WO2024242174A1 - Single-round infectious rotavirus and use thereof - Google Patents

Abstract

The present invention provides a single-round infectious rotavirus characterized by having a mutation in at least one viral protein of the rotavirus, which is selected from the group consisting of VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3, and NSP4. The single-round infectious rotavirus according to the present invention can be used in a rotavirus vaccine, a rotavirus neutralization test method, and the like.

Description

Single infectious rotavirus and its uses

The present invention relates to a single-infectious rotavirus, a rotavirus vaccine containing the single-infectious rotavirus, and a rotavirus neutralization test method using the single-infectious rotavirus.

Rotavirus, which belongs to the Reoviridae family, is known as a virus that causes diarrhea in infants and young children. Infants between 6 months and 2 years of age are most susceptible to rotavirus infection and disease, with nearly 100% of people infected by the age of 5. A live attenuated rotavirus vaccine has been developed and is designated as a routine vaccination in Japan under the Vaccination Act. The preventive effect of the live attenuated rotavirus vaccine has been confirmed, but there have been reports of the virus spreading into the environment and the virus reverting to pathogenicity.

Single-transmissible viruses are not only useful for basic research such as elucidating the viral life cycle, but are also expected to be applied to the development of safer next-generation vaccines. The present inventors have developed a practical method for artificially synthesizing rotavirus (reverse genetics system) (Patent Document 1, Non-Patent Document 1), and this technology has made it possible to produce artificial recombinant rotaviruses with any mutation or artificial recombinant rotaviruses carrying foreign genes. However, there have been no reported cases of single-transmissible rotaviruses.

International Publication WO2018/062199

Kanai et al., Proc Natl Acad Sci U S A. 2017 Feb 28; 114(9):2349-2354

The objective of the present invention is to provide a single-infectious rotavirus. A further objective of the present invention is to provide a rotavirus vaccine containing a single-infectious rotavirus and a rotavirus neutralization test method using a single-infectious rotavirus.

In order to solve the above problems, the present invention includes the following inventions.
[1] A monoinfectious rotavirus characterized by having a mutation in at least one viral protein selected from the group consisting of rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3 and NSP4.
[2] The mono-infectious rotavirus described in [1] above, which has a mutation that induces dysfunction of a structural protein.
[3] A monoinfectious rotavirus described in [1] or [2] above, having a mutation in VP6.
[4] A monoinfectious rotavirus described in [3] above, having a mutation in domain H of VP6.
[5] The mono-infectious rotavirus described in [4] above, which has a mutation in the A'-A'' loop, the D'-D'' loop or the αA-I loop of domain H of VP6.
[6] A monoinfectious rotavirus described in [1] or [2] above, having a mutation in VP4.
[7] A mono-infectious rotavirus described in [6] above, having a mutation that deletes the lectin domain and/or the β barrel domain of VP4.
[8] A monoinfectious rotavirus described in [1] or [2] above, having a mutation in VP7.
[9] A mono-infectious rotavirus described in [8] above, having a mutation that deletes domain II of VP7.
[10] The mono-infectious rotavirus according to any one of [1] to [9], which is a reassortant.
[11] A mono-infectious rotavirus described in any of [1] to [9], which is a reassortant having a segmented RNA genome encoding structural proteins of a different genotype from that of the parent strain or structural proteins of a different strain from that of the parent strain.
[12] The mono-infectious rotavirus according to any one of [1] to [9], which is a reassortant having a segmented RNA genome encoding the structural proteins of human rotavirus.
[13] The mono-infectious rotavirus according to any one of [1] to [12] above, which expresses a foreign gene.
[14] The mono-infectious rotavirus described in [13], wherein the foreign gene is a gene encoding an antigenic protein of a virus or pathogenic microorganism other than rotavirus.
[15] The mono-infectious rotavirus described in [13] above, wherein the foreign gene is a reporter gene.
[16] The mono-infectious rotavirus described in [15] above, wherein the reporter gene is a gene encoding a fluorescent protein or a chemiluminescent protein.
[17] A vaccine comprising the monoinfectious rotavirus described in any one of [1] to [14].
[18] The vaccine described in [17] above, which is a rotavirus vaccine.
[19] A method for neutralizing a rotavirus, comprising using a monoinfectious rotavirus according to any one of [1] to [16] above.

The present invention can provide a single-infectious rotavirus. The present invention can also provide a rotavirus vaccine containing a single-infectious rotavirus, and a rotavirus neutralization test method using a single-infectious rotavirus.

FIG. 1 shows the domain structure of VP6 and the mutated region of the mutant VP6 used in Example 1. FIG. 1 shows the results of measuring the viral titers of 18 types of artificial recombinant rotaviruses in which mutations have been introduced into VP6. FIG. 1 shows the results of comparing the proliferation ability of nine types of artificial recombinant rotaviruses that showed high titers in Example 1 when they were infected into cells that persistently express VP6 and cells that do not express VP6. This figure shows the results of infecting the artificial recombinant rotavirus Δ169-174 + linker selected in Example 2 into VP6-sustained expressing cells and cells that do not express VP6, and comparing their proliferation ability; (A) shows the results for cells that do not express VP6, and (B) shows the results for cells that sustainably express VP6. FIG. 1 shows the results of infecting cells that do not express VP6 with the artificial recombinant rotavirus Δ169-174+linker and examining the proliferation ability of the virus after five repeated passages. FIG. 1 shows the results of infecting cells that do not express VP6 with the monoinfectious rotavirus Δ169-174+linker and a wild-type simian rotavirus, and observing the expression of NSP4 and VP6 by immunofluorescence antibody analysis. This figure shows the results of infecting cells persistently expressing VP6 with a green fluorescent protein-expressing single-infectious rotavirus, which was prepared by inserting a gene encoding green fluorescent protein (ZsG) into the segmented RNA genome encoding NSP1 in the single-infectious rotavirus Δ169-174 + linker, and with a single-infectious rotavirus Δ169-174 + linker that does not express green fluorescent protein, and observing the expression of ZsG and NSP4. This figure shows the results of infecting VP6 persistently expressing cells with a luciferase-expressing single-infectious rotavirus prepared by inserting a gene encoding luciferase (NLuc) into the segmented RNA genome encoding NSP1 in the single-infectious rotavirus Δ169-174 + linker, and with a single-infectious rotavirus Δ169-174 + linker that does not express luciferase, and measuring luciferase activity over time. This figure shows the results of neutralization tests of luciferase-expressing, single-infectious rotavirus and luciferase-expressing, self-replicating rotavirus without a mutation in VP6, using mouse serum immunized with wild-type simian rotavirus SA11 strain. FIG. 1 shows the results of producing five types of single-infectious rotaviruses Δ169-174+linker having a segmented RNA genome encoding human rotavirus VP7 and measuring the viral titer. FIG. 1 shows the results of a neutralization test of single-infectious rotaviruses having segmented RNA genomes encoding four types of human rotavirus VP7, using mouse serum immunized with wild-type simian rotavirus SA11 strain. [0046] FIG. 1 shows the results of examining the vaccine effect by orally administering single-infectious rotavirus Δ169-174+linker to mice three times, and subjecting the blood samples collected two weeks after each administration to a neutralization test to obtain sera. FIG. 1 shows the structure of rotavirus (A) and the structure of VP6 (B).

