A kind of infectious spleen and kidney necrosis virus attenuated vaccine strain and its application

Abstract

The present application relates to the field of virus vaccine technology, and in particular to an attenuated vaccine strain of infectious spleen and kidney necrosis virus and its application. The present application selected the virulent strain ISKNV DW and continuously propagated it in CPB cells for 55 generations. It was found that ISKNV-p55 has no pathogenicity to mandarin fish and can provide 100% immune protection. The above results show that this attenuated ISKNV-p55 strain can be used as an attenuated vaccine to prevent and control the outbreak of ISKNV.

Description

Technical Field

[0001] The present application relates to the technical field of virus vaccines, and in particular to an attenuated vaccine strain of infectious spleen and kidney necrosis virus and its application. Background Art

[0002] In 1994, researcher Wu Shuqin first reported the virus disease of mandarin fish. Subsequently, Professor He Jianguo's team named it infectious spleen and kidney necrosis virus (ISKNV) based on its spleen and kidney necrosis as the main pathological characteristics. It has a wide spectrum of infection and a high mortality rate. It is still the main pathogen endangering the mandarin fish farming industry. Infectious spleen and kidney necrosis virus (ISKNV) is a representative member of the genus Iridoviridae. It is an enveloped intracellular icosahedral virus. my country classifies it as a Class II animal epidemic disease, which is the highest level of epidemic prevention and control for aquatic animals. There is no specific drug for the treatment and prevention and control of viral diseases, and vaccines are still an important means of preventing and controlling viral diseases.

[0003] At present, the cell-inactivated vaccine of ISKNV (NH0618 strain) of Sun Yat-sen University has been applied to the market, and its virus-encoded protein subunit vaccine and virus DNA vaccine are under development. The ISKNV and SCRV bivalent cell-inactivated vaccine developed by the Pearl River Institute has completed laboratory research. Virus protein subunit vaccines and virus DNA vaccines have the disadvantages of low immune efficacy and high production costs. Inactivated vaccines can only induce humoral immunity but not cellular immunity, and need to be injected for immunization. They are more suitable for large-sized fish species, and the operation is relatively cumbersome. At the same time, they will cause certain stress to mandarin fish. Immersion immunization is simple to operate and can be used for large-scale immunization of small fish fry. It will be an important research direction for aquatic vaccines. Attenuated vaccines can induce humoral immunity and stimulate cellular immunity. They have a small inoculation dose, good immune effect, and can also be used for immersion immunization. Although Sun Yat-sen University has constructed a virus strain lacking virulence genes based on known virulence genes, the strain still has certain virulence.

[0004] At present, there is no attenuated vaccine for infectious spleen and kidney necrosis disease, so the development of ISKNV attenuated vaccine is a scientific problem that needs to be urgently solved in the current mandarin fish farming industry. Summary of the invention

[0005] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide an attenuated vaccine strain of infectious spleen and kidney necrosis virus and its application. This application selected the virulent strain ISKNV DW and continuously propagated it in CPB cells for 55 generations. It was found that ISKNV-p55 has no pathogenicity to mandarin fish and can provide 100% immune protection. The above results show that this attenuated ISKNV-p55 strain can be used as an attenuated vaccine to prevent and control the outbreak of ISKNV.

[0006] To achieve the above purpose, the technical solution adopted by this application is:

[0007] The present application provides an attenuated vaccine strain of infectious spleen and kidney necrosis virus, the attenuated vaccine strain of infectious spleen and kidney necrosis virus is named ISKNV-p55, the preservation classification name is Megalocytivirus, the preservation number is CCTCC NO: V2023117, and the preservation time is December 27, 2023.

[0008] The present invention obtains the ISKNV-p55 of the present invention by passage of the strong strain ISKNV-DW (NCBI accession number: HQ317465). The cell adaptability of ISKNV-p55 is enhanced, and the virus titer can reach TCID 50 =10 -7.96 The ISKNV-p55 of the present application is no longer pathogenic to Siniperca chuatsi and can provide 100% protection against the attack of strong toxins. The results of the virulence reversion experiment show that ISKNV-p55 is relatively stable in the sensitive host Siniperca chuatsi and is not easy to reversion. Its minimum immune dose is TCID 50 =10 -5.96 The above experimental results show that ISKNV-p55 can be used as a live attenuated vaccine to prevent and control ISKNV.

