Peste des petits ruminants virus strain and application thereof

Abstract

The invention discloses a peste des petits ruminants virus strain with a microbial preservation number of CCTCC (China Center For Type Culture Collection) NO: V202487. A DNA vaccine and an RNA vaccine which are prepared by taking proteins coded by the H gene and the F gene of the strain as immunogens have good immunogenicity and immune protection capacity. The strain has an application prospect.

Description

Technical Field

[0001] The invention belongs to the field of preventive veterinary medicine and relates to a peste des petits ruminants virus strain and an application thereof. Background Art

[0002] Peste des Petits Ruminants (PPR) is an acute, contagious viral disease of domestic sheep and wild small ruminants, caused by the Peste des Petits Ruminants virus (PPRV). Clinical symptoms include high fever, pneumonia, diarrhea, increased ocular and nasal discharge, stomatitis, and various erosive lesions of the mucous membranes. Due to its rapid spread and high mortality rate, the World Organization for Animal Health (OIE) has classified PPR as a notifiable major animal disease. PPR is one of the most economically destructive diseases affecting small ruminants. With the development of the global economy, people's living standards have greatly improved, and the demand for mutton and goat milk products is increasing. If the epidemic is not effectively controlled, the direct and indirect economic consequences will be immeasurable. Therefore, in 2015, the Food and Agriculture Organization of the United Nations (FAO) and the OIE endorsed the Global Strategy for the Control and Eradication of PPR, aiming to completely eradicate PPR by 2030. Therefore, the development of a PPR vaccine, particularly one that can distinguish between natural infection and vaccine immunity (DIVA vaccine), is crucial. The PPRV genome is a single-stranded, negative-sense, non-segmented RNA approximately 16 kb long. From the 3' to the 5' end of the RNA chain, six genes, 3'-NPMFHL-5', are arranged sequentially. These genes encode, in order, six structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin (H), and large protein (L). Furthermore, the P gene can produce two non-structural proteins, C and V, through mRNA editing. The H and F proteins possess excellent immunogenicity. The F protein, comprising 546 amino acids and a molecular weight of approximately 59.3 kDa, is a type I membrane protein that forms the outer surface spikes of the viral envelope and is responsible for mediating binding between the virus and host cell receptors and fusion with the cell membrane, thereby completing viral invasion. In addition to its cell fusion activity, the F protein also possesses hemolytic activity, causing lysis of chicken red blood cells.

[0003] The H protein, consisting of 609 amino acids and a molecular weight of approximately 69 kDa, is a type II membrane glycoprotein responsible for viral binding to host cell receptors. PPRV utilizes the signaling lymphocyte activation molecule (SLAM / CD150) as a lymphocyte receptor and the lectin cell adhesion molecule 4 (nectin-4) as its receptor on epithelial cells. The PPRV H protein possesses both hemagglutinin and neuraminidase activities, hence its designation as the hemagglutinin-neuraminidase protein. The H protein is not highly conserved among different members of the Morbillivirus genus and is a key factor in the diverse cell tropisms within the genus. It is also a key factor in the cross-species pathogenicity of the lagomorphic rinderpest virus. Furthermore, the H protein is immunogenic, inducing downregulation of CD46, participating in host cell complement regulation, and inducing higher levels of neutralizing antibodies than the F protein. Therefore, H and F proteins can be used as immunogens in vaccine development. Examples include sheeppox virus-vectored vaccines, subunit vaccines, and adenovirus-vectored vaccines. In December 2020, the US FDA granted emergency approval for two mRNA COVID-19 vaccines, the world's first commercially available mRNA vaccines. Due to their effectiveness, safety, high production efficiency, and affordability, mRNA vaccines are considered the future of vaccinology. Thanks to their exceptional performance, mRNA vaccine developers Moderna and BioNTech have amassed market capitalizations of $160 billion and $80 billion, respectively. However, due to their high cost and poor stability, mRNA vaccines are currently unsuitable for cost-sensitive animal vaccines and those used in harsh environments.

[0004] DNA is easier to synthesize than mRNA, making it easier to produce and less expensive. DNA is also more stable, making it easier to store and transport. mRNA vaccines usually need to be stored at -80°C, while DNA vaccines can be stored at room temperature. DNA vaccines can remain stable for more than one year at room temperature and can be stored for five years at standard refrigeration temperatures (2°C-8°C), while mRNA vaccines can only be stored for five days under standard refrigeration conditions. Therefore, the use of DNA vaccine technology to develop animal vaccines not only has the advantages of mRNA vaccines, such as a short development cycle and ease of responding to disease mutations, but also has advantages that inactivated vaccines, attenuated vaccines, recombinant protein vaccines, and viral vector vaccines do not have, such as safety, simple production, and low cost.

[0005] However, at present, DNA vaccines mainly use cytokines, gene guns, intradermal or intramuscular electric shock technology, needle-free syringes, rotary tattooing devices (tattooing), magnetic perforation / acoustic perforation / photoperforation technology, skin microneedle injection technology, DNA binding proteins and other methods to enhance immunity. The above methods have disadvantages such as high cost, complex use, and unstable effects, and are not suitable for animal vaccines.

[0006] According to the World Organization for Animal Health (WOAH) manual, virus-neutralizing antibodies are a key indicator for evaluating PPR. Furthermore, while mRNA vaccines are more expensive than DNA vaccines, they offer greater immune efficacy due to the elimination of DNA transcription and translation processes, lack integration risk, and offer improved safety, similarly promising market prospects. Summary of the Invention

[0007] The present invention first systematically identified and evaluated the virulence of the PPRV field strain isolated in 2020, confirming that it is a super virulent strain, indicating that the prevention and control of PPR in my country cannot be relaxed. At the same time, the creative use of LNP as a delivery medium has overcome the technical defects of existing DNA vaccines and has been successfully used to construct a DNA vaccine for peste des petits ruminants. The H and F genes of the super virulent strain isolated on the field are then used as antigens, which greatly improves the immune efficacy and protective efficacy against the field strain. The neutralizing antibodies of the H single-gene DNA vaccine after two immunizations can reach about 1900-11000, which is higher than the immune efficacy of the attenuated vaccine strain. It is easy to use and has low cost, overcoming the defect that the attenuated vaccine can be used as a DIVA vaccine. In addition, an mRNA vaccine for PPR was constructed, which also induced high levels of antibodies after immunization. Both of these nucleic acid vaccines can be used as DIVA vaccines for the purification and eradication of peste des petits ruminants in my country and around the world, and have good application prospects.

