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WO1998035023A1 - Procede servant a cultiver le virus du syndrome respiratoire et reproductif porcin afin de l'utiliser en tant que vaccin et dans des essais diagnostiques - Google Patents

Procede servant a cultiver le virus du syndrome respiratoire et reproductif porcin afin de l'utiliser en tant que vaccin et dans des essais diagnostiques Download PDF

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Publication number
WO1998035023A1
WO1998035023A1 PCT/US1998/002044 US9802044W WO9835023A1 WO 1998035023 A1 WO1998035023 A1 WO 1998035023A1 US 9802044 W US9802044 W US 9802044W WO 9835023 A1 WO9835023 A1 WO 9835023A1
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Prior art keywords
prrsv
monocytes
virus
cell line
cytokine
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PCT/US1998/002044
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English (en)
Inventor
Michael D. Sussman
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Origen, Inc.
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Publication of WO1998035023A1 publication Critical patent/WO1998035023A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/38011—Tombusviridae
    • C12N2770/38051—Methods of production or purification of viral material

Definitions

  • the present invention relates to a method for propagating porcine reproductive and respiratory syndrome virus (PRRSV) for the manufacture of PRRSV vaccines, use in diagnostic assays, and the generation of cell lines.
  • PRRSV porcine reproductive and respiratory syndrome virus
  • the method describes production of macrophages from monocytes of porcine origin by treatment with cytokines to propagate PRRSV.
  • the method is also useful for the production of PRRSV for vaccines and for the replication of PRRSV for diagnostic assays.
  • the method of the present invention also provides a means for the establishment of a continuous cell line for PRRSV production for vaccines and diagnostic assays.
  • PRRSV Porcine reproductive and respiratory syndrome virus
  • the disease agent has been identified as a member of the Arterivirus genus within the Togaviridae family.
  • the PRRSV is a small enveloped spherical positive strand RNA virus, with an average virion diameter of 62 nm and a 25-30 nm core surrounded by an envelope (Goyal, J Vet Diagn Invest Vol. 5 pages 656- 664) (1993) .
  • the disease is characterized by reproductive failure (abortions) , respiratory disease and variable clinical signs that include anorexia, fever, dyspnea, and neurological signs. Macrophages of the lung appear to be the primary type of cell that becomes infected.
  • PRRSV porcine alveolar macrophages
  • PAM porcine alveolar macrophages
  • Vero simian cell lines derived from African green monkey cells
  • 9009B U.S. Patent 5,510,258
  • MA-104 U.S. Patent 5,476,778
  • MARC-145 Kerm et al., 1993, Arch Virol Vol. 133 pages 477-483.
  • the extent to which any particular isolate replicates to high titers on any of these Vero derived cell lines is unique to the specific PRRSV isolate.
  • PAMs are the only known primary cells that have a high sensitivity to PRRSV infection and can produce relatively high yields of PRRSV. PAMs are the natural host for PRRSV.
  • PAMS are the natural host for PRRSV.
  • disadvantages to using PAMS such as difficulty in obtaining PAMs of consistent high quality, the difficulty of harvesting PAMs by pulmonary lavage and the risk of contamination by other organisms present in the lung, and the high cost associated with maintaining a sufficient number of swine to provide the quantities of PAMs needed for large scale vaccine production or diagnostics.
  • Pulmonary lavage requires anesthetizing the animal thereby incurring further expense since a veterinarian is required to monitor the procedure and animal use permits are required.
  • the preferred method for propagating PRRSV would be a cell method that utilized a porcine macrophage cell line or primary porcine macrophages that could be obtained more readily than PAMs. Such cells would be similar to the natural PRRSV host and virus replication in such cells would be more efficient and less prone to selection of PRRSV with accumulated mutations. It is well known to those skilled in the art that propagation of viruses in heterologous host cells results in viruses that accumulate mutations that do not occur in viruses propagated in their natural host cell. While macrophage cell lines have been created from other species, notably the rat and the mouse (Mbawuike et al., 1989, J Leuk Biol Vol. 46 pages 119-127) no porcine macrophage cell line exists. However, PAMs like all macrophages are derived from undifferentiated macrophages called monocytes.
