggt ggctggagta agcaatttcg agctcatccc gaatcctgag ctcgctaaaa 2100

atcttgtgac cgagtatgga aggttcgacc ctggcgcaat gaattacaca aagctgattc 2160

tgtccgaacg ggataggctg ggtatcaaga cagtttggcc cacgcgcgaa tacacagatt 2220

tcagggagta ctttatggag gtcgcagatt tgaatagccc acttaagatc gctggagcat 2280

ttggctttaa ggatattatc cgcgcaatca gaaggtagtg tgccttctag ttgccagcca 2340

tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 2400

ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 2460

gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 2520

ggggatgcgg tgggctctat ggtgtacata attcttattt atctttaatc catgaggagc 2580

atttttattt taaaaatgtc agccgccagc cctataaccc tagatcgcaa ctgatcccta 2640

gtctgcgtta tttgtcttgc aatcttttcg cacgcctttg tgagtgcata caatgccccc 2700

ctgctcgctt ttctgaaatc gcgtcgggtc attaatgtgt cggctatcac aatgcgagat 2760

gtactcgaca tgtccgtgtc tgtactattg ggattgtaaa tagtcgaccg cgaatcatca 2820

gagtcggaat ctgtaaagga tacagattcc gactctgagc gcttatgaat gggatccact 2880

cggacgttgt tgaacttccg ttcggattct gcttcagtca acaccggccc ccgatagcta 2940

ctaaggttgg ggggtttgtg ggttgtttgt gaaactgctt tgcggtgtgc attaccacgg 3000

ggggtgtggg gaagtatctg tttccacgat gcgataacgt tcggtggcgg agggggcgat 3060

tcattctcta gtgtacgcgt ttcaacttca ggaacgtgat tatttctttc aggacactct 3120

ttccaatttc cttcttcctt cacttcgggt acaggtatat tcttaatgtt tacatacatg 3180

tcgtctgctc gtctcaactg cggggttatg atgggtggtg gtgacagtct ctccgaatga 3240

tcg 3243

<210> 28

<211> 4124

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-IBD #34

<400> 28

atgggctccc gctgttcaaa aaaatcagcg tgcgtcgata agactttggt gcagtctctt 60

cggggtcgca atttagattt gccgcatgga gggtatctgg ggatttttgc caatgctgga 120

gcgacgactg tacgattcgt cccatcggga tctagcagac caatgatgtt gacacacatc 180

ggccatgcat gtacggacgg tctattgcgc gagtttgtta ttttcgaagg acaagatgga 240

agtgtatatg gaaccgacaa taatgttagt ttgcatttct tagggcggaa tctacatgat 300

atcttatcca agcggggtat gagccagaga gatgtgatgg tcataaaggg taaatttttt 360

agatctgaaa taacgcagtt gcccaaacaa cgatcgcgat taaaagaaaa atcggatggt 420

tcaattagga catgcatgga ttctgtgcgc ataaaccata accgcagcac tgttgggcac 480

ttcggtaact caaatgcgaa gcgttgcacg tctgcgataa ctacgcctac tatgcacatt 540

gttactcctg catcttaaaa atatatcctg tagtaatttt cacagcaatg tcataacatc 600

atctcgctaa agaatgacct gggattggag aagtaatgaa tatttgcaac caatgcattg 660

aataaactaa cattaaactc gaggtgagcc ccacgttctg cttcactctc cccatctccc 720

ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 780

gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg 840

gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt 900

atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc 960

gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc gccccggctc 1020

tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1080

aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga aagccttaaa 1140

gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt 1200

gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc 1260

ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc 1320

cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg 1380

gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc 1440

gagttgctga gcacggcccg gcttcggggtg cggggctccg tgcggggcgt ggcgcggggc 1500

tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc 1560

gggccgggga gggctcgggg gaggggcgcg gcggccccgg agcgccggcg gctgtcgagg 1620

cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc 1680

tttgtcccaa atctggcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg 1740

cgcgggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1800

cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg ctgccgcagg gggacggctg 1860

ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggggttta 1920

tatcttccct tctctgttcc tccgcagcca gccatggcca ccatgacaaa cctgcaagat 1980

caaacccaac agattgttcc gttcatacgg agccttctga tgccaacaac cggaccggcg 2040

tccattccgg acgacaccct ggagaagcac actctcaggt cagagacctc gacctacaat 2100

ttgactgtgg gggacacagg gtcagggcta attgtctttt tccctggatt ccctggctca 2160

attgtgggtg ctcactacac actgcagagc aatgggaact acaagttcga tcagatgctc 2220

ctgactgccc agaacctacc ggccagctac aactactgca gactagtgag tcggagtctc 2280

acagtgaggt caagcacact ccctggtggc gtttatgcac taaacggcac cataaacgcc 2340

gtgaccttcc aaggaagcct gagtgaactg acagatgtta gctacaatgg gttgatgtct 2400

gcaacagcca acatcaacga caaaattggg aatgtcctgg taggggaagg ggtcactgtc 2460

ctcagcctac ccacatcata tgatcttggg tatgtgaggc ttggtgaccc cattcccgct 2520

atagggcttg acccaaaaat ggtagctaca tgcgacagca gtgacaggcc cagagtctac 2580

accataactg cagccgatga ttaccaattc tcatcacagt accaaccagg tggggtaaca 2640

atcacactgt tctcagccaa cattgatgct atcacaagcc tcagcattgg gggagagctc 2700

gtgtttcaaa caagcgtcca aggccttgta ctgggcgcca ccatctacct tataggcttt 2760

gatgggactg cggtaatcac cagagctgta gccgcagata atgggctgac ggccggcacc 2820

gacaatctta tgccattcaa tcttgtcatt ccaaccaatg agataaccca gccaatcaca 2880

tccatcaaac tggagatagt gacctccaaa agtggtggtc aggcagggga tcagatgtca 2940

tggtcggcaa gtgggagcct agcagtgacg atccatggtg gcaactatcc aggggccctc 3000

cgtcccgtca cactagtagc ctacgaaaga gtggcaacag gatccgtcgt tacggtcgct 3060

ggggtgagta acttcgagct gattccaaat cctgaactag caaagaacct ggttacagaa 3120

tacggccgat ttgacccagg agccatgaac tacacaaaat tgatactgag tgagagggac 3180

cgtcttggca tcaagaccgt ctggccaaca agggagtaca ctgattttcg tgagtacttc 3240

atggaggtgg ccgacctcaa ctctcccctg aagattgcag gagcatttgg cttcaaagac 3300

ataatccggg ctataaggag gtaagctgtg ccttctagtt gccagccatc tgttgtttgc 3360

ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 3420

aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 3480

gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg 3540

ggctctatgg tcaattattt tatttaataa catatagccc aaagacctct atgaacattt 3600

agtttcccgt atactcaacg gcgcgtgtac acacgcatct ctttgcatag cgatgaagtt 3660

tgttcggcag cagaaaatgc agatatccaa caatctggag aaaacttatc atcacagtgg 3720

cagtggaaac ataccccctc tatattcatg gtataattat cgtctacagc gtccaggata 3780

gtggcgtgag aaaatggaga tctgcagccc tcctttccat ggcatgccgc tttattgttc 3840

attaaacgca caatggtctc aacgccagat atgggcatag attctgaaga acccgttgac 3900

aatccgaaga agaaggcgtg caggtctttg gaagactcgc acgttggtct tataatgtat 3960

gatcgagatg tcaccctaat gccacatggt acaggcttat cgcggtcatg gcgatcggac 4020

ttgtaatttg caacgatggg caaaggatcg acgacatgcc aaacattctg aacccgtaga 4080

gatgttaacg atgacgagga tgaatatccc atgctcgctg ccat 4124

<210> 29

<211> 4572

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #38

<400> 29

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat agaattcact 540

agtggatccc ccaactccgc ccgttttatg actagaacca atagttttta atgccaaatg 600

cactgaaatc ccctaatttg caaagccaaa cgccccctat gtgagtaata cggggacttt 660

ttacccaatt tcccaagcgg aaagccccct aatacactca tatggcatat gaatcagcac 720

ggtcatgcac tctaatggcg gcccataggg actttccaca tagggggcgt tcaccatttc 780

ccagcatagg ggtggtgact caatggcctt tacccaagta cattgggtca atgggaggta 840

agccaatggg tttttcccat tactggcaag cacactgagt caaatgggac tttccactgg 900

gttttgccca agtacattgg gtcaatggga ggtgagccaa tgggaaaaac ccattgctgc 960

caagtacact gactcaatag ggactttcca atgggttttt ccattgttgg caagcatata 1020

aggtcaatgt gggtgagtca atagggactt tccattgtat tctgcccagt acataaggtc 1080

aataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgcg tcaataggga 1140

ctttccatg ggttttgccc agtacataag gtcaataggg gatgagtcaa tgggaaaaac 1200

ccattggagc caagtacact gactcaatag ggactttcca ttgggttttg cccagtacat 1260

aaggtcaata gggggtgagt caacaggaaa gtcccattgg agccaagtac attgagtcaa 1320

tagggacttt ccaatgggtt ttgcccagta cataaggtca atgggaggta agccaatggg 1380

tttttcccat tactggcacg tatactgagt cattagggac tttccaatgg gttttgccca 1440

gtacataagg tcaatagggg tgaatcaaca ggaaagtccc attggagcca agtacactga 1500

gtcaataggg actttccatt gggttttgcc cagtacaaaa ggtcaatagg gggtgagtca 1560

atgggttttt cccattattg gcacgtacat aaggtcaata ggggtgagtc attgggtttt 1620

tccagccaat ttaattaaaa cgccatgtac tttcccacca ttgacgtcaa tgggctattg 1680

aaactaatgc aacgtgacct ttaaacggta ctttcccata gctgattaat gggaaagtac 1740

cgttctcgag ccaatacacg tcaatgggaa gtgaaagggc agccaaaacg taacaccgcc 1800

ccggttttcc cctggaaatt ccatattggc acgcattcta ttggctgagc tgcgttctac 1860

gtgggtataa gaggcgcgac cagcgtcggt accgtcgcag tcttcggtct gaccaccgta 1920

gaacgcagag ctcctcgctg caggcggccg ctctagaact cgtcgatcgc agcgatgggc 1980

tccagatctt ctaccaggat cccagtacct ctgatgctga ccgtccgaat catgttggca 2040

ctgagttgcg tctgtccgac cagctccctt gatggcaggc ctcttgcagc tgcagggatt 2100

gtggtaacag gagacaaagc agtcaacata tacacctcat ctcagacagg gtcaatcata 2160

atcaagttac tcccaaatat gcccaaggat aaagaggcgt gtgcaaaagc cccattggaa 2220

gcatacaaca ggacattgac tactttgctc accccccttg gtgattctat ccgtaggata 2280

caagagtctg tgaccacatc cggaggaggg aaacagggac gtcttatagg cgccattatc 2340

ggtggtgtag ctctcggggt tgcaaccgct gcacagataa cagcagcctc ggctctgata 2400

caagccaatc aaaatgctgc caacatcctc cggctcaaag agagcattgc tgcaaccaat 2460

gaggctgtgc acgaggtcac tgacggatta tcacaactag cagtggcagt tgggaagatg 2520

cagcaatttg ttaatgacca gtttaataaa acagctcagg aattggactg tataaaaatt 2580

acacagcagg ttggtgtaga actcaacctg tacctaactg aattgactac agtattcggg 2640

ccacaaatca cttcccctgc cttaactcag ctgactatcc aggcgcttta caatctagct 2700

ggtgggaata tggattactt gttgactaag ttaggtgtag gaaacaacca actcagctca 2760

ttaattggta gtggcctgat taccggcaac cctatcctgt acgactcaca gactcaactc 2820

ttgggtatac aggtcaccct accctcagtc gggaatctaa ataatatgcg tgccacctac 2880

ctggaaacct tgtctgtaag tacaaccaaa ggatttgcct cagcacttgt cccaaaagta 2940

gtgacacagg ttggttccgt gatagaagag cttgacacct cgtactgtat cgagaccgat 3000

ttggacctat attgtacaag aatagtgaca ttccctatgt ctcctggtat ttattcctgt 3060

ttgagtggca atacatctgc ttgcatgtat tcaaagactg aaggcgcact cactacgccg 3120

tatatgaccc tcaaaggctc agttattgcc aactgtaaga tgacaacatg tagatgtgca 3180

gaccccccgg gtatcatatc gcagaattat ggagaagctg tgtctctaat agataggcaa 3240

tcatgcaata tcttatcctt agacgggata actttgaggc tcagtgggga atttgatgca 3300

acttatcaaa agaatatctc aatacaagat tctcaagtaa tagttacagg caatcttgac 3360

atctcgactg agcttgggaa tgtcaacaac tcgataagta atgctttgga taagttagag 3420

gaaagcaaca gcaaactaga caaggtcaat gttaaactga ccagcacatc cgctcttatt 3480

acctatatcg ttttaactgt catatctctt gtatgtggta tacttagcct ggttctagca 3540

tgctacctga tgtacaagca aaaggcgcaa cagaagacct tgttgtggct tgggaataat 3600

accctagacc agatgagggc cactacaaaa atgtagcttg atctagagcg gccgcgggga 3660

tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 3720

aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 3780

caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 3840

gtgggaggtt ttttcggatc ctctagagtc gagtacataa ttcttattta tctttaatcc 3900

atgaggagca tttttatttt aaaaatgtca gccgccagcc ctataaccct agatcgcaac 3960

tgatccctag tctgcgttat ttgtcttgca atcttttcgc acgcctttgt gagtgcatac 4020

aatgcccccc tgctcgcttt tctgaaatcg cgtcgggtca ttaatgtgtc ggctatcaca 4080

atgcgagatg tactcgacat gtccgtgtct gtactattgg gattgtaaat agtcgaccgc 4140

gaatcatcag agtcggaatc tgtaaaggat acagattccg actctgagcg cttatgaatg 4200

ggatccactc ggacgttgtt gaacttccgt tcggattctg cttcagtcaa caccggcccc 4260

cgatagctac taaggttggg gggtttgtgg gttgtttgtg aaactgcttt gcggtgtgca 4320

ttaccacggg gggtgtgggg aagtatctgt ttccacgatg cgataacgtt cggtggcgga 4380

gggggcgatt cattctctag tgtacgcgtt tcaacttcag gaacgtgatt atttctttca 4440

ggacactctt tccaatttcc ttcttccttc acttcgggta caggtatatt cttaatgttt 4500

acatacatgt cgtctgctcg tctcaactgc ggggttatga tgggtggtgg tgacagtctc 4560

tccgaatgat cg 4572

<210> 30

<211> 4402

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #39

<400> 30

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat atcgaggtga 540

gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt 600

tatttatttt ttaattattt tgtgcagcga tggggggcggg gggggggggg gcgcgcgcca 660

ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc 720

aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc 780

tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgt tgccttcgcc ccgtgccccg 840

ctccgcgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 900

agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctc 960

gtttcttttc tgtggctgcg tgaaagcctt aaagggctcc gggagggccc tttgtgcggg 1020

ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg ggggagcgccg cgtgcggccc 1080

gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcgtg 1140

tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cgggggggct gcgaggggaa 1200

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcggcggt 1260

cgggctgtaa cccccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg 1320

gtgcggggct ccgtgcgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc 1380

aggtggggggt gccggggcggg gcggggccgc ctcggggccgg ggagggctcg ggggaggggc 1440

gcggcggccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1500

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctggc ggagccgaaa 1560

tctgggaggc gccgccgcac cccctctagc gggcgcgggc gaagcggtgc ggcgccggca 1620

ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccatc 1680

tccagcctcg gggctgccgc agggggacgg ctgccttcgg gggggacggg gcagggcggg 1740

gttcggcttc tggcgtgtga ccggcggggt ttatatcttc ccttctctgt tcctccgcag 1800

ccagccatgg ccaccatggg ctccagatct tctaccagga tcccagtacc tctgatgctg 1860

accgtccgaa tcatgttggc actgagttgc gtctgtccga ccagctccct tgatggcagg 1920

cctcttgcag ctgcagggat tgtggtaaca ggagacaaag cagtcaacat atacacctca 1980

tctcagacag ggtcaatcat aatcaagtta ctcccaaata tgcccaagga taaagaggcg 2040

tgtgcaaaag ccccattgga agcatacaac aggacattga ctactttgct cacccccctt 2100

ggtgattcta tccgtaggat acaagagtct gtgaccacat ccggaggagg gaaacaggga 2160

cgtcttatag gcgccattat cggtggtgta gctctcgggg ttgcaaccgc tgcacagata 2220

acagcagcct cggctctgat acaagccaat caaaatgctg ccaacatcct ccggctcaaa 2280

gagagcattg ctgcaaccaa tgaggctgtg cacgaggtca ctgacggatt atcacaacta 2340

gcagtggcag ttgggaagat gcagcaattt gttaatgacc agtttaataa aacagctcag 2400

gaattggact gtataaaaat tacacagcag gttggtgtag aactcaacct gtacctaact 2460

gaattgacta cagtattcgg gccacaaatc acttcccctg ccttaactca gctgactatc 2520

caggcgcttt acaatctagc tggtgggaat atggattact tgttgactaa gttaggtgta 2580

ggaaacaacc aactcagctc attaattggt agtggcctga ttaccggcaa ccctatcctg 2640

tacgactcac agactcaact cttgggtata caggtcaccc taccctcagt cgggaatcta 2700

aataatatgc gtgccaccta cctggaaacc ttgtctgtaa gtacaaccaa aggatttgcc 2760

tcagcacttg tcccaaaagt agtgacacag gttggttccg tgatagaaga gcttgacacc 2820

tcgtactgta tcgagaccga tttggaccta tattgtacaa gaatagtgac attccctatg 2880

tctcctggta tttattcctg tttgagtggc aatacatctg cttgcatgta ttcaaagact 2940

gaaggcgcac tcactacgcc gtatatgacc ctcaaaggct cagttattgc caactgtaag 3000

atgacaacat gtagatgtgc agaccccccg ggtatcatat cgcagaatta tggagaagct 3060

gtgtctctaa tagataggca atcatgcaat atcttatcct tagacgggat aactttgagg 3120

ctcagtgggg aatttgatgc aacttatcaa aagaatatct caatacaaga ttctcaagta 3180

atagttacag gcaatcttga catctcgact gagcttggga atgtcaacaa ctcgataagt 3240

aatgctttgg ataagttaga ggaaagcaac agcaaactag acaaggtcaa tgttaaactg 3300

accagcacat ccgctcttat tacctatatc gttttaactg tcatatctct tgtatgtggt 3360

atacttagcc tggttctagc atgctacctg atgtacaagc aaaaggcgca acagaagacc 3420

ttgttgtggc ttgggaataa taccctagac cagatgaggg cactacaaa aatgtagtgt 3480

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 3540

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 3600

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 3660

agacaatagc aggcatgctg gggatgcggt gggctctatg gtgtacataa ttcttattta 3720

tctttaatcc atgaggagca tttttatttt aaaaatgtca gccgccagcc ctataaccct 3780

agatcgcaac tgatccctag tctgcgttat ttgtcttgca atcttttcgc acgcctttgt 3840

gagtgcatac aatgcccccc tgctcgcttt tctgaaatcg cgtcgggtca ttaatgtgtc 3900

ggctatcaca atgcgagatg tactcgacat gtccgtgtct gtactattgg gattgtaaat 3960

agtcgaccgc gaatcatcag agtcggaatc tgtaaaggat acagattccg actctgagcg 4020

cttatgaatg ggatccactc ggacgttgtt gaacttccgt tcggattctg cttcagtcaa 4080

caccggcccc cgatagctac taaggttggg gggtttgtgg gttgtttgtg aaactgcttt 4140

gcggtgtgca ttaccacggg gggtgtgggg aagtatctgt ttccacgatg cgataacgtt 4200

cggtggcgga gggggcgatt cattctctag tgtacgcgtt tcaacttcag gaacgtgatt 4260

atttctttca ggacactctt tccaatttcc ttcttccttc acttcgggta caggtatatt 4320

cttaatgttt acatacatgt cgtctgctcg tctcaactgc ggggttatga tgggtggtgg 4380

tgacagtctc tccgaatgat cg 4402

<210> 31

<211> 4413

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #40

<400> 31

gccactgtat gggccattta tgtttatcga gtctaaaagt cgtggaagcg cctggccatc 60

gccagtctcg acctgccact gctgagttcg gcttcaattc cagcatccca gaaatgtcgc 120

agtcaccatc acgccgtgca tcgcttccta cgaggccttt tgacgcttct gatttgggca 180

catacaccct ggacatactc caccgctatt cgctcgtaga tttagtacaa ctactgaatg 240

acttgccgcg taacattacc tccacgcccg cttctaatgt agaaaccatg gcaaaaatta 300

atgttttaag ggccatttgc gtaggatttg ccgaggtccg tcgccacaac gacgcgcgaa 360

ctttacagcg aacggcaatg tttgccgccg acgacgtcgc atcacggatc agaccatcca 420

ttggattaaa gcgcacctac ccaccgggta tattttccac agctattacc gtatctaatt 480

ccgaggatga agagcgaaat tcgtgatcgt aaaaataaaa aatacaagat atcgaggtga 540

gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt 600

tatttatttt ttaattattt tgtgcagcga tggggggcggg gggggggggg gcgcgcgcca 660

ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc 720

aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc 780

tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgt tgccttcgcc ccgtgccccg 840

ctccgcgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 900

agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctc 960

gtttcttttc tgtggctgcg tgaaagcctt aaagggctcc gggagggccc tttgtgcggg 1020

ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg ggggagcgccg cgtgcggccc 1080

gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcgtg 1140

tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cgggggggct gcgaggggaa 1200

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcggcggt 1260

cgggctgtaa cccccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg 1320

gtgcggggct ccgtgcgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc 1380

aggtggggggt gccggggcggg gcggggccgc ctcggggccgg ggagggctcg ggggaggggc 1440

gcggcggccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1500

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctggc ggagccgaaa 1560

tctgggaggc gccgccgcac cccctctagc gggcgcgggc gaagcggtgc ggcgccggca 1620

ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccatc 1680

tccagcctcg gggctgccgc agggggacgg ctgccttcgg gggggacggg gcagggcggg 1740

gttcggcttc tggcgtgtga ccggcggggt ttatatcttc ccttctctgt tcctccgcag 1800

ccagccatgg ccaccatggg ctccagatct tctaccagga tcccagtacc tctgatgctg 1860

accgtccgaa tcatgttggc actgagttgc gtctgtccga ccagctccct tgatggcagg 1920

cctcttgcag ctgcagggat tgtggtaaca ggagacaaag cagtcaacat atacacctca 1980

tctcagacag ggtcaatcat aatcaagtta ctcccaaata tgcccaagga taaagaggcg 2040

tgtgcaaaag ccccattgga agcatacaac aggacattga ctactttgct cacccccctt 2100

ggtgattcta tccgtaggat acaagagtct gtgaccacat ccggaggagg gaaacaggga 2160

cgtcttatag gcgccattat cggtggtgta gctctcgggg ttgcaaccgc tgcacagata 2220

acagcagcct cggctctgat acaagccaat caaaatgctg ccaacatcct ccggctcaaa 2280

gagagcattg ctgcaaccaa tgaggctgtg cacgaggtca ctgacggatt atcacaacta 2340

gcagtggcag ttgggaagat gcagcaattt gttaatgacc agtttaataa aacagctcag 2400

gaattggact gtataaaaat tacacagcag gttggtgtag aactcaacct gtacctaact 2460

gaattgacta cagtattcgg gccacaaatc acttcccctg ccttaactca gctgactatc 2520

caggcgcttt acaatctagc tggtgggaat atggattact tgttgactaa gttaggtgta 2580

ggaaacaacc aactcagctc attaattggt agtggcctga ttaccggcaa ccctatcctg 2640

tacgactcac agactcaact cttgggtata caggtcaccc taccctcagt cgggaatcta 2700

aataatatgc gtgccaccta cctggaaacc ttgtctgtaa gtacaaccaa aggatttgcc 2760

tcagcacttg tcccaaaagt agtgacacag gttggttccg tgatagaaga gcttgacacc 2820

tcgtactgta tcgagaccga tttggaccta tattgtacaa gaatagtgac attccctatg 2880

tctcctggta tttattcctg tttgagtggc aatacatctg cttgcatgta ttcaaagact 2940

gaaggcgcac tcactacgcc gtatatgacc ctcaaaggct cagttattgc caactgtaag 3000

atgacaacat gtagatgtgc agaccccccg ggtatcatat cgcagaatta tggagaagct 3060

gtgtctctaa tagataggca atcatgcaat atcttatcct tagacgggat aactttgagg 3120

ctcagtgggg aatttgatgc aacttatcaa aagaatatct caatacaaga ttctcaagta 3180

atagttacag gcaatcttga catctcgact gagcttggga atgtcaacaa ctcgataagt 3240

aatgctttgg ataagttaga ggaaagcaac agcaaactag acaaggtcaa tgttaaactg 3300

accagcacat ccgctcttat tacctatatc gttttaactg tcatatctct tgtatgtggt 3360

atacttagcc tggttctagc atgctacctg atgtacaagc aaaaggcgca acagaagacc 3420

ttgttgtggc ttgggaataa taccctagac cagatgaggg cactacaaa aatgtagctt 3480

gatctagagc ggccgcgggg atccagacat gataagatac attgatgagt ttggacaaac 3540

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 3600

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 3660

gtttcaggtt cagggggagg tgtgggaggt tttttcggat cctctagagt cgagtacata 3720

attcttattt atctttaatc catgaggagc atttttattt taaaaatgtc agccgccagc 3780

cctataaccc tagatcgcaa ctgatcccta gtctgcgtta tttgtcttgc aatcttttcg 3840

cacgcctttg tgagtgcata caatgccccc ctgctcgctt ttctgaaatc gcgtcgggtc 3900

attaatgtgt cggctatcac aatgcgagat gtactcgaca tgtccgtgtc tgtactattg 3960

ggattgtaaa tagtcgaccg cgaatcatca gagtcggaat ctgtaaagga tacagattcc 4020

gactctgagc gcttatgaat gggatccact cggacgttgt tgaacttccg ttcggattct 4080

gcttcagtca acaccggccc ccgatagcta ctaaggttgg ggggtttgtg ggttgtttgt 4140

gaaactgctt tgcggtgtgc attaccacgg ggggtgtggg gaagtatctg tttccacgat 4200

gcgataacgt tcggtggcgg agggggcgat tcattctcta gtgtacgcgt ttcaacttca 4260

ggaacgtgat tatttctttc aggacactct ttccaatttc cttcttcctt cacttcgggt 4320

acaggtatat tcttaatgtt tacatacatg tcgtctgctc gtctcaactg cggggttatg 4380

atgggtggtg gtgacagtct ctccgaatga tcg 4413

<210> 32

<211> 3849

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #41

<400> 32

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg ttgacattga ttattgacta 600

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 660

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 720

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 780

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 840

gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 900

tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 960

tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 1020

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 1080

actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 1140

ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc 1200

ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1260

gcttaccgcc accatgggct ccagatcttc taccaggatc ccagtacctc tgatgctgac 1320

cgtccgaatc atgttggcac tgagttgcgt ctgtccgacc agctcccttg atggcaggcc 1380

tcttgcagct gcagggattg tggtaacagg agacaaagca gtcaacatat acacctcatc 1440

tcagacaggg tcaatcataa tcaagttact cccaaatatg cccaaggata aagaggcgtg 1500

tgcaaaagcc ccattggaag catacaacag gacattgact actttgctca ccccccttgg 1560

tgattctatc cgtaggatac aagagtctgt gaccacatcc ggaggaggga aacagggacg 1620

tcttataggc gccattatcg gtggtgtagc tctcggggtt gcaaccgctg cacagataac 1680

agcagcctcg gctctgatac aagccaatca aaatgctgcc aacatcctcc ggctcaaaga 1740

gagcattgct gcaaccaatg aggctgtgca cgaggtcact gacggattat cacaactagc 1800

agtggcagtt gggaagatgc agcaatttgt taatgaccag tttaataaaa cagctcagga 1860

attggactgt ataaaaatta cacagcaggt tggtgtagaa ctcaacctgt acctaactga 1920

attgactaca gtattcgggc cacaaatcac ttcccctgcc ttaactcagc tgactatcca 1980

ggcgctttac aatctagctg gtgggaatat ggattacttg ttgactaagt taggtgtagg 2040

aaacaaccaa ctcagctcat taattggtag tggcctgatt accggcaacc ctatcctgta 2100

cgactcacag actcaactct tgggtataca ggtcacccta ccctcagtcg ggaatctaaa 2160

taatatgcgt gccacctacc tggaaacctt gtctgtaagt acaaccaaag gatttgcctc 2220

agcacttgtc ccaaaagtag tgacacaggt tggttccgtg atagaagagc ttgacacctc 2280

gtactgtatc gagaccgatt tggacctata ttgtacaaga atagtgacat tccctatgtc 2340

tcctggtatt tattcctgtt tgagtggcaa tacatctgct tgcatgtatt caaagactga 2400

aggcgcactc actacgccgt atatgaccct caaaggctca gttattgcca actgtaagat 2460

gacaacatgt agatgtgcag accccccggg tatcatatcg cagaattatg gagaagctgt 2520

gtctctaata gataggcaat catgcaatat cttatcctta gacgggataa ctttgaggct 2580

cagtggggaa tttgatgcaa cttatcaaaa gaatatctca atacaagatt ctcaagtaat 2640

agttacaggc aatcttgaca tctcgactga gcttgggaat gtcaacaact cgataagtaa 2700

tgctttggat aagttagagg aaagcaacag caaactagac aaggtcaatg ttaaactgac 2760

cagcacatcc gctcttatta cctatatcgt tttaactgtc atatctcttg tatgtggtat 2820

acttagcctg gttctagcat gctacctgat gtacaagcaa aaggcgcaac agaagacctt 2880

gttgtggctt gggaataata ccctagacca gatgaggggcc actacaaaaa tgtagcttga 2940

tctagagcgg ccgcggggat ccagacatga taagatacat tgatgagttt ggacaaacca 3000

caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat 3060

ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt 3120

ttcaggttca gggggaggtg tgggaggttt tttcggatcc tctagagtcg agtttaatgt 3180

tagtttattc aatgcattgg ttgcaaatat tcattacttc tccaatccca ggtcattctt 3240

tagcgagatg atgttatgac attgctgtga aaattactac aggatatatt tttaagatgc 3300

aggagtaaca atgtgcatag taggcgtagt tatcgcagac gtgcaacgct tcgcatttga 3360

gttaccgaag tgcccaacag tgctgcggtt atggtttatg cgcacagaat ccatgcatgt 3420

cctaattgaa ccatccgatt tttcttttaa tcgcgatcgt tgtttgggca actgcgttat 3480

ttcagatcta aaaaatttac cctttatgac catcacatct ctctggctca taccccgctt 3540

ggataagata tcatgtagat tccgccctaa gaaatgcaaa ctaacattat tgtcggttcc 3600

atatacactt ccatcttgtc cttcgaaaat aacaaactcg cgcaatagac cgtccgtaca 3660

tgcatggccg atgtgtgtca acatcattgg tctgctagat cccgatggga cgaatcgtac 3720

agtcgtcgct ccagcattgg caaaaatccc cagataccct ccatgcggca aatctaaatt 3780

gcgaccccga agagactgca ccaaagtctt atcgacgcac gctgattttt ttgaacagcg 3840

ggagcccat 3849

<210> 33

<211> 4599

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the original transfer plasmid for HVT-ND #42

<400> 33

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg gaattcacta gtggatcccc 600

caactccgcc cgttttatga ctagaaccaa tagtttttaa tgccaaatgc actgaaatcc 660

cctaatttgc aaagccaaac gccccctatg tgagtaatac ggggactttt tacccaattt 720

cccaagcgga aagcccccta atacactcat atggcatatg aatcagcacg gtcatgcact 780

ctaatggcgg cccataggga ctttccacat agggggcgtt caccatttcc cagcataggg 840

gtggtgactc aatggccttt acccaagtac attgggtcaa tgggaggtaa gccaatgggt 900

ttttcccatt actggcaagc acactgagtc aaatgggact ttccactggg ttttgcccaa 960

gtacattggg tcaatgggag gtgagccaat gggaaaaacc cattgctgcc aagtacactg 1020

actcaatagg gactttccaa tgggtttttc cattgttggc aagcatataa ggtcaatgtg 1080

ggtgagtcaa tagggacttt ccattgtatt ctgcccagta cataaggtca atagggggtg 1140

aatcaacagg aaagtcccat tggagccaag tacactgcgt caatagggac tttccattgg 1200

gttttgccca gtacataagg tcaatagggg atgagtcaat gggaaaaacc cattggagcc 1260

aagtacactg actcaatagg gactttccat tgggttttgc ccagtacata aggtcaatag 1320

ggggtgagtc aacaggaaag tcccattgga gccaagtaca ttgagtcaat agggactttc 1380

caatgggttt tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt 1440

actggcacgt atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt 1500

caataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga 1560

ctttccatg ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc 1620

ccattattgg cacgtacata aggtcaatag gggtgagtca ttgggttttt ccagccaatt 1680

taattaaaac gccatgtact ttcccaccat tgacgtcaat gggctattga aactaatgca 1740

acgtgacctt taaacggtac tttcccatag ctgattaatg ggaaagtacc gttctcgagc 1800

caatacacgt caatgggaag tgaaagggca gccaaaacgt aacaccgccc cggttttccc 1860

ctggaaattc catattggca cgcattctat tggctgagct gcgttctacg tgggtataag 1920

aggcgcgacc agcgtcggta ccgtcgcagt cttcggtctg accaccgtag aacgcagagc 1980

tcctcgctgc aggcggccgc tctagaactc gtcgatcgca gcgatgggct ccagatcttc 2040

taccaggatc ccagtacctc tgatgctgac cgtccgaatc atgttggcac tgagttgcgt 2100

ctgtccgacc agctcccttg atggcaggcc tcttgcagct gcagggattg tggtaacagg 2160

agacaaagca gtcaacatat acacctcatc tcagacaggg tcaatcataa tcaagttact 2220

cccaaatatg cccaaggata aagaggcgtg tgcaaaagcc ccattggaag catacaacag 2280

gacattgact actttgctca ccccccttgg tgattctatc cgtaggatac aagagtctgt 2340

gaccacatcc ggaggaggga aacagggacg tcttataggc gccattatcg gtggtgtagc 2400

tctcggggtt gcaaccgctg cacagataac agcagcctcg gctctgatac aagccaatca 2460

aaatgctgcc aacatcctcc ggctcaaaga gagcattgct gcaaccaatg aggctgtgca 2520

cgaggtcact gacggattat cacaactagc agtggcagtt gggaagatgc agcaatttgt 2580

taatgaccag tttaataaaa cagctcagga attggactgt ataaaaatta cacagcaggt 2640

tggtgtagaa ctcaacctgt acctaactga attgactaca gtattcgggc cacaaatcac 2700

ttcccctgcc ttaactcagc tgactatcca ggcgctttac aatctagctg gtgggaatat 2760

ggattacttg ttgactaagt taggtgtagg aaacaaccaa ctcagctcat taattggtag 2820

tggcctgatt accggcaacc ctatcctgta cgactcacag actcaactct tgggtataca 2880

ggtcacccta ccctcagtcg ggaatctaaa taatatgcgt gccacctacc tggaaacctt 2940

gtctgtaagt acaaccaaag gatttgcctc agcacttgtc ccaaaagtag tgacacaggt 3000

tggttccgtg atagaagagc ttgacacctc gtactgtatc gagaccgatt tggacctata 3060

ttgtacaaga atagtgacat tccctatgtc tcctggtatt tattcctgtt tgagtggcaa 3120

tacatctgct tgcatgtatt caaagactga aggcgcactc actacgccgt atatgaccct 3180

caaaggctca gttattgcca actgtaagat gacaacatgt agatgtgcag accccccggg 3240

tatcatatcg cagaattatg gagaagctgt gtctctaata gataggcaat catgcaatat 3300

cttatcctta gacgggataa ctttgaggct cagtggggaa tttgatgcaa cttatcaaaa 3360

gaatatctca atacaagatt ctcaagtaat agttacaggc aatcttgaca tctcgactga 3420

gcttgggaat gtcaacaact cgataagtaa tgctttggat aagttagagg aaagcaacag 3480

caaactagac aaggtcaatg ttaaactgac cagcacatcc gctcttatta cctatatcgt 3540

tttaactgtc atatctcttg tatgtggtat acttagcctg gttctagcat gctacctgat 3600

gtacaagcaa aaggcgcaac agaagacctt gttgtggctt gggaataata ccctagacca 3660

gatgaggggcc actacaaaaa tgtagcttga tctagagcgg ccgcggggat ccagacatga 3720

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 3780

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 3840

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt 3900

tttcggatcc tctagagtcg agtttaatgt tagtttattc aatgcattgg ttgcaaatat 3960

tcattacttc tccaatccca ggtcattctt tagcgagatg atgttatgac attgctgtga 4020

aaattactac aggatatatt tttaagatgc aggagtaaca atgtgcatag taggcgtagt 4080

tatcgcagac gtgcaacgct tcgcatttga gttaccgaag tgcccaacag tgctgcggtt 4140

atggtttatg cgcacagaat ccatgcatgt cctaattgaa ccatccgatt tttcttttaa 4200

tcgcgatcgt tgtttgggca actgcgttat ttcagatcta aaaaatttac cctttatgac 4260

catcacatct ctctggctca taccccgctt ggataagata tcatgtagat tccgccctaa 4320

gaaatgcaaa ctaacattat tgtcggttcc atatacactt ccatcttgtc cttcgaaaat 4380

aacaaactcg cgcaatagac cgtccgtaca tgcatggccg atgtgtgtca acatcattgg 4440

tctgctagat cccgatggga cgaatcgtac agtcgtcgct ccagcattgg caaaaatccc 4500

cagataccct ccatgcggca aatctaaatt gcgaccccga agagactgca ccaaagtctt 4560

atcgacgcac gctgattttt ttgaacagcg ggagcccat 4599

<210> 34

<211> 3974

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the cloning plasmid for pSiteB