[0008] A first aspect of the present invention provides a peste des petits ruminants virus strain, wherein the peste des petits ruminants virus strain is selected from S1 and S2;

[0009] S1: Peste des petits ruminants virus strain with microbiological accession number CCTCC NO: V202487;

[0010] S2: passaged virus strain of peste des petits ruminants virus with microbiological deposit number CCTCC NO: V202487;

[0011] During the passage process of the passaged virus strain of the peste des petits ruminants virus strain with the microbial preservation number CCTCC NO: V202487, the transcriptional regulatory activity of all genes in the genome, the transcription initiation and transcription termination of all genes in the genome, the amino acid sequence of the proteins encoded by all genes in the genome, the clinical pathogenicity of the virus, the immunogenicity of the virus and the viral reproduction ability did not change compared with the peste des petits ruminants virus strain with the microbial preservation number CCTCC NO: V202487.

[0012] In some embodiments, in S1 and / or S2, the amino acid sequence of the protein encoded by the H gene of the Peste des petits ruminants virus strain is shown as SEQ ID NO.15; the amino acid sequence of the protein encoded by the F gene of the Peste des petits ruminants virus strain is shown as SEQ ID NO.17.

[0013] The second aspect of the present invention provides a method for culturing Peste des Petits Ruminants virus, which comprises inoculating the Peste des Petits Ruminants virus strain described in the first aspect of the present invention into Peste des Petits Ruminants virus-susceptible cells, and performing proliferation culture of the Peste des Petits Ruminants virus strain to obtain a proliferated Peste des Petits Ruminants virus strain.

[0014] In some embodiments, the peste des petits ruminants virus susceptible cells are Vero cells or Vero cells expressing goat signaling lymphocyte activation molecule receptor.

[0015] A third aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P8 and P9;

[0016] P1: Protein

[0017] The protein is a first protein, a second protein, or a combination of the first protein and the second protein;

[0018] The amino acid sequence of the first protein is shown in SEQ ID NO.15;

[0019] The amino acid sequence of the second protein is shown in SEQ ID NO.17;

[0020] P2: fusion protein

[0021] The fusion protein is the first fusion protein, the second fusion protein, or a combination of the first fusion protein and the second fusion protein;

[0022] The amino acid sequence of the first fusion protein contains the amino acid sequence of a tag peptide and / or a signal peptide used for separating and purifying the protein and the amino acid sequence shown in SEQ ID NO.15;

[0023] The amino acid sequence of the second fusion protein contains the amino acid sequence of a tag peptide and / or a signal peptide used for separating and purifying the protein and the amino acid sequence shown in SEQ ID NO.17;

[0024] P3: RNA

[0025] The RNA is a first RNA, a second RNA, or a combination of the first RNA and the second RNA;

[0026] The first RNA can be translated to produce the first protein described in P1 or the first fusion protein described in P2;

[0027] The second RNA can be translated to produce the second protein described in P1 or the second fusion protein described in P2;

[0028] P4: Gene

[0029] The gene is the first gene, the second gene, or a combination of the first gene and the second gene;

[0030] The coding sequence of the first gene is capable of encoding the first protein described in P1 or the first fusion protein described in P2;

[0031] The coding sequence of the second gene is capable of encoding the second protein described in P1 or the second fusion protein described in P2;

[0032] P5: Gene expression cassette

[0033] The gene expression cassette is a first gene expression cassette, a second gene expression cassette, or a combination of the first gene expression cassette and the second gene expression cassette;

[0034] The gene expression product in the first gene expression cassette is the first RNA described in P3;

[0035] The gene expression product in the second gene expression cassette is the second RNA described in P3;

[0036] P6: Genetic Engineering Vectors

[0037] The genetic engineering vector is the first genetic engineering vector, the second genetic engineering vector, the third genetic engineering vector, or a combination of the first genetic engineering vector and the second genetic engineering vector;

[0038] The first genetic engineering vector contains the first gene expression cassette described in P5;

[0039] The second genetic engineering vector contains the second gene expression cassette described in P5;

[0040] The third genetic engineering vector contains the first gene expression cassette and the second gene expression cassette described in P5;

[0041] P7: Cells

[0042] The cell is a first cell, a second cell, a third cell, or a combination of the first cell and the second cell;

[0043] The first cell contains the first genetic engineering vector described in P6;

[0044] The second cell contains the second genetic engineering vector described in P6;

[0045] The third cell contains the third genetic engineering vector described in P6;

[0046] The coding gene in the gene expression cassette of the first genetic engineering vector is constitutively expressed or artificially induced;

[0047] The coding gene in the gene expression cassette of the second genetic engineering vector is constitutively expressed or artificially induced;

[0048] The coding gene in the gene expression cassette of the third genetic engineering vector is constitutively expressed or artificially induced;

[0049] P8: Composition

[0050] The composition contains the protein described in P1, the fusion protein described in P2, the RNA described in P3, the genetic engineering vector described in P6 or the cell described in P7; and

[0051] P9: Test kit

[0052] The kit contains the protein described in P1, the fusion protein described in P2, the RNA described in P3, the genetic engineering vector described in P6 or the cells described in P7.

[0053] In some embodiments, it is selected from the following A1, A2, A3, A4, A5:

[0054] A1: The promoter in the gene expression cassette is a T7 promoter;

[0055] A2: The backbone vector of the genetic engineering vector is pBlue or pCAGGS;

[0056] A3: The cells are Escherichia coli cells;

[0057] A4: The coding sequence in the first RNA is shown as SEQ ID NO. 14 or SEQ ID NO. 18;

[0058] A5: The coding sequence in the second RNA is shown as SEQ ID NO.16 or SEQ ID NO.19.

[0059] The fourth aspect of the present invention provides the use of the PPR virus strain described in the first aspect of the present invention or the biological material described in the third aspect of the present invention in preparing a preparation for preventing, mitigating or controlling PPR.

[0060] In some embodiments, the peste des petits ruminants is the peste des petits ruminants caused by the peste des petits ruminants virus strain described in the first aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 These are morphological photos of China / HLJ / 2020 (C20) infected Vero and Vero-gSLAM cells.

[0062] Figure 2 This is the growth curve of the China / HLJ / 2020 strain on Vero and Vero-gSLAM cells.

[0063] Figure 3 These are photos of immunofluorescence detection of China / HLJ / 2020 (C20) infection in Vero and Vero-gSLAM cells.

[0064] Figure 4 Statistical information on clinical signs of goats infected with China / HLJ / 2020, (A) clinical score, (B) body temperature curve, (C) survival rate curve, and (D) clinical symptoms.

[0065] Figure 5 Statistical charts of viral load in nasal swabs (A), anal swabs (B), blood (C), and tissues (D) after infection with China / HLJ / 2020.

[0066] Figure 6 Photos of pathological changes in goats after infection with China / HLJ / 2020.

