Attenuated virus strain of largemouth bass rhabdovirus and its application

Abstract

The present invention relates to the field of viruses, and in particular to a largemouth bass rhabdovirus attenuated virus strain and its application. The provided largemouth bass rhabdovirus attenuated virus strain has a deposit number of CCTCC NO: V202412. The virus strain has weak toxicity and good immunogenicity, can be used for the development of largemouth bass rhabdovirus attenuated vaccine, and has important significance for the prevention and treatment of fish rhabdovirus.

Description

Technical Field

[0001] The invention relates to the field of viruses, and in particular to an attenuated virus strain of largemouth bass rhabdovirus and an application thereof. Background Art

[0002] Largemouth bass rhabdovirus (Micropterus salmoides Rhabdovirus, MsRV) can cause explosive deaths of famous fish such as mandarin fish and seabass. It is extremely susceptible to infection from the seedling stage to the breeding process, and the mortality rate can be as high as over 90%. In particular, the mortality rate of seedling infection is almost 100%. There is no effective prevention and control method, and the epidemic is becoming more and more widespread, causing huge economic losses to the aquaculture industry. It has become one of the main bottlenecks in the development of aquaculture.

[0003] At present, there is a lack of commercial attenuated vaccines for largemouth bass rhabdovirus. The inactivated vaccine has the disadvantages of short-lived antibody levels and causes adverse stress responses in the body. The attenuated vaccine can still grow and reproduce in the body after vaccination, and produces immunity quickly and strongly, which can stimulate the body's lasting immunity to pathogens. Moreover, the immunization routes are diversified, such as immersion, nasal drops, eye drops, etc.

[0004] In the early stage of vaccine research, the main immunization route was injection of vaccines, which was complicated to operate and had increased cost, making it difficult to promote and use. In addition, injection of vaccines can only be performed when the fish body is about 5 cm long, and it cannot be used for seedlings and fry. Rhabdoviruses are extremely harmful to seedlings and fry, with a very high mortality rate. Therefore, the development of effective immersion immunization has important application prospects. Summary of the invention

[0005] In view of the above problems, the present invention provides a largemouth bass rhabdovirus strain, which is a weak strain with good immunogenicity and can be used as a candidate strain for a weak rhabdovirus vaccine.

[0006] The invention aims to provide a largemouth bass rhabdovirus attenuated virus strain and application thereof.

[0007] The technical solution adopted by the present invention is:

[0008] A attenuated largemouth bass rhabdovirus strain, named as largemouth bass rhabdovirus MsRV-0509, has been deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: V 202412.

[0009] The virus solution containing the above-mentioned attenuated virus strain of largemouth bass rhabdovirus also belongs to the protection scope of the present invention.

[0010] The use of the above-mentioned attenuated largemouth bass rhabdovirus strain in the preparation of anti-fish rhabdovirus vaccine also falls within the protection scope of the present invention.

[0011] Furthermore, the fish is at least one of sea bass, mandarin fish, snakehead fish and bamboo fish.

[0012] The use of the above-mentioned attenuated largemouth bass rhabdovirus strain in the preparation of a preparation for preventing and treating fish rhabdovirus diseases also falls within the protection scope of the present invention.

[0013] The present invention also provides an anti-largemouth bass rhabdovirus vaccine, wherein the active component of the vaccine comprises the largemouth bass rhabdovirus attenuated virus strain.

[0014] Furthermore, the vaccine is an attenuated vaccine.

[0015] Furthermore, the vaccine is an immersion immunization vaccine.

[0016] The present invention also provides a method for preparing the above-mentioned anti-largemouth bass rhabdovirus vaccine, comprising the steps of: inoculating the largemouth bass rhabdovirus attenuated virus strain described in claim 1 into CPB cells for culture, obtaining a purified clone strain through 5 rounds of plaque purification, and then extracting the virus liquid.

[0017] Furthermore, the culture medium used in the culture is L15 culture medium containing 2% low melting point agarose and 5% FBS.

