Rotavirus reassortment attenuated strain LG12 and application thereof

Abstract

The invention discloses a rotavirus reassortment attenuated strain LG12 and application of the rotavirus reassortment attenuated strain LG12. The rotavirus reassortment attenuated strain LG12 comprises: i) a coding gene of a structural protein VP7 of a human G12 type rotavirus strain G12P [6]; and ii) coding genes of structural proteins VP1, VP2, VP3, VP4 and VP6 and non-structural proteins NSP1, NSP2, NSP3, NSP4 and NSP5 of the sheep rotavirus strain LLR. The reassortment strain provided by the invention has the safety of animal strains and the immunogenicity of human G12 type RV, can effectively prevent infection of human G12 type wild strains, and can have different degrees of cross protection on infection of other various G type RV wild strains; the method has great significance for developing RV vaccines which cover more G and P genotypes of Chinese epidemic RVA, have stronger prevention pertinence and provide a better protection effect on Chinese infantile diarrhea.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of classical gene reassortment, and particularly relates to a rotavirus reassortant attenuated strain LG12 and application. BACKGROUND

[0002] Rotavirus gastroenteritis (RVGE) is a global common infectious disease caused by rotavirus (RV) infection. RVGE is highly prevalent in children under 5 years old, and almost every child is infected with RV at least once before the age of 3-5 years. From 2016 to 2020, 89.7% of children hospitalized due to gastroenteritis in China were caused by RV [1] . Population-based surveillance shows that the annual incidence of RV gastroenteritis in children under 5 years old is 50 / 1000, accounting for about 20% of all etiological gastroenteritis in children [2] . RV is a double-stranded RNA virus belonging to the Reoviridae family, and its genome consists of 11 double-stranded RNA segments, encoding structural proteins VP1-4, VP6, and VP7. VP4 determines the P genotype, and VP7 determines the G genotype. The main RV genotypes that infect humans worldwide include G9P[8], G8P[8], G3P[8], G1P[8], G2P[4], G4P[8], etc. [3-4] The G12 type RV was first discovered in the Philippines in 1987, and no other cases were found until it was reported in 1998 [5] . In the 1990s, the se585 strain was reported in the United States [6] , and the G12P[9] strain Arg720 was detected in Argentina in 1999 [7] . From 2002 to 2008, G12-type RV spread and prevailed in Asian countries [8-20] . From 2009 to 2018, the prevalence of G12-P[8] / P[6] human rotavirus increased globally, and was reported in the United States

[21] , Nigeria

[22] , St. Louis

[23] , Nepal

[24] , Brazil

[25] , Bangladesh

[26] , and Mozambique

[27] . Currently, G12-type RV is recognized as the sixth important genotype associated with human infection [28-30] , and in 2019, the first G12P[6] strain was discovered in Wuhan, China

[31] , indicating that the virus has the potential to spread in China.

[0003] Currently, RV vaccination is one of the most effective and cost-efficient methods for preventing RVGE. While the mainstream Rotarix and RotaTeq vaccines are more effective for children in Europe and America, their effectiveness and cost-effectiveness in developing countries in Asia and Africa are relatively low.

[32] Domestically produced RV vaccines, including LLR® and LLR3® developed by Lanzhou Institute of Biological Products Co., Ltd., and Wusheng Erlunbao developed by Wuhan Institute of Biological Products Co., Ltd., have been approved for marketing and use. To date, none of the globally registered oral attenuated RV vaccines contain the G12 strain; therefore, China currently lacks an ideal vaccine strain that can effectively prevent human G12 wild-type virus infection. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides a rotavirus reassortant attenuated strain LG12 and its application. Based on the first G12P[6] type RV positive fecal sample obtained in China in 2024, this invention successfully isolated and purified the RV G12P[6] strain, which can be stably passaged in vitro. A safe and efficient gene reassortant attenuated strain was constructed using this G12P[6] wild-type strain and the sheep LLR strain as a vaccine candidate.

[0005] In one aspect, the present invention provides a rotavirus reassortant attenuated strain LG12, comprising: i) the gene encoding the structural protein VP7 of human G12 rotavirus strain G12P[6]; and ii) the genes encoding the structural proteins VP1, VP2, VP3, VP4, VP6 and the non-structural proteins NSP1, NSP2, NSP3, NSP4, NSP5 of sheep rotavirus strain LLR; the human G12 rotavirus strain G12P[6] is deposited under the accession number CCTCC NO:V202577.

[0006] In some implementations, the rotavirus reassortant attenuated strain LG12 is preserved under the accession number CCTCC NO: V202578.

[0007] In some implementations, the method for constructing the rotavirus reassortant attenuated strain LG12 includes: co-infecting monolayer MA-104 cells with human G12 type rotavirus strain G12P[6] and sheep rotavirus strain LLR; harvesting the mixed culture when the monolayer MA-104 cells are ≥75% cytopathic; and screening the target gene reassortant strain from the mixed culture by limiting dilution and plaque assay.

[0008] In another aspect, the present invention provides the application of the aforementioned rotavirus reassortant attenuated strain LG12 in the preparation of rotavirus vaccines.

[0009] In some implementations, rotavirus vaccines are used to prevent disease caused by infection with wild-type human rotavirus strains of type G12, G1, G2, G4, or G9.

[0010] In another aspect, the present invention provides the application of the aforementioned rotavirus reassortant attenuated strain LG12 in the preparation of rotavirus diagnostic reagents.

[0011] In another aspect, the present invention provides a vaccine comprising an immunogenically effective amount of the aforementioned rotavirus reassortant attenuated strain LG12.

[0012] In some implementations, the vaccine also contains at least one acceptable adjuvant.

[0013] In some implementations, the vaccine also contains at least one pharmaceutically acceptable carrier or diluent.

[0014] In some implementations, the vaccine is available in oral or injectable form.

[0015] In another aspect, the present invention provides a diagnostic reagent containing the aforementioned rotavirus reassortant attenuated strain LG12.

[0016] The beneficial effects of this invention are as follows: This invention successfully constructed a rotavirus reassortant attenuated strain LG12 with sheep rotavirus strain as the backbone, containing human G12-VP7 gene and capable of stable passage in Vero cells. This reassortant attenuated strain carries the VP7 gene of human G12 type rotavirus strain G12P[6] (which stimulates human G12 type specific neutralizing antibody), and the other 10 gene fragments are all from sheep rotavirus strain LLR. It has both the safety of animal strains and the immunogenicity of human G12 type RV, which can effectively prevent infection of human G12 type wild virus strains, and can have different degrees of cross-protection against infection of various G type RV wild virus strains including G1, G2, G4 or G9. It is of great significance for developing RV vaccines that cover more G and P genotypes of RVA prevalent in China, have stronger preventive targeting, and provide better protection against infant diarrhea in China. Attached Figure Description