[0067] Figure 7 Figure 1 shows the pathogenicity of the PPRV China / HLJ / 2013 strain in goats. (A) Temperature curve. (B) Survival rate curve. (C) Nasal swab viral load. (D) Tissue viral load.

[0068] Figure 8 The figure shows the immunization results of 500 μg dose, the dotted line is the lower limit of detection, and VNT represents virus neutralizing antibodies.

[0069] Figure 9 The figure shows the immunization results of 50 μg dose, the dotted line is the lower limit of detection, and VNT represents virus neutralizing antibodies.

[0070] Figure 10These are the results of analysis of body temperature, clinical symptom scores and survival rate after PPR challenge.

[0071] Figure 11 Figure 2 shows the clinical symptoms and autopsy analysis after PPR challenge. H is the immunized group and Control is the non-immunized control group. DETAILED DESCRIPTION

[0072] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0073] Materials and instruments not described in the present invention are conventional materials and instruments in the field. Operation details not described in the present invention are conventional operations in the field. The software used in the present invention is operated by conventional methods with reference to the instructions of the software provider. The kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0074] Vero-gSLAM cells expressing goat SLAM (Signaling lymphocyte activation molecule) receptors are preserved by the laboratory of Harbin Veterinary Research Institute. The construction method of Vero-gSLAM cells can be found in the literature Construction of recombinant adenovirus expressing goat SLAM receptor and its application in enhancing PPRV infection in sheep primary cells, Chen Hefeng et al., Chinese Journal of Preventive Veterinary Medicine, December 2020, Volume 42, Issue 12.

[0075] During the passage process, the passaged virus strain of the PPR virus strain with the microbial deposit number CCTCC NO: V202487 has no changes in the transcriptional regulatory activity of all genes in the genome, the transcription initiation and termination of all genes in the genome, the amino acid sequence of the proteins encoded by all genes in the genome, the clinical pathogenicity of the virus, the immunogenicity of the virus, and the reproductive capacity of the virus compared to the PPR virus strain with the microbial deposit number CCTCC NO: V202487. This includes but is not limited to the following:

[0076] During the passage of the C20 strain, the viral reproductive capacity did not change, which mainly refers to the fact that the genome sequence did not undergo any mutations and maintained its original reproductive capacity (no mutation-induced lethality, etc.), or the slight change in reproductive capacity was within the error range of the detection system of conventional technology or the degree of change that could not be detected by conventional technology. According to common sense in the field, even for two progeny viruses isolated from a monoclonal plaque of the C20 strain, there are systematic errors in the results of the reproductive capacity index measurement, and there will be certain differences in the measurements of different batches or different operators. Obviously, minor gene mutations inevitably occur during the passage of the virus. Synonymous mutations in the coding sequence or minor mutations in the non-coding region of the genome that are not involved in the genetic regulation of the virus do not affect the reproductive capacity of the virus or its biological activity. This passaged strain falls within the scope of the substantial technical contribution of the deposited bacterial strain (C20 strain) and is the inevitable reasonable variation range of the direct technical contribution of the present invention. This range will minimize the obvious separation between the content protected by the patent, the content of actual use, and the content defined in the claims. Similarly, no change in the clinical pathogenicity and immunogenicity of the virus also means that it does not exceed the systematic error of the detection method, the difference is not statistically significant, or the difference value is slightly higher than, approximately equal to, or less than the standard deviation of the measurement method.

[0077] During the propagation of the C20 strain, the transcriptional regulatory activity of all genes did not change. This mainly refers to the fact that the pattern of all transcriptional regulatory elements accepting gene expression regulation from endogenous viruses or host sources did not change, the pattern of responding to external signals did not undergo detectable changes, and the host signal transduction pathways and metabolic regulation patterns in which the virus participates did not undergo detectable changes. Minor mutations that do not participate in regulation, such as minor changes in non-coding new sequence bases, did not affect the expression regulation pattern of the corresponding genes, and fell within the range of no change in transcriptional regulatory activity.

[0078] During the passage of the C20 strain virus, the transcription initiation and termination of all genes did not change, which mainly means that the pattern of gene expression regulation did not change, and there was no detectable significant change in the transcriptional regulation of the genes.

[0079] During the propagation of the C20 strain virus, the amino acid sequences of all proteins did not change. This mainly means that during the propagation of the strain, the amino acid sequences of all proteins did not change. Synonymous mutations of genes will not affect gene function, and mutations during the virus propagation process are inevitable. Therefore, the progeny viruses whose amino acid sequences of all proteins have not changed are still within the scope of the technical contribution of the preserved strain.

[0080] The experimental animals in this invention are goats and sheep aged about 4-6 months. All experimental sheep were isolated and observed 3 days before the experiment, including temperature measurement and clinical manifestation observation. All experimental sheep were screened for brucellosis and tested for PPRV antibodies before the experiment. All experimental sheep were negative for PPRV antibodies and brucellosis. The experimental plan was reviewed and approved by the Animal Experiment Ethics Committee of Harbin Veterinary Research Institute. All experiments were conducted in a Class III biosafety laboratory (BSL3).

[0081] Example 1. Acquisition, isolation, and identification of the China / HLJ / 2020 strain of Peste des Petits Ruminants virus

[0082] 1. Source of Cases

[0083] The present invention collected a number of goat spleens for epidemiological investigations of several pathogens. Targeting PPR virus, primers F1 and R1 (for N gene amplification) and probe P1 were designed. Quantitative polymerase chain reaction (qPCR) was used to detect the presence of PPR virus in spleen samples. A PPR virus amplification signal was detected in goat spleen a, leading to the preliminary conclusion that spleen a contained PPR virus and that the goat from which spleen a originated was suffering from PPR.

[0084] F1 sequence (SEQ ID NO. 1): 5'-AACTGAGAAGGTGGGTTAAATACAC-3'.

[0085] R1 sequence (SEQ ID NO. 2): 5'-ACAATATAGTTGTCAATGTCGCAGA-3'.

[0086] P1 sequence (partial nucleic acid sequence named SEQ ID NO. 3): FAM-5'-CAAGTATGAGAGATACCATGAACCGCCG-3'-Tamra.