  • Monocytes are precursor cells that are found systemically in the blood. During the course of development monocytes leave blood circulation and enter tissues that have become infected. Binding of specific cytokines, notably macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) , to monocytes induces them to transform into macrophages. The presence of these cytokines in blood and tissues results from secretions of lymphocytes and leucocytes. M-CSF added to macrophages isolated from blood of porcine origin has been shown to serve as both a differentiating and longevity factor.
  • M-CSF macrophage colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Live and killed virus vaccines to control PRRS have been available for several years, however these vaccines have been effective in less than 50% of herds, this is significant in view that most herds are vaccinated 3 to 5 times a year. While killed vaccines are safe, they are less effective than live virus vaccines. However, live virus vaccines cannot be used in pregnant sows as vaccine induced viremia may be transferred to offspring, resulting in disease symptoms. In recent months, new variants of PRRSV have appeared which overcome any protection provided by current vaccines. Killed virus vaccines have been prepared from
  • PRRSV isolated from PAMs infected with PRRSV While the PRRSV genome appears to be stabile when propagated on PAMs obtained from the lungs of pigs by pulmonary lavage both the process of PAM production and the high cost has precluded use of the procedure for large scale production of PRRSV vaccines.
  • killed virus vaccines occupy a niche market whereby PRRSV from a particular farm is isolated and used to produce a vaccine specific for the PRRSV isolate currently found on the farm.
  • Live virus vaccines are prepared from PRRSV isolated from infected simian cell lines (United States Patents 5,476,778 and 5,510,258). While passage of PRRSV on simian cell lines can result in attenuated virus suitable for use as a vaccine, the inherent instability of the virus genome when propagated on said cell lines makes it difficult to maintain virus at the appropriate level of attenuation and in some cases may produce a virus at a higher passage level that is more virulent than a virus at a lower level. The instability of the PRRS virus genome in these cells makes it unlikely that a vaccine virus can be produced that contained markers that would clearly distinguish the vaccine virus from naturally occurring virus. This has prevented development of serological and PCR based diagnostics that distinguish swine that have been vaccinated against PRRS from swine that have been infected with a naturally occurring PRRSV.
  • the present invention relates to a method for production of macrophages from monocytes of porcine origin to propagate PRRSV.
  • the present invention relates to a method of growing and isolating porcine reproductive and respiratory syndrome virus (PRRSV) which comprises: isolating monocytes, treating said monocytes with a cytokine in a suitable growth, medium, inoculating the cytokine treated monocytes with PRRSV and incubating the inoculated cytokine treated monocytes at about 31°C to 37 °C until cytopathic effect is observed.
  • the present invention further relates to a method for producing a hybridoma cell line susceptible to PRRSV infection which comprises: fusing monocytes or macrophages with a cell line and then growing of the hybridoma in a suitable medium.
  • the present invention also relates to a hybridoma cell line susceptible to PRRSV infection comprising monocytes or macrophages fused to a cell line .
  • the present invention describes a method for the isolation of a specific subset of blood cells from swine called monocytes and treating said cells with cytokines to render the cells susceptible to PRRSV infection and replication.
  • the method is useful for the production of PRRSV for vaccines and for the replication of PRRSV for diagnostic assays.
  • the method of the present invention also provides a means for the establishment of a continuous cell line for PRRSV production for vaccines and diagnostic assays.
  • HBSS Hank's Balanced Salt Solution
  • Dextran T-500 Pulcoa
  • the mixture of blood and HBSS/Dextran was mixed by inversion and incubated for two hours at 37° C. in an atmosphere of 5% C0 2 .
  • the lower layer consisted mostly of erythrocytes while the upper layer became enriched for leucocytes.
