Bovine infectious rhinotracheitis virus HeNan 01 strain and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a bovine infectious rhinotracheitis virus HeNan01 strain and application thereof. The bovine infectious rhinotracheitis virus HeNan01 strain has a preservation number of CCTCC NO:V2024110, and is a bovine infectious rhinotracheitis virus BHV-1.2 subtype strain and a currently popular dominant strain. After an animal is immunized by using a vaccine composition prepared from the HeNan01 strain, the animal body can rapidly produce a humoral immune response and high-level antibodies, and has a good prevention and control effect on current popular bovine infectious rhinotracheitis virus infection.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to a bovine infectious rhinotracheitis virus strain HeNan01 and its applications. Background Technology

[0002] Infectious Bovine Rhinotracheitis Virus (IBRV), also known as Bovine Herpesvirus 1 (BoHV-1), belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, and genus Varicellovirus. This virus is a significant pathogen in cattle, causing a series of disease syndromes. It is an acute, contagious disease characterized by fever, difficulty breathing, nasal redness and discharge, respiratory mucosal edema, hemorrhage, necrosis, and erosion. The World Organisation for Animal Health classifies it as a Category B infectious disease, posing a significant threat and economic loss to global livestock farming.

[0003] For the prevention and control of IBRV, vaccination is a good option. In previous studies, the dominant genotype of IBRV infecting beef cattle was BHV-1.1, while new research reports that the prevalent IBRV strain in my country is the BHV-1.2 subtype.

[0004] Therefore, it is urgent to isolate the prevalent strains of bovine infectious rhinotracheitis virus (IBRV) in my country to lay the foundation for precise prevention and control. Thus, screening for highly immunogenic IBRV strains from prevalent strains and developing vaccines to prevent the spread and outbreak of the disease, and to reduce the economic losses it causes to the cattle industry, is of great significance.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] To address the problems existing in current IBRV prevention, this application aims to provide a Bovine Infectious Rhinotracheitis Virus strain HeNan01 and its application, which can effectively prevent Bovine Infectious Rhinotracheitis Virus infection.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] A strain of infectious bovine rhinotracheitis virus, HeNan01, was deposited at the China Center for Type Culture Collection (CCTCC) on November 13, 2024, with accession number CCTCCNO:V2024110, at Wuhan University, Wuhan, China.

[0009] Application of HeNan01 strain in the preparation of a vaccine to prevent bovine infectious rhinotracheitis virus disease.

[0010] Furthermore, the vaccine is a whole-virus inactivated vaccine, a subunit vaccine, a live attenuated vaccine, a genetically engineered vaccine, or a DNA vaccine.

[0011] Application of HeNan01 strain in the preparation of diagnostic reagents for bovine infectious rhinotracheitis.

[0012] A vaccine composition for the prevention of bovine infectious rhinotracheitis virus disease, the vaccine composition comprising an immunizing dose of bovine infectious rhinotracheitis virus antigen and a pharmaceutically acceptable vector, wherein the bovine infectious rhinotracheitis virus antigen comprises an inactivated antigen of the bovine infectious rhinotracheitis virus HeNan01 strain or a culture thereof of the present application.

[0013] Furthermore, the bovine infectious rhinotracheitis virus HeNan01 strain was ≥10 μmol / L before inactivation. 7.0 TCID 50 / ml, preferably 10 7.0 ~10 8.0 TCID 50 / ml, more preferably 10 7.5 TCID 50 / ml.

[0014] Furthermore, the pharmaceutically acceptable carrier includes at least one of adjuvants, lyophilization protectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

[0015] Preferably, the adjuvant comprises one or more of the following: aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206, Montanide ISA 201, or gel adjuvant.

[0016] Preferably, the adjuvant is a biphasic adjuvant used to prepare an oil-in-water emulsion.

[0017] Furthermore, the concentration range of the adjuvant is from 5 wt% to 50 wt%, preferably from 30 wt% to 50 wt%, more preferably 50 wt%.

[0018] Furthermore, the freeze-drying protectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

[0019] Furthermore, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-2.

[0020] Compared with existing technologies, the HeNan01 strain isolated in this application has the following characteristics:

[0021] (1) The HeNan01 strain is a subtype of bovine infectious rhinotracheitis virus BHV-1.2, which is currently the dominant circulating strain.

[0022] (2) The gC protein of the HeNan01 strain has unique amino acid site mutations in domain 2 and domain 4: 124P-G, 125R-G, 130S-R, and 278Q-R, for a total of 4 mutations.