[0018] The invention also provides a preparation for preventing and treating largemouth bass rhabdovirus, wherein the preparation contains the largemouth bass rhabdovirus strain.

[0019] The attenuated virus strain of largemouth bass rhabdovirus provided by the present invention has been patented and the deposit information is as follows:

[0020] Biological material (strain): Perch rhabdovirus MsRV-0509

[0021] Classification name: Rhabdovirus

[0022] Deposit date: February 28, 2024

[0023] Deposit number: CCTCC NO:V202412

[0024] Depository: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province

[0025] Address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province (opposite the main entrance of the First Affiliated Primary School).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The invention obtains a largemouth bass rhabdovirus strain MsRV-0509 by isolating it from nature, purifies it through plaques, and conducts biological characteristic research to confirm that the MsRV-0509 strain is a weak strain with good immunogenicity, and has the advantages of long-lasting immunity, good effect, safety and reliability, etc.; it can be used as a candidate strain for a largemouth bass rhabdovirus attenuated vaccine. The results of the immune research show that both intraperitoneal injection and immersion immunization can play an effective protective role, and the immersion immunization has a better immune effect, and can also effectively protect seedlings after the virus is attacked, which provides technical support for the development of an effective rhabdovirus immersion vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of plaque purification;

[0029] Figure 2 This is the optimization result of the proliferation conditions of MsRV-0509;

[0030] Figure 3 is the growth curve of MsRV-0509;

[0031] Figure 4 The results of the virulence reversion test of MsRV-0509;

[0032] Figure 5 The tissue distribution and growth and decline patterns of MsRV-0509 in the body;

[0033] Figure 6 The safety evaluation results of MsRV-0509;

[0034] Figure 7 The results of intraperitoneal vaccination of MsRV-0509;

[0035] Figure 8 Immersion immunization results for MsRV-0509. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with examples. It should be understood that the following examples are only used as explanations and illustrations of the present invention and do not limit the scope of the present invention in any way.

[0037] Biomaterials:

[0038] MsRV virus strain: that is, largemouth bass rhabdovirus, isolated and preserved by the laboratory of the Fish Disease Department of the Pearl River Fisheries Research Institute.

[0039] CPB cells: infectious spleen and kidney necrosis virus, can be isolated in the laboratory or purchased. This embodiment is isolated and stored in the laboratory of the fish disease room of the Pearl River Fisheries Research Institute.

[0040] The sea bass was purchased from Guangdong Dequan Fishery Development Co., Ltd.

[0041] Reagents: All are commercially available.

[0042] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0043] Example 1 Isolation, culture and identification of MsRV-0509 virus strain

[0044] 1.1 Separation

[0045] The kidneys and spleens of the diseased fish were ground and centrifuged at 5000 rpm / min for 5 min. The supernatant was filtered and inoculated into a monolayer of CPB cells. After incubation for 1 h, L15 culture medium containing 2% FBS was added and placed in a 28°C incubator for culture. The cells were observed under a microscope every day. If more than 90% of the cells had lesions and cell detachment, they were collected and placed in a low-temperature refrigerator for later use.

[0046] 1.2 Plaque purification

[0047] CPB cells were seeded into 6-well plates and grown overnight until 90% monolayer was achieved. The virus was diluted to 10 -4 , 10 -5 , 10 -6 The plate was inoculated into a 6-well plate, 500 μl / well, with 2 replicates for each dilution, and two blank controls were set; after incubation in a 28°C incubator for 1 hour, the virus solution was aspirated, and L15 culture medium containing 2% low melting point agarose and 5% FBS was added, 1 ml / well, and sealed with a sealing film after solidification, and cultured in a 28°C incubator for 4 days; 0.1% neutral red stock solution was diluted to a final concentration of 1 / 10000-1 / 5000, and then stained in a dark place for 1-3 hours; a single plaque was selectively picked with a 200 μl pipette tip, the picked plaque was added to L15 for dilution, and then inoculated on CPB for amplification; the supernatant of the cells with lesions was frozen and thawed twice, and the next round of infection and plaque isolation was carried out according to the same method, and a total of five rounds of plaque purification were repeated.

