Human rotavirus strain RV009 and separation, culture and identification method thereof

Abstract

The invention discloses a human rotavirus strain and a method for separating, culturing and identifying the human rotavirus strain, the human rotavirus strain is a rotavirus RV009 strain, the human rotavirus strain is submitted to China General Microbiological Culture Collection Center (CGMCC) on July 22, 2024, and the preservation number is CGMCC No.45999. The invention also discloses a method for separating, culturing and identifying the human rotavirus strain. The separation, culture and identification method of the human rotavirus strain RV009 provided by the invention is beneficial to rapid virus amplification, production cycle shortening and low-cost obtaining of high-yield viruses, and is beneficial to vaccine production, virology research and development of diagnostic reagents.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to a human rotavirus strain RV009 and its isolation, culture and identification methods. Background Art

[0002] G9 rotavirus belongs to a genotype of human rotavirus. Like other types of rotaviruses, it poses a threat to human health. According to the latest research, G9 rotavirus is one of the epidemic strains similar to the Wa strain, with a genotype of G9P. Together with the epidemic strains similar to the DS-1 strain and the AU-1 strain, they constitute the main epidemic genotypes of human rotaviruses.

[0003] Rotavirus infections are particularly common in children. Almost every child has been infected with rotavirus, including G9 rotavirus, before the age of 3 - 5. The clinical manifestations after infection vary in severity, and the main symptoms are diarrhea, vomiting, fever, and dehydration. Although these symptoms are relatively common in infants and young children, persistent vomiting and watery diarrhea can easily lead to dehydration and electrolyte disorders, and in severe cases, shock or even death may occur.

[0004] The best way to prevent rotavirus infection is to vaccinate. It can not only effectively reduce the infection risk but also alleviate the severity of symptoms. Although a few children may have adverse reactions such as mild diarrhea after vaccination, these reactions are usually transient, and the safety of the vaccine has been verified by clinical trials.

[0005] In summary, G9 rotavirus is one of the main currently prevalent rotaviruses and poses a threat to children's health. By vaccinating and strengthening public health measures, the transmission of rotavirus can be effectively prevented and controlled. Therefore, the isolation, culture of G9 rotavirus strains and subsequent vaccine research are in line with epidemiological significance and have application prospects.

[0006] Currently prevalent G9 strains cannot be directly purchased from ATCC. Wild-type G9 strains can only be obtained through self-isolation and purification for the subsequent research and development of G9 rotavirus vaccines. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the object of the present invention is to provide a wild-type single purified strain RV009 of human G9 rotavirus and to provide the isolation, culture and identification methods of the above strain. This strain has strong virus replication ability and good genetic stability, and can be applied to the production of virus seeds for the development of human rotavirus vaccines. The applicable vaccine types include attenuated rotavirus oral vaccines and inactivated human rotavirus injection vaccines.

[0008] To achieve the above object, the present invention provides a human rotavirus strain, which is the human G9 rotavirus RV009. Its classification and naming: Rotavirus; Depositary Institution: China General Microbiological Culture Collection Center (CGMCC); Deposit Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit Date: July 22, 2024; Deposit Number: CGMCC No. 45999.

[0009] The present invention also provides a method for isolating the above human rotavirus strain, which specifically includes the following steps:

[0010] A1. Obtain the original virus sample: Collect the diarrhea excrement specimens of infants and young children with diarrhea in the pediatrics department of the hospital as the original virus sample;

[0011] B1. Colloidal gold detection: Use a colloidal gold detection kit to detect the original sample to obtain a sample with a positive colloidal gold retest;

[0012] C1. RNA-PAGE detection: Add the samples with positive colloidal gold retest into a phosphate buffer solution with a concentration of 0.005 - 0.01 mol / L and a pH of 7.0 - 7.2 respectively, stir to obtain a suspension, centrifuge at 10000 - 12000 g for 15 - 30 min to take the supernatant, and use a 0.22 μm filter to sterilize and filter, and store it at -18°C to -20°C for standby to obtain a fecal treatment sample with a positive colloidal gold result; Extract the viral genomic RNA from the fecal treatment sample with a positive colloidal gold result, perform non-denaturing PAGE gel electrophoresis for 8 - 10 h, take a photo after silver nitrate staining, and detect the band pattern of the extracted viral nucleic acid sample to determine its basic characteristics;

[0013] D1. RT-PCR detection: Extract the viral genomic RNA, perform RT-PCR with specific primers for the VP7 gene sequence of rotavirus, sequence the obtained PCR products, and compare the sequencing results with the VP7 gene published in Genbank to determine the type of the virus sample;

[0014] E1. Screening and determination of candidate strains: After confirmation by the above colloidal gold detection, RNA-PAGE detection and RT-PCR detection, select the G9 type strain with epidemiological significance from all the detected positive samples to obtain a positive specimen of the rotavirus seed strain;

[0015] F1. Obtaining virus samples: Resuspend the positive specimens of rotavirus strains determined by screening in step E1 in a solution with a concentration of 0.005 - 0.01 mol / L and a pH of 7.0 - 7.2, centrifuge at 10000 - 12000 g for 15 - 30 min, take the supernatant, and filter it through a 0.22 μm filter to remove bacteria, then store it at -18°C to -20°C for later use, obtaining a diluted solution of diarrhea samples from children infected with rotavirus;

[0016] G1. Adaptation of the virus to MA104 cells: Digest the confluent MA104 cells with 0.25% trypsin - 0.03% EDTA, resuspend the cells with MEM - 10% NBCS, and then inoculate 10 5.0 cells per flask into a small square flask, and culture at 37°C for 3 - 4 days until the cells form a monolayer again; Inoculate the diluted solution of diarrhea samples from children infected with rotavirus obtained in step F1 onto the confluent MA104 cells, and adsorb at 37°C for 60 - 100 min; After adsorption, remove the virus solution, add MEM cell maintenance medium without newborn bovine serum, continue to culture at 37°C for 5 - 7 days and observe the cytopathic effect. After harvesting the culture, continue to inoculate MA104 cells in the same way for passage or freeze - store for later use. Finally, screen out the RV009 strain, obtaining the harvested solution of RV009 rotavirus that has been adapted and cultured on MA104 cells;

[0017] H1. Cloning and purification of the virus: Dilute the harvested solution of RV009 rotavirus that has been adapted and cultured on MA104 cells by a 10 - fold gradient, and take the virus solutions at five dilution degrees of 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 and inoculate them into six - well plates of confluent MA104 cells respectively, adsorb at 37°C for 60 - 100 min, add MEM medium containing 0.4 - 0.7% agarose, wait for the agar to solidify, then invert and culture at 37°C for 2 - 4 days; Then add MEM medium containing 0.4 - 0.7% agarose and 0.05 - 0.15% neutral red, continue to culture for 2 - 4 days, and observe the formation of plaques; Use a Pasteur pipette to pick a single plaque, transfer it to a 1.5 mL centrifuge tube containing 1 mL MEM medium, and ensure the uniform mixing of the virus and the medium by vortex oscillation; After freeze - thawing, centrifuge the mixture at a speed of 4500 - 8000 g at 3 - 5°C for 25 - 35 min, take the supernatant, and immediately inoculate it onto a new monolayer of MA104 cells, culture at 37°C for 3 - 4 days, and harvest the culture after the cells show cytopathic effect; After harvesting the culture, continue to inoculate MA104 cells in the same way for passage or freeze - store for later use, obtaining the virus solution.

