Porcine rotavirus live attenuated vaccine strain without trypsin dependence and application thereof

Abstract

The application discloses a pancreatin-independent porcine rotavirus attenuated live vaccine strain and application thereof, and takes a porcine rotavirus virulent strain HLJ / 2021 isolated in Heilongjiang province as a research object, and obtains an attenuated live vaccine strain PoRV-F100 (namely HLJ / 2021-F100) with reduced pathogenicity to piglets, good immunogenicity and in-vitro passage not dependent on pancreatin by continuously passing 100 generations on MA104 cells. Experiments prove that the attenuated live vaccine strain PoRV-F100 has significantly enhanced cell adaptability, can stably replicate in vitro without pancreatin, has no obvious pathogenicity to 5-day-old piglets, retains good immunogenicity, can induce high-level neutralizing antibodies in mice, can activate mucosal immune response by oral immunization, can produce porcine rotavirus specific sIgA, and can provide effective protection for piglets by maternal passive immunization. The attenuated live vaccine strain PoRV-F100 can be used for preparing a porcine rotavirus attenuated live vaccine, and provides a new means for prevention and control of porcine rotavirus.

Description

Technical Field

[0001] This invention relates to a live attenuated porcine rotavirus vaccine strain and its application. This invention belongs to the fields of microbiology and veterinary medicine. Background Technology

[0002] Porcine rotavirus (PoRV) belongs to the genus Rotavirus in the family Reoviridae. It is one of the core pathogens causing viral diarrhea in piglets, especially 1-4 week old suckling piglets. Since its first isolation in 1975, the virus has spread widely around the world, becoming one of the important diseases affecting the healthy development of the pig industry.

[0003] PoRV is primarily transmitted via the fecal-oral route. The virus particles are highly resistant to the external environment, to acids, alkalis, and common disinfectants, and can survive for extended periods in pig farm environments, forming a continuous cycle. Its incubation period is short (12-24 hours), and it spreads rapidly among susceptible populations, infecting the entire herd within days. Infected piglets typically exhibit symptoms such as acute watery diarrhea, vomiting, anorexia, and dehydration, which can lead to death in severe cases, especially in piglets under one week old, where morbidity can reach over 80% and mortality can reach 100%. Furthermore, PoRV often co-infects with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), further aggravating the condition and increasing piglet mortality, causing significant economic losses to the pig farming industry.

[0004] In recent years, with the increasing scale and intensification of pig farming in my country, the prevalence of porcine reproductive and respiratory syndrome (PoRV) has become increasingly severe, with its detection rate showing a year-on-year upward trend. Meanwhile, PoRV exhibits diverse genotypes and is prone to mutation; currently, 11 groups (A to K) have been identified, with group A being the main pathogen causing diarrhea in piglets, accounting for over 90% of clinical isolates. Furthermore, based on sequence differences between the outer capsid glycoprotein VP7 (G genotyping) and the spike protein VP4 (P genotyping), a G / P bigenotyping system has been established. Currently, multiple combinations of G and P genotypes have been discovered, and the prevalent genotypes vary across different regions, with new genotypes constantly emerging, posing a significant challenge to PoRV control.

[0005] Vaccination is one of the main methods for preventing and controlling PoRV infection. Currently, the PoRV vaccines approved for marketing both domestically and internationally mainly include inactivated vaccines and live attenuated vaccines. Inactivated vaccines are usually prepared using traditional strains and have the advantage of high safety, but their immunogenicity is relatively weak, requiring multiple immunizations to produce sufficient protection. Furthermore, the induced mucosal immune response is weak, making it difficult to effectively prevent intestinal infection and viral shedding. Live attenuated vaccines, on the other hand, have advantages such as strong immunogenicity, long duration of immunity, and the ability to induce mucosal immune responses. They can better mimic natural infection and stimulate a comprehensive immune response in the body, thus having broader application prospects in PoRV prevention and control.

[0006] However, existing live attenuated PoRV vaccines still have some problems. On the one hand, the immunoprotective rate of some vaccine strains is not high, making it difficult to provide effective cross-protection against different PoRV genotypes; on the other hand, some vaccine strains may pose a risk of virulence reversion during use, bringing potential safety hazards to the pig industry. In addition, traditional PoRV vaccine strains usually need to be cultured in a medium containing trypsin, which not only increases the cost of vaccine production but may also introduce exogenous contaminants, affecting the safety and quality stability of the vaccine.

[0007] Cell attenuation through passage is a commonly used method for developing viral vaccines. Its core mechanism involves changing the viral replication site from the natural host's intestinal tissue to in vitro cultured cells, altering the viral selection pressure and replication environment. This induces genetic mutations in the virus during adaptation to cell culture, ultimately allowing for the selection of attenuated variants that retain good immunogenicity, replicate independently of pancreatic enzymes, and lack intestinal pathogenicity. Currently, this method has been successfully applied to the development of various viral vaccines, such as polio, measles, and hepatitis A vaccines. In rotavirus vaccine development, widely used commercial vaccines both domestically and internationally, such as Rotarix® and RotaTeq®, all utilize in vitro cell adaptation passage to achieve viral attenuation, thereby obtaining vaccine strains.

[0008] Although cell passaging attenuation technology has achieved remarkable results in the development of viral vaccines, there are still relatively few studies on cell passaging attenuation for PoRV, and the attenuated live vaccine strains obtained in existing studies often have problems such as decreased immunogenicity and incomplete elimination of pancreatic enzyme dependence.

[0009] Therefore, developing a safe, effective, and pancreatic enzyme-independent live attenuated PoRV vaccine strain is of great significance for improving the immunoprotective effect of PoRV vaccines, reducing vaccine production costs, and ensuring vaccine safety. Summary of the Invention

[0010] The purpose of this invention is to provide a live attenuated porcine rotavirus vaccine strain HLJ / 2021-F100, which has good immunogenicity and safety, and whose replication process does not depend on pancreatic enzymes, and can be used to prepare a live attenuated porcine rotavirus vaccine.

[0011] Another object of the present invention is to provide a vaccine composition comprising the above-mentioned porcine rotavirus attenuated live vaccine strain HLJ / 2021-F100 and its application.

[0012] To achieve the above objectives, the present invention employs the following technical means: This invention uses the previously isolated and identified G5P[7] type PoRV virulent strain HLJ / 2021 as the research object. The PoRVHLJ / 2021 strain was continuously passaged in MA104 cells. It was found that with the increase of passage number, the adaptability of the virus to the cells was significantly enhanced, the time of cytopathic effect was shortened, and the viral titer gradually increased. After 80 passages, the viral titer could be stably maintained at 10. 8.5 TCID 50 The cell count was above 100 / mL; one-step growth curves showed that the HLJ / 2021-F100 strain proliferated faster in MA104 cells than the HLJ / 2021-F5 strain, and reached the stationary phase in a shorter time. Plaque assay results showed that the average plaque diameter of the HLJ / 2021-F100 strain was significantly larger than that of the HLJ / 2021-F5 strain. Trypsin-dependent assay results showed that the HLJ / 2021-F100 strain could be stably passaged without the addition of trypsin.

[0013] To determine the pathogenicity of the HLJ / 2021-F100 strain, this invention used 5-day-old piglets as a model, orally inoculating them with either the HLJ / 2021-F100 or HLJ / 2021-F5 strains, with the PBS group serving as a healthy control group. Results showed that HLJ / 2021-F5 infection caused piglets to develop severe watery diarrhea, significant atrophy and loss of ileal villi, and substantial changes in the lungs; infected piglets shed the virus for up to 90 hours. In contrast, no obvious clinical symptoms were observed in piglets infected with the HLJ / 2021-F100 strain, and no significant pathological damage was observed in the intestines or lungs. Viral shedding was detectable, but lasted only 54 hours, and the viral load and tissue viral load were significantly reduced. These results indicate that after 100 consecutive passages, the HLJ / 2021-F100 strain no longer exhibits significant pathogenicity in piglets.

[0014] To determine whether the immunogenicity of the HLJ / 2021-F100 strain had changed, 6-8 day old Kunming mice were used as a model and orally and intraperitoneally injected with HLJ / 2021-F100, HLJ / 2021-F5, and PBS, respectively. The results showed that both oral and intraperitoneal injections of HLJ / 2021-F100 and HLJ / 2021-F5 induced the production of PoRV-specific IgG and neutralizing antibodies in Kunming mice, but only the oral immunization group showed detectable production of specific sIgA. Results of immunization experiments in pregnant mice showed that specific IgG and sIgA were detectable in colostrum after immunization with the HLJ / 2021-F100 strain, with sIgA detected only in the oral immunization group. Challenge experiments on pups with the HLJ / 2021-F5 strain showed that pups in the unimmunized control group experienced diarrhea and weight loss, while only a few pups in the intraperitoneal immunization group experienced mild diarrhea. No obvious diarrhea was observed in the oral immunization group. The weight gain of pups in both the oral and intraperitoneal immunization groups was not significantly different from that in the healthy control group. In conclusion, the HLJ / 2021-F100 strain exhibits good immunogenicity and can provide effective protection for pups through maternal passive immunization. Oral immunization is more advantageous in inducing mucosal immunity and providing passive protection.

