Passage attenuating method of bovine nodular skin disease virus and application of passage attenuating method

Abstract

The invention provides a bovine nodular dermatosis virus passage attenuating method and application thereof.The method comprises the steps that a bovine nodular dermatosis virus LSDV NM2020 strain serves as a parent strain, 25 generations of continuous passage is conducted through 9-day-old SPF chick embryo chorioallantoic membrane, 30 generations of continuous passage is conducted in Vero E6 cells in a gradual cooling mode, and finally the attenuated strain is obtained through plaque cloning and purification; the strain is named as an LSDV-Z strain. The low virulent strain shows good safety in cattle while keeping the growth titer close to that of a parent strain, does not cause clinical symptoms and does not have an external detoxification phenomenon. A virus attack protection test proves that the LSDV-Z strain can provide 100% immune protection for virulent virus attack. The method disclosed by the invention has the advantages of short attenuation period, high efficiency, no need of transgenic operation and the like, and the obtained LSDV-Z strain can be used as a safe and effective candidate vaccine strain and is used for preventing and controlling bovine nodular dermatosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal epidemic prevention, and particularly relates to a passage attenuation method of bovine lumpy skin disease virus and application thereof. BACKGROUND

[0002] Bovine lumpy skin disease (LSD) is an acute and severe infectious disease caused by bovine lumpy skin disease virus (LSDV), which is listed as a reportable animal disease by the World Organization for Animal Health (WOAH) and belongs to a class of animal diseases. The disease is characterized by fever, extensive nodules on the skin and mucosa, and superficial lymph node enlargement, often leading to decreased milk production, abortion, skin lesions, secondary infection, and even death, causing serious economic losses to the cattle industry. LSDV is mainly transmitted by arthropod vectors, and the epidemic spreads rapidly, making it difficult to control.

[0003] Currently, the international community mainly relies on vaccination to prevent and control LSD. Foreign countries have commercialized homologous live vaccines based on the Neethling attenuated strain. Although these vaccines have certain protective effects, the attenuation of the virus strain is often insufficient, and side effects such as local reactions at the injection site, fever, and temporary decrease in milk production may occur after immunization. Even a small number of cattle may exhibit mild "Neethling disease" symptoms, and the safety needs to be improved. Clinically, heterologous immunization with goatpox live vaccine is mainly used. However, although goatpox virus and LSDV belong to the same genus of goatpox virus, there are genetic differences, and the cross-protection effect is not complete. Long-term heterologous immunization may accelerate virus recombination and variation, increasing the complexity of prevention and control.

[0004] In terms of vaccine strain development technology, traditional attenuation methods rely on long-term continuous passage in a single host cell or animal, which is time-consuming and inefficient, with a long process (usually several years) and a high degree of randomness in the direction and degree of attenuation.

[0005] Therefore, in view of the current situation of insufficient safety of homologous LSD vaccines, limited protection of heterologous vaccines, long cycle of traditional attenuation technology, and slow approval of genetically engineered vaccines, it is urgent to develop a new passage attenuation method based on non-transgenic means to quickly obtain a safe and highly immunogenic LSDV attenuated strain, to meet the urgent needs of high-efficiency and safe LSD prevention and control products in China and around the world. SUMMARY

[0006] This invention provides a method for attenuating bovine nodular dermatitis virus through passage and its application, resulting in a safe, non-virulent, and 100% immune-protective attenuated live virus strain LSDV-Z, which can be used as a candidate vaccine strain for the prevention of bovine nodular dermatitis.

[0007] On the one hand, the present invention provides a passaged attenuated strain of bovine nodular dermatitis virus, employing the following technical solution: A passaged attenuated strain of bovine nodular dermatitis virus, named LSDV-Z. Its microbial preservation number is: CGMCC No. 46987; Name of the depository: China General Microbiological Culture Collection Center, China Committee on the Preservation of Microbial Cultures; Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing; Preservation period: December 23, 2025.

[0008] Preferably, the attenuated strain LSDV-Z has a 12-nucleotide deletion in the ORF148 gene.