[0018] In the rotavirus isolation method of the present invention, by improving the traditional rotavirus isolation method, a relative centrifugal force of 10,000 - 12,000 g is selected for centrifugation. After centrifugation for 15 - 30 minutes, the supernatant is taken and filtered through a filter to remove bacteria, obtaining a higher virus recovery rate and better impurity removal effect. In the method provided by the present invention, MEM cell maintenance medium without newborn bovine serum is used to culture and observe cytopathic effects at 37°C, further improving the primary culture efficiency of rotavirus. The harvested solution of RV009 rotavirus that has been adaptively cultured on MA104 cells is diluted and then inoculated onto MA104 cells again for culturing to clone and purify the RV009 strain virus. Through gradient dilution and multiple clone purifications, it is beneficial to obtain a virus strain with good genetic stability and strong infectivity, which is beneficial for subsequent virus research and vaccine development.

[0019] The present invention also provides a culture method for the above-mentioned human rotavirus strain, which specifically includes the following steps:

[0020] Adaptation of the virus on Vero cells: Digest the confluent Vero cells with 0.25% trypsin - 0.03% EDTA and resuspend the cells with DMEM - 10% NBCS. Inoculate small square bottles at 10 5.0 cells / bottle. Culture at 37°C and 5.0% CO2 for 3 - 4 days until a confluent cell monolayer is formed, then inoculate 1 mL of the virus solution, and the inoculation amount is 10 4.5 -10 6.5 CCID50 / mL. Adsorb at 37°C for 1 - 2 hours, then add DMEM cell maintenance medium without newborn bovine serum, culture at 37°C and observe cytopathic effects. Harvest the culture, and the harvested culture can be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0021] Preferably, the culture method for the human rotavirus strain can also be:

[0022] Digest the confluent Vero cells with 0.25% trypsin - 0.03% EDTA and resuspend the cells with DMEM - 10% NBCS. Inoculate small square bottles at 10 5.0 cells / bottle. Culture at 37°C and 5.0% CO2 for 3 - 4 days until a confluent cell monolayer is formed; then inoculate 1 mL of the virus solution, and the inoculation amount is 10 4.5 -10 6.5 CCID 50 / mL, where the virus solution contains additives, polyethylene glycol, and polymers. Adsorb at 37°C for 1 - 2 hours; then add DMEM cell maintenance medium without newborn bovine serum, culture at 37°C and observe cytopathic effects. Harvest the culture, and the harvested culture can be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0023] Preferably, the virus solution is the above-mentioned human rotavirus RV009 virus solution of G9 type.

[0024] Preferably, the final concentration of the additive in the virus solution is 1-6 mmol / mL; the final concentration of the polyethylene glycol is 1-10 mmol / mL; the final concentration of the polymer is 1-5 mmol / mL.

[0025] More preferably, the additive is selected from one of calcium chloride and magnesium chloride.

[0026] More preferably, the polymer is selected from one of poly(L-lysine), poly(L-arginine) and poly(L-glutamic acid).

[0027] The rotavirus culture method involved in the present invention is an improvement adapted to the new genotype human rotavirus based on the method for preparing the seed virus of the traditional virus-type vaccine.

[0028] The present invention also provides a method for identifying the above-mentioned human rotavirus strain, which specifically includes the following steps:

[0029] Virus titer determination: Measured by the CCID 50 method. The virus sample is activated in a water bath at 37 °C for 0.5-1 h, and the activated virus sample is diluted at a 10-fold dilution; in a 96-well plate with MA104 cells grown into a confluent monolayer, the residual maintenance fluid is removed, and 95-105 μL of the virus dilution is added to each well, and two columns of wells are used as cell control wells, and maintenance fluid without serum is added; then the plate is placed in an incubator at 37 °C and 5.0% CO2 for 6-8 days; after that, the 96-well plate is frozen and thawed, and then the solution in each well is completely transferred to a 96-well plate pre-coated with goat anti-rotavirus immunopolyclonal antibody (diluted 1:2000), and incubated at 37 °C for 0.5-1.5 h to allow the antibody to bind to the virus; after washing the plate, HRP-labeled goat anti-rotavirus detection antibody diluted 1:2000 is added, and incubated again at 37 °C for 1 h; after washing the plate, TMB chromogenic solution is added, and the color is developed in the dark at room temperature for 4-6 min, the color development reaction is terminated by adding the termination solution, and then the optical absorption value is detected with an enzyme-linked immunosorbent assay instrument at wavelengths of 450 nm and 650 nm; calculate the virus titer.

[0030] Preferably, the virus sample is the virus solution harvested after the adaptation culture of the above-mentioned human rotavirus RV009 virus of G9 type on Vero cells.

[0031] For further illustration of the present invention, the present invention also performs whole-genome sequencing on the above-mentioned human rotavirus strain. The total RNA of the human rotavirus strain RV009 is entrusted to TaKaRa Biotechnology Co., Ltd. (Dalian, China) for sequencing, and sequence analysis and comparison are performed on the sequencing results.

[0032] The rotavirus strain identification involved in the present invention is an improvement adapted to human rotavirus based on the method for preparing traditional virus-type vaccine strains. The detection method for virus titer is CCID 50 detection method.

[0033] Advantages of the present invention:

[0034] 1. Compared with the prior art, the present invention successfully isolates a G9 wild-type single purified strain, the RV009 strain, which has strong virus replication ability and good genetic stability and can be used as the production strain for developing rotavirus vaccines, applicable to rotavirus attenuated oral vaccines and rotavirus inactivated injectable vaccines. At the same time, the method for isolating, culturing and identifying human rotavirus provided by the present invention can provide strong support for the research and clinical application of human rotavirus.

