Use of prasrjvibacter in the preparation of a product against porcine reproductive and respiratory syndrome virus

Abstract

The application provides application of a prasrachta bacterium in preparation of a product against a porcine reproductive and respiratory syndrome virus, and belongs to the technical field of prevention and control of the porcine reproductive and respiratory syndrome virus (PRRSV). The application first proposes that the prasrachta bacterium is used for prevention and control of the PRRSV, the prasrachta bacterium has a persistent and stable disinfection effect on the PRRSV, and the stable PRRSV inactivation effect is maintained within an action time of 24 hours. Moreover, the prasrachta bacterium has a non-cytotoxicity characteristic, while realizing persistent and efficient virus inactivation, damage of a chemical disinfectant to host cells is avoided. In addition, the prasrachta bacterium is used for prevention and control of the PRRSV, is environment-friendly, provides a safer and more persistent biological disinfection scheme for the PRRSV prevention and control, and highlights the application potential of the prasrachta bacterium in breeding environment disinfection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of prevention and control of porcine reproductive and respiratory syndrome virus, and particularly relates to application of Clostridium piliforme in preparation of an anti-porcine reproductive and respiratory syndrome virus product. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and has become one of the most destructive diseases in global intensive pig production. Since the first outbreak, PRRSV has evolved from the classic form to the highly pathogenic strain, causing huge economic losses to the global pig industry. PRRSV is mainly transmitted through aerosols, but its survival ability on non-living surfaces (such as metal, plastic and rubber) further increases the risk of transmission. Studies have shown that PRRSV can survive for several hours to several days under different environmental conditions, depending on factors such as temperature, humidity and surface material. Therefore, facilities and tools contaminated with the virus can become important transmission media, increasing the difficulty of prevention and control.

[0003] To combat the spread of PRRSV, the World Organization for Animal Health (OIE) and various national agricultural departments recommend the use of highly effective chemical disinfectants in high-risk breeding environments. However, the application of existing chemical disinfectants has significant limitations. Although chemical disinfectants can quickly inactivate PRRSV in the environment, their effective period is usually short, making it difficult to achieve sustained interruption of the virus transmission chain in breeding environments, especially under high-density breeding conditions, the virus can continue to spread through personnel, equipment and air, making the surface after disinfection may be quickly contaminated again, becoming a new source of virus transmission. More seriously, long-term and large-scale use of chemical disinfectants can harm the health of practitioners through respiratory irritation and skin contact, and can also have negative impacts on the surrounding environment and ecosystem, including soil and water pollution and potential harm to wildlife. In addition, some disinfectants can induce or select pathogens with antimicrobial drug resistance (AMR), which not only exacerbates the difficulty of PRRSV prevention and control, but also can have a chain reaction on the management of other animal diseases. Given that the AMR problem has triggered a serious animal and human health crisis worldwide, the misuse of disinfectants in PRRSV prevention and control can further exacerbate the severity of this problem. Therefore, the development of an efficient, long-lasting, environmentally friendly and economically viable disinfectant has become an urgent need in current PRRSV prevention and control strategies.

[0004] In recent years, probiotics have shown great potential as an eco-friendly alternative in pathogen prevention and control. Faecalibacterium prausnitzii, as a functional probiotic, has been proven to have a significant effect on regulating the intestinal microbiota. However, it is still unclear whether F. prausnitzii can be used as a potential ecological disinfectant, and there is no report on any related research. SUMMARY

[0005] Therefore, the purpose of the present application is to provide the application of F. prausnitzii in the preparation of products against porcine reproductive and respiratory syndrome virus, to provide an efficient and sustainable alternative for the prevention and control of PRRSV, and to provide a feasible solution to reduce the negative impact of traditional disinfectants on the environment and microbiota.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0007] The present application provides the application of F. prausnitzii in the preparation of products against porcine reproductive and respiratory syndrome virus.

[0008] The present application also provides the application of F. prausnitzii in the preparation of inactivated porcine reproductive and respiratory syndrome virus products.

[0009] The present application also provides the application of F. prausnitzii bacterial suspension in the preparation of products for preventing and controlling porcine reproductive and respiratory syndrome virus.