[0017] Figure 1A Photographs of MA-104 cells infected with G12P[6] for 5 days (×100); Figure 1B Photographs of MA-104 cells infected with G12P[6] for 6 days (×100); Figure 1C Photograph of MA-104 cells after 6 days of culture (×100). Figure 2A Photographs of Vero cells infected with G12P[6] for 5 days (×100); Figure 2B Photographs of Vero cells infected with G12P[6] for 6 days (×100); Figure 2C Photograph of Vero cells after 6 days of culture (×100). Figure 3 G12P[6] forms plaques on MA-104 cells; Figure 4 The nucleic acid banding pattern of the G12P[6] virus RNA-PAGE genome; A is G12P[6] (MA-104 cells); L is LLR control; B is G12P[6] (Vero cells); Figure 5 The RNA-PAGE genome of the gene reassortment mixture is shown as the nucleic acid banding pattern; lanes 1-5 are the gene reassortment mixtures; G12 is the G12P[6] control; L is the LLR control; Figure 6 The plaques formed on MA-104 cells by gene reassortment mixtures; Figure 7 The nucleic acid banding patterns of RNA-PAGE genomes of different clones; lanes 1-10 represent different clones; G12 is the G12P[6] control; L is the LLR control; Figure 8 The nucleic acid banding of the RNA-PAGE genome of the LG12 reassortant attenuated strain; lanes 1 and 2 are MA-104 virus harvest fluid, lanes 3 and 4 are Vero virus harvest fluid, G12 is G12P[6] control; L is LLR control; Figure 9 Agarose gel electrophoresis image of PCR products; lanes 1 and 2, cDNA from fragment 1; lanes 3 and 4, cDNA from fragment 2; lanes 5 and 6, cDNA from fragment 3; lanes 7 and 8, cDNA from fragment 4; lane 9, cDNA from fragment 6; lane 10, cDNA from fragment 9; lane 11, cDNA from fragment 5; lane 12, cDNA from fragment 7; lane 13, cDNA from fragment 8; lane 14, cDNA from fragment 10; lane 15, cDNA from fragment 11. Figure 10A The amino acid sequence consistency scores for VP7 and VP4 fragments of LLR, LG12, and G12 are shown. Figure 10B The similarity score results for the amino acid sequences of VP7 and VP4 fragments of LLR, LG12, and G12; Figure 11 The amino acid sequence alignment results for the VP7 fragments of LG12 and G12; Figure 12 The amino acid sequence alignment results for the VP4 fragment of LG12 and LLR; Figure 13A LG12 Mycoplasma test results - VIC quality control channel; Figure 13B For LG12 mycoplasma test results - FAM detection channel; Figure 14A Photographs of MA-104 cells 6 days after LG12 infection (×100). Figure 14B Photograph of MA-104 cells after 6 days of culture (×100). Figure 15A Photographs of Vero cells 6 days after LG12 infection (×100). Figure 15B Photograph of Vero cells after 6 days of culture (×100). Figure 16 The replication curve of LG12 virus; Figure 17A The image shows the RNA-PAGE pattern of LG12 passaged in MA-104; lanes 1-20 contain the LG12 virus harvested from 1-20 consecutive passages; G12 is the G12P[6] control; L is the LLR control; Figure 17B The RNA-PAGE pattern of Vero passaged LG12 is shown; lanes 1-20 are the LG12 virus harvested from 1-20 consecutive passages; G12 is the G12P[6] control; L is the LLR control; Figure 18 The viral titers of LG12 at different passages in two cell cultures; Figure 19 The average body temperature change of mice in each group within 56 days after immunization; Figure 20 The mean body weight change of mice in each group within 56 days after immunization; Figure 21A The results are from an ELISA assay for IgG antibody response in mouse serum, where “****” indicates a highly statistically significant difference. Figure 21B The results are from an ELISA assay for IgA antibody response in mouse serum. "***" indicates a statistically significant difference, and "****" indicates an extremely statistically significant difference. Figure 22A The results show the serum neutralizing antibody titers of LG12-immunized mice, where "****" indicates a highly statistically significant difference. Figure 22BThe results of cross-neutralizing antibody titers between serum from LG12-immunized mice and other G-type RV wild-type strains; Figure 23 Results of SIgA titer assay in fecal samples from LG12-immunized mice; Figure 24 The mean body temperature changes of mice in each group within 15 days after challenge. Figure 25 The mean body weight change of mice in each group within 15 days after challenge. Figure 26 The results show the SIgA titer in fecal samples from each group of mice within 15 days after challenge. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Materials and Methods 1. Cells: Rhesus monkey kidney cells (MA-104, P30); African green monkey kidney cells (Vero, P135) were preserved and provided by the Viral Vaccine Project Team of the Project Management Office of Lanzhou Institute of Biological Products Co., Ltd.

[0020] 2. Virus strains and G12P[6] samples: Sheep strains LLR, G1, G2, G3, G4 and G9 human RV were all preserved and provided by the Viral Vaccine Project Team of the Project Management Office of Lanzhou Institute of Biological Products Co., Ltd.; G12P[6] virus positive fecal samples were collected and provided by Wuhan Center for Disease Control and Prevention (CDC).

[0021] 3. Major Reagents and Instruments: RPMI-1640, MEM, and trypsin were purchased from Gibco (USA); newborn calf serum was purchased from Lanzhou Rongye Biotechnology Co., Ltd.; low-melting-point agarose, horseradish peroxidase (HRP)-labeled anti-mouse IgG (H+L), and anti-mouse IgA were purchased from Sigma (USA); the total RNA extraction kit RNeasyMini Kit and the Agarose Gel DNA recovery kit were purchased from Qiagent (Germany); the One-Step PrimeScript RT-PCR Kit (Perfect Real...) was also purchased. The Time-based ELISA kit was purchased from TaKaRa Corporation, Japan; the rotavirus ELISA kit was provided by the Diagnostic Supplies Department of Lanzhou Institute of Biological Products Co., Ltd. (National Drug Approval Number: S10870016); the Mycoplasma DNA Extraction and Purification Kit (2G) (Magnetic Bead Method) (Ⅰ), Mycoplasma DNA Extraction and Purification Kit (2G) (Magnetic Bead Method) (Ⅱ), Mycoplasma DNA Extraction and Purification Kit (2G) (Magnetic Bead Method) (Ⅲ), and Mycoplasma DNA Detection Kit (2G) (PCR-Fluorescent Probe Method) were all purchased from Huzhou Shenke Biotechnology Co., Ltd.; the fluorescence cell analyzer was purchased from Shanghai Ruiyu Biotechnology Co., Ltd.; the CO2INC-108 incubator was purchased from Memmert GmbH, Germany; the XD-30 inverted microscope was purchased from Ningbo Sunny Instruments Co., Ltd.; the SpectraMax M3 microplate reader was purchased from Molecular Corporation, USA; the S1000 Thermal Cycler PCR amplification instrument was purchased from BIO-RAD Corporation, USA; and the ImageQuant LAS 4000 gel imaging system was purchased from General Electric (GE), USA.

[0022] Example 1: Isolation of G12P[6] virus strain 1. MA-104 cell culture: Remove MA-104 cell cryovials from liquid nitrogen and thaw them in a 37°C water bath. Culture MA-104 cells in RPMI-1640 (containing 10% newborn calf serum) for 24 hours, then replace with the corresponding culture medium (containing 5% newborn calf serum). Continue culturing for 72 hours. Once the cells have grown to a monolayer, discard the growth medium and digest the cells with 0.25% Trypsin-0.02% EDTA at a rate of 5 × 10⁻⁶ cells / mL. 4 T25 cell culture flasks were seeded at a concentration of 10 mL / flask and incubated at 37°C for 96 h. The cells were observed to form a monolayer under a microscope. MA-104 cells were washed twice with RPMI-1640 and then used for further processing.

[0023] 2. Isolation and culture of G12P[6] virus strain: G12P[6] type RV positive fecal specimens were resuspended in PBS, vortexed until the feces dispersed, centrifuged, and the supernatant was sterilized by filtration through a 0.22μm filter. 400μL was placed in a centrifuge tube, and trypsin (concentration: 300μg / mL) was added to a final concentration of 15μg / mL. After treatment in a 37.0℃ water bath for 60min, the supernatant was inoculated into T25 cell flasks that had grown into monolayers of MA-104 cells. The cells were adsorbed at 37.0℃ for 1h, the supernatant was discarded, and trypsin and Plasmocin were added. TM Treatment with RPMI-1640 culture medium (final concentration of trypsin 0.5 μg / mL, Plasmocin) TM The final concentration of the treatment was 25 μg / mL. The cells were cultured in a 5% CO2 incubator at 36.5℃ for 5-7 days. The cell status and cytopathic effect (CPE) were observed daily. When the enzyme-linked immunosorbent assay (ELISA) was positive and CPE ≥ 75%, the cells were harvested and the G12P[6] virus isolate was initially obtained. The harvested solution was passaged for 5 generations using the same method. The 5th generation (P5) was passaged to T75-FLASK, and the virus titer was determined. The P5 harvested solution was purified by plaque cloning.