[0015] In summary, this invention has successfully obtained a live attenuated PoRV vaccine strain HLJ / 2021-F100 that is not significantly pathogenic to piglets, has good immunogenicity, and does not require the addition of pancreatic enzymes for in vitro culture. This strain meets the core characteristics of a live attenuated vaccine candidate strain and can provide important materials and theoretical support for the subsequent development of an oral live attenuated PoRV vaccine.

[0016] Based on the above research, this invention provides a pancreatic enzyme-free, attenuated live porcine rotavirus vaccine strain, named PoRV-F100. This strain was obtained by passaged porcine rotavirus strain HLJ / 2021 for 100 generations in MA104 cells, and is therefore also called HLJ / 2021-F100. This strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.47079.

[0017] Preferably, the porcine rotavirus attenuated live vaccine strain can replicate stably in vitro without relying on trypsin, and the viral titer is stably maintained at 10. 8.5 TCID 50 / mL or more.

[0018] Furthermore, the present invention also proposes the application of the described live attenuated porcine rotavirus vaccine strain in the preparation of a vaccine for the prevention of porcine rotavirus infection.

[0019] Furthermore, the present invention also proposes a vaccine composition comprising the aforementioned live attenuated porcine rotavirus vaccine strain.

[0020] Preferably, the vaccine composition further comprises a pharmaceutically acceptable carrier or adjuvant.

[0021] Furthermore, the present invention also proposes the use of the vaccine composition in the preparation of a vaccine for the prevention of porcine rotavirus infection.

[0022] Preferably, the vaccine is a live attenuated vaccine.

[0023] Compared with the prior art, the beneficial effects of the present invention are: This invention uses the highly virulent PoRV strain HLJ / 2021 isolated in Heilongjiang Province as the research object. Through continuous in vitro passage, a strain with reduced pathogenicity to piglets, good immunogenicity, and in vitro passage independent of trypsin was obtained. Specifically: 1. The live attenuated porcine rotavirus vaccine strain HLJ / 2021-F100 of the present invention has no obvious pathogenicity to 5-day-old piglets. No clinical symptoms such as diarrhea and weight loss were observed in piglets after vaccination. The intestinal tissue pathological damage was slight, the viral shedding time was short and the viral shedding amount was low, and it has good safety.

[0024] 2. The attenuated live vaccine strain HLJ / 2021-F100 of the present invention retains good immunogenicity and can induce mice to produce highly efficient neutralizing antibodies and IgG. Oral immunization can activate the intestinal mucosal sIgA response and can provide effective protection for offspring mice through maternal passive immunization. After challenge, offspring mice have mild diarrhea symptoms, normal weight gain, shortened shedding time and reduced shedding amount.

[0025] 3. The attenuated live vaccine strain HLJ / 2021-F100 of the present invention can stably replicate in vitro without relying on trypsin, and the viral titer is stably maintained at 10. 8.5 With a TCID level above 50 / mL, this characteristic helps reduce vaccine production costs and the risk of exogenous contamination, which is of great practical significance for the industrial production of vaccines.

[0026] In summary, the porcine rotavirus HLJ / 2021-F100 attenuated live vaccine strain of the present invention has good safety, immunogenicity and genetic stability, and can be used to prepare porcine rotavirus attenuated live vaccine, providing a new and effective means for the prevention and control of porcine rotavirus. Attached Figure Description

[0027] Figure 1 CPE for each generation of viruses; Figure 2 The results of IFA identification for each generation of the virus; Figure 3 TCID for different generations of HLJ / 2021 strain 50 ; Figure 4 The growth characteristics of the HLJ / 2021-F5 and HLJ / 2021-F100 strains; Among them, a) growth curves of HLJ / 2021-F5 and HLJ / 2021-F100 strains; b) plaque results of HLJ / 2021-F5 and HLJ / 2021-F100 strains; Figure 5 For HLJ / 2021, with / without IFA and TCID. 50 Test results; Among them, a) IFA results with / without pancreatic enzyme PoRV infection; b) with / without pancreatic enzyme TCID 50 Test results; Figure 6 Stability analysis of trypsin-free passage of HLJ / 2021-F100 strain; Among them, a) IFA test results for stability without trypsin passage; b) virus titer test results for virus without trypsin passage; Figure 7 Growth characteristics of HLJ / 2021-F100 strain with / without trypsin addition; Among them, a) growth curves of HLJ / 2021-F100 strain with and without trypsin; b) plaque diagrams of HLJ / 2021-F100 strain with and without trypsin; Figure 8 Western blot analysis was performed to determine the integrity of the VP4 protein. Where M represents the protein molecular weight marker; in lane 1, under the condition of adding trypsin, the VP4 protein of HLJ / 2021-F100 strain was effectively cleaved into two characteristic bands; in lane 2, under the condition of not adding trypsin, the VP4 protein of HLJ / 2021-F100 strain existed in its intact form; in lane 3, when trypsin was added, the VP4 protein of HLJ / 2021-F5 was effectively cleaved into two characteristic bands; Figure 9 These are clinical symptoms in piglets; Among them, a) diarrhea symptoms in piglets; b) severity of diarrhea in piglets; c) weight changes in piglets; and d) body temperature changes in piglets. Figure 10 Pathological changes in the intestines of piglets upon necropsy; Figure 11 Viral load in piglet tissues; Figure 12 HE staining results for the ileum and lungs; Figure 13 The detoxification status of piglets; Figure 14 The titer of mouse serum IgG antibodies (log2) is given. Figure 15The titer of neutralizing antibodies in mouse serum (log2); Figure 16 The titer of mouse intestinal sIgA antibody (log2); Figure 17 The results of antibody titer testing in colostrum; Among them, a) the IgG antibody titer (log2) in colostrum; b) the sIgA antibody titer (log2) in colostrum. Figure 18 These are clinical symptoms in baby mice; Among them, a) scores of baby mouse feces; b) diarrhea status of baby mice; c) changes in average weight of baby mice; Figure 19 Images of necropsy procedures performed on baby mice; Figure 20 The viral load of enteroviruses in baby mice; Figure 21 The detoxification status of baby mice; Figure 22 Comparison of protein conformations between HLJ / 2021-F5 and HLJ / 2021-F100 strains.

[0028] Information on strain preservation: Name: PoRV-F100 Classification and nomenclature: Porcine rotavirus (Porcin rotavirus) Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Accession number: CGMCC NO.47079 Preservation period: July 20, 2026 Detailed Implementation

[0029] The present invention will be further described below with reference to specific examples and accompanying drawings. The advantages and features of the present invention will become clearer with this description. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0030] Example 1: Preparation of a pancreatic enzyme-free, attenuated live porcine rotavirus vaccine 1. Experimental Materials 1.1 Cells and viruses MA104 cells were purchased from the American Type Culture Collection (ATCC) and preserved in liquid nitrogen in our laboratory; the PoRVHLJ / 2021 strain (described in the following literature: Isolation, Identification and Pathogenicity Analysis of Porcine Rotavirus, Wang Yanan et al., Journal of Animal Science and Veterinary Medicine, 2025, 56(5), 2259-2269) was preserved and provided by Northeast Agricultural University.

[0031] 1.2 Laboratory Animals Six 5-day-old piglets used in the experiment were purchased from a pig farmer in Harbin; 6-8 week old SPF-grade Kunming mice and 7-day-old pregnant mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the "Charter of the Experimental Animal Ethics Committee of Northeast Agricultural University".

[0032] 1.3 Main Reagents Cell culture: The cell culture medium was DMEM containing 10% newborn fetal bovine serum (BioChannel), and the basal DMEM was purchased from CEA. Trypsin was prepared as a 2.5% stock solution (dissolved in PBS) and diluted 10-fold before use.