[0035] 2. In the isolation and culture of the human rotavirus strain RV009 involved in the present invention, it is found that adding calcium chloride, polyethylene glycol and polymers to the rotavirus solution of the present invention for Vero cell culture adsorption is beneficial to shortening the cytopathic time, enhancing the infectivity of rotavirus and increasing the yield, optimizing the isolation and culture conditions. Description of the drawings

[0036] Figure 1 Morphology of rotavirus under electron microscope;

[0037] Figure 2 Colloidal gold detection pictures of group A rotavirus in excreta of some diarrhea cases;

[0038] Figure 3 PAGE electrophoresis detection map of rotavirus genome in excreta of some diarrhea cases;

[0039] Figure 4 PAGE electrophoresis detection map of the genome of the isolated strain RV009 after passage;

[0040] Figure 5 Agarose gel electrophoresis detection map of amplified rotavirus VP7 gene of some samples;

[0041] Figure 6 Partial excerpt of VP7 gene sequence alignment of G9 type samples. Detailed implementation manners

[0042] Parameters and sources of specific chemical substances used.

[0043] Polyethylene glycol, molecular weight: 2000;

[0044] Poly(L-lysine), molecular weight: 80000;

[0045] Poly(L-arginine), molecular weight: 80,000;

[0046] Poly(L-glutamic acid), molecular weight: 80,000.

[0047] The cell seed of MA104 cells is derived from the China Center for Type Culture Collection (CCTCC) and is the 11th passage;

[0048] The cell seed of Vero cells is derived from the American Type Culture Collection (ATCC) and is the 123rd passage.

[0049] Human rotavirus belongs to the Reoviridae family. Its mature virus particles are smooth wheel-shaped under the electron microscope, with a diameter of about 65 - 75 nm. It has three layers of protein capsids. The innermost nucleocapsid encloses the viral genome and the enzymes necessary for viral replication; the middle shell is composed of radially arranged subunits; the outer shell is smooth and has spikes. The three layers of protein capsids enclose 11 double-stranded RNA viral genomes, with a genome size of 18,555 base pairs. Each fragment is basically a gene. The 11 genes encode the structural proteins and non-structural proteins of the virus respectively. VP7 belongs to one of the structural proteins. The VP7 protein is a glycoprotein that constitutes the outermost surface of the virus and determines the G serotype of rotavirus (G-serotypes), which is an important basis for rotavirus typing.

[0050] Example 1

[0051] A method for the isolation, culture and identification of a human rotavirus strain RV009, comprising the following steps:

[0052] (1) Isolation method of human rotavirus strain RV009

[0053] 1.1 Obtaining the original virus sample

[0054] The original sample collected the diarrhea excrement specimens of hospitalized and outpatient cases of children with diarrhea in Taixing People's Hospital and Taixing Second People's Hospital in Jiangsu Province in 2022. The RV009 strain was derived from a diarrhea sample of a 2-year-old male. The sample was collected on January 29, 2022, with no previous medical history. Rotavirus antigen was detected positive in the excrement, and the confirmed case was diarrhea caused by rotavirus infection;

[0055] 1.2 Colloidal gold detection

[0056] Collect the diarrhea excrement specimens of children with diarrhea in the pediatrics department of the hospital. Samples retested as positive by the Group A rotavirus colloidal gold detection kit (WanTai Biological) were continued for subsequent experiments, and samples with negative retest results were discarded;

[0057] 1.3 RNA-PAGE detection

[0058] Samples with positive colloidal gold retest results were added to 0.01 mol / L phosphate buffer solution (pH 7.2) respectively and stirred to obtain a suspension. After centrifuging at 12,000 g for 20 min, the supernatant was taken, and sterilizing filtration was carried out using a 0.22 μm filter (from Millipore Corporation), and it was stored at -20 °C for standby use, obtaining fecal treatment samples with positive colloidal gold results; Viral genomic RNA was extracted from the fecal treatment samples with positive colloidal gold results (operated according to the operation manual of Thermo Scientific GeneJET Viral DNA and RNA Purification Kit), non-denaturing PAGE gel electrophoresis was carried out for 9 h, and after silver nitrate staining, it was photographed, and the band pattern of the extracted viral nucleic acid samples was detected to determine their basic characteristics;

[0059] 1.4 RT-PCR Detection

[0060] Viral genomic RNA was extracted (operated according to the operation manual of Thermo Scientific GeneJET Viral DNA and RNA Purification Kit), and RT-PCR was carried out using specific primers for the rotavirus VP7 gene sequence (TaKaRa Prime ScriptTM One Step RT-PCR Kit Ver.2 kit). The obtained PCR products were entrusted to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were compared with the VP7 gene published in Genbank to determine the type of the viral sample;

[0061] 1.5 Screening and Determination of Candidate Strains

[0062] After confirmation by the above-mentioned colloidal gold detection, RNA-PAGE detection and RT-PCR detection, from all the detected positive samples, G9 type strains with epidemiological significance were selected, that is, positive samples of rotavirus seed strains were obtained;

[0063] 1.6 Obtaining Viral Samples

[0064] The positive specimens of rotavirus seed strains obtained in step 1.5 above were resuspended in phosphate buffer solution with a concentration of 0.01 mol / L and a pH of 7.2. After centrifuging at 12,000 g for 20 min, the supernatant was taken, and sterilizing filtration was carried out using a 0.22 μm filter, and it was stored at below -20 °C for standby use, obtaining a dilution of diarrhea samples of children infected with rotavirus;

[0065] 1.7 Adaptation of the Virus to MA104 Cells

[0066] Digest the MA104 cells that have formed a monolayer with 0.25% trypsin (from Gibco) - 0.03% EDTA, resuspend the cells with MEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and then inoculate them into small square bottles at 10 5.0 cells / bottle and culture at 37°C for 3 days until the cells form a monolayer again; inoculate the diluted diarrhea sample of the rotavirus-infected child obtained in step 1.6 onto the MA104 cells that have formed a monolayer, and adsorb at 37°C for 90 min; after adsorption, remove the virus solution, add MEM (from Gibco) cell maintenance medium without newborn bovine serum, continue to culture at 37°C for 6 days and observe cytopathic effects. After the cells show cytopathic effects, harvest the culture; after harvesting the culture, continue to inoculate onto MA104 cells according to the same method for passage culture or cryopreservation for later use to obtain the RV009 rotavirus harvest solution that has been adapted to culture on MA104 cells;