[0048] Purified clones were obtained by 5 rounds of plaque purification. Figure 1 As shown, Figure 1 A is an example of a virus plaque. Figure 1 B is the virus titer of each clone strain, and the titer of each strain was measured separately, and finally MsRV-0509 with a higher virus titer was selected for subsequent research.

[0049] 1.3 Virus strain sequencing

[0050] Extract RNA from the virus liquid and perform reverse transcription to obtain cDNA, and then perform PCR amplification with the following primers. Recover the PCR bands after electrophoresis, connect to the T vector, clone and transform, and send the obtained positive colonies for sequencing. At least 3 samples are sent for each fragment. The full-length sequence of the strain is obtained after comparison of the sequencing results.

[0051] Table 1 Sequencing primers

[0052]

[0053] Example 2 Biochemical and immunological tests of MsRV-0509 virus strain

[0054] 2.1 Optimization of proliferation conditions of MsRV-0509

[0055] CPB cells were cultured overnight to grow to 90% monolayer, and infected with virus at different multiplicity of infection (MOI: 1, 0.5, 0.1, 0.01, 0.001, 0.0001). The virus was collected when the cell lesion rate reached 80% to 90%. The titer was measured after repeated freezing and thawing twice, and the virus copy number was measured by fluorescence quantitative PCR. Figure 2 shown.

[0056] It can be seen that the virus titer is highest when MsRV is inoculated with an MOI of 0.0001, and subsequent experiments can be based on this condition.

[0057] 2.2 Growth curve of MsRV-0509

[0058] The prepared monolayer CPB cells were infected with the virus at 0.0001 MOI (multipicity of infection), and samples were collected every 12 hours after inoculation. After repeated freezing and thawing twice, the samples were aliquoted and stored at -80°C. The virus titer was determined and the virus growth curve was drawn. Figure 3 shown.

[0059] It can be seen that the titer of MsRV-0509 virus was the highest at 96h after infection, which was 10 8.571 TCID50 / mL, after which the virus titer begins to decrease, so the best time to collect the virus is around 96 hours after infection.

[0060] 2.3 Virulence determination of MsRV-0509

[0061] The known strong strains MsRV-SS and MsRV-0509 were diluted 10 times and inoculated into the peritoneum of sea bass to determine the LD of the virus. 50 .

[0062] The LD50 of the highly virulent MsRV-GM strain is 10 -6 / ml, and the highest concentration of MsRV-0509 was 10 -1 The mortality rate after vaccination was around 10%, which did not reach the half mortality rate, and it also clearly confirmed that the virulence of the MsRV-0509 strain was weak and it was a weak strain.

[0063] 2.4 Virulence reversion test of MsRV-0509

[0064] MsRV-0509 was soaked and injected intraperitoneally into sea bass, and 48 hours after inoculation, the liver, spleen, kidney and brain of sea bass were taken and ground to obtain the diseased F1 generation, and then the F1 was centrifuged and filtered to make tissue toxin for blind transmission experiment. The obtained tissue toxin was diluted 4 times and then soaked and injected intraperitoneally into sea bass, and then passed to F5 generation in sequence, and the tissue samples of each generation were tested. Figure 4 As shown, M.DL2000; 1-5: tissue samples of F1 to F5 California seabass infected with MsRV; 6: negative control; (a)(b) are the qPCR and conventional PCR test results of the immersion infection group; (c)(d) are the qPCR and conventional PCR test results of the intraperitoneal injection infection group.

[0065] There were no deaths among the blind-transmitted largemouth bass of five generations infected with the virus by immersion and intraperitoneal injection during the observation period; the virus was detected only in the body tissues of the blind-transmitted F1 fish in the immersion infection group, while the virus was no longer detected in the F2 largemouth bass infected with tissue virus; a small amount of virus was detected in the F1 and F2 generations of the intraperitoneal injection infection group, indicating that the vaccine strain will not experience reversion to virulence.