[0087] 2. Virus Isolation and Culture

[0088] The supernatant of the sheep spleen (spleen a) that was positive in the quantitative PCR test was taken and inoculated into Vero-gSLAM cells with a cell density of 80%-90% cultured in DMEM containing 2% FBS. After 2 hours of induction, the supernatant was discarded and DMEM containing 2% FBS was added to continue culturing and observing until cell pathological changes occurred. When the lesion reached about 90%, the diseased cells and supernatant were collected, frozen and thawed three times, centrifuged at 3000 rpm at 4°C for 5 minutes, and the supernatant virus liquid (virus liquid b) was aspirated and packaged and stored at -80°C. The virus strain contained in the virus liquid b was named PPRV China / HLJ / 2020 (abbreviated as C20)

[0089] 3. Amplification and pathological changes of local strains in Vero and Vero-gSLAM cells

[0090] Vero and Vero-gSLAM cells cultured in DMEM medium containing 2% FBS were infected with C20 strain at an MOI of 0.1, and the Vero and Vero-gSLAM cells were observed under a microscope 72 hours after infection. Uninfected cells were used as negative controls.

[0091] The results are as follows Figure 1 As shown in the figure, the photograph shows obvious CPE, manifested by large areas of cell rounding, fusion, and shedding, without the typical pathological changes seen in PPRV-infected Vero-gSLAM. However, the Vero cells showed almost no changes, and the negative control cells showed no changes. This is consistent with the biological characteristics of the wild-type strain cultured in vitro, indicating successful virus isolation.

[0092] IV. Plotting the Growth Curve of the Local Strain

[0093] Virus titer detection method: Use DMEM medium containing 10% FBS to plate Vero cells or Vero-gSLAM cells on a 96-well cell culture plate one day in advance, 100 μL per well. After overnight, when the cell confluence is about 80%-90%, discard the supernatant and add 100 μL of diluted virus solution to each well. The virus solution dilution method is as follows: thaw the virus solution on ice, dilute it 10-fold with DMEM medium containing 2% FBS, and dilute the virus solution at 10 -1 -10 -6 Dilution, each dilution was repeated 8 times, and the cells were placed in a 37°C incubator with 5% CO2 for 5 days. CPE was observed every day and recorded. Virus titer was calculated according to the Reed-Muench method, and the virus titer was expressed as TCID 50 Vero and Vero-gSLAM cells were infected with C20 strain at an MOI of 0.1 (culture medium was DMEM containing 2% FBS), and samples were taken for detection on days 1, 2, 3, and 4 after infection. 50 Methods The virus titer was determined, and the growth kinetics curves of PPRV on Vero and Vero-gSLAM were drawn.

[0094] The results are as follows Figure 2 As shown. It can be seen that the C20 strain can grow on both Vero and Vero-gSLAM cells, but the infection peak is 48h on Vero-gSLAM, and the highest virus titer is about 10 7 TCID 50 / mL; the infection peak of China / HLJ / 2020 on Vero was around 96h, and the highest virus value was about 10 5 TCID 50 The virus growth titer on Vero-gSLAM was much higher than that on Vero cells, indicating that SLAM expression promoted the replication of C20 on Vero cells.

[0095] 5. Indirect Immunofluorescence (IFA)

[0096] To verify the specificity of the China / HLJ / 2020 strain for the PPRV-N monoclonal antibody, indirect immunofluorescence assays were performed on the isolated strain using the PPRV-N monoclonal antibody. Vero cells and Vero-gSLAM cells cultured in DMEM medium containing 2% FBS were infected with China / HLJ / 2020 (MOI = 0.1). 36 h after infection, the cells were fixed, permeabilized, blocked, and incubated with PPRV-N monoclonal antibody (1:100 dilution ratio, mouse monoclonal antibody IgG prepared by hybridoma method using the N protein of the PPRV N75 / 1 vaccine strain; the full-length cDNA clone of the PPRV / N75 / 1 vaccine strain is described in patent application number CN201010559545.8). FITC-labeled goat anti-mouse IgG (1:100 dilution ratio, Sigma) was used as the secondary antibody. After cell nuclei were stained with DAPI, fluorescence was observed under an inverted fluorescence microscope and photographed.

[0097] Photos such as Figure 3 As shown, specific green fluorescence can be detected on both Vero and Vero-gSLAM cells inoculated with the virus China / HLJ / 2020, but the fluorescence on Vero-gSLAM cells is significantly greater than that on Vero cells, indicating that the isolated virus is PPRV and Vero-gSLAM is more susceptible to PPRV than Vero cells.

[0098] 6. Pathogenicity Test of Isolated Strains

[0099] In order to evaluate the pathogenicity of PPRV China / HLJ / 2020 strain to goats, 10 3.3 TCID 50 (n=6), 10 4.3 TCID 50 (n=4) and 10 5.3 TCID 50The goats were infected with the infection dose of 10 μg / mL intranasal drops and 1 mL subcutaneous injection (n=4). In order to compare the virulence difference with the previously isolated strains, a control group of C13 strain (microorganism collection number: CCTCC NO: V202135) was set up at the same time. The infection dose was 10 μg / mL and 10 μg / mL respectively. 4.3 TCID 50 (n=4) and 10 5.3 TCID 50 (n=5), infected in the same way.

[0100] After infection, body temperature was measured daily, and clinical symptoms such as animal behavior and mental state were observed and scored (clinical symptom scoring criteria are shown in Table 1 below). The observation period after infection was 21 days, and animals that did not die on day 21 were euthanized. Whole blood, anticoagulated blood, and nasal swabs were collected on days 0, 2, 5, 8, 11, 14, 17, and 21 after infection. Tissues such as the lungs, spleen, colon, and mesenteric lymph nodes were collected from euthanized or morbidly deceased sheep.

[0101] Table 1. Clinical symptom scoring criteria

[0102]

[0103] After infection, all animals were observed for clinical symptoms every day until the end of the experiment. Figure 4 As shown in A, symptoms begin to appear on day 3, reach a peak on day 7-10, and then begin to decline. Figure 4 As shown in B, some infected goats in each group developed high fever (≥40℃) 3 days after infection, and the average body temperature peaked 5-8 days later, and then the body temperature dropped until death; the mortality after infection is as follows Figure 4 As shown in C, all sheep infected with all doses became ill and died within 13 days after infection. Diarrhea began in some sheep 6 days after infection, and the diarrhea worsened with the increase in infection time, from soft stool to watery stool. On the 6th day after infection, the test sheep began to show respiratory symptoms, including coughing and sneezing. The results of the quantitative scoring of behavioral performance are shown in Figure 4 A. Three days after infection, some sheep began to have mucous discharge from the eyes and nose. As the infection time increased, all sheep began to have mucous discharge from the eyes and nose. The amount of discharge increased with the progression of the disease, and some sheep developed purulent nasal discharge ( Figure 4 D). As the disease progresses, fever persists, diarrhea worsens, and infected sheep develop symptoms such as decreased appetite and depression, eventually leading to death ( Figure 4 A).