[0009] Preferably, the ORF152 gene of the attenuated strain LSDV-Z has deletions at positions 146815-146858 bp, 146879-146893 bp, 148046-148086 bp, and 148107-148156 bp.

[0010] On the other hand, the present invention also provides a method for attenuating bovine nodular dermatitis virus through passage, employing the following technical solution: A method for attenuating bovine nodular dermatitis virus through passage includes the following steps: (1) The wild-type strain of bovine nodular dermatosis virus was continuously passaged on the chorioallantoic membrane of chicken embryos. (2) The virus obtained in step (1) was inoculated into Vero E6 cells and passaged continuously under the condition of gradually decreasing culture temperature; (3) The virus obtained in step (2) is purified by plaque cloning to obtain a strain with reduced virulence.

[0011] The preferred wild-type strain is LSDV NM2020.

[0012] Preferably, in step (1), the chicken embryo is a 9-day-old SPF chicken embryo; Subcultured at 35℃ for 5 days; The number of generations is 25.

[0013] Preferably, the conditions for gradually reducing the culture temperature in step (2) are: starting from 37℃, reducing by 1-1.5℃ with each generation, and finally reducing to 30℃.

[0014] The present invention also provides the use of the attenuated strain LSDV-Z in the preparation of drugs or vaccines for the prevention of bovine nodular skin disease.

[0015] In summary, the beneficial effects of the present invention are as follows: This invention provides a method for attenuating bovine nodular dermatitis virus strains through passage and its application. This method innovatively employs a strategy combining "chicken embryo passage" and "cell cryogenic adaptation passage," significantly shortening the attenuation period compared to traditional long-term passage methods using a single host. It also directionally induces attenuated mutations in the virus, enabling the rapid development of candidate vaccine strains against newly emerging strains and effectively addressing the control pressures brought about by viral mutations.

[0016] The attenuated live virus strain LSDV-Z, successfully obtained using this method, has been experimentally proven to possess excellent biological characteristics. While maintaining good proliferative capacity, this strain is highly safe for cattle, causing no clinical symptoms, fever, or viral shedding after inoculation. More importantly, it induces robust immune protection, providing 100% protection against virulent virus challenge in tests. Therefore, the LSDV-Z strain is a safe, effective, and highly promising candidate vaccine strain, offering a new solution for the prevention and control of bovine nodular skin disease. Attached Figure Description

[0017] Figure 1 Image showing lesions in the chorioallantoic membrane of a chicken embryo; Figure 2 The results are PCR identification of purified LSDV NM2020-E25-C30 plaques. Figure 3 CPE observation results for F1, F15, F30 and F55 generation viruses; Figure 4 This is a one-step growth curve of LSDV-Z strain; Figure 5 The viral load curve for LSDV-Z strain is shown. Figure 6 The image shows the body temperature measurement results of cattle in the safety test of LSDV-Z strain; Figure 7 Figure showing the inoculation site observation results of cattle in the safety test of LSDV-Z strain; Figure 8 A graph showing the body temperature observation results of cattle in the efficacy test; Figure 9 The image shows the results of the inoculation site observation in cow number 454 of the control group; Figure 10 The image shows the results of the inoculation site observation in cow number 464 of the control group; Figure 11 The image shows the results of the inoculation site observation for cow No. 458 in the immunization group; Figure 12 This is a diagram showing the results of the inoculation site observation for cow No. 481 in the immunization group. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the embodiments.