  • the leucocytes consisted primarily of lymphocytes, polymorphonuclear cells, and monocytes.
  • the leucocyte enriched supernatant was separated from the lower layer with a 10 ml serological pipette with an attached suction device and layered upon a 10 ml step cushion consisting of 5.7 % Ficoll and 9 % aqueous sodium diatrizoate in H 2 0 contained in a 50 ml conical centrifuge tube.
  • the tubes were spun at 2,000 rpm for 2 hours at 4° C. Afterwards, granulocytes and erythrocytes were separated from lymphocytes and monocytes which were found in a band at the top of the cushion.
  • the layer above the lymphocytes comprising HBSS and porcine serum was removed and the band containing the monocytes and lymphocytes was transferred to a new tube.
  • Monocyte enriched bands from several tubes were combined. The cells were pelleted in a centrifuge at 2,000 rpm for 5 min and washed 3 times with HBSS at 4° C.
  • the cells were resuspended in medium that comprised either RPMI 1640 (Gibco, Grand Island, New York) that contained 10% fetal bovine serum or macrophage serum free medium (Mf-SFM, Gibco BRL.). Medium used to cultivate macrophages was supplemented with 10% L929 conditioned medium.
  • L929 (American Type Culture Collection (ATCC) CCL-1) cells are a murine cell line that constitutively secretes murine M-CSF into the tissue culture medium. L929 cells were grown steadily in either RPMI 1640 medium containing 10 % fetal bovine serum or Mf-SFM at 37° C. in 5% C0 2 .
  • M-CSF macrophage colony stimulating factor
  • M-CSF may be produced synthetically or expressed biochemically in bacteria.
  • the aforementioned monocytes and lymphocytes were counted and then plated in suitable cell culture vessels, for example flasks, dishes or roller bottles at a density of approximately 4,000 cells per square centimeter. The cultures are then incubated overnight at 37° C. in 5 % C0 2 . Monocytes adhered to the culture vessel whereas lymphocytes did not. The cultures were washed with medium to remove the unattached lymphocytes. The cultures now comprised primarily monocytes. Approximately, 4 xlO 8 monocytes were obtained per liter of blood. The monocytes were either frozen for future use or treated with M-CSF and infected with PRRSV.
  • monocytes to M-CSF Exposure of monocytes to M-CSF induced their dif erentiation into macrophages.
  • monocytes isolated and treated as described above were infected with • PRRSV at a range of multiplicities of infection (MOI) . Cytopathic effect, indicative of PRRSV infection, was evident within one day post infection with PRRSV, even at MOIs of 0.1. At all MOIs, PRRSV replicated and titers exceeding 10 5 TCID j o/ml (10,000 virions per cell) were obtained.
  • PRRSV is heat labile at 37°C but relatively stable for long periods of time at 4°C. and -70° C.
  • the thermolability of the virus at 37° C. suggests that propagation of the virus at temperatures lower than 37° C. will produce virus titers higher than 10 5 TCID 50 /ml.
  • temperature sensitive or cold-sensitive PRRSV can be propagated in the present invention. Therefore, in the aforementioned invention the PRRSV infection is practiced at temperatures within the range of 25° C. to 37° C.
  • the invention may be used to produce any PRRS virus isolate or strain for purposes of vaccine production or diagnostics. This method is useful because it is likely that virus isolated from this culturing system will not have significant genomic rearrangements.
  • the present invention is used to produce killed vaccines that are used to protect sows and gilts against PRRS infection.
  • the antigenic mass of PRRSV propagated by the present invention is used for vaccines. Once PRRSV is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods practiced by those skilled in the art. For example, the virus antigenic mass can be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain virus antigenic mass to produce vaccines.
  • the PRRSV is grown according to the method of the present invention to a titer of 10 5 TCID 50 /ml.
  • the virus is then isolated by art-known methods and inactivated by treatment with formalin or with binary ethyleneimine (BEI) , both methods are well known to those skilled in the art.
