Carp herpesvirus type 2 attenuated strain for preparing a koi haematopoietic organ necrosis disease vaccine

Abstract

The application discloses a weak strain of Cyprinid herpesvirus 2 (CyHV-2) G-RP7 for preparing a common carp hematopoietic organ necrosis disease vaccine, and the preservation number is CGMCC No. 24099. The weak strain is used to infect a goldfish fin cell line (RyuF-2), and after 4-5 days, typical cytopathic effect (CPE) is observed on the cells, and the continuous passage can stably cause the cytopathic effect. The weak strain is sensitive to the RyuF-2 cell line, realizes in-vitro stable culture and amplification of the weak strain, and the TCID50 of the virus suspension is determined by a Reed-Muench method 50 up to 10 5.9 / ml. The virulence of the weak strain does not return to be strong after continuous in-vitro and in-vivo passages. The weak strain can be used for preparing the common carp hematopoietic organ necrosis disease vaccine, and both immersion immunization and intraperitoneal injection immunization can stimulate the fish to produce specific immune response, and have good anti-CyHV-2 infection effect.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, and in particular to a type II attenuated strain of carp herpesvirus used to prepare a vaccine for crucian carp hematopoietic organ necrosis disease. Background Technology

[0002] Vaccination is the most effective means of preventing and controlling viral diseases in aquatic animals. Various vaccines are available, including inactivated vaccines, subunit vaccines, DNA vaccines, and live attenuated vaccines. Immunization methods include injection and immersion immunization. Immersion immunization is considered the most practical method due to its simplicity, high efficiency, low cost, and minimal harm to aquatic animals. However, because immersion immunization requires a much higher viral load than injection immunization, existing live vaccines, subunit vaccines, and DNA vaccines can only be administered via injection. Only some live attenuated vaccines can be used for immersion immunization, which is one of their advantages. However, a drawback of live attenuated vaccines is the tendency for the virulence of the attenuated strain to revert to its previous form. Therefore, attenuated strains that do not revert to their previous form after in vivo passage and are sensitive to cell lines (producing a high viral load) are essential for preparing live attenuated vaccines and achieving immersion immunization.

[0003] Crucian carp is a common freshwater aquaculture species in my country, accounting for approximately 12% of the total freshwater fish production. Cyprinid herpesvirus 2 (CyHV-2) infection in crucian carp causes hematopoietic organ necrosis disease, which is highly contagious and has a mortality rate as high as 90-100%. Research on vaccines for crucian carp hematopoietic organ necrosis disease is underway. Chinese patent application number 202010183827.6 discloses a cyprinid herpesvirus 2 attenuated strain and its application. The cyprinid herpesvirus 2 attenuated strain is CyHV-2DX2019 isolated from silver carp, and its application as a cyprinid herpesvirus 2 attenuated vaccine. However, the patent application specification (0018-0027) states that CyHV-2 DX2019 was inoculated into crucian carp brain tissue cells (GiCB) and cultured blindly for 10 consecutive generations. During this infection process, the cells did not exhibit typical pathological effects. Only the culture of the 10th generation of blindly passaged cells was subjected to viral DNA amplification, proving that the 10th generation of blindly passaged cells contained the attenuated CyHV-2 DX2019 type II carp herpesvirus. The viral concentration was determined to be ≥5×10⁻⁶ using PCR technology. 5 The number of copies / ml indicates that the carp herpesvirus type II attenuated strain CyHV-2 DX2019 is insensitive to the crucian carp brain tissue cell (GiCB) line, making large-scale culture of the strain difficult. Therefore, the vaccine prepared using the carp herpesvirus type II attenuated strain CyHV-2DX2019 can only stimulate an immune response in fish through intraperitoneal injection, and the minimum dose injection only achieves an immunization protection rate of 92.85%. Summary of the Invention

[0004] This invention aims to address the problems existing in the prior art by providing a carp herpesvirus type II attenuated strain for preparing a vaccine against crucian carp hematopoietic organ necrosis. The carp herpesvirus type II attenuated strain (Cyprinid herpesvirus 2, CyHV-2) was deposited on January 17, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Datun Road, Chaoyang District, Beijing; accession number: CGMCC NO: 24099.