[0033] Antibody and Immunoassay: The primary antibody was PoRV VP6 monoclonal antibody stored in our laboratory; the secondary antibodies included FITC-labeled (Zhongshan Jinqiao) and HRP-labeled goat anti-mouse IgG (Zhongshan Jinqiao) and HRP-goat anti-mouse IgA (Abcam). The ELISA coating buffer was pH 9.6 carbonate buffer, the washing buffer was PBS containing 0.05% Tween-20, the blocking buffer was 5% skim milk powder-PBS, TMB was a two-component mixture for color development, and the stop solution was 10% sulfuric acid.

[0034] Plaque assay: Low melting point agarose (Solarbio), 0.5% crystal violet solution.

[0035] Protein electrophoresis: Commercially available 15% / 5% precast gels (Novizan) were used. The electrophoresis buffer contained Tris-Base, glycine, and SDS; the wet transfer buffer contained an additional 20% methanol and was prepared fresh for each use.

[0036] Animal experiments: The intestinal lavage fluid was PBST containing protease inhibitors, and the colostrum lysate was 2% BSA-PBS containing protease inhibitors.

[0037] 1.4 Preparation of anti-PoRV VP6 monoclonal antibody Anti-PoRV VP6 monoclonal antibody has been documented in the following literature: Meng Liu. Preparation of anti-porcine rotavirus monoclonal antibody and preliminary establishment of double-antibody sandwich ELISA detection method [D]. Northeast Agricultural University, 2014. The specific preparation method is as follows: 1) Antigen preparation: Recombinant PoRV HLJ / 2021 strain VP6 protein was expressed in prokaryotes and purified by affinity chromatography to obtain recombinant VP6 protein with a purity >90% as an immunogen.

[0038] 2) Mouse immunization: 6-8 week old female BALB / c mice were immunized subcutaneously at multiple sites using recombinant VP6 protein in combination with Freund's adjuvant; a total of 3 immunizations were performed, with an interval of 2 weeks between each immunization, and the mouse spleen cells were collected 3 days after the last immunization.

[0039] 3) Cell fusion and screening: Mouse spleen cells and SP2 / 0 myeloma cells were fused using PEG-mediated cell fusion; the fused cells were plated and cultured, and the indirect ELISA method was used for initial screening. The microplate was coated with recombinant VP6 protein to screen for positive hybridoma cell wells; the positive wells were subcloned using the limiting dilution method, and three rounds of subcloning were performed to finally obtain a hybridoma cell line that stably secretes anti-PoRV VP6 antibody.

[0040] 4) Ascites preparation and purification: BALB / c mice were sensitized by intraperitoneal injection of liquid paraffin, and hybridoma cells were injected intraperitoneally 7 days later; ascites was collected after the mice's abdomens were significantly distended; the ascites was crudely extracted by precipitation with saturated ammonium sulfate, and then purified by Protein G affinity chromatography to obtain purified VP6 monoclonal antibody, which was identified as specifically recognizing PoRV VP6 protein.

[0041] 1.5 Primers Based on the PoRV whole genome reference sequence published in GenBank, this invention used software to design and commissioned the synthesis of 12 pairs of specific primers (11 pairs for amplifying each gene fragment and 1 pair for virus identification). The primer sequences are detailed in Table 1.

[0042] Table 1. Primer sequence information for the entire PoRV genome. ; 2 Experimental Methods 2.1 In vitro culture of PoRV 2.1.1 PoRV succession When the confluence of MA104 cells reached 80%–90%, the frozen PoRV HLJ / 2021 strain was taken from the laboratory and slowly thawed at 4°C. Trypsin was added to a final concentration of 30 μg / mL, mixed well, and incubated at 37°C for 1 h to complete virus activation. The cells were washed twice with sterile PBS, and the activated virus solution was inoculated into a cell monolayer. The cells were incubated at 37°C for 1 h for adsorption, with gentle shaking every 20 min to ensure uniform virus adsorption. After adsorption, the virus adsorption solution was discarded, and the cells were washed twice more with PBS. DMEM maintenance medium containing 1 μg / mL trypsin was added, and the cells were incubated in an incubator for further culture.

[0043] Cell culture time was controlled at over 48 hours, and the inoculation ratio was flexibly adjusted according to the rate of cytopathic progression to prolong the viral replication cycle within the cells, increase the number of replications, and thus increase the probability of viral gene mutation. When the proportion of cultured cells detached reached over 70%, the virus solution was collected and subjected to three freeze-thaw cycles. After sterilization by filtration through a 0.22 μm filter, it was passaged again using the same method described above, and the above operations were repeated to complete continuous passage. Starting from F40, domesticated strains were selected every 10 generations, passaged three times consecutively in a trypsin-free system, and the viral titer was measured to assess the trypsin dependence of the strain.

[0044] 2.1.2 Indirect Immunofluorescence Assay (IFA) Key passage viruses (F5, F10, F20…F100) from the continuous passage process were selected and named HLJ / 2021-F5, HLJ / 2021-F10, HLJ / 2021-F20…HLJ / 2021-F100, respectively. These were seeded at an infection multiple of 0.01 MOI into 12-well culture plates with 80% cell confluence, following the specific procedures outlined in section 2.1.1. After 36 h of virus culture, the cytopathic effect (CPE) was observed and recorded. The cell culture medium was then discarded, and each well was filled with pre-cooled anhydrous ethanol and fixed at room temperature for 30 min. The cells were then washed three times with PBS for 5 min each time, gently to prevent cell detachment. 500 μL of anti-PoRV VP6 monoclonal antibody was added to each well, and the plates were incubated at 37°C for 1 h. The cells were then washed three times with PBS for 5 min each time, protected from light, and any remaining liquid was patted dry. Subsequently, 500 μL of FITC-labeled goat anti-mouse IgG and DAPI were added to each well, and the mixture was incubated at 37°C in the dark for 30 min. The wells were then washed three times with PBST in the dark, for 5 min each time. Finally, the specific fluorescence was observed and recorded under a fluorescence microscope.

[0045] 2.1.3 TCID 50 Measurement To clarify the variation pattern of viral titer during continuous passage of PoRV, viral solutions from passages F10, F20...F100 were selected and serially diluted 10-fold (dilution factor 10). -1 ~10 ⁻10 The cells were then inoculated into 96-well culture plates containing MA104 cells, following the specific procedures outlined in section 2.1.1. After static incubation for 36 h, IFA detection was performed according to the method described in section 2.1.2. The cells showing specific fluorescence were recorded in each well. Finally, the TCID of each passage of virus samples was calculated and determined using the Reed-Muench method. 50 .

[0046] 2.1.4 Plotting the PoRV growth curve HLJ / 2021-F5 and HLJ / 2021-F100 strains (PoRV-F100, hereinafter the same) were selected and inoculated into MA104 cells at a MOI of 0.01, with three biological replicates per group. After inoculation, 200 μL of cell supernatant was collected every 12 h until 96 h of culture. The TCID values ​​of the collected viral supernatant samples at each time point were determined according to the method described in section 2.1.3. 50 Based on the measurement results, one-step growth curves of MA104 cells infected by HLJ / 2021-F5 strain and HLJ / 2021-F100 strain were plotted, and the differences in proliferation kinetics of different generations of the virus were compared and analyzed.

[0047] 2.1.5 Plaque Assay HLJ / 2021-F5 and HLJ / 2021-F100 strains were selected. MA104 cells were cultured in 6-well plates to form a dense monolayer. After that, they were injected with 200, 100, 50, and 10 TCID50, respectively. 50 Cells were inoculated with HLJ / 2021-F5 or HLJ / 2021-F100 strain virus at an infection rate of / mL. After adsorption at 37°C for 1 hour, the virus solution was aspirated, and the cells were washed 2-3 times with PBS. Then, 2mL of fresh DMEM covering medium (containing 1μg / mL trypsin, 1% penicillin-dextrose antibody, and 0.75% low-melting-point agarose) was added to each well, and the cells were incubated upside down in a 37°C, 5% CO2 incubator for maintenance. On day 5 post-infection, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Plaque formation was observed and compared. Subsequently, 10 plaques were randomly selected, and the diameter of each plaque was measured and recorded using ImageJ software for subsequent statistical analysis of plaque size.

[0048] 2.2 Detection of biological characteristics of HLJ / 2021-F100 strain 2.2.1 Detection of trypsin dependence in HLJ / 2021-F100 strain After passages of PoRV HLJ / 2021 to the F100 generation, inoculation was performed without trypsin and under otherwise unchanged culture conditions, following the steps outlined in section 2.1.1. Subsequently, HLJ / 2021-F5 and HLJ / 2021-F100 viruses were inoculated into MA104 cells at a multiple of infection (MOI) of 0.1, with and without trypsin, respectively. After 36 h of culture, viral fluorescence signals were detected using the IFA method. Simultaneously, viral fluids from each group were collected, and TCID assays were performed according to the methods outlined in section 2.1.3. 50 Measurement.