[0023] (3) The vaccine composition prepared with HeNan01 strain can prevent the spread of bovine infectious rhinotracheitis virus. Furthermore, after immunizing animals with the vaccine composition containing this strain, the animals can quickly produce a humoral immune response and generate high levels of antibodies. It has a good preventive and control effect on the currently prevalent bovine infectious rhinotracheitis virus infection and has good biosafety. Attached Figure Description

[0024] The various technical features of this application and their relationships will be further explained below with reference to the accompanying drawings. The drawings are exemplary; some technical features are not shown to scale, and some drawings may omit technical features commonly used in the art to which this application pertains that are not essential for understanding and implementing this application, or additionally show technical features that are not essential for understanding and implementing this application. In other words, the combination of various technical features shown in the drawings is not intended to limit this application. Furthermore, throughout this application, the same reference numerals refer to the same things. Specific descriptions of the drawings are as follows:

[0025] Figure 1 This is a diagram of the antigen detection of different generations of bovine infectious rhinotracheitis virus HeNan01 strain by qPCR in 2.2 of Example 2;

[0026] Figure 2This is a negative-stained electron microscope image of the HeNan01 strain of bovine infectious rhinotracheitis virus in Example 2, Section 2.3;

[0027] Figure 3 This is a nucleotide homology matrix of the gC gene of the bovine infectious rhinotracheitis virus HeNan01 strain in Example 2, section 2.4.

[0028] Figure 4 This is a phylogenetic tree analysis of the gC gene of the bovine infectious rhinotracheitis virus strain HeNan01 in section 2.4 of Example 2. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0030] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0031] In this application, "preferred," "preferred," and "ideal" refer to options that are optional, meaning they are selected from either "with" or "without." If multiple "preferred" options appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferred" option is independent.

[0032] The term "infectious bovine rbinotracheitis virus" (IBRV) belongs to the Herpesviridae family, Alphaherpesvirinae subfamily. It contains 138 kb of linear double-stranded DNA, and its genome encodes at least 70 proteins, including 11 structural proteins that are glycoproteins. The gC glycoprotein plays an important role in viral adsorption, entry, and intercellular diffusion. Furthermore, the gC protein is one of the main antigenic proteins that stimulate the host's immune response, stimulating the body to produce neutralizing antibodies and causing lysis of infected cells in the presence of complement.

[0033] Unless otherwise specified in this application, the term "infectious bovine rhinotracheitis virus (IBRV)" refers to wild-type IBRV, virulent IBRV, and wild-type IBRV, and these terms can be used interchangeably.

[0034] This application describes the independent isolation of a bovine infectious rhinotracheitis virus strain HeNan01, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:V2024110, located at Wuhan University, Wuhan, China, on November 13, 2024.

[0035] Experimental results show that this strain possesses strong toxicity and good immunogenicity. Based on this, it can be used to prepare a vaccine for the prevention of bovine infectious rhinotracheitis virus disease, and also to prepare diagnostic reagents for bovine infectious rhinotracheitis.

[0036] In some implementations, the vaccine is a whole-virus inactivated vaccine, a subunit vaccine, a live attenuated vaccine, a genetically engineered vaccine, or a DNA vaccine.

[0037] This application also provides a vaccine composition comprising an immunizing dose of bovine infectious rhinotracheitis virus antigen and a pharmaceutically acceptable vector, wherein the bovine infectious rhinotracheitis virus antigen comprises an inactivated antigen of the bovine infectious rhinotracheitis virus HeNan01 strain or a culture thereof as described in this application. The vaccine composition is prepared from a virulent strain, exhibits good immunogenicity, and provides complete protection to cattle.

[0038] The term "vaccine composition," also known as an immunogenic composition, refers to an immunogenic preparation, including whole cells, inactivated or attenuated, live viruses or bacteria, or polysaccharides, or combinations thereof, which are administered to stimulate a humoral and cellular immune response in a receptor to one or more antigens present in the immunogenic composition. Immunization is the process of administering a vaccine composition and stimulating an immune or immunogenic response to an antigen in a host, preferably an animal such as a cow.

[0039] The term "immune dose" should be understood as "immunely effective dose," also known as immune protective dose or effective dose to elicit an immune response. It refers to the amount of antigen that can effectively induce an immune response in a recipient, sufficient to prevent or improve the signs or symptoms of disease, including adverse health effects or complications thereof. This immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications of infection caused by a pathogen. Humoral immunity or cell-mediated immunity, or both, can be induced. An animal's immune response to an immunogenic composition can be indirectly assessed, for example, by measuring antibody titers, lymphocyte proliferation analysis, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain. The protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs in the subject such as mortality, reduction in morbidity, temperature values, overall physiological status, and overall health and performance. The immune response may include, but is not limited to, the induction of cellular and / or humoral immunity.