[0005] The technical solution of this invention is a carp herpesvirus type II attenuated strain for preparing a vaccine against crucian carp hematopoietic organ necrosis disease. It is carp herpesvirus type II (Cyprinid herpesvirus 2, CyHV-2) G-RP7, with the accession number CGMCC No.24099.

[0006] The carp herpesvirus type II G-RP7 provided by this invention is a type II attenuated strain of carp herpesvirus isolated from goldfish. When this attenuated strain was used to infect the goldfish fin cell line (RyuF-2), typical cytopathic effects (CPE) were observed in the cells after 4-5 days, and the cytopathic effect was stably induced in consecutive passages. The strain was sensitive to the RyuF-2 cell line, achieving stable in vitro culture and amplification of the attenuated strain. The TCID of the virus suspension was determined by the Reed-Muench method. 50 Up to 10 5.9 / ml, and this attenuated strain did not show virulence reversion after continuous in vitro and in vivo passages. It can be used to prepare a vaccine for crucian carp hematopoietic organ necrosis disease. Both immersion immunization and intraperitoneal injection immunization can stimulate a specific immune response in fish, showing good efficacy against carp herpesvirus type II infection. Injection immunization has a protection rate as high as 100%, significantly higher than the protection rate of existing injection immunization techniques. In the minimum immunization dose study, immersion immunization showed that when the virus suspension TCID... 50 2×10 2 At a concentration of 100 ml, the immune protection rate can reach 94%. This immune protection rate is also higher than that of existing injection immunization techniques, even when the immunization route is superior to injection immunization. Attached Figure Description

[0007] Figure 1 These are images of normal RyuF-2 cells and cytopathic effects after inoculation with G-RP7 under an optical microscope, as shown in the embodiments of the present invention.

[0008] Figure 2 This is a schematic diagram illustrating the protection rate of heterotrophic silver carp at different immunization concentrations in embodiments of the present invention.

[0009] Figure 3 This is a schematic diagram of the viral antibody level (ELISA) after 21 days of immersion immunization with G-RP7 in an embodiment of the present invention.

[0010] Figure 4 This is a schematic diagram of the viral antibody level (ELISA) 21 days after G-RP7 injection immunization according to an embodiment of the present invention.

[0011] Deposit date: January 17, 2022;

[0012] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0013] Address: No. 3, Jia, Datun Road, Chaoyang District, Beijing;

[0014] Accession number: CGMCC NO: 24099. Detailed Implementation

[0015] 1. Isolation, identification, and culture of carp herpesvirus type II G-RP7 strain

[0016] (1) Isolation and identification of carp herpesvirus type II G-RP7 strain

[0017] The carp herpesvirus type II G-RP7 strain in this invention is derived from diseased goldfish. Kidney tissue from diseased, dying goldfish was washed with M199 medium containing a 1% penicillin-streptomycin-amphotericidal B solution, homogenized, and centrifuged at 12,000 rpm / min for 10 min. The supernatant was sterilized through a 0.22 μm filter and inoculated into RyuF-2 fin cells of gilt goldfish. The cells were cultured at 25°C in M199 medium supplemented with 5% fetal bovine serum and 1% triple antibiotics (penicillin, streptomycin, and gentamicin). Cytopathic effects were continuously observed, and the cell culture supernatant (cell culture) was collected after 10 days and stored at -80°C. o C. The cell culture was amplified using primers CyHV-2Hel-F: 5'-GGGGACTTGCGAAGAGTTTGATTTCTAC-3' and CyHV-2Hel-R: 5'-CCATAGTCACCATCGTCTCATC-3'. The amplified product was approximately 360 bp as shown by agarose gel electrophoresis, which was consistent with the size of the positive target fragment band of carp herpesvirus type II. Sequence alignment results showed that it was a CyHV-2 sequence.