[0049] 2.2.2 Stability assay of HLJ / 2021-F100 strain without trypsin passage HLJ / 2021-F100 virus was further propagated for 10 passages without trypsin. The F1, F2, F3, and F4 virus solutions were inoculated into 12-well plates at 0.1 MOI. After 36 hours, the virus was analyzed using IFA (Infectious Disease Assay), following procedure 2.1.2. The F1, F2, F3, and F4 virus solutions were then inoculated into T25 cell flasks at 0.01 MOI. After 48 hours, the virus titer was measured after three freeze-thaw cycles at -80°C, following procedure details in 2.1.3.

[0050] 2.2.3 Plotting the trypsin-free growth curve of HLJ / 2021-F100 strain MA104 cells were infected with HLJ / 2021-F100 at a trypsin-free MOI of 0.01, with three biological replicates per group. Supernatant was collected every 12 h post-infection up to 96 h, and TCID values ​​were determined at each time point according to method 2.1.3. 50 Repeat the process three times independently and plot the one-step growth curve.

[0051] 2.2.4 Characteristics of HLJ / 2021-F100 strain with / without trypsin plaque formation Following the method described in 2.1.5, HLJ / 2021-F100 plants were treated with 10-200 TCID50. 50 / mL was seeded into 6-well culture plates containing a dense monolayer of MA104 cells. After adsorption, agarose covering material containing 1 μg / mL trypsin or without trypsin was added. After culturing for 5 days, the cells were fixed and stained to observe and compare plaque formation. Subsequently, 10 plaques were randomly selected, and the diameter of each plaque was measured and recorded using ImageJ software for subsequent statistical analysis of plaque size.

[0052] 2.2.5 Detection of VP4 protein by Western Blot HLJ / 2021-F100 virus was seeded at 0.1 MOI into 12-well culture plates with suitable cell confluence. Two groups were set up: one with trypsin and one without. F5 generation virus was used as a blank control. After 36 h of cell culture, the old culture medium was discarded, and RIPA cell lysis buffer containing protease inhibitors was added. Cells were thoroughly scraped using a cell scraper, and total protein samples were collected (all operations were performed on ice). 5× SDS loading buffer was added to the protein samples and diluted to 1× concentration. After mixing, the samples were heated in a boiling water bath for 10 min to fully denature the proteins. After separation by SDS-PAGE for 3 h, the protein samples were transferred to methanol-activated PVDF membranes and transferred at 120 V for 30 min, followed by 80 V for 60 min. After transfer, the membranes were blocked with 5% skim milk prepared with PBS at 37°C for 1 h; washed several times with PBST on a shaker. Mouse anti-PoRV VP6 primary antibody was then added, and the membranes were incubated at room temperature for 2 h; followed by washing with PBST. Add HRP-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 60 min; wash with PBST and then perform colorimetric detection.

[0053] 2.3 Pathogenicity analysis in piglets 2.3.1 Grouping and Infection of Experimental Animals Six 5-day-old healthy piglets with negative PoRV test results were randomly divided into 3 groups (n=2). The HLJ / 2021-F5 group was orally inoculated with 5 mL of F5 virus solution (10... 7.5 TCID 50 / mL), the HLJ / 2021-F100 group was orally inoculated with 5 mL of F100 virus solution (10 7.5 TCID 50 The healthy control group was inoculated with an equal volume of sterile cell culture medium ( / mL). All piglets in each group were fed milk powder every 3 hours.

[0054] 2.3.2 Observation of clinical symptoms in piglets The severity of diarrhea in piglets was observed and recorded every 24 hours, while their weight and body temperature were measured.

[0055] 2.3.3 Detoxification Detection Anal swab samples were collected from piglets every 6 hours and incubated overnight at 4°C using a shaker. After shaking, the samples were centrifuged at 3000 rpm for 10 min, and the supernatant was collected, aliquoted into sterile centrifuge tubes, and stored at -80°C for later use. 100 µL of the anal swab supernatant was used to extract total RNA using an RNA extraction kit, strictly following the instructions of the Feijie Bio RNA extraction kit. The extracted RNA was immediately subjected to reverse transcription, and any unused RNA samples were stored at -80°C.

[0056] The reverse transcription system consists of: template RNA 1 μL, RRI 1 μL, RTAce 1 μL, buffer 4 μL, DNTPS 2 μL, Pandom Primer 1 μL, and Nucleasa-Free Water 20 μL.

[0057] The reverse transcription reaction program was set as follows: 75℃ pre-denaturation for 10 min; 42℃ reverse transcription reaction for 1 h; 72℃ termination reaction for 10 min. The reverse transcription products were detected by qPCR using the identification primers (PoRV-F / PoRV-R) in Table 1. The detection system included: 10 μL F488 SYBR qPCR Mix, 0.8 μL PoRV-F (10 μmol / L), 0.8 μL PoRV-R (10 μmol / L), 2 μL cDNA template, and 6.4 μL enzyme-free sterile water, for a total volume of 20 μL.

[0058] The reaction conditions were set as follows: 95℃ pre-denaturation for 30 s (for enzyme activation); followed by 40 cycles, each cycle consisting of 95℃ denaturation for 5 s and 60℃ annealing extension for 30 s. After the qPCR reaction, the viral copy number in the sample was calculated based on the detected CT value, thereby quantifying the viral load in the piglets.

[0059] 2.3.4 Necropsy of piglets After piglets showed obvious clinical symptoms after infection, piglets in each group were euthanized and dissected at the same time. The remaining piglets were fed until no virus shedding was detected within 48 hours and then euthanized. The intestinal pathological changes of the piglets were observed after dissection.

[0060] 2.3.5 Detection of viral load in tissues Intestinal, stomach, and lung tissues were collected from each group, ground with liquid nitrogen, resuspended in physiological saline, centrifuged, and the supernatant was collected. The viral load of the tissue supernatant from the intestinal, stomach, and lung tissues was detected using qRT-PCR. For specific operating procedures, please refer to 2.3.3.

[0061] 2.3.6 Pathological examination of intestinal tissue sections Ileal and lung tissues were collected from piglets and fixed in 4% paraformaldehyde for 48 hours. The fixed samples were then sent to Nanjing Friss Medical Technology Co., Ltd. for routine dehydration, paraffin embedding, sectioning (approximately 4-5 μm thick), and HE staining. After staining, the sections were scanned using a pathological slide scanner to convert the tissue sections into high-resolution digital images. Finally, the digital images were observed on a computer terminal to assess the pathological changes in each tissue.

[0062] 2.4 Immunogenicity detection of HLJ / 2021-F100 strain 2.4.1 Animal Immunization and Grouping An immunization model was established using 6-8 week old Kunming mice, which were randomly divided into 6 groups: HLJ / 2021-F5 oral immunization group, HLJ / 2021-F5 intraperitoneal immunization group, HLJ / / 2021-F100 oral immunization group, HLJ / / 2021-F100 intraperitoneal immunization group, saline oral control group, and saline intraperitoneal control group, with 8 mice in each group. Before immunization, the viral fluid was filtered through a 0.22 μm filter for sterilization. Immunization was carried out via both oral gavage and intraperitoneal injection, with an immunization dose of 200 μL per mouse. The viral titer of both HLJ / 2021-F5 and HLJ / / 2021-F100 strains was 10. 7 TCID 50 / mL. Immunize according to the basic immunization + booster immunization schedule, with a 2-week interval between the two immunizations.

[0063] 2.4.2 Detection of mouse-specific IgG antibodies Mouse serum samples were collected and serially diluted at ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. The titer of specific IgG antibodies in the serum was determined using an indirect ELISA method. PoRV virus suspension was added to 100 μL / well of an ELISA plate and coated at 37°C for 2 h. The plate was washed five times with PBST for 5 min each time. Then, 5% BSA (prepared in PBS) was added for blocking, and the plate was incubated at 37°C for 2 h, followed by the same washing steps. Serially diluted test serum was added, along with a negative mouse serum control group and a PBS blank group. Each dilution was in triplicate. The plates were incubated at 37°C for 2 h and then washed. HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:8000 was added, and the plate was incubated at 37°C for 60 min, followed by thorough washing with PBST. Add an equal proportion of TMB chromogenic solution, incubate at 37°C in the dark for 15 min, terminate the reaction with H2SO4, and measure the absorbance at 450 nm using a microplate reader. A sample is considered antibody-positive when the P / N ratio is ≥ 2.1.