[0009] The present application also provides the use of the above-mentioned attenuated vaccine strain of infectious spleen and kidney necrosis virus in the preparation of a live vaccine against infectious spleen and kidney necrosis virus in fish.

[0010] As a preferred embodiment of the application described in this application, the minimum immunization dose of the attenuated vaccine strain of infectious spleen and kidney necrosis virus is TCID 50 =10 -5.96 / tail.

[0011] The present application also provides the use of the above-mentioned attenuated vaccine strain of infectious spleen and kidney necrosis virus in the preparation of drugs or preparations for preventing and treating infectious spleen and kidney necrosis virus in fish.

[0012] As a preferred embodiment of the application described in the present application, the fish includes mandarin fish.

[0013] The present application also provides a method for producing the attenuated vaccine strain of infectious spleen and kidney necrosis virus, the production method comprising:

[0014] ISKNV DW was cultured with CPB cells. After inoculation of the virus, 2% FBS L-15 maintenance solution (2 mL FBS + 98 mL L-15) was added for continuous culture. When the cell pathological rate was 80-90%, the virus was collected by freezing and thawing twice. The virus was continuously passaged for 55 generations to obtain the infectious spleen and kidney necrosis virus attenuated vaccine strain.

[0015] Preferably, the concentration of L-15 in the FBS L-15 maintenance solution is 2%.

[0016] In the production method, the dosage of ISKNV DW is 10 4 TCID 50 .

[0017] This application selected a strong strain (ISKNV DW) and evaluated the growth characteristics, safety and immunity of ISKNV-p55 after 55 consecutive passages on CPB cells. The results showed that after 55 consecutive passages on CPB cells, the cell adaptability of the virus was enhanced and the virus titer could reach TCID 50 =10 -7.96 .

[0018] Animal experiment results showed that ISKNV-p55 is no longer pathogenic to Siniperca chuatsi and can provide 100% protection against strong toxins. The results of virulence reversion experiments showed that ISKNV-p55 is relatively stable in the sensitive host Siniperca chuatsi and is not easy to reversion. Its minimum immune dose is TCID 50 =10 -5.96 The above experimental results show that ISKNV-p55 can be used as a live attenuated vaccine to prevent and control ISKNV.

[0019] The present application also provides a live vaccine against infectious spleen and kidney necrosis virus, wherein the live vaccine comprises the above-mentioned attenuated vaccine strain of infectious spleen and kidney necrosis virus.

[0020] As a preferred embodiment of the live vaccine against infectious spleen and kidney necrosis virus described in the present application, the minimum immunizing dose of the attenuated vaccine strain of infectious spleen and kidney necrosis virus is TCID 50 =10 -5.96 / tail.

[0021] The present application also provides a drug or preparation for preventing and treating infectious spleen and kidney necrosis virus, wherein the drug or preparation comprises the above-mentioned attenuated vaccine strain of infectious spleen and kidney necrosis virus.

[0022] As a preferred embodiment of the drug or preparation for preventing and treating infectious spleen and kidney necrosis virus described in the present application, the drug or preparation also includes a pharmaceutically acceptable carrier.

[0023] Compared with the prior art, this application has the following beneficial effects:

[0024] The present application provides an attenuated vaccine strain of infectious spleen and kidney necrosis virus and its application. The present application selects the virulent strain ISKNV DW and continuously propagates it in CPB cells for 55 generations. It is found that ISKNV-p55 has no pathogenicity to mandarin fish and can provide 100% immune protection against the attack of virulent virus. The above results show that this attenuated ISKNV-p55 strain can be used as an attenuated vaccine to prevent and control the outbreak of ISKNV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure shows the nucleic acid identification results of p0, p10, p20, p40 and p55 generations;

[0026] Figure 2 One-step growth curves of ISKNV p0, ISKNV p25, and ISKNV-p55;