[0024] 3. Plaque cloning and purification of G12P[6] virus strain: Take the P5 harvested solution of G12P[6] virus strain and perform a 10-fold serial dilution. 3 ~10 8Each dilution was seeded into a 6-well plate containing MA-104 cells, with 0.9 mL seeded per well and two replicates per dilution. After seeding, the plate was incubated at 37.0℃ in a 5% CO2 cell culture incubator for 4 hours. The supernatant was discarded, and 2 mL of 1.0% low-melting-point agarose prepared with RPMI-1640 was added to each well. After solidification at room temperature, the plate was incubated at 37.0℃ in a 5% CO2 cell culture incubator for 5 days. Then, 2 mL of 1.0% low-melting-point agarose prepared with RPMI-1640 containing neutral red was added to each well. After solidification at room temperature, the plate was incubated at 37.0℃ in a 5% CO2 cell culture incubator, and plaques were observed from 4 to 24 hours. Pick a single plaque from the high dilution wells and inoculate it onto MA-104 cells in a 24-well plate. Culture at 37.0℃ and 5% CO2 for 6 days. Harvest when CPE ≥ 75%. After ELISA detection, clone the sample from the strongest positive well again. Repeat the cloning process 5 times. Select the clone strain with the highest viral titer for amplification culture. Complete the strain identification, gene sequencing identification and viral purity check. Complete the adaptation culture and various tests on Vero cells and preserve the strain. The G12P[6] virus strain is preserved at the China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit date: October 31, 2025, deposit number: CCTCC NO: V202577, classification name: Human rotavirus strain G12P[6].

[0025] 4. Determination and analysis of whole genome nucleotide sequence: Whole genome sequencing identification was performed using next-generation sequencing (the specific process was completed by Shanghai Berger Medical Technology Co., Ltd.).

[0026] 5. Results: 5.1 Morphological changes of G12P[6] cells during culture: The filtrate of G12P[6] positive clinical samples was passaged in MA-104 cells. Under a microscope, obvious CPE was observed in the cells. The infected cells became hypertrophic and elongated. After 6 days of culture, the intercellular spaces became loose, and most cells shrank, lysed and detached, forming large patches of empty plaques. The detached cells aggregated into a large number of suspended clumps ( Figure 1A , Figure 1B The control group cells grew well, and no changes were observed in the cells. Figure 1C ). G12P[6] isolates were inoculated into a monolayer of Vero cells, and CPE was observed under a microscope starting from P2. With prolonged culture time, the cells began to shrivel and lyse at 5 days. Figure 2A At 6 days, some cells elongated. Figure 2B The culture supernatant showed reduced light transmittance; the control group cells grew well, and no CPE was observed. Figure 2C ).

[0027] 5.2 Plaque purification results of G12P[6] isolate: The plaque test results showed that G12P[6] formed typical plaques after adsorbing on MA-104 cells for 4 h and culturing at 37℃ with 5% CO2 for 5 d. The plaques were round or nearly round with neat edges and clear transparency. The number of plaques gradually decreased with the increase of virus dilution. Figure 3 Single plaques were selected for amplification and culture. After ELISA detection, the strongest positive wells were cloned again. This cloning process was repeated 5 times. The single plaque formed at the highest dilution was selected to obtain a high-titer clone strain, which was then further amplified and cultured to preserve the strain.

[0028] 5.3 Polyacrylamide gel electrophoresis assay of G12P[6] viral RNA: The results showed that the electrophoretic pattern of G12P[6] was a unique group A rotavirus pattern (11 bands arranged in a manner similar to LLR, showing a typical 4:2:3:2): 4 high molecular weight dsRNA segments (gene fragments 1-4); 5 medium-sized segments (gene fragments 5-9), of which 7-9 together form a characteristic trisomy; 2 small fragments (gene fragments 10 and 11), and the electrophoretic patterns of the virus cultured in Vero cells and MA-104 cells were consistent. See Figure 4 .

[0029] 5.4 Determination and sequence analysis of the whole genome nucleotide sequence of G12P[6]: The results of second-generation sequencing showed that all 11 full-length gene fragments of G12P[6] were in a large branch with group A RV, belonging to human rotavirus, and were homologous with the G12P[6] strain uploaded to GenBank by Wuhan CDC in 2019, as shown in Table 1.

[0030] Table 1. Alignment results of 11 full-length gene sequences with GenBank accession sequences.

[0031] Example 2: Construction of the LG12 strain 1. Classical gene reassortment 1.1 MA-104 cell culture: After MA-104 cells have grown to a monolayer, discard the growth medium, digest the cells with 0.25% Trypsin-0.02% EDTA, and culture at a rate of 5 × 10⁻⁶ cells / year. 4 Inoculate 24-well plates at a concentration of 1 mL / well and incubate at 37°C for 96 h. After observing the cells forming a monolayer under a microscope, wash twice with RPMI-1640 medium and set aside.

[0032] 1.2 Reassortment of G12P[6] with LLR viral genes: Trypsin with a final concentration of 15 μg / mL was added to the parental strains G12P[6] and LLR, respectively, and incubated in a water bath at 37.0℃ for 60 min. Then, a 10-fold serial dilution was performed, starting from dilution 10. 4 For inoculation, two strains were mixed and used to infect MA-104 cells. The cells were incubated at 37.0℃ with 5.0% CO2 for 2 hours, gently shaken once every 20 minutes, and the adsorbed solution was discarded after 2 hours. The cells were washed twice with RPMI-1640 solution, and serum-free RPMI-1640 culture medium containing 0.5 μg / mL was added to 1 mL / well. The cells were then incubated at 37.0℃ with 5.0% CO2. The mixed culture was harvested when the CPE was observed to be ≥75% under a microscope.

[0033] 2. Isolation and screening of reassortant attenuated strains 2.1 Isolation of reassortant attenuated strains: The target strain was isolated using the plaque assay and limiting dilution method, i.e., the mixed culture was serially diluted 10-fold to 10^ ... 9 Dilute to 10 times 4 ~10 9 Virus solution was inoculated into 6-well plates containing MA-104 cells, with two replicates per dilution. 0.9 mL was inoculated into each well and incubated at 37.0℃ with 5% CO2 for 4 hours. The supernatant was discarded, and 2 mL of 1% low-melting-point agarose (RPMI-1640) was added per well. After solidification at room temperature, the plates were incubated. After 4-5 days, based on the CPE (Critical Positive Precipitation), 2 mL of 1% low-melting-point agarose (RPMI-1640) containing neutral red was added per well. After solidification at room temperature, the plates were incubated for 4-24 hours, observing for plaques. Individual plaques were picked and seeded onto 24-well MA-104 cells, one plaque per well. The plates were incubated at 37.0℃ with 5% CO2 for 4-6 days. Harvesting was performed when CPE ≥ 75%, and samples were collected for analysis.

[0034] 2.2 RNA-PAGE detection: Plaques were transferred to the harvested medium in a 24-well plate, and 400 μL was extracted from each well. Viral genomic RNA was extracted using the phenol-chloroform extraction method. Electrophoresis was performed according to the standard polyacrylamide gel electrophoresis (PAGE) procedure. The gel concentration was 10%, and the sample loading volume was 10 μL / lane. G12P[6] and LLR lanes were added as controls. The target gene reassortant progeny strains were initially selected with the VP7 band consistent with the G12P[6] control and the other bands consistent with the LLR strain. The target gene reassortant progeny strains were cloned again. This cloning was repeated 6 times. The positive well with the strongest OD value detected by ELISA in the 6th plaque clone was selected, enlarged, cultured, and the virus was preserved after passing the test. The target gene reassortant attenuated strain was named LG12. Deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on October 31, 2025, accession number: CCTCC NO:V202578, classified and named as LG12 Rotavirus reassortant attenuated strain.

[0035] 3. Results 3.1 RNA-PAGE electrophoresis of the gene reassortment mixture: The results showed that the mixture contained all gene segments of both viral strains, indicating that the two strains can coexist in a suitable ratio. (See [link to relevant documentation]). Figure 5 .

[0036] 3.2 Plaque purification of reassortant attenuated strains: Plaque assay results showed that the virus mixture formed typical plaques on MA-104 cells, which were round or nearly round with neat edges and clear transparency; the number of plaques gradually decreased with increasing viral dilution. Figure 6 Single plaques were selected and cultured in 24-well plates. After ELISA detection, the culture medium from the positive wells was sampled and RNA-PAGE was used for preliminary identification of the virus strain.

[0037] 3.3 Preliminary screening of monoclonal reassortant attenuated strains: Based on the band positions in the RNA-PAGE map, clones in lanes 7, 9, and 10 were selected as the target reassortant attenuated strains. Their VP7 gene band was consistent with G12P[6], and the other bands were consistent with LLR. See Figure 7 After six consecutive rounds of plaque purification, a pure reassortant attenuated strain was obtained.