[0040] The term "bovine infectious rhinotracheitis virus antigen" refers to any composition containing at least one form of bovine infectious rhinotracheitis virus antigen that can induce, stimulate, or resist an immune response to bovine infectious rhinotracheitis virus infection. The antigen form includes, but is not limited to, inactivated, attenuated, or subunit antigens. Inactivated antigens can be inactivated and maintain their immunogenicity by various methods, including chemical treatment, physical treatment (such as acoustic treatment, irradiation, heat), or any other commonly used method sufficient to inhibit the replication or growth of the organism. Preferably, the pathogen is inactivated after collection and optionally subjected to clarification and purification. Chemical treatment uses, for example, formalin or formaldehyde, β-propiolactone, ethyleneimine, diethyleneimine BEI, thimerosal, etc. Inactivation methods are well known to those skilled in the art, such as by β-propiolactone (Plana-Duran et al., Vet. Microbiol., 1997, 55:361-370) or by BEI treatment (US5587164).

[0041] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent in the vaccine composition of this application that does not irritate the body or impede the biological activity and properties of the compound, except for the bovine infectious rhinotracheitis virus antigen.

[0042] In some embodiments, pharmaceutically acceptable carriers include at least one of adjuvants, lyophilization protectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

[0043] The term "adjuvant" may include aluminum gel adjuvants; saponins, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), and GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; and compounds selected from copolymers of maleic anhydride and alkenyl derivatives.

[0044] The term "emulsion" may be particularly based on light liquid paraffin oils (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oils, especially isobutylene or decanene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / decanoate), glyceryl tri-(octanoate / decanoate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, especially esters of sorbitan, esters of mannitol (such as anhydrous mannitol oleate), esters of aliphatic glycols, esters of polyglycerol, esters of propylene glycol, and esters of oleic acid, isostearic acid, castor oil, or hydroxystearic acid. These may be ethoxylated, as well as polyoxypropylene-polyoxyethylene block copolymers, especially Pluronic products, particularly L121. See Hunter et al., *The theory and practical application of adjuvants* (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and Todd et al., *Vaccine* (1997, 15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of "Vaccine design, the Subunit and adiuvant approach" (Plenum Press, 1995) by Powell M and Newman M can be used.

[0045] The term "polymer of acrylic acid or methacrylic acid" preferably refers to crosslinked acrylic acid or methacrylic acid polymers, especially those crosslinked with polyolefin ethers or polyols of sugar, compounds known as Carbomer (trade name Carbopol) (Phameuropa, 1996, 8(2)). Those skilled in the art may also refer to U.S. Patent US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three hydroxyl groups, preferably no more than eight, wherein the hydrogen atoms of at least three hydroxyl groups are replaced by unsaturated aliphatic radicals having at least two carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl, and other ethylenically unsaturated groups. These unsaturated groups may themselves contain other substituents, such as methyl groups. These products are sold under the name Carbopol (BF Goodrich, Ohio, USA), which is particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, Capop 974P, 934P and 971P can be mentioned, with Capop 971P being the preferred choice.

[0046] The term "copolymer of maleic anhydride and alkenyl derivatives" may also refer to the copolymer of maleic anhydride and ethylene, EMA (Monsanto), which dissolves in water to produce an acidic solution, which is then neutralized, preferably to physiological pH, to produce an adjuvant solution into which immunogenic, immunizing, or vaccine compositions can be incorporated.

[0047] The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin (recombinant or other), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, etc.

[0048] In a preferred embodiment, the adjuvant includes one or more of the following: mineral oil, aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin of Escherichia coli, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206, Montanide ISA 201, or gel adjuvant.

[0049] Preferably, the adjuvant is a biphasic adjuvant used to prepare an oil-in-water emulsion.

[0050] In some embodiments, the concentration of the adjuvant ranges from 5 wt% to 50 wt%, preferably from 30 wt% to 50 wt%, and more preferably 50 wt%.

[0051] The concentration range of the adjuvant may be, but is not limited to, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt%.

[0052] The term "lyophilization protectant" refers to a component, other than excipients, that protects the efficacy of a drug's active ingredient during the freeze-drying process and subsequent storage. Lyophilization protectants can be selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

[0053] In some embodiments, the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-2.

[0054] In a preferred embodiment of this application, the inactivated antigen content of the bovine infectious rhinotracheitis virus HeNan01 strain or its culture in the vaccine composition described in this application is ≥10% before inactivation. 7.0 TCID 50 / ml.

