G10P[11] bovine rotavirus HSX252 strain capable of stable subculture and application thereof

Abstract

The application discloses a G10P[11] bovine rotavirus HSX252 strain capable of stable subculture and application thereof, and belongs to the field of veterinary microbiology and biotechnology. The strain is isolated from the feces of a diarrheal calf, and has been preserved in the China General Microbiological Culture Collection Center on June 23, 2026, with a preservation number of CGMCC No. 47073. The strain can stably proliferate in MA104 cells, produce typical cytopathic effects after optimization of trypsin activation conditions, and can be continuously subcultured to the 20th generation, with a virus titer of 10 6.80 TCID 50 / mL. Whole genome sequencing determines that the genotype of the strain is G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H3, and the genetic stability is good. The application also provides application of the strain in bovine rotavirus pathogen research, establishment of an isolation and culture system, genetic evolution analysis and construction of an animal pathogenic model. The application strain has a clear source, a clear genetic background and stable culture characteristics, and provides a valuable standard strain resource for basic research and related biological product research and development of bovine rotavirus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a stable cultureable G10P

[11] bovine rotavirus HSX252 strain and its application. Background Technology

[0002] Bovine rotavirus ( Bovine Rotavirus Rotavirus (BRV), belonging to the genus Rotavirus in the family Reoviridae, is a major pathogen causing diarrhea in newborn calves. This disease primarily affects calves under one month old, clinically manifesting as severe diarrhea, dehydration, growth retardation, and even death, causing significant economic losses to dairy and beef cattle farming. The rotavirus genome consists of 11 segmented double-stranded RNAs, encoding six structural proteins (VP1–VP4, VP6, and VP7) and five non-structural proteins (NSP1–NSP5). Among these, the outer capsid proteins VP7 and VP4 are the main determinants of viral neutralizing antigens. Based on their genetic differences, rotaviruses are classified into G and P types, respectively. Currently, over 40 G types and more than 30 P types have been identified globally. This diversity of genotype combinations provides an important molecular basis for viral classification and epidemiological surveillance.

[0003] In cattle herds, the reported prevalent genotypes mainly include G6, G8, G10 and other G types, as well as P[1], P[5], P

[11] and other P types. Among them, G10P

[11] is one of the more common dominant genotypes in calves in my country, playing an important role in the outbreak and spread of calf diarrhea. In recent years, with the continuous deepening of molecular epidemiological research, genetic variations and gene reassortment phenomena of bovine rotavirus from different geographical sources have been continuously discovered, suggesting that the molecular evolutionary dynamics and genetic diversity of this virus are more complex than previously thought. However, the current monitoring data of bovine rotavirus is still relatively limited, and the distribution and evolutionary patterns of prevalent strains in different regions are still unclear, which restricts the scientific assessment of the epidemic trend of this virus.

[0004] Currently, most of the publicly reported research on bovine rotavirus focuses on epidemiological surveys and genotyping analysis, while standardized strains available for laboratory research are relatively scarce. In particular, there is still a severe shortage of G10P

[11] bovine rotavirus strains that can stably proliferate in vitro, be continuously passaged, and have a clear genetic background. Existing virus isolation and culture processes generally suffer from problems such as low virus isolation success rate, poor cell adaptability, difficulty in continuous passage, and unstable virus titer. This directly limits the in-depth development of applied basic research work such as research on viral pathogenic characteristics, exploration of pathogenic mechanisms, evaluation of diagnostic reagents, and screening of vaccine candidate strains.

[0005] Therefore, obtaining a standard strain of G10P

[11] bovine rotavirus with a clear origin, well-defined genetic background, and the ability to replicate efficiently and be stably passaged in vitro is the key to solving the above-mentioned research bottlenecks. This strain can not only provide stable experimental materials for the study of bovine rotavirus gene function, antigenic epitope analysis, and the analysis of genetic evolution, but also provide important support for the development of prevention and control technologies for calf rotavirus disease. Based on this, this invention isolated a stable passaged G10P

[11] bovine rotavirus HSX252 strain and systematically identified its biological characteristics and performed whole-genome genetic analysis to make up for the lack of existing standard bovine rotavirus strain resources. Summary of the Invention

[0006] This invention provides a G10P

[11] bovine rotavirus HSX252 strain with accession number CGMCC No.47073. This strain can stably proliferate in MA104 cells and be passaged continuously to the F20 generation, with a viral titer of 10. 6.80 TCID 50 / mL, the whole genome genotype is G10-P

[11] -I2-R2-C2-M2-A3-N2-T6-E2-H3, with good genetic stability, and can be used for bovine rotavirus etiology research, isolation and culture system establishment, genetic evolution analysis and animal pathogenicity model construction.