[0038] Example 3: Detection of LG12 reassortant attenuated strain 1. Virus titer determination: After freezing and thawing, the virus titer was determined according to the efficacy test section of the 2000 edition of the "Chinese Biological Products Regulations" (2002 supplement), using the cell culture infectious dose (CCID) 50%. 50 Viral titers were determined using ELISA and the Karber method to calculate the corresponding lgCCID. 50 / mL.

[0039] 2. RNA-PAGE assay: Take 400 μL of LG12 virus solution, extract viral genomic RNA using phenol-chloroform extraction method, detect viral nucleic acid banding by PAGE and silver staining, and preserve dry gel.

[0040] 3. Amplification and sequencing of 11 gene fragments: Based on the RV gene sequence in GenBank, specific primers for each gene fragment were designed using Primer 5.0 software. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. 120.0 μL of LG12 virus solution was used to extract total RNA according to the instructions of the total RNA extraction kit, followed by reverse transcription-polymerase chain reaction (RT-PCR). Following the instructions of the reverse transcription kit, a 25.0 μL system was used: 10.0 μL of RNA template was added, followed by 12.5 μL of one-step buffer (2×), 1.0 μL each of the upstream and downstream primers, and 0.5 μL of the One-Step PrimeScript RT-PCR Kit, for a total of 25.0 μL. The following program was used in a PCR amplification instrument: reverse transcription at 48℃ for 30 min, pre-denaturation at 94℃ for 2 min, denaturation at 94℃ for 30 sec, annealing at 52℃ for 30 sec, annealing at 68℃ for 2 min, amplification for 40 cycles, and extension at 68℃ for 5 min. After the reaction, 5.0 μL of the PCR amplification product was subjected to electrophoresis on a 1% agarose gel at 120V for 30 min, and the pattern was observed and saved. The PCR product was purified by gel electrophoresis and then sequenced.

[0041] 4. Whole genome sequencing identification: LG12 virus P5 harvest fluids from MA-104 cells and Vero cells were subjected to second-generation whole genome sequencing (the specific process was completed by Shanghai Berger Medical Technology Co., Ltd.), and homology analysis was performed on the sequencing results.

[0042] 5. Sequence alignment of antigen proteins: The nucleic acid sequences of VP7 and VP4 of RV G12P[6], LG12 and LLR were aligned using the Expasy Translate tool.

[33] (https: / / web.expasy.org / translate / ) was translated into an amino acid sequence using ESPript3 (https: / / espript.ibcp.fr / ESPript / ESPript / index.php).

[34] Multiple sequence alignment and alignment results were performed using the msa package in R (version 4.4.2).

[35] The pheatmap package is used to visualize the results of calculating sequence consistency and similarity.

[0043] 6. Sterility test: Perform sterility test according to the 2020 edition (Part III) of the Chinese Pharmacopoeia.

[0044] 7. Mycoplasma test: The MycoSHENTEK® Mycoplasma DNA Extraction and Purification Kit (2G) (Magnetic Bead Method) is used in conjunction with the MycoSHENTEK® Detection Kit (2G). Step 1: Extract trace amounts of mycoplasma DNA from the sample to be tested. First, prepare reagents and instruments: Prepare various liquids and related instruments according to the kit instructions. Second, sample processing: (1) Cell removal: Centrifuge 1 mL of LG12 sample at 72×g for 5 min, remove cell pellet, and transfer the supernatant to a new centrifuge tube. (2) Concentration centrifugation: Centrifuge the transferred sample supernatant at 18000×g for 30 min, discard part of the supernatant, and keep 400 μL. (3) Sample processing solution: Add sample processing solution (sample to processing solution volume ratio of 10:1) to the concentrated sample, vortex to mix, treat at 25℃ for 5 min, and centrifuge briefly for 3 sec. The same method is used to process the negative control sample (NCS) and the positive control (PCS). (4) Sample digestion: Add 10 μL of 5M NaCl and 10 μL of IC to all samples, vortex to mix, and centrifuge briefly for 3 seconds. Add 20 μL of proteinase K and 100 μL of lysis buffer to all samples, vortex to mix, centrifuge briefly for 3 seconds, then incubate at 55°C for 60 min. Add 200 μL of working binding buffer, vortex to mix, and centrifuge briefly for 3 seconds. Add 200 μL of isopropanol and 30 μL of magnetic beads to the sample mixture, vortex for 5 min, centrifuge for 10 seconds, and then place on a magnetic separator for 3-5 min. Carefully remove the supernatant with a pipette tip. Remove the centrifuge tube from the magnetic separator and wash it successively with 700 μL of washing buffer A and washing buffer B, vortexing for 30 seconds to mix the magnetic beads and washing buffer. After rapid centrifugation for 10 seconds, return the centrifuge tube to the magnetic separator. Once the magnetic beads have completely separated, remove the supernatant with a pipette tip to complete the washing process. Remove the centrifuge tube from the magnetic separator, open the cap, and dry at room temperature for 30 seconds to 3 minutes to remove residual ethanol. Add 50 μL of preheated elution buffer, vortex for 5 seconds to mix the magnetic beads and elution buffer, and incubate in a 70°C metal bath for 7 minutes. Centrifuge the centrifuge tube rapidly for 1 minute, and place it on the magnetic separator. After the magnetic beads have separated, carefully transfer the solution to a clean centrifuge tube with a pipette tip. This process can be repeated once to obtain the purified sample solution. The second step involves PCR-fluorescent probe detection. Prepare the qPCR reaction solution according to the kit instructions. Calculate the required number of reaction wells based on the number of samples to be tested; generally, two replicate wells are used. Number of reaction wells = (1 positive control PC + 1 template-free control NTC + 1 negative control NCS + 1 positive control PCS + N test samples) × 2; Calculate the total amount of MIX required based on the number of reaction wells: MIX = (number of reaction wells + 2) × 10 μL (including the loss from 2 wells).After thoroughly mixing all solutions by vortexing, add the samples to eight-tube strips according to the pre-defined layout. Include one positive control (PC), one template-free control (NTC), one negative control (NCS), and one positive control (PCS). The sample volume is 30 μL per well. Perform two replicates for each assay. After adding the samples, seal the eight-tube strips tightly with the provided caps and place them in a PCR tube shaker to mix. Centrifuge briefly for 10 seconds and then transfer to a qPCR instrument. Set the three-step reaction program: 25℃ UNG enzyme for 10 min; 95℃ pre-denaturation for 10 min; 95℃ for 15 sec, 62℃ for 30 sec, 72℃ for 1.5 min (read fluorescence), 45 cycles, reaction volume 30 μL. Result determination: The sample containing phosphoramide (VIC) channel CT value ≤35 and the amplification curve shows effective "S"-shaped amplification, the hydroxyfluorescein (FAM) channel CT value >40 and the amplification curve has no obvious peak, and both the negative and positive controls meet the expectations, the result is determined to be negative for mycoplasma.

[0045] 8. Results: 8.1 Virus titer determination: Calculated using the Karber method formula: lgCCID 50 = L - d ( s -0.5), where L =Logarithm of the highest dilution d =The difference between the logarithms of dilution, s =The total percentage of positive tubes, the viral titer of LG12 was 7.25~8.50 lg CCID. 50 / mL.

[0046] 8.2 RNA-PAGE assay: PAGE showed that LG12 was elongated, with 11 bands exhibiting the unique 4:2:3:2 arrangement of group A rotavirus. Only the VP7 gene segment was parallel to G12P[6]-VP7, while the remaining segments were parallel to the LLR segment. The electrophoretic patterns of the virus cultured in Vero cells and MA-104 cells were consistent, and the bands were all very clear. See Figure 8 .

[0047] 8.3 RT-PCR products of 11 gene fragments of LG12: RT-PCR amplification products were analyzed by 1% agarose gel electrophoresis. Specific target bands for each fragment were visible, and their sizes were consistent with expectations. (See attached image) Figure 9 .