[0104] Serum separation: Place the centrifuge tube containing non-anticoagulant blood in a 37°C incubator for 2 hours, take it out and place it in a 4°C refrigerator for 1 hour. After the serum is precipitated, balance the centrifuge tube and place it in a 4°C centrifuge, centrifuge at 3000 rpm for 10 minutes, take the supernatant into a new centrifuge tube, mix well, divide it into portions, mark them and store them at -20°C.

[0105] Viral genomic RNA was extracted from tissue, swab, and blood samples using the RNA Easy Fast Tissue / Cell Kit, the TIANamp Virus DNA / RNA Kit, and the RNAprep Pure Blood Kit, respectively. RNA was reverse transcribed into cDNA using reverse transcriptase according to the manufacturer's instructions. qPCR was performed using primers F1 and R1 and probe P1 as described above. Samples with a Ct value ≥40 were considered negative.

[0106] The results are as follows Figure 5 As shown, A shows the N gene copy number in nasal swabs, B shows the N gene copy number in anal swabs, C shows the N gene copy number in blood, and D shows the N gene copy number in spleen, mesenteric lymph, lung, and colon after death or euthanasia. Viruses can be detected on the 5th day after infection, and the viral load reaches a peak on the 8th day; except for 10 5.3 TCID 50 Except for low levels of viral load detected on the second day after the dose, higher viral load levels were detected on days 5 and 8 ( Figure 5 C), the viral load level is consistent with the clinical symptoms. The infected sheep died, and the spleen, mesenteric lymph, lung, colon, and other tissues were collected and ground and RNA was extracted for RT-qPCR. The viral load in the organs of the test sheep in each group was positive ( Figure 5 D) Overall, viral loads in swabs, blood, and organs across the different dose groups showed no clear correlation with the infectious dose. Observation of the aforementioned organs revealed typical PPR lesions, including: pulmonary congestion and consolidation, mesenteric lymphadenopathy, and colonic congestion and petechial hemorrhages.

[0107] For photos of pathological changes in goats infected with the virus, see China / HLJ / 2020. Figure 6 , which shows the tissue dissection photos of the lungs, colon, and lymph nodes under three doses of inoculation. All tissues showed typical small ruminant plague lesions such as bleeding and swelling. This shows that the strain of virus successfully caused the disease and showed strong virulence.

[0108] PPRV China / HLJ / 2013 is divided into 10 5.3 TCID 50 and 10 4.3 TCID 50After the goats were infected by combined intranasal and subcutaneous injection, the two groups of goats showed obvious fever on the 4th day after infection (average body temperature ≥ 40℃), and the body temperature gradually decreased 8-10 days after infection ( Figure 7 A). However, the mortality rate of goats in both groups did not exceed 50%, of which 10 4.3 TCID 50 The survival rate of goats in the dose group was 70%, 5.3 TCID 50 The survival rate of goats in the dose group was 80% ( Figure 7 B). In addition, the viral load of nasal swabs of infected sheep reached its peak on the 8th day after infection, and the virus could be detected in the spleen, colon, and mesenteric lymph nodes ( Figure 7 C and D).

[0109] The infection test results of the two isolates showed that the pathogenicity of the C13 strain was poor, and its 1000 LD50 infection dose was 10 8.49 TCID 50 The C20 strain is more pathogenic. Since the lower limit of virulence has not been determined, it is estimated that the infectious dose of 1000 LD50 is ≤10 5.80 TCID 50 , which is more than 490 times more toxic than C13.

[0110] 7. Viral genome sequencing

[0111] RNA was extracted from viral fluid b and reverse-transcribed into cDNA using random primers and reverse transcriptase. PCR amplification was then performed using the five primer pairs shown in Table 2. The PCR reaction system consisted of 50 μL: 2×Phanta Max Master Mix 25 μL; 2 μL each of the upstream and downstream primers (10 μM); 3 μL of template; and the volume was made up to 50 μL with ddH2O. The fragments obtained by PCR were recovered and purified, and then commissioned to Jilin Kumei Biotechnology Co., Ltd. for Sanger sequencing. The resulting sequencing results were assembled using DNASTAR to generate the genomic sequence. BLAST comparison analysis was performed in NCBI to confirm that it was the PPRV genomic sequence. The sequences of each primer pair are recorded in Table 2.

[0112] Table 2. Primers for amplifying the full-length cDNA of the PPRV genome

[0113]

[0114]

[0115] The genomic segment sequence of the H gene of strain C20 is (SEQ ID NO.14):

[0116]

[0117] The amino acid sequence of the H protein of strain C20 is (SEQ ID NO.15):

[0118] MSAQRERINAFYKDNPHNKNHRVILDRERLVIERPYILLGVLLVMFLSLIGLLAIAGIRLHRATVGTSEIQSRLNTNIKLTESIDHQTKDVLTPLFKIIGDEVGIRIPQKFSDLVKFISDKIKFLNPDREYDFRDLRWCMNPPERVKINFDQFCEYKAAGKSIEHIFESPLNKSKKLQSLTLGPGTGCLGRTVTRAHFSELTMTLMDLDLEMKHNVSSVFTVVEEGLFGRTYTVWRSDARDPSTDLGMGHFLRVFEIGLVRDLGLGPPVFHMTNYLTVNMSDDYRRCLLAVGELKLIALCTSSETVTLSERGVPRREPLVVVILNLAGPTLGGELYSVLPTSGLMVEKLYLSSHRGIIKDDEANWVVPSTDVRDLQNKGECLVEACKTRPPSFCNGTGSGPWSEGRIPAYGVIRVSLDLASDPDVVITSVFGPLIPHLSGMDLYNNPFSKAVWLAVPPYEQSFLGMINTIGFPNRAEVMPHILTTEIRGPRGRCHVPIELSRRADDDIKIGSNMVILPTMDLRYITATYDVSRSEHAIVYYIYDTSRSSSYFYPVRLNFKGNPLSLRIECFPWRHKVWCYHDCLIYNTITGEEVHTRGLTGIEVTCNPV

[0119] The genomic segment sequence of the F gene of strain C20 is (SEQ ID NO.16):

[0120]

[0121] The amino acid sequence of the F protein of strain C20 is (SEQ ID NO.17):

[0122] MTRVAILTFLFLFPNVVTCQIHWGNLSKIGIVGTGSASYKVMTRPSHQTLVIKLMPNITAIDNCTKSEIAEYKRLLITVLKPVEDALSVITKNVRPIQTLTPGRRTRRFAGAVLAGVALGVATAAQITAGVALHQS LMNSQAIESLKTSLEMSNQAIEEIRLANKETILAVQGVQDYINNELVPSVHRMSCELVGHKLGLKLLRYYTEILSIFGPSLRDPISAEISIQALSYALGGDINKILDKLGYSGGDFLAILESKGIKARVTYVDTRDY FIILSIAYPTLSEIKGVIVHKIEAITYNIGAQEWYTTIPKYVATQGYLISNFDETSCVFTPEGTVCSQNALYPMSPLLQECFRGSTKSCARTLVSGTISNRFILSKGNLIANCASVLCKCYTTETVISQDPDKLLT VVASDKCPVVEVDGVTIQVGSREYPDSVYLHKIDLGPAISLEKLDVGTNLGNAVTRLENAKELLDASDQILKTVKGVPFSGNMYIALAACIGVSLGLVTLICCCKGRCKNKEIPISKINPGLKPDLTGTSKSYVRSL

[0123] Furthermore, the genomic segments encoding amino acid sequences of the six genes in the C20 strain were compared with the genes in GenBank, and the names and homology percentages of the strains with the highest homology to each segment were recorded in Table 3 below.