This figure shows the results of orally administering a single infectious rotavirus Δ169-174 + linker or wild-type rotavirus to mice, recovering the intestines two days later, and examining the viral copy number and the presence or absence of infectious virus. (A) shows the results of measuring the viral copy number in the cecum, and (B) shows the results of infecting VP6-expressing MA104 cells with a cecal homogenate and measuring the viral titer. FIG. 1A is a diagram showing the structure of rotavirus VP7, and FIG. 1B is a diagram showing the mutated region of the mutant VP7 used in Example 12. This figure shows the results of comparing the proliferation ability of cells that persistently express VP7 and cells that do not express VP7 infected with the artificial recombinant rotavirus VP7-ΔDomain II produced in Example 12, where (A) shows the result for cells that do not express VP7, and (B) shows the result for cells that persistently express VP7. This figure shows the results of infecting cells that do not express VP7 with artificial recombinant rotavirus VP7-ΔDomain II and examining the proliferation ability of the virus after five repeated passages. FIG. 1A is a diagram showing the structure of rotavirus VP4, and FIG. 1B is a diagram showing the mutated region of the mutant VP4 used in Example 15. This figure shows the results of infecting the artificial recombinant rotavirus VP4-120bp prepared in Example 15 into VP4-sustained expressing cells and cells that do not express VP4, and comparing their proliferation ability; (A) shows the results for cells that do not express VP4, and (B) shows the results for cells that sustainably express VP4. FIG. 1 shows the results of infecting cells that do not express VP4 with an artificial recombinant rotavirus VP4-120bp, and examining the proliferation ability of the virus after five passages. This figure shows the results of infecting cells that do not express VP4 with a green fluorescent protein-expressing single-infectious rotavirus, which was created by inserting a gene encoding green fluorescent protein (ZsG) into the segmented RNA genome of a single-infectious rotavirus VP4-120bp in which most of the VP4 gene had been deleted, and a single-infectious rotavirus (VP4-120bp) that does not express green fluorescent protein, and observing the expression of ZsG and NSP4. This figure shows the results of infecting cells that do not express VP4 with a luciferase-expressing single-infectious rotavirus prepared by inserting a gene encoding luciferase (NLuc) into the segmented RNA genome of the single-infectious rotavirus VP4-120bp in which most of the VP4 gene has been deleted, and a single-infectious rotavirus (VP4-120bp) that does not express luciferase, and measuring luciferase activity over time. This figure shows the results of infecting cells that do not express VP4 with an RBD-expressing single-infectious rotavirus (VP4-120bp-RBD) created by inserting a gene encoding the receptor binding domain (RBD) of the spike protein of the novel coronavirus (SARS-CoV-2) into the segmented RNA genome of the single-infectious rotavirus VP4-120bp in which most of the VP4 gene has been deleted, and a single-infectious rotavirus that does not express RBD (VP4-120bp), and observing the expression of RBD and NSP3 by Western blotting.

The present invention provides a mono-infectious rotavirus. In this specification, "mono-infectious rotavirus" means a rotavirus that is unable to form infectious virus particles after a single infection, or whose production amount of infectious virus particles is suppressed after a single infection. In addition, "mono-infectious rotavirus" means a rotavirus whose viral titer decreases by subculture. When a mono-infectious rotavirus infects a cell expressing a normal viral protein corresponding to a viral protein into which a mutation has been introduced, it can form infectious virus particles and grow, but it cannot grow when it infects normal cells, so it is safe and does not spread in the environment.

Rotavirus is a non-enveloped virus belonging to the Reoviridae family. The virus particle has a triple structure consisting of an outer coat, an inner coat, and a core protein, and contains a double-stranded RNA genome consisting of 11 segments (see Figure 13 (A)). These 11 gene segments code for six structural proteins (VPs) and six non-structural proteins (NSPs) (Table 1).

The single-infectious rotavirus of the present invention may have a mutation in at least one viral protein selected from the group consisting of rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3, and NSP4. As shown in Table 1, VP1, VP2, VP3, VP4, VP6, and VP7 are structural proteins of rotavirus, and NSP2, NSP3, and NSP4 are non-structural proteins. The single-infectious rotavirus of the present invention may have a mutation in a structural protein of rotavirus, or may have a mutation that induces dysfunction of a structural protein.

The number of viral proteins having a mutation may be two or more, or may be one, but is preferably one. The structural protein having a mutation may be VP6 (inner coat protein). If the structural protein having a mutation is VP6, it is preferable that the mutation is in a region that interacts with VP7 (outer coat protein). The region that interacts with VP7 may be domain H of VP6. Domain H contains three loop structures (A'-A'' loop, D'-D'' loop, and αA-I loop), and any one of these loops may have a mutation.

The mutated structural protein may be VP4 (spike protein). When the mutated structural protein is VP4, the mutated VP4 may be a deletion of the lectin domain, a deletion of the β-barrel domain, or a deletion of both the lectin domain and the β-barrel domain. The larger the deleted region, the less likely reversion mutation will occur and the easier it will be to introduce a foreign gene, so it is preferable that the mutated VP4 be a deletion of both the lectin domain and the β-barrel domain. A mutant VP4 having deletions of both the lectin domain and the β-barrel domain of VP4 may be a mutant VP4 in which at least 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp or more remain on the N-terminus and at least 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp or more remain on the C-terminus in the base sequence of the segmented genome encoding VP4. The number of bases remaining on the N-terminus and C-terminus is not particularly limited, and the number of bases on both sides does not need to be the same.

The structural protein having a mutation may be VP7 (coat protein). When the structural protein having a mutation is VP7, it may be a deletion of domain II. A mutant VP7 having a deletion of domain II of VP7 may be a mutant VP7 having at least 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, 250 bp or more remaining on the N-terminus and at least 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, 250 bp or more remaining on the C-terminus. The number of bases remaining on the N-terminus and C-terminus is not particularly limited, and the number of bases on both sides does not need to be the same.

The type of mutation is not particularly limited, and examples include amino acid deletion and amino acid substitution. The number of amino acids to be deleted or substituted is not particularly limited, and may be 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. The amino acid substitution may be an alanine substitution, or may be any amino acid linker. Mutations can be introduced into structural genes using known gene recombination techniques.

The monoinfectious rotavirus of the present invention can be produced using a known method for producing an artificial recombinant rotavirus. For example, the method described in Patent Document 1 (WO2018/062199) or Non-Patent Document 1 (Kanai et al., Proc Natl Acad Sci USA. 2017 Feb 28; 114(9):2349-2354), or a method partially modified from these methods, can be suitably used. It is preferable to introduce a normal protein expression vector corresponding to the mutated viral protein into the host cell. A capping enzyme expression vector, a FAST (fusion-associated small transmembrane) protein expression vector, a rotavirus NSP2 expression vector, a rotavirus NSP5 expression vector, or the like may be appropriately selected and introduced into the host cell. For example, the method for producing an artificial recombinant rotavirus described in Example 1 of the present application may be used.

As the capping enzyme, for example, the capping enzyme of vaccinia virus can be used. As the FAST protein, p10 of Nelson Bay reovirus, p10 of avian reovirus, etc. can be used. As the host cell, a cell that is highly sensitive to rotavirus and has a high gene transfer efficiency can be preferably used. Examples include BHK cells, MA104 cells, COS7 cells, CV1 cells, Vero cells, L929 cells, 293T cells, A549 cells, etc.

　Whether the artificial recombinant rotavirus produced is a monoinfectious rotavirus can be determined by infecting the produced artificial recombinant rotavirus into cells expressing a normal structural protein corresponding to the structural protein into which a mutation has been introduced and cells not expressing said structural protein, and confirming that the virus grows in the former cells and does not grow in the latter cells.

The monoinfectious rotavirus of the present invention may be a reassortant. A rotavirus reassortant is a rotavirus having a new genotype configuration resulting from recombination of segmented RNA genomes between different species or strains of rotavirus. Reassortants are useful for functional analysis of segmented genomes and are extremely useful as artificial recombinant rotaviruses as vaccine candidates. The segmented RNA genome to be recombined may be a segmented RNA genome encoding a structural protein of a genotype different from that of the parent strain. There are many genotypes in each of the 11 gene segments of rotavirus, and as an example, 42 types of G type (VP7) and 58 types of P type (VP4) have been reported. These genotypes will continue to increase in the future. The segmented RNA genome to be recombined may be a segmented RNA genome encoding a structural protein of a strain different from that of the parent strain. The genotype of the structural protein of the strain different from that of the parent strain may be the same as or different from that of the parent strain. Furthermore, the segmented RNA genome to be recombined may be a segmented RNA genome that encodes a structural protein of a human or non-human mammalian or avian rotavirus, or may be a segmented RNA genome that encodes a structural protein of a genotype different from that of the parent strain of a human or non-human mammalian or avian rotavirus.

The structural proteins of the heterologous or heterologous strain of rotavirus used to prepare the reassortants may be structural proteins other than the structural proteins having a mutation in the parent strain. For example, in the case of a single-infectious rotavirus having a mutation in VP6 (inner capsid protein), it is preferable to use a reassortant having a segmented RNA genome encoding VP7 (outer capsid protein) and/or VP4 (spike protein) of the heterologous or heterologous strain of rotavirus. In the case of a single-infectious rotavirus having a mutation in VP4, it is preferable to use a reassortant having a segmented RNA genome encoding VP7 and/or VP6 of the heterologous or heterologous strain of rotavirus. In the case of a single-infectious rotavirus having a mutation in VP7, it is preferable to use a reassortant having a segmented RNA genome encoding VP4 and/or VP6 of the heterologous or heterologous strain of rotavirus. The heterologous rotavirus is preferably a human rotavirus. By recombining with a segmented RNA genome that codes for the structural proteins of human rotavirus, it is possible to produce a single-infectious rotavirus having the antigenicity of various genotypes (serotypes) of human rotavirus. Such single-infectious rotavirus having the antigenicity of various genotypes (serotypes) of human rotavirus is very useful as a vaccine antigen for rotavirus vaccines. It is also very useful as an antigen for neutralization tests.