[0019] Example Example 1

[0020] The optimization of chicken embryo and cell passage conditions for bovine nodular dermatitis virus was carried out through the following steps: (1) Determination of the optimal inoculation age of LSDV NM2020 strain for SPF chicken embryos Healthy SPF chicken embryos at 7, 9, and 11 days of normal development were collected. The embryos were held upright (air cell facing upwards). A small hole was made in the center of the eggshell in the air cell (the hole must pierce both the shell and shell membrane). The embryo was then laid flat with the front side (where the chorioallantoic membrane vessels are well-developed and relatively thick) facing upwards. The hole was made again, avoiding the blood vessels, breaking only the shell and shell membrane without damaging the chorioallantoic membrane. Air was drawn from the original air cell using a bulb syringe to create negative pressure. This caused the chorioallantoic membrane on the front side of the embryo to separate from the shell membrane and sink, forming an artificial air cell. A diluted LSDV NM2020 strain virus suspension was sprayed onto the chorioallantoic membrane of the artificial air cell, 0.2 mL per embryo. After inoculation, the holes in both the original and artificial air cells were sealed sequentially with white glue. Chicken embryos were placed flat at 37℃ for further incubation and observed for 5 days. The embryos were candled once a day, and dead embryos were discarded. Then, the chicken embryos were dissected under sterile conditions to observe their chorioallantoic membranes. The diseased chorioallantoic membranes were added to PBS containing 300 U / ml penicillin and 0.3 mg / ml streptomycin at a volume ratio of 1:3. The mixture was then ground into a tissue homogenate in a tissue homogenizer, centrifuged at 3000 r / min for 10 min, and the supernatant was collected for virus content detection.

[0021] Viral content was determined using the real-time quantitative RT-PCR method (LightCycler® 480Ⅱ) previously developed in our laboratory, as shown in Table 1. The primers were LSDV-qPCR-F / LSDV-qPCR-R, and the probe was LSDV-qPCR-P. The 5' end of the probe was labeled with FAM, and the 3' end with MGB. The target fragment length was 192 bp. The reaction mixture consisted of 10 μL of 2×Premix Ex Taq enzyme, 2 μL of DNA template, 1.2 μL each of forward and reverse primers (10 μmol / L), 0.8 μL of probe (10 μmol / L), and 4.8 μL of PCR-grade deionized water, totaling 20 μL. The reaction conditions were set as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ extension for 30 s, for a total of 40 cycles.

[0022] Table 1 Primer sequences used in this invention

[0023] The chorioallantoic membrane lesions and Ct values ​​of chicken embryos are shown in Table 2 below. The virus content of LSDV NM2020 strain was the highest after inoculation into 9-day-old SPF chicken embryos, with a Ct value of 27.81±1.39. Therefore, the optimal inoculation age of LSDV NM2020 strain into SPF chicken embryos was determined to be 9 days.

[0024] Table 2. Virus content detection results of SPF chicken embryos of different ages after inoculation with LSDV NM2020 strain.

[0025] (2) Determination of the minimum culture temperature for inoculating SPF chicken embryos with LSDV NM2020 strain To determine the minimum culture temperature for LSDV NM2020 strain after inoculation with SPF chicken embryos, 15 9-day-old SPF chicken embryos were inoculated with LSDV NM2020 strain virus solution via the chorioallantoic membrane and cultured at 33℃, 35℃, and 37℃, with 5 embryos at each temperature. The embryos were observed for 5 days post-inoculation, with candling performed daily. Dead embryos were discarded, and the embryos were then dissected under aseptic conditions to observe the chorioallantoic membrane. Diseased chorioallantoic membranes were added to PBS containing 3% antibiotics at a volume ratio of 1:3 and homogenized in a tissue homogenizer. The homogenate was centrifuged at 3000 r / min for 10 min, and the supernatant was used for virus content detection. The Ct values ​​of the chorioallantoic membranes harvested at 33℃, 35℃, and 37℃ were 34.36±3.87, 28.32±2.47, and 27.62±1.76, respectively. This indicates that culture at 33℃ is not conducive to the proliferation of LSDV NM2020 strain, while the virus content detected at 35℃ is only slightly lower than that at 37℃. Therefore, the minimum culture temperature for inoculating LSDV NM2020 strain into SPF chicken embryos was determined to be 35℃.

[0026] (3) Determination of the optimal time for virus harvesting from SPF chicken embryos inoculated with LSDV NM2020 strain To determine the optimal time for harvesting LSDV NM2020 strain after inoculation with SPF chicken embryos, 15 9-day-old SPF chicken embryos were inoculated with LSDV NM2020 strain virus via chorioallantoic membrane. Five embryos were harvested at each time point: 5 days, 7 days, and 9 days post-inoculation. The diseased chorioallantoic membranes were then homogenized in PBS containing 300 U / ml penicillin and 0.3 mg / mL streptomycin at a volume ratio of 1:3 using a tissue homogenizer. The homogenate was centrifuged at 3000 r / min for 10 min, and the supernatant was used for virus content analysis.