<400> 34

tctgcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta 60

tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag 120

ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata 180

caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg 240

acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca 300

ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt 360

gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga 420

atcgagtgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca 480

ggatattctt ctaatacctg gaacgctgtt tttccgggga tcgcagtggt gagtaaccat 540

gcatcatcag gagtacggat aaaatgcttg atggtcggaa gtggcataaa ttccgtcagc 600

cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc 660

agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc 720

ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat 780

cgcggcctcg acgtttcccg ttgaatatgg ctcatattct tcctttttca atattattga 840

agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 900

aaacaaatag gggtcagtgt tacaaccaat taaccaattc tgaacattat cgcgagccca 960

tttatacctg aatatggctc ataacacccc ttgcagtgcg actaacggca tgaagctcgt 1020

cggggctgga tcgcttcgta ttgcgagctg tgcggctgag ttgacgtatc tgtgctggat 1080

gattactcat aacggcaccg ctatcaaacg tgccacgttc atgtccgtgt cgcccttcgc 1140

tgaaactagt ctctacactt cttgttaaat ggaaagtgca tttgcttgtt cttacaatcg 1200

gcccgagtct cgttcacagc gcctcgttca cacttaaacc acaaatagtc tacaggctat 1260

atgggagcca gactgaaact cacatatgac taatattcgg gggtgttagt cacgtgtagc 1320

ccattgtgtg catataacga tgttggacgc gtccttattc gcggtgtact tgatactatg 1380

gcagcgagca tgggatattc atcctcgtca tcgttaacat ctctacgggt tcagaatgtt 1440

tggcatgtcg tcgatccttt gcccatcgtt gcaaattaca agtccgatcg ccatgaccgc 1500

gataagcctg taccatgtgg cattagggtg acatctcgat catacattat aagaccaacg 1560

tgcgagtctt ccaaagacct gcacgccttc ttcttcggat tgtcaacggg ttcttcagaa 1620

tctatgccca tatctggcgt tgagaccatt gtgcgtttaa tgaacaataa agcggcatgc 1680

catggaaagg agggctgcag atctccattt tctcacgcca ctatcctgga cgctgtagac 1740

gataattata ccatgaatat agagggggta tgtttccact gccactgtga tgataagttt 1800

tctccagatt gttggatatc tgcattttct gctgccgaac aaacttcatc gctatgcaaa 1860

gagatgcgtg tgtacacgcg ccgttgagta tacgggaaac taaatgttca tagaggtctt 1920

tgggctatat gttattaaat aaaataattg ggcgcgccac cggtacgagt cactggatcc 1980

tctagtcagc ctcgagtgac tagcgtgcta gcagtggccg gccgtttaat gttagtttat 2040

tcaatgcatt ggttgcaaat attcattact tctccaatcc caggtcattc tttagcgaga 2100

tgatgttatg acattgctgt gaaaattact acaggatata tttttaagat gcaggagtaa 2160

caatgtgcat agtaggcgta gttatcgcag acgtgcaacg cttcgcattt gagttaccga 2220

agtgcccaac agtgctgcgg ttatggttta tgcgcacaga atccatgcat gtcctaattg 2280

aaccatccga tttttctttt aatcgcgatc gttgtttggg caactgcgtt atttcagatc 2340

taaaaaattt accctttatg accatcacat ctctctggct cataccccgc ttggataaga 2400

tatcatgtag attccgccct aagaaatgca aactaacatt attgtcggtt ccatatacac 2460

ttccatcttg tccttcgaaa ataacaaact cgcgcaatag accgtccgta catgcatggc 2520

cgatgtgtgt caacatcatt ggtctgctag atcccgatgg gacgaatcgt acagtcgtcg 2580

ctccagcatt ggcaaaaatc cccagatacc ctccatgcgg caaatctaaa ttgcgacccc 2640

gaagagactg caccaaagtc ttatcgacgc acgctgattt ttttgaacag cgggagccca 2700

ttatcttcag tggagcgtag acgggcgagg ctaattatgt gacatagcaa cactgcatgt 2760

atgtttttat aaatcaataa gagtacataa tttattacgt atcatttccg tttgtaatat 2820

actcctgcag gcgtcaaaag ggcgacacac tgtcattagc aactccttgt ccttcgatct 2880

cgtcaacaac agcttgcagt tcaaatacaa gacccagaag gcgactattc tggaagcgag 2940

cttgaagcca gacgctgagt acgaaaaggg gcccgagctt aagactggcc gtcgttttac 3000

aacacagaaa gagtttgtag aaacgcaaaa aggccatccg tcaggggcct tctgcttagt 3060

ttgatgcctg gcagttccct actctcgcct tccgcttcct cgctcactga ctcgctgcgc 3120

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 3180

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 3240

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 3300

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 3360

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 3420

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 3480

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 3540

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 3600

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 3660

ggtgctacag agttcttgaa gtggtgggct aactacggct acactagaag aacagtattt 3720

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 3780

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 3840

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 3900

aacgacgcgc gcgtaactca cgttaaggga ttttggtcat gagcttgcgc cgtcccgtca 3960

agtcagcgta atgc 3974

<210> 35

<211> 7246

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the final transfer plasmid for pSiteB #42

<400> 35

tctgcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta 60

tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag 120

ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata 180

caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg 240

acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca 300

ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt 360

gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga 420

atcgagtgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca 480

ggatattctt ctaatacctg gaacgctgtt tttccgggga tcgcagtggt gagtaaccat 540

gcatcatcag gagtacggat aaaatgcttg atggtcggaa gtggcataaa ttccgtcagc 600

cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc 660

agaaacaact ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc 720

ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat 780

cgcggcctcg acgtttcccg ttgaatatgg ctcatattct tcctttttca atattattga 840

agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 900

aaacaaatag gggtcagtgt tacaaccaat taaccaattc tgaacattat cgcgagccca 960

tttatacctg aatatggctc ataacacccc ttgcagtgcg actaacggca tgaagctcgt 1020

cggggctgga tcgcttcgta ttgcgagctg tgcggctgag ttgacgtatc tgtgctggat 1080

gattactcat aacggcaccg ctatcaaacg tgccacgttc atgtccgtgt cgcccttcgc 1140

tgaaactagt ctctacactt cttgttaaat ggaaagtgca tttgcttgtt cttacaatcg 1200

gcccgagtct cgttcacagc gcctcgttca cacttaaacc acaaatagtc tacaggctat 1260

atgggagcca gactgaaact cacatatgac taatattcgg gggtgttagt cacgtgtagc 1320

ccattgtgtg catataacga tgttggacgc gtccttattc gcggtgtact tgatactatg 1380

gcagcgagca tgggatattc atcctcgtca tcgttaacat ctctacgggt tcagaatgtt 1440

tggcatgtcg tcgatccttt gcccatcgtt gcaaattaca agtccgatcg ccatgaccgc 1500

gataagcctg taccatgtgg cattagggtg acatctcgat catacattat aagaccaacg 1560

tgcgagtctt ccaaagacct gcacgccttc ttcttcggat tgtcaacggg ttcttcagaa 1620

tctatgccca tatctggcgt tgagaccatt gtgcgtttaa tgaacaataa agcggcatgc 1680

catggaaagg agggctgcag atctccattt tctcacgcca ctatcctgga cgctgtagac 1740

gataattata ccatgaatat agagggggta tgtttccact gccactgtga tgataagttt 1800

tctccagatt gttggatatc tgcattttct gctgccgaac aaacttcatc gctatgcaaa 1860

gagatgcgtg tgtacacgcg ccgttgagta tacgggaaac taaatgttca tagaggtctt 1920

tgggctatat gttattaaat aaaataattg ggcgcgccaa ctccgcccgt tttatgacta 1980

gaaccaatag tttttaatgc caaatgcact gaaatcccct aatttgcaaa gccaaacgcc 2040

ccctatgtga gtaatacggg gactttttac ccaatttccc aagcggaaag ccccctaata 2100

cactcatatg gcatatgaat cagcacggtc atgcactcta atggcggccc atagggactt 2160

tccacatagg gggcgttcac catttcccag cataggggtg gtgactcaat ggcctttacc 2220

caagtacatt gggtcaatgg gaggtaagcc aatgggtttt tcccattact ggcaagcaca 2280

ctgagtcaaa tgggactttc cactgggttt tgcccaagta cattgggtca atgggaggtg 2340

agccaatggg aaaaacccat tgctgccaag tacactgact caatagggac tttccaatgg 2400

gtttttccat tgttggcaag catataaggt caatgtgggt gagtcaatag ggactttcca 2460

ttgtattctg cccagtacat aaggtcaata gggggtgaat caacaggaaa gtcccattgg 2520

agccaagtac actgcgtcaa tagggacttt ccattgggtt ttgcccagta cataaggtca 2580

ataggggatg agtcaatggg aaaaacccat tggagccaag tacactgact caatagggac 2640

tttccattgg gttttgccca gtacataagg tcaatagggg gtgagtcaac aggaaagtcc 2700

cattggagcc aagtacattg agtcaatagg gactttccaa tgggttttgc ccagtacata 2760

aggtcaatgg gaggtaagcc aatgggtttt tcccattact ggcacgtata ctgagtcatt 2820

agggactttc caatgggttt tgcccagtac ataaggtcaa taggggtgaa tcaacaggaa 2880

agtcccattg gagccaagta cactgagtca atagggactt tccattgggt tttgcccagt 2940

acaaaaggtc aatagggggt gagtcaatgg gtttttccca ttattggcac gtacataagg 3000

tcaatagggg tgagtcattg ggtttttcca gccaatttaa ttaaaacgcc atgtactttc 3060

ccaccattga cgtcaatggg ctattgaaac taatgcaacg tgacctttaa acggtacttt 3120

cccatagctg attaatggga aagtaccgtt ctcgagccaa tacacgtcaa tgggaagtga 3180

aagggcagcc aaaacgtaac accgccccgg ttttcccctg gaaattccat attggcacgc 3240

attctattgg ctgagctgcg ttctacgtgg gtataagagg cgcgaccagc gtcggtaccg 3300

tcgcagtctt cggtctgacc accgtagaac gcagagctcc tcgctgcagg cggccgctct 3360

agaactcgtc gatcgcagcg atgggctcca gatcttctac caggatccca gtacctctga 3420

tgctgaccgt ccgaatcatg ttggcactga gttgcgtctg tccgaccagc tcccttgatg 3480

gcaggcctct tgcagctgca gggattgtgg taacaggaga caaagcagtc aacatataca 3540

cctcatctca gacagggtca atcataatca agttactccc aaatatgccc aaggataaag 3600

aggcgtgtgc aaaagcccca ttggaagcat acaacaggac attgactact ttgctcaccc 3660

cccttggtga ttctatccgt aggatacaag agtctgtgac cacatccgga ggagggaaac 3720

agggacgtct tataggcgcc attatcggtg gtgtagctct cggggttgca accgctgcac 3780

agataacagc agcctcggct ctgatacaag ccaatcaaaa tgctgccaac atcctccggc 3840

tcaaagagag cattgctgca accaatgagg ctgtgcacga ggtcactgac ggattatcac 3900

aactagcagt ggcagttggg aagatgcagc aatttgttaa tgaccagttt aataaaacag 3960

ctcaggaatt ggactgtata aaaattacac agcaggttgg tgtagaactc aacctgtacc 4020

taactgaatt gactacagta ttcgggccac aaatcacttc ccctgcctta actcagctga 4080

ctatccaggc gctttacaat ctagctggtg ggaatatgga ttacttgttg actaagttag 4140

gtgtaggaaa caaccaactc agctcattaa ttggtagtgg cctgattacc ggcaacccta 4200

tcctgtacga ctcacagact caactcttgg gtatacaggt caccctaccc tcagtcggga 4260

atctaaataa tatgcgtgcc acctacctgg aaaccttgtc tgtaagtaca accaaaggat 4320

ttgcctcagc acttgtccca aaagtagtga cacaggttgg ttccgtgata gaagagcttg 4380

acacctcgta ctgtatcgag accgatttgg acctatattg tacaagaata gtgacattcc 4440

ctatgtctcc tggtatttat tcctgtttga gtggcaatac atctgcttgc atgtattcaa 4500

agactgaagg cgcactcact acgccgtata tgaccctcaa aggctcagtt attgccaact 4560

gtaagatgac aacatgtaga tgtgcagacc ccccgggtat catatcgcag aattatggag 4620

aagctgtgtc tctaatagat aggcaatcat gcaatatctt atccttagac gggataactt 4680

tgaggctcag tggggaattt gatgcaactt atcaaaagaa tatctcaata caagattctc 4740

aagtaatagt tacaggcaat cttgacatct cgactgagct tgggaatgtc aacaactcga 4800

taagtaatgc tttggataag ttagaggaaa gcaacagcaa actagacaag gtcaatgtta 4860

aactgaccag cacatccgct cttattacct atatcgtttt aactgtcata tctcttgtat 4920

gtggtatact tagcctggtt ctagcatgct acctgatgta caagcaaaag gcgcaacaga 4980

agaccttgtt gtggcttggg aataataccc tagaccagat gagggccact acaaaaatgt 5040

agcttgatct agagcggccg cggggatcca gacatgataa gatacattga tgagtttgga 5100

caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 5160

gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 5220

tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt cggatcctct agagtcgagc 5280