The single-infectious rotavirus of the present invention may be a single-infectious rotavirus expressing a foreign gene. The single-infectious rotavirus expressing a foreign gene can be produced by introducing a foreign gene into at least one of the segmented RNA genomes so that the foreign gene can be expressed. The foreign gene is not particularly limited, and a single-infectious rotavirus expressing any foreign gene can be produced. For example, the single-infectious rotavirus produced can be used as a vaccine by expressing a foreign gene encoding an antigen protein of a virus or pathogenic microorganism other than rotavirus. Examples of viruses and pathogenic microorganisms other than rotavirus include norovirus, adenovirus, hepatitis A virus, sapovirus, hand, foot and mouth disease virus, enterovirus, HIV, coronavirus, novel coronavirus (SARS-CoV-2), Salmonella, Campylobacter, Vibrio parahaemolyticus, Escherichia coli O157, Vibrio cholerae, Salmonella typhi, Shigella, etc. These vaccine antigens may be epitope peptides. A vaccine may be constructed by combining multiple artificial recombinant rotaviruses that express different foreign vaccine antigens.

The foreign gene may be an antigenic protein (e.g., VP4, VP7, etc.) of a heterologous or heterologous rotavirus. By expressing one or more antigenic proteins of a heterologous or heterologous rotavirus, a monoinfectious rotavirus that can be used as a polyvalent rotavirus vaccine can be provided. The antigenic protein may be an epitope peptide.

The foreign gene may be a reporter gene. There is no particular limitation on the reporter gene, so long as it is a commonly used one. Examples include genes encoding fluorescent proteins, chemiluminescent proteins, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, alkaline phosphatase, peroxidase, etc. The reporter gene may be a gene encoding a fluorescent protein or a chemiluminescent protein.

Fluorescent proteins include, but are not limited to, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow-green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, etc., and any known fluorescent protein can be suitably used. Chemiluminescent proteins include, but are not limited to, luciferase, aequorin, obelin, etc., and any known chemiluminescent protein can be suitably used.

　Single-infectious rotaviruses that express a reporter gene allow easy visualization of the amount of virus, and can be used for rotavirus neutralization tests and screening of anti-rotavirus drugs, which can be performed more safely and simply than using self-replicating rotaviruses.

〔vaccine〕
The single-infectious rotavirus of the present invention can be used as a vaccine. That is, the present invention provides a vaccine using the single-infectious rotavirus of the present invention. As described above, the single-infectious rotavirus of the present invention, the single-infectious rotavirus having a segmented RNA genome encoding a structural protein of human rotavirus (reassortant), and the single-infectious rotavirus expressing a foreign gene encoding an antigen protein of a heterologous or heterologous rotavirus strain can be suitably used as a rotavirus vaccine. In addition, a polyvalent rotavirus vaccine can be constructed by mixing multiple single-infectious rotaviruses expressing antigen proteins of rotaviruses of different genotypes (serotypes).

As mentioned above, single-infectious rotaviruses that express foreign genes encoding antigenic proteins of viruses and pathogenic microorganisms other than rotaviruses can be suitably used as vaccines against various viruses and pathogenic microorganisms. A vaccine may be constructed by combining multiple single-infectious rotaviruses that express different foreign antigenic proteins.

[Neutralization test method]
The single-infectious rotavirus of the present invention can be used in a rotavirus neutralization test. That is, the present invention provides a method for a rotavirus neutralization test using the single-infectious rotavirus of the present invention. The rotavirus neutralization test can be carried out by mixing serially diluted test serum and the single-infectious rotavirus of the present invention to carry out an antigen-antibody reaction, inoculating the mixture into culture cells on a microplate and culturing the mixture, and then measuring the number of virus-infected cells.

The neutralization test method of the present invention preferably uses a single-infectious rotavirus that expresses a reporter gene. By using a single-infectious rotavirus that expresses a reporter gene, the number of virus-infected cells can be measured based on the expression level of the reporter protein, making it possible to safely carry out a simple neutralization test with little variation.

The present invention will be explained in detail below with reference to examples, but the present invention is not limited to these.

Example 1: Creation of a single-infectious rotavirus by VP6 mutation introduction
Materials and Methods
(1) Virus The simian rotavirus SA11 strain (hereinafter referred to as "SA11 strain") was used (see Table 1).

(2) Plasmids containing each segmented RNA genome expression cassette of the SA11 strain (segmented RNA genome expression vectors)
According to the method described in WO2018/062199 (Patent Document 1), a plasmid containing cDNA of 11 segmented RNA genomes of SA11 strain was prepared. RT-PCR was performed using double-stranded RNA extracted from the virus as a template and specific primers based on the base sequence of each segmented RNA genome. The obtained RT-PCR product (cDNA of each segmented RNA genome) was inserted between the T7 promoter sequence and the Hepatitis D virus (HDV) ribozyme sequence of the p3E5 plasmid to obtain a plasmid containing an expression cassette of each segmented RNA genome. The expression cassette of each segmented RNA genome has a structure in which the T7 promoter sequence is adjacent to the cDNA of each segmented RNA genome on the 5' side and the HDV ribozyme sequence is adjacent to the 3' side, and the T7 terminator sequence is located downstream of the T7 promoter sequence. The constructed plasmids (segmented RNA genome expression vectors) are designated pT7-VP1SA11, pT7-VP2SA11, pT7-VP3SA11, pT7-VP4SA11, pT7-VP6SA11, pT7-VP7SA11, pT7-NSP1SA11, pT7-NSP2SA11, pT7-NSP3SA11, pT7-NSP4SA11 and pT7-NSP5SA11, respectively.

(3) Capping enzyme expression vector As the capping enzyme expression vector, as described in Kanai et al. (Proc Natl Acad Sci USA 2017 Feb 28; 114(9): 2349-2354), the protein coding region DNA of the vaccinia virus D1R gene (GenBank ACCESSION No.: NC006998 positions 93948 to 96482) and the protein coding region DNA of the vaccinia virus D12L gene (GenBank ACCESSION No.: NC006998 positions 107332 to 108195) were inserted into the Bg1II cleavage site of the pCAGGS plasmid to prepare pCAG-D1R (vaccinia virus mRNA capping enzyme large subunit expression vector) and pCAG-D12L (vaccinia virus mRNA capping enzyme small subunit expression vector).

(4) Rotavirus VP6 expression vector The VP6 expression vector was prepared by inserting the protein coding region DNA of the VP6 gene (SEQ ID NO: 6) of SA11 strain into the BamHI and EcoRI cleavage sites of the pcDNA3.1(+) plasmid to prepare pcDNA3-VP6. The coding region DNA was synthesized by an artificial gene synthesis service (GenScript).

(5) Rotavirus NSP2 and NSP5 Expression Vectors The NSP2 and NSP5 expression vectors were prepared by inserting the protein coding region DNA of the SA11 strain NSP2 gene (SEQ ID NO: 8) and the protein coding region DNA of the NSP5 gene (SEQ ID NO: 11) into the EcoRI cleavage site of the pCAGGS plasmid, respectively, to prepare pCAG-NSP2 and pCAG-NSP5. Each coding region DNA was synthesized by an artificial gene synthesis service (Eurofins Genomics). Each synthetic DNA was inserted into the EcoRI cleavage site of the pCAGGS plasmid, respectively, to prepare pCAG-NSP2 and pCAG-NSP5.

(6) VP6 Mutation-Introduced Plasmids Eighteen types of plasmids were constructed by introducing various mutations into the cDNA of the RNA genome of rotavirus VP6 contained in pT7-VP6SA11 using known gene recombination techniques. The domain structure of rotavirus VP6 and the various mutated regions are shown in Figure 1. Specific details of the mutations are shown in Table 2.

(7) T7 RNA polymerase persistent expression cells BHK-T7/P5 cells persistently expressing T7 RNA polymerase were used. BHK-T7/P5 cells were prepared by transfecting BHK cells (Baby Hamster Kidney Cells) with pCAGGS plasmid, in which DNA encoding T7 RNA polymerase was inserted downstream of the CAG promoter, and culturing and selecting in an antibiotic-containing medium.