[0048] 8.4 Whole-genome sequencing identification: The sequence of VP1 is shown in SEQ ID NO: 1, the sequence of VP2 is shown in SEQ ID NO: 2, the sequence of VP3 is shown in SEQ ID NO: 3, the sequence of VP4 is shown in SEQ ID NO: 4, the sequence of NSP1 is shown in SEQ ID NO: 5, the sequence of VP6 is shown in SEQ ID NO: 6, the sequence of NSP3 is shown in SEQ ID NO: 7, the sequence of NSP2 is shown in SEQ ID NO: 8, the sequence of VP7 is shown in SEQ ID NO: 9, the sequence of NSP4 is shown in SEQ ID NO: 10, and the sequence of NSP5 is shown in SEQ ID NO: 11. The results of second-generation sequencing showed that among the 11 full-length gene fragments of the LG12 virus strain, VP7 and G12P[6] had 99.53% homology, and the remaining fragments had ≥99.47% homology with sheep RV. This indicates that the LLR-VP7 gene and the G12P[6]-VP7 gene have been exchanged, and a human-sheep rotavirus gene reassortant strain containing the G12 type VP7 gene was successfully obtained, as shown in Table 2.

[0049] Table 211 shows the alignment results of 11 full-length gene sequences with GenBank accessed sequences.

[0050] 8.5 Comparison of antigen protein amino acid sequences: The results of the comparison between the parental strains LLR and G12P[6] and the gene reassortant strain LG12 showed that the VP7 amino acid sequence of LG12 and G12P[6] was highly consistent, and the amino acid sequence similarity score was significantly higher than that of LG12 and LLR; the VP4 protein sequence of LG12 and LLR was highly consistent, and the amino acid sequence similarity score was significantly higher than that of LG12 and G12P[6]. See Figure 10A , 10B .

[0051] The amino acid sequence alignment results showed that the amino acid sequences of RV G12P[6] and LG12-VP7 were basically identical. Figure 11 This indicates that the RV G12P[6]-VP7 gene and the LLR-VP7 gene fragment have undergone gene reassortment, while the VP4 gene fragment has not undergone gene reassortment. Figure 12 ).

[0052] 8.6 Sterility test: After 14 days of incubation, the positive control tube was turbid, while the test sample (LG12 virus suspension) tube and the negative control tube were clear. The results indicated that the LG12 sterility test was qualified.

[0053] 8.7 Mycoplasma testing: Real-time quantitative PCR results showed that the quality control results were normal, and the CT value of mycoplasma in the sample was >45, which was considered negative (see Table 3).Figure 13A , Figure 13B .

[0054] Table 3 Results of Real-time Quantitative Detection of Fluorescence

[0055] Example 4: Adaptive culture of LG12 on Vero cells 1. Vero cell culture: Vero cells (passage 135) were removed from liquid nitrogen and thawed in a 37°C water bath. They were then cultured in MEM (containing 10% newborn calf serum) for 24 hours, followed by replacement with the appropriate culture medium (containing 5% newborn calf serum). The cells were cultured for another 72 hours. Once a monolayer had formed, the growth medium was discarded, and the cells were digested with 0.25% Trypsin-0.02% EDTA at a concentration of 1×10⁻⁶. 5 Cells were seeded at a concentration of 10 mL / mL into T25 cell flasks and incubated at 37°C for 96 h. The cells were observed to form a monolayer under a microscope, washed once with MEM, and then used for further processing.

[0056] 2. Adaptation culture of LG12 on Vero cells: Take 1 mL of LG12 P5 harvest medium from MA-104 cell culture, add 50 μL of 300 μg / mL trypsin to make a final concentration of 15 μg / mL, incubate at 37℃ for 60 min to activate, filter sterilize with a 0.22 μm filter, and inoculate into a bottle of Vero cells that have grown into a monolayer. Incubate at 37.0℃ for 1 h for adsorption, discard the adsorption medium, add MEM maintenance medium to 10 mL (final trypsin concentration of 0.5 μg / mL), and incubate at 36.5℃ in a 5% CO2 incubator. Observe the cell status and CPE daily. Harvest according to CPE after 5-7 days of culture, and use the harvest medium from the previous generation as seed. Repeat this process for more than 5 passages. 3. Results: 3.1 Morphological changes in MA-104 cells infected with LG12: After 3 days of infection with LG12, MA-104 cells were observed under a microscope to be enlarged and elongated. By day 6 of culture, the intercellular spaces became loose, and some cells shrank, lysed, and detached, forming large patches of empty plaques. Figure 14A The light transmittance of the culture supernatant was reduced; the control group cells grew well, and no changes were observed in the cells. Figure 14B ).

[0057] 3.2 Changes in the morphology of Vero cells infected with LG12: Microscopic observation revealed that after 6 days of culture, a large number of LG12-infected Vero cells detached, elongated, and some cells lysed. Figure 15A The detached and lysed cells clumped together and floated, causing decreased light transmittance of the liquid; the control group cells grew well, and no CPE was observed. Figure 15B ).

[0058] Example 5: Proliferation curves of LG12 in MA-104 and Vero cells LG12 virus was cultured continuously for 8 days on MA-104 and Vero cells, respectively, and CCID was obtained. 50 The viral titer of daily samples was detected using a method to plot viral proliferation curves, see [link / reference]. Figure 16 As the number of culture days increased, the viral titer gradually increased, with the highest viral infection titer at 5-8 days. The proliferation trend of LG12 on the two cell types was basically the same.

[0059] Example 6: Study on the passaging stability of LG12 1. The LG12 virus was diluted 30 times and inoculated into MA-104 and Vero cells. It was passaged for 20 generations. Samples were taken from each generation for RNA-PAGE detection. Samples from P5, P7 and P9 were randomly selected every day to detect the virus titer and plot the virus proliferation curve. The virus titer of the single harvest fluid was determined for 20 consecutive generations.

[0060] 2. Results: 2.1 RNA-PAGE analysis showed that the banding patterns of the LG12 viral genome at different passages were consistent in both cell matrices. Figure 17A , Figure 17B .

[0061] 2.2 The results of next-generation sequencing showed that the 11 genes of the virus passaged from the two cell lines were homologous, as shown in Table 4.

[0062] Table 4. Homology comparison results of LG12 gene fragments in two cell cultures

[0063] CCIDs passaged 20 times in two cell types 50 The results showed that the LG12 virus titer remained between 7.25 and 8.25 lg CCID in Vero cells. 50 / mL, the LG12 virus titer was maintained at 7.75~8.75 lg CCID in MA-104 cells. 50 / mL, the viral titer of LG12 in MA-104 cells was 0.50~1.00 lg CCID higher than that in Vero cells. 50 / mL, and maintained good proliferation stability in both cell types, see Figure 18 .

[0064] Example 6: LG12 Immunogenicity Test 1. Mouse Immunization: Thirty 3-week-old BALB / c mice were randomly divided into three groups of 10 each. Blood was collected before immunization. Group 1 was immunized by gavage (simulating oral vaccination) with 200 μL / mouse; Group 2 was immunized by intraperitoneal injection with 200 μL / mouse; Group 3 served as the negative control group and was immunized by gavage with MEM culture medium, 200 μL / mouse. Body temperature and body weight of each mouse were measured daily after immunization. After 28 days, all groups were immunized with the same second dose. Tail blood was collected before each immunization (diluted 4-fold at the time of collection); after immunization, the mice were observed daily for their reaction, mental state, and whether they had symptoms such as ruffled hair, hair loss, and loss of appetite; because rotavirus infection initially presents with symptoms such as watery diarrhea, fever, and vomiting, and diarrhea symptoms can last for 4-5 days, the characteristics of mouse feces were observed 0-7 days after immunization in this experiment. Fecal samples were collected daily starting 4 hours after immunization, and fresh feces were collected every 24 hours. After resuspending and mixing with PBS, the supernatant was collected by centrifugation, and 50 μL of each mouse was extracted. The suspensions of fecal samples from the same mouse from 0-7 days were combined and tested by ELISA. The microplate reader readings were set to zero with the blank wells and each well was read at a wavelength of 450 nm. A positive result was defined as an OD value (S) of the fecal supernatant well / OD value (N) of the negative control well ≥2.1, which determined whether the mouse corresponding to the fecal sample had shed the virus during the observation period. 2. Serum IgG and IgA antibody detection: The titers of serum IgG and IgA antibodies were detected using an indirect ELISA method. Plate coating: LG12 virus antigen (MA-104 cell culture) was diluted to a viral titer of 5.5 g CCI. 50 Coat the microplate with 100 μL / well, incubate at 37°C for 2 h, then at 4°C overnight. Block with 20% newborn calf serum, incubate at 37°C for 2 h, then at 4°C overnight. After drying, store at -20°C for later use. Serially dilute mouse serum (100 μL / well, 2 wells per dilution) and add it to the microplate. Incubate at 37°C for 1 h, wash 5 times with PBST, add 100 μL / well of HRP-labeled anti-mouse IgG (1:2000) or IgA antibody (1:2000), incubate at 37°C for 1 h, wash 5 times with PBST, add 50 μL / well of the chromogenic substrate H2O2 / TMB, incubate at 37°C for 10 min, and then stop the reaction. Detect the OD value using a microplate reader. Antibody titer is determined by the highest serum dilution where the serum OD value (S) / negative serum OD value (N) is ≥2.1 times. The limit of detection (LOD) is defined as the initial dilution factor of serum. All samples below the LOD are assigned a value of 1 / 2 LOD.