tagcagtggc cggccgttta atgttagttt attcaatgca ttggttgcaa atattcatta 5340

cttctccaat cccaggtcat tctttagcga gatgatgtta tgacattgct gtgaaaatta 5400

ctacaggata tatttttaag atgcaggagt aacaatgtgc atagtaggcg tagttatcgc 5460

agacgtgcaa cgcttcgcat ttgagttacc gaagtgccca acagtgctgc ggttatggtt 5520

tatgcgcaca gaatccatgc atgtcctaat tgaaccatcc gatttttctt ttaatcgcga 5580

tcgttgtttg ggcaactgcg ttatttcaga tctaaaaaat ttacccttta tgaccatcac 5640

atctctctgg ctcatacccc gcttggataa gatatcatgt agattccgcc ctaagaaatg 5700

caaactaaca ttattgtcgg ttccatatac acttccatct tgtccttcga aaataacaaa 5760

ctcgcgcaat agaccgtccg tacatgcatg gccgatgtgt gtcaacatca ttggtctgct 5820

agatcccgat gggacgaatc gtacagtcgt cgctccagca ttggcaaaaa tccccagata 5880

ccctccatgc ggcaaatcta aattgcgacc ccgaagac tgcaccaaag tcttatcgac 5940

gcacgctgat ttttttgaac agcgggagcc cattatcttc agtggagcgt agacgggcga 6000

ggctaattat gtgacatagc aacactgcat gtatgttttt ataaatcaat aagagtacat 6060

aatttattac gtatcatttc cgtttgtaat atactcctgc aggcgtcaaa agggcgacac 6120

actgtcatta gcaactcctt gtccttcgat ctcgtcaaca acagcttgca gttcaaatac 6180

aagacccaga aggcgactat tctggaagcg agcttgaagc cagacgctga gtacgaaaag 6240

gggcccgagc ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa 6300

aaaggccatc cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc 6360

cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 6420

cagctcactc aaaggcggta atacggttat cccagaatc aggggataac gcaggaaaga 6480

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 6540

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 6600

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 6660

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 6720

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 6780

ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 6840

actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 6900

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg 6960

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 7020

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 7080

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 7140

tgatcttttc tacggggtct gacgctcagt ggaacgacgc gcgcgtaact cacgttaagg 7200

gattttggtc atgagcttgc gccgtcccgt caagtcagcg taatgc 7246

<210> 36

<211> 4440

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #44

<400> 36

atggcagcga gcatgggata ttcatcctcg tcatcgttaa catctctacg ggttcagaat 60

gtttggcatg tcgtcgatcc tttgcccatc gttgcaaatt acaagtccga tcgccatgac 120

cgcgataagc ctgtaccatg tggcattagg gtgacatctc gatcatacat tataagacca 180

acgtgcgagt cttccaaaga cctgcacgcc ttcttcttcg gattgtcaac gggttcttca 240

gaatctatgc ccatatctgg cgttgagacc attgtgcgtt taatgaacaa taaagcggca 300

tgccatggaa aggagggctg cagatctcca ttttctcacg ccactatcct ggacgctgta 360

gacgataatt ataccatgaa tatagagggg gtatgtttcc actgccactg tgatgataag 420

ttttctccag attgttggat atctgcattt tctgctgccg aacaaacttc atcgctatgc 480

aaagagatgc gtgtgtacac gcgccgttga gtatacggga aactaaatgt tcatagaggt 540

ctttgggcta tatgttatta aataaaataa ttgaccagtg tcgaggtgag ccccacgttc 600

tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt 660

taattatttt gtgcagcgat gggggcgggg gggggggggg cgcgcgccag gcggggcggg 720

gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg 780

cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg 840

aagcgcgcgg cgggcgggag tcgctgcgtt gccttcgccc cgtgccccgc tccgcgccgc 900

ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga 960

cggcccttct cctccggggct gtaattagcg cttggtttaa tgacggctcg tttcttttct 1020

gtggctgcgt gaaagcctta aagggctccg ggagggccct ttgtgcgggg gggagcggct 1080

cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggcccg cgctgcccgg 1140

cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcgtgt gcgcgagggg 1200

agcgcggccg ggggcggtgc cccgcggtgc gggggggctg cgaggggaac aaaggctgcg 1260

tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac 1320

ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc 1380

cgtgcggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg 1440

ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc 1500

ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt 1560

gcgagaggc gcagggactt cctttgtccc aaatctggcg gagccgaaat ctgggaggcg 1620

ccgccgcacc ccctctagcg ggcgcgggcg aagcggtgcg gcgccggcag gaaggaaatg 1680

ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccatct ccagcctcgg 1740

ggctgccgca ggggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct 1800

ggcgtgtgac cggcggggtt tatatcttcc cttctctgtt cctccgcagc cagccatggc 1860

caccatgggc tccagatctt ctaccaggat cccagtacct ctgatgctga ccgtccgaat 1920

catgttggca ctgagttgcg tctgtccgac cagctccctt gatggcaggc ctcttgcagc 1980

tgcagggatt gtggtaacag gagacaaagc agtcaacata tacacctcat ctcagacagg 2040

gtcaatcata atcaagttac tcccaaatat gcccaaggat aaagaggcgt gtgcaaaagc 2100

cccattggaa gcatacaaca ggacattgac tactttgctc accccccttg gtgattctat 2160

ccgtaggata caagagtctg tgaccacatc cggaggaggg aaacagggac gtcttatagg 2220

cgccattatc ggtggtgtag ctctcggggt tgcaaccgct gcacagataa cagcagcctc 2280

ggctctgata caagccaatc aaaatgctgc caacatcctc cggctcaaag agagcattgc 2340

tgcaaccaat gaggctgtgc acgaggtcac tgacggatta tcacaactag cagtggcagt 2400

tgggaagatg cagcaatttg ttaatgacca gtttaataaa acagctcagg aattggactg 2460

tataaaaatt acacagcagg ttggtgtaga actcaacctg tacctaactg aattgactac 2520

agtattcggg ccacaaatca cttcccctgc cttaactcag ctgactatcc aggcgcttta 2580

caatctagct ggtgggaata tggattactt gttgactaag ttaggtgtag gaaacaacca 2640

actcagctca ttaattggta gtggcctgat taccggcaac cctatcctgt acgactcaca 2700

gactcaactc ttgggtatac aggtcaccct accctcagtc gggaatctaa ataatatgcg 2760

tgccacctac ctggaaacct tgtctgtaag tacaaccaaa ggatttgcct cagcacttgt 2820

cccaaaagta gtgacacagg ttggttccgt gatagaagag cttgacacct cgtactgtat 2880

cgagaccgat ttggacctat attgtacaag aatagtgaca ttccctatgt ctcctggtat 2940

ttattcctgt ttgagtggca atacatctgc ttgcatgtat tcaaagactg aaggcgcact 3000

cactacgccg tatatgaccc tcaaaggctc agttattgcc aactgtaaga tgacaacatg 3060

tagatgtgca gaccccccgg gtatcatatc gcagaattat ggagaagctg tgtctctaat 3120

agataggcaa tcatgcaata tcttatcctt agacgggata actttgaggc tcagtgggga 3180

atttgatgca acttatcaaa agaatatctc aatacaagat tctcaagtaa tagttacagg 3240

caatcttgac atctcgactg agcttgggaa tgtcaacaac tcgataagta atgctttgga 3300

taagttagag gaaagcaaca gcaaactaga caaggtcaat gttaaactga ccagcacatc 3360

cgctcttatt acctatatcg ttttaactgt catatctctt gtatgtggta tacttagcct 3420

ggttctagca tgctacctga tgtacaagca aaaggcgcaa cagaagacct tgttgtggct 3480

tgggaataat accctagacc agatgagggc cactacaaaa atgtagcttg atctagagcg 3540

gccgcgggga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa 3600

tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 3660

ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc 3720

agggggaggt gtgggaggtt ttttcggatc ctctagagtc gagtttaatg ttagtttatt 3780

caatgcattg gttgcaaata ttcattactt ctccaatccc aggtcattct ttagcgagat 3840

gatgttatga cattgctgtg aaaattacta caggatatat ttttaagatg caggagtaac 3900

aatgtgcata gtaggcgtag ttatcgcaga cgtgcaacgc ttcgcatttg agttaccgaa 3960

gtgcccaaca gtgctgcggt tatggtttat gcgcacagaa tccatgcatg tcctaattga 4020

accatccgat ttttctttta atcgcgatcg ttgtttgggc aactgcgtta tttcagatct 4080

aaaaaattta ccctttatga ccatcacatc tctctggctc ataccccgct tggataagat 4140

atcatgtaga ttccgcccta agaaatgcaa actaacatta ttgtcggttc catatacact 4200

tccatcttgt ccttcgaaaa taacaaactc gcgcaataga ccgtccgtac atgcatggcc 4260

gatgtgtgtc aacatcattg gtctgctaga tcccgatggg acgaatcgta cagtcgtcgc 4320

tccagcattg gcaaaaatcc ccagataccc tccatgcggc aaatctaaat tgcgaccccg 4380

aagagactgc accaaagtct tatcgacgca cgctgatttt tttgaacagc gggagcccat 4440

<210> 37

<211> 3832

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #45

<400> 37

atgggctccc gctgttcaaa aaaatcagcg tgcgtcgata agactttggt gcagtctctt 60

cggggtcgca atttagattt gccgcatgga gggtatctgg ggatttttgc caatgctgga 120

gcgacgactg tacgattcgt cccatcggga tctagcagac caatgatgtt gacacacatc 180

ggccatgcat gtacggacgg tctattgcgc gagtttgtta ttttcgaagg acaagatgga 240

agtgtatatg gaaccgacaa taatgttagt ttgcatttct tagggcggaa tctacatgat 300

atcttatcca agcggggtat gagccagaga gatgtgatgg tcataaaggg taaatttttt 360

agatctgaaa taacgcagtt gcccaaacaa cgatcgcgat taaaagaaaa atcggatggt 420

tcaattagga catgcatgga ttctgtgcgc ataaaccata accgcagcac tgttgggcac 480

ttcggtaact caaatgcgaa gcgttgcacg tctgcgataa ctacgcctac tatgcacatt 540

gttactcctg catcttaaaa atatatcctg tagtaatttt cacagcaatg tcataacatc 600

atctcgctaa agaatgacct gggattggag aagtaatgaa tatttgcaac caatgcattg 660

aataaactaa cattaaactt gacattgatt attgactagt tattaatagt aatcaattac 720

ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 780

cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 840

catagtaacg ccaataggga ctttccatg acgtcaatgg gtggagtatt tacggtaaac 900

tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 960

tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 1020

ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 1080

catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 1140

cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 1200

ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 1260

agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca 1320

ctatagggag acccaagctg gctagcgttt aaacttaagc ttaccgccac catgggctcc 1380

agatcttcta ccaggatccc agtacctctg atgctgaccg tccgaatcat gttggcactg 1440

agttgcgtct gtccgaccag ctcccttgat ggcaggcctc ttgcagctgc agggattgtg 1500

gtaacaggag acaaagcagt caacatatac acctcatctc agacagggtc aatcataatc 1560

aagttactcc caaatatgcc caaggataaa gaggcgtgtg caaaagcccc attggaagca 1620

tacaacagga cattgactac tttgctcacc ccccttggtg attctatccg taggatacaa 1680

gagtctgtga ccacatccgg aggagggaaa cagggacgtc ttataggcgc cattatcggt 1740

ggtgtagctc tcggggttgc aaccgctgca cagataacag cagcctcggc tctgatacaa 1800

gccaatcaaa atgctgccaa catcctccgg ctcaaagaga gcattgctgc aaccaatgag 1860

gctgtgcacg aggtcactga cggattatca caactagcag tggcagttgg gaagatgcag 1920

caatttgtta atgaccagtt taataaaaca gctcaggaat tggactgtat aaaaattaca 1980

cagcaggttg gtgtagaact caacctgtac ctaactgaat tgactacagt attcgggcca 2040

caaatcactt cccctgcctt aactcagctg actatccagg cgctttacaa tctagctggt 2100

gggaatatgg attacttgtt gactaagtta ggtgtaggaa acaaccaact cagctcatta 2160

attggtagtg gcctgattac cggcaaccct atcctgtacg actcacagac tcaactcttg 2220

ggtatacagg tcaccctacc ctcagtcggg aatctaaata atatgcgtgc cacctacctg 2280

gaaaccttgt ctgtaagtac aaccaaagga tttgcctcag cacttgtccc aaaagtagtg 2340

acacaggttg gttccgtgat agaagagctt gacacctcgt actgtatcga gaccgatttg 2400

gacctatatt gtacaagaat agtgacattc cctatgtctc ctggtattta ttcctgtttg 2460

agtggcaata catctgcttg catgtattca aagactgaag gcgcactcac tacgccgtat 2520

atgaccctca aaggctcagt tattgccaac tgtaagatga caacatgtag atgtgcagac 2580

cccccgggta tcatatcgca gaattatgga gaagctgtgt ctctaataga taggcaatca 2640

tgcaatatct tatccttaga cgggataact ttgaggctca gtggggaatt tgatgcaact 2700

tatcaaaaga atatctcaat acaagattct caagtaatag ttacaggcaa tcttgacatc 2760

tcgactgagc ttgggaatgt caacaactcg ataagtaatg ctttggataa gttagaggaa 2820

agcaacagca aactagacaa ggtcaatgtt aaactgacca gcacatccgc tcttattacc 2880

tatatcgttt taactgtcat atctcttgta tgtggtatac ttagcctggt tctagcatgc 2940

tacctgatgt acaagcaaaa ggcgcaacag aagaccttgt tgtggcttgg gaataatacc 3000

ctagaccaga tgaggggccac tacaaaaatg tagctgtgcc ttctagttgc cagccatctg 3060

ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 3120

cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 3180

gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 3240

atgcggtggg ctctatggtc aattatttta tttaataaca tatagcccaa agacctctat 3300

gaacatttag tttcccgtat actcaacggc gcgtgtacac acgcatctct ttgcatagcg 3360

atgaagtttg ttcggcagca gaaaatgcag atatccaaca atctggagaa aacttatcat 3420

cacagtggca gtggaaacat accccctcta tattcatggt ataattatcg tctacagcgt 3480

ccaggatagt ggcgtgagaa aatggagatc tgcagccctc ctttccatgg catgccgctt 3540

tattgttcat taaacgcaca atggtctcaa cgccagatat gggcatagat tctgaagaac 3600

ccgttgacaa tccgaagaag aaggcgtgca ggtctttgga agactcgcac gttggtctta 3660

taatgtatga tcgagatgtc accctaatgc cacatggtac aggcttatcg cggtcatggc 3720