(8) VP6 persistent expression cells The rotavirus VP6 gene was introduced into the pLVSIN-CMV-Neo vector (Takara Bio) and co-transfected with psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into 293T cells to produce lentivirus. The lentivirus was then infected into African green monkey MA104 cells (ATCC CRL-2378.1), and drug selection was performed with G418 (800 μg/mL) two days later. The cells expressing VP6 most strongly were cloned from the VP6-expressing MA104 cells obtained by drug selection using limiting dilution. Limiting dilution was performed four times to obtain MA104 cells that persistently express VP6.

(9) Preparation of artificial recombinant viruses On the day before transfection, BHK-T7/P5 cells were seeded in a 12-well culture plate at 5 × ¹⁰⁴ cells/well. The next day, 10 types of segmented RNA genome expression vectors (pT7-VP1SA11, pT7-VP2SA11, pT7-VP3SA11, pT7-VP4SA11, pT7-VP7SA11, pT7-NSP1SA11, pT7-NSP2SA11, pT7-NSP3SA11, pT7-NSP4SA11, and pT7-NSP5SA11), mutated pT7-VP6SA11, capping enzyme expression vector (pCAGD1R and A total of 16 types of plasmids, including pCAG-D12L, pCAG-NSP2 expression vector (pCAG-NSP2SA11), pCAG-NSP5 expression vector (pCAG-NSP5SA11), and pCAG-VP6 expression vector (pcDNA3-VP6), were introduced into BHK-T7/P5 cells at 0.125 μg each (total 2 μg) using a transfection reagent (TransIT-LT1 (trade name), Mirus). 2 μL of transfection reagent was used per μg of DNA. BHK-T7/P5 cells were cultured in DMEM medium containing 5% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% _CO2 . 24 hours after transfection, the medium was replaced with FBS-free, trypsin-containing medium, and VP6-sustained MA104 cells were added at 2× ¹⁰⁵ cells/well. After co-culture for 5 days, the medium and cells were harvested. The harvested medium and cells were frozen and thawed three times to prepare a cell lysate. 10% of the obtained cell lysate was added to freshly prepared VP6-sustained MA104 cells (2× ¹⁰⁵ cells/well, 12-well culture plate), and the cells were further cultured for 5 days. The medium and cells were frozen and thawed three times to prepare a cell lysate. The collected medium and cells were frozen and thawed three times to prepare a cell lysate. The collected medium and cells were added to VP6-sustained MA104 cells (1× ¹⁰⁴ cells/well, 96-well culture plate), and the cells were fixed with formalin 18 hours later. After membrane permeabilization with 0.5% Triton-X, the infected cells were visualized by immunofluorescence antibody technique. The primary antibody used in the immunofluorescence antibody technique was rabbit anti-rotavirus NSP4 antibody (J Virol. 2023 Jan 31;97(1):e0186122.), and the secondary antibody was CF488-labeled anti-rabbit IgG antibody (Biotum). If infected cells were confirmed, it was determined that the recombinant virus had been produced.

(10) Measurement of virus titer MA104 cells expressing VP6 were seeded at 1 x ¹⁰⁴ cells/well in a 96-well culture plate and cultured overnight. 15 μL of cell lysate was added to 135 μL of saline and stirred to prepare a 10-fold dilution. This procedure was repeated to prepare serially diluted virus samples. 50 μL/well of serially diluted virus samples were added to MA104 cells expressing VP6, and the cells were fixed with formalin after 18 hours. Membrane permeabilization was performed with 0.5% Triton-X, and infected cells were visualized by immunofluorescence antibody method. The primary antibody for the immunofluorescence antibody method was the same rabbit-derived anti-rotavirus NSP4 antibody as in (9) above, and the secondary antibody was a CF488-labeled anti-rabbit IgG antibody. The number of infected cells was counted to calculate the virus titer.

<Results>
The results are shown in Figure 2. We were able to create artificial recombinant viruses using 18 types of VP6 mutation-introduced plasmids that introduced mutations into VP6. Among the three types of loop structures of rotavirus VP6 that were introduced with partial deletion mutations, alanine substitution mutations, or substitution mutations with linker sequences, some viruses had higher viral titers than others.

Example 2: Examination of the proliferation ability of VP6 mutant rotaviruses
The nine types of VP6 mutant rotaviruses that showed high titers in Example 1 were compared in terms of their proliferation ability in MA104 cells that persistently express VP6 and in MA104 cells that do not express VP6 (hereinafter referred to as "normal MA104 cells").
Materials and Methods
VP6-sustained MA104 cells and normal MA104 cells were seeded in a 24-well culture plate at 7.5 x ¹⁰⁴ cells/well and cultured overnight. The nine types of viruses selected in Example 1 were infected at an MOI of 0.01, and 24 hours after infection, the medium and cells were frozen and thawed three times to prepare cell lysates, and the virus titer was measured. A wild-type virus without VP6 mutation was used as a control. The virus titer was measured in the same manner as in Example 1.

<Results>
The results are shown in Figure 3. All nine types of viruses used showed higher virus titers in MA104 cells with persistent VP6 expression than in normal MA104 cells. Among them, the virus (Δ169-174 + linker) that showed low proliferation in normal MA104 cells but efficiently propagated in MA104 cells with persistent VP6 expression was used in subsequent experiments.

Example 3: Comparison of proliferation ability between normal MA104 cells and MA104 cells that continuously express VP6
The proliferation ability of the VP6 mutant rotavirus Δ169-174+linker selected in Example 2 was compared between normal MA104 cells and MA104 cells that persistently express VP6.
Materials and Methods
Normal MA104 cells and MA104 cells expressing VP6 were seeded in a 24-well culture plate at 7.5 x ¹⁰⁴ cells/well and cultured overnight. Δ169-174 + linker was infected at an MOI of 0.01, and the medium and cells were frozen and thawed three times at 0, 24, 48, and 72 hours after infection to prepare cell lysates and measure the virus titer. A wild-type virus without VP6 mutation was used as a control. The virus titer was measured in the same manner as in Example 1.

<Results>
The results are shown in Figure 4. (A) shows the results for normal MA104 cells, and (B) shows the results for MA104 cells with sustained VP6 expression. Δ169-174 + linker hardly grew in normal MA104 cells, but grew efficiently in MA104-VP6 cells.

Example 4: Examination of proliferation ability in normal MA104 cells
To further confirm that the VP6 mutant rotavirus Δ169-174 + linker was unable to grow in normal MA104 cells, it was subcultured in normal MA104 cells.
Materials and Methods
Normal MA104 cells were seeded at 3 x ¹⁰⁵ cells/well in a 6-well culture plate and cultured overnight. Δ169-174 + linker was infected at MOI 1.0, and after one week of culture, the medium and cells were collected and frozen and thawed to prepare a cell lysate. 10% of the obtained cell lysate was added to new normal MA104 cells and cultured for one week. The virus was passaged five times, and the virus titer was measured at each passage using the same method as in Example 1.

<Results>
The results are shown in Figure 5. The titer of Δ169-174+linker decreased with passage, and the virus was not detected after the third passage. From this result, it was determined that Δ169-174+linker is a single-pass infectious rotavirus that cannot grow in normal cells that do not express VP6.

Example 5: Examination of viral protein expression in normal cells
In order to use a single-infectious virus as a vaccine, it is necessary to express viral proteins, even though infectious viral particles are not formed in normal cells that do not express VP6. Therefore, we infected normal MA104 cells with Δ169-174+linker, which was determined to be a single-infectious rotavirus in Example 3, and observed the expression of viral proteins.

Materials and Methods
Normal MA104 cells were seeded at 7.5 × ¹⁰⁴ cells/well in a 24-well culture plate containing a coverslip and cultured overnight. Δ169-174 + linker and wild-type simian rotavirus (SA11 strain) were infected at an MOI of 1.0, and the cells were fixed with formalin 8 hours later. The cells were permeabilized with 0.5% Triton-X and immunostained with rabbit anti-NSP4 and mouse anti-VP6 antibodies as primary antibodies, and CF488-labeled anti-rabbit IgG antibodies (Biotum) and CF594-labeled anti-mouse IgG antibodies (Biotum) as secondary antibodies. Nuclei were stained with Hoechst and observed under a fluorescent microscope.