[0067] 1.8 Cloning and purification of the virus

[0068] Dilute the RV009 rotavirus harvest solution that has been adapted to culture on MA104 cells obtained in the above step 1.7 by a 10-fold gradient, and take 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 virus solutions of five dilution factors and inoculate them into six-well plates of MA104 cells that have formed a monolayer, and adsorb at 37°C for 90 min; after the adsorption is completed, add MEM medium (from Gibco) containing 0.6% agarose (from Lonza) to each well. After the agar solidifies, place the six-well plates upside down in an incubator at 37°C and continue to culture; after 3 days, add MEM medium (from Gibco) containing 0.6% agarose (from Lonza) and 0.1% neutral red (from SIGMA) to each well, and continue to culture for about 3 days. Observe the formation of plaques using a microscope; use a Pasteur pipette to pick a single plaque and transfer it to a 1.5 mL centrifuge tube containing 1 mL MEM medium (from Gibco), and ensure the virus is evenly mixed with the medium by vortex oscillation; after alternating freezing and thawing 3 times at -20°C and room temperature, centrifuge the mixture at 5000 g at 4°C for 30 min to precipitate impurities, take the supernatant, and immediately inoculate it onto a new monolayer of MA104 cells and culture at 37°C for 3 days. After the cells show cytopathic effects, harvest the culture. After harvesting the culture, continue to inoculate onto MA104 cells according to the same method for virus growth and passage to obtain the virus solution;

[0069] (2) Culture method of human rotavirus strain RV009

[0070] 2.1 Adaptation of the virus on Vero cells

[0071] Use 0.25% trypsin (from Gibco) - 0.03% EDTA to digest the Vero cells that have formed a monolayer, resuspend the cells with DMEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and inoculate small square bottles at 10 5.0 cells / bottle. Incubate at 37°C and 5% CO2 for 3 days until a dense cell monolayer is formed, then inoculate 1 mL of the virus solution obtained in step 1.8 above, with an inoculation amount of 10 5.5 CCID50 / mL, adsorb at 37°C for 90 min; then add DMEM cell maintenance medium without newborn bovine serum (from Gibco), continue to culture at 37°C and observe cytopathic effect; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can continue to be passaged on Vero cells or stored frozen for later use to obtain the virus solution;

[0072] (3) Identification method for human rotavirus strain RV009

[0073] 3.1 Virus titer determination

[0074] Use the CCID 50 method for determination: Take the virus solution obtained in step 2.1 above as the virus sample, add 2.5 mg / mL trypsin (from Gibco) (to make the final concentration of trypsin 20 μg / mL), activate it in a 37°C water bath for 1 h, and perform 10-fold serial dilution of the activated virus sample; in a 96-well plate of MA104 cells that have grown into a dense monolayer, remove the residual maintenance medium, add 100 μL of the virus dilution to each well, and use two columns of wells as cell control wells, adding maintenance medium without serum; seal the 96-well plate with a sealing film, then place the plate in an incubator at 37°C and 5% CO2 for 7 days; after 7 days, freeze-thaw the 96-well plate, and then completely transfer the solution in each well to a 96-well plate pre-coated with a sheep anti-rotavirus immunopolyclonal antibody (diluted 1:1000) manufactured by Millipore, incubate at 37.0°C for 1 h to allow the antibody to bind to the virus; after washing the plate, add the HRP-labeled goat anti-rotavirus detection antibody prepared by Jiangsu Liweisi Biotechnology Co., Ltd. diluted 1:2000, and incubate again at 37.0°C for 1 h; after washing the plate, add TMB chromogenic solution (Beyotime), develop color in the dark at room temperature for 5 min, add the stop solution to terminate the color reaction, and then detect the optical absorbance with an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm (main wavelength) and 650 nm (calibration wavelength).

[0075] Use the Karber method to calculate the virus titer, and the calculation formula is as follows (when the sample volume is 100 μl):

[0076] lgCCID 50 = X m −1 / 2d + d·∑pi / 100

[0077] X m is the logarithm of the highest dilution multiple with positive virus antigen;

[0078] D is the logarithm of the dilution factor (multiple);

[0079] ∑pi is the sum of the highest complete antigen positive detection concentration and the lesion percentages of each dilution after that;

[0080] 3.2 Whole genome sequencing of RV009 virus

[0081] The total RNA of the rotavirus strain (RV009) was entrusted to TaKaRa Biotechnology Co., Ltd. (Dalian, China) for sequencing; using the total RNA of rotavirus as a template, 11 gene-specific primers were designed and synthesized; 2 μL of PrimeScript 1-step enzyme mix, 2×1-step buffer (25 μL), 1 μL of 20 μM F / R primer, 20 μL of RNase-free dH2O, and 1 μL of template RNA were transferred to a PCR amplifier and RT-PCR amplification was carried out according to the following program: the reverse transcription reaction was incubated at 50 °C for 30 min, and then initial denaturation at 94 °C for 2 min; subsequent steps included denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 3 min, for a total of 35 cycles; 5 μL of each PCR product was subjected to 1% agarose gel electrophoresis, and the gel imaging was used to take pictures and observe; 2000 bp DNA ladder marker was used to estimate the product size; the PCR amplicons were purified using the Takara Mini BEST Agarose Gel DNA Extraction Kit Ver. 4.0 (Takara CodeNo. 9762) and sequenced using an automatic DNA sequencer (ABI3730, USA), and the sequencing results were subjected to sequence analysis and alignment. Through sequence analysis, the whole genome type of the RV009 strain was determined to be G9-P

[10] -I1-R3-C3-M3-A9-N1-T3-E3-H6.

[0082] Full gene sequence of the human rotavirus strain RV009:

[0083] The full gene sequence of the NSP1 gene is shown in SEQ ID NO: 1;

[0084] The full gene sequence of the NSP2 gene is shown in SEQ ID NO: 2;

[0085] The full gene sequence of the NSP3 gene is shown in SEQ ID NO: 3;

[0086] The full sequence of the NSP4 gene is shown in SEQ ID NO: 4;

[0087] The full sequence of the NSP5 gene is shown in SEQ ID NO: 5;

[0088] The full sequence of the VP1 gene is shown in SEQ ID NO: 6;

[0089] The full sequence of the VP2 gene is shown in SEQ ID NO: 7;

[0090] The full sequence of the VP3 gene is shown in SEQ ID NO: 8;

[0091] The full sequence of the VP4 gene is shown in SEQ ID NO: 9;

[0092] The full sequence of the VP6 gene is shown in SEQ ID NO: 10;

[0093] The full sequence of the VP7 gene is shown in SEQ ID NO: 11.