[0027] Figure 3 This is a diagram showing the safety and immune efficacy of ISKNV-p55; ( Figure 3 -A is the pathogenicity of ISKNV-p55; Figure 3 -B is the amount of virus in each organ of the ISKNV-p55 and ISKNV p0 challenge groups; Figure 3 -C is the ISKNV-p55 immune efficacy; Figure 3 -D is the autopsy changes of ISKNV-p55, ISKNV p0 and L-15 groups);

[0028] Figure 4 The results of the virulence reversal experiment of ISKNV-p55 are shown in Figure ( Figure 4 -A is the virus copies in the spleens of three groups of mandarin fish: a, b, and c; Figure 4 -B is the necropsy changes of Siniperca chuatsi in groups a, b, c, d, e and f);

[0029] Figure 5 This is the result graph of the minimum immune dose of ISKNV-p55. DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0032] In the following examples, CPB (Chinese perch cell, CPB) cells were isolated, domesticated and preserved in this experiment, and ISKNV DW strain was isolated and frozen in this experiment (Xiaozhe Fu, Ningqiu Li, Lihui Liu, Qiang Lin, Fang Wang, Yingtiao Lai, Haiming Jiang, Houjun Pan, Cunbin Shi, Shuqin Wu. Genotype and host range analysis of infectious spleen and kidney necrosis virus (ISKNV). Virus Genes (2011) 42: 97-109).

[0033] Example 1

[0034] 1. Virus passage and detection:

[0035] Take the CPB that has grown a monolayer of cells, discard the 8% FBS L-15 medium, rinse the cells twice with PBS, and add 10 4 TCID 50 The virus solution (ISKNV DW strain) was continuously cultured in L-15 maintenance medium containing 2% FBS. When the cell lesions were 80-90%, the virus was collected by freezing and thawing twice. The above method was used for continued subculture, and PCR identification was performed every 10 generations (refer to Liang Hongru, Ma Siaya, Fu Xiaozhe, Lin Qiang, Liu Lihui, Niu Yinjie, Huang Zhibin, Lin Li, Li Ningqiu. Establishment of triple PCR detection method for infectious spleen and kidney necrosis virus, mandarin fish frog virus and mandarin fish rhabdovirus. Journal of Northwest A&F University (Natural Science Edition) 2021.49.11).

[0036] When the strain reached the 25th generation, it was named ISKNV-p25;

[0037] When it was passaged to the 55th generation, the strain was named ISKNV-p55 and deposited in the China Center for Type Culture Collection. The preservation classification name is Megalocytivirus, the preservation number is CCTCC NO: V2023117, the preservation time is December 27, 2023, the preservation unit is the China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0038] The pathological changes of p0-p55 generations were basically the same, with no significant difference. The pathological changes were basically stable at 6-7 days. PCR was used to measure p0, p10, p20, p40 and p55 generations, and specific bands appeared in each generation ( Figure 1The above results indicate that ISKNV-p55 did not undergo significant changes during the passage of ISKNV DW.

[0039] Example 2. Growth characteristics of ISKNV DW, ISKNV p25 and ISKNV-p55

[0040] A 12-well plate covered with a monolayer of CPB cells was inoculated with three generations of viruses (ISKNVDW, ISKNV p25 and ISKNV-p55 obtained in Example 1), and the cell pathological effect was observed every 24 h after inoculation. When the cell pathological effect was 80-90%, the virus was collected at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h after inoculation, and the TCID 50 Determine the virus titer of each generation and the TCID of the collected samples 50 , the production curves of ISKNV DW, ISKNV p25 and ISKNV-p55 were drawn and analyzed.

[0041] The one-step growth curves of ISKNV DW, ISKNV p25 and ISKNV-p55 were determined, and it was found that ISKNV p25 and ISKNV-p55 had similar growth kinetics to the ISKNV p0 original strain.

[0042] When the virus infected cells for 24-96h, the pathological changes and proliferation rates of ISKNV p25 and ISKNV-p55 were slightly faster than those of the original strain ISKNV p0, and their virus titers were also correspondingly higher than those of ISKNV p0. However, the final virus titers of ISKNV p25 and ISKNV-p55 were not significantly different from those of ISKNV p0 ( Figure 2 The above results indicate that with the passage of the virus, ISKNV-p55 becomes more adaptable to CPB cells.