<Results>
The results are shown in Figure 6. When Δ169-174+linker was infected into normal MA104 cells, viral protein expression was confirmed. This result indicates that Δ169-174+linker is promising for use as a vaccine.

Example 6: Construction of a single-infectious rotavirus containing a reporter gene
6-1 Single-infectious rotavirus expressing green fluorescent protein <Materials and methods>
(1) Green fluorescent protein gene-inserted NSP1 expression plasmid The ZsGreen (hereinafter referred to as "ZsG") gene was used as the green fluorescent protein gene. The ZsG gene-inserted NSP1 gene expression plasmid was pT7-NSP1-ZsG-Δ722 described in Kanai et al. (J Virol. 2019 Feb 15; 93(4): e01774-18.). pT7-NSP1-ZsG-Δ722 is a plasmid in which the ZsG gene is inserted between positions 111 and 112 of the NSP1 gene of pT7-NSP1SA11, and 722 nt (positions 134 to 855) are deleted from the NSP1 gene.

(2) Preparation of single-infectious rotavirus expressing green fluorescent protein Among the 11 types of segmented RNA genome expression vectors prepared in Example 1, pT7-VP6SA11/Δ169-174+linker was used instead of pT7-VP6SA11, and pT7-NSP1-ZsG-Δ722 was used instead of pT7-NSP1SA11. In addition to these, capping enzyme expression vectors (pCAGD1R and pCAG-D12L), rotavirus NSP2 expression vector (pCAG-NSP2SA11), rotavirus NSP5 expression vector (pCAG-NSP5SA11), and rotavirus VP6 expression vector (pcDNA3-VP6) were also introduced into BHK-T7/P5 cells to prepare single-infectious rotavirus expressing green fluorescent protein in the same manner as in “Materials and Methods” (9) of Example 1. As a control, pT7-NSP1SA11 lacking ZsG was used instead of pT7-NSP1-ZsG-Δ722 to generate a single-infectious rotavirus that does not express green fluorescent protein.

(3) Confirmation of ZsG expression The two types of viruses prepared were infected into MA104 cells that continuously express VP6 at an MOI of 1.0 and cultured for 8 hours. To confirm that the cells that emitted green fluorescence were virus-infected cells, the expression of virus-derived NSP4 was confirmed. That is, after 8 hours of culture, the cells were fixed with formalin. The membrane was permeabilized with 0.5% Triton-X, and stained with rabbit-derived anti-NSP4 antibody as the primary antibody and CF594-labeled anti-rabbit IgG antibody (Biotum) as the secondary antibody. Nuclei were stained with Hoechst and observed under a fluorescent microscope.

<Results>
The results are shown in Figure 7. It was confirmed that cells infected with the ZsG-introduced, single-infectious rotavirus (Δ169-174 + linker) emitted green fluorescence.

6-2 Luciferase-expressing single-infectious rotavirus <Materials and methods>
(1) Luciferase gene-inserted NSP1 expression plasmid As the luciferase gene, the NanoLuc (hereinafter referred to as "NLuc") gene, which is a luciferase gene derived from Oplophorus gracilirostris, was used. As the NLuc gene-inserted NSP1 gene expression plasmid, pT7-NSP1-NLuc-Δ722 described in Kanai et al. (J Virol. 2019 Feb 15; 93(4): e01774-18.) was used. pT7-NSP1-NLuc-Δ722 is a plasmid in which the NLuc gene is inserted between positions 111 and 112 of the NSP1 gene of pT7-NSP1SA11, and 722 nt (positions 134 to 855) are deleted from the NSP1 gene.

(2) Preparation of luciferase-expressing, single-infectious rotavirus Among the 11 types of segmented RNA genome expression vectors prepared in Example 1, pT7-VP6SA11/Δ169-174+linker was used instead of pT7-VP6SA11, and pT7-NSP1-NLuc-Δ722 was used instead of pT7-NSP1SA11. In addition to these, capping enzyme expression vectors (pCAGD1R and pCAG-D12L), rotavirus NSP2 and NSP5 expression vectors (pCAG-NSP2SA11 and pCAG-NSP5SA11), and rotavirus VP6 expression vector (pcDNA3-VP6) were also introduced into BHK-T7/P5 cells to prepare NLuc-expressing, single-infectious rotaviruses in the same manner as in “Materials and Methods” (9) of Example 1. As a control, pT7-NSP1SA11, which does not contain NLuc, was used instead of pT7-NSP1-NLuc-Δ722.

(3) Luciferase activity measurement MA104 cells expressing VP6 were seeded in a 96-well culture plate at 1 × ¹⁰⁴ cells/well and cultured overnight. The cells were infected with the NLuc-expressing single-infective rotavirus prepared in (2) at an MOI of 0.01, and the medium and cells were collected over time and frozen and thawed to prepare cell lysates. The luciferase activity of the cell lysates was measured using the Nano-Glo Luciferase Assay System (trade name, Promega).

<Results>
The results are shown in Figure 8. It was shown that the luciferase activity of the NLuc-expressing, single-infectious rotavirus increased over time after infection.

Example 7: Neutralization test using luciferase-expressing single-infectious rotavirus
Materials and Methods
(1) Luciferase-expressing, single-infectious rotavirus The NLuc-expressing, single-infectious rotavirus prepared in Example 6-2 was used. As a control, a NLuc-expressing, self-replicating rotavirus was used, which was prepared by replacing pT7-VP6SA11/Δ169-174+linker with pT7-VP6SA11, in which no mutation was introduced into VP6, in the NLuc-expressing, single-infectious rotavirus prepared in Example 6-2. The viral titers of both viruses were measured, and viruses with a titer of 100 FFU were prepared for each.

(2) Serum Anti-rSA11, as described by Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.), was used. Anti-rSA11 was obtained by orally administering wild-type SA11 strain at 1.0 × 10 ⁶ FFU/mouse to BALB/c mice (male, 3 weeks old) and repeating the administration 7 times at 2-week intervals, followed by blood sampling.

(3) Neutralization test method MA104 cells expressing VP6 were seeded at 1 x ¹⁰⁴ cells/well in a 96-well culture plate and cultured overnight. The serum from (2) was serially diluted and mixed with 100 FFU of the virus prepared in (1) and incubated at 37°C for 1 hour, after which each virus-serum mixture was added to the cells and cultured for 24 hours. The medium and cells were collected and frozen and thawed, and the luciferase activity of the prepared cell lysate was measured using the Nano-Glo Luciferase Assay System. The reduction rate of luciferase activity compared to the luciferase activity of the control without serum was calculated as the neutralization rate.

<Results>
The results are shown in Figure 9. The results of the neutralization test of the luciferase-expressing, singly infectious rotavirus were equivalent to those of the luciferase-expressing, self-replicating rotavirus.
In conventional neutralization tests, the number of virus-infected cells is generally measured visually under a microscope, which makes the operation complicated, time-consuming, and subjective to the large variation in the results depending on the person performing the measurement. In addition, since an infectious virus is used, a P2 level facility is required. From the results of this example, it has been shown that a simple neutralization test with little variation can be safely performed by using a single-infectious rotavirus expressing a reporter gene in the neutralization test.

Example 8: Preparation of a single-infectious rotavirus having a human rotavirus coat protein
Although the SA11 strain is a simian rotavirus, when a single-infectious rotavirus is actually used as a vaccine or an antigen for neutralization tests, it is desirable to have the human rotavirus envelope protein. Therefore, Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.) attempted to create a single-infectious rotavirus carrying a segmented RNA genome encoding VP7 of human rotavirus isolated from the stool of nine children with acute gastroenteritis in Japan. VP7 is an envelope protein located in the outermost layer of the virus particle and is a protein that determines the antigenicity and immunogenicity of the virus (see Figure 13 (A)).

Materials and Methods
Among the 11 types of segmented RNA genome expression vectors prepared in Example 1, pT7-VP6Δ169-174+linker encoding a mutated VP6 was used instead of pT7-VP6SA11, and pT7-hVP7, a segmented RNA genome expression vector encoding VP7 of human rotavirus, was used instead of pT7-VP7SA11. In addition to these, capping enzyme expression vectors (pCAGD1R and pCAG-D12L), rotavirus NSP2, NSP5 expression vectors (pCAG-NSP2SA11 and pCAG-NSP5SA11), and rotavirus VP6 expression vector (pcDNA3-VP6) were also introduced into BHK-T7/P5 cells to prepare single-infectious rotaviruses having VP7 of human rotavirus in the same manner as in "Materials and Methods" (9) of Example 1. Five types of human rotaviruses with VP7 genotypes (serotypes) G1, G2, G3, G8, and G9 were used, and segmented RNA genomes encoding VP7 obtained from each of these five types of human rotaviruses were used (Kanai et al., J Virol. 2020 Dec 22;95(2):e01374-20.).