  • BEI binary ethyleneimine
  • the aforementioned inactivated virus is mixed with any of the art-known adjuvants.
  • the resulting vaccine formulations are administered to swine by methods familiar to those skilled in the art.
  • An example of inactivation by formalin is mixing the virus suspension with 37 % formaldehyde to a final formaldehyde concentration of 0.05%.
  • the virus- formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature.
  • the inactivated virus mixture is tested for residual live virus by assaying for growth on M-CSF treated monocytes of the present invention.
  • inactivation by BEI is mixing the virus suspension with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM.
  • the virus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours.
  • the inactivated virus mixture is tested for residual live virus by assaying for growth on M-CSF treated monocytes of the present invention.
  • the present invention is used to produce live vaccines to protect swine against PRRS. Attenuated strains of PRRSV that are no longer pathogenic in swine are propagated as described in Example 1.
  • Virus prepared as described in Example 1 may be concentrated, frozen, and stored at -70° C. or freeze-dried and stored at 4° C.
  • Virus for vaccination can be mixed with a saline solution to the appropriate dosage and administered either by oralnasal exposure or by injection.
  • PRRSV Genetically engineered PRRSV is propagated according to the present invention.
  • PRRSV genetically engineered to be attenuated by deletion of viral sequences or insertion of non-PRRSV sequences or containing sequences of other related arteritis viruses are propagated by the method of the present invention.
  • An example of a genetically engineered PRRSV with a deletion is a virus with open reading frame 5 deleted or deleted and replaced with an appropriate marker or stuffer fragment.
  • An example of a genetically engineered PRRSV containing sequences from a related arteritis virus is a PRRSV whereby its polymerase gene is replaced with the polymerase gene from equine arteritis virus.
  • PRRSV are also useful for tagging a vaccine virus with a specific sequence marker that would enable distinguishing vaccinated swine from swine infected with naturally occurring field strains.
  • the tag may comprise a sequence that encoded a polypeptide, not present in swine, that is assayed by an immunologically-based assay such as an ELISA assay that utilized an antibody that reacted against the encoded polypeptide.
  • the tag may comprise a unique sequence not found in PRRSV that is identifiable by the polymerase chain reaction.
  • the narrow host tropism of PRRSV to PAMs indicates to one skilled in the art that specific host factors or receptors must be required for permissivity of PRRSV infection and replication.
  • cells of the monocyte/macrophage lineage of the present invention such genes can be temporally regulated and induced at the time of infection to express proteins that support PRRSV binding, cell penetration, and virus replication. Therefore cells of the present invention can be useful for providing active genes • in the production of hybridomas that would also support the growth of PRRSV.
  • Such hybridomas would consist of cells of the present invention fused by methods known to those skilled in the art to a stable porcine cell lines such as swine testes (ATCC CRL-1746) or pig kidney cells.
  • the present invention therefore includes the production of hybridomas comprising fusions of monocytes/macrophages with a stabile porcine cell line to create a new stable cell line for the propagation of PRRSV.
  • Example 7 An Example of diagnostic use 4 x 10 3 monocytes isolated as described can be used to determine the presence of PRRSV in blood or tissues of animals suspected of being infected with PRRSV either by virus isolation serological methods or PCR. The method can use microtiter plates with the cells in the wells to which the tissue is added. Summary
  • the present invention is useful for the production of inactivated vaccines that provide protection against specific PRRS isolates or strains. Wherein specific strains affect certain herds, the specific strains may be isolated and grown in by the method of the present invention to high titers. The resulting virus may then be inactivated with formalin or BEI, mixed with an appropriate adjuvant and intramuscularly inoculated into pigs providing immunity to that specific PRRSV strain or isolate.
  • the present invention is useful for the production of live PRRSV vaccines comprising an attenuated PRRSV strain or a PRRSV that has been genetically engineered to be- attenuated or a PRRSV genetically engineered to contain a marker gene or genetically engineered to delete a nonessential gene.