[0066] 2.5 Tissue distribution and growth and decline of MsRV-0509 in vivo

[0067] Largemouth bass were immunized by intraperitoneal injection with a dose of 5×10 2 TCID 50 / tail, the brain, heart, liver, spleen, head kidney, intestine, gill, stomach and muscle of 3 largemouth bass were weighed on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 14th day after immunization, and stored at -80℃. The virus content in the tissues was detected. The results were as follows: Figure 5 shown.

[0068] MsRV-0509 was detected in the brain, heart, liver, spleen, head kidney, intestine, gills, stomach and muscle tissues of largemouth bass. The viral expression levels in all tissues reached the highest on the first day, began to decline on the second day, and were undetectable in all tissues on the seventh day.

[0069] 2.6 Safety evaluation of MsRV-0509 strain

[0070] Largemouth bass weighing 3±0.5 g and 5±0.5 cm in length were immunized by immersion and intraperitoneal injection, respectively, and observed for 14 days, and the data were recorded.

[0071] The results are as follows Figure 6 As shown, Figure (a) is immersion immunization, and Figure (b) is intraperitoneal injection immunization. There was no death in the immersion immunization group during the observation period, and the vaccine strain was safe and reliable through immersion immunization; in the intraperitoneal injection group, the immunization dose was 5×10 3 TCID50, 5×10 2 The survival rates of largemouth bass at TCID50 were 93% and 96% respectively. The vaccine strain was immunized by intraperitoneal injection and was safe and reliable.

[0072] 2.7 Safety evaluation of overdose inoculation of MsRV-0509 in seabass

[0073] Largemouth bass weighing 3±0.5g and 5±0.5cm in length were immunized by immersion or intraperitoneal injection in three batches. The samples were observed for 10 consecutive days and the data were recorded.

[0074] The results are shown in Tables 2 and 3. There was no death during the observation period of the single-dose immunization, indicating that the vaccine strain was safe and reliable through immersion immunization. There were 3 deaths in one batch during the observation period of the single-dose immunization by intraperitoneal injection, with a mortality rate of 10%. There was no death in the other two batches during the observation period, indicating that the vaccine strain was safe and reliable through injection immunization.

[0075] Table 2 Excessive dosage for one soak

[0076]

[0077] Table 3 Intraperitoneal injection of a single superdose

[0078]

[0079] 2.8 Intraperitoneal immunization of MsRV-0509

[0080] Largemouth sea bass weighing 3±0.5g and 5±0.5cm in length were immunized by intraperitoneal injection. Strong toxin was injected intraperitoneally 21 and 28 days after immunization to verify the efficacy of the immunity. The fish were observed for 10 consecutive days, and the data were recorded to calculate the relative immune protection rate (RPS).

[0081] The results are as follows Figure 7 As shown, (a) shows the cumulative mortality of largemouth bass injected with strong toxins intraperitoneally 21 days after immunization; (b) shows the cumulative mortality of largemouth bass injected with strong toxins intraperitoneally 28 days after immunization.

[0082] The challenge group showed that the immune dose was 5×10 4 TCID 50, 5×10 3 TCID 50 , 5×10 2 TCID 50 The relative immune protection rate of largemouth bass was 100%, and the immune dose was 5×10 1 TCID 50 , 5TCID 50 , the relative immune protection rates of largemouth bass were 79.2% and 66.7% respectively;

[0083] The challenge group showed that the immune dose was 5×10 3 TCID 50 , 5×10 2 TCID 50 The relative immune protection rate of largemouth bass was 100%, and the immune dose was 5×10 1 TCID 50 , 5TCID 50 , 5×10 -1 TCID 50 The relative immune protection rates of largemouth bass were 83.0%, 53.4% ​​and 56.5% respectively.

[0084] 2.9MsRV-0509 immersion immunity

[0085] Largemouth bass weighing 3±0.5g and 5±0.5cm in length were immunized by immersion. Strong toxin was injected intraperitoneally 21 and 28 days after immunization to verify the efficacy of the immunity. The fish were observed for 10 consecutive days, and the data were recorded to calculate the relative immune protection rate (RPS).

