Virus-attenuated bovine nodular skin disease virus LSDV-75 and application thereof

Abstract

The invention discloses a virulence-attenuated bovine nodular skin disease virus LSDV-75 and application thereof, and belongs to the technical field of attenuated strains. The invention aims to provide a bovine nodular skin disease virus vaccine strain which is sufficient in attenuation and good in immune effect. The invention provides a virulence-attenuated bovine nodular skin disease virus strain, which is named as sheep pox virus LSDV-75, has the preservation number of CCTCC NO: V2024114, and is preserved in the China Center for Type Culture Collection on December 2, 2024. The vaccine strain provided by the invention can be used as a vaccine candidate strain for safely and effectively preventing and controlling bovine nodular dermatosis.

Description

Technical Field

[0001] The invention belongs to the technical field of attenuated virus strains, and particularly relates to a virulence-attenuated bovine lumpy skin disease virus LSDV-75 and an application thereof. Background Art

[0002] Lumpy skin disease (LSD) is a major infectious disease in cattle. It is caused by the lumpy skin disease virus (LSDV), which belongs to the Poxviridae family and, along with sheeppox virus (SPPV) and goatpox virus (GTPV), forms the Capripoxvirus genus. Currently, there are no commercially available vaccines for LSD, and most research focuses on inactivated vaccines. However, these vaccines are costly, have poor immune efficacy, and require human intervention for inactivation. Therefore, a virulently attenuated vaccine strain with enhanced immune efficacy is needed. Summary of the Invention

[0003] The purpose of the present invention is to provide a bovine lumpy skin disease virus vaccine strain with sufficient attenuation and good immune effect.

[0004] The present invention provides a bovine lumpy skin disease virus strain (Lumpy skin disease virus) LSDV-75 with attenuated virulence, named as capripox virus LSDV-75, with a preservation number of CCTCC NO: V2024114, and deposited in the China Center for Type Culture Collection, with a preservation date of December 2, 2024.

[0005] The present invention provides an attenuated bovine lumpy skin disease virus vaccine, which comprises the bovine lumpy skin disease virus strain.

[0006] It is further defined that the vaccine further comprises a pharmaceutical carrier or a pharmaceutically acceptable excipient.

[0007] It is further defined that the vaccine is in the form of an injection.

[0008] It is further defined that the content of bovine lumpy skin disease virus is 10 3 TCID 50 -10 6 TCID 50 .

[0009] The present invention provides an application of the bovine lumpy dermatitis virus strain in preparing a vaccine for preventing or treating bovine lumpy dermatitis.

[0010] It is further defined that the content of the bovine lumpy skin disease virus is 10 3 TCID 50 -10 6 TCID 50 .

[0011] Beneficial effect: a safe and weakened bovine lumpy skin disease virus was obtained; the weakened bovine lumpy skin disease virus was immunized (10 5 TCID 50 ) cattle did not get sick and the nucleic acid and antibody of the cohabiting cattle were negative, indicating that there was no horizontal transmission phenomenon and the strain was a fully attenuated vaccine strain; the challenge experiment showed that the attenuated bovine nodular skin disease virus could 6 TCID 50 ~10 8.5 TCID 50 The virulent strain provides 100% immune protection and can be used as a safe and effective vaccine to prevent and control bovine nodular dermatitis.

[0012] [Biological Deposit Information]: A virulent strain of bovine lumpy skin disease virus (Lumpy skin disease virus) LSDV-75, named sheep pox virus LSDV-75, with the deposit number CCTCC NO: V2024114, deposited in the China Center for Type Culture Collection, the deposit date is December 2, 2024, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the growth curve result of bovine lumpy skin disease virus; Figure 2 The left figure is a comparison of the plaque formation of the highly virulent strain LSDV-XJ and the right figure is a comparison of the plaque formation of the newly isolated strain LSDV-75. Figure 3 This is a graph showing the changes in body temperature of cattle after inoculation with bovine lumpy skin disease virus; Figure 4 This is the detoxification results chart; Figure 5 This is the result diagram of IgG antibody production after vaccination; Figure 6 This is a graph showing the results of neutralizing antibody production after immunization; Figure 7 is 10 6 TCID 50 Body temperature results after virus attack; Figure 8 is 10 6 TCID50 Results of detoxification after attack; Figure 9 is 10 7.5 TCID 50 Body temperature results after virus attack; Figure 10 is 10 7.5 TCID 50 Results of detoxification after attack; Figure 11 is 10 8.5 TCID 50 Body temperature results after virus attack; Figure 12 is 10 8.5 TCID 50 Results of toxin excretion after attack; DETAILED DESCRIPTION LSDV-XJ records the first isolation and identification of bovine lumpy skin disease virus in my country in the article, Zhang Minmin, 2020, which is the LSDV / China / Xinjiang / 2019 strain (Xinjiang / 2019), also called wtLSDV in this application.

[0014] Example 1. Isolation of bovine lumpy skin disease virus strain LSDV-75 1. The LSDV-XJ virus strain was serially subcultured 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 passage 50, plaque purification was performed again. Continued subculture was performed. At passage 75, plaque purification was performed again. If multiple small plaques were observed, these small plaques were selected 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-75, designated capripox virus LSDV-75, and stored frozen in aliquots at -70°C. A virulent strain of bovine lumpy skin disease virus (Lumpy skin disease virus) LSDV-75 was named as capripox virus LSDV-75, with the deposit number CCTCC NO: V2024114, and deposited in the China Center for Type Culture Collection on December 2, 2024, at Wuhan University, Wuhan, China.