  • the present invention is useful for diagnostic purposes and the identification of PRRSV strains, especially PRRSV strains that may not grow in simian derived cell lines such as MA-104 or MARC-145.
  • the present invention also includes the production of hybridomas comprising fusions of monocytes/macrophages with a stabile porcine cell line to create a new stable cell line for the propagation of PRRSV. It is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the hereinafter appended claims.

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Abstract

L'invention concerne un procédé servant à produire des macrophages à partir de monocytes d'origine porcine afin de propager le virus du syndrome respiratoire et reproductif porcin (PRRSV). Ce procédé consiste à isoler un sous-ensemble spécifique de cellules sanguines du porc appelées monocytes et à les traiter avec des cytokines, ce qui les expose à l'infection par PRRSV et les rend sujettes à la réplication. Ce procédé est utile afin de produire PRRSV dans le but de le mettre en application dans des vaccins et des essais diagnostiques. De plus, ce procédé permet d'obtenir des cellules susceptibles d'être infectées par PRRSV, qui sont appropriées pour établir une ligne cellulaire continue servant à cultiver PRRSV, de manière à produire des vaccins.
PCT/US1998/002044 1997-02-07 1998-02-04 Procede servant a cultiver le virus du syndrome respiratoire et reproductif porcin afin de l'utiliser en tant que vaccin et dans des essais diagnostiques WO1998035023A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008089094A2 (fr) 2007-01-12 2008-07-24 The Board Of Trustees Of The University Of Illinois Cellules non simiennes permettant de développer le virus du syndrome dysgénésique et respiratoire du porc (prrs)
WO2009059411A1 (fr) * 2007-11-06 2009-05-14 Valorisation-Recherche, Limited Partnership Lignée de cellules épithéliales de poumon de porc et son utilisation dans la production et la détection du virus du syndrome reproducteur et respiratoire porcin
WO2012110490A1 (fr) * 2011-02-17 2012-08-23 Boehringer Ingelheim Vetmedica Gmbh Procédé de production de prrsv à l'échelon commercial
US8383131B2 (en) 2004-09-21 2013-02-26 Boehringer Ingelheim Vetmedica, Inc. Porcine reproductive and respiratory syndrome isolates and methods of use
US8546124B2 (en) 1996-10-30 2013-10-01 Boehringer Ingelheim Vetmedica Gmbh Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof
US8741309B2 (en) 1999-04-22 2014-06-03 The United States Of America As Represented By The Secretary Of Agriculture Porcine reproductive and respiratory syndrome vaccine based on isolate JA-142
US8765142B2 (en) 2011-02-17 2014-07-01 Boehringer Ingelheim Vetmedica Gmbh European PRRSV strain
US8790656B2 (en) 1996-10-30 2014-07-29 Boehringer Ingelheim Vetmedica Gmbh PRRSV vaccines
EP1742658B1 (fr) * 2004-04-23 2014-12-24 Zoetis P&U LLC Facteur de permissivite cellulaire pour virus, et son utilisation
US9080143B2 (en) 2005-06-24 2015-07-14 University Of Minnesota PRRS viruses, infectious clones, mutants thereof, and method of use
US9187731B2 (en) 2011-07-29 2015-11-17 Boehringer Ingelheim Vetmedica Gmbh PRRS virus inducing type I interferon in susceptible cells
US9315781B2 (en) 2011-07-29 2016-04-19 Boehringer Ingelheim Vetmedica Gmbh Infectious CDNA clone of european PRRS virus and uses thereof
US9561270B2 (en) 2009-09-02 2017-02-07 Boehringer Ingelheim Vetmedica, Inc. Methods of reducing virucidal activity in PCV-2 compositions and PCV-2 compositions with an improved immunogenicity
US9579373B2 (en) 2013-03-15 2017-02-28 Boehringer Ingelheim Vetmedica, Inc. Porcine reproductive and respiratory syndrome virus, compositions, vaccine and methods of use