[0007] On the one hand, the present invention provides a stable subculture-compatible G10P

[11] bovine rotavirus HSX252 strain, which adopts the following technical solution: A stable cultureable G10P

[11] bovine rotavirus strain ( Bovine Rotavirus The HSX252 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 47073.

[0008] Preferably, the HSX252 strain can stably proliferate in MA104 cells and be continuously passaged up to the 20th generation.

[0009] Preferably, the viral titer of the HSX252 strain in MA104 cells is 10. 5.50 ~10 6.80 TCID 50 / mL.

[0010] Preferably, the whole genome genotype of the HSX252 strain is G10-P

[11] -I2-R2-C2-M2-A3-N2-T6-E2-H3.

[0011] Preferably, the VP4 protein of the HSX252 strain has one or more dominant antigenic epitope regions as shown in SEQ ID NO:50-53; the VP7 protein of the HSX252 strain has one or more dominant antigenic epitope regions as shown in SEQ ID NO:54-57.

[0012] On the other hand, the present invention provides the application of the above-mentioned stable subculture G10P

[11] bovine rotavirus HSX252 strain in establishing a bovine rotavirus isolation and culture system.

[0013] The present invention also provides the application of the above-mentioned stable subculture G10P

[11] bovine rotavirus HSX252 strain in constructing a bovine rotavirus phylogenetic tree.

[0014] The present invention also provides the application of the above-mentioned stable cultureable G10P

[11] bovine rotavirus HSX252 strain in the preparation of a kit for constructing a bovine rotavirus animal pathogenic model.

[0015] Preferably, the animal is a suckling mouse.

[0016] The present invention uses the G10P

[11] bovine rotavirus strain isolated from fecal samples of 5-day-old diarrheal calves. A bovine rotavirus strain that can be stably passaged and cultured, with the genotype G10-P

[11] -I2-R2-C2-M2-A3-N2-T6-E2-H3, named rotavirus group A HSX252, was deposited on June 23, 2026, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 47073, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0017] In summary, the beneficial effects of the present invention are as follows: This invention obtained a G10P

[11] type bovine rotavirus HSX252 strain with a clear origin, complete preservation, and clear genetic background. This strain overcomes the technical bottlenecks commonly found in existing bovine rotavirus isolation and culture, such as low virus isolation success rate, poor cell adaptability, difficulty in continuous passage, and unstable virus titer. It can stably proliferate in MA104 cells and be continuously passaged to the 20th generation, with a virus titer of up to 10. 6.80 TCID 50 The strain exhibits good cell adaptability and in vitro replication ability. Genetic stability analysis showed that the genetic distance of the VP6 gene sequence between generations was only 0.000–0.010, with only 0–2 base differences, demonstrating that the strain can maintain high genetic stability during long-term passage, providing reliable experimental material for subsequent research.

[0018] The strain has completed whole-genome sequencing and genotyping, and its genome configuration has been identified as G10-P

[11] -I2-R2-C2-M2-A3-N2-T6-E2-H3, which enriches the genomic data resources of bovine rotavirus and can provide important references for molecular epidemiological monitoring, genetic variation analysis and evolutionary tracing of bovine rotavirus. At the same time, the strain showed good pathogenicity in suckling mouse animal models and could stably induce typical diarrhea symptoms, which can be used for the study of the pathogenic mechanism of bovine rotavirus and the standardization of animal infection models. In addition, the successful prediction of the dominant antigenic epitopes of VP4 and VP7 proteins has laid the foundation for the application of this strain in subunit vaccine development, diagnostic antigen screening and serological detection methods. The HSX252 strain provided by this invention, as a standardized biological material, can effectively make up for the lack of standard strains of G10P

[11] bovine rotavirus in my country, and has an important supporting role and application prospect for promoting basic research on bovine rotavirus and the development of related biological products. Attached Figure Description