[0043] Example 3: Safety and immune efficacy experiments of ISKNV-p55

[0044] 80 8-10 cm mandarin fish were selected and divided into 4 groups, 20 in each group. 50 =10 -5.5 ), a group of ISKNV-p55 collected propagation stock solution (TCID 50 =10 -7.69) and two PBS control groups (one group as PBS positive control for subsequent challenge, and one group as PBS negative control). After one week of temporary storage, 100 μL / tail was injected intraperitoneally. The symptoms of disease were observed and recorded every day after inoculation, and the number of deaths was counted. After inoculation, the observation was continued for 21 days. All live fish in the ISKNV-p55 group and one of the PBS positive control groups were challenged with ISKNV p0, and a group of control groups were inoculated with PBS to evaluate the immune efficacy of ISKNV-p55.

[0045] In order to evaluate the pathogenicity of the passaged virus ISKNV-p55 to Siniperca chuatsi, a challenge experiment was conducted. Figure 3 As shown in the figure, after the inoculation of ISKNV-p55 and the PBS negative control group, all the mandarin fish were normal and no clinical symptoms of ISKNV appeared. However, one mandarin fish died 8 days after the inoculation of the original ISKNV p0 strain, and the peak of death appeared at 9-10 days. At 12 days, all mandarin fish died ( Figure 3 -A).

[0046] The autopsy results showed that the ISKNV p0 group had typical clinical symptoms of ISKNV, such as blood loss and whitening of the liver and gills, a large amount of yellow ascites in the abdominal cavity, and congestion and enlargement of the spleen; while the ISKNV-p55 and control groups showed no clinical symptoms in any organ ( Figure 3 -D).

[0047] The viral loads of various organs of Siniperca chuatsi in the ISKNV p0 and ISKNV-p55 groups were analyzed. It was found that the viral copies of the liver, spleen, kidney and intestine of the ISKNV p0 group were significantly higher than those of the ISKNV-p55 group, and the highest viral load was in the spleen in both groups ( Figure 3 -B).

[0048] A group of negative PBS control group and ISKNV-p55 group of live Siniperca chuatsi were challenged with virulent ISKNV DW. The Siniperca chuatsi in the negative PBS control group died 100%, while all the Siniperca chuatsi in the ISKNV-p55 group survived ( Figure 3 -C). The above results indicate that ISKNV-p55 can be used as a candidate strain for attenuated vaccine to prevent and control ISKNV.

[0049] Example 4: ISKNV-p55 re-enhancement experiment

[0050] 120 8-10 cm mandarin fish were divided into 6 groups, 20 in each group, 5 experimental groups (a, b, c, d, e), and 1 L-15 negative control group (f). Group a was intraperitoneally injected with cell culture ISKNVp55 stock solution (TCID 50 =10 -7.96), 100 μL / tail. Seven days after the infection, two fish were dissected to observe the changes in various organs, determine the virus content in each organ, and grind the tissue fluid. The supernatant of the tissue fluid was injected intraperitoneally into group B mandarin fish. The above operation steps were repeated 5 times to perform the re-strengthening experiment to evaluate the stability of the virulence of ISKNV-p55.

[0051] In order to explore whether the virulence of ISKNV-p55 would be re-enhanced in its susceptible host, Siniperca chuatsi, the virus fluid ISKNV-p55 harvested from cells was blindly propagated for 5 generations.

[0052] The results show that if Figure 4 As shown in the figure, all the five generations of blind transmission of Siniperca chuatsi were healthy and alive, without clinical symptoms of ISKNV; the autopsy results also showed that the tissues and organs of each blind transmission generation were normal ( Figure 4 -A). At the fourth blind passage, the virus could no longer be detected by fluorescent quantitative PCR ( Figure 4 -B). The above results indicate that the virulence of ISKNV-p55 is relatively stable and is not prone to relapse.