[0027] The chorioallantoic membrane lesions and Ct value measurements of chicken embryos are shown in Table 3 below. The highest virus content of LSDV NM2020 strain was observed 5 days after inoculation of 9-day-old SPF chicken embryos, with a Ct value of 29.84±3.62. Therefore, the optimal time for harvesting LSDV NM2020 strain after inoculation of 9-day-old SPF chicken embryos was determined to be 5 days.

[0028] Table 3. Virus content detection results at different time points after 9-day-old SPF chicken embryos were inoculated with LSDV NM2020 strain.

[0029] (4) Determination of the optimal passage of LSDV NM2020 strain To determine the optimal passaged cells for LSDV NM2020 strain, MDBK cells, BHK-21 cells, Vero cells, and Vero E6 cells with well-developed monolayers were inoculated with LSDV NM2020 strain. The cells were harvested 5 days after inoculation, frozen 3 times, centrifuged at 3000 r / min for 10 min, and the supernatant was collected for virus content detection.

[0030] The Ct values ​​after inoculation of different cells are shown in Table 4 below. The viral load of LSDV NM2020 strain was high in both Vero and Vero E6 cells. Considering that CPE was more obvious after inoculation in Vero E6 cells, Vero E6 cells were determined to be the best passaged cell for LSDV NM2020 strain.

[0031] Table 4. Results of LSDV NM2020 strain virus content detection after inoculation into different generations of cells.

[0032] Example 2 The virulent strain LSDV NM2020 of bovine nodular dermatitis virus was passaged continuously, and the specific steps are as follows: (1) LSDV NM2020 strain was continuously passaged on chicken embryos for 25 generations. Chicken embryos were inoculated with the chorioallantoic membrane using the method described in Example 1. After inoculation, the embryos were placed flat and incubated at 35°C for 5 days. Candling was performed once daily, and dead embryos were discarded. The embryos were then dissected under aseptic conditions to observe the chorioallantoic membrane. Diseased chorioallantoic membranes were added to PBS containing 300 U / ml penicillin and 0.3 mg / ml streptomycin at a volume ratio of 1:3. The mixture was homogenized in a tissue homogenizer, centrifuged at 3000 r / min for 10 min, and the supernatant was aliquoted. (Refer to...) Figure 1 The image shows the diseased chorioallantoic membrane.

[0033] The chorioallantoic membranes from healthy 9-day-old SPF chicken embryos with normal development were re-inoculated, and passaged continuously for 25 generations using the same method. The 25th generation chorioallantoic membranes with lesions were added to PBS solution containing 300 U / mL penicillin and 0.3 mg / mL streptomycin at a volume ratio of 1:3, and homogenized in a tissue homogenizer. The homogenate was centrifuged at 3000 r / min for 10 min, and the supernatant was aliquoted and named LSDV NM2020-E25.

[0034] (2) LSDV NM2020-E25 was continuously passaged in Vero E6 cells for 30 generations. LSDV NM2020-E25 virus homogenate was inoculated into Vero E6 cells that had grown into a well-developed monolayer. The cell culture incubator temperature was adjusted to 35℃, and the virus was harvested after 3-5 days of culture. The cells were then subjected to three freeze-thaw cycles for further passage. When the virus adapted to this temperature and could induce cytopathic effects in over 80% of the cells within approximately 96 hours, the incubator temperature was lowered by 1-2℃. This process was repeated, gradually lowering the culture temperature to 30℃. This method was used for 30 passages. After 3-5 days of culture, the 30th passage cell virus was subjected to three freeze-thaw cycles, centrifuged at 3000 rpm for 10 minutes, and the supernatant was aliquoted and named LSDV NM2020-E25-C30.