[0018] (2) Culture of carp herpesvirus type II G-RP7 strain

[0019] Will be saved at -80 o 100 μl of cell culture from cell C was thawed at room temperature and seeded into a 25 cm² cell culture medium containing a monolayer of RyuF-2 cells. 3Cells were cultured in M199 medium with a serum concentration of 5% in cell culture flasks. The cytopathic effect was observed daily under a light microscope, and the results are as follows: Figure 1 As shown in A and B, Figure 1 A is a normal RyuF-cell optical microscope image below; Figure 1 B shows a light microscope image of cells 5 days after inoculation with the G-RP7 strain. The results indicate that RyuF- cells showed significant cytopathic effects 5 days after inoculation with the G-RP7 strain. Cultures were collected and passaged repeatedly 10 times, consistently showing stable cytopathic effects. PCR verification and sequence alignment of the collected cell cultures confirmed that the carp herpesvirus type II G-RP7 strain could be stably amplified in RyuF-2 cells and produce significant cytopathic effects. The TCID of the virus suspension obtained from each passage was determined using the Reed-Muench method. 50 10 3.9-5.9 / ml.

[0020] 2. Experiment on pathogenicity and protective rate of carp herpesvirus type II G-RP7 strain against hybrid silver carp.

[0021] Three hundred crucian carp with an average weight of 52.0±5.0g and an average body length of 16.0±2.0cm were selected. Before the experiment, the crucian carp underwent viral nucleic acid testing to confirm they were negative for carp herpesvirus type II. The crucian carp were randomly divided into two groups according to the immunization method: immersion immunization and intraperitoneal injection immunization. Each immunization method included a G-RP7 experimental group and an M199 control group; each experimental group and control group was further divided into an observation group (for recording survival rate) and a challenge group (for calculating protection rate). The TCID of the CyHV-2G-RP7 strain used for immersion immunization was [not specified in the original text]. 50 10 2 / ml, soaked at 25℃ for 2 h and then fed in circulating water; TCID of CyHV-2 G-RP7 strain used for immunization was injected. 50 10 3 / ml, 100µl per fish was injected intraperitoneally, and the fish were housed in recirculating water. Twenty-one days after immunization, the surviving fish were challenged with the virus, and the survival rate of each group was recorded. The results are shown in Table 1.

[0022] Table 1: Safety and protection rate results of CyHV-2 G-RP7 strain

[0023]

[0024] The results showed that the survival rate of CyHV-2 G-RP7 strain immersion immunization and injection immunization groups was as high as 100% within 25 days. After 21 days, the results of the challenge experiment showed that the protection rates of immersion immunization and injection immunization were 92% and 100%, respectively.

[0025] Thus, this invention has obtained a carp herpesvirus type II attenuated strain that stably proliferates in RyuF-2 and is non-pathogenic to crucian carp. This strain was sent to the China Culture Collection Center on January 17, 2022, and classified as: Carp Herpesvirus Type II G-RP7 strain, accession number: CGMCC NO: 24099, address: No. 3, Datun Road, Chaoyang District, Beijing.

[0026] 3. Preparation of live attenuated vaccine for crucian carp hematopoietic organ necrosis

[0027] CyHV-2 G-RP7 was cultured in RyuF-2 cells to obtain a viral suspension, which was then inoculated into 75 cm cells containing a monolayer of RyuF-2 cells. 3 In cell culture flasks, after more than 50% cytopathic effects appeared, the cultures were frozen at -80°C and thawed at room temperature, repeated three times. Subsequently, the cultures were centrifuged at 4000 rpm / min for 30 min at 4°C. The concentration of the collected virus suspension was determined using the Reed-Muench method. The suspension was diluted with M199 medium to prepare a live attenuated vaccine against crucian carp hematopoietic organ necrosis. The vaccine contained carp herpesvirus type II G-RP7 strain, with a TCID value of [missing information]. 50 10 4 / mL.

[0028] 4. Application of live attenuated vaccine against crucian carp hematopoietic organ necrosis

[0029] (1) Study on the minimum immunization dose of live vaccine against crucian carp hematopoietic organ necrosis disease

[0030] The attenuated live vaccine for crucian carp hematopoietic organ necrosis prepared above was diluted to a concentration of 2×10⁻⁶. 0 / mLTCID 50 2×10 1 / mL TCID 50 2×10 2 / mL TCID 50 Each group consisted of 30 fish, which were immunized by soaking in water at 25°C for 2 hours. The fish were then challenged with the virus 21 days post-immunization, and the protection rate was calculated. The results are as follows: Figure 2 As shown.

[0031] The results showed that the CyHV-2 G-RP7 content was 2×10 2 / mL TCID 50 The immunization protection rate is 94%, and the protection rate decreases as the vaccine concentration decreases.