The virus titers were calculated for the five types of single-infectious rotaviruses (Δ169-174 + linker/G1, Δ169-174 + linker/G2, Δ169-174 + linker/G3, Δ169-174 + linker/G8, Δ169-174 + linker/G9) with segmented RNA genomes encoding VP7 of the human rotaviruses produced, and the single-infectious rotavirus SA11 strain (Δ169-174 + linker) using the same method as in "Materials and Methods" (10) of Example 1.

<Results>
The results are shown in Figure 10. It was demonstrated that single-infectious rotaviruses could be produced using plasmids carrying VP7 from human rotaviruses of each genotype.

Example 9: Neutralization test using single-infectious rotavirus expressing VP7 of human rotavirus
To analyze whether single-infectious rotaviruses expressing VP7 from human rotaviruses of different genotypes (serotypes) have different antigenicities, neutralization tests were performed.

Materials and Methods
(1) Single-episode infectious rotaviruses having human rotavirus VP7 As single-episode infectious rotaviruses expressing human rotavirus VP7, Δ169-174+linker/G1, Δ169-174+linker/G3, Δ169-174+linker/G8, and Δ169-174+linker/G9, which have different VP7 genotypes (serotypes) prepared in Example 8, were used. As controls, Δ169-174+linker, which is a single-episode infectious rotavirus of the SA11 strain, and wild-type SA11 strain were used. The viral titer of each virus was measured, and a virus with a titer of 100 FFU was prepared for each virus.

(2) Serum Anti-rSA11 described in Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.) was used. Anti-rSA11 was obtained by orally administering wild-type SA11 strain to BALB/c mice (male, 3 weeks old) at 1.0 × 10 ⁶ FFU/mouse, repeating the administration 7 times at 2-week intervals, and then collecting blood serum. The VP7 genotype (serotype) of the wild-type SA11 strain is G3.

(3) Neutralization test method MA104 cells expressing VP6 were seeded at 1 x ¹⁰⁴ cells/well in a 96-well culture plate and cultured overnight. The serum from (2) was serially diluted and mixed with 100 FFU of the virus prepared in (1) and incubated at 37°C for 1 hour, after which each virus-serum mixture was added to the cells to start culture. After 18 hours, the cells were fixed with formalin, permeabilized with 0.5% Triton-X, and the infected cells were visualized by immunofluorescence antibody technique and the number of infected cells was counted. The primary antibody for the immunofluorescence antibody technique was rabbit-derived anti-rotavirus NSP4 antibody, and the secondary antibody was CF488-labeled anti-rabbit IgG antibody (Biotum). The reduction rate of the number of infected cells compared to the number of infected cells in the control without serum was calculated as the neutralization rate.

<Results>
The results are shown in FIG. 11. The wild-type SA11 strain (genotype G3), the single-infectious rotavirus Δ169-174+linker (genotype G3) expressing VP7 of the SA11 strain, and the single-infectious rotavirus Δ169-174+linker/G3 expressing human VP7 of genotype G3 were efficiently neutralized by serum immunized with the wild-type SA11 strain (genotype G3). On the other hand, the single-infectious rotavirus expressing human VP7 of a genotype other than G3 had low neutralizing activity. This result indicates that the single-infectious rotaviruses expressing VP7 of human rotaviruses with different genotypes have different antigenicity. Therefore, it was confirmed that single-infectious rotaviruses with different antigenicity can be produced.

Example 10: Vaccine effect of single-infectious rotavirus
Materials and Methods
(1) Vaccine administration to mice Three groups were established: a group administered Δ169-174 + linker, a single infectious rotavirus of the SA11 strain, a group administered wild-type SA11 strain (positive control), and a group administered PBS (negative control). Concentrated virus (1.0 × ¹⁰⁷ FFU/mouse) or PBS was orally administered to BALB/c mice (female, 4 weeks old, n = 5-6/group). Mice were immunized three times at 3-week intervals, and blood was collected two weeks after each immunization, and the serum was used for neutralization tests.

(2) Neutralization test method Normal MA104 cells were seeded at 1 x ¹⁰⁴ cells/well in a 96-well culture plate and cultured overnight. Serially diluted mouse serum and 100FFU of NLuc-expressing self-replicating rotavirus (see Example 7) were mixed and incubated at 37°C for 1 hour, after which the virus-serum mixture was added to the cells and cultured for 24 hours. The medium and cells were collected and frozen and thawed, and the luciferase activity of the prepared cell lysate was measured using the Nano-Glo Luciferase Assay System. The reduction rate of luciferase activity compared to the luciferase activity of the control without serum was calculated as the neutralization rate. In addition, the serum dilution showing 50% neutralization (NT50) was calculated. The higher the NT50 value, the higher the neutralization activity. The Mann-Whitney U test was performed for statistical analysis. A P value of 0.05 or less was determined to be significant.

<Results>
The results are shown in Figure 12. (A) shows the NT50 of the serum after one immunization, (B) shows the NT50 of the serum after two immunizations, and (C) shows the NT50 of the serum after three immunizations. At any time point, there was no significant difference between the single-infectious rotavirus group and the wild-type SA11 strain group, demonstrating that the single-infectious rotavirus exerts a vaccine effect equivalent to that of the wild-type rotavirus.

Example 11: Growth of single-infectious rotavirus in mice
Materials and Methods
Two groups were established: one group administered Δ169-174 + linker, a single infectious rotavirus of the SA11 strain (single infectious virus group), and the other group administered wild-type SA11 strain (wild-type virus group). Concentrated virus (1 × ¹⁰⁷ FFU/mouse) was orally administered to BALB/c mice (female, 3 weeks old, n = 18-19/group). After 2 days, the intestines were collected, and the cecum was excised and processed with a bead homogenizer. RNA was extracted from the obtained cecal samples using Trizol reagent (Invitrogen), and the number of viral RNA copies was measured by qRT-PCR. For qRT-PCR, a Thunderbird probe One-step qRT-PCR kit (Toyobo) was used. The following primer-probe set was used to detect the VP1 segment of rotavirus.
Forward: AGGCAAACCATTGGAGGCAGAC (SEQ ID NO: 14)
Reverse: CCAACCAGAACATGACTGCATT (SEQ ID NO: 15)
Probe: FAM-TCCAACAGCGGAGGAATATACGGAC-TAMRA (SEQ ID NO: 16)

In addition, cecal samples were added to the culture medium of VP6-expressing MA104 cells to infect them with the virus, and viral titers were measured. A t-test was performed for the number of viral RNA copies, and a Mann-Whitney U test was performed for the viral titers. A P value of 0.05 or less was considered to indicate a significant difference.

<Results>
The results are shown in Figure 14. (A) shows the RNA copy number of the virus, and (B) shows the results of infectious virus detection. When comparing the wild-type virus group (WT) and the single-infectious virus group (Δ169-174 + linker), there was no statistically significant difference in the copy number, but the wild-type virus tended to have a higher copy number, suggesting that the virus was proliferating. In the detection of infectious virus, the wild-type virus had a significantly higher virus titer than the single-infectious virus. In detail, the number of individuals in which infectious virus was detected was 7/19 in the wild-type virus group and 0/18 in the single-infectious virus group. This result suggested that the single-infectious virus cannot proliferate in mice.

Example 12: Creation of a single-infectious rotavirus by VP7 mutation introduction
Materials and Methods
In the method for producing a single-infectious rotavirus in Example 1, the mutated gene was changed from VP6 to VP7, and the VP6 expression vector was changed to a VP7 expression vector to produce a single-infectious rotavirus. The materials not used in Example 1 are shown below.

(1) Rotavirus VP7 expression vector The VP7 expression vector was prepared by inserting the protein coding region DNA of the VP7 gene (SEQ ID NO: 9) of SA11 strain into the BamHI and EcoRI cleavage sites of the pcDNA3.1(+) plasmid to prepare pcDNA3-VP7. The coding region DNA was synthesized by an artificial gene synthesis service (GenScript).

(2) VP7 Mutation-Introduced Plasmids Five types of plasmids were constructed by introducing deletion mutations into the cDNA of the rotavirus VP7 RNA genome contained in pT7-VP7SA11 using known gene recombination techniques. The structure of rotavirus VP7 and the various mutated regions are shown in Figure 15.