[0019] Figure 1 A comparison chart of cytopathic effects; in, Figure 1 Cell A in the middle is a normal cell; Figure 1 Cell B is inoculated with BRV virus; Figure 2 The image shows an electrophoresis diagram of VP6 gene amplification, where M is the DL 500 DNA marker, 1 is the target fragment of the VP6 gene from the HSX252 strain, 2 is the positive control, and 3 is the negative control. Figure 3 The image shows an electrophoresis diagram of VP7 gene amplification, where M is the DL 1500 DNA marker, 1 is the negative control, 2 is the VP7 gene positive control, and 3 is the target fragment of VP7 amplification using HSX252. Figure 4 The results are for G-type identification amplification. Among them, M is the DL 1000 DNA marker, 1 is the target fragment for HSX252 G10 amplification, 2 is the target fragment for isolated strain G5 amplification, 3 is the target fragment for isolated strain G6 amplification, 4 is the target fragment for isolated strain G8 amplification, 5 is the target fragment for isolated strain G9 amplification, 6 is the positive control for G10 amplification, and 7 is the negative control. Figure 5 This is a growth curve of the F10 generation virus. Figure 6 Pairwise genetic distance analysis was performed on the VP6 gene sequences of different generations of the HSX252 strain; in, Figure 6The sequence alignment results of the F5-F20 generation VP6 products of the HSX252 strain are shown in Figure A. Figure 6 B represents the genetic distance of the sequencing sequences of VP6 products from generations F5 to F20 of the HSX252 strain; Figure 7 This is a nucleotide homology analysis diagram between HSX252 and the reference strain VP1; Figure 8 This is a phylogenetic tree diagram of the HSX252 VP1 gene; Figure 9 This is a nucleotide homology analysis diagram between HSX252 and the reference strain VP2; Figure 10 Genetic phylogenetic tree of HSX252 VP2 gene; Figure 11 This is a nucleotide homology analysis diagram between HSX252 and the reference strain VP3; Figure 12 The phylogenetic tree of the HSX252 VP3 gene; Figure 13 This is a nucleotide homology analysis diagram between HSX252 and the reference strain VP4; Figure 14 The phylogenetic tree of the HSX252 VP4 gene; Figure 15 This is a nucleotide homology analysis diagram between HSX252 and the reference strain VP6; Figure 16 Genetic phylogenetic tree of the HSX252 VP6 gene; Figure 17 Nucleotide homology analysis between HSX252 and the reference strain VP7; Figure 18 This is a phylogenetic tree of the HSX252 VP7 gene; Figure 19 This is a nucleotide homology analysis diagram between HSX252 and the reference strain NSP1; Figure 20 Genetic phylogenetic tree of HSX252 NSP1 gene; Figure 21 This is a nucleotide homology analysis diagram between HSX252 and the reference strain NSP2; Figure 22 Genetic phylogenetic tree of HSX252 NSP2 gene; Figure 23 This is a nucleotide homology analysis diagram between HSX252 and the reference strain NSP3; Figure 24 Genetic phylogenetic tree of HSX252 NSP3 gene; Figure 25This is a nucleotide homology analysis diagram between HSX252 and the reference strain NSP4; Figure 26 Genetic phylogenetic tree of HSX252 NSP4 gene Figure 27 This is a nucleotide homology analysis diagram between HSX252 and the reference strain NSP5; Figure 28 Genetic phylogenetic tree of HSX252 NSP5 gene Figure 29 Prediction of VP4 and VP7 protein epitopes in HSX252 strain; in, Figure 29 A is a predicted epitope of the HSX252 VP4 protein. Figure 29 B is a predicted epitope of the HSX252 VP7 protein antigen; Figure 30 Clinical diarrhea symptoms in suckling mice after HSX2525 challenge; Figure 31 The results show the amplification of the VP6 gene in the feces of suckling mice. M is the DL 500 DNA marker, 1 is the negative control, 2 is the positive control, and 3 is the target fragment of the VP6 gene from the HSX252 strain. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example 1 The specific steps for isolating and culturing the HSX252 strain are as follows: S1. Sample pretreatment and virus isolation Fresh fecal samples were collected from 5-day-old calves with spontaneous diarrhea at a large-scale cattle farm in Bayannur City, Inner Mongolia Autonomous Region. The sick calves showed signs of depression, decreased feed intake, diarrhea, and excretion of yellowish-white, thin, watery feces.

[0022] a. Sample pretreatment: Place 0.2g of feces into a 3mL sterile centrifuge tube, add 1mL of pre-cooled PBS, mix thoroughly using a vortex mixer, and centrifuge at 4℃ and 10000 rpm / min for 3 min. Collect the supernatant and filter it sequentially through 0.45 μm and 0.22 μm sterile filter membranes. Use the filtrate for subsequent separation.