[0094] Example 2

[0095] A method for isolation, culture and identification of a human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the culture method of the human rotavirus strain RV009 in step (2) is as follows:

[0096] Use 0.25% trypsin (from Gibco) - 0.03% EDTA to digest the Vero cells that have formed a monolayer, resuspend the cells with DMEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and inoculate small square bottles at 10 5.0 cells / bottle. After culturing at 37°C and 5% CO2 for 3 days until a confluent cell monolayer is formed, inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, wherein the virus solution contains calcium chloride with a final concentration of 5 mmol / mL, polyethylene glycol with a final concentration of 8 mmol / mL, and poly(L-lysine) with a final concentration of 4 mmol / mL. Adsorb at 37°C for 90 min; then add a DMEM cell maintenance solution (from Gibco) without newborn bovine serum, and continue to culture and observe the cytopathic effect at 37°C; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can continue to be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0097] Example 3

[0098] A method for isolating, culturing and identifying human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the (2) culturing method of human rotavirus strain RV009 is as follows:

[0099] Digest the confluent monolayer of Vero cells with 0.25% trypsin (from Gibco)-0.03% EDTA, resuspend the cells with DMEM (from Gibco)-10% NBCS (from Zhejiang Tianhang), and inoculate small square flasks at 10 5.0 cells / flask. Incubate at 37°C and 5% CO2 for 3 days until a confluent cell monolayer is formed, then inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, where the virus solution contains calcium chloride at a final concentration of 5 mmol / mL, polyethylene glycol at a final concentration of 8 mmol / mL, and poly(L-arginine) at a final concentration of 4 mmol / mL. Adsorb at 37°C for 90 min; then add DMEM cell maintenance medium (from Gibco) without newborn bovine serum, continue to culture at 37°C and observe for cytopathic effects; when the cytopathic effects become obvious, harvest the culture, and the harvested culture can be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0100] Example 4

[0101] A method for isolating, culturing and identifying human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the (2) culturing method of human rotavirus strain RV009 is as follows:

[0102] Digest the confluent monolayer of Vero cells with 0.25% trypsin (from Gibco)-0.03% EDTA, resuspend the cells with DMEM (from Gibco)-10% NBCS (from Zhejiang Tianhang), and inoculate small square flasks at 10 5.0 cells / flask. Incubate at 37°C and 5% CO2 for 3 days until a confluent cell monolayer is formed, then inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, where the virus solution contains calcium chloride at a final concentration of 5 mmol / mL, polyethylene glycol at a final concentration of 8 mmol / mL, and poly(L-glutamic acid) at a final concentration of 4 mmol / mL. Adsorb at 37°C for 90 min; then add DMEM cell maintenance medium (from Gibco) without newborn bovine serum, continue to culture at 37°C and observe for cytopathic effects; when the cytopathic effects become obvious, harvest the culture, and the harvested culture can be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0103] Example 5

[0104] A method for isolating, culturing and identifying human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the (2) culturing method of human rotavirus strain RV009 is as follows:

[0105] Use 0.25% trypsin (from Gibco) - 0.03% EDTA to digest the Vero cells that have formed a monolayer, resuspend the cells with DMEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and inoculate small square bottles at 10 5.0 cells / bottle. After culturing at 37°C and 5% CO2 for 3 days until a dense cell monolayer is formed, inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, wherein the virus solution contains magnesium chloride with a final concentration of 5 mmol / mL, polyethylene glycol with a final concentration of 8 mmol / mL, and poly(L-lysine) with a final concentration of 4 mmol / mL. Adsorb at 37°C for 90 min; then add DMEM cell maintenance solution (from Gibco) without newborn bovine serum, continue to culture at 37°C and observe the cytopathic effect; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can continue to be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0106] Comparative Example 1

[0107] A method for isolating, culturing and identifying human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the (2) culturing method of human rotavirus strain RV009 is as follows:

[0108] Use 0.25% trypsin (from Gibco) - 0.03% EDTA to digest the Vero cells that have formed a monolayer, resuspend the cells with DMEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and inoculate small square bottles at 10 5.0 cells / bottle. After culturing at 37°C and 5% CO2 for 3 days until a dense cell monolayer is formed, inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, wherein the virus solution contains calcium chloride with a final concentration of 5 mmol / mL and polyethylene glycol with a final concentration of 8 mmol / mL. Adsorb at 37°C for 90 min; then add DMEM cell maintenance solution (from Gibco) without newborn bovine serum, continue to culture at 37°C and observe the cytopathic effect; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can continue to be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0109] Comparative Example 2

[0110] A method for isolating, culturing and identifying human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the (2) culturing method of human rotavirus strain RV009 is as follows:

[0111] Use 0.25% trypsin (from Gibco) - 0.03% EDTA to digest the Vero cells that have formed a monolayer, resuspend the cells with DMEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and inoculate small square bottles at 10 5.0 cells / bottle. After culturing at 37°C and 5% CO2 for 3 days until a confluent cell monolayer is formed, inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, wherein the virus solution contains a final concentration of 8 mmol / mL polyethylene glycol and a final concentration of 4 mmol / mL poly(L-lysine), and adsorb at 37°C for 90 min; then add DMEM cell maintenance solution (from Gibco) without newborn bovine serum, continue to culture at 37°C and observe cytopathic effect; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0112] Comparative Example 3

[0113] A method for isolating, culturing and identifying human rotavirus strain RV009, which is different from Example 1 in that the specific operation of the (2) culturing method of human rotavirus strain RV009 is as follows:

[0114] Use 0.25% trypsin (from Gibco) - 0.03% EDTA to digest the Vero cells that have formed a monolayer, resuspend the cells with DMEM (from Gibco) - 10% NBCS (from Zhejiang Tianhang), and inoculate small square bottles at 10 5.0 cells / bottle. After culturing at 37°C and 5% CO2 for 3 days until a confluent cell monolayer is formed, inoculate 1 mL of the virus solution obtained in step 1.8 above, and the inoculation amount is 10 5.5 CCID 50 / mL, wherein the virus solution contains a final concentration of 5 mmol / mL calcium chloride and a final concentration of 4 mmol / mL poly(L-lysine), and adsorb at 37°C for 90 min; then add DMEM cell maintenance solution (from Gibco) without newborn bovine serum, continue to culture at 37°C and observe cytopathic effect; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can be passaged on Vero cells or stored frozen for later use to obtain the virus solution.