[0032] (2) Report on humoral immune response induced by live vaccine against crucian carp hematopoietic organ necrosis

[0033] The above-prepared attenuated live vaccine for crucian carp hematopoietic organ necrosis was diluted to the appropriate concentration and injected separately (10...3 / mLTCID 50 ) Immunization and soaking (10) 2 / mL TCID 50 Immunize hybrid silver carp with IgM antibodies. After 21 days, 20 fish from each group were collected and treated with anti-carp IgM. + Specific monoclonal antibodies were used to determine the antiviral antibody levels in the serum of immunized crucian carp immunized via injection and immersion immunization using an indirect enzyme-linked immunosorbent assay (ELISA). Unimmunized fish served as a negative control. The results are as follows: Figure 3 , Figure 4 As shown.

[0034] The results showed that the levels of antiviral antibodies in the G-RP7 immersion immunization and injection immunization groups were higher than those in the negative control group. Compared with immersion immunization, the increase in antibody levels in the injection immunization group was more significant.

[0035] (3) Evaluation report on the virulence reversion of live vaccine against crucian carp hematopoietic organ necrosis

[0036] ① In vitro passage experiment of carp herpesvirus type II G-RP7

[0037] At 25 cm 3 In cell culture flasks, the goldfish fin cell line (RyuF-2) was cultured in M199 medium containing 5% fetal bovine serum and 1% triple antibodies. After the cells grew into a monolayer (24-48 h), 0.1 ml of the CyHV-2G-RP7 strain (denoteed as P1) frozen at -80℃ was added to each flask. After culturing in a 25℃ incubator for 4-6 days, typical cytopathic effects (CPE) appeared in the cells. The culture was collected, centrifuged at 4000 rpm for 30 min at 4℃, and the supernatant was collected and stored at -80℃, designated as P2. P2 was then seeded into RyuF-2 cells, and the above operation was repeated 15 times. P1, P5, P10, and P15 were then diluted to 10⁻⁶. 3 / mL TCID 50 Inject 30 crucian carp per group, each receiving 0.1 ml; observe continuously for 21 days and record survival rate. 21 days after immunization, challenge P1, P5, P10, and P15 were administered via intraperitoneal injection, and observed continuously for 25 days, recording infection status and survival rate.

[0038] The results are shown in Table 2.

[0039] Table 2. Safety and protection rate of in vitro passage of attenuated live attenuated vaccine for crucian carp hematopoietic organ necrosis.

[0040]

[0041] The results showed that the G-RP7 strain, as an attenuated vaccine, could be stably passaged in RyuF-2, and it remained non-pathogenic to crucian carp after passage. The protection rate against challenge was 100% 21 days after immunization.

[0042] ② In vivo passage experiment of carp herpesvirus type II G-RP7

[0043] The above-prepared attenuated live attenuated vaccine for crucian carp hematopoietic organ necrosis was diluted to TCID10. 50 10 3 / mL, healthy hybrid crucian carp were injected with the vaccine at a dose of 0.1 ml / fish. Twenty fish were used to observe infection status and calculate survival rate. On the 5th day after inoculation, the spleen and kidney of 5 fish were collected, mixed and ground, and about 0.1 g of tissue homogenate was added to 1 ml of M199 medium. The mixture was centrifuged at 4000 rpm for 30 min at 4℃. The supernatant was collected and filtered through a 0.22 μm filter membrane. The supernatant of the tissue homogenate was injected into a new group of healthy crucian carp. The above operation was repeated 5 times. The crucian carp injected with the attenuated live vaccine were designated as P1, and the other groups were designated as P2, P3, P4, P5, and P6 respectively. The results are shown in Table 3.

[0044] Table 3. Survival rate of live attenuated vaccine against crucian carp hematopoietic organ necrosis in vivo after passage.

[0045]

[0046] The results showed that none of the above-mentioned P1, P2, P3, P4, P5, and P6 exhibited significant reversion to toxicity after in vivo passage.

Claims

1. A type II attenuated strain of carp herpesvirus for preparing a vaccine against crucian carp hematopoietic organ necrosis, characterized in that... Cyprinid herpesvirus type II (CyHV-2) G-RP7, with accession number CGMCC No. 24099.

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