[0064] 2.4.3 Detection of mouse-specific sIgA antibodies To detect the titer of secretory immunoglobulin sIgA in the mouse intestinal mucosa, the mouse intestines were irrigated with a 2% BSA solution containing a protease inhibitor. The entire procedure was performed on ice, and the irrigation was repeated twice to ensure thorough irrigation. The collected intestinal irrigated fluid was serially diluted 2-fold, with an initial dilution ratio of 1:2. Intestinal irrigated fluid from control mice was used as a negative control. The sIgA antibody titer was detected according to the ELISA procedure described in 2.4.2, using HRP-labeled goat anti-mouse IgA antibody as the secondary antibody at a dilution ratio of 1:8000. A sample was considered positive if the p / n ratio was ≥2.1.

[0065] 2.5 Protection Experiment for Young Rats Against Virus Challenge 2.5.1 Animal Immunization and Grouping A pregnancy-induced immunization model was established using Kunming mice at 7 days of gestation. These mice were randomly divided into four groups: oral immunization group, intraperitoneal immunization group, non-immunized challenge group, and healthy control group, with two mice in each group. Before immunization, the HLJ / 2021-F100 strain of virus was sterilized by filtration through a 0.22 μm filter. Immunization was administered via oral gavage and intraperitoneal injection. The immunization dose per pregnant mouse was 200 μL, and the viral titer of the HLJ / 2021-F100 strain used was 10. 7 TCID 50 / mL; Immunize according to the procedure of basic immunization combined with booster immunization, with an interval of 1 week between the two immunizations.

[0066] 2.5.2 Detection of colostrum IgG and sIgA Newborn mice were necropsized, and their stomach tissue was completely dissected and removed. The chyme from the stomach was collected into sterile centrifuge tubes, resuspended in 2% BSA solution containing protease inhibitors, and incubated at 4°C with gentle shaking for 4 hours to fully lyse and dissolve the antibody proteins in the chyme, ensuring the accuracy of subsequent detection. The levels of IgG and sIgA antibodies in the chyme samples were detected according to the methods described in Sections 2.2.3 and 2.2.4, respectively.

[0067] 2.5.3 Infected mice with the virus When the pups reached 5 days of age, the PoRV challenge experiment was conducted. Before challenge, the virus solution was filtered through a 0.22 μm filter for sterilization. Challenge was performed orally via gavage: the oral immunization group, the intraperitoneal immunization group, and the non-immunized challenge group were all inoculated by gavage with 100 μL / puppy of virulent PoRV virus solution at a viral titer of 10. 7.5 TCID 50 / mL; healthy control group mice were administered an equal volume of sterile DMEM cell culture medium by gavage as a blank control.

[0068] 2.5.4 Observation of clinical symptoms in baby mice The severity of diarrhea in the pups was observed and recorded every 24 hours, and the total weight of the litter of pups was measured to determine the average weight change.

[0069] 2.5.5 Detoxification Detection Perianal swabs were collected from experimental mice at 24-hour intervals and incubated on a shaker at 4°C for 8 hours. The samples were then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected, and 100 µL was used to extract RNA and reverse transcribe it into cDNA. The procedure was the same as described in Section 2.3.3.

[0070] 2.5.6 Necropsy of baby mice After the pups showed obvious clinical symptoms after infection, the oral immunization group, the intraperitoneal immunization group, the non-immunized challenge group, and the healthy control group were euthanized at the same time and dissected. The intestinal pathological changes of the pups were observed after dissection.

[0071] 2.5.7 Detection of viral load in the intestines of baby mice Intestinal tissue was collected from pups, ground with liquid nitrogen, resuspended in physiological saline, and centrifuged at 12,000 r / min for 15 min at 4°C. The supernatant was collected after centrifugation, and the viral load in the intestinal tissue of pups was detected according to the method described in Section 2.3.3.

[0072] 2.5.8 Genome sequencing and analysis of HLJ / 2021-F100 strain 2.5.8.1 Whole genome amplification of HLJ / 2021-F100 strain RNA was extracted from HLJ / 2021-F100 viral fluid using an RNA extraction kit and reverse transcribed into cDNA (steps refer to the Phanta Max DNA / RNA Extraction Kit Instructions). Using cDNA as a template, PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase high-fidelity enzyme. Specific steps and reaction systems are detailed in the high-fidelity enzyme instructions. The primers used were the PoRV fragment-specific primers in Table 1. Amplified fragments of 11 genes, namely VP1, VP2, VP3, VP4, VP6, VP7, and NSP1~NSP5, were obtained.

[0073] 2.5.8.2 Gene sequencing of HLJ / 2021-F100 strain The PCR products amplified with high-fidelity enzymes were recovered using an OMEGA gel extraction kit. The recovery procedure was performed according to the gel extraction kit instructions. The purified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing using third-generation nanopore sequencing, yielding the sequences of 11 genes: VP1, VP2, VP3, VP4, VP6, VP7, and NSP1–NSP5.

[0074] 2.5.8.3 Nucleotide and amino acid sequence alignment and homology analysis The sequences of each gene fragment of HLJ / 2021-F100 obtained from sequencing were compared with the corresponding gene fragment sequences of HLJ / 2021-F5 obtained from previous sequencing. Nucleotide and amino acid sequence alignment was performed using MEGA 7.0 software and the ESPript online website, and nucleotide and amino acid homology were calculated. The variation characteristics at the nucleotide and amino acid levels were analyzed. This clarified the variation of nucleotide and amino acid sites during passage, laying the foundation for subsequent analysis of the association between variation and viral biological characteristics.

[0075] 2.5.8.4 Protein conformation analysis The VP4, VP7, and NSP4 proteins, which are closely related to viral immunogenicity and pathogenicity, were selected. AlphaFold2 was used to predict and simulate the three-dimensional structures of the corresponding proteins in the HLJ / 2021-F5 and HLJ / 2021-F100 strains. Furthermore, PyMOL software was used to label the mutation sites of the corresponding proteins in the two viruses. By comparing the superposition of their spatial conformations, the structural differences of key proteins during attenuation through passage were analyzed.

[0076] 2.6 Data Statistical Analysis Animal experimental data and ELISA experimental data were statistically analyzed using GraphPad Prism 8 statistical software, where * indicates significant differences (…). P＜0.05 ** indicates a highly significant difference () P＜0.01 ); *** indicates significant difference ( P＜0.001 **** indicates a highly significant difference ( P＜0.0001 (); where the same letter indicates no significant difference, and different letters indicate a significant difference.

[0077] 3 Results 3.1 Analysis of in vitro passage and growth characteristics of PoRV 3.1.1 Results of continuous in vitro passage of the HLJ / 2021-F5 strain To reduce the pathogenicity of the PoRV HLJ / 2021-F5 strain to piglets, this invention continuously passaged it in MA104 cells. Observations were made on the cells after each passage inoculation. It was found that obvious cytopathic effects, such as cell shrinkage, aggregation, and detachment, were observed 24-36 hours after inoculation with each passage. Furthermore, the time to onset of cytopathic effects gradually shortened with increasing passage number. Figure 1 ). Specific fluorescent signals were observed in all generations of the virus when detected using IFA 24 hours after inoculation. Figure 2 ) TCID for each generation of viruses 50The test results showed that the viral titer gradually increased with the number of passages, reaching 10 for the F10 generation. 6.5 TCID 50 / mL, the titer of F70 generation can reach 10. 8 TCID 50 / mL, F80-F100 maintained at 10 8.5 TCID 50 / mL ( Figure 3 These results indicate that the cell adaptability of PoRV HLJ / 2021 gradually increased during in vitro passage.

[0078] 3.1.2 Analysis of the growth characteristics of the passaged strains To investigate the differences in proliferation kinetics among different generations of the virus strain, this invention inoculated MA104 cells with HLJ / 2021-F5 and HLJ / 2021-F100 strains at a MOI of 0.01. Supernatants were collected at different time points after infection, and TCID was measured. 50 Plot a one-step growth curve. The results are as follows: Figure 4 As shown in a and b, the HLJ / 2021-F100 strain achieved 10 mmol / L saturation 24 hours after inoculation. 9.0 TCID 50 / mL, reaching a peak of 10 at 36 h. 9.7 TCID 50 The titer was 10 / mL, then plateaued and slowly decreased; the HLJ / 2021-F5 strain multiplied relatively slowly, with a 24-hour titer of 10. 4 TCID 50 / mL, reaching a peak value of approximately 10 at 60 h. 7.1 TCID 50 / mL ( Figure 4 a). To further understand the differences in growth characteristics between HLJ / 2021-F100 and HLJ / 2021-F5 strains, this invention detected viral proliferation characteristics using a plaque assay. The results showed that the average plaque diameter of HLJ / 2021-F5 strain was 1.43±0.26 mm, while the average plaque diameter of HLJ / 2021-F100 strain was 2.52±0.56 mm, significantly larger than that of HLJ / 2021-F5 strain. Figure 4 (b) Combined with the growth kinetics results, it was found that the HLJ / 2021-F100 strain exhibited a more significant proliferative advantage compared to the HLJ / 2021-F5 strain, characterized by a shorter latency period, faster proliferation rate, and higher peak viral titer. These results indicate that the HLJ / 2021 strain showed significantly improved proliferation efficiency and enhanced cell adaptability during continuous in vitro passage.