[0055] As a more preferred embodiment of this application, in the vaccine composition described in this application, the inactivated antigen content of the bovine infectious rhinotracheitis virus HeNan01 strain or its culture is 10% of the inactivated antigen. 7.0 -10 8.0 TCID 50 / ml.

[0056] In a preferred embodiment of this application, the vaccine composition described herein contains an inactivated antigen content of the bovine infectious rhinotracheitis virus HeNan01 strain or its culture of 10-1. 7.5 TCID 50 / ml.

[0057] The term "prevention" in relation to bovine infectious rhinotracheitis virus (BVR) infection refers to inhibiting the replication of BVR, inhibiting the spread of BVR, preventing BVR from settling in its host, and alleviating the symptoms of disease or symptom of BVR infection.

[0058] This application also relates to the use of the described vaccine composition in the preparation of a medicament for the prevention of diseases related to bovine infectious rhinotracheitis virus infection.

[0059] The present application is further described below with reference to specific embodiments. The advantages and features of the present application will become clear from the description. The embodiments described are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0060] All chemical reagents used in the embodiments of this application are of analytical grade.

[0061] To make this application easier to understand, specific embodiments are described below to further illustrate this application. Unless otherwise specified, the experimental methods described in this application are conventional methods; and unless otherwise specified, the biological materials described are all commercially available.

[0062] Example 1: Virus Isolation

[0063] 1.1 Sample Information

[0064] In a cattle farm in Henan Province, a sick cow developed a fever that rose above 40°C for two consecutive days, along with viscous purulent nasal discharge, coughing, and difficulty breathing, which were suspected symptoms of IBRV. Nasal swab samples were collected.

[0065] 1.2 Virus Isolation

[0066] Nasal swabs were placed in sterilized EP tubes, 1 ml of PBS solution was added, and the mixture was thoroughly mixed. The mixture was then subjected to three freeze-thaw cycles, centrifuged at 5000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm filter for sterilization. The sterilized supernatant was aliquoted, and after passing sterility testing, it was stored for later use. The filtered supernatant was diluted 5-fold and 10-fold using sterile DMEM medium and inoculated into T25 cell culture flasks containing monolayers of MDBK cells, 1 mL / flask. The flasks were incubated at 37°C with 5% CO2 for 1 h for adsorption, then discarded, and 5 mL of 2% cell maintenance medium was added. A negative cell control was also included. Cells were observed daily; cytopathic effects were observed within 48 h, and approximately 85% cytopathic effect was observed after 67 h of culture. The cell flasks were then frozen at -80°C, and the culture medium was harvested. The harvested cell-virus solution was subjected to three freeze-thaw cycles and then passaged into MDBK cells for three consecutive generations, all of which showed significant and stable cytopathic effects.

[0067] Example 2: Identification of isolates

[0068] 2.1 Test for the purity of the virus strain

[0069] The obtained isolated viral strains were subjected to exogenous pathogen detection. The isolated viral strains were passaged three times in MDBK cells, and the purity of the isolated viral strains was detected by PCR, primarily for mycoplasma and bovine viral diarrhea virus (BVM). The results showed that neither mycoplasma nor BVM was detected in the viral fluid of the isolated viral strains.

[0070] 2.2 PCR Amplification and Identification

[0071] The isolated viral strain was passaged continuously in MDBK cells to the F3 generation. During this process, 200 μl of cell culture from each generation was collected, and viral nucleic acid was extracted using a viral nucleic acid extraction kit. IBRV primers and probes were synthesized according to GB / T 27981-2011 "Real-time Fluorescent PCR Detection Method for Bovine Infectious Rhinotracheitis Virus," and were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer and probe sequences are as follows:

[0072] Upstream primer: TGTGGACCTAAACCTCACGGT;

[0073] Downstream primer: GTAGTCGAGCAGACCCGTGTC;

[0074] Probe primers: FAM-AGGACCGCGAGTTCTTGCCGCBHQ-1.

[0075] The reaction system consisted of: 12.5 μl of qPCR reaction solution, 0.5 μl of 10 μM / ml forward and reverse primers, 1 μl of probe primers, 2 μl of DNA, and deionized water to a final volume of 25 μl. The mixture was thoroughly mixed. The PCR reaction conditions were as follows: 95℃ for 90 seconds, 95℃ for 10 seconds, 60℃ (fluorescence acquisition) for 30 seconds, for 40 cycles. The results are as follows: Figure 1 As shown, IBRV was detected in cell culture samples from all three generations of the isolated strain. This indicates that the isolated strain is bovine infectious rhinotracheitis virus.