gatcggactt gtaatttgca acgatgggca aaggatcgac gacatgccaa acattctgaa 3780

cccgtagaga tgttaacgat gacgaggatg aatatcccat gctcgctgcc at 3832

<210> 38

<211> 4592

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #46

<400> 38

atgggctccc gctgttcaaa aaaatcagcg tgcgtcgata agactttggt gcagtctctt 60

cggggtcgca atttagattt gccgcatgga gggtatctgg ggatttttgc caatgctgga 120

gcgacgactg tacgattcgt cccatcggga tctagcagac caatgatgtt gacacacatc 180

ggccatgcat gtacggacgg tctattgcgc gagtttgtta ttttcgaagg acaagatgga 240

agtgtatatg gaaccgacaa taatgttagt ttgcatttct tagggcggaa tctacatgat 300

atcttatcca agcggggtat gagccagaga gatgtgatgg tcataaaggg taaatttttt 360

agatctgaaa taacgcagtt gcccaaacaa cgatcgcgat taaaagaaaa atcggatggt 420

tcaattagga catgcatgga ttctgtgcgc ataaaccata accgcagcac tgttgggcac 480

ttcggtaact caaatgcgaa gcgttgcacg tctgcgataa ctacgcctac tatgcacatt 540

gttactcctg catcttaaaa atatatcctg tagtaatttt cacagcaatg tcataacatc 600

atctcgctaa agaatgacct gggattggag aagtaatgaa tatttgcaac caatgcattg 660

aataaactaa cattaaacga attcactagt ggatccccca actccgcccg ttttatgact 720

agaaccaata gtttttaatg ccaaatgcac tgaaatcccc taatttgcaa agccaaacgc 780

cccctatgtg agtaatacgg ggacttttta cccaatttcc caagcggaaa gccccctaat 840

acactcatat ggcatatgaa tcagcacggt catgcactct aatggcggcc catagggact 900

ttccacatag ggggcgttca ccatttccca gcataggggt ggtgactcaa tggcctttac 960

ccaagtacat tgggtcaatg ggaggtaagc caatgggttt ttcccattac tggcaagcac 1020

actgagtcaa atgggacttt ccactgggtt ttgcccaagt acattgggtc aatgggaggt 1080

gagccaatgg gaaaaaccca ttgctgccaa gtacactgac tcaataggga ctttccaatg 1140

ggtttttcca ttgttggcaa gcatataagg tcaatgtggg tgagtcaata gggactttcc 1200

attgtattct gcccagtaca taaggtcaat agggggtgaa tcaacaggaa agtcccattg 1260

gagccaagta cactgcgtca atagggactt tccattgggt tttgcccagt acataaggtc 1320

aataggggat gagtcaatgg gaaaaaccca ttggagccaa gtacactgac tcaataggga 1380

ctttccatg ggttttgccc agtacataag gtcaataggg ggtgagtcaa caggaaagtc 1440

ccattggagc caagtacatt gagtcaatag ggactttcca atgggttttg cccagtacat 1500

aaggtcaatg ggaggtaagc caatgggttt ttcccattac tggcacgtat actgagtcat 1560

tagggacttt ccaatgggtt ttgcccagta cataaggtca ataggggtga atcaacagga 1620

aagtcccatt ggagccaagt acactgagtc aatagggact ttccatggg ttttgcccag 1680

tacaaaaggt caatagggg tgagtcaatg ggtttttccc attattggca cgtacataag 1740

gtcaataggg gtgagtcatt gggtttttcc agccaattta attaaaacgc catgtacttt 1800

cccaccattg acgtcaatgg gctattgaaa ctaatgcaac gtgaccttta aacggtactt 1860

tcccatagct gattaatggg aaagtaccgt tctcgagcca atacacgtca atgggaagtg 1920

aaagggcagc caaaacgtaa caccgccccg gttttcccct ggaaattcca tattggcacg 1980

cattctattg gctgagctgc gttctacgtg ggtataagag gcgcgaccag cgtcggtacc 2040

gtcgcagtct tcggtctgac caccgtagaa cgcagagctc ctcgctgcag gcggccgctc 2100

tagaactcgt cgatcgcagc gatgggctcc agatcttcta ccaggatccc agtacctctg 2160

atgctgaccg tccgaatcat gttggcactg agttgcgtct gtccgaccag ctcccttgat 2220

ggcaggcctc ttgcagctgc agggattgtg gtaacaggag acaaagcagt caacatatac 2280

acctcatctc agacagggtc aatcataatc aagttactcc caaatatgcc caaggataaa 2340

gaggcgtgtg caaaagcccc attggaagca tacaacagga cattgactac tttgctcacc 2400

ccccttggtg attctatccg taggatacaa gagtctgtga ccacatccgg aggagggaaa 2460

cagggacgtc ttataggcgc cattatcggt ggtgtagctc tcggggttgc aaccgctgca 2520

cagataacag cagcctcggc tctgatacaa gccaatcaaa atgctgccaa catcctccgg 2580

ctcaaagaga gcattgctgc aaccaatgag gctgtgcacg aggtcactga cggattatca 2640

caactagcag tggcagttgg gaagatgcag caatttgtta atgaccagtt taataaaaca 2700

gctcaggaat tggactgtat aaaaattaca cagcaggttg gtgtagaact caacctgtac 2760

ctaactgaat tgactacagt attcggggcca caaatcactt cccctgcctt aactcagctg 2820

actatccagg cgctttacaa tctagctggt gggaatatgg attacttgtt gactaagtta 2880

ggtgtaggaa acaaccaact cagctcatta attggtagtg gcctgattac cggcaaccct 2940

atcctgtacg actcacagac tcaactcttg ggtatacagg tcaccctacc ctcagtcggg 3000

aatctaaata atatgcgtgc cacctacctg gaaaccttgt ctgtaagtac aaccaaagga 3060

tttgcctcag cacttgtccc aaaagtagtg acacaggttg gttccgtgat agaagagctt 3120

gacacctcgt actgtatcga gaccgatttg gacctatatt gtacaagaat agtgacattc 3180

cctatgtctc ctggtattta ttcctgtttg agtggcaata catctgcttg catgtattca 3240

aagactgaag gcgcactcac tacgccgtat atgaccctca aaggctcagt tattgccaac 3300

tgtaagatga caacatgtag atgtgcagac cccccgggta tcatatcgca gaattatgga 3360

gaagctgtgt ctctaataga taggcaatca tgcaatatct tatccttaga cgggataact 3420

ttgaggctca gtggggaatt tgatgcaact tatcaaaaga atatctcaat acaagattct 3480

caagtaatag ttacaggcaa tcttgacatc tcgactgagc ttgggaatgt caacaactcg 3540

ataagtaatg ctttggataa gttagaggaa agcaacagca aactagacaa ggtcaatgtt 3600

aaactgacca gcacatccgc tcttattacc tatatcgttt taactgtcat atctcttgta 3660

tgtggtatac ttagcctggt tctagcatgc tacctgatgt acaagcaaaa ggcgcaacag 3720

aagaccttgt tgtggcttgg gaataatacc ctagaccaga tgaggggccac tacaaaaatg 3780

tagcttgatc tagagcggcc gcggggatcc agacatgata agatacattg atgagtttgg 3840

acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 3900

tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 3960

ttttatgttt caggttcagg gggaggtgtg ggaggttttt tcggatcctc tagagtcgac 4020

aattatttta tttaataaca tatagcccaa agacctctat gaacatttag tttcccgtat 4080

actcaacggc gcgtgtacac acgcatctct ttgcatagcg atgaagtttg ttcggcagca 4140

gaaaatgcag atatccaaca atctggagaa aacttatcat cacagtggca gtggaaacat 4200

accccctcta tattcatggt ataattatcg tctacagcgt ccaggatagt ggcgtgagaa 4260

aatggagatc tgcagccctc ctttccatgg catgccgctt tattgttcat taaacgcaca 4320

atggtctcaa cgccagatat gggcatagat tctgaagaac ccgttgacaa tccgaagaag 4380

aaggcgtgca ggtctttgga agactcgcac gttggtctta taatgtatga tcgagatgtc 4440

accctaatgc cacatggtac aggcttatcg cggtcatggc gatcggactt gtaatttgca 4500

acgatgggca aaggatcgac gacatgccaa acattctgaa cccgtagaga tgttaacgat 4560

gacgaggatg aatatcccat gctcgctgcc at 4592

<210> 39

<211> 4433

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of transfer plasmid for HVT-ND #48

<400> 39

atgggctccc gctgttcaaa aaaatcagcg tgcgtcgata agactttggt gcagtctctt 60

cggggtcgca atttagattt gccgcatgga gggtatctgg ggatttttgc caatgctgga 120

gcgacgactg tacgattcgt cccatcggga tctagcagac caatgatgtt gacacacatc 180

ggccatgcat gtacggacgg tctattgcgc gagtttgtta ttttcgaagg acaagatgga 240

agtgtatatg gaaccgacaa taatgttagt ttgcatttct tagggcggaa tctacatgat 300

atcttatcca agcggggtat gagccagaga gatgtgatgg tcataaaggg taaatttttt 360

agatctgaaa taacgcagtt gcccaaacaa cgatcgcgat taaaagaaaa atcggatggt 420

tcaattagga catgcatgga ttctgtgcgc ataaaccata accgcagcac tgttgggcac 480

ttcggtaact caaatgcgaa gcgttgcacg tctgcgataa ctacgcctac tatgcacatt 540

gttactcctg catcttaaaa atatatcctg tagtaatttt cacagcaatg tcataacatc 600

atctcgctaa agaatgacct gggattggag aagtaatgaa tatttgcaac caatgcattg 660

aataaactaa cattaaactc gaggtgagcc ccacgttctg cttcactctc cccatctccc 720

ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 780

gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg 840

gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt 900

atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc 960

gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc gccccggctc 1020

tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1080

aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga aagccttaaa 1140

gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt 1200

gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc 1260

ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc 1320

cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg 1380

gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc 1440

gagttgctga gcacggcccg gcttcggggtg cggggctccg tgcggggcgt ggcgcggggc 1500

tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc 1560

gggccgggga gggctcgggg gaggggcgcg gcggccccgg agcgccggcg gctgtcgagg 1620

cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc 1680

tttgtcccaa atctggcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg 1740

cgcgggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1800

cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg ctgccgcagg gggacggctg 1860

ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggggttta 1920

tatcttccct tctctgttcc tccgcagcca gccatggcca ccatgggctc cagatcttct 1980

accaggatcc cagtacctct gatgctgacc gtccgaatca tgttggcact gagttgcgtc 2040

tgtccgacca gctcccttga tggcaggcct cttgcagctg cagggattgt ggtaacagga 2100

gacaaagcag tcaacatata cacctcatct cagacagggt caatcataat caagttactc 2160

ccaaatatgc ccaaggataa agaggcgtgt gcaaaagccc cattggaagc atacaacagg 2220

acattgacta ctttgctcac cccccttggt gattctatcc gtaggataca agagtctgtg 2280

accacatccg gaggagggaa acagggacgt cttataggcg ccattatcgg tggtgtagct 2340

ctcggggttg caaccgctgc acagataaca gcagcctcgg ctctgataca agccaatcaa 2400

aatgctgcca acatcctccg gctcaaagag agcattgctg caaccaatga ggctgtgcac 2460

gaggtcactg acggattatc acaactagca gtggcagttg ggaagatgca gcaatttgtt 2520

aatgaccagt ttaataaaac agctcaggaa ttggactgta taaaaattac acagcaggtt 2580

ggtgtagaac tcaacctgta cctaactgaa ttgactacag tattcggggcc acaaatcact 2640

tcccctgcct taactcagct gactatccag gcgctttaca atctagctgg tgggaatatg 2700

gattacttgt tgactaagtt aggtgtagga aacaaccaac tcagctcatt aattggtagt 2760

ggcctgatta ccggcaaccc tatcctgtac gactcacaga ctcaactctt gggtatacag 2820

gtcaccctac cctcagtcgg gaatctaaat aatatgcgtg ccacctacct ggaaaccttg 2880

tctgtaagta caaccaaagg atttgcctca gcacttgtcc caaaagtagt gacacaggtt 2940

ggttccgtga tagaagagct tgacacctcg tactgtatcg agaccgattt ggacctatat 3000

tgtacaagaa tagtgacatt ccctatgtct cctggtattt attcctgttt gagtggcaat 3060

acatctgctt gcatgtattc aaagactgaa ggcgcactca ctacgccgta tatgaccctc 3120

aaaggctcag ttattgccaa ctgtaagatg acaacatgta gatgtgcaga ccccccgggt 3180

atcatatcgc agaattatgg agaagctgtg tctctaatag ataggcaatc atgcaatatc 3240

ttatccttag acgggataac tttgaggctc agtggggaat ttgatgcaac ttatcaaaag 3300

aatatctcaa tacaagattc tcaagtaata gttacaggca atcttgacat ctcgactgag 3360

cttgggaatg tcaacaactc gataagtaat gctttggata agttagagga aagcaacagc 3420

aaactagaca aggtcaatgt taaactgacc agcacatccg ctcttattac ctatatcgtt 3480

ttaactgtca tatctcttgt atgtggtata cttagcctgg ttctagcatg ctacctgatg 3540

tacaagcaaa aggcgcaaca gaagaccttg ttgtggcttg ggaataatac cctagaccag 3600

atgaggggcca ctacaaaaat gtagcttgat ctagagcggc cgcggggatc cagacatgat 3660

aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 3720

ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 3780

taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 3840

ttcggatcct ctagagtcga caattatttt atttaataac atatagccca aagacctcta 3900

tgaacattta gtttcccgta tactcaacgg cgcgtgtaca cacgcatctc tttgcatagc 3960

gatgaagttt gttcggcagc agaaaatgca gatatccaac aatctggaga aaacttatca 4020

tcacagtggc agtggaaaca taccccctct atattcatgg tataattatc gtctacagcg 4080

tccaggatag tggcgtgaga aaatggagat ctgcagccct cctttccatg gcatgccgct 4140

ttattgttca ttaaacgcac aatggtctca acgccagata tgggcataga ttctgaagaa 4200

cccgttgaca atccgaagaa gaaggcgtgc aggtctttgg aagactcgca cgttggtctt 4260

ataatgtatg atcgagatgt caccctaatg ccacatggta caggcttatc gcggtcatgg 4320

cgatcggact tgtaatttgc aacgatgggc aaaggatcga cgacatgcca aacattctga 4380

acccgtagag atgttaacga tgacgaggat gaatatccca tgctcgctgc cat 4433

<210> 40

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of primer 1 for mutagenesis of the located

above 5' of the Sbfi gfp gene

<400> 40

ggctagcgtt taaacttaag cttacctgca ggccaccatg gtgagcaagg gcgccgagc 59

<210> 41

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of primer 2 for mutagenesis of the located

above 5' of the Sbfi gfp gene

<400> 41

gctcggcgcc cttgctcacc atggtggcct gcaggtaagc ttaagtttaa acgctagcc 59

<210> 42

<211> 61

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of primer 1 for mutagenesis of the located

below 3' gene Sbfi gfp

<400> 42

cacggcatgg atgagctgta caagtgacct gcaggtgtgc cttctagttg ccagccatct 60

g 61

<210> 43

<211> 61

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of primer 2 for mutagenesis of the located

below 3' gene Sbfi gfp

<400> 43

cagatggctg gcaactagaa ggcacacctg caggtcactt gtacagctca tccatgccgt 60

g 61

<210> 44

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the upstream primer UL55-gene 3