[0079] 3.2 Trypsin-dependent analysis of HLJ / 2021 passaged strains 3.2.1 Results of the detection of trypsin dependence in HLJ / 2021 passage strains The test results showed that the F40–F70 generation strains still strictly depended on exogenous trypsin for proliferation, while the F80 generation strain had stably acquired a heritable trypsin-free proliferation phenotype. To further purify the viral population, solidify the mutant traits, and ensure the genetic stability of the strain phenotype, this study continued to passage the strain to the F100 generation while maintaining a constant low trypsin pressure, based on the emergence of the target phenotype in the F80 generation, ultimately obtaining a phenotypically stable trypsin-free domesticated strain.

[0080] During continuous passage at a constant low trypsin level of 1 μg / mL, the viral titer was initially low, but gradually increased with passage accumulation, eventually evolving the ability to proliferate without exogenous trypsin. To determine whether PoRV's dependence on trypsin decreased while its cellular adaptability improved during passage, MA104 cells were infected with HLJ / 2021-F5 and HLJ / 2021-F100 strains at 0.1 MOI under trypsin-containing and trypsin-free conditions, respectively. IFA and TCID were measured 24 h later. 50 Measurements were taken. Results are as follows: Figure 5 As shown in a and b, strain HLJ / 2021-F5 exhibits strict trypsin dependence and cannot replicate without trypsin; strain HLJ / 2021-F100 showed no significant difference in replication with or without trypsin. To further determine whether strain HLJ / 2021-F100 can be stably passaged without trypsin, this invention continuously passaged it without adding trypsin, and performed testing every 3 generations. IFA test results are as follows. Figure 6 As shown in a and b, stable virus protein-specific fluorescent signals were detectable in HLJ / 2021-F100 strains after passage to the 10th generation under trypsin-free conditions. Figure 6 a) Virus titers remained at 10 at each generation. 7.5 -10 8.5 TCID 50 / mL, and there was no significant difference ( Figure 6 b). The above results demonstrate that the HLJ / 2021-F100 strain can stably proliferate in in vitro cell lines without relying on trypsin.

[0081] 3.2.2 Effects of trypsin on the proliferation characteristics of the HLJ / 2021-F100 strain To further determine whether trypsin affects the proliferation characteristics of the HLJ / 2021-F100 cell line, HLJ / 2021-F100 cells were infected with MA104 cells at a MOI of 0.01 under trypsin-containing and trypsin-free conditions, and growth curves were plotted. The results are as follows: Figure 7As shown in a and b, the growth curves of both groups of viruses are consistent with the viral proliferation pattern. The main difference is that the virus in the group without added trypsin reached its peak at 60 h after infection, while the group with added trypsin reached its peak at 36 h. This indicates that adding trypsin can cause HLJ / 2021-F100 to enter the plateau phase earlier. Figure 7 a). The plaque formation ability of strain HLJ / 2021-F100 under different pancreatic enzyme conditions was compared and analyzed. The results showed that the average plaque diameter of this strain was 2.52±0.56 mm when pancreatic enzyme was added, and 2.38±0.37 mm when pancreatic enzyme was not added. The plaque size was similar between the two groups, with no significant difference. Figure 7 (b) indicates that the HLJ / 2021-F100 strain has low dependence on pancreatic enzymes during in vitro culture, and can proliferate efficiently and form typical plaques under both pancreatic enzyme and non-pancreatic enzyme conditions, demonstrating good in vitro adaptability.

[0082] 3.2.3 Preliminary study on the cell invasion mechanism of HLJ / 2021-F100 strain To investigate the cell invasion mechanism of the HLJ / 2021-F100 strain under trypsin-independent conditions, this invention used Western blotting to detect the expression and integrity of VP4 protein in cells infected by this strain under trypsin-free and pancreatic enzyme-free conditions. The results are as follows: Figure 8 As shown, in a culture system with added trypsin, the VP4 protein of strain HLJ / 2021-F100 was effectively cleaved into two characteristic bands (lane 1); while in the absence of trypsin, the VP4 protein remained intact (lane 2). HLJ / 2021-F5 also exhibited typical VP4 cleavage bands when trypsin was added (lane 3). These experimental results confirm that enzymatic cleavage of the VP4 protein is not a necessary pathway for PoRV to invade cells, suggesting that the virus may have alternative invasion mechanisms independent of trypsin activation.

[0083] 3.3 Pathogenicity evaluation of HLJ / 2021-F100 strain 3.3.1 Clinical symptoms of infected piglets To evaluate the pathogenicity of HLJ / 2021-F100 strain to piglets, this invention uses 5 mL of 10 7.5 TCID 50 Piglets were administered HLJ / 2021-F100 strain viral fluid at a concentration of / mL via gavage. Piglets administered HLJ / 2021-F5 strain served as a virulent control group, while piglets administered PBS served as a healthy control group. Changes in diarrhea, body weight, body temperature, and other clinical symptoms were continuously monitored. Results are as follows: Figure 9 As shown in the ad, piglets in the HLJ / 2021-F5 group began to show typical symptoms such as watery diarrhea, anal redness and swelling, and perianal contamination 36 hours after infection. Figure 9 a) The diarrhea score gradually increased from 24 h, peaked at 72 h, and remained at a high level from 48 to 96 h. P<0.0001 Symptoms subsided after 120 hours and recovered after 144 hours; however, the HLJ / 2021-F100 group and the healthy control group did not experience diarrhea throughout the entire process, their stool characteristics were normal, and their scores remained at 0 ( Figure 9 b). Furthermore, the body weight of piglets in the HLJ / 2021-F5 group began to decrease 48 hours after infection, and from 72 hours onwards, the difference compared to the HLJ / 2021-F100 group and the healthy control group was significant ( P<0.0001 The weight of piglets in the HLJ / 2021-F100 group decreased to its lowest value at 120 h, and then slowly recovered; the trend of weight change in the HLJ / 2021-F100 group was consistent with that in the healthy control group. P>0.05 () Figure 9 c). Piglets in the HLJ / 2021-F5 group showed elevated body temperature 24-96 hours after viral challenge; the body temperature of the HLJ / 2021-F100 group and the control group did not show significant changes. Figure 9 d). Furthermore, no piglets died in the experiment. These results indicate that the HLJ / 2021-F5 strain is highly pathogenic, while after 100 consecutive passages, the HLJ / 2021-F100 strain showed no significant pathogenicity in infected piglets.

[0084] 3.3.2 Changes observed during necropsy of infected piglets To investigate the intestinal pathological changes in piglets infected with HLJ / 2021-F5 and HLJ / 2021-F100 strains, this invention performed necropsy on piglets on days 5 and 21 post-infection to observe gross pathological changes in the intestines and related organs. The results are as follows: Figure 10 As shown, compared with the control group: on day 5 post-infection, piglets in the HLJ / 2021-F5 infected group showed typical intestinal lesions, characterized by significantly thinned and transparent intestinal walls, decreased elasticity, and watery contents filling the intestinal lumen; solid lesions were also observed in the lungs of piglets. In contrast, no obvious pathological changes were observed in the intestines or organs of piglets in the HLJ / 2021-F100 infected group. On day 21 post-infection, no significant gross pathological changes were observed in piglets in either the HLJ / 2021-F5 or HLJ / 2021-F100 infected groups, showing no significant difference compared to the control group. These results suggest that the HLJ / 2021-F5 strain can still induce acute intestinal injury in piglets, while the pathogenicity of the HLJ / 2021-F100 strain has been significantly weakened, and infection with it did not induce obvious organ lesions.