[0010] Preferably, the concentration of the F. prausnitzii bacterial suspension is 1 mg / mL or more.

[0011] Preferably, the preparation method of the F. prausnitzii bacterial suspension comprises the following steps: inoculating F. prausnitzii into a modified enhanced Clostridium broth medium and culturing.

[0012] Preferably, the preparation method of the F. prausnitzii bacterial suspension with a concentration of 1 mg / mL or more comprises the following steps: inoculating F. prausnitzii into a modified enhanced Clostridium broth medium, culturing to an OD 600 value of 0.6-0.7, separating and collecting the bacterial body precipitate and the fermentation liquor, and mixing the fermentation liquor and the bacterial body precipitate to a concentration of the F. prausnitzii bacterial suspension of 1 mg / mL or more.

[0013] Preferably, the product comprises a disinfectant.

[0014] The present application also provides a disinfectant for preventing and controlling porcine reproductive and respiratory syndrome virus, wherein the active ingredient of the disinfectant comprises F. prausnitzii or F. prausnitzii bacterial suspension.

[0015] Preferably, the concentration of the F. prausnitzii bacterial suspension is 1 mg / mL or more.

[0016] Preferably, the volume ratio of the Clostridium bolteae bacterial suspension to the porcine reproductive and respiratory syndrome virus liquid is (1:1)~(32:1), and the titer of the porcine reproductive and respiratory syndrome virus liquid is 10 5.5 TCID 50 / mL.

[0017] The beneficial effects of the present application are:

[0018] The present application first proposes to use Clostridium bolteae for the prevention and control of PRRSV, and Clostridium bolteae has a persistent and stable disinfection effect on PRRSV, which maintains stable PRRSV inactivation effect within 24h of action time. Moreover, it has non-cytotoxicity characteristics, which, while achieving persistent and efficient virus inactivation, avoids damage to host cells by chemical disinfectants. In addition, using Clostridium bolteae for the prevention and control of PRRSV is environmentally friendly, and provides a safer and more persistent biological disinfection solution for PRRSV prevention and control, highlighting the application potential of Clostridium bolteae in breeding environment disinfection. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The colony morphology of the six bacteria is shown in Figure 1;

[0020] Figure 2 The growth curves of the six bacteria are shown in Figure 2;

[0021] Figure 3 The lesion observation results of PRRSV-infected Marc-145 cells are shown in Figure 3, wherein A is a negative control well without virus inoculation, and B is a lesion well inoculated with virus;

[0022] Figure 4 The absolute quantitative RT-qPCR results of PRRSV are shown in Figure 4, wherein A is the dissolution curve of the standard plasmid, and B is the standard curve of real-time fluorescent quantitative PCR of PRRSV;

[0023] Figure 5 The PRRSV viral load results of different bacteria detected by RT-qPCR are shown in Figure 5, wherein *** indicates p<0.001, and **** indicates p<0.0001;

[0024] Figure 6 The indirect immunofluorescence results of different bacteria against PRRSV are shown in Figure 6;

[0025] Figure 7 The RT-qPCR detection results of the disinfection effect of Clostridium bolteae bacterial suspension, bacterial body precipitation and fermentation liquid on PRRSV are shown in Figure 7, wherein **** indicates p<0.0001;

[0026] Figure 8 The indirect immunofluorescence detection results of the disinfection effect of Clostridium bolteae bacterial suspension, bacterial body precipitation and fermentation liquid on PRRSV are shown in Figure 8;

[0027] Figure 9 RT-qPCR detection results of the optimal working concentration of Prasvirus suspension for disinfection, wherein *** represents p<0.001, and **** represents p<0.0001;

[0028] Figure 10 Indirect immunofluorescence detection results of the optimal working concentration of Prasvirus suspension for disinfection;

[0029] Figure 11 RT-qPCR detection results of the antiviral effect of Prasvirus suspension and PRRSV at different mixing ratios, wherein ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001;

[0030] Figure 12 Indirect immunofluorescence detection results of the antiviral effect of Prasvirus suspension and PRRSV at different mixing ratios;

