Novel bovine nodular skin disease virus LSDV-125 strain and application thereof in vaccine preparation

Abstract

The invention provides a novel bovine nodular skin disease virus LSDV-125 strain and application thereof in vaccine preparation, and belongs to the technical field of virology. The problem that a novel vaccine strain which is clear in genetic background, stably weakened in virulence and capable of inducing broad-spectrum immune response is urgently needed is solved. The invention relates to a sheep pox virus strain, in particular to a sheep pox virus LSDV-125 which is named sheep pox virus LSDV-HLJ-2023, has the preservation number of CCTCC (China Center for Type Culture Collection) NO: V202537, and is preserved in China Center for Type Culture Collection on May 13, 2025. The method is mainly used for developing efficient and safe attenuated live vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of virology, and in particular relates to a novel bovine lumpy skin disease virus LSDV-125 strain and application thereof in vaccine preparation. Background Art

[0002] Bovine lumpy skin disease (LSD) is an acute, febrile, contagious disease caused by LSDV (poxvirus) in the poxvirus family, primarily affecting cattle. The disease spreads rapidly, causing significant economic losses. Existing live vaccines primarily include attenuated Neethling strain vaccines, but these have the following drawbacks: 1. High risk of reversion to virulence: Some vaccine strains may revert to virulence in the body, posing a safety risk; 2. Doubts about cross-protection against emerging variants: Due to the highly conserved LSDV genome, the whole genome homology between published strains is greater than 99%, making it difficult for existing vaccines to effectively cover newly emerging variants; 3. Lack of new strain resources with significant genetic differences: Lack of low-virulence, representative new LSDV strains for new vaccine development; 4. Complex genome structure: The LSDV genome consists of variable regions at both ends and a conserved region in the middle. The ends contain inverted repeat sequences, and related virulence genes and immune regulatory genes are mostly distributed in the variable regions, which further increases the complexity of strain screening.

[0003] Therefore, there is an urgent need for a new LSDV strain with a clear genetic background, stably reduced virulence, and good immunogenicity for the development of an efficient and safe live attenuated vaccine. Summary of the Invention

[0004] In view of this, the present invention aims to propose a new bovine lumpy skin disease virus LSDV-125 strain and its application in vaccine preparation, so as to solve the problem of the urgent need for a new vaccine strain with a clear genetic background, stable and weakened virulence and the ability to induce a broad-spectrum immune response.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a bovine lumpy skin disease virus strain (Lumpy skin disease virus) LSDV-125, which is named as capripox virus LSDV-HLJ-2023, with a preservation number of CCTCC NO: V202537, and is deposited in the China Center for Type Culture Collection on May 13, 2025.

[0006] It is further defined that the bovine lumpy skin disease virus strain LSDV-125 is a natural recombinant between the African Neethling strain (KX764644.1) and the Morocco strain (MW631933.1).

[0007] The present invention provides an attenuated bovine lumpy skin disease virus vaccine, which comprises the bovine lumpy skin disease virus strain LSDV-125.

[0008] It is further defined that the vaccine further comprises a pharmaceutically acceptable excipient.

[0009] It is further defined that the vaccine is in the form of an injection.

[0010] It is further defined that the content of bovine lumpy skin disease virus is 10 4 TCID 50 -10 5 TCID 50 .

[0011] The present invention provides an application of the bovine lumpy skin disease virus LSDV-125 in preparing a vaccine for preventing bovine lumpy skin disease.

[0012] It is further defined that the content of bovine lumpy skin disease virus is 10 4 TCID 50 -10 5 TCID 50 .

[0013] The present invention provides an application of the bovine lumpy skin disease virus LSDV-125 in preparing a vaccine for treating bovine lumpy skin disease.

[0014] It is further defined that the content of bovine lumpy skin disease virus is 10 4 TCID 50 -10 5 TCID 50 .

[0015] Compared with the prior art, the present invention has the following advantages: high doses of and The LSDV-125 strain showed no adverse clinical manifestations such as high fever (body temperature < 40°C) and systemic nodular reaction after vaccination, indicating that the strain is safe. 4 TCID 50 / head and 10 5 TCID 50 / head inoculated, the experimental cattle did not show clinical symptoms, such as high fever >40℃ and no systemic nodules, indicating that the LSDV-125 strain is fully attenuated.