[0023] b. Virus isolation: Observe the growth status of MA104 cells. When the cells cover 80%–90% of the bottom of the culture flask, discard the culture medium and wash three times with PBS. Take 500 μL of the filtrate and add trypsin (containing EDTA) to a final concentration of 25 μg / mL, and activate in a 37℃ water bath for 20 min. Inoculate the activated filtrate into MA104 cells and incubate at 37℃ with 5% CO2 for 1.5 h for adsorption. Gently shake the culture flask every 30 min to ensure uniform virus contact with the cells. A negative control is also provided. After adsorption, add DMEM maintenance medium containing 1% penicillin and trypsin (containing EDTA) to a final concentration of 1 μg / mL, and incubate at 37℃ with 5% CO2 to observe and record cytopathic effects.

[0024] The results are as follows Figure 1 As shown, after the second blind passage, cells began to exhibit typical cytopathic effects, including cell shrinkage, increased refractive index, and vacuolation. The cytopathic effect rate reached 85%–90% 72–96 h post-infection. Virus harvesting was performed when CPE reached 85%–90%, involving three freeze-thaw cycles, followed by centrifugation at 4°C and 10,000 rpm / min for 3 min to collect the supernatant. The collected supernatant was stored as viral fluid and passaged using the method described above.

[0025] S2, Optimization of trypsin activation conditions Viral suspensions were treated with trypsin (containing EDTA) at final concentrations of 5, 10, 15, 20, 25, and 30 μg / mL, respectively. After activation at 37℃ for 20 min, the suspensions were inoculated into monolayers of MA104 cells. Each group was configured with three replicates, and a control group without trypsin treatment was included. Cytopathic effects (CPE) were observed and recorded. The results are shown in Table 1. When the trypsin concentration reached 25 μg / mL, CPE was most pronounced and the cell condition was good. At a trypsin concentration of 30 μg / mL, although CPE still occurred, some non-specific cell damage and detachment were observed.

[0026] Therefore, 25 μg / mL was determined to be the optimal activation concentration for the isolation and culture of the HSX252 strain.

[0027] Table 1. Effects of different trypsin concentrations on the isolation and culture efficiency of HSX252.

[0028] S3 and HSX252 strains were continuously passaged. The HSX252 strain was continuously passaged using the culture system established in step S1. Viruses were harvested at 85%–90% cytopathic effect rate in each generation, and the supernatant was inoculated into new monolayers of MA104 cells after three freeze-thaw cycles. After continuous passage to the 20th generation (F20), the virus still stably produced typical cytopathic effects. Viral titers were measured in generations F5, F6, F7, F8, F9, F10, F15, and F20, and the results showed that the viral titer remained at 10. 5.50 ~10 6.80 TCID 50 Within the range of / mL, the fluctuation was small. The results indicate that the HSX252 strain can stably proliferate and be continuously passaged in MA104 cells, exhibiting good cell adaptability and culture stability, and can be used for subsequent etiological studies and the establishment of animal infection models.

[0029] Example 2 The specific steps for identifying the HSX252 strain are as follows: a. PCR identification: Viral RNA was extracted and reverse transcribed into cDNA. Referencing "Chang Jitao. Epidemiological investigation of bovine rotavirus pathogen, construction of gene reassortant strains and immunogenicity evaluation of bivalent attenuated strains [D]. Chinese Academy of Agricultural Sciences, 2009," primers for VP6 and VP7 genes were synthesized, and PCR amplification was performed using a PCR instrument (SensoQuest, Germany). VP6 gene was used to identify BRV, and VP7 gene was used for G-type identification. Primer sequences are shown in Tables 2 and 5, and PCR amplification procedures and reaction systems are shown in Tables 3, 4, 6, and 7. Results are as follows: Figures 2-4 As shown, the expected fragments were successfully amplified. The VP6 gene is approximately 207 bp in size, and the VP7 gene is approximately 1062 bp in size. Figure 4 The results showed that the HSX252 strain only amplified the G10 type specific band (715 bp), and did not amplify the G5, G6, G8 and G9 type specific bands, indicating that the strain belongs to the G10 type bovine rotavirus.