[0031] Figure 13 RT-qPCR detection results of PRRSV viral load at different times of each disinfectant, wherein * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, and ns represents p>0.05;

[0032] Figure 14 PI dead cell staining results after treatment with different disinfectants;

[0033] Figure 15 RT-qPCR detection results of PRRSV inactivation on a glass surface by different disinfectants, wherein * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, and ns represents p>0.05;

[0034] Figure 16 PI dead cell staining results on a glass surface after treatment with different disinfectants. DETAILED DESCRIPTION

[0035] The application provides application of Prasvirus in preparation of inactivated porcine reproductive and respiratory syndrome virus products or in preparation of anti-porcine reproductive and respiratory syndrome virus products.

[0036] In some embodiments of the application, the Prasvirus is Faecalibacterium prausnitziiStrain A2-165, purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), Leibniz Institute, with the accession number of DSM 17677. In the present application, the product preferably comprises a disinfectant.

[0037] The present application also provides a use of a suspension of Faecalibacterium prausnitzii in the preparation of a product for preventing and controlling porcine reproductive and respiratory syndrome virus.

[0038] In the present application, the concentration of the suspension of Faecalibacterium prausnitzii is preferably 1 mg / mL or higher, and in some embodiments, the concentration of the suspension of Faecalibacterium prausnitzii is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or 30 mg / mL. In the present application, the method for preparing the suspension of Faecalibacterium prausnitzii preferably comprises the following steps: inoculating Faecalibacterium prausnitzii into a modified reinforced clostridial broth medium and culturing. The culture is preferably anaerobic static culture, and the culture temperature is preferably 37°C. The present application does not have special limitations on the specific source of the modified reinforced clostridial broth medium, and a conventional commercially available product can be used.

[0039] In the present application, the method for preparing a suspension of Faecalibacterium prausnitzii with a concentration of 1 mg / mL or higher preferably comprises the following steps: inoculating Faecalibacterium prausnitzii into a modified reinforced clostridial broth medium and culturing to an OD 600 value of 0.6-0.7, separating and collecting the bacterial precipitate and the liquid (the liquid is referred to as fermentation broth), and mixing the fermentation broth with the bacterial precipitate to obtain a suspension of Faecalibacterium prausnitzii with a concentration of 1 mg / mL or higher. In the present application, the separation method is preferably centrifugation, and the centrifugation conditions are preferably 4°C, 5000 rpm for 8 min.

[0040] The present application also provides a disinfectant for preventing and controlling porcine reproductive and respiratory syndrome virus, wherein the active ingredient of the disinfectant comprises Faecalibacterium prausnitzii or a suspension of Faecalibacterium prausnitzii.

[0041] In the present application, the concentration of the suspension of Pr. acidipropionici is preferably 1 mg / mL or more. In some embodiments, the concentration of the suspension of Pr. acidipropionici is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or 30 mg / mL. In the present application, the preparation method of the suspension of Pr. acidipropionici is as described above, which is not repeated here. In the present application, the volume ratio of the suspension of Pr. acidipropionici to the PRRSV liquid is preferably (1:1)~(32:1), and in some embodiments of the present application, the volume ratio of the suspension of Pr. acidipropionici to the PRRSV liquid is 1:1, 4:1, 8:1, 16:1 or 32:1. In the present application, the titer of the PRRSV liquid is preferably 10 5.5 TCID 50 / mL.

[0042] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0043] In the following examples, all the methods are conventional unless otherwise specified.

[0044] In the following examples, all the materials and reagents used are commercially available unless otherwise specified.

[0045] In the following examples, the Pr. acidipropionici is Faecalibacterium prausnitzii strain A2-165, purchased from the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ), with the accession number DSM 17677.

[0046] In the following examples, the modified reinforced Clostridium broth is purchased from Shandong Top Bioengineering Co., Ltd., and the modified Clostridium agar plate is prepared by adding 2% agar powder (mass / volume) to the modified reinforced Clostridium broth.