[0053] Example 5. Determination of the minimum immunizing dose of ISKNV-p55

[0054] 100 8-10cm mandarin fish were selected and divided into 5 groups (A, B, C, D, E), with 20 fish in each group. The three immunization groups were ISKNV-p55 10 -1 A(TCID 50 =10 -6.96 ), ISKNV-p55 10 -2 B (TCID 50 =10 -5.96 ),CISKNV-p55(TCID 50 =10 -4.96 ) and D, E (PBS), 100 μL / tail were immunized by intraperitoneal injection. 21 days after immunization, A, B, C, and D were challenged with ISKNV p0, 100 μL (TCID 50 =10 -6.5 ) / tail, and group E was inoculated with PBS 100 μL / tail. The mice were observed for 21 days after the challenge, and the clinical changes were observed and recorded, and the incidence and mortality were counted.

[0055] To determine the minimum immunizing dose of ISKNV-p55, 10 -1 , 10 -2 and 10 -3 Siniperca chuatsi were immunized with three concentrations (100 μL / tail). -1 and 10 -2 All the Mandarin fish survived healthily without any clinical symptoms.-3 Four Siniperca chuatsi died in the immunization group, and all Siniperca chuatsi died in the control group ( Figure 5 The above results indicate that the minimum immune dose of ISKNV-p55 is TCID 50 =10 -5.96 / tail.

[0056] ISKNV is the main virus in the mandarin fish farming industry, and its epidemic has caused huge economic losses to fish farming. There are still no effective prevention and control measures for viral diseases, and vaccination is still the main means of prevention and control. At present, the inactivated ISKNV vaccine has achieved certain results, but it has certain limitations in actual aquatic applications, such as the need for repeated vaccinations, the use of adjuvants and the difficulty in large-scale application. The live attenuated vaccine can achieve a good immune effect with a single vaccination, without the need to add adjuvants and can be used for soaking immunization to expand the scale of immunization. The team of Professor He from Sun Yat-sen University constructed two attenuated strains lacking virulence genes, but they both have certain virulence to mandarin fish. This application is based on CPB cells. After 55 consecutive generations of the strain ISKNV, it has no pathogenicity to mandarin fish. Using ISKNV p0 to attack mandarin fish vaccinated with ISKNV-p55 can provide 100% protection. The minimum immune dose of ISKNV-p55 was evaluated, and its TCID 50 =10 -4.96 It can still provide 100% protection against ISKNV. The present application obtains a weak strain ISKNV-p55 that can provide 100% protection against ISKNV virulent attack, which can be used as a candidate strain for ISKNV attenuated live vaccine.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. An attenuated vaccine strain of infectious spleen and kidney necrosis virus, characterized in that: The infectious spleen and kidney necrosis virus attenuated vaccine strain is named ISKNV-p55, the preservation number is CCTCCNO: V2023117, and the preservation time is December 27, 2023.

2. Use of the attenuated vaccine strain of infectious spleen and kidney necrosis virus as claimed in claim 1 in the preparation of a live vaccine against infectious spleen and kidney necrosis virus in fish.

3. The use according to claim 2, characterized in that The minimum immunizing dose of the attenuated vaccine strain of infectious spleen and kidney necrosis virus is TCID 50 =10 -5.96 / tail.

4. Use of the attenuated vaccine strain of infectious spleen and kidney necrosis virus as claimed in claim 1 in the preparation of drugs for preventing and treating infectious spleen and kidney necrosis virus in fish.

5. The use according to any one of claims 2 to 4, characterized in that: The fish includes mandarin fish.

6. A live vaccine against infectious spleen and kidney necrosis virus, characterized in that: The live vaccine comprises the attenuated vaccine strain of infectious spleen and kidney necrosis virus as described in claim 1.

7. The anti-infective spleen and kidney necrosis virus live vaccine according to claim 6, characterized in that: The minimum immunizing dose of the attenuated vaccine strain of infectious spleen and kidney necrosis virus is TCID 50 =10 -5.96 / tail.

8. A drug for preventing and treating infectious spleen and kidney necrosis virus, characterized in that: The medicine comprises the attenuated vaccine strain of infectious spleen and kidney necrosis virus as claimed in claim 1.

9. The drug for preventing and treating infectious spleen and kidney necrosis virus as claimed in claim 8, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

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