A method for establishing a grass carp GCRV infection model
By using the infection method of mixing virus feed in grass carp, the problems of unnatural infection pathways and unclear dose quantification in the existing technology were solved, more accurate and stable experimental results were achieved, and the natural virus transmission process was simulated, providing a more effective strategy