[0047] In the following examples, all the data are analyzed using GraphPad Prism software and selecting mean ± standard error (mean ± SEM) for statistical processing. Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 and ns p>0.05 respectively represent statistical difference, statistically significant difference, statistically extremely significant difference, statistically extremely significant difference and no statistical difference.

[0048] Example 1

[0049] (1) Cultivation of Faecalibacterium prausnitzii and other five bacteria

[0050] Plate cloning:

[0051] The six bacteria preserved in glycerol were recovered on the corresponding solid medium plates using the four-zone division method. Specifically, Faecalibacterium prausnitzii (F. prausnitzii) was used on a modified Clostridium agar plate, Bacillus licheniformis (B. licheniformis), Bacillus subtilis (B. subtilis), and Bacillus megaterium (B. megaterium) were used on LB agar plates, and Lactobacillus plantarum and Lactobacillus fermentum were used on MRS agar plates. F. prausnitzii plates were placed in an anaerobic workstation and incubated at 37°C for 24 hours, and the other bacterial plates were placed in a 37°C incubator and incubated for 24 hours. The colony morphology was observed daily until typical, well-defined single colonies were obtained. The results are shown in Table 1. Figure 1 Table 1: Colony morphology of the six bacteria on their respective solid medium plates

[0052] Single colonies were picked and inoculated into the corresponding liquid medium. F. prausnitzii was inoculated into a modified Clostridium liquid medium and incubated anaerobically at 37°C, B. licheniformis, B. subtilis, and B. megaterium were inoculated into LB liquid medium and incubated at 37°C with 200 rpm shaking, and L. plantarum and L. fermentum were inoculated into MRS liquid medium and incubated at 37°C.

[0053] 16S rRNA verification: The bacterial liquid was subjected to PCR amplification using universal primers 27F: 5'-AGAGTTTGATCCTGGCCTCA-3' (SEQ ID NO. 1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2) under the same conditions as above. The PCR product was sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing, and the sequencing results were analyzed by NCBI BLAST comparison to verify the consistency of the gene sequence. The results showed that the 16S rRNA gene sequences of each bacterial species were more than 99% identical to their corresponding reference sequences. The above molecular identification results were consistent with the morphological observation of the colonies, and the taxonomic status of the bacteria used in the experiment was determined.

[0054] (2) Growth curve determination of F. prausnitzii and other five bacteria

[0055] Each bacterial liquid was inoculated into the corresponding liquid medium at a volume percentage of 2%, cultured in the corresponding culture conditions for 24 h, 3 parallel groups were set for each group, the inoculated well plate was placed in the enzyme marker and the relevant parameters were set, OD 600 was measured every 2 h until 12 h, and the growth curve of the bacteria was drawn according to the OD 600 value.

[0056] The results are shown in Figure 2 Each bacteria showed significant differences in growth rate under suitable culture conditions. Specifically, Prasvillus showed relatively slow growth characteristics as a strict anaerobic bacteria. In contrast, the aerobic Bacillus species showed a rapid growth pattern, and Lactobacillus plantarum and Lactobacillus fermentum showed a medium growth rate as facultative anaerobic bacteria.

[0057] (3) Virus amplification and titer determination

[0058] Marc-145 cells frozen in liquid nitrogen were quickly placed in a 37°C water bath for 1-2 min, then the cell suspension was transferred to a 1.5 mL EP tube, 1 mL of DMEM complete medium was added to gently resuspend the cells, and after centrifugation at 800 rpm for 3 min, the supernatant was discarded. The cells were inoculated into a 100 mm dish and placed in a 37°C, 5% CO2 incubator for incubation. PRRSV virus was inoculated into Marc-145 cells with a single layer density of 80%-90%, and DMEM medium containing 2% fetal bovine serum (FBS) was used for culture. When 80% of the cells showed obvious cytopathic effect (CPE), the virus liquid was collected and freeze-thawed 3 times, the supernatant was centrifuged, aliquoted and stored at -80°C for use.