[0076] 2.3 Negative staining electron microscopy observation of the virus

[0077] Take 20 μl of IBRV-positive virus solution, add an appropriate amount to the copper grid of a Formav membrane, and incubate for 5 min. Absorb the residual liquid with filter paper. After negative staining with 2% phosphotungstic acid, observe the morphology of the virus particles using a transmission electron microscope. The results show that the virus particles are spherical, enveloped, and approximately 170 nm in diameter. Figure 2 ).

[0078] 2.4 Sequence analysis of the main functional gC gene of the virus

[0079] A pair of primers was designed based on the gC gene of IBRV (accession number MK654724.1) published in GenBank. The primer sequences are as follows:

[0080] Upstream primer gC-F: ACCACGAAAGCACAAAACGGAC;

[0081] Downstream primer gC-R: GCGACGACTACGAAAACTACGA.

[0082] The reaction mixture consisted of: 25 μl of 2×PCR buffer for KOD FX, 10 μl of 2 mM dNTPs, 1.5 μl of upstream primer, 1.5 μl of downstream primer, 1 μl of KOD FX, 1 μl of template, and deionized water to a final volume of 50 μl. The mixture was thoroughly mixed. The PCR conditions were: 94℃ for 2 minutes, 98℃ for 10 seconds, 68℃ for 2 minutes, 30 cycles, followed by a final cycle of 25℃ for 5 minutes.

[0083] Sequencing results showed that the amino acid sequence encoded by the gC gene of this isolated strain is SEQ ID NO: 1, and the specific sequence is as follows:

[0084] MGPLGRAWLIAAIFAWALLSARRGLAEEAEASPSPPPSP SPTETESSAGTTGATPPTPNSPDATPEDSTPGATTPGGTPGPPSVSEHDPPVTNSTPPPAPPEDGRPGGAGNASRDGRPSGGGRGGPPRPRKAPPKERKWMLCEREAVAASYAEPLYV HCGVADNATGGARLELWFQRVGRFRSTRGDDEAVRNPFPRAPPVLLFAAQNGSVAYRSAELGGNYIFPSPADPRNLPLTVRSLTAATEGVYTWRRDMGAKSQRKVVTVTTHRAPAVSV EPRPALEGAGYAAVCRAAEYYPPRSTRLRWFRNGYPVEARHARDVFTVDDSGLFSRTSVLTLEDATPTAHPPNLRCDVSWFQSANMERRFYAAGTPAVYRPPELRVYFEGGEAVCEAR CVPEGRVSLRWTVRDGIAPSRTEQTGVCAERPGLVNLRGVRLLSTTDGPVDYTCTATGYPAPLPEFSATATYDASPGLIGSPVLVSVVAVACGLGAVGLLLVAASCLRRKARARL(SEQ ID NO: 1).

[0085] Further homology comparisons were performed between SEQ ID NO: 1 and the gC gene sequences of other bovine infectious rhinotracheitis strains in MegAlign, and the results are shown below. Figure 3 .

[0086] Based on IBRV antigenicity and genomic analysis, it can be divided into two subtypes: BHV-1.1 and BHV-1.2. Recent research reports indicate that the BHV-1.2 subtype is the currently circulating strain in my country. Figure 4 It can be seen that this isolate belongs to BHV-1.2. This indicates that the isolated strain in this application is a circulating strain of bovine infectious rhinotracheitis virus (BHV) type BHV-1.2. Sequence alignment results show that the gC protein sequence of this isolate shares approximately 97.8% homology with that of the BHV-1.1 strain, indicating a significant genetic distance; the gC protein sequence of this isolate shares 98.2% homology with the K22 strain published by NCBI.

[0087] Protein sequence alignment results showed that, compared with the BHV-1.2 strain, the IBRV isolate exhibited the following mutation in domain 2 of the gC protein: at 124P→ G, 125R→G, 130S→R; The following mutation occurs in domain 4: 278Q→R, for a total of 4 mutations. Domain 2 of the gC protein constitutes a highly antigenic region, possibly an immunodominant region, because most specific epitopes between residues 22-150 exhibit significant hydrophilicity and extensive glycan modification, thereby regulating biological activity. Mutations at positions 124, 125, and 130 may affect viral immunogenicity. The heparin-binding domain of IBRV gC is located within domain 4, and viral adsorption to cells requires synergistic interaction with cellular heparin-like receptors; therefore, the mutation at position 278 may affect heparin binding, thereby affecting viral adsorption to cells.