<400> 44

ccaccgggta tattttccac agc 23

<210> 45

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the lower primer UL55-gene 3

<400> 45

gacccgacgc gatttcaga 19

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the upper primer for PCR UL55-gene 3

<400> 46

actgccactg tgatgataag 20

<210> 47

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the lower primer for PCR UL55-gene 3

<400> 47

ctcgctaaag aatgacctg 19

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within the coding region of IBD VP2

<400> 48

ggccgacctc aactctccac tcaa 24

<210> 49

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below within HVT IBD #1

<400> 49

ttcacagcgc ctcgttcaca ctta 24

<210> 50

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above the connection

HVT IBD #1 Transfer Plasmid Insertion Site

<400> 50

aatattcggg ggtgttagtc 20

<210> 51

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer within

coding region of IBDV VP2

HVT IBD #1

<400> 51

tgtggttggc atcagaag 18

<210> 52

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer within

coding region of IBDV VP2

<400> 52

cggcgccatg aactacacaa aact 24

<210> 53

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the site inserts UL35/36

HVT-IBD #5

<400> 53

ggggcggaaa caaataaact ctcg 24

<210> 54

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper insertion site primer

UL35/36

HVT IBD #5

<400> 54

actcggcggt aggggcattt g 21

<210> 55

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer within

hCMV HVT IBD promoter

No. 5

<400> 55

gggtcgttgg gcggtcagc 19

<210> 56

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upstream promoter located

above the integration site

HVT IBD #6a

<400> 56

tagactcggc ggtaggggca tttg 24

<210> 57

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the pec promoter

HVT IBD #6a

<400> 57

gggggtcgtt gggcggtcag from 21

<210> 58

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within the coding region of IBD VP2

HVT IBD #6a

<400> 58

cggcgccatg aactacacaa aact 24

<210> 59

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the site inserts UL35/36

<400> 59

ggggcggaaa caaataaact ctcg 24

<210> 60

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upstream promoter located

above the integration site

UL55/gen 3 for HVT IBD No. 9

<400> 60

actgccactg tgatgataag 20

<210> 61

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the integration site

UL55/gene for HVT IBD No. 9

<400> 61

ctcgctaaag aatgacct 18

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for insertion of upper primer orientation,

surrounding upstream HVT IBD #9 VP2 gene insertion junction

<400> 62

aatattcggg ggtgttagtc 20

<210> 63

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for insertion of downstream primer orientation,

located within

IBDV VP2 coding region for HVT IBD #9

<400> 63

ctcgcttctc agtgtatgtt tttc 24

<210> 64

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

below the site that defines the proper

insertion of the IBDV VP2 coding region for HVT IBDV #9

<400> 64

atcctgagct cgctaaaaat cttg 24

<210> 65

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the site that defines the proper

insertion of the IBDV VP2 coding region for HVT IBDV #9

<400> 65

cctcgcccgt ctacgctcca ct 22

<210> 66

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for the located

above the integration site area

UL55 gene for HVT IBD No. 30

<400> 66

tagcgacccg agtagga 17

<210> 67

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer for the located

above the integration site area

UL55 gene for HVT IBD No. 30

<400> 67

cgcccacggt caaattgtat gtag 24

<210> 68

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for confirmation

proper orientation

VP2 insertion surrounding the 3' insertion site junction

HVT IBD #30

<400> 68

ttatccgcgc aatcagaagg 20

<210> 69

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for confirmation

proper orientation

VP2 insertion surrounding the 3' insertion site junction

HVT IBD #30

<400> 69

atggggcgga aacaaataaa ct 22

<210> 70

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for confirmation

proper orientation of integration of the VP2 insert outside the expression cassette

HVT IBD #30

<400> 70

ccaccgggta tattttccac agc 23

<210> 71

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for confirmation

proper orientation of the VP2 insert integration

outside the HVT IBD expression cassette #30

<400> 71

gacccgacgc gatttcaga 19

<210> 72

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for confirmation

proper orientation

VP2 insert located above the UL35/36 HVT IBD integration site #31

No. 31

<400> 72

ctcggcggta ggggcatttg ataa 24

<210> 73

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for confirmation

proper orientation

VP2 insertion located within the chicken beta-actin promoter

HVT IBD #31

<400> 73

agatggggag agtgaagcag aacg 24

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for confirmation

VP2 inserts,

located within the coding region of IBDV VP2

<400> 74

ttatccgcgc aatcagaagg 20

<210> 75

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for confirmation

proper orientation

VP2 inserts located below the UL35/36 HVT integration site

IBD #31

<400> 75

atggggcgga aacaaataaa ct 22

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer targeting the located

below

VP2 HVT IBD insertion site #31, located within

VP2 inserts

<400> 76

ttatccgcgc aatcagaagg 20

<210> 77

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the site UL35/36

HVT IBD #31

<400> 77

atggggcgga aacaaataaa ct 22

<210> 78

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for the located

above the integration site area

gene 3-UL55 for HVT-IBD No. 34

<400> 78

cctcgcccgt ctacgctcca ctga 24

<210> 79

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the chicken beta-actin promoter

for HVT-IBD #34

<400> 79

ccgcccccat cgctgcacaa aata 24

<210> 80

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within the coding region of IBDV VP2

for HVT-IBD #34

<400> 80

ggccctccgt cccgtcac 18

<210> 81

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the insertion site of gene 3-UL55

for HVT IBD #34

<400> 81

ttcacagcgc ctcgttcaca ctta 24

<210> 82

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

outside the VP2 expression cassette

HVT IBD #34

<400> 82

ctcgctaaag aatgacctg 19

<210> 83

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

outside the VP2 expression cassette

HVT IBD #34

<400> 83

actgccactg tgatgataag 20

<210> 84

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for the located

above the integration site area

UL35-UL36 HVT ND No. 38

<400> 84

tgtttaaggc attttcaagt 20

<210> 85

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> The DNA sequence for the downstream primer, which is located

within the coding region of NDV F

HVT ND #38

<400> 85

attcggacgg tcagcatca 19

<210> 86

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer surrounding the 3' junction

insertion located within the coding region of NDV F HVT ND #38

<400> 86

accagatgag ggccactaca aaaa 24

<210> 87

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the site inserts UL35-UL36

HVT ND #38

<400> 87

atggggcgga aacaaataaa ct 22

<210> 88

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

HVT ND #38

<400> 88

gaggtccgtc gccacaa 17

<210> 89

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

HVT ND #38

<400> 89

gcaagacaaa taacgcagac ta 22

<210> 90

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above the integration site area

UL35-UL36 for HVT-ND No. 39

<400> 90

ctcggcggta ggggcatttg ataa 24

<210> 91

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the chicken beta-actin promoter

HVT-ND #39

<400> 91

agatggggag agtgaagcag aacg 24

<210> 92

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer surrounding the located

below is the connection

inserts located within the poly A region HVT-ND #39

<400> 92

tggggatgcg gtgggctcta 20

<210> 93

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located below

site inserts UL35-UL36

HVT-ND #39

<400> 93

ggggcggaaa caaataaact ctcg 24

<210> 94

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

HVT-ND #39

<400> 94

ccaccgggta tattttccac agc 23

<210> 95

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

HVT-ND #39

<400> 95

gacccgacgc gatttcaga 19

<210> 96

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above the integration site

UL35/36 for HVT ND No. 40

<400> 96

ctcggcggta ggggcatttg ataa 24

<210> 97

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the chicken beta-actin promoter

for HVT ND No. 40

<400> 97

agatggggag agtgaagcag aacg 24

<210> 98

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within the coding region of NDVF

for HVT ND No. 40

<400> 98

accagatgag ggccactaca aaaa 24

<210> 99

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the connection

insertion site for HVT ND No. 40

<400> 99

atggggcgga aacaaataaa ctct 24

<210> 100

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

for HVT ND No. 40

<400> 100

ccaccgggta tattttccac agc 23

<210> 101

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

for HVT No. 40

<400> 101

gacccgacgc gatttcaga 19

<210> 102

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for PCR amplification of the cassette

for HVT ND No. 42

<400> 102

tacttagtca taggcgcgcc aactccgccc gttttatgac tagaacca 48

<210> 103

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for PCR amplification of the cassette

for HVT ND No. 42

<400> 103

tgacagtatc tagctagctc gactctagag gatccgaaaa aacctccca 49

<210> 104

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

outside the cassette for HVT

ND No. 42

<400> 104

actgccactg tgatgataag 20

<210> 105

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

outside the cassette for HVT

ND No. 42

<400> 105

ctcgctaaag aatgacctg 19

<210> 106

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

for HVT ND No. 42

<400> 106

ttcacagcgc ctcgttcaca ctta 24

<210> 107

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region ND F

for HVT ND No. 42

<400> 107

ttcggacggt cagcatcaga ggta 24

<210> 108

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

for HVT ND No. 42

<400> 108

gcctcggcgt ggtagttctc 20

<210> 109

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region ND F

for HVT ND No. 42

<400> 109

attcggacgg tcagcatca 19

<210> 110

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

for HVT ND No. 42

<400> 110

ttcacagcgc ctcgttcaca ctta 24

<210> 111

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region of NDV F

for HVT ND No. 42

<400> 111

gcttttgcac acgcctcttt atcc 24

<210> 112

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

<400> 112

ttcacagcgc ctcgttcaca ctta 24

<210> 113

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region ND F

for HVT ND No. 42

<400> 113

ttcggacggt cagcatcaga ggta 24

<210> 114

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

for HVT ND No. 42

<400> 114

accagatgag ggccactaca aaaa 24

<210> 115

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region ND F

for HVT ND No. 42

<400> 115

cgcccgtcta cgctccactg a 21

<210> 116

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above UL55 for HVT

ND No. 44

<400> 116

aatattcggg ggtgttag 18

<210> 117

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the chicken beta-actin promoter

for HVT ND No. 44

<400> 117

ggggggagatg ggggagatga 20

<210> 118

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above UL55 for HVT

ND No. 44

<400> 118

gcctcggcgt ggtagttctc 20

<210> 119

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the chicken beta-actin promoter

for HVT ND No. 44

<400> 119

ccgcccccat cgctgcacaa aata 24

<210> 120

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within the sequence encoding the NDV F gene,

for HVT ND No. 44

<400> 120

accagatgag ggccactaca aaaa 24

<210> 121

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the UL55-gene 3 insertion site

for HVT ND No. 44

<400> 121

ctcgcccgtc tacgctccac tgaa 24

<210> 122

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

for HVT ND No. 44

<400> 122

actgccactg tgatgataag 20

<210> 123

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

for HVT #44

<400> 123

ctcgctaaag aatgacctg 19

<210> 124

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

for HVT #45

<400> 124

actgccactg tgatgataag 20

<210> 125

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

for HVT #45

<400> 125

ctcgctaaag aatgacctg 19

<210> 126

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

for HVT ND No. 45

<400> 126

ctcgcccgtc tacgctccac tgaa 24

<210> 127

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region ND F

for HVT ND No. 45

<400> 127

gcttttgcac acgcctcttt atcc 24

<210> 128

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond the expression cassette

for HVT ND No. 45

<400> 128

cacgctgcta ttgtaacg 18

<210> 129

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the coding region ND F

for HVT ND No. 45

<400> 129

actgggatcc tggtagaag 19

<210> 130

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer surrounding the located

below is the connection

insertion site for HVT ND No. 45

<400> 130

accagatgag ggccactaca aaaa 24

<210> 131

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the insertion site of gene 3-UL55