[0035] (3) Plaque cloning purification of LSDV NM2020-E25-C30 was carried out. The specific method was as follows: the harvested virus solution was diluted 1:100 and 1 mL of virus solution was inoculated into each well of a 6-well cell culture plate into Vero E6 cells. After incubation at 30℃ for 1 h, the virus solution was aspirated and culture medium containing 1% low melting point agarose was spread on the plate. When obvious plaques appeared at about 96 h, a single plaque was picked, and the viral genome was extracted. The virus was amplified using a pair of specific primers LSDV-F / R with the viral genome as a template. The result was that a gene fragment of about 376 bp was successfully amplified. The purified plaque virus was inoculated into Vero E6 cells for proliferation culture. When more than 90% of the cells showed cytopathic effects, the cytotoxicity was harvested, frozen and thawed 3 times, centrifuged at 3000 r / min for 10 min, the supernatant was collected, aliquoted and frozen at -80℃, and named LSDV strain Z.

[0036] Reference Figure 2 The image shows the agarose gel electrophoresis results of the amplified fragments. M represents the DL2000 DNA Marker; 1 represents the 25th generation of LSDV NM2020 chicken embryos; 2 represents the negative control; and 3 represents the positive control.

[0037] Example 3

[0038] The biological characteristics of LSDV strain Z were studied using the following steps: (1) Genome analysis of LSDV Z strain The complete genome sequences of LSDV strain Z and its parental wild-type strain LSDV NM2020 were determined using high-throughput sequencing (next-generation sequencing). The results showed that the LSDV Z genome was approximately 151,000 bp in length, essentially the same as the parental wild-type strain LSDVNM2020. Compared to the parental strain, the LSDV Z genome did not exhibit large-scale deletions or rearrangements, and its overall genome composition remained highly stable. This indicates that alternating passages and low-temperature adaptation did not cause structural damage to the viral genome, but rather induced mainly point mutations and small fragment deletions.

[0039] Mutation analysis: Specific mutations were found in several genes related to virulence and immune regulation in the LSDV Z strain. Compared with the parental strain LSDV NM2020, the LSDV Z strain exhibited a single base deletion in its ORF148 gene (encoding a protein belonging to the Ankyrin repeat superfamily and related to viral virulence), with a deletion of 12 nucleotides, potentially leading to reduced virulence. The LSDV Z strain's ORF152 gene (encoding a protein belonging to the Ankyrin repeat superfamily and related to viral virulence) showed four significant deletion mutations in its coding region, located at 146815-146858 bp (45 bp), 146879-146893 bp (15 bp), 148046-148086 bp (41 bp), and 148107-148156 bp (50 bp). This resulted in the mutated ORF152 gene exhibiting significantly lower virulence compared to the LSDV strain. The NM2020 strain contains a 151-nucleotide deletion in the corresponding gene, representing a significant structural truncation mutation. This mutation may severely alter the protein's three-dimensional conformation and the integrity of its functional regions, causing the protein to lose or significantly weaken its ability to participate in host immune regulation. This gene deletion may significantly reduce viral pathogenicity without significantly affecting viral replication in cell culture systems.

[0040] Table 5 Deletion gene sequences

[0041] In addition, compared with the parental strain LSDV NM2020, LSDV Z strain has five point mutations. Among them, the G→A mutation at 56014 bp causes a non-synonymous substitution, changing the amino acid from Cys to Tyr. There are also four ambiguous base mutations: A→R (A / G) at 7901 bp, A→R at 77745 bp, G→K (G / T) at 111086 bp, and G→R (G / A) at 146445 bp. Due to the uncertain base composition, the corresponding amino acid changes cannot be uniquely determined. These ambiguous sites suggest the possible presence of nucleotide polymorphisms or sequencing signal contamination in the sample, and their impact on protein translation and function is still difficult to accurately determine.

[0042] (2) Cytopathic effect (CPE) of LSDV Z strain The F1, F15, F30 and F55 generations of viruses from the passage process were taken and inoculated into primary sheep testicular cells that had grown into a good monolayer with the same viral content (MOI=1.0). The cells were observed under a microscope on days 1 to 5 after inoculation, and the pathological changes of the cells were recorded.