[0065] 3. Detection of anti-LG12 neutralizing antibodies: A microneutralization assay (MN) was used, in which MA-104 cells were prepared at a concentration of 1×10⁻⁶ cells / mL.5 Inoculate 96-well plates with a concentration of [number] cells / mL. After 72 hours, allow the plate to form a monolayer, then wash once with washing buffer. Perform serial 2-fold dilutions of the serum to be tested using PBS, starting with a 1:8 ratio and ending at 1:16 384. Different serum dilutions were then compared with trypsin-activated serum and serum diluted to a viral titer of 3.01 g CCID. 50 Mix equal volumes of LG12 virus solution ( / mL) and incubate at 37℃ with 5% CO2 for 1 hour to neutralize. Then, inoculate the mixture into prepared 96-well plates at 100 μL / well for each dilution, with two replicates per sample. Simultaneously, add 3.0 g cCCID to two replicates in the last column of each plate. 50 / mL LG12 virus solution was used as a positive control, blank mouse serum (1:4 diluted serum) as a negative control, and RPMI-1640 as a cell control. The cells were incubated at 37℃ in a 5% CO2 incubator. Results were observed and recorded daily after 2 days, generally for 4-5 days. The cells were then freeze-thawed once at -20℃. The culture medium was then aspirated and processed according to the literature.

[36] ELISA testing was performed, and the highest dilution of serum that could protect 50% of cultured cells from lesions was used as the 50% neutralizing titer (PD) of that serum. 50 Since the volume of mouse serum is limited and retesting is not possible, if the dilution exceeds the upper limit and no endpoint value is detected, the upper limit set in the experiment shall be used for the value. If the dilution is less than 1:8, it shall be judged as negative and the value shall be assigned as 1:4. Cross-neutralizing antibody detection between LG12-immunized mouse serum and other human G-type RV wild-type strains: Serum obtained from LG12-immunized mice was mixed with trypsin-activated serum and diluted to a viral titer of 3.0 lg CCID. 50 / mL of G1, G2, G3, G4, G9 and G12P[6] wild-type viruses were neutralized in vitro, and the neutralization titer of the heterotype virus was determined according to the above operation procedure.

[0066] 4. Immunization and challenge test in mice: All mice were challenged with a viral titer of 5.5 g CCCID on day 28 after the second immunization. 50 / mL G12P[6] wild-type virus solution was administered via gavage to challenge mice (simulating fecal-oral infection), 500μL / mouse. The body temperature, body weight, mental state, diet, and fecal characteristics of the mice were recorded daily for 15 days after challenge. Feces were collected from 4 hours to 15 days, and collected once every 24 hours. The virus shedding status of each group of mice after challenge was detected in the same way as in step 1.

[0067] 5. Mouse intestinal IgA (SIgA) detection: From 4 hours after the first immunization to 28 days after the second immunization (a total of 56 days), and from 4 hours after challenge to 15 days after challenge, 10 fresh fecal samples were collected daily from each mouse and stored at -20℃. The samples were then analyzed at the end of the experiment. The detection method was as follows: The samples were soaked in 1.2 mL PBS for 1 hour, resuspended, centrifuged, and the supernatant was collected. The supernatant was then used to coat an ELISA plate (step 2), and the OD value of IgA antibody in each group of mouse fecal samples was detected in two replicates. A According to the instructions for the rotavirus detection kit (ELISA), a positive result for IgA antibody is determined by a fecal OD value (S) / negative control OD value (N) ≥ 2.1 times.

[0068] 6. Statistical Analysis: OriginPro 2018 software was used to perform statistical analysis on the experimental data of different experimental groups and control groups. P A difference of <0.05 is statistically significant. 7. Results 7.1 Observation results of clinical symptoms in mice after immunization: After two doses of antigen were administered, neither group 1 nor group 2 mice showed any abnormal symptoms such as vomiting, loss of appetite, slow movement, ruffling, hair loss, or diarrhea. There were no significant differences between the mice in the experimental group and the control group during the experiment.

[0069] 7.2 Virus shedding in mice after immunization: ELISA results of resuspended fecal samples showed that RV was not detected in the feces of mice in experimental groups 1 and 2 and the control group after immunization with 1 and 2 doses.

[0070] 7.3 Changes in body temperature and body weight in mice after immunization: After LG12 immunization, the body temperature and body weight of mice in the experimental and control groups were measured daily for 56 days. Results showed that the body temperature of mice in all three groups fluctuated within the normal range within 56 days after vaccination. Analysis of variance using OriginPro software showed that ( F =1.505, P =0.2267) The difference was not statistically significant, see Figure 19 The body weight of mice in all three groups continued to increase after immunization, with rapid weight gain in the early stages of the juvenile mice. There was no statistically significant difference in body weight changes among the groups during the experiment. F =1.011, P =0.3659), see Figure 20 .

[0071] 7.4 Analysis of serum antibody immune response in immunized mice: Purified LG12 virus was plated, and the IgG and IgA antibody titers in the serum of immunized mice were measured by indirect ELISA. The seroconversion rates of IgG and IgA antibodies in the serum of mice in both experimental groups 1 and 2 were 100%. 28 days after one dose of immunization, the average IgG antibody titer in the serum of experimental group 1 was 3.2577 lg, which was statistically significant compared with the control group. t =11.98, P <0.0001), the average IgG antibody titer in the two experimental groups was 3.2577lg, which was statistically significant compared with the control group. t =11.98, P <0.0001), there was no statistically significant difference between experimental group 1 and experimental group 2. P >0.9999, t =0.000); 28 days after immunization with two doses, the average serum IgG antibody titer in group 1 was 4.7930 lg, and the average titer in group 2 was 4.5220 lg, with statistically significant differences compared to the first dose (respectively...). t =7.759 and t =6.414, P <0.0001), there was no statistically significant difference between experimental group 1 and experimental group 2. P =0.1016, t =1.725). 28 days after one dose of immunization, the average serum IgA antibody titer in group 1 was 3.1072 lg, which was statistically significant compared to the control group. t =16.43, P <0.0001); the average IgA antibody titer in experimental group 2 was 2.7761lg, which was statistically significant compared with the control group. t =12.94, P <0.0001), there was no statistically significant difference between experimental group 1 and experimental group 2. t =2.091, P =0.0510); 28 days after the second dose of immunization, the average serum IgA antibody titer in group 1 was 3.7393lg, which was statistically significant compared with the first dose. t =7.759, P <0.0001); the average IgA antibody titer in the two experimental groups was 3.4082lg, which was statistically significant compared with the first dose. t =4.163, P <0.05); there was no statistically significant difference between experimental group 1 and experimental group 2 ( t =1.766, P =0.0944), see Figure 21A ,Figure 21B .

[0072] 7.5 Analysis of neutralizing antibodies in mouse serum after immunization: MN results showed that the seroconversion rate of antibodies in immunized mice was 100% fourfold. With increasing immunization doses, the level of neutralizing antibodies showed a significant dose-dependent effect. The difference in neutralizing antibody titers between the first and second doses in experimental group 1 was statistically significant. t =8.647, P <0.0001), the difference in neutralizing antibody titer between the first and second doses in the two experimental groups was statistically significant ( t =10.37, P <0.0001), the GMT of the neutralizing antibody in the second dose was 29.86 times and 26.00 times that of the first dose, respectively. In the control group mice, the serum neutralizing antibody titers after both the first and second doses were below the detection limit and were therefore considered negative. See Figure 22A .