[0088] 2.5 Virus content detection

[0089] A suspension of well-grown MDBK cells was seeded onto 96-well cell culture plates. When the cells reached a monolayer, the IBRV isolate was serially diluted 10-fold with cell maintenance medium containing 2% serum, with each dilution seeded into 8 wells. Negative and positive control wells were also included. The plates were incubated at 37°C in a 5% CO2 incubator, and cytopathic effects were observed daily. After 3-5 days of continuous culture, viral infection was observed, and viral titers were calculated. The number of wells showing CPE was counted, and the TCID of the cytotoxicity was calculated using the Reed-Muench method. 50 The titer of this bovine infectious rhinotracheitis virus strain was 10. 8.71 TCID 50 / ml. This indicates that the bovine infectious rhinotracheitis virus strain can be stably passaged in MDBK cells and a high titer of virus solution can be obtained, making it a candidate strain for bovine infectious rhinotracheitis virus vaccine. The bovine infectious rhinotracheitis virus strain was named Bovine Infectious Rhinotracheitis Virus HeNan01 strain and has been deposited.

[0090] Example 3: Pathogenicity study of bovine infectious rhinotracheitis virus strain HeNan01

[0091] Ten healthy 5-month-old IBRV antigen-antibody negative calves were randomly divided into two groups of 5. Group 1 was inoculated with bovine infectious rhinotracheitis virus strain HeNan01. 7.0 TCID 50 / head; Group 2 was the blank control group, inoculated with an equal volume of DMEM culture medium. Clinical symptoms and body temperature changes were observed daily for 14 days. No clinical symptoms were observed in the blank control group (Group 2); 5 cattle in Group 1 developed varying degrees of fever after inoculation, with persistent high temperature for more than 3 days, nasal redness, tearing, purulent eye discharge, and decreased appetite. Results are shown in Table 1.

[0092] Table 1. Pathogenicity test results of bovine infectious rhinotracheitis virus strain HeNan01. Group Animal numbers Inoculation dosage Number of cases 1 5 2ml / head 5 / 5 2 5 2ml culture medium / head 0 / 5

[0093] After cattle in the challenge group developed clinical symptoms, nasal swabs were collected, viral nucleic acid was extracted, and RT-PCR was performed. The virus was then isolated, inoculated into MDBK cells, and cytopathic effects were observed.

[0094] RT-PCR analysis of nasal swab samples showed a positive result for IBRV. Simultaneous inoculation with MDBK cells resulted in the same cytopathic effect. This indicates that the virus causing fever, nasal congestion, tearing, purulent eye discharge, and loss of appetite in cattle in this experiment was indeed bovine infectious rhinotracheitis virus (BRV). The isolate HeNan01 described in this application is a virulent strain of BRV.

[0095] Example 4: Suspension culture of bovine infectious rhinotracheitis virus HeNan01 strain cells

[0096] MDBK cells (bovine kidney cells) in the logarithmic growth phase were harvested and stored at an initial cell density of 1.0 × 10⁻⁶ cells / year. 6 ~1.1×10 6 MDBK cells were seeded at a density of 1.8 × 10⁶ cells / ml into a 3L reactor. 6 ~2.0×10 6 At a cell density of 1,000 cells / ml, inoculate with IBRV strain F5 seed at an MOI of 0.01. Set the temperature to 37°C and the pH to 7.0–7.2. Set the dissolved oxygen (DO) value to 40%–60%, with DO control correlated to agitation speed and bottom aeration rate. Maintain DO at the set values ​​through agitation and aeration. Agitation speed of 50–60 rpm and aeration rate of 0.05–0.2 vvm were used. Harvest the cell culture after 120 hours of culture.

[0097] Example 5: Preparation of inactivated vaccine of bovine infectious rhinotracheitis virus HeNan01 strain

[0098] 5.1 Preparation of Viral Fluid

[0099] The viral supernatant from Example 4 was used as the antigen for the vaccine.

[0100] 5.2 Preparation of inactivated virus vaccines

[0101] Add a filtered, sterile 0.1 mol / L BEI solution to the virus supernatant to a final concentration of 0.003 mol / L, stirring continuously. Start timing when the temperature reaches 30°C and maintain this temperature for 28 hours. After 28 hours of inactivation treatment, add a filtered, sterile 1 mol / L sodium thiosulfate solution to the inactivated virus solution, ensuring the final volume of sodium thiosulfate is 10% of the volume of the added BEI solution. Mix thoroughly and store at 2–8°C. Simultaneously, take samples for inactivation testing.