[0030] Table 2 Primer sequences for the VP6 gene

[0031] Table 3. RT-PCR reaction system for VP6 gene

[0032] Table 4. RT-PCR reaction procedure for VP6 gene

[0033] Table 5 Primer sequences for G-type identification

[0034] Table 6. PCR reaction system for G-type identification

[0035] Table 7. PCR reaction procedure for G-type identification

[0036] b. Half-maximum tissue culture infection dose (TCID) 50 Measurement and growth curve plotting: F10 generation virus was inoculated into 6-well cell plates. Cells and supernatants were collected at 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, 66 h, and 72 h, and the cells were subjected to three freeze-thaw cycles to measure the TCID of the virus at different time points. 50 The virus was serially diluted 10-fold, and each 10-fold dilution was seeded into a monolayer of MA104 cells in 96-well plates. Each dilution was replicated in 8 wells, with 100 μL of virus seeded into each well. A negative control was also included. The cells were incubated at 37°C in a 5% CO2 incubator and observed for 5 days. The TCID of the virus was determined using the Reed-Muench method. 50 , is 10 6.81 TCID 50 / mL, growth curve as shown Figure 5 As shown, the HSX252 strain can continuously proliferate after inoculation into MA104 cells, reaching its peak proliferation 60 hours post-infection, with a viral titer of 10. 6.80 TCID 50 The viral titer was measured at 1 / mL, after which it stabilized and remained at a high level until 72 hours later. These results indicate that the HSX252 strain exhibits good cell adaptability and in vitro proliferation ability, and can stably replicate in MA104 cells to achieve a high viral titer.

[0037] c. Gene stability analysis across different generations: Pairwise genetic distance analysis of the VP6 gene sequences of the HSX252 strain in generations F5, F6, F7, F8, F9, F10, F15, and F20 was performed using MEGA software. Figure 6 As shown, the genetic distance between generations ranged from 0.000 to 0.010 (average approximately 0.005 to 0.007), with only 0-2 base differences, indicating that the virus maintained high genetic stability in MA104 cells through continuous passage to the F20 generation. These results demonstrate that the HSX252 strain exhibits long-term genetic stability in MA104 cells, providing molecular-level evidence for the long-term culture stability of the virus and its subsequent applications.

[0038] Example 3 The whole genome characterization of the HSX252 strain was performed using the following steps: To clarify the genetic characteristics of the HSX252 strain, its whole genome was amplified, sequenced, and genotype analyzed. RNA was extracted and cDNA was reverse-engineered from the F10 generation virus. Based on the G10 BRV whole genome sequence already registered in GenBank, primers covering 11 genes (VP1-VP4, VP6, VP7, NSP1-NSP5) were designed using SnapGene software and PCR amplification was performed. The primer sequences are shown in Table 8. Electrophoresis was performed on a 2% agarose gel at a constant voltage of 200 V for 20 min. After electrophoresis, the gel imaging results were observed, and the target fragment was purified using a PCR purification kit. The PCR products were then sequenced.

[0039] The obtained sequences were assembled and homology analyzed using MEGA 11 software. A phylogenetic tree was constructed using the maximum likelihood method, with a guide value set to 1000. Based on the classification criteria established by the Rotavirus Classification Working Group (RCWG), nucleotide homology of 11 gene segments was used as the criterion. The homology cutoff values ​​for each gene segment were 80%, 80%, 85%, 83%, 84%, 81%, 79%, 85%, 85%, 85%, and 91%, respectively. Genotyping was performed using the Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx naming format. Simultaneously, the genotype of the HSX252 strain was determined using the automatic genotyping tool provided by ViPR (https: / / www.viprbrc.org / ) combined with the NCBI online comparison function. The results showed that the complete genome sequence of the HSX252 strain was successfully amplified, and its genotype composition was determined to be: G10-P

[11] -I2-R2-C2-M2-A3-N2-T6-E2-H3. The results of the genome feature analysis are as follows: Figures 7-28As shown, homology analysis results indicated that the VP1 and NSP4 genes shared the highest homology with HB-3 strain (ON711384.1), at 97.9% and 98.9%, respectively. The VP2 gene shared the highest homology with JS31 strain (PQ332928.1) and Y3 strain (OM212040.1), at 96.6%. The VP3 gene shared the highest homology with MMC88 strain (HQ641366.1), at 96.3%. The VVP4 and NSP2 genes shared the highest homology with JS31 strain (PQ332930.1), at 97.4% and 97.5%, respectively. The VP6 gene showed the highest homology with the Y3 strain (OM212043.1), at 98.6%; the VP7 gene showed the highest homology with the SD1 strain (PQ332943.1), at 97.4%; the NSP1 gene showed the highest homology with the SX1 strain (PQ332955.1), at 98.7%; the NSP3 gene showed the highest homology with the CR231 strain (AB748600.1), at 97.5%; and the NSP5 gene showed high homology with the JS31 strain (PQ332923.1) and HeB1 (PQ332904.1), both at 99.7%. Genetic evolution analysis showed that 11 gene segments of the HSX252 strain clustered in the same evolutionary branch as bovine rotavirus strains, indicating that this strain belongs to the typical bovine G10P

[11] type bovine rotavirus.