[0085] 3.3.3 Detection of viral load in infected piglet tissues To clarify the tissue tropism and viral load distribution characteristics of HLJ / 2021-F5 and HLJ / 2021-F100 strains in piglets after artificial infection, this experiment collected tissues from different intestinal segments and major organs of piglets on day 5 post-challenge. The porcine rotavirus (PoRV) genome copy number in each tissue was detected and compared using RT-qPCR. Results are as follows: Figure 11 As shown, both strains can effectively replicate in intestinal tissues such as the duodenum, jejunum, ileum, cecum, colon, and rectum, and viral nucleic acid can be detected. Among them, the HLJ / 2021-F5 group had the highest viral load in the ileum tissue, reaching log 10 The viral load in the HLJ / 2021-F5 group was 4.89 RNA copies / μL, which was significantly higher than that in the HLJ / 2021-F100 infection group; at the same time, the viral load in the duodenum, jejunum, ileum, and rectum was also significantly higher in the HLJ / 2021-F100 group. P＜0.05 Furthermore, PoRV nucleic acid was also detected in stomach and lung tissues, with the viral load in the lungs of the HLJ / 2021-F5 group reaching as high as log [missing information]. 10 7.32 RNA copies / μL, significantly higher than the HLJ / 2021-F100 group ( P＜0.0001 The above results indicate that the HLJ / 2021-F5 strain has a significantly stronger proliferation capacity in the intestinal and lung tissues of piglets than the HLJ / 2021-F100 strain, suggesting that the replication capacity of this strain in vivo decreases significantly with continuous in vitro passage.

[0086] 3.3.4 Observation of pathological changes in the intestinal tissue of infected piglets To investigate the pathological damage to the intestinal and lung tissues of piglets caused by infection with HLJ / 2021-F5 and HLJ / 2021-F100 strains, this invention collected ileum and lung tissues from piglets on day 5 post-infection, prepared pathological sections, stained with hematoxylin and eosin (HE), and performed histopathological observation of the tissues. The results are as follows: Figure 12As shown, the HLJ / 2021-F5 infected piglets exhibited significant pathological damage in the ileum, characterized by severe atrophy and even disintegration of intestinal villi; the lung tissue showed consolidation changes, accompanied by alveolar stenosis, inflammatory cell infiltration, and widening of alveolar septa. In the HLJ / 2021-F100 infected piglets, only slight rupture of the ileum villi was observed, with no significant difference compared to the control group; the lung tissue showed only slight inflammatory cell infiltration, with minimal damage. The ileum and lung tissues of the control group piglets showed normal morphology and structure, without any pathological damage. These results indicate that the HLJ / 2021-F5 strain can cause significant pathological damage to the ileum and lung tissues, demonstrating strong pathogenicity; while the HLJ / 2021-F100 strain, after continuous passage, caused very slight damage to the ileum and lung tissues, with no significant difference compared to the control group, indicating that the pathogenicity of the HLJ / 2021-F100 strain has been significantly weakened.

[0087] 3.3.5 Detection of virus shedding in piglets To investigate the dynamic patterns of viral shedding in piglets using the parental PoRV strain HLJ / 2021-F5 and the passaged HLJ / 2021-F100 strain, anal swab samples were collected from piglets every 6 hours after oral administration of the virus. RT-qPCR was used to quantitatively detect viral load, and the onset time, peak level, and duration of viral shedding for both strains were systematically analyzed. Results are as follows: Figure 13 As shown, PoRV RNA was detectable in all infected piglets 6 hours after challenge, with higher viral load in the HLJ / 2021-F5 group than in the LJ / 2021-F100 group. The duration of viral shedding differed significantly between the two groups. Viral RNA was undetectable in the HLJ / 2021-F100 group 54 hours after challenge, while shedding in the HLJ / 2021-F5 group lasted up to 90 hours after challenge, and the shedding time in the HLJ / 2021-F100 group was shortened by 36 hours. No PoRV RNA was detected in anal swabs from the control group. These results indicate that the replication capacity of the HLJ / 2021-F100 strain in piglets significantly decreased after continuous in vitro passage, manifested as a lower peak viral load, reduced viral shedding levels, and a shorter duration of shedding.

[0088] 3.4 Immunogenicity of HLJ / 2021-F100 strain 3.4.1 Determination of serum-specific IgG antibody levels in immunized mice To systematically evaluate whether the immunogenicity of the HLJ / 2021-F100 strain had changed, HLJ / 2021-F100 and HLJ / 2021-F5 strains were immunized via different routes (oral and intraperitoneal injection). Peripheral blood was collected from mice at 7, 14, 21, and 28 days after the initial immunization, and serum was separated. Serum IgG levels were detected by ELISA. The results are as follows: Figure 14As shown in the figure, 7 days after the initial immunization, specific IgG was detectable only in the intraperitoneal injection group, indicating that in the early stages of immunization, the intraperitoneal injection route can initiate the body's systemic humoral immune response more quickly, while the difference in strain generation has no significant effect on early antibody production. At 21 days (7 days after booster immunization) and 28 days (14 days after booster immunization) after the initial immunization, the specific IgG titers in all immunization groups remained at a high level, and the intraperitoneal injection group was higher than the oral immunization group at all time points. No significant difference in antibody titers was observed between the HLJ / 2021-F5 strain and the HLJ / 2021-F100 strain under the same immunization route.

[0089] 3.4.2 Determination of neutralizing antibody titer in mouse serum The level of neutralizing antibodies in the serum of immunized mice was detected using a virus neutralization assay. Results are as follows: Figure 15 As shown, neutralizing antibodies were detected in the intraperitoneal immunization group as early as 7 days post-immunization. Neutralizing antibodies were also detected in all immunization groups and 14 days post-initial immunization. The intraperitoneal immunization group of strain HLJ / 2021-F5 showed the highest levels of neutralizing antibodies at all time points post-immunization. These results indicate that both oral and intraperitoneal immunization can induce the production of neutralizing antibodies, but intraperitoneal immunization induces higher levels of neutralizing antibodies in serum. The neutralizing antibody titer produced by strain HLJ / 2021-F5 is higher than that of strain HLJ / 2021-F100.

[0090] 3.4.3 Measurement of mouse-specific sIgA antibody levels To evaluate the effects of different immunization routes on inducing intestinal mucosal immunity, the level of sIgA in the intestinal lavage fluid of immunized mice was detected by ELISA. The results are as follows: Figure 16 As shown, sIgA antibodies were detectable in the oral immunization group 7 days after immunization, peaked at 21 days after the first immunization, and decreased slightly at 28 days but remained at a high level; no sIgA antibodies were detected in the intraperitoneal immunization group throughout the entire testing period. The results indicate that both HLJ / 2021-F5 and HLJ / 2021-F100 strains can induce high levels of sIgA production after oral immunization, with a significant difference between the two only at 21 days. p＜0.05 Intraperitoneal immunity cannot induce an intestinal sIgA response.

[0091] 3.5. Evaluation of protection against virus challenge using HLJ / 2021-F100 3.5.1 Levels of PoRV-specific IgG and sIgA in colostrum from pregnant mice To determine the levels of IgG and sIgA in the colostrum of HLJ / 2021-F100 pregnant mice immunized orally and intraperitoneally, gastric chyme was collected from the stomachs of pups at 1, 4, 7, and 11 days postpartum for IgG and sIgA testing. Results are as follows: Figure 17As shown in a and b, both the oral immunization group and the intraperitoneal immunization group produced IgG, with no significant difference; only the oral immunization group showed detectable sIgA. The sIgA antibody titer in breast milk remained at a high level from 1 to 7 days postpartum, and decreased significantly at 11 days. p＜ 0.05 ).

[0092] 3.5.2 Clinical symptoms of passively immunized mouse pups after challenge with the virus 100 microliters 10 7.5 TCID 50 After administering / mL PoRV virus solution to immunized female mice and their offspring, the infected piglets were observed for clinical symptoms and their feces were scored every 24 hours. Results were as follows: Figure 18 As shown in AC, 24 hours after challenge, the suckling mice developed diarrhea. Among them, the unimmunized challenge group had watery stools and significantly higher diarrhea scores than the other groups. P＜ 0.001 The mice in the intraperitoneal immunization group recovered to normal 96 hours after challenge; a few suckling mice in the intraperitoneal immunization group had loose stools after challenge, but returned to normal defecation within 72 hours; the mice in the oral immunization group had soft stools 24 hours after challenge, but returned to normal within 48 hours. Figure 18 a, b). The average body weight of suckling mice in each group increased slowly within 48 hours after challenge compared to the negative control group; the average body weight of mice in the non-immunized challenge group was significantly lower than that of the immunized groups after challenge. Figure 18 c).

[0093] 3.5.3 Results of intestinal necropsy in passively immunized pups after viral challenge To investigate the pathological changes in the intestinal tissue of pups after passive immunization in pregnant mice and subsequent challenge with a virus, pups on the second day after challenge were dissected to observe the pathological changes in their intestines. The results are as follows: Figure 19 As shown, compared with the control group, at 2 days, the intestinal wall of the unimmunized mice showed obvious pathological changes, becoming thinner and transparent, and lacking elasticity, with watery contents; the oral immunization group and the intraperitoneal immunization group showed no significant changes compared with the healthy control group.