[0016] [Biological Deposit Information]: A virulent strain of bovine lumpy skin disease virus (Lumpy skin disease virus), named sheep pox virus LSDV-HLJ-2023, with the deposit number CCTCC NO: V202537, deposited in the China Center for Type Culture Collection on May 13, 2025, at Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings: Figure 1 Vaccinate cattle for 10 4 TCID 50 Figure 2 shows the results of body temperature changes after LSDV-125 strain; Figure 2 Cattle vaccination 10 5 TCID 50 Figure 2 shows the results of body temperature changes after LSDV-125 strain; Figure 3 Vaccinate cattle for 10 4 TCID 50 Results of neutralizing antibody production after infection with LSDV-125 strain; Figure 4 Vaccinate cattle for 10 5 TCID 50 Results of neutralizing antibody production after infection with LSDV-125 strain; Figure 5 Vaccinate cattle for 10 5 TCID 50 Results of IL-6 production after infection with LSDV-125 strain; Figure 6 The attack dose is 10 3 TCID 50 Results of post-neutralizing antibody production; Figure 7 The attack dose is 10 6.5 TCID 50 The result diagram of the body temperature change of the cow; Figure 8 The attack dose is 10 7.5 TCID 50 The result diagram of the body temperature change of the cow; Figure 9 The attack dose is 10 8.5 TCID 50 The result diagram of the body temperature change of the cow; Figure 10 The attack dose is 10 6.5 TCID 50 The results of the changes in the detoxification of the cows are shown in the figure; Figure 11 The attack dose is 10 7.5 TCID 50 The results of the changes in the detoxification of the cows are shown in the figure; Figure 12 The attack dose is 10 8.5 TCID 50 The results of the changes in the detoxification of the cows are shown in the figure; Figure 13 For Attack 10 8.5 TCID 50 The LSDV antibody results were then obtained using the IPMA detection method. DETAILED DESCRIPTION

[0018] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely explain the technical solutions in the embodiments of the present invention. It should be noted that the embodiments of the present invention and the features therein can be combined with each other in the absence of conflict, and the embodiments described are only part of the embodiments of the present invention, not all of the embodiments.

[0019] LSDV-XJ is recorded in the article [Zhang Minmin, Sun Yajie, Liu Wenxing, et al. The first isolation and identification of bovine lumpy skin disease virus in my country [J]. Chinese Journal of Preventive Veterinary Medicine, 2020(010):042.] and is recorded in the article as LSDV / China / Xinjiang / 2019 strain (Xinjiang / 2019).

[0020] LSDV-75 is disclosed in patent document application number 202510065202.2.

[0021] Example 1. Obtaining bovine lumpy skin disease virus LSDV-125 strain 1. Serial subculture of the LSDV-XJ virus strain on Vero cells: Specifically, the LSDV-XJ virus strain was inoculated into Vero cells at a volume ratio of 1:100. Five days after inoculation, the cell culture was frozen and thawed three times, and then fresh Vero cells were inoculated at a volume ratio of 1:100. At passage 25, the virus was plaque-purified and the size of the plaques formed was observed. Continued subculture was performed at passages 50 and 75, and plaque purification was performed again. Continued subculture was performed at passage 125, and plaque purification was performed again. If multiple small plaques were observed, these small plaques were picked and propagated. Plaque purification was continued until small plaques were consistently formed. The resulting virus was then propagated to obtain a weakened bovine lumpy skin disease virus strain, LSDV-125, designated capripox virus LSDV-125, and stored frozen in aliquots at -70°C. Also named sheeppox virus LSDV-HLJ-2023, the deposit number is CCTCC NO: V202537, deposited in the China Center for Type Culture Collection, the deposit date is May 13, 2025, and the deposit address is Wuhan University, Wuhan, China.

[0022] 2. Genomic Analysis Whole genome sequence: The LSDV-125 strain genome is approximately 151,000 bp in length.

[0023] Recombination analysis: Recombination analysis using RDP and SimPlot software revealed that the strain was a natural recombinant between the African Neethling strain (KX764644.1) and the Morocco strain (MW631933.1). The recombinant regions were primarily located in the LSDV007–LSDV010 gene region, corresponding to nucleotide positions 4428–7708 bp and 134903–139888 bp.

[0024] Mutation analysis: Specific mutations were found in multiple genes related to virulence and immune regulation in the LSDV-125 strain, especially in the inverted repeat region. In addition: (1) Results The protein encoded by the ORF005 gene did not terminate at amino acid 108, encoding 63 more amino acids than the strain LSDV-75; (2) Results Compared with the LSDV-75 strain, the ORF055 gene showed premature termination, encoding 7 fewer amino acids, which would lead to a weakening of virulence.