<400> 131

ttcacagcgc ctcgttcaca ctta 24

<210> 132

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer surrounding the located

below is the connection

insertion site for HVT ND No. 45

<400> 132

gcacatccgc tcttattacc tat 23

<210> 133

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the site

insertions of gene 3-UL55

<400> 133

ttcacagcgc ctcgttcaca ctta 24

<210> 134

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

for HVT ND No. 46

<400> 134

tagagggggt atgtttccac tgc 23

<210> 135

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

for HVT ND No. 46

<400> 135

gtcataacat catctcgcta aag 23

<210> 136

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above and beyond

Integration site for HVT ND #46

<400> 136

cacgctgcta ttgtaacg 18

<210> 137

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the mCMV promoter

for HVT ND No. 46

<400> 137

ggtgaacgcc ccctatgtgg a 21

<210> 138

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within a sequence,

encoding the NDV F gene, for HVT ND No. 46

<400> 138

accagatgag ggccactaca aaaa 24

<210> 139

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below and beyond

expression cassettes for HVT ND #46

<400> 139

ttcacagcgc ctcgttcaca ctta 24

<210> 140

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within a sequence,

encoding the NDV F gene, for HVT ND No. 46

<400> 140

gcacatccgc tcttattacc tat 23

<210> 141

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below and beyond

expression cassettes for HVT ND #46

<400> 141

ttcacagcgc ctcgttcaca ctta 24

<210> 142

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within a sequence,

encoding the NDV F gene, for HVT ND No. 46

<400> 142

atgtggtata cttagcctgg ttc 23

<210> 143

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below and beyond

expression cassettes for HVT ND #46

<400> 143

aatattcggg ggtgttag 18

<210> 144

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

within a sequence,

encoding the NDV F gene, for HVT ND No. 46

<400> 144

ggaataatac cctagaccag atg 23

<210> 145

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below and beyond

expression cassettes for HVT ND #46

<400> 145

gtcataacat catctcgcta aag 23

<210> 146

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

above the area

integration site gene 3-UL55 for HVT ND #48

<400> 146

cctcgcccgt ctacgctcca ctga 24

<210> 147

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

within the chicken beta-actin promoter

for HVT ND No. 48

<400> 147

ccgcccccat cgctgcacaa aata 24

<210> 148

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer surrounding the located

below is the insert site connection for HVT ND #48

<400> 148

accagatgag ggccactaca aaaa 24

<210> 149

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

below the insertion site of gene 3-UL55

for HVT ND No. 48

<400> 149

ttcacagcgc ctcgttcaca ctta 24

<210> 150

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer surrounding the located

below is the insert site connection for HVT ND #48

<400> 150

gcacatccgc tcttattacc tat 23

<210> 151

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located below

insertion site of gene 3-UL55

for HVT ND No. 48

<400> 151

ttcacagcgc ctcgttcaca ctta 24

<210> 152

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer located

beyond the expression cassette

for HVT ND No. 48

<400> 152

actgccactg tgatgataag 20

<210> 153

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the downstream primer located

beyond the expression cassette

for HVT ND No. 48

<400> 153

ctcgctaaag aatgacctg 19

<210> 154

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for cassette amplification

NDV F based expression

HVT ND No. 42 for HVT-IBD-ND No. 42-No. 30

<400> 154

tacttagtca taggcgcgcc aactccgccc gttttatgac tagaacca 48

<210> 155

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for cassette amplification

NDV F based expression

HVT ND No. 42 for HVT-IBD-ND No. 42-No. 30

<400> 155

tgacagtatc tagctagctc gactctagag gatccgaaaa aacctccca 49

<210> 156

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the upper primer for cassette amplification

IBD gene based expression

plasmids No. 30 for HVT-IBD-ND No. 42-No. 30

<400> 156

ctgtcagacg agtaccggtt tgacattgat tattgactag ttattaatag ta 52

<210> 157

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the lower primer for cassette amplification

IBD gene based expression

plasmids No. 30 for HVT-IBD-ND No. 42-No. 30

<400> 157

tctagtactg atggtaccac catagagccc accgcatccc ca 42

<210> 158

<211> 570

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence for the RSV LTR promoter

<400> 158

actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt 60

gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg 120

accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta 180

cgggccagat atacgcgtat ctgaggggac tagggtgtgt ttaggcgaaa agcggggctt 240

cggttgtacg cggttaggag tcccctcagg atatagtagt ttcgcttttg catagggagg 300

gggaaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc 360

aacatgcctt acaaggag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 420

acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg 480

aattccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgccattt 540

gaccattcac cacattggtg tgcacctcca 570

<---

Claims (66)

1. A recombinant turkey herpes virus (HVT) genome encoding and expressing an antigenic polypeptide of a pathogenic agent, wherein said genome contains one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL 35/UL 36 intergenic loci in a unique long (UL) region of the HVT genome. 2. A recombinant turkey herpes virus (HVT) genome encoding and expressing an antigenic polypeptide of a pathogenic agent, wherein said genome comprises one or more nucleotide sequences encoding one or more heterologous antigens inserted into the intergenic loci UL 35/UL 36 in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL55/gene 3 site of the unique long region (UL) of the HVT genome. 3. The recombinant HVT genome according to claim 1 or 2, characterized in that one or more of the said heterologous antigens are characterized by a protective effect against an avian pathogen(s) selected from the group consisting of: infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). 4. A recombinant HVT genome according to any of the preceding claims, characterized in that one or more of said heterologous antigens are selected from the group consisting of: a) VP2, VP3 or VP4 proteins of infectious bursal disease virus (IBDV); b) VP1 or VP2 proteins of chicken anemia virus (CAV); c) the chimeric F/HN protein or the F, NP, P, M, HN or L protein of Newcastle disease virus (NDV); d) proteins S1, S2 or M of infectious bronchitis virus (IBV); e) proteins gB, gC, gD, gE, gH, gl or gL of infectious laryngotracheitis virus (ILTV); and f) any of the HA, NA, NP or M proteins of avian influenza virus (AIV). 5. A recombinant HVT genome according to any of the preceding claims, characterized in that one or more of said heterologous antigens are characterized by a protective effect against IBDV. 6. The recombinant HVT genome according to claim 5, characterized in that one or more of said heterologous antigens are the VP2 protein of IBDV. 7. The recombinant HVT genome according to claim 6, characterized in that said VP2 protein sequence is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence containing SEQ ID NO. 5 or SEQ ID NO. 10. 8. The recombinant HVT genome according to claim 6, characterized in that said VP2 protein is encoded by a nucleotide sequence containing SEQ ID NO. 5 or SEQ ID NO. 10. 9. A recombinant HVT genome according to any one of claims 1-4, characterized in that one or more heterologous antigens are characterized by a protective effect against Newcastle disease virus (NDV). 10. The recombinant HVT genome according to claim 9, characterized in that one or more of said heterologous antigens are the NDV F protein. 11. The recombinant HVT genome according to claim 10, characterized in that said NDV F protein is encoded by a nucleotide sequence that is at least 80% identical to the sequence containing SEQ ID NO. 3. 12. The recombinant HVT genome according to claim 10, characterized in that said F NDV protein is encoded by a nucleotide sequence containing SEQ ID NO. 3. 13. A recombinant HVT genome according to any of the preceding claims, characterized in that one or more of said heterologous antigens have a protective effect against NDV and IBDV. 14. The recombinant HVT genome according to claim 13, characterized in that at least one of said heterologous antigens comprises the NDV F protein and the IBDV VP2 protein. 15. The recombinant HVT genome according to claim 14, characterized in that said NDV F protein is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence containing SEQ ID NO. 3, and said IBDV VP2 protein is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequences containing SEQ ID NO. 5 or SEQ ID NO. 10. 16. The recombinant HVT genome according to claim 14 or 15, characterized in that said NDV F protein is encoded by a nucleotide sequence containing SEQ ID NO. 3, and the VP2 protein from IBDV is encoded by nucleotide sequences containing SEQ ID NO. 5 or SEQ ID NO. 10. 17. A recombinant HVT genome according to any of the preceding claims, comprising a genome comprising one or more expression cassettes comprising one or more nucleotide sequences that encode one or more heterologous antigens. 18. The recombinant HVT genome according to claim 17, characterized in that said expression cassette contains one or more promoters. 19. The recombinant HVT genome of claim 18, wherein one or more of said promoters are selected from the group consisting of: the human cytomegalovirus (hCMV) immediate early promoter; the guinea pig CMV immediate early promoter; the mouse CMV immediate early promoter; the Rec promoter; the chicken β-actin promoter; the SV40 promoter; the pseudorabies virus glycoprotein X promoters; the herpes simplex virus-1 alpha 4 promoter; the promoters of the glycoproteins gA, gC, gB, gE or gI of the Marek's disease virus; the promoters of the glycoprotein gB, gE, gI, gD of the infectious laryngotracheitis viruses; and the VP8 promoter of the bovine herpes virus 1/1. 20. The recombinant HVT genome according to claim 19, characterized in that one or more of said promoters contain the human CMV promoter. 21. The recombinant HVT genome according to claim 19, characterized in that one or more of said promoters contain the mouse CMV promoter. 22. The recombinant HVT genome according to claim 19, characterized in that one or more of the said promoters contain the hCMV and mCMV promoter. 23. Isolated DNA encoding a recombinant HVT genome according to any one of claims 1 and 3-22. 24. Isolated DNA encoding the recombinant HVT genome according to any one of claims 2-22. 25. An immunogenic composition comprising a recombinant HVT according to any one of claims 1-22, further comprising a pharmaceutically acceptable carrier, excipient or adjuvant. 26. A vaccine composition comprising a recombinant HVT according to any one of claims 1-22, further comprising a pharmaceutically acceptable carrier, excipient or adjuvant. 27. The vaccine composition according to claim 26, further comprising an additional Marek's disease virus (MDV) selected from the group consisting of: the naturally attenuated strain MDV-1 Rispens (CVI-988); or the herpes virus Gallid 3 strain SB-1. 28. The vaccine composition according to claim 27, characterized in that said additional MDV contains a recombinant genome. 29. The vaccine composition according to claim 28, characterized in that said recombinant MDV genome contains one or more nucleotide sequences encoding one or more heterologous antigens that have a protective effect against one or more avian pathogens. 30. A vaccine composition according to any one of claims 26-29 for use in vaccinating poultry against one or more diseases caused by one or more avian pathogens. 31. A vaccine composition according to any one of claims 26 to 29 for use in protecting poultry from clinical symptoms caused by one or more avian pathogens. 32. A vaccine composition according to any one of claims 26 to 29 for use in protecting poultry against clinical symptoms caused by Marek's disease virus and clinical symptoms caused by one or more avian pathogens. 33. The vaccine composition according to any one of claims 30-32, characterized in that one or more of the said avian pathogens are selected from the group consisting of: infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). 34. The vaccine composition according to claim 33, characterized in that one or more of the said avian pathogens include the Newcastle disease virus. 35. The vaccine composition according to claim 33, characterized in that one or more of the said avian pathogens include infectious bursal disease virus (IBDV). 36. The vaccine composition according to claim 33, characterized in that one or more of the said avian pathogens include Newcastle disease virus and infectious bursal disease virus (IBDV). 37. A vaccine composition according to any one of claims 26 to 36 for use in vaccinating birds, wherein the vaccine composition is administered by at least one or more administrations of the vaccine composition by spraying, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration or a combination thereof. 38. A vaccine composition according to claim 37, characterized in that said vaccine composition is administered by in ovo administration. 39. The vaccine composition according to claim 38, characterized in that said in ovo administration occurs into an egg cell with a developing embryo in a period between about 16 to 22 days of development. 40. A vaccine composition according to any one of claims 37-39, characterized in that said in ovo administration occurs into an egg cell with a developing embryo during a period of about 18 days of development. 41. A vaccine composition according to any one of claims 37-40, characterized in that said administration of the vaccine composition comprises administration in ovo followed by administration of a spray. 42. The vaccine composition according to claim 37, characterized in that said administration of the vaccine composition includes administration of a spray. 43. A method for vaccinating poultry for the treatment or prevention of Marek's disease and one or more poultry diseases caused by one or more poultry pathogens, comprising the step of administering an effective amount of a vaccine composition according to claims 26-36. 44. The method according to claim 43, characterized in that one or more of said avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). 45. The method according to claim 44, characterized in that one or more of said avian pathogens include infectious bursal disease virus (IBDV). 46. The method according to claim 44, characterized in that one or more of the said bird pathogens include Newcastle disease virus (NDV). 47. The method according to claim 44, characterized in that one or more of said avian pathogens comprise infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). 48. A method for inducing an immune response in an avian animal to Marek's disease virus and one or more avian pathogens, comprising the step of administering to the bird an effective amount of the immunogenic composition according to claim 25 or the vaccine composition according to any one of claims 26-36. 49. The method of claim 48, wherein one or more of said avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV); Newcastle disease virus (NDV); infectious bronchitis virus (IBV); infectious laryngotracheitis virus (ILTV); chicken anemia virus (CAV); and avian influenza virus (AIV). 50. The method according to claim 49, characterized in that one or more of said avian pathogens include infectious bursal disease virus (IBDV). 51. The method according to claim 49, characterized in that one or more of the said bird pathogens include Newcastle disease virus (NDV). 52. The method according to claim 49, characterized in that one or more of said avian pathogens comprise infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). 53. The method according to any one of paragraphs 43-52, characterized in that said administration is carried out by spraying, in ovo administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration. 54. The method according to claim 53, characterized in that the said route of administration includes administration in ovo. 55. The method according to claim 54, characterized in that said in ovo administration occurs into an egg cell with a developing embryo in the period between about 16 to 22 days of development. 56. The method according to any one of paragraphs 53-55, characterized in that said in ovo administration occurs into an egg cell with a developing embryo during a period of about 18 days of development. 57. The method according to paragraphs 53-56, characterized in that the said route of administration includes administration in ovo followed by administration of a spray. 58. The method according to paragraph 53, characterized in that the said route of administration includes administration of a spray. 59. The method according to any one of paragraphs 45-58, characterized in that said bird is selected from the group consisting of chicken, turkey, goose, duck, pheasant, ostrich, pigeon and quail. 60. The method according to paragraph 59, characterized in that said bird is a chicken.