[0015] 2. After obtaining the bovine lumpy skin disease virus strain LSDV-75, we conducted a comparative study of its biological characteristics with its parent strain, LSDV XJ strain. The bovine lumpy skin disease virus strain LSDV-75 was compared with its parent strain, LSDV XJ strain. Both strains were infected at an MOI of 0.05, and samples were collected and measured at different time points. Finally, the growth curves of the bovine lumpy skin disease virus strain LSDV-75 and the parent strain were measured ( Figure 1 ) The results showed that the strain had a similar growth trend to the parent strain, but the corresponding titer was significantly decreased, with titers of 10 5.25 TCID 50 / 0.1ml and 10 4.5 TCID 50 / 0.1ml. The results of the plaque assay were similar. The plaques formed by the bovine lumpy skin disease virus strain LSDV-75 were significantly smaller than those of the parent strain. The plaque sizes of the bovine lumpy skin disease virus strain LSDV-75 and the parent strain were 370 μm and 1200 μm, respectively ( Figure 2 ), the bovine lumpy skin disease virus strain LSDV-75 has small plaques and its replication ability is weaker than that of the parental strain.

[0016] Example 2. Application of bovine lumpy skin disease virus strain LSDV-75 1. Safety experiment: The bovine lumpy skin disease virus strain LSDV-75 obtained in Example 1 and the parent strain were inoculated intradermally into cattle (n=4, n=5) at a dose of 10 5 TCID 50 Compared with the parent strain, the bovine lumpy skin disease virus strain LSDV-75 did not develop typical clinical symptoms after inoculation ( Figure 3 ), and the detoxification amount was significantly lower than that of the parent strain group ( Figure 4 The virulence of the attenuated strain was significantly reduced, the cattle did not become ill, and the nucleic acid and antibody tests of the cohabiting cattle were negative, indicating that there was no horizontal transmission phenomenon and it was a fully attenuated vaccine strain. At the same time, the production of LSDV antibodies induced by the bovine nodular skin disease virus strain LSDV-75 was also tested. Figure 5 shown.

[0017] 2. Immunity experiments: Results: The bovine lumpy skin disease virus strain LSDV-75 and PBS were subcutaneously inoculated into the experimental cattle (n=4) at an immunization dose of 10 3 TCID 50 , blood was collected every 7 days for antibody testing. Figure 6 As shown, the bovine lumpy skin disease virus strain LSDV-75 can produce a neutralizing antibody titer of about 1:16 at most.

[0018] 3. Challenge experiment: Challenge was conducted 21 days after immunization, with a challenge dose of 10 6 TCID 50 , 10 7.5 TCID 50 , 10 8.5 TCID 50 The results showed that the animals immunized with the bovine lumpy skin disease virus strain LSDV-75 were protected and did not develop clinical symptoms of bovine lumpy skin disease, while the PBS control group showed typical clinical symptoms. Figure 7 , 9, 11, the body temperature of the immunized group cattle was within the normal range, while the PBS control group had a high fever for 4 days, with the highest temperature exceeding 40°C. In addition, the amount and time of toxin excretion in the immunized group were significantly lower than those in the control group, as shown in Figure 8 , 10, 12, the attenuated bovine lumpy skin disease virus can 6 to 10 8.5 TCID 50 The virulent strain provides 100% immune protection and can be used as a safe and effective vaccine to prevent and control bovine nodular dermatitis.

[0019] Example 3. Preparation of live vaccines Live vaccine: The bovine lumpy skin disease virus strain LSDV-75 was used as a live vaccine, and LT cells were infected with 0.5 MOI to reproduce the virus. The vaccine strains that were reproduced in large quantities were all bovine lumpy skin disease virus strains.

[0020] Example 4. Preparation of freeze-dried virus vaccine strains The bovine lumpy skin disease virus strain LSDV-75 was used to infect LT cells at an MOI of 0.5 for virus propagation. The virus was collected after 5-7 days, and the cells were repeatedly frozen and thawed three times at -70°C. The cells were then centrifuged at 5000 rpm to remove cell debris. The remaining supernatant was mixed with a lyophilization protectant in a ratio of 1:1, divided into penicillin bottles, and placed at -20°C for 6 hours, at -80°C for 4 hours, and then placed in a freeze dryer (-40°C) for 24 hours to obtain the freeze-dried virus vaccine strain.

Claims

1. A strain of attenuated bovine lumpy skin disease virus, characterized in that: It is named sheeppox virus LSDV-75, with the deposit number of CCTCC NO: V2024114, and is deposited in the China Center for Type Culture Collection, with the deposit date of December 2, 2024.

2. A bovine lumpy skin disease virus attenuated vaccine, characterized in that: The vaccine comprises the bovine lumpy skin disease virus strain of claim 1.

3. The vaccine according to claim 2, characterized in that The vaccine also includes pharmaceutically acceptable excipients.

4. The vaccine according to claim 2, characterized in that The vaccine is in the form of an injection.

5. The vaccine according to claim 2, characterized in that The content of bovine lumpy skin disease virus is 10 3 TCID 50 -10 6 TCID 50 .

6. Use of the bovine lumpy skin disease virus according to claim 1 in the preparation of a vaccine for preventing or treating bovine lumpy skin disease.

7. The use according to claim 6, characterized in that The content of the bovine nodular skin disease virus is 10 3 TCID 50 -10 6 TCID 50 .

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