[0115] Test Example 1

[0116] The adaptive culture of the human rotavirus strain RV009 on Vero cells was observed separately according to the methods of Examples 1-5 and Comparative Examples 1-3. The cytopathic effect was observed, and the time when obvious cytopathic changes occurred in the cells during the culture of the RV009 strain virus in Vero cells in each example and comparative example was recorded as the passage number increased. The specific situation is shown in Table 1 below;

[0117] CCID 50 (Cell culture median infective dose) is an experimental unit for measuring virus infectivity, indicating the minimum amount of virus required to infect 50% of the cells. CCID 50 The lower the value, the stronger the virus infectivity, that is, a smaller amount of virus can infect a large number of cells. Therefore, CCID 50 value is inversely proportional to the virus infectivity. A low CCID 50 value means that the virus has a high infection efficiency; the virus infectivity titers (lgCCID 50 ) of the virus strains RV009 in Examples 1-5 and Comparative Examples 1-3 at 48 h after the 10th passage of adaptive culture on Vero cells were recorded respectively, as shown in Table 2 below;

[0118] Table 1

[0119]

[0120] Table 2

[0121]

[0122] As can be seen from Table 1, by comparing Examples 1-5 and Comparative Examples 1-3, it was found that as the passage number increased, the time for obvious cytopathic changes to occur in the cells of Examples 2-5 and Comparative Examples 1-3 was shorter than that of Example 1; by comparing Examples 2-5 and Comparative Examples 1-3, it was found that the time for obvious cytopathic changes to occur in the cells of Examples 2-5 was shorter than that of Comparative Examples 1-3. Among them, the time of Example 2 was the shortest, and obvious cytopathic changes could be observed after culturing for 76 h, 44 h, and 22 h in the 3rd, 4th, and 5th passages respectively.

[0123] As can be seen from Table 2, by comparing Examples 1-5 and Comparative Examples 1-3, it was found that after the 10th passage of adaptive culture of the RV009 strain virus on Vero cells, the virus infectivity titers at 48 h of Examples 2-5 and Comparative Examples 1-3 were higher than that of Example 1; by comparing Examples 2-5 and Comparative Examples 1-3, it was found that the virus infectivity titers of Examples 2-5 were higher than those of Comparative Examples 1-3. Among them, the virus infectivity titer of Example 2 was the highest, reaching 7.20 lgCCID 50 / mL.

[0124] The analysis results of Table 1 and Table 2 show that in the virus culture step, the combined use of additives, polyethylene glycol, and polymers in the virus solution helps to shorten the time of cytopathic effect and increase the infectious titer of the virus. Moreover, the change in the types of additives and polymers has an obvious impact on the infection efficiency of human rotavirus strain RV009 on Vero cells. Among them, choosing calcium chloride as the additive and poly(L-lysine) as the polymer is more conducive to shortening the virus infection time, improving the virus infection efficiency, and increasing the infectious titer of the virus compared to choosing magnesium chloride as the additive and poly(L-arginine) or poly(L-glutamic acid) as the polymers, and the virus shows higher infectivity. The possible reason for the analysis is that in this invention, the combined use of calcium chloride, polyethylene glycol, and poly(L-lysine) is introduced into the virus culture step. The three may produce a synergistic effect during the virus culture adsorption process to promote the contact infection of human rotavirus strain RV009 on Vero cells and increase the virus infection efficiency. During the culture adsorption process of the virus solution and Vero cells, the calcium ions in calcium chloride may interact with the surface proteins of human rotavirus strain RV009 or certain receptors on the Vero cell membrane, promoting the tight binding of virus particles to the cell membrane, thereby increasing the possibility of virus infection. And calcium ions may play a regulatory role through their signal transduction during the virus replication and proliferation process, which is beneficial to enhancing the virus replication and proliferation ability, thus promoting the virus infection of Vero cells. At the same time, polyethylene glycol can promote the aggregation of virus particles, reduce the diffusion of the virus, thereby increasing the contact opportunity between the virus and the cells, being conducive to the adsorption of the virus to the cells, and can reduce the influence of impurities in the culture medium on virus infection, improving the virus infection efficiency. When calcium chloride and polyethylene glycol are used in combination, calcium chloride can promote the fusion of the virus and the cell membrane by generating calcium ions, while polyethylene glycol promotes the aggregation of virus particles physically. The combined action of the two can promote the fusion and infection of the virus and the cells, thereby further improving the virus infection efficiency. Poly(L-lysine) is a cationic polymer that can interact with the negatively charged cell surface through ionic adsorption. This property enables it to interact with the negative charges on the virus and the cell membrane, promoting virus adsorption and fusion, thus promoting the binding of human rotavirus strain RV009 to Vero cells, improving the infectivity of the virus and the ability of the virus to invade cells, and thus possibly improving the virus infection efficiency. At the same time, its cationic property also enables it to protect the virus from adverse factors in the environment, such as degradation by nucleases or oxidative stress. This protective effect helps the virus maintain the integrity of its structure and function during cell culture, thereby enhancing the stability of virus infection and indirectly reducing the probability of gene mutation of the virus during the culture process, being beneficial to increasing the adaptability of human rotavirus strain RV009 to Vero cells and enhancing the genetic stability of human rotavirus strain RV009.Moreover, calcium chloride is used in combination with poly(L-lysine). Calcium ions can promote the interaction between poly(L-lysine) and viruses or cell membranes, thereby further enhancing the virus infection efficiency and shortening the time of cytopathic effect. Compared with poly(L-arginine) and poly(L-glutamic acid), poly(L-lysine) has a higher positive charge density, which enables it to interact more effectively with the negative charges on the virus surface and the cell membrane, thus promoting the fusion of the virus and the cell membrane. Therefore, poly(L-lysine) has a better effect on improving the virus infection efficiency. In the present invention, calcium chloride, polyethylene glycol and poly(L-lysine) are used in combination in the adaptive culture process of human rotavirus strain RV009 on Vero cells. The synergistic effect of the three may enhance the adsorption of the virus to Vero cells, protect the virus structure, improve the virus replication ability and promote the fusion of the virus and the cell membrane, thereby enhancing the infection efficiency and genetic stability of human rotavirus strain RV009.

[0125] In summary, the method for isolation, culture and identification of a human rotavirus strain RV009 provided by the present invention helps to rapidly amplify the virus, shorten the production cycle and obtain a high yield of virus at a low cost, which is beneficial to vaccine production, virological research and the development of diagnostic reagents.