[0059] To determine the virus titer, Marc-145 cells were inoculated into a 96-well plate and incubated overnight, then washed with sterile PBS. The virus liquid was diluted by 10 times, and each dilution was inoculated into a vertical row of wells, with a negative control without virus inoculation. The 96-well plate was placed in a 37°C, 5% CO2 incubator for culture, and the cytopathic effect was observed and recorded daily. When no new cytopathic wells were added to the cells, the Reed-Muench method was used to calculate the median tissue culture infectious dose (TCID 50 ) of the virus.

[0060] The results of PRRSV virus amplification are shown in Figure 3The results are shown in the figure. In which A is the negative control group without virus inoculation, Marc-145 cells maintain typical epithelial morphology, cells are arranged closely, cells maintain normal morphology, monolayer integrity is good, cell fusion degree is greater than 95%, and no pathological changes are observed. In contrast, the virus inoculation group shown in B shows typical CPE characteristics, about 90% of the cells have morphological changes, showing cell shrinkage, rounding off, aggregation, cytoplasmic granulation, increased intercellular space, and ultimately leading to monolayer integrity destruction. The half number of tissue culture infectious dose of virus was calculated by Reed-Muench method to be 10 5.5 TCID 50 / mL. The results show that the virus has a certain infection titer, which is suitable for subsequent experiments.

[0061] (4) Establishment of absolute quantitative RT-qPCR identification method of PRRSV

[0062] Preparation of recombinant plasmid standard:

[0063] The viral DNA extraction kit was used to extract the whole genome of PRRSV as a template, and the primers designed in Table 1 (the primer pair of P-O5FR-F and P-O5FR-R in Table 1 is designed for the junction product, i.e. a sequence on the ORF5 gene of PRRSV; the primer pair of M13-F and M13-R is a target primer on the pEASY-Blunt vector, used to verify whether the fragment is successfully inserted into the vector) was used for amplification of the target fragment. The target band was recovered by gel recovery, and the recovered product was ligated with the pEASY-Blunt vector (a product of Transgene) and transformed into Trans-T1 competent cells. It was placed in a 37°C incubator until single colonies appeared. The positive recombinant plasmid colonies were selected and cultured, and the correct recombinant plasmid was sequenced. The plasmid copy number was calculated according to the following formula, and was stored at -20°C for standby.

[0064]

[0065] Table 1 PCR primers

[0066]

[0067] Establishment of standard curve:

[0068] The prepared standard plasmid was diluted by 10 times, a total of 6 gradients, in order of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6, as the standard template for the RT-qPCR reaction. Each dilution gradient of the standard plasmid was set in triplicate to ensure the reliability and repeatability of the data. Meanwhile, a negative control group was set to exclude the possibility of non-specific amplification or contamination. The viral nucleic acid copy number at each dilution gradient was detected using RT-qPCR, and the Ct value was recorded. By fitting the Ct value with the known concentration of the standard plasmid logarithm (Log10 copies / μL), a standard curve was plotted.

[0069] The standard curve of real-time fluorescent quantitative PCR for PRRSV was successfully established by amplifying the gradient-diluted recombinant plasmid standard. The melting curve, as shown in A of Figure 4 , showed a single peak shape, indicating good primer specificity and no non-specific amplification products. The standard curve, as shown in B of Figure 4 , had an equation of y = -3.9657x + 38.498, where y represented the Ct value and x represented the logarithm of the template concentration (Log10 copies / μL). The correlation coefficient R² of the equation was 0.999, indicating that the RT-qPCR method had excellent linear relationship within the detection range and could accurately reflect the quantitative relationship between the template concentration and the Ct value.

[0070] (5) Indirect immunofluorescence detection of viral titer

[0071] After the cells were treated accordingly, the culture medium in the 96-well plate was discarded, and the cells were gently washed twice with pre-cooled PBS (3 min / time). Then 4% paraformaldehyde was added for room temperature fixation for 20 min. After fixation, the cells were washed 3 times with PBST containing 0.1% Tween-20 (3 min / time). To block non-specific binding, 3% BSA was added for room temperature blocking for 2 h, and after blocking, the cells were washed repeatedly 3 times with PBST (3 min / time). Rabbit-derived anti-PRRS virus nucleocapsid protein primary antibody (1:500) was added for 4°C incubation overnight. The next day, the primary antibody was discarded and the cells were washed thoroughly 3 times with PBST (3 min / time). Then FITC-labeled goat anti-rabbit IgG secondary antibody was added for room temperature incubation in the dark for 2 h. After the secondary antibody incubation, the cells were washed 3 times with PBST in the dark (3 min / time). Finally, the cells were observed under an inverted fluorescence microscope and images were captured.

