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 specifically relates to a virulence-attenuated bovine lumpy skin disease virus LSDV-75 and an application thereof. Background Art

[0002] Lumpy skin disease (LSD) is an important infectious disease of cattle. The disease is caused by Lumpy skin disease virus (LSDV), which belongs to the Poxviridae family and forms the Capripoxvirus genus with Sheeppox virus (SPPV) and Goatpox virus (GTPV). Currently, there is no commercialized vaccine for this disease, and most research is focused on inactivated vaccines. However, inactivated vaccines have the problems of high cost and poor immune effect, and require human intervention for inactivation, so it is necessary to provide a vaccine strain with good immune effect. Summary of the invention

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

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

[0005] The present invention provides an attenuated vaccine of bovine nodular dermatitis virus, which comprises the bovine nodular dermatitis virus strain.

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

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

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

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

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

[0011] Beneficial effect: A safe and weakened bovine nodular skin disease virus was obtained; the weakened bovine nodular 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 it was a fully attenuated vaccine strain; the challenge experiment showed that the attenuated bovine nodular skin disease virus could be effective against 10 6 TCID 50 The strong strain provides 100% immune protection and can be used as a safe and effective vaccine for the prevention and control of bovine nodular dermatitis.

[0012] [Biological Deposit Information]: A weakly virulent bovine lumpy skin disease virus strain (Lumpy skin diseasevirus) LSDV-75, named sheeppox virus LSDV-75, with the deposit number of 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 figure is a comparison of the plaque formation results of the newly isolated strain LSDV-75 and the highly virulent strain LSDV-XJ; the left figure is the plaque result of the highly virulent strain LSDV-XJ, and the right figure is the plaque result of the newly isolated strain LSDV-75; Figure 3 This is the result of temperature change after inoculation of bovine lumpy skin disease virus; Figure 4 This is the result chart of detoxification; 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 Temperature results chart during clinical detoxification after challenge; Figure 8 Time results of clinical detoxification after challenge. DETAILED DESCRIPTION

[0014] 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.

[0015] Example 1. Isolation of bovine lumpy dermatosis virus strain LSDV-75 1. The virus strain LSDV-XJ was continuously subcultured on Vero cells. The specific operation was as follows: the virus strain LSDV-XJ was inoculated into Vero cells at a volume ratio of 1:100. On the 5th day after inoculation, the cell culture was frozen and thawed three times, and then new Vero cells were inoculated at a volume ratio of 1:100. When the virus strain LSDV-XJ was subcultured to the 25th generation, the virus was plaque purified, and the size of the plaque formation was observed. At the same time, the subculture was continued. At the 50th generation, the virus was plaque purified again. At the same time, the subculture was continued. At the 75th generation, the virus was plaque purified again. It was observed that there were many small plaques formed. The small plaques were picked, and the virus was propagated. The virus was then plaque purified again until the small plaques could be formed stably and continuously. After that, the corresponding virus was propagated to obtain a toxicity-weakened bovine nodular skin disease virus strain (Lumpy skin disease virus) LSDV-75, named sheep pox virus LSDV-75, which was packaged and frozen in a -70 refrigerator. A weakened bovine lumpy skin disease virus strain (Lumpy skin disease virus) LSDV-75 was named sheeppox 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.

[0016] 2. After obtaining the bovine nodular skin disease virus strain LSDV-75, we conducted a comparative study on its biological characteristics with its parent strain LSDV XJ strain, and compared the bovine nodular skin disease virus strain LSDV-75 with its parent strain LSDV XJ strain. The two strains were infected at 0.05 MOI, and samples were collected and measured at different time points. Finally, the growth curves of the bovine nodular 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 reduced, with titers of 10 5.25 TCID 50 / 0.1ml and 10 4.5 TCID 50 / 0.1ml. The plaque assay results were similar. The plaques formed by the bovine nodular skin disease virus strain LSDV-75 were significantly smaller than those of the parent strain. The plaque sizes of the bovine nodular skin disease virus strain LSDV-75 and the parent strain were 370 microns and 1200 microns, 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.

[0017] Example 2. Application of bovine nodular dermatosis virus strain LSDV-75 1. Safety experiment: The bovine nodular dermatosis 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 nodular 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 get sick, and the nucleic acid and antibody 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 bovine nodular skin disease virus strain LSDV-75 was also tested for induction of LSDV antibody production, such as Figure 5 shown.

[0018] 2.Immunological experiments: Results: The bovine nodular skin disease virus strain LSDV-75 and PBS were subcutaneously inoculated into the experimental cattle (n=4) at a dose of 10 3 TCID 50 Blood samples were collected every 7 days for antibody testing. Figure 6 As shown, the bovine lumpy dermatitis virus strain LSDV-75 can produce a maximum neutralizing antibody titer of about 1:16.

[0019] 3. Virus challenge experiment: 21 days after immunization, the virus challenge dose was 10 6 TCID 50 The results showed that animals immunized with the bovine nodular dermatosis virus strain LSDV-75 were protected from clinical symptoms of bovine nodular dermatosis, while the PBS control group showed typical clinical symptoms. Figure 7 As shown, 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 fever 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, such as Figure 8 As shown, attenuated bovine lumpy skin disease virus can 6 TCID 50The strong strain provides 100% immune protection and can be used as a safe and effective vaccine for the prevention and control of bovine nodular dermatitis.

[0020] Example 3. Preparation of live vaccines Live vaccine: The bovine lumpy dermatitis 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 dermatitis virus strains.

[0021] Example 4. Preparation of freeze-dried virus vaccine strains The bovine nodular dermatitis 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. Then, the cells were centrifuged at 5000 rpm to remove the cell debris. The remaining supernatant was mixed with a lyophilizing protective agent in a ratio of 1:1, divided into penicillin bottles, first 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, the deposit number is CCTCC NO: V2024114, and it is deposited in the China Center for Type Culture Collection, and the deposit date is December 2, 2024.

2. A bovine nodular dermatosis virus attenuated vaccine, characterized in that: The vaccine comprises the bovine nodular dermatitis 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 bovine nodular skin disease virus is 10 3 TCID 50 -10 6 TCID 50 .

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