[0073] Cross-neutralization tests with various G-type RV wild-type strains (G1, G2, G3, G4, G9, and G12) showed that the neutralizing antibody titers of mouse serum against the parental human G12P[6] rotavirus after LG12 immunization were all higher than the neutralizing antibody titers against the corresponding heterotype viruses. The average neutralizing antibody titer against the G1 wild-type strain was 1.2342lg; the average neutralizing antibody titer against the G2 wild-type strain was 2.7693lg; and the average neutralizing antibody titer against the G3 wild-type strain was... The mean neutralizing antibody titer against G4 wild-type virus was 0.9031 lg; the mean neutralizing antibody titer against G9 wild-type virus was 2.4985 lg; the mean neutralizing antibody titer against G12 wild-type virus was 1.5653 lg; the mean neutralizing antibody titer against G12 wild-type virus was 3.9134 lg; the LOD value was 0.9031 lg; and the negative assignment value was 0.6021 lg. This indicates that LG12 immunization provides varying degrees of cross-protection against other G-type wild-type RV strains, but the neutralizing titer against G3 is the lowest. Figure 22B .

[0074] 7.6 Results of mouse intestinal IgA (SIgA) detection: IgA antibodies were detected in fecal samples 3 days after the first immunization of mice. The titer of IgA antibodies increased over time. As shown in the figure, the titer of IgA antibodies in fecal samples reached a plateau at 5 days. There was no statistically significant difference in IgA titer between experimental group 1 (gavage immunization group) and experimental group 2 (intraperitoneal injection group). t =0.5511, P >0.05), see Figure 23 .

[0075] 7.7 Results of the challenge test 7.7.1 Clinical symptoms and toxin shedding in mice after challenge: According to visual observation, mice in experimental group 1, experimental group 2 and control group did not show diarrhea, ruffled hair, loss of appetite and lethargy, and their mental state was good.

[0076] The results of ELISA detection of fecal samples from mice after resuspension challenge showed that RV was not detected in the feces of mice in both the experimental and control groups. This indicates that the reassortant attenuated strains LG12 and G12P[6] may both be attenuated strains in mice.

[0077] 7.7.2 Changes in body temperature and body weight of mice in each group within 1 week after challenge: The results showed that within 3 days after challenge with the G12P[6] wild-type strain, most mice in the control group had elevated body temperature, with the average body temperature slightly higher than that in the experimental group. After 3 days, the body temperature of mice in all three groups fluctuated within the normal range. See Figure 24 .

[0078] Within 5 days after challenge, the average body weight of mice in both groups increased slowly, with no significant decrease in body weight. As shown in the figure, the average body weight of mice in the control group decreased slightly 1 day after challenge, but all three groups showed stable weight gain after one week. Statistical analysis showed no statistically significant difference in body weight changes between the two experimental groups. t =0.02533, P =0.9800), there were no statistically significant differences in body weight changes between experimental groups 1 and 2 and the control group (respectively). t =0.3179 and 0.3322, P >0.05) See Figure 25 .

[0079] 7.7.3 Results of intestinal IgA (SIgA) detection in mice after challenge: In the control group, IgA antibody levels in fecal samples gradually increased from negative to high levels, reaching a peak 5 days after challenge; in the experimental group, IgA titers in fecal samples did not decrease significantly due to challenge and remained relatively stable at a certain level. Figure 26 .

[0080] References [1] LI F, GUO L, LI Q, et al. Changes in the epidemiology and clinical characteristics of viral gastroenteritis among hospitalized children in the Mainland of China: a retrospective study from 2016 to 2020[J]. BMCPediatr, 2024, 24(1): 303. [2] WANG J X,ZHOU H L, MO Z J, et al. Burden of viral gastroenteritisin children living in rural China: populationbased surveillance[J]. Int JInfect Dis, 2020, 90: 151-160. [3] Matthijnssens J, Ciarlet M, McDonald SM, et al. Uniformity ofrotavirus strain nomenclature proposed by the Rotavirus ClassificationWorking Group (RCWG) [J]. Arch Virol, 2011, 156(8): 1397-1413. [4] Gibory M,Bruun T, Flem E,et al. Genetic diversity of rota virusstrains circulating in Norway before and after the introduction of rotavirusvaccination in children [J]. J Med Virol, 2022, 94(6): 2624 2631. [5] Taniguchi, K.; Urasawa, T.; Kobayashi, N.; Gorziglia, M.;Urasawa, S. Nucleotide sequence of VP4 and VP7 genes of human rotaviruseswith subgroup I specificity and long RNA pattern: Implication for new Gserotype specificity. J. Virol. 1990, 64, 5640–5644. [6] Griffin, D.D.; Nakagomi, T.; Hoshino, Y.; Nakagomi, O.; Kirkwood,C.D.; Parashar, U.D.; Glass, R.I.; Gentsch, J.R. National RotavirusSurveillance System. Characterization of nontypeable rotavirus strains fromthe United States: Identification of a new rotavirus reassortant (P2A[6],G12)and rare P3[9] strains related to bovine rotaviruses. Virology 2002, 294,256–269. [7] Castello, A.A.; Argüelles, M.H.; Rota, R.P.; Olthoff, A.; Jiang,B.; Glass, R.I.; Gentsch, J.R.; Glikmann, G. Molecular epidemiology of groupA rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to2003 and emergence of the infrequent genotype G12. J. Clin. Microbiol. 2006,44, 2046–2050. [8] Ratho, R.K.; Chhotray, G.P.; et al. Emergence of novel humangroup A rotavirus G12 strains in India. J. Clin. Microbiol. 2003, 41, 2760–2762. [9] Wakuda, M.; Nagashima, S.; Kobayashi, N.; Pongsuwanna, Y.;Taniguchi, K. Serologic and genomic characterization of a G12 human rotavirusin Thailand. J. Clin. Microbiol. 2003, 41, 5764–5769.

[10] Shinozaki, K.; Okada, M.; Nagashima, S.; Kaiho, I.; Taniguchi,K. Characterization of human rotavirus strains with G12 and P[9]detected inJapan. J. Med. Virol. 2004, 73, 612–616.

[11] Samajdar, S.; Varghese, V.; Barman, P.; Ghosh, S.; Mitra, U.;Dutta, P.; Bhattacharya, S.K.; Narasimham, M.V.; Panda, P.; Krishnan, T.; etal. Changing pattern of human group A rotaviruses: Emergence of G12 as animportant pathogen among children in eastern India. J. Clin. Virol. 2006, 36,183–188.

[12] . Uchida , R. ; Pandey, BD; Sherchand , JB ; Ahmed, K.; Yokoo,M.; Nakagomi, T.; Caves, LE; Cunliffe, NA; Hart, CA; Nakagomi, O.Molecular epidemiology of rotavirus diarrhea among children and adults inNepal: Detection of G12 strains with P[6]or P[8] and a G11P

[25] strain. J.Clin. Microbiol. Rev. 2006, 44, 3499–3505.

[13] Banerjee, I.; Ramani, S.; Primrose, B.; Iturriza-Gomara , M. ;Gray , JJ ; Brown , DW ; Kang, G. Modification of rotavirus multiplex RT-PCR for the detection of G12 strains based on characterization of emerging G12rotavirus strains from South India. J. Med. Virol. Rev. 2007, 79, 1413–1421.

[14] Pun, SB; Nakagomi, T.; Sherchand , JB ; Pandey, BD; Caves,LE; Cunliffe, NA; Hart, CA; Nakagomi, O. Detection of G12 humanrotaviruses in Nepal. Emerg. Infect. This. 2007, 13, 482–484. [CrossRef] [PubMed]

[15] Rahman, M.; Matthijnssens , J. ; Yang, X.; Delbeke, T.; Arijs , I. ;Taniguchi , K. ; Iturriza-Gomara, M.; Iftekharuddin , N. ; Azim, T.; Van Ranst,M. Evolutionary history and global spread of the emerging g12 human rotaviruses. J. Virol. Rev. 2007, 81, 2382–2390. [CrossRef]

[16] Ramani, S.; Banerjee, I.; Gladstone , BP ; Sarkar , R. ;Selvapandian , D. ; Le Fevre, AM; Jaffar , S. ; Iturriza-Gomara, M.; Gray ,JJ ; Estes, MK; et al. Geographic information systems and genotyping inidentification of rotavirus G12 infections in residents of an urban slum withsubsequent detection in hospitalized children: Emergence of G12 genotype inSouth India. J. Clin. Microbiol. Rev. 2007, 45, 432–437.