[0072] (6) Comparison of the anti-PRRSV effects of P. praevaleus and other bacterial species

[0073] P. praevaleus, B. licheniformis, B. subtilis, B. megaterium, L. plantarum, and L. fermentum were selected for anti-PRRSV activity evaluation. Each bacterial species was inoculated into the corresponding liquid medium and cultured under anaerobic or aerobic conditions at 37°C. When the OD 600When the value is 0.6-0.7 (the subsequent experiments uniformly use this range), equal amounts of bacterial suspensions are taken in each group, centrifuged at 4°C and 5000 rpm for 8 min, the supernatant is discarded, and the bacterial pellet is resuspended with sterile PBS buffer to the original volume. PRRSV virus liquid (10 5.5 TCID 50 / mL, and the subsequent experiments all use this virus titer) is mixed with the resuspended liquid of each group at a ratio of 1:1, and the mixture is incubated at room temperature for 0.5 h, while setting the virus liquid without inoculating bacteria as a negative control. Virus load detection is performed by real-time fluorescent quantitative PCR (specific method same as step (4) above) and indirect immunofluorescence (IFA) (specific method same as step (5) above). For IFA detection, the virus-bacteria co-incubation liquid is mixed with the basic culture medium at a ratio of 1:1 and inoculated into a 96-well plate with Marc-145 cells, which is then cultured at 37°C and 5% CO2 for 48 h. After that, the mixture is observed and images are collected under uniform exposure parameters using a fluorescence microscope. By comparing the virus copy number (RT-qPCR) and cytopathic effect inhibition rate of each treatment group, the best bacterial species for anti-virus effect is screened.

[0074] The results are shown in Figure 5 and Figure 6 . According to the results of RT-qPCR and IFA, Prevotella plara shows the best anti-PRRSV effect among all the tested bacteria. Based on this finding, Prevotella plara is selected as the research object in subsequent experiments.

[0075] Example 2

[0076] Comparison of PRRSV disinfection effects of Prevotella plara bacterial suspension, bacterial pellet and fermentation liquid:

[0077] To evaluate the anti-virus effect of Prevotella plara bacterial suspension, bacterial pellet and fermentation liquid, OD 600 value of 0.6-0.7 (bacterial suspension preparation method same as step (1) of Example 1) is centrifuged at 4°C and 5000 rpm for 8 min to separate the bacterial pellet and the supernatant fermentation liquid. The bacterial pellet is resuspended with sterile PBS buffer to the original volume to ensure that the bacterial concentration is consistent with the original bacterial suspension. Then, equal volumes of bacterial suspension, resuspended bacterial pellet and fermentation liquid are taken and mixed with PRRSV virus liquid at a ratio of 1:1, and the mixture is incubated at room temperature for 0.5 h. By comparing the differences between the bacterial suspension group, the bacterial pellet group and the fermentation liquid group through real-time fluorescent quantitative PCR (specific method same as step (4) of Example 1) and indirect immunofluorescence (specific method same as step (5) of Example 1), it is evaluated whether the disinfection effect of Prevotella plara is dominated by the bacterial itself or relies on the synergistic effect of other components in the fermentation liquid.

[0078] The results are shown in Figure 7 and Figure 8As shown, by Figure 7 It can be seen that the viral copy number decreased most significantly in the bacterial suspension group. The bacterial suspension group contained both bacterial cell precipitation and fermentation broth, and the two may have significantly enhanced the antiviral effect through synergistic effects. The viral copy number in the fermentation broth group increased by 0.8% compared with the control group, suggesting that the fermentation broth alone not only failed to inhibit viral replication, but may have even promoted viral proliferation under certain conditions. Figure 8 The indirect immunofluorescence results further validated the above findings. This finding provides an important basis for developing antiviral strategies based on whole-bacterial formulations.