(3) VP7 persistent expression cells The rotavirus VP7 gene was introduced into the pLVSIN-CMV-Neo vector (Takara Bio) and co-transfected with psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into 293T cells to produce lentivirus. The lentivirus was then infected into African green monkey MA104 cells (ATCC CRL-2378.1), and after 2 days, drug selection was performed with G418 (800 μg/mL). From the VP7-expressing MA104 cells obtained by drug selection, cells that persistently and strongly expressed VP7 were cloned by limiting dilution.

<Results>
The results are shown in Table 3. An artificial recombinant virus was successfully produced using a VP7 mutation introduction plasmid (pT7-VP7SA11-Δdomain II) in which domain II of VP7 was deleted, and a VP7 mutation introduction plasmid (pT7-VP7SA11-250bp) in which 250bp at each end of the VP7 gene was left intact. The virus titer was higher for the artificial recombinant virus (VP7-ΔDomain II) in which domain II of VP7 was deleted. VP7-ΔDomain II was used in subsequent experiments.

Example 13: Comparison of proliferation ability between normal MA104 cells and MA104 cells continuously expressing VP7
The VP7 mutant rotavirus VP7-ΔDomain II prepared in Example 12 was compared for its proliferation ability in normal MA104 cells and in MA104 cells that persistently express VP7.
Materials and Methods
Normal MA104 cells and MA104 cells expressing VP7 were seeded at 7.5 x ¹⁰⁴ cells/well in a 24-well culture plate and cultured overnight. VP7-ΔDomain II was infected at an MOI of 0.01, and the medium and cells were frozen and thawed three times at 0, 24, 48, and 72 hours after infection to prepare cell lysates and measure the virus titer. A wild-type virus without VP7 mutation was used as a control. The virus titer was measured in the same manner as in Example 1.

<Results>
The results are shown in Figure 16. (A) shows the results for normal MA104 cells, and (B) shows the results for MA104 cells continuously expressing VP7. VP7-ΔDomain II hardly grew in normal MA104 cells, but grew efficiently in MA104-VP7 cells.

Example 14: Examination of proliferation ability in normal MA104 cells
To further confirm that the VP7 mutant rotavirus VP7-ΔDomain II cannot grow in normal MA104 cells, it was subcultured in normal MA104 cells.
Materials and Methods
Normal MA104 cells were seeded at 1.5 x ¹⁰⁵ cells/well in a 12-well culture plate and cultured overnight. VP7-ΔDomain II was infected at MOI 1.0, and after one week of culture, the medium and cells were collected and frozen and thawed to prepare a cell lysate. 10% of the obtained cell lysate was added to new normal MA104 cells and cultured for one week. The virus was passaged five times, and the virus titer was measured at each passage using the same method as in Example 1.

<Results>
The results are shown in Figure 17. The titer of VP7-ΔDomain II decreased with passage, and the virus was not detected after the second passage. From this result, it was determined that VP7-ΔDomain II is a single-pass infectious rotavirus that cannot grow in normal cells that do not express VP7.

Example 15: Creation of a single-infectious rotavirus by VP4 mutation introduction
Materials and Methods
In the method for producing a single-infectious rotavirus in Example 1, the mutated gene was changed from VP6 to VP4, and the VP6 expression vector was changed to a VP4 expression vector to produce a single-infectious rotavirus. The materials not used in Example 1 are shown below.
(1) Rotavirus VP4 expression vector The VP4 expression vector was prepared by inserting the protein coding region DNA of the VP4 gene (SEQ ID NO: 4) of SA11 strain into the BamHI and EcoRI cleavage sites of the pcDNA3.1(+) plasmid to prepare pcDNA3-VP4. The coding region DNA was synthesized by an artificial gene synthesis service (GenScript).

(2) VP4 Mutation-Introduced Plasmid Three types of plasmids were constructed by introducing deletion mutations into the cDNA of the RNA genome of rotavirus VP4 contained in pT7-VP4SA11 using known gene recombination techniques. The structure of rotavirus VP4 and the various mutated regions are shown in Figure 18.

(3) VP4 persistent expression cells The rotavirus VP4 gene was introduced into the pLVSIN-CMV-Neo vector (Takara Bio) and co-transfected with psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into 293T cells to produce lentivirus. The lentivirus was then infected into African green monkey MA104 cells (ATCC CRL-2378.1), and after 2 days, drug selection was performed with G418 (800 μg/mL). From the VP4-expressing MA104 cells obtained by drug selection, cells that persistently and strongly expressed VP4 were cloned by limiting dilution.

<Results>
The results are shown in Table 4. Using the VP4 mutation plasmid (pT7-VP4SA11-ΔLectin) lacking the VP4 lectin domain, the VP4 mutation plasmid (pT7-VP4SA11-Δβ-Barrel) lacking the VP4 β-Barrel domain, and the VP4 mutation plasmid (pT7-VP4SA11-120bp) with 120bp at each end of the VP4 gene remaining, we were able to create artificial recombinant viruses. The larger the deletion region, the less likely it is that reversion will occur and the easier it is to introduce a foreign gene, so we used VP4-120bp in the subsequent experiments.

Example 16: Comparison of proliferation ability between normal MA104 cells and MA104 cells continuously expressing VP4
The VP4 mutant rotavirus VP4-120bp prepared in Example 15 was compared in terms of proliferation ability in normal MA104 cells and in MA104 cells that persistently express VP4.
Materials and Methods
Normal MA104 cells and MA104 cells expressing VP4 were seeded in a 24-well culture plate at 7.5 x ¹⁰⁴ cells/well and cultured overnight. VP4-120bp was infected at an MOI of 0.01, and the medium and cells were frozen and thawed three times at 0, 24, 48, and 72 hours after infection to prepare cell lysates and measure the virus titer. A wild-type virus without VP4 mutation was used as a control. The virus titer was measured in the same manner as in Example 1.

<Results>
The results are shown in Figure 19. (A) shows the results for normal MA104 cells, and (B) shows the results for MA104 cells continuously expressing VP4. VP4-120bp hardly grew in normal MA104 cells, but grew efficiently in MA104-VP4 cells.

Example 17: Examination of proliferation ability in normal MA104 cells
To further confirm that the VP4 mutant rotavirus VP4-120bp was unable to grow in normal MA104 cells, it was subcultured in normal MA104 cells.
Materials and Methods
Normal MA104 cells were seeded at 1.5 x ¹⁰⁵ cells/well in a 12-well culture plate and cultured overnight. VP4-120bp was infected at MOI 1.0, and after one week of culture, the medium and cells were collected and frozen and thawed to prepare a cell lysate. 10% of the obtained cell lysate was added to new normal MA104 cells and cultured for one week. The virus was passaged five times, and the virus titer was measured at each passage using the same method as in Example 1.

<Results>
The results are shown in Figure 20. The titer of VP4-120bp decreased with passage, and the virus was not detected after the second passage. From this result, it was determined that VP4-120bp is a single-pass infectious rotavirus that cannot grow in normal cells that do not express VP4.

Example 18: Preparation of a single-infectious rotavirus carrying a foreign gene in the VP4 segment
18-1 Single-infectious rotavirus expressing green fluorescent protein <Materials and methods>
(1) Preparation of green fluorescent protein-expressing single-infectious rotavirus The ZsG gene was used as the green fluorescent protein gene. A ZsG expression plasmid was prepared by inserting the foot-and-mouth disease virus FMDV 2A sequence (self-cleavage sequence) and the ZsG gene immediately after the 5'-end 120bp of the pT7-VP4SA11-120bp plasmid. A ZsG-expressing single-infectious rotavirus was prepared in the same manner as in Example 15. As a control, a wild-type artificial recombinant rotavirus was prepared using a plasmid (pT7-VP4SA11) encoding a wild-type VP4 segment, and a VP4-deleted artificial recombinant rotavirus (single-infectious rotavirus) was prepared using pT7-VP4SA11-120bp without the ZsG gene inserted therein.

(2) Confirmation of ZsG expression The prepared virus was infected into normal MA104 cells at MOI 1.0 and cultured for 8 hours. To confirm that the cells emitting green fluorescence were virus-infected cells, the expression of virus-derived NSP4 was confirmed. That is, after 8 hours of culture, the cells were fixed with formalin. The membrane was permeabilized with 0.5% Triton-X, and stained with rabbit-derived anti-NSP4 antibody as the primary antibody and CF594-labeled anti-rabbit IgG antibody (Biotum) as the secondary antibody. Nuclei were stained with Hoechst and observed under a fluorescent microscope.