[0126] Test Example 2

[0127] According to the test results of Test Example 1, it can be seen that the culture method of the human rotavirus strain RV009 provided in Example 2 of the present invention can significantly improve the rotavirus infection efficiency and virus infectivity. The human rotavirus strain RV009 of the present invention and the commercially available rotavirus A strain WI61 (from the American Type Culture Collection (ATCC), VR-3392) were respectively cultured by the virus culture method provided in Example 2 of the present invention, and the virus solution was inoculated onto Vero cells for adaptive culture:

[0128] Vero cells forming a monolayer were digested with 0.25% trypsin (from Gibco)-0.03% EDTA, and the cells were resuspended with DMEM (from Gibco)-10% NBCS (from Zhejiang Tianhang). The cells were inoculated into small square bottles at a density of 10 5.0 cells / bottle. After culturing at 37 °C and 5% CO2 for 3 days until a dense cell monolayer was formed, 1 mL of the activated rotavirus solution (human rotavirus strain RV009 or commercially available rotavirus A strain WI61) was inoculated, and the inoculation amount was 10 5.5 CCID 50 / mL, wherein the virus solution contains calcium chloride at a final concentration of 5 mmol / mL, polyethylene glycol at a final concentration of 8 mmol / mL, and poly(L-lysine) at a final concentration of 4 mmol / mL, and adsorb at 37 °C for 90 min; then add DMEM cell maintenance medium without newborn bovine serum (from Gibco), continue to culture at 37 °C and observe cytopathic effect; when the cytopathic effect becomes obvious, harvest the culture, and the harvested culture can continue to be passaged on Vero cells or stored frozen for later use;

[0129] Record the time when obvious cytopathic effect appears in the infected cells of the two viruses respectively, and use CCID 50 method to determine the virus infectivity titer (lgCCID 50 ) of the two virus strains after adapting and culturing on Vero cells according to the virus culture method of Example 2 of the present invention at the 48th h after the 10th passage. The specific test results are shown in Table 3 below:

[0130] Table 3

[0131]

[0132] As can be seen from Table 3, by comparing the rotavirus strain RV009 provided by the present invention with the commercially available rotavirus strain WI61, it is found that with the increase of the passage generation times, the time for obvious cytopathic effect to appear in the rotavirus strain RV009 provided by the present invention is shorter than that of the commercially available rotavirus strain WI61. Obvious cytopathic effect can be observed in the 3rd, 4th, and 5th generations of the rotavirus strain RV009 provided by the present invention after culturing for 76 h, 44 h, and 22 h respectively, while obvious cytopathic effect can be observed in the 4th and 5th generations of the commercially available rotavirus strain WI61 after culturing to 70 h and 45 h respectively; by comparing the rotavirus strain RV009 provided by the present invention with the commercially available rotavirus strain WI61, it is found that the virus infectivity titer of the strain RV009 at the 48th h is significantly higher than that of the commercially available rotavirus strain WI61. The virus infectivity titer of the rotavirus strain RV009 provided by the present invention reaches 7.20 lgCCID 50 / mL, while the commercially available rotavirus strain WI61 is only 5.40 lgCCID 50 / mL; It shows that the culturing method provided in Example 2 of the present invention is conducive to improving the viral infectivity of the rotavirus strain RV009 of the present invention, while culturing the commercially available rotavirus strain WI61 using the culturing method provided in Example 2 of the present invention does not show good viral infection efficiency. The reason for the analysis may be that for the commercially available rotavirus strain WI61, its sensitivity to the concentration or combination of calcium chloride, polyethylene glycol, and poly(L-lysine) introduced in the culturing method provided by the present invention is different from that of the rotavirus strain RV009 of the present invention. The concentration and ratio of these substances may be more optimal for the RV009 strain, while they may inhibit the infection efficiency of the WI61 strain. Moreover, different rotavirus strains may have different biological characteristics and host ranges. The WI61 strain may not be adapted to the culturing conditions provided by the present invention, thus affecting the receptor-binding ability of the WI61 strain to Vero cells and resulting in a low infection efficiency of the WI61 strain to Vero cells, leading to poor infection efficiency.

Claims

1. A human rotavirus strain, characterized in that: This strain is human G9 rotavirus RV009, and its classification name is: Rotavirus; preservation unit: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); preservation date: July 22, 2024; preservation number: CGMCC No.45999.

2. The human rotavirus strain according to claim 1, characterized in that The complete gene sequence of the human G9 rotavirus RV009 is as follows: the complete gene sequence of the NSP1 gene is shown in SEQ ID NO: 1; the complete gene sequence of the NSP2 gene is shown in SEQ ID NO: 2; the complete gene sequence of the NSP3 gene is shown in SEQ ID NO: 3; the complete gene sequence of the NSP4 gene is shown in SEQ ID NO: 4; the complete gene sequence of the NSP5 gene is shown in SEQ ID NO: 5; the complete gene sequence of the VP1 gene is shown in SEQ ID NO: 6; the complete gene sequence of the VP2 gene is shown in SEQ ID NO: 7; the complete gene sequence of the VP3 gene is shown in SEQ ID NO: 8; the complete gene sequence of the VP4 gene is shown in SEQ ID NO: 9; the complete gene sequence of the VP6 gene is shown in SEQ ID NO: 10; and the complete gene sequence of the VP7 gene is shown in SEQ ID NO:

11.