[0079] Example 3

[0080] The optimal working concentration for Clostridium plasmid suspension to exert its disinfection effect:

[0081] The inactivation effect of the system on PRRSV was evaluated by setting different bacterial cell concentration gradients. OD was collected. 600 The *Clostridium pluvialis* bacterial suspension with a concentration of 0.6-0.7 (prepared as in step (1) of Example 1) was centrifuged at 4°C and 5000 rpm for 8 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in the original fermentation broth and prepared into bacterial suspensions with concentrations of 1 mg / mL, 5 mg / mL, 15 mg / mL, and 25 mg / mL, respectively. The suspensions were thoroughly mixed to ensure uniform dispersion of the bacteria. The bacterial suspensions of different concentrations were mixed with PRRSV virus solution at a 1:1 ratio and incubated at room temperature for 0.5 h by rotation. The changes in the number of virus particles at different bacterial concentrations were compared by real-time quantitative PCR (the specific method is the same as step (4) of Example 1) and indirect immunofluorescence (the specific method is the same as step (5) of Example 1) to determine the optimal working concentration of *Clostridium pluvialis* bacterial suspension to achieve the best disinfection effect.

[0082] The results are as follows Figure 9 and Figure 10 As shown, RT-qPCR and IFA results jointly confirmed that 25 mg / mL Clostridium pluvialis suspension can most effectively inhibit PRRSV infection and replication, protecting host cells from viral pathological damage.

[0083] Example 4

[0084] Based on the results of the optimal working concentration of Clostridium praosporum suspension in Example 3, the suspension was mixed with PRRSV virus solution at ratios of 1:1, 4:1, 8:1, 16:1, and 32:1 at this concentration and incubated at room temperature for 0.5 h by rotation. The changes in the number of virus particles at different ratios were compared by real-time quantitative PCR (specific method as in step (4) of Example 1) and indirect immunofluorescence (specific method as in step (5) of Example 1) to determine the optimal working ratio of Clostridium praosporum suspension, which is convenient for subsequent comparison with other disinfection products.

[0085] Results as shown in Figure 11 and Figure 12 , qPCR and IFA results from the molecular level and cellular level together confirmed that the Prasvillus bacteria suspension reached the best anti-PRRSV effect at a ratio of 16:1, while too high a ratio could lead to factors such as bacterial aggregation or excessive concentration of metabolic products, which would otherwise affect its anti-viral activity.

[0086] Example 5

[0087] Under the optimal working concentration of 25 mg / mL of the Prasvillus bacteria suspension in Example 3 and the optimal working ratio (disinfectant: virus liquid = 16:1) in Example 4, the Prasvillus bacteria suspension, 75% ethanol (C2H5OH), 0.1% sodium hypochlorite (NaClO), and 0.5% hydrogen peroxide (H2O2) were mixed with PRRSV virus liquid at a ratio of 1:1, respectively, and incubated for 0.5 h, 1 h, 4 h, 8 h, and 24 h at room temperature. Virus load was detected by real-time fluorescent quantitative PCR (the specific method is the same as step (4) in Example 1) and PI dead cell staining. For PI dead cell staining, the virus-bacteria co-incubation liquid of each group at 24 h was inoculated into a 96-well plate with Marc-145 cells at a ratio of 1:1, and incubated in a 37°C, 5% CO2 incubator. At 12 h, 24 h, 48 h, and 96 h, the culture medium in the 96-well plate was removed, and the residual medium was removed by gentle washing with pre-cooled PBS once. Then, 100 μL of PI staining working solution was added to each well, and incubated at 37°C for 45 min in the dark. After incubation, the PI staining solution was removed, and the background fluorescence was reduced by washing once with PBS. Then, the cell staining was observed immediately using an inverted fluorescence microscope, and images were collected.