[0040] The results showed that the HSX252 strain has a stable and complete genome structure. Its genotype is G10-P

[11] -I2-R2-C2-M2-A3-N2-T6-E2-H3. Eleven gene sequences have been uploaded to NCBI GenBank with accession numbers PZ660301-PZ660311. It can be used as a standard reference strain for bovine rotavirus etiology research, genetic evolution analysis and vaccine evaluation research.

[0041] Table 8 Primer sequences for whole-genome amplification

[0042] Example 4 The antigenicity analysis of the HSX252 strain follows these steps: Five classic algorithms—Kolaskar-Tongaonkar antigenicity, Parker hydrophilicity, Chou-Fasman β-turn, Karplus-Schulz flexibility, and Emini surface accessibility—were used to predict the antigenicity of the amino acid sequences of VP4 and VP7 proteins in the HSX252 strain. Through comprehensive interpretation of the multi-parameter prediction maps, such as… Figure 29 As shown in Table 9, several dominant antigenic epitope regions of the VP4 and VP7 proteins were identified. These predicted epitope regions are located in... Figure 29 It exhibits a significant positive peak with highly overlapping multiple prediction curves and is rich in hydrophilic, charged and polar amino acid residues, demonstrating excellent surface exposure and immunogenicity.

[0043] Table 9. Dominant antigenic epitope regions of VP4 and VP7 proteins in HSX252 strain.

[0044] Example 5 The pathogenicity test of the HSX252 strain follows these steps: Twenty 5-7 day old suckling mice were randomly divided into an infection group and a control group. Each suckling mouse in the infection group was injected with 100 μL of F10 generation virus solution by gavage, while each suckling mouse in the control group was injected with 100 μL of DMEM culture medium by gavage. The fecal condition of the suckling mice was observed and recorded daily.

[0045] After being challenged with the virus, the suckling mice developed diarrhea, such as... Figure 30 As shown, yellow, watery feces adhered to the perianal area and tail base. The feces were thin in consistency, and the skin around the anus was moist and visibly contaminated. BRV VP6 PCR detection of the feces in suckling mice was performed. Figure 31 This further confirms viral infection. The above results directly reflect the pathogenicity of the HSX252 strain, providing a reliable reference for subsequent experimental research.

[0046] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A stable subcultureable G10P[11] bovine rotavirus HSX252 strain, characterized in that, The HSX252 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 47073.

2. The G10P[11] bovine rotavirus HSX252 strain that can be stably passaged according to claim 1, characterized in that, The HSX252 strain was able to proliferate stably in MA104 cells and be continuously passaged up to the 20th generation.

3. The G10P[11] bovine rotavirus HSX252 strain that can be stably passaged according to claim 2, characterized in that, The viral titer of the HSX252 strain in MA104 cells was 10. 5.50 ~10 6.80 TCID 50 / mL.

4. The G10P[11] bovine rotavirus HSX252 strain that can be stably passaged according to claim 1, characterized in that, The complete genome genotype of the HSX252 strain is G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H3.

5. The G10P[11] bovine rotavirus HSX252 strain that can be stably passaged according to claim 1, characterized in that, The VP4 protein of the HSX252 strain has one or more dominant antigenic epitope regions as shown in SEQ ID NO:50-53; the VP7 protein of the HSX252 strain has one or more dominant antigenic epitope regions as shown in SEQ ID NO:54-57.

6. The application of the stable passageable G10P[11] bovine rotavirus HSX252 strain as described in any one of claims 1-5 in the establishment of a bovine rotavirus isolation and culture system.

7. The application of the stable subcultured G10P[11] bovine rotavirus HSX252 strain as described in any one of claims 1-5 in constructing a bovine rotavirus phylogenetic tree.

8. The use of the stable passageable G10P[11] bovine rotavirus HSX252 strain as described in any one of claims 1-5 in the preparation of a kit for constructing a bovine rotavirus animal pathogenic model.

9. The application according to claim 8, characterized in that, The animal in question is a suckling mouse.

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