[17] Ray, P.; Sharma, S.; Agarwal, R.K.; Longmei, K.; Gentsch, J.R.;Paul, V.K.; Glass, R.I.; Bhan, M.K. First detection of G12 rotaviruses innewborns with neonatal rotavirus infection at all India Institute of MedicalSciences, New Delhi, India. J. Clin. Microbiol. 2007, 45, 3824–3827.[CrossRef][PubMed]

[18] Le, V.P.; Kim, J.Y.; Cho, S.L.; Nam, S.W.; Lim, I.; Lee, H.J.;Kim, K.; Chung, S.I.; Song, W.; Lee, K.M.; et al. Detection of unusualrotavirus genotypes G8P[8]and G12P[6] in South Korea. J. Med. Virol. 2008,80, 175–182. [CrossRef] [PubMed]

[19] Sharma, S.; Ray, P.; Gentsch, J.R.; Glass, R.I.; Kalra, V.;Bhan, M.K. Emergence of G12 rotavirus strains in Delhi, India, in 2000 to2007. J. Clin. Microbiol. 2008, 46, 1343–1348. [CrossRef]

[20] Alam, M.M.; Malik, S.A.; Shaukat, S.; Naeem, A.; Sharif, S.;Angez, M.; Rana, M.S.; Khurshid, A.; Zaidi, S.Z. Genetic characterization ofrotavirus subtypes in Pakistan-first report of G12 genotype from Pakistanunder WHO-Eastern Mediterranean region. Virus Res. 2009, 144, 280–284.

[21] Fedorova, N.B.; Halpin, R.A.; et al. Multiple introductions andantigenic mismatch with vaccines may contribute to increased predominance ofG12P[8]rotaviruses in the United States. J. Virol. 2018, 93, e01476-18.[CrossRef] [PubMed]

[22] Japhet, M.O.; Famurewa, O.; Iturriza-Gómara, M.; Adesina, O.A.;Opaleye, O.O.; Niendorf, S.; Bock, C.T.; Mas Marques, A. Group A rotavirusescirculating prior to a national immunization programme in Nigeria: Clinicalmanifestations, high G12P[8]frequency, intra-genotypic divergence of VP4 andVP7. J. Med. Virol. 2018, 90, 239–249. [CrossRef]

[23] Wylie , KM ; Stanley , KM ; TeKippe , EM ; Mihindukulasuriya,K.; Storch, GA Resurgence of rotavirus genotype G12 in St. Louis, Missouri. Louis during the2014–2015 rotavirus season. J. Pediatric. Infect. This. Soc. 2017, 6, 346–351.

[24] Dhital, S.; Sherchand , JB ; Pokhrel , BM ; Parajuli, K.; Shah,N.; Mishra, SK; Sharma, S.; Kattel, HP; Khadka, S.; Khatiwada, S.; et al.Molecular epidemiology of Rotavirus causing diarrhea among children less thanfive years of age visiting national level pediatric hospitals, Nepal. BMCPediatr. 2017, 17, 101. [CrossRef]

[25] Da Silva, MF; Fumian , TM ; de Assis , RM ; Fialho, AM;Carvalho-Costa, FA; da Silva Ribeiro de Andrade, J.; Leite, JP Genetic characterization of VP7 andVP8* of group A rotavirus genotype G12P[8]:Emergence and spread in the eastern Brazilian coast in 2014. J. Med.Virol. Rev. 2017, 89, 64–70. [CrossRef] [PubMed]

[26] Tanmoy, A.M.; Ahmed, A.N.; Arumugam, R.; Hossain, B.; Marzan,M.; Saha, S.; Arifeen, S.E.; Baqui, A.H.; Black, R.E.; Kang, G.; et al.Rotavirus surveillance at a WHO-coordinated invasive bacterial diseasesurveillance site in Bangladesh: A feasibility study to integrate twosurveillance systems. PLoS ONE 2016, 11, e0153582. [CrossRef]

[27] Langa, J.S.; Thompson, R.; Arnaldo, P.; Resque, H.R.; Rose, T.;Enosse, S.M.; Fialho, A.; de Assis, R.M.; da Silva, M.F.; Leite, J.P.Epidemiology of rotavirus A diarrhea in Chokwe, Southern Mozambique, fromFebruary to September, 2011. J. Med. Virol. 2016, 88, 1751–1758. [CrossRef]

[28] Bányai, K.; László, B.; Duque, J.; Steele, A.D.; Nelson, E.A.S.;Gentsch, J.R.; Parashar, U.D. Systematic review of regional and temporaltrends in global rotavirus strain diversity in the pre rotavirus vaccine era:Insights for understanding the impact of rotavirus vaccination programs.Vaccine 2012, 30, A122–A130. [CrossRef]

[29] Rahman, M.; Sultana, R.; Ahmed, G.; Nahar, S.; Hassan, ZM; Saiada, F.; Podder, G.; Faruque, AS; Siddique, AK; Sack, DA; et al. [CrossRef]

[30] Gómez, MM; Resque, HR; Volotão Ede, M.; Rose, TL; daSilva, MF; Heylen, E.; Zeller, M.; Matthijnssens, J.; Leite, JP 2014, 28, 385–388. [CrossRef]

[31] Zhou X, Wang YH, Pang BB, Chen N, Kobayashi N. Surveillance ofHuman Rotavirus in Wuhan, China (2011-2019): Predominance of G9P[8]andEmergence of G12. Pathogens. 2020 Oct 2;9(10):810.

[32] He Lianhua, Li Jinsong, Duan Zhaojun. Research progress on the efficacy and safety of rotavirus vaccines in China [J]. Chinese Journal of Vaccines and Immunization, 2023, 29(2): 239-245.

[33] The SIB Swiss Institute of Bioinformatics' resources: focus oncurated databases [J].Nucleic Acids Res, 2016, 44(D1): D27-37.

[34] Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server [J].Nucleic Acids Research,2014,42(W1): W320-W324.

[35] Bodenhofer U, Bonatesta E, Horejš-Kainrath C, et al.msa: an Rpackage for multiple sequence alignment [J].Bioinformatics,2015,31(24): 3997-3999.

[36] China National Committee for Standardization of Biological Products. Chinese Biological Products Regulations [S]. 2000 edition, 2002 supplement. Beijing: Chemical Industry Press, 2002: 8-10.

Claims

1. A rotavirus reassortant attenuated strain LG12, characterized in that: The rotavirus reassortant attenuated strain LG12 comprises: i) a coding gene of structural protein VP7 of human G12 type rotavirus strain G12P[6]; and ii) coding genes of structural proteins VP1, VP2, VP3, VP4, VP6 and non-structural proteins NSP1, NSP2, NSP3, NSP4, NSP5 of sheep rotavirus strain LLR; The human G12 type rotavirus strain G12P[6] has a preservation number of CCTCC NO: V202577.

2. The rotavirus reassortant attenuated strain LG12 according to claim 1, characterized by: The rotavirus reassortant attenuated strain LG12 has a preservation number of CCTCC NO: V202578.

3. Use of the rotavirus reassortant attenuated strain LG12 of claim 1 or 2 in the preparation of a rotavirus vaccine.

4. Use according to claim 3, characterized in that: The rotavirus vaccine is used for preventing diseases caused by infection of human G12, G1, G2, G4 or G9 type rotavirus wild strains.

5. Use of the rotavirus reassortant attenuated strain LG12 of claim 1 or 2 in the preparation of a rotavirus diagnostic reagent.

6. A vaccine, characterized in that: The vaccine comprises an immunogenically effective amount of the rotavirus reassortant attenuated strain LG12 of claim 1 or 2.

7. The vaccine of claim 6, characterized in that: The vaccine further comprises at least one acceptable adjuvant.

8. The vaccine of claim 6, characterized in that: The vaccine further comprises at least one pharmaceutically acceptable carrier or diluent.

9. The vaccine of claim 6, characterized in that: The vaccine is in the form of an oral dosage form or an injection dosage form.

10. A diagnostic reagent, characterized by: The diagnostic reagent contains the rotavirus reassortant attenuated strain LG12 of claim 1 or 2.

Images