[0088] Results as shown in Figure 13 and Figure 14 , from Figure 13 It can be seen that with the increase of time, the virus copy number in the Prasvillus bacteria suspension steadily decreased, while the virus copy number of other disinfectants all showed different degrees of rebound, suggesting that the inactivation effect weakened with time. From the PI dead cell staining results of Figure 14 , it can be seen that the three groups of traditional chemical disinfectants all caused 100% cell death after 96 h. In contrast, the cell death rate of the Prasvillus treatment group remained at a physiological level throughout the experimental period. These results collectively show that the Prasvillus bacteria suspension achieves sustained and efficient virus inactivation through a non-cytotoxic biological mechanism, avoiding the common problem of host cell damage caused by chemical disinfectants, and exhibiting significant biological safety advantages and application potential.

[0089] Example 6

[0090] Antiviral activity: surface test method as follows:

[0091] Take 50 μL PRRSV-JXA1 virus liquid evenly on the glass surface of 5x5 cm, dry at room temperature, then cover with 50 μL different disinfectants, collect samples at 0.5h, 1h, 8h, 24h. For sample collection, use sterile cotton swab to sample, first wet the sterile cotton swab in sterile PBS to maintain microbial activity and improve sampling efficiency, then use the sterile cotton swab to evenly wipe in the sampling area with an "S" trajectory, while applying moderate pressure to ensure that the microorganisms are effectively adsorbed. Each surface sampling time is controlled within 30 seconds to avoid cotton swab drying. A separate sterile cotton swab is used for each sampling area to avoid cross contamination between samples. After sampling, immediately place the cotton swab in a centrifuge tube containing sterile PBS, shake well for 1 min to elute the microorganisms. Take 200 μL from the eluate into a new sterile centrifuge tube, then add 500 μL Trizol, store at -80°C for subsequent extraction of sample RNA and RT-qPCR viral load detection (RT-qPCR detection method same as step (4) of Example 1). The eluate of each group at 24h is inoculated into the 96-well plate coated with Marc-145 cells at a ratio of 1:1, and placed in a 37°C, 5% CO2 incubator for incubation. At 12h, 24h, 48h and 96h, observe the PI dead cell staining under a fluorescence microscope (PI dead cell staining test method same as Example 5).

[0092] The results are shown in Figure 15 and Figure 16 According to the comprehensive RT-qPCR and PI dead cell staining results, Prevotella albensis exhibits a significant performance advantage over traditional chemical disinfectants in glass surface disinfection applications. Its stable inactivation effect and extremely low cytotoxicity make it particularly suitable for application scenarios that require long-term surface disinfection.

[0093] The above only describes the preferred embodiments of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. The application of Clostridium pluvialis in the preparation of products against porcine reproductive and respiratory syndrome virus, characterized in that, The aforementioned Clostridium is Faecalibacterium prausnitziistrain A2-165, deposited at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, accession number DSM17677.

2. The application of Clostridium pluvialis in the preparation of inactivated porcine reproductive and respiratory syndrome virus products, characterized in that, The aforementioned Clostridium is Faecalibacterium prausnitziistrain A2-165, deposited at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, accession number DSM17677.

3. The application of Clostridium praosporum suspension in the preparation of products for the prevention and control of porcine reproductive and respiratory syndrome virus, characterized in that, The aforementioned Clostridium is Faecalibacterium prausnitziistrain A2-165, deposited at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, accession number DSM 17677.

4. The application according to claim 3, characterized in that, The concentration of the Clostridium praosporum suspension is above 1 mg / mL.

5. The application according to claim 3, characterized in that, The method for preparing the Clostridium praosporum suspension includes the following steps: inoculating Clostridium praosporum into a modified enhanced Clostridium broth medium and culturing it.

6. The application according to claim 4, characterized in that, The method for preparing Clostridium praosporum suspensions with a concentration of 1 mg / mL or higher includes the following steps: inoculating Clostridium praosporum into a modified enhanced Clostridium broth medium and culturing until OD500. 600 The concentration was 0.6~0.

7. The bacterial precipitate and fermentation broth were separated and collected. The fermentation broth and bacterial precipitate were mixed until the concentration of the Clostridium praosporum suspension was above 1 mg / mL.

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