[0124] Table 3. Homology analysis of C20 strain

[0125]

[0126] 8. Preservation of Microbial Materials

[0127] The virus strain isolated in the present invention was deposited with a patent procedure-approved depository institution. The depository is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The microbial accession number is CCTCC NO: V202487. The culture is named Peste des petits ruminants virus (PPRV / China / HLJ / 2020). The Chinese classification name is "Pesticide of Petits Ruminants Virus" and the English classification name is "Peste des petits ruminants virus." The deposit date is December 30, 2024, and the identified survival date is December 31, 2024.

[0128] Example 2: Preparation of DNA vaccine and immune performance testing

[0129] 1. Construction of recombinant plasmid

[0130] The H gene sequence of the PPRV / China2020 strain was optimized according to the sheep codon bias to obtain a codon-optimized H gene coding sequence. The H gene fragment was artificially synthesized, and the coding sequence is as follows (SEQ ID NO.18):

[0131]

[0132] The synthetic H gene fragment was cloned into the kanamycin-resistant pCAGGS vector using a homologous recombination kit (purchased from Nanjing Novozymes). (The construction method of the pCAGGS vector is clearly disclosed in the literature, Niwa H., Yamamura K., Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector [J]. Gene, 1991, 108: 193–199.)) Homologous recombination was performed according to the kit instructions. The recombinant vector was transformed into Escherichia coli DH5α strain, and plasmids were prepared in large quantities using plasmid Giga Kits (Qiagen). The extracted plasmid was sequenced by Sanger sequencing to verify the correct recombination. The recombinant plasmid was named pCAGGS-PPRVH. The F gene sequence of the PPRV / China2020 strain was optimized according to sheep codon bias to obtain a codon-optimized F gene coding sequence. The F gene fragment was synthesized, and the sequence is as follows (SEQ ID NO. 19):

[0133]

[0134] The artificially synthesized F gene fragment was cloned into the kanamycin-resistant pCAGGS vector using a homologous recombination kit (purchased from Nanjing Novozymes) and homologous recombination was performed according to the steps in the kit instructions. The recombinant vector was transformed into Escherichia coli DH5α strain, and plasmid Giga Kits (Qiagen) were used to prepare plasmids in large quantities. The plasmids were extracted and sequenced by Sanger method to verify the correct recombination. The recombinant plasmid was named pCAGGS-PPRVF.

[0135] 2. LNP Encapsulation of DNA Vaccines

[0136] SM-102 (cationic lipid, purchased from MedChemExpress, USA, 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octylnonyl ester, CAS No. 2089251-47-6), DSPC (neutral phosphatidylcholine, purchased from MedChemExpress, USA, distearoylphosphatidylcholine, CAS No. 816-94-4), cholesterol (purchased from MedChemExpress, USA) and DMG-PEG were prepared by ethanol. 2000 (polyethylene glycol derivative, purchased from MedChemExpress, USA, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol-2000, molecular weight 2526, CAS No. 160743-62-4), and then the lipid components were mixed according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000=50:10:38.5:1.5 to obtain a lipid mixture (prepared into an ethanol solution with a total concentration of four lipid materials of 13.6 mg / mL).

[0137] Plasmid pCAGGS-PPRVH or pCAGGS-PPRVF was dissolved in 100 mM citrate buffer (pH 4.0) to a concentration of 180 μg / ml. The lipid mixture and plasmid solution were mixed in a microfluidic device (purchased from Shanghai Myanna Instrument Technology Co., Ltd.) at a volume ratio of 1:3 and a nitrogen to phosphorus ratio of 1:6 to obtain a LNP solution. The ethanol and citrate buffer in the prepared LNP solution were replaced with an equal volume of Tris-NaAc buffer (pH 7.4) using a Milipore 100 kD ultrafiltration tube by centrifugation (3000 g). The solution was sterile filtered, aliquoted, and stored at -20°C.

[0138] The LNPs were taken out from -20 °C and the particle size and PDI of the LNPs were measured using a Nano ZS / ZS90 analyzer (Malvern). The encapsulation efficiency and concentration were measured using a Quant-iT RiboGreen DNA kit (Thermo Fisher Scientific, USA). After all indicators were qualified, they were used in downstream experiments.

[0139] The results showed that the encapsulation efficiencies of pCAGGS-PPRVH and pCAGGS-PPRVF were 94.3% and 95.2%, respectively. The LNP nanoparticle diameters of pCAGGS-PPRVH and pCAGGS-PPRVF were 106 nm and 105 nm, respectively, and the particle size distribution indexes (PDI) were 0.110 and 0.107, respectively.

[0140] 3. DNA vaccine immune efficacy determination

[0141] Immunization design and grouping were performed according to Table 4. The weight of the dose refers to the mass of the plasmid (not the mass of the composition). The delivery material was not represented by dissolving the plasmid in PBS. The delivery material LNP was the LNP used in step 2. The plasmids used were two different LNPs, and a mixture of two LNPs was used.

[0142] All immunization groups received two intramuscular immunizations, with a booster immunization 21 days after the first immunization. Blood was collected at 3, 5, and 13 weeks (500 μg group only) and 67 weeks (50 μg group only) after the first immunization, and serum was separated and tested for PPR neutralizing antibodies according to the method reported in reference 1 (Hu, Q., et al., Rescue of recombinant peste des petits ruminants virus: creation of a GFP-expressing virus and application in rapid virus neutralization test. VetRes, 2012. 43(1): p. 48.) (the same below).