[0043] Experimental results are as follows Figure 3As shown, the parental virus strain caused milder cytopathic effects, slower cell morphological changes, and significant cell aggregation. In contrast, the passaged attenuated virus strain exhibited increasingly pronounced CPE effects with each passage, suggesting that as the number of passages increases, the strain becomes more adapted to cell culture conditions and may be more suitable as a vaccine candidate strain.

[0044] (3) Determination of one-step growth curve of LSDV Z strain The proliferation characteristics of the parental strain and the attenuated LSDV Z strain were compared by measuring viral proliferation in cells. In this invention, the virus was inoculated into PLT cells at an MOI of 1.0, and samples were collected at multiple time points (24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours) for virus titer determination. Virus titer was detected after three freeze-thaw cycles, and a one-step growth curve of the virus was plotted.

[0045] The results are as follows Figure 4 As shown, the one-step growth curves of the parent strain and the LSDV Z strain exhibit similar trends, with no significant difference in viral load at the highest point, indicating that viral attenuation through passage does not have a significant negative impact on viral replication.

[0046] (4) Determination of viral load curve of LSDV strain Z Whole genome extraction was performed on viral fluids from different infection time points. The viral load at different time points was determined using a real-time quantitative RT-PCR method previously developed in our laboratory. Primers were LSDV-qPCR-F / LSDV-qPCR-R, and the probe was LSDV-qPCR-P. The 5' end of the probe was labeled with FAM, and the 3' end with MGB. The target fragment length was 192 bp. The reaction system consisted of 10 μL of 2×Premix Ex Taq enzyme, 2 μL of DNA template, 1.2 μL each of forward and reverse primers (10 μmol / L), 0.8 μL of probe (10 μmol / L), and 4.8 μL of PCR-grade deionized water, totaling 20 μL. The reaction conditions were set as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ extension for 30 s, for a total of 40 cycles. Viral load curves were plotted based on the established standard curve and calculation method. The results are shown below. Figure 5 As shown, there was no significant difference in the growth trends between the parental virus strain and the LSDV Z strain.

[0047] Example 4

[0048] The safety test of the passaged attenuated strain LSDV Z in cattle was conducted using the following steps: (1) Four healthy, susceptible LSD-negative cattle aged 6-12 months were selected and randomly divided into two groups: an LSDV Z strain immunization group and a control group, with two cattle in each group. The two cattle in the immunization group were numbered 454 and 464, respectively. 0.5 mL of virus solution (virus content of 105.2 TCID 50 / mL) was inoculated intradermally at two points on the hairless area inside the tail of the immunized cattle. The two cattle in the control group, numbered 457 and 481, were inoculated intradermally at two points on the tail with 0.5 mL of DMEM / F12 medium.

[0049] (2) After immunization, the cattle in the immunized group and the control group were observed for 21 consecutive days. The mental state, drinking water and feed intake of the experimental cattle were observed daily. The injection site was observed daily for symptoms such as swelling, ulceration, necrosis and nodules. Oral swabs, nasal swabs and anal swabs of the experimental cattle were collected daily for LSDV virus detection. The body temperature of the experimental cattle was measured daily (the average body temperature of the 3 days before immunization was used as the baseline body temperature). Serum and whole blood samples of the experimental cattle were collected before immunization and on the 14th and 21st day after immunization.

[0050] The results are as follows Figure 6 As shown, none of the two experimental cattle immunized with LSDV strain Z and the two control cattle exhibited obvious fever symptoms during the 21-day observation period after immunization, and their body temperatures remained within the normal range. Figure 7 As shown, no obvious swelling, ulceration, necrosis, or nodules were observed at the inoculation site or on the skin throughout the body, and no viral shedding was detected in oral, nasal, or anal swabs. This indicates that the LSDV Z strain was sufficiently attenuated and has good safety for the experimental cattle.

[0051] Example 5

[0052] The potency test of the passaged attenuated strain LSDV Z followed these steps: (1) After the above safety test, the hair of four experimental cattle in the immunization group and the control group was shaved in four random areas on the abdomen and the inside of the tail and wiped with alcohol. Four intradermal points on the abdomen and two intradermal points on the tail were inoculated with 0.5 mL of virulent strain virus solution (virus content of 105.6 TCID / mL) at each point, and 2 mL of virulent strain virus solution was injected into the jugular vein at the same time.