[0102] Take the inactivated virus solution and inoculate it into one flask (25cm) of MDBK cells at a volume ratio of 1 / 10. 2 / bottle), after adsorption at 37℃ for 1 hour, discard the culture medium, add DMEM maintenance medium, and simultaneously set up one bottle of normal cell control. Culture at 37℃ for 2-4 days; no cytopathic effect should appear. Harvest the culture, freeze-thaw it, and then sample and inoculate it into MDBK cells for two blind passages; no cytopathic effect should appear in either case, indicating that the tested inactivated virus solution is completely inactivated. Finally, slowly add the inactivated IBRV antigen to the adjuvant under low-speed conditions, mix well, and stir at 250 rpm for 10 minutes. The adjuvant suitable for this application can be any adjuvant known to those skilled in the art. In this application, a biphasic adjuvant (water-in-oil-in-water emulsion) is selected, such as Montanide ISA 206 adjuvant. Specific ratios are shown in Table 2. After emulsification, the vaccine is tested for dosage form as follows: take a clean pipette, draw a small amount of vaccine, and drop it onto a clean, cold water surface; if it diffuses in a cloud-like manner, the vaccine dosage form is qualified. After emulsification, the vaccine was tested for stability. The procedure was as follows: 10 ml of vaccine was added to a centrifuge tube and centrifuged at 3000 r / min for 15 minutes. No stratification or emulsion breaking was observed, and the vaccine stability was qualified.

[0103] Table 2. Ratio of Bovine Infectious Rhinotracheitis Virus HeNan01 Strain Inactivated Vaccine

[0104] Example 6: Safety test of inactivated vaccine against bovine infectious rhinotracheitis virus HeNan01 strain

[0105] The inactivated bovine infectious rhinotracheitis virus strain HeNan01 vaccine prepared in Example 5 was subjected to laboratory tests for sterility, stability, and morphology, in accordance with the current Chinese Veterinary Pharmacopoeia. All tests were qualified.

[0106] 6.1 Safety Experiment in Calves

[0107] Fifteen 5-month-old calves that were negative for IBRV antigen and antibody were selected for safety testing. Each calf received a double dose (4 ml / calves) of immunization via neck muscle. A control group of three calves received no treatment. The results are shown in Table 3.

[0108] Table 3: Safety Test Results of Overdose for Bovine Infectious Rhinotracheitis Virus HeNan01 Strain Inactivated Vaccine (Calves)

[0109] After calves in each immunization group were vaccinated with the inactivated bovine infectious rhinotracheitis virus HeNan01 strain vaccine prepared in this invention, they were observed for 14 consecutive days. Their body temperature, mental state, and appetite were normal, and no IBRV-related clinical symptoms were observed, indicating that the vaccine is safe for calves.

[0110] 6.2 Safety trials of fattening cattle

[0111] Fifteen 2-year-old fattening cattle that were negative for IBRV antigen and antibody were selected for safety testing. The fattening cattle were immunized with a double dose of 4 ml / head via neck muscle immunization. A control group of 3 cattle received no treatment. The experimental results are shown in Table 4.

[0112] Table 4: Safety Test Results of Overdose of Bovine Infectious Rhinotracheitis Virus HeNan01 Strain Inactivated Vaccine (Fattening Cattle)

[0113] Five fattening cattle in each immunization group were vaccinated with the inactivated bovine infectious rhinotracheitis virus HeNan01 strain vaccine prepared according to this invention. After 14 consecutive days of observation, their body temperature, mental state, and appetite were normal, and no IBRV-related clinical symptoms were observed, indicating that the vaccine is safe for fattening cattle.

[0114] Example 7: Efficacy test of inactivated bovine infectious rhinotracheitis virus strain HeNan01 vaccine

[0115] 7.1 Test of the immunogenicity of the inactivated bovine infectious rhinotracheitis virus strain HeNan01 vaccine in guinea pigs

[0116] Forty healthy guinea pigs weighing 350-400g were selected for this experiment. The guinea pigs were randomly divided into four groups of ten each. Group 1 was immunized with vaccine 1, Group 2 with vaccine 2, and Group 3 with vaccine 3, with each guinea pig receiving an intramuscular injection of 1ml of PBS in the leg. Group 4 was the blank control group, receiving an equal volume of sterile PBS intramuscularly in the leg. A second immunization was administered 21 days after the first immunization. Post-immunization, inflammation at the injection site and clinical symptoms of the guinea pigs were observed. Starting two weeks after the first immunization, blood samples were collected weekly to detect the serum IBRV neutralizing antibody titer. Results showed that two weeks after the second immunization, the serum IBRV neutralizing antibody level in all guinea pigs in the immunized groups was not lower than 1:45, and the IBRV neutralizing antibody level reached its peak two weeks after the second immunization, with a titer ranging from 1:50.12 to 1:177.8. The serum IBRV neutralizing antibody level in the control group remained at 0.

[0117] Immunization experiments showed that the HeNan01 strain of bovine infectious rhinotracheitis virus (BVRV) effectively stimulated a strong humoral immune response and can be used as a candidate strain for vaccine development to prevent BVRV infection. See Table 5 for details.