[0143] Table 4. DNA vaccine immunization sheep experimental design and grouping

[0144]

[0145]

[0146] The statistical results of neutralizing antibodies in Experiment 1 are as follows Figure 8As shown in the figure, two weeks after the sheep were immunized twice with "H / LNP" (500 μg) and "H+F / LNP" (500 μg H LNP and 500 μg F LNP mixed together), the average PPRV neutralizing antibody levels were 10 3.8 and 10 3.3 The conversion rate was 100%, while the neutralizing antibody in the control group was only 10 1.4 The conversion rate was 75%, and the neutralizing antibody level in the control group was much lower than that in the LNP group. Ten weeks after the second vaccination (13 weeks after the first vaccination), the average PPRV neutralizing antibody level in the LNP "H / LNP" (500μg) and "H+F / LNP" (500μgH and 500μgF) groups decreased to 10 3.1 and 10 2.5 , the conversion rate remained 100%; however, the neutralizing antibody in the control group dropped below 10, and the conversion rate dropped to 0%.

[0147] The 500 μg dose trial (Experiment 1) demonstrated that the LNP and DNA combination induced highly potent neutralizing antibodies, significantly higher than the control group, suggesting potential for lowering the immunization dose to reduce production costs. Therefore, Experiment 2 (see Table 4 for immunization design and grouping) employed a 50 μg dose and supplemented the F gene alone immunization trial.

[0148] The statistical results of neutralizing antibodies in Experiment 2 are as follows Figure 9 As shown in Figure 2, 3 weeks after goats and sheep were immunized with "H / LNP" (50 μg) and "H+F / LNP" (50 μg H and 50 μg F), the average PPRV neutralizing antibody levels were higher than 10 1.9 The conversion rate was 100%, while the average neutralizing antibody in the F immunization group was only about 10 1.0 The conversion rate was 50%, which was much lower than that of the "H / LNP" and "H+F / LNP" groups. Since F was not the best choice, no booster immunization was performed for the F group. Two weeks after the booster immunization of "H / LNP" (50μg) and "H+F / LNP" (50μg H and 50μg F) in goats and sheep (5 weeks after the first immunization), the average neutralizing antibody level of PPRV was higher than 10 3.3 , the conversion rate was 100%. Further continuous monitoring of antibody levels in goats and sheep groups immunized with H alone showed that their antibodies could be maintained for at least about 67 weeks, and the neutralizing antibody positivity rate remained at 100%, demonstrating the good sustained immune efficacy of the vaccine.

[0149] Goats immunized with H alone and controls (CTL, unvaccinated goats) at 67 weeks post-immunization were subjected to a PPR virulent challenge test with the C20 strain. 1 mL of C20 was inoculated intranasally (in) and subcutaneously. The total challenge dose for each goat was 10 6.3 TCID50 . Within 21 days after inoculation (pi), clinical symptoms and body temperature were monitored every day. According to the severity of clinical symptoms, including behavior, anorexia, fever, eye and nasal secretions, salivation, acute respiratory symptoms and diarrhea, scores were given (Table 1). All dead animals were autopsied. The results showed that the control sheep showed typical clinical symptoms of PPR, including fever, depression, loss of appetite, mucus or purulent secretions from the eyes and nose, diarrhea, and some had symptoms of coughing and difficulty breathing; after autopsy, organs such as the lungs, mesenteric lymph nodes, and colon showed swelling, congestion, bleeding and other lesions, and the mortality rate reached 100% ( Figure 10 and Figure 11 The goats in the H / LNP immunization group showed no typical clinical symptoms of PPR and organ lesions, and the survival rate was 100% ( Figure 10 and Figure 11 ).

[0150] Example 3: Preparation of RNA vaccine and immune performance testing

[0151] 1. Construction of mRNA vaccine recombinant vector

[0152] According to the methods of literature 2 and 3 (Xia, X., Detailed Dissection and Critical Evaluation of the Pfizer / BioNTech and Moderna mRNA Vaccines. Vaccines (Basel), 2021.9 (7); Geng C, Zhou K, Yan Y, et al. A Preparation Method for mRNA-LNPs with Improved Properties [J]. Journal of Controlled Release, 2023, 364: 632-643.), the sequences of H and F optimized according to goat-biased codons (sequences see SEQ ID NO. 18 and SEQ ID NO. 19 in Example 2) were inserted between the 5'-UTR and 3'-UTR.

[0153] The 5'-UTR (from human α-globin), kozak sequence (GCCACC), optimized H gene sequence from PPRV C20 strain (sequence see SEQ ID NO.20 in Example 2) or optimized F gene sequence from PPRV C20 strain (sequence see SEQ ID NO.21 in Example 2), 3'-UTR (from human α-globin), and ployA (100 nt) sequence were artificially synthesized and cloned downstream of the T7 promoter of the pBlue vector to obtain plasmids pBm-PPRVH and pBm-PPRVF.

[0154] 5'-UTR (SEQ ID NO.20) sequence:

[0155] GCTAGCATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC

[0156] 3'-UTR (SEQ ID NO.21) sequence:

[0157] GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGT ACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA

[0158] ployA(100nt)(SEQ ID NO.22 sequence):

[0159] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0160] 2. Encapsulation of mRNA vaccines

[0161] The pBm-PPRVH and pBm-PPRVF plasmids were extracted using the Qiagen endotoxin-free plasmid extraction kit and transcribed using in vitro transcription kits (T7 High Yield RNA Transcription Kit, N'-Me-Pseudo UTP, Nanjing Novozymes Co., Ltd.). The in vitro transcripts were capped using a capping kit (mRNA Cap-2'-0-Methyltransferase, mRNACAP-2'-O-methyltransferase, Nanjing Novozymes Co., Ltd.). After capping using the capping kit, the capped mRNA was purified using an RNA purification kit (VAHTS RNA Clean Beads, Nanjing Novozymes Co., Ltd.).

[0162] Purified PPRVH or PPRVF capped mRNA was dissolved in 100 mM citrate buffer (pH 4.0) to an mRNA concentration of 150 μg / mL. The lipid components were mixed at a molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 50:10:38.5:1.5 to create a lipid mixture (prepared with a total concentration of 9.86 mg / mL of the four lipid materials in ethanol). The lipid mixture was mixed with the mRNA solution at a volume ratio of 1:3 and a nitrogen to phosphorus ratio of 1:6 in a microfluidic device (purchased from Shanghai Maianna Instrument Technology Co., Ltd.) to create a LNP solution. The ethanol and citrate buffer in the prepared LNP solution were replaced with an equal volume of Tris-NaAc buffer (pH 7.4) using Milipore 100 kD ultrafiltration tubes by centrifugation (3000 g). The resulting LNPs were sterile filtered, aliquoted, and stored at -20°C.