[0086] The results are as follows Figure 8 As shown, (a) the cumulative mortality of largemouth bass injected with strong venom 21 days after immunization; (b) the cumulative mortality of largemouth bass injected with strong venom 28 days after immunization.

[0087] The challenge group 21 days after immunization showed that the relative immune protection rates of largemouth bass were 100%, 75.0%, 52.3%, 9.5%, and 14.3% at immunization doses of 106TCID50 / mL, 105TCID50 / mL, 104TCID50 / mL, 103TCID50 / mL, and 102TCID50 / mL, respectively;

[0088] The challenge group showed that the relative immune protection rates of largemouth bass were 100%, 26.7%, 26.0%, and 0 when the immunization doses were 106TCID50 / mL, 105TCID50 / mL, 104TCID50 / mL, and 103TCID50 / mL, respectively.

[0089] 2.10 Determination of neutralizing antibodies after MsRV-0509 immunization

[0090] 14 days after the seabass was immunized with MsRV-0509, blood was collected and serum was prepared, which was treated in a 56°C water bath for 30 minutes and then packaged. After the serum was diluted 2-fold in series, 100 μl of serum was taken and added with an equal volume of virus solution, incubated for 1 hour, and inoculated into CPB cells that had grown to a monolayer, with 4 replicates for each sample; virus control wells and cell control wells were set up; the cells were cultured in a 28°C incubator; and the serum neutralization effect was observed after 10 days.

[0091] The results showed that 14 days after the sea bass was immunized with MsRV-0509, blood was collected and serum was prepared. The neutralizing effective titer of the serum after 2-fold serial dilution was 1:2 8 , which proves that immunization can induce the body to produce effective protective antibodies.

[0092] In summary, the present invention can obtain a largemouth bass rhabdovirus strain MsRV-0509 by isolating it from nature, purifying it through plaques, and conducting biological property studies to confirm that the MsRV-0509 strain is a weak strain with good immunogenicity, and has the advantages of long-lasting immunity, good effect, safety and reliability; it can be used as a candidate strain for largemouth bass rhabdovirus attenuated vaccine. Further immune research results show that both intraperitoneal injection and immersion immunization can play an effective protective role, and immersion immunization has a better immune effect, and can also effectively protect seedlings after attacking the virus, providing technical support for the development of an effective rhabdovirus immersion vaccine.

[0093] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0094] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A attenuated virus strain of largemouth bass rhabdovirus, characterized in that: It was named as sea bass rhabdovirus MsRV-0509 and has been deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: V202412.

2. Use of the attenuated largemouth bass rhabdovirus strain according to claim 1 in the preparation of an anti-fish rhabdovirus vaccine, wherein the vaccine is an attenuated vaccine.

3. The use according to claim 2, wherein the fish is at least one of sea bass, mandarin fish, snakehead fish, and bamboo fish.

4. Use of the attenuated largemouth bass rhabdovirus strain according to claim 1 in the preparation of a preparation for preventing and treating fish rhabdovirus diseases.

5. An anti-largemouth bass rhabdovirus vaccine, characterized in that: The active ingredients of the vaccine include the attenuated virus strain of largemouth bass rhabdovirus according to claim 1, and the vaccine is an attenuated vaccine.

6. The vaccine according to claim 5, characterized in that The vaccine is an immersion immunization vaccine.

7. The method for preparing a vaccine according to claim 6, characterized in that: Includes steps: The attenuated virus strain of largemouth bass rhabdovirus according to claim 1 is inoculated into CPB cells for culture, and purified by plaque purification for 5 rounds to obtain a purified clone strain, and then the virus liquid is extracted.

8. The method for preparing a vaccine according to claim 7, characterized in that: The culture medium used in the culture is L15 medium containing 2% low melting point agarose and 5% FBS.

9. A preparation for preventing and treating largemouth bass rhabdovirus, characterized in that: The preparation contains the largemouth bass rhabdovirus strain according to claim 1.

Images