<Results>
The results are shown in Figure 21. It was confirmed that cells infected with the ZsG-introduced, single-infectious rotavirus (VP4-120-ZsG) emitted green fluorescence.

18-2 Luciferase-expressing single-infectious rotavirus <Materials and methods>
(1) Luciferase gene-inserted NSP1 expression plasmid The NLuc gene was used as the luciferase gene. An NLuc expression plasmid was prepared by inserting the foot-and-mouth disease virus FMDV 2A sequence (self-cleavage sequence) and the NLuc gene immediately after the 5'-end 120 bp of the pT7-VP4SA11-120bp plasmid. A NLuc-expressing single-infectious rotavirus was prepared in the same manner as in Example 15. As a control, a VP4-deleted single-infectious rotavirus was prepared using pT7-VP4SA11-120bp without the NLuc gene inserted therein.

(2) Luciferase activity measurement Normal MA104 cells were seeded at 1 × ¹⁰⁴ cells/well in a 96-well culture plate and cultured overnight. The prepared NLuc-expressing single-infectious rotavirus and VP4-deleted single-infectious rotavirus were infected at MOI 0.1, respectively, and the medium and cells were collected over time and frozen and thawed to prepare cell lysates. In addition, MA104 cells cultured in a medium containing trypsin (VP4-120-NLuc+Trypsin) were added as the MA104 cells to be infected with the NLuc-expressing single-infectious rotavirus. Luciferase activity of the cell lysate was measured using Nano-Glo Luciferase Assay System (trade name, Promega).

<Results>
The results are shown in Figure 22. It was shown that the luciferase activity of the NLuc-expressing, single-infectious rotavirus increased over time after infection. There was also no difference in luciferase activity in the presence or absence of trypsin. Trypsin is an enzyme necessary for making rotavirus infective. In the case of a replicative virus, secondary and tertiary infections occur in the presence of trypsin, and luciferase activity increases in the presence of trypsin more than in the absence of trypsin 24 hours after infection. However, in this experiment, there was no difference even after 24 hours, so it is considered that the virus is single-infectious, with no infectious virus being produced.

Example 19: Construction of VP4-deleted, single-infectious rotavirus having human rotavirus coat protein
As in Example 8, Kanai et al. (J Virol. 2020 Dec 22;95(2):e01374-20.) produced a single-infectious rotavirus having a segmented RNA genome encoding VP7 of human rotavirus isolated from the stool of nine children with acute gastroenteritis in Japan.

Materials and Methods
Of the 11 types of segmented RNA genome expression vectors prepared in Example 1, pT7-VP4SA11-120bp encoding the mutated VP4 was used instead of pT4-VP6SA11, and pT7-hVP7, a segmented RNA genome expression vector encoding VP7 of human rotavirus, was used instead of pT7-VP7SA11. In addition to these, capping enzyme expression vectors (pCAGD1R and pCAG-D12L), rotavirus NSP2, NSP5 expression vectors (pCAG-NSP2SA11 and pCAG-NSP5SA11), and rotavirus VP4 expression vector (pcDNA3-VP4) were also introduced into BHK-T7/P5 cells to prepare VP4-deleted single-infectious rotavirus having VP7 of human rotavirus in the same manner as in "Materials and Methods" (9) of Example 1. Four types of human rotaviruses with VP7 genotypes (serotypes) G1, G2, G8 and G9 were used as human rotaviruses, and segmented RNA genomes encoding VP7 obtained from each of these four types of human rotaviruses were used (Kanai et al., J Virol. 2020 Dec 22;95(2):e01374-20.). The viral titers were calculated in the same manner as in Example 1 for the four types of VP4-deleted single-infectious rotaviruses (VP4-120bp/G1, VP4-120bp/G2, VP4-120bp/G8, VP4-120bp/G9) having segmented RNA genomes encoding the VP7 of the human rotaviruses produced.

<Results>
The results are shown in Table 5. Using the plasmids carrying VP7 from each genotype of human rotavirus, VP4-deleted single-infectious rotavirus carrying the coat protein of human rotavirus could be produced. These single-infectious rotaviruses expressing VP7 from human rotaviruses of different genotypes are single-infectious rotaviruses with different antigenicity (see Example 9).

Example 20: Preparation of a single-infectious rotavirus carrying the spike protein gene of the new coronavirus in the VP4 segment
Materials and Methods
(1) Preparation of a single-infectious rotavirus expressing the spike protein of the new coronavirus As a foreign gene, a gene encoding the receptor binding domain (RBD) of the spike protein of the new coronavirus (SARS-CoV-2) was used. The new coronavirus RBD gene was amplified from the spike protein expression plasmid described in Minami et al. (Microbiol Spectr. 2024 Apr 2;12(4):e0285923.). A plasmid was prepared by inserting the foot-and-mouth disease virus FMDV 2A sequence (self-cleavage sequence) and the new coronavirus RBD gene immediately after the 5'-end 120 bp of the pT7-VP4SA11-120bp plasmid. A single-infectious rotavirus expressing the new coronavirus RBD was prepared in the same manner as in Example 15. As a control, a VP4-deleted single-infectious rotavirus was prepared using pT7-VP4SA11-120bp without the RBD gene inserted.

(2) Confirmation of RBD expression The prepared virus was infected into normal MA104 cells at MOI 1.0 and cultured for 16 hours. After culture, cell lysates were prepared using RIPA buffer, and protein expression was confirmed by Western blotting. Proteins were separated by SDS-PAGE using 10% polyacrylamide gel and transferred to a PVDF membrane. After blocking with 3% skim milk, rabbit anti-SARS-CoV-2 RBD antibody (Sino Biological), rabbit anti-rotavirus NSP3 antibody, or mouse anti-β-actin antibody (Sigma Aldrich) were reacted as primary antibodies. HRP-labeled anti-rabbit IgG antibody (Nacalai Tesque) or HRP-labeled anti-mouse antibody (Nacalai Tesque) were reacted as secondary antibodies. Chemi-Lumi-One Ultra (trade name, Nacalai Tesque) was used for color development. Images were taken using a chemiluminescence imaging device.

<Results>
The results are shown in Figure 23. It was confirmed that cells infected with a single-infectious rotavirus (VP4-single-RBD) carrying the receptor-binding domain (RBD) of the spike protein of the novel coronavirus (SARS-CoV-2) expressed RBD.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. In addition, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims (19)

A single-episode infectious rotavirus characterized by having a mutation in at least one viral protein selected from the group consisting of rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP2, NSP3 and NSP4. The mono-infectious rotavirus of claim 1, which has a mutation that induces dysfunction of a structural protein. The mono-infectious rotavirus of claim 1, having a mutation in VP6. The monoinfectious rotavirus of claim 3, having a mutation in domain H of VP6. The monoinfectious rotavirus according to claim 4, which has a mutation in the A'-A'' loop, the D'-D'' loop, or the αA-I loop of domain H of VP6. The mono-infectious rotavirus of claim 1, having a mutation in VP4. The monoinfectious rotavirus of claim 6, which has a mutation that deletes the lectin domain and/or the β-barrel domain of VP4. The mono-infectious rotavirus of claim 1, having a mutation in VP7. The monoinfectious rotavirus of claim 8, having a mutation that deletes domain II of VP7. The monoinfectious rotavirus of claim 1, which is a reassortant. The monoinfectious rotavirus of claim 1, which is a reassortant having a segmented RNA genome encoding structural proteins of a different genotype from the parent strain or structural proteins of a different strain from the parent strain. The monoinfectious rotavirus of claim 1, which is a reassortant having a segmented RNA genome encoding the structural proteins of human rotavirus. The monoinfectious rotavirus of claim 1, which expresses a foreign gene. The monoinfectious rotavirus according to claim 13, wherein the foreign gene is a gene encoding an antigenic protein of a virus or a pathogenic microorganism other than rotavirus. The monoinfectious rotavirus of claim 13, wherein the foreign gene is a reporter gene. The monoinfectious rotavirus of claim 15, wherein the reporter gene is a gene encoding a fluorescent protein or a chemiluminescent protein. A vaccine containing a single-dose infectious rotavirus according to any one of claims 1 to 14. The vaccine according to claim 17, which is a rotavirus vaccine. A method for neutralizing a rotavirus, comprising using a single-infectious rotavirus according to any one of claims 1 to 16.