[0025] Example 2. Safety and immunogenicity studies The experimental group was mainly divided into two groups, each group had 4 LSDV serum negative black and white dairy cows, which were injected with LSDV-125 Group, injected with LSDV-125 group; cattle were inoculated with LSDV-125 strain; the control group was the PBS group, cattle were inoculated with the same dose of PBS.

[0026] 1. Safety Assessment High doses of and The LSDV-125 strain, the results are as follows Figure 1 and 2 As shown in the figure, there were no adverse clinical manifestations such as high fever (body temperature < 40°C) and systemic nodular reactions after vaccination, indicating that the strain is safe. and After vaccination, the experimental cattle showed no clinical symptoms, such as high fever >40°C and no systemic nodules, indicating that the LSDV-125 strain was fully attenuated.

[0027] 2. Immunogenicity Assessment Vaccination 10 4 TCID 50 or 10 5 TCID 50 The results of the 4 experimental cows were LSDV-125, the control group was PBS, and the control group was LSDV-75. Figure 3-5 As shown, LSDV neutralizing antibodies can be produced on the 10th day. The neutralizing antibody level detected by serum neutralization test method reaches a peak value of 1:75 at the 4th week of vaccination. At the same time, the changes in immune factors in the serum were detected, and the results showed that the level of inflammatory factor IL-6 was significantly lower than that of the parent strain LSDV-75 control group. These data indicate that the LSDV-125 strain produces a lower inflammatory response than its parent strain LSDV-75. 4 TCID 50 or 10 5 TCID 50 LSDV-125 can cause cattle to produce high levels of neutralizing antibodies.

[0028] Example 3. Evaluation of protective power LSDV seronegative cattle were used for the challenge experiment, and the experimental animals were divided into 4 groups, 4 in each group, including the LSDV-125 vaccination group and the control group (PBS group). 3 TCID 50 Four weeks after immunization with the dose, the neutralizing antibody titer was measured as Figure 6 As shown, different doses of challenge experiments were then carried out ( or or The virus used for the challenge was LSDV / China / Xinjiang / 2019. After the challenge, the body temperature changes, clinical symptoms and detoxification were observed. The results are as follows: Figure 9-12 As shown in the figure, the body temperature of the LSDV-125 immunized group remained within the normal range, and no typical nodule symptoms were observed; the amount of toxin excretion was significantly lower than that of the non-immunized control group; the overall LSDV antibody production was measured by the IPMA method as shown in the figure. Figure 6-13 As shown in the figure, before the infection, the antibody titer could reach 1:1094, and after the infection, the antibody titer increased to a maximum of 1:2080. These data indicate that LSDV-125 can produce a good immune response and therefore has good protection.

[0029] The specific embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The specific embodiments do not describe all details in detail, nor do they limit the invention to the specific embodiments described. Numerous modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention.

Claims

1. A bovine lumpy skin disease virus strain LSDV-125, characterized in that: It is named sheeppox virus LSDV-HLJ-2023, the deposit number is CCTCC NO: V202537, and it is deposited in the China Center for Type Culture Collection, and the deposit date is May 13, 2025.

2. The bovine lumpy skin disease virus LSDV-125 according to claim 1, characterized in that The bovine lumpy skin disease virus strain LSDV-125 is a natural recombinant between the African Neethling strain and the Morocco strain; the gene ID of the African Neethling strain is KX764644.1, and the gene ID of the Morocco strain is MW631933.

1.

3. An attenuated bovine lumpy skin disease virus vaccine, characterized in that: The vaccine comprises the bovine lumpy skin disease virus strain LSDV-125 according to claim 1.

4. The vaccine according to claim 3, characterized in that The vaccine also includes pharmaceutically acceptable excipients.

5. The vaccine according to claim 3, characterized in that The vaccine is in the form of an injection.

6. The vaccine according to claim 3, characterized in that The content of bovine lumpy skin disease virus is 10 4 TCID 50 -10 5 TCID 50 .

7. Use of the bovine lumpy skin disease virus LSDV-125 according to any one of claims 1 to 3 in the preparation of a vaccine for preventing bovine lumpy skin disease.

8. The use according to claim 7, characterized in that The content of bovine lumpy skin disease virus is 10 4 TCID 50 -10 5 TCID 50 .

9. Use of the bovine lumpy skin disease virus LSDV-125 according to any one of claims 1 to 3 in the preparation of a vaccine for treating bovine lumpy skin disease.

10. The use according to claim 9, characterized in that The content of bovine lumpy skin disease virus is 10 4 TCID 50 -10 5 TCID 50 .

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