3. A method for isolating human rotavirus strains as claimed in claim 1, characterized in that: The specific steps include: A1. Obtaining original virus samples: Collect stool samples from children with diarrhea in the hospital as original virus samples; B1. Colloidal gold detection: Use the colloidal gold detection kit to detect the original sample and obtain the sample with positive colloidal gold retest; C1. RNA-PAGE detection: add the samples with positive colloidal gold retest to a phosphate buffer solution with a concentration of 0.005-0.01 mol / L and a pH of 7.0-7.2, stir to obtain a suspension, centrifuge at 10000-12000g for 15-30min, take the supernatant, sterilize and filter with a 0.22μm filter, and store at -18℃ to -20℃ for later use to obtain fecal processing samples with positive colloidal gold results; extract viral genomic RNA from fecal processing samples with positive colloidal gold results, perform non-denaturing PAGE gel electrophoresis for 8-10h, take photos after silver nitrate staining, and perform band type detection on the extracted viral nucleic acid samples to determine their basic characteristics; D1. RT-PCR detection: Extract viral genomic RNA, perform RT-PCR using specific primers for the rotavirus VP7 gene sequence, sequence the resulting PCR product, and compare the sequencing results with the VP7 gene published in Genbank to determine the type of the virus sample; E1. Screening and determination of candidate strains: After confirmation by the above-mentioned colloidal gold test, RNA-PAGE test and RT-PCR test, G9 strains with epidemiological significance are selected from all positive samples detected, and positive specimens of rotavirus strains are obtained; F1. Obtain virus samples: resuspend the positive specimens of the rotavirus strains screened and determined in step E1 in a solution with a concentration of 0.005-0.01 mol / L and a pH of 7.0-7.2, centrifuge at 10000-12000 g for 15-30 min, obtain the supernatant, sterilize and filter using a 0.22 μm filter, and store at -18°C to -20°C for later use, to obtain a dilution of diarrhea samples of children infected with rotavirus; G1. Virus adaptation on MA104 cells: MA104 cells that had formed a monolayer were digested with 0.25% trypsin-0.03% EDTA, and the cells were resuspended in MEM-10% NBCS and then heated for 10 min. 5.0 The cells / bottle are inoculated into a small square bottle, and cultured at 37° C. for 3-4 days until the cells form a monolayer again; the diluted solution of the diarrhea sample of the rotavirus-infected child obtained in step F1 is inoculated onto the MA104 cells that have formed a monolayer, and adsorbed at 37° C. for 60-100 minutes; after adsorption, the virus solution is removed, and MEM cell maintenance solution without newborn calf serum is added, and the culture is continued at 37° C. for 5-7 days and the lesions are observed, and after the culture is harvested, the MA104 cells are inoculated in the same way for passage or frozen for standby use, and the RV009 strain is finally screened to obtain the RV009 rotavirus harvest solution that has been adapted to culture on MA104 cells; H1. Virus cloning and purification: dilute the RV009 rotavirus harvested from MA104 cells in a 10-fold gradient. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Five dilutions of virus solution were inoculated into six-well plates of MA104 cells that had formed a monolayer, adsorbed at 37°C for 60-100 minutes, and MEM medium containing 0.4-0.7% agarose was added. After the agar solidified, it was inverted and cultured at 37°C for 2-4 days; then MEM medium containing 0.4-0.7% agarose and 0.05-0.15% neutral red was added, and the culture was continued for 2-4 days to observe the formation of plaques; a single plaque was picked up with a Pasteur pipette and transferred to a plate containing 1 mL In a 1.5 mL centrifuge tube containing MEM medium, vortex oscillation is used to ensure that the virus and medium are evenly mixed; after freezing and thawing, the mixture is centrifuged at 4500-8000g at 3-5°C for 25-35 minutes, the supernatant is taken, and then inoculated onto a new MA104 cell monolayer, cultured at 37°C for 3-4 days, and the culture is harvested after the cell pathological changes; after harvesting the culture, continue to inoculate MA104 cells in the same way for passage or freeze for later use to obtain RV009 rotavirus liquid.

4. A method for culturing a human rotavirus strain as claimed in claim 1, characterized in that: The specific steps include: Virus adaptation on Vero cells: Vero cells that had formed a monolayer were digested with 0.25% trypsin-0.03% EDTA and resuspended in DMEM-10% NBCS. 5.0 Cells / bottle were inoculated into small square bottles and cultured at 37°C, 5.0% CO2 for 3-4 days until a dense cell monolayer was formed. Then 1 mL of virus solution was inoculated. The inoculation volume was 10 4.5 -10 6.5 CCID 50 / mL, adsorb at 37℃ for 1-2h, then add DMEM cell maintenance medium without newborn calf serum, culture at 37℃ and observe the cytopathic effect, harvest the culture, and the harvested culture can be further passaged on Vero cells or frozen for future use to obtain the virus liquid.

5. The method for culturing a human rotavirus strain according to claim 4, characterized in that: The method for culturing the human rotavirus strain may also be: The Vero cells that had formed a monolayer were digested with 0.25% trypsin-0.03% EDTA and resuspended in DMEM-10% NBCS. 5.0 Cells / bottle were inoculated into small square bottles and cultured at 37°C, 5.0% CO2 for 3-4 days until a dense cell monolayer was formed; then 1 mL of virus solution was inoculated, with an inoculum of 10 4.5 -10 6.5 CCID 50 / mL, wherein the virus solution contains additives, polyethylene glycol and polymers, and is adsorbed at 37°C for 1-2h; then, DMEM cell maintenance solution without newborn calf serum is added, and the cells are cultured at 37°C and the cell lesions are observed, and the culture is harvested. The harvested culture can be further passaged on Vero cells or frozen for later use to obtain the virus solution.

6. The method for culturing a human rotavirus strain according to claim 4 or 5, characterized in that: The virus liquid is the human G9 rotavirus RV009 virus liquid as described in claim 1.

7. The method for culturing a human rotavirus strain according to claim 5, characterized in that: The final concentration of the additive in the virus solution is 1-6 mmol / mL; the final concentration of the polyethylene glycol is 1-10 mmol / mL; the final concentration of the polymer is 1-5 mmol / mL; the additive is selected from one of calcium chloride and magnesium chloride; the polymer is selected from one of poly(L-lysine), poly(L-arginine) and poly(L-glutamic acid).

8. A method for identifying human rotavirus strains as claimed in claim 1, characterized in that: The specific steps include: Virus titer determination: CCID 50 The virus sample was activated in a water bath at 37°C for 0.5-1h, and the activated virus sample was diluted 10 times; in a 96-well plate of MA104 cells that had grown into a dense monolayer, the residual maintenance solution was removed, and 95-105 μL of virus dilution solution was added to each well, and two columns of wells were used as cell control wells, and maintenance solution without serum was added; the 96-well plate was sealed with a sealing film, and then the plate was placed in an incubator at 37°C and 5.0% CO2 for 6-8 days; after that, the 96-well plate was frozen and thawed, and the solution in each well was completely dried. Transfer all the samples to a 96-well plate pre-coated with goat anti-rotavirus immune polyclonal antibody (1:2000 dilution), incubate at 37°C for 0.5-1.5h to allow the antibody to bind to the virus; after washing the plate, add 1:2000 diluted HRP-labeled goat anti-rotavirus detection antibody, and incubate again at 37°C for 1h; after washing the plate, add TMB colorimetric solution, develop the color at room temperature in the dark for 4-6min, add stop solution to terminate the color development reaction, and then use an enzyme reader to detect the light absorption value at 450nm and 650nm wavelengths; calculate the virus titer.

9. The method for identifying human rotavirus strains according to claim 8, characterized in that: The virus sample is the virus liquid harvested after the human G9 rotavirus RV009 virus according to claim 1 is adapted and cultured on Vero cells.

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