[0053] (2) After challenge, cattle in the immunized group and the control group were observed for 21 consecutive days. The mental state, drinking water, and feed intake of the experimental cattle were observed daily. The inoculation site was observed daily for symptoms such as swelling, ulceration, necrosis, and nodules. The observations were taken and recorded, and the clinical symptoms were systematically scored. Oral swabs, nasal swabs, and anal swabs of the experimental cattle were collected daily for LSDV virus detection, and the body temperature of the experimental cattle was measured daily. Serum and whole blood samples were collected from the experimental cattle before challenge and on days 3, 5, 7, 14, and 21 after challenge. On day 21 after challenge, all experimental cattle were anesthetized, bled to death, and dissected to observe whether the skin and internal organs showed symptoms such as swelling, ulceration, necrosis, and nodules.

[0054] The results are as follows Figure 8 As shown, during the 21-day observation period after challenge, both experimental cattle in the control group exhibited significant body temperature increases from day 1 to day 3 after challenge, reaching a maximum of 41.5℃, and their body temperature gradually returned to normal after 5 days of challenge; Figures 9-12 As shown, both experimental cattle exhibited typical symptoms of bovine nodular dermatitis, with obvious, hard nodules at the injection site, reaching up to approximately 9 cm in diameter, accompanied by redness, swelling, and ulceration. Simultaneously, numerous secondary nodules were observed on the skin around the thighs and anus. Virus shedding was detected in oral, nasal, and anal swabs starting on day 5 post-challenge. In contrast, both experimental cattle in the immunization group had normal body temperatures and did not exhibit obvious fever symptoms; no swelling, ulceration, necrosis, or nodules were observed at the injection site or on the skin throughout the body; and no virus shedding was detected in oral, nasal, or anal swabs.

[0055] The above results indicate that the LSDV Z strain has good immunogenicity and can protect experimental cattle 100% against the attack of virulent strains of bovine nodular dermatitis virus, and has the potential to become a candidate strain for an excellent bovine nodular dermatitis vaccine.

[0056] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A passaged, attenuated, strain of bovine dermatophilus virus, which is characterized in that, The attenuated strain of the passage-attenuated bovine dermal nodular disease virus is named LSDV-Z, and the microbial preservation number of the attenuated strain LSDV-Z is CGMCC NO. 46987.

2. A passaged, attenuated, strain of bovine dermatophilus virus according to claim 1, characterised in that, The ORF148 gene of the attenuated strain LSDV-Z is deleted by 12 nucleotides.

3. A passaged attenuated strain of bovine dermatophilus virus according to claim 1, characterised in that, The ORF152 gene of the attenuated strain LSDV-Z is deleted at positions 146815-146858 bp, 146879-146893 bp, 148046-148086 bp and 148107-148156 bp.

4. A method for the passage attenuation of a bovine nodular dermatopathy virus according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) continuously passing a wild strain of bovine dermal nodular disease virus on a chicken embryo chorioallantoic membrane; (2) inoculating the virus obtained in step (1) into Vero E6 cells and continuously passing under the condition of gradually reducing the culture temperature; (3) carrying out plaque clone purification on the virus obtained in step (2) to obtain a virulence-reduced strain.

5. The method of passage-attenuation of bovine papular dermatosis virus according to claim 4, characterized in that, The wild strain is LSDV NM2020 strain.

6. The method of passage-attenuation of bovine papular dermatosis virus according to claim 4, characterized in that, In step (1), the chicken embryo is a 9-day-old SPF chicken embryo. The passage is cultured at 35℃ for 5 days. The continuous passage is 25 passages.

7. The method of passage-attenuation of bovine papular dermatosis virus according to claim 4, characterized in that, In step (2), the condition of gradually reducing the culture temperature is that the culture temperature is reduced by 1-1.5℃ per passage from 37℃ to 30℃.

8. Use of the attenuated strain of the passage-attenuated bovine dermal nodular disease virus according to any one of claims 1-3 in the preparation of a medicine or vaccine for preventing bovine dermal nodular disease.

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