[0118] Table 5 Results of antibody tests for guinea pig immunogenicity

[0119] 7.2 Testing the immunogenicity of the inactivated bovine infectious rhinotracheitis virus HeNan01 strain vaccine in calves

[0120] Twenty healthy 5-month-old IBRV antigen- and antibody-negative calves were selected for this experiment. The calves were randomly divided into four groups of five each. Group 1 was immunized with vaccine 1, Group 2 with vaccine 2, and Group 3 with vaccine 3, receiving 2 ml / calves via intramuscular injection in the neck. Group 4 served as a blank control group, receiving the same volume of sterile PBS via intramuscular injection in the neck. A second immunization was administered 21 days after the first immunization. Post-immunization, inflammation at the injection site and clinical symptoms of the calves were observed. Blood samples were collected two weeks after the second immunization to detect the serum IBRV neutralizing antibody titer. Results showed that the IBRV neutralizing antibody titers in the immunized groups two weeks after the second immunization were all above 1:64, while the serum neutralizing antibody titers in the control group were all below 1:2. See Table 6 for details.

[0121] Table 6 Results of antibody tests for calf immunization efficacy

[0122] Two weeks after the second immunization, all calves were challenged with the virus at a dose of 10 μL of the HeNan01 strain of bovine infectious rhinotracheitis virus. 7.0 TCID 50 / head, clinical symptoms and body temperature changes were observed daily after challenge for 14 days. 5 / 5 of the immunized group were protected, while 5 / 5 of the challenged control group developed the disease. The results of the immunized group's protection are shown in Table 7.

[0123] Table 7 Results of virus challenge protection in calves after immunization.

[0124] The results showed that at the challenge dose, none of the five cattle in each of the vaccine immunization groups (Vaccine 1, Vaccine 2, and Vaccine 3) exhibited clinical symptoms, and all three vaccines achieved a 100% protection rate. In contrast, the five cattle in the control group developed varying degrees of fever after challenge, with persistent high temperature lasting more than three days, nasal congestion, tearing, purulent eye discharge, and decreased appetite. The 100% protection rate of vaccines 1, 2, and 3 in cattle indicates that the inactivated vaccines prepared using this strain have a good immunoprotective effect against bovine infectious rhinotracheitis virus infection.

[0125] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

[0126] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. A strain of bovine infectious rhinotracheitis virus, HeNan01, characterized in that, It was deposited on November 13, 2024 at the China Center for Type Culture Collection, with accession number CCTCCNO:V2024110, and the deposit address is Wuhan University, Wuhan, China.

2. The use of the HeNan01 strain according to claim 1 in the preparation of a vaccine for the prevention of bovine infectious rhinotracheitis virus disease.

3. The application according to claim 2, characterized in that, The vaccine may be a whole-virus inactivated vaccine, a subunit vaccine, a live attenuated vaccine, a genetically engineered vaccine, or a DNA vaccine.

4. The use of the HeNan01 strain according to claim 1 in the preparation of diagnostic reagents for bovine infectious rhinotracheitis.

5. A vaccine composition for the prevention of bovine infectious rhinotracheitis virus disease, characterized in that, The vaccine composition comprises an immunizing dose of bovine infectious rhinotracheitis virus antigen and a pharmaceutically acceptable carrier, wherein the bovine infectious rhinotracheitis virus antigen comprises an inactivated antigen of the bovine infectious rhinotracheitis virus HeNan01 strain or a culture thereof as described in claim 1.

6. The vaccine composition according to claim 5, characterized in that, The bovine infectious rhinotracheitis virus HeNan01 strain was ≥10 before inactivation. 7.0 TCID 50 / ml, preferably 10 7.0 ~10 8.0 TCID 50 / ml, more preferably 10 7.5 TCID 50 / ml.

7. The vaccine composition according to claim 5 or 6, characterized in that, The pharmaceutically acceptable carriers include at least one of the following: adjuvants, lyophilization protectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. Preferably, the adjuvant comprises one or more of the following: aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, MontanideISA 206, MontanideISA 201, or Gel adjuvant. Preferably, the adjuvant is a biphasic adjuvant used to prepare an oil-in-water emulsion.

8. The vaccine composition according to claim 7, characterized in that, The concentration range of the adjuvant is from 5 wt% to 50 wt%, preferably from 30 wt% to 50 wt%, more preferably 50 wt%.

9. The vaccine composition according to claim 7, characterized in that, The freeze-drying protectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

10. The vaccine composition according to claim 7, characterized in that, The immunostimulants include α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-2.

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