[0094] 3.5.4 Viral load in the intestines of passively immunized baby mice after challenge with the virus Intestinal tissue was collected from suckling mice on day 2 after challenge with the virus, and the viral load of PoRV in the intestinal segments was detected by RT-qPCR. The results are as follows: Figure 20 As shown, viral nucleic acid was not detected in the intestinal tissue of the negative control group; PoRV was detected in the intestines of the oral immunization group, intraperitoneal immunization group, and positive control group, with the highest viral load in the non-immunized challenge group, reaching log 10 4.17 RNA copies / μL ( P< 0.0001The enterovirus load in the intraperitoneal immunization group was significantly higher than that in the oral immunization group. P<0.05 The above results indicate that passive immunization in pregnant mice can effectively reduce the enterovirus load in suckling mice after challenge, and the maternal antibodies induced by oral immunization have a significantly better protective effect on suckling mice than those induced by intraperitoneal immunization.

[0095] 3.5.5 Virus shedding in passively immunized offspring mice after viral challenge To investigate the dynamic pattern of viral shedding after passive immunization in pups, this invention collected perianal swabs every 24 hours after viral challenge in suckling mice and detected viral nucleic acid using RT-PCR. The results are as follows: Figure 21 As shown, PoRV RNA was detected in all infected pups 24 h after challenge. Pups in the oral and intraperitoneal immunization groups stopped shedding the virus 96 h after challenge, while pups in the positive control group stopped shedding the virus 120 h after challenge. Regarding viral shedding levels, the viral load in the unimmunized challenge group was significantly higher than that in the immunized group, and the viral load in the intraperitoneal immunization group was significantly higher than that in the oral immunization group. P＜0.05 These results indicate that the oral route-induced mucosal immune response can more effectively inhibit viral replication and excretion in the intestine, providing stronger protection for challenged mice.

[0096] 3.6 Genome sequence analysis of HLJ / 2021-F100 strain To obtain the complete genome sequence of the HLJ / 2021-F100 strain, viral RNA was first extracted, reverse transcribed, and then PCR was performed using the primers in Table 1. The amplified fragment sizes all matched the target gene. The PCR products were sent to the company's third-generation nanopore sequencing, and the sequences of 11 genes, namely VP1, VP2, VP3, VP4, VP6, VP7, and NSP1-NSP5, were obtained, as shown in SEQ ID NO. 25-35, respectively.

[0097] 3.7 Nucleotide Change Analysis of HLJ / 2021-F100 Strain Alignment analysis of the coding nucleotide sequences of the whole genome of HLJ / 2021-F100 strain showed that the average nucleotide homology between HLJ / 2021-F100 and HLJ / 2021-F5 strains was 99.46%, with homology of individual genes ranging from 97.27% to 100.00%. Specifically, VP3, VP6, and NSP3 genes showed 100% homology and highly stable sequences; VP1, VP2, VP4, VP7, NSP1, NSP2, and NSP5 genes all showed homology above 99.48%, with only minor nucleotide variations; while NSP4 showed the lowest homology at only 97.27%, with significantly higher variation than other genes (Table 2). The statistical analysis of nucleotide mutations in each gene showed that the VP4 and NSP4 genes had the most abundant nucleotide mutations, with 16 and 12 mutation sites respectively, and a very high proportion of missense mutations, 10 in each gene. The VP1 and VP2 genes each contained 6 mutation sites, with VP1 containing 5 missense mutations and VP2 containing 3. The VP7 gene contained 5 mutation sites, 3 of which were missense mutations. The NSP1 and NSP2 genes each contained 4 mutation sites, with 3 and 1 missense mutations respectively. The NSP5 gene contained 3 mutation sites, with only 1 missense mutation. These results indicate that the degree of variation in different regions of the HLJ / 2021 strain's genome differed significantly during continuous in vitro passages. The high missense mutation rates in the VP4 and NSP4 genes, along with the low homology level of NSP4, suggest a direct correlation with changes in viral pathogenicity.

[0098] ; 3.8 Analysis of Amino Acid Changes in HLJ / 2021-F100 Strain The results of amino acid sequence alignment analysis of proteins from strain HLJ / 2021-F100 showed that its average whole-genome amino acid homology with strain HLJ / 2021-F5 was 98.95%, with individual proteins exhibiting amino acid homology ranging from 94.33% to 100.00%. Specifically, VP3, VP6, and NSP3 showed 100% amino acid homology, indicating highly stable protein conformations; VP1, VP2, VP4, VP7, NSP1, NSP2, and NSP5 all showed amino acid homology above 98.00%, with minor amino acid variations; while NSP4 showed the lowest amino acid homology at only 94.33%, with a significantly higher degree of variation than the other proteins (Table 3). The statistical analysis of amino acid mutation counts in each protein showed that VP4 and NSP4 proteins had the most amino acid mutations, both with 10. VP1 protein contained 5 amino acid mutation sites, VP2 protein contained 3, VP7 protein contained 3, NSP1 protein contained 3, NSP2 protein contained 1, and NSP5 protein contained 1 (Table 4). These results indicate that the overall amino acid sequences of the HLJ / 2021 strain were highly conserved during continuous in vitro passages, but the degree of variation among different proteins differed significantly. VP4 and NSP4 proteins had the most amino acid mutations, and combined with their low homology, this suggests that they may be directly related to changes in viral pathogenicity.

[0099] ; Table 4. Amino acid changes of structural proteins in HLJ / 2021 ; ; 3.9 Conformational Analysis of Key Proteins in HLJ / 2021-F100 Strain To further analyze the impact of amino acid mutations on protein conformation, this study used AlphaFold to perform three-dimensional structural simulations of key proteins in strains HLJ / 2021-F100 and HLJ / 2021-F5, and then used PyMOL to visualize and analyze the protein spatial conformations. The results showed that the RMSD values ​​of VP4 and VP7 proteins in strains HLJ / 2021-F100 and HLJ / 2021-F5 were 0.055 Å and 0.555 Å, respectively, far below 1 Å. This indicates that although missense mutations exist in the VP4 and VP7 genes, these mutations did not affect the overall three-dimensional structure of the proteins. The spatial conformation of strain HLJ / 2021-F100 almost completely overlapped with that of strain HLJ / 2021-F5, indicating a high degree of structural conservation. This suggests that the three-dimensional folding state of VP4 and VP7 proteins remained highly stable during passage. The RMSD value of the NSP4 protein was as high as 12.777 Å, indicating that the NSP4 protein of the HLJ / 2021-F100 strain underwent a significant and substantial change in three-dimensional conformation compared to the HLJ / 2021-F5 strain. This substantial conformational shift suggests that the spatial structural stability or functional domain folding state of the NSP4 protein is severely affected by missense mutations, which may directly impact the protein's biological activity (such as toxicity-related functions). Figure 22 In summary, the core structures of VP4 and VP7 proteins are highly conserved, and mutations have not disrupted the spatial structure of their key antigenic epitopes, indicating that their immunogenicity is preserved. However, the conformation of the NSP4 protein has undergone significant changes, which may be directly related to its functional alterations (such as decreased pathogenicity). These results suggest that VP4 / VP7 can still serve as effective protective antigens during passage, but mutations in VP4 and NSP4, along with conformational changes, may be key factors contributing to viral attenuation.

Claims

1. A live attenuated porcine rotavirus vaccine strain that is non-trypsin-dependent, characterized in that, The porcine rotavirus attenuated live vaccine strain is named PoRV-F100 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.47079.

2. The porcine rotavirus attenuated live vaccine strain as described in claim 1, characterized in that, The porcine rotavirus attenuated live vaccine strain described above can replicate in vitro without the need for trypsin, and the viral titer is maintained at 10. 8.5 TCID 50 / mL or more.

3. The use of the live attenuated porcine rotavirus vaccine strain of claim 1 in the preparation of a vaccine for the prevention of porcine rotavirus infection.

4. A vaccine composition, characterized in that, It includes the live attenuated porcine rotavirus vaccine strain as described in claim 1.

5. The vaccine composition according to claim 4, characterized in that, It also includes pharmaceutically acceptable carriers or adjuvants.

6. The use of the vaccine composition according to claim 4 or 5 in the preparation of a vaccine for the prevention of porcine rotavirus infection.

7. The application according to claim 6, characterized in that, The vaccine in question is a live attenuated vaccine.

Images