[0163] Aliquots of LNPs were removed from -20°C and measured for particle size and particle size distribution index (PDI) using a Nano ZS / ZS90 analyzer (Malvern). Encapsulation efficiency and concentration were determined using a Quant-iT RiboGreen RNA assay kit (Thermo Fisher Scientific, USA). Once qualified, the LNPs were used in downstream experiments. The results showed that the LNP encapsulation efficiency of PPRVH and PPRVF mRNA was 95.3% and 96.1%, respectively. The LNP nanoparticle diameters of PPRVH and PPRVF mRNA were 75 nm and 73 nm, respectively, with particle size distribution indexes (PDIs) of 0.08 and 0.09, respectively.

[0164] 3. PPRV mRNA vaccine can induce sufficient PPRV neutralizing antibodies

[0165] The encapsulated mRNA was injected intramuscularly into mice at a dose of 10 μg (mRNA weight) per mouse, and two immunizations were performed with an interval of 3 weeks. After immunization, blood was collected to detect PPRV neutralizing antibodies.

[0166] PPRV neutralizing antibody detection was performed on the mouse serum 14 days after booster immunization with LNP-encapsulated PPRV mRNA vaccine (H and F antigens). The results showed (Table 5) that 14 days after the second immunization, the PPRV neutralizing antibody levels of mice vaccinated with H or F antigen mRNA vaccine were all higher than 100, and 100% of the antibodies turned positive (≥10), while the PPRV neutralizing antibody levels of the PBS-injected control group were all negative.

[0167] Table 5. Neutralizing antibodies induced by PPRV mRNA vaccines

[0168]

[0169] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A peste des petits ruminants virus strain, wherein the peste des petits ruminants virus strain is selected from S1 and S2; S1: Peste des petits ruminants virus strain with microbiological accession number CCTCC NO: V202487; S2: passaged virus strain of peste des petits ruminants virus with microbiological deposit number CCTCC NO: V202487; During the passage process of the passaged virus strain of the peste des petits ruminants virus strain with the microbial preservation number CCTCC NO: V202487, the transcriptional regulatory activity of all genes in the genome, the transcription initiation and transcription termination of all genes in the genome, the amino acid sequence of the proteins encoded by all genes in the genome, the clinical pathogenicity of the virus, the immunogenicity of the virus and the viral reproduction ability did not change compared with the peste des petits ruminants virus strain with the microbial preservation number CCTCC NO: V202487.

2. The peste des petits ruminants virus strain according to claim 1, characterized in that In S1 and / or S2, the amino acid sequence of the protein encoded by the H gene of the Peste des petits ruminants virus strain is shown as SEQ ID NO.15; the amino acid sequence of the protein encoded by the F gene of the Peste des petits ruminants virus strain is shown as SEQ ID NO.

17.

3. A method for culturing PPR virus, comprising: inoculating the PPR virus strain according to claim 1 or 2 into PPR virus-susceptible cells, and performing a proliferation culture of the PPR virus strain to obtain a proliferated PPR virus strain.

4. The culture method according to claim 3, wherein The peste des petits ruminants virus susceptible cells are Vero cells or Vero cells expressing goat signaling lymphocyte activation molecule receptors.

5. A biomaterial, which is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P8 and P9; P1: Protein The protein is a first protein, a second protein, or a combination of the first protein and the second protein; The amino acid sequence of the first protein is shown in SEQ ID NO.15; The amino acid sequence of the second protein is shown in SEQ ID NO.17; P2: fusion protein The fusion protein is the first fusion protein, the second fusion protein, or a combination of the first fusion protein and the second fusion protein; The amino acid sequence of the first fusion protein contains the amino acid sequence of a tag peptide and / or a signal peptide used for separating and purifying the protein and the amino acid sequence shown in SEQ ID NO.15; The amino acid sequence of the second fusion protein contains the amino acid sequence of a tag peptide and / or a signal peptide used for separating and purifying the protein and the amino acid sequence shown in SEQ ID NO.17; P3: RNA The RNA is a first RNA, a second RNA, or a combination of the first RNA and the second RNA; The first RNA can be translated to produce the first protein described in P1 or the first fusion protein described in P2; The second RNA can be translated to produce the second protein described in P1 or the second fusion protein described in P2; P4: Gene The gene is the first gene, the second gene, or a combination of the first gene and the second gene; The coding sequence of the first gene is capable of encoding the first protein described in P1 or the first fusion protein described in P2; The coding sequence of the second gene is capable of encoding the second protein described in P1 or the second fusion protein described in P2; P5: Gene expression cassette The gene expression cassette is a first gene expression cassette, a second gene expression cassette, or a combination of the first gene expression cassette and the second gene expression cassette; The gene expression product in the first gene expression cassette is the first RNA described in P3; The gene expression product in the second gene expression cassette is the second RNA described in P3; P6: Genetic Engineering Vectors The genetic engineering vector is the first genetic engineering vector, the second genetic engineering vector, the third genetic engineering vector, or a combination of the first genetic engineering vector and the second genetic engineering vector; The first genetic engineering vector contains the first gene expression cassette described in P5; The second genetic engineering vector contains the second gene expression cassette described in P5; The third genetic engineering vector contains the first gene expression cassette and the second gene expression cassette described in P5; P7: Cells The cell is a first cell, a second cell, a third cell, or a combination of the first cell and the second cell; The first cell contains the first genetic engineering vector described in P6; The second cell contains the second genetic engineering vector described in P6; The third cell contains the third genetic engineering vector described in P6; The coding gene in the gene expression cassette of the first genetic engineering vector is constitutively expressed or artificially induced; The coding gene in the gene expression cassette of the second genetic engineering vector is constitutively expressed or artificially induced; The coding gene in the gene expression cassette of the third genetic engineering vector is constitutively expressed or artificially induced; P8: Composition The composition contains the protein described in P1, the fusion protein described in P2, the RNA described in P3, the genetic engineering vector described in P6 or the cell described in P7; and P9: Test kit The kit contains the protein described in P1, the fusion protein described in P2, the RNA described in P3, the genetic engineering vector described in P6 or the cells described in P7.

6. The biomaterial according to claim 4, selected from the following A1, A2, A3, A4, A5: A1: The promoter in the gene expression cassette is a T7 promoter; A2: The backbone vector of the genetic engineering vector is pBlue or pCAGGS; A3: The cells are Escherichia coli cells; A4: The coding sequence in the first RNA is shown as SEQ ID NO. 14 or SEQ ID NO. 18; A5: The coding sequence in the second RNA is shown as SEQ ID NO.16 or SEQ ID NO.

19.

7. Use of the peste des petits ruminants virus strain according to claim 1 or 2 or the biological material according to claim 5 or 6 in the preparation of a preparation for preventing, alleviating or controlling peste des petits ruminants.

8. The use according to claim 7, characterized in that The peste des petits ruminants is the peste des petits ruminants virus strain described in claim 1 or 2.

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