CN118791578A (zh) * 2024-07-29 2024-10-18 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 猪繁殖与呼吸综合征病毒nsp12蛋白的抗原表位、单克隆抗体及应用

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US5510258A (en) * 1993-02-08 1996-04-23 Bayer Corporation Porcine reproductive and respiratory syndrome virus antigen and processes for the preparation and use of said antigen in vaccines and diagnostics

Patent Citations (1)

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US5510258A (en) * 1993-02-08 1996-04-23 Bayer Corporation Porcine reproductive and respiratory syndrome virus antigen and processes for the preparation and use of said antigen in vaccines and diagnostics

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8546124B2 (en) 1996-10-30 2013-10-01 Boehringer Ingelheim Vetmedica Gmbh Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof
US8790656B2 (en) 1996-10-30 2014-07-29 Boehringer Ingelheim Vetmedica Gmbh PRRSV vaccines
US8747859B2 (en) 1999-04-22 2014-06-10 The United States Of America, As Represented By The Secretary Of Agriculture Porcine reproductive and respiratory syndrome vaccine based on isolate JA-142
US8741309B2 (en) 1999-04-22 2014-06-03 The United States Of America As Represented By The Secretary Of Agriculture Porcine reproductive and respiratory syndrome vaccine based on isolate JA-142
NO341211B1 (no) * 2004-04-23 2017-09-11 Zoetis Services Llc In vitro fremgangsmåte for å fremme infeksjon i en virveldyrcelle med PRRSV og en fremgangsmåte for å måle tilbøyeligheten en testcellelinje har for å tillate PRRSV-infeksjon
EP2520310B1 (fr) * 2004-04-23 2016-03-30 Zoetis Services LLC Facteur de permissivite cellulaire pour virus, et son utilisation
EP1742658B1 (fr) * 2004-04-23 2014-12-24 Zoetis P&U LLC Facteur de permissivite cellulaire pour virus, et son utilisation
US8383131B2 (en) 2004-09-21 2013-02-26 Boehringer Ingelheim Vetmedica, Inc. Porcine reproductive and respiratory syndrome isolates and methods of use
US9080143B2 (en) 2005-06-24 2015-07-14 University Of Minnesota PRRS viruses, infectious clones, mutants thereof, and method of use
US8663984B2 (en) 2007-01-12 2014-03-04 The Board Of Trustees Of The University Of Illinois Non-simian cells for growth of porcine reproductive and respiratory syndrome (PRRS) virus
US8202717B2 (en) * 2007-01-12 2012-06-19 The Board Of Trustees Of The University Of Illinois Non-simian cells for growth of porcine reproductive and respiratory syndrome (PRRS) virus
WO2008089094A2 (fr) 2007-01-12 2008-07-24 The Board Of Trustees Of The University Of Illinois Cellules non simiennes permettant de développer le virus du syndrome dysgénésique et respiratoire du porc (prrs)
US20140162362A1 (en) * 2007-01-12 2014-06-12 The Board Of Trustees Of The University Of Illinois Non-Simian Cells for Growth of Porcine Reproductive and Respiratory Syndrome (PRRS) Virus
US9169465B2 (en) * 2007-01-12 2015-10-27 The Board Of Trustees Of The University Of Illinois Non-simian cells for growth of porcine reproductive and respiratory syndrome (PRRS) virus
WO2009059411A1 (fr) * 2007-11-06 2009-05-14 Valorisation-Recherche, Limited Partnership Lignée de cellules épithéliales de poumon de porc et son utilisation dans la production et la détection du virus du syndrome reproducteur et respiratoire porcin
US9561270B2 (en) 2009-09-02 2017-02-07 Boehringer Ingelheim Vetmedica, Inc. Methods of reducing virucidal activity in PCV-2 compositions and PCV-2 compositions with an improved immunogenicity
WO2012110490A1 (fr) * 2011-02-17 2012-08-23 Boehringer Ingelheim Vetmedica Gmbh Procédé de production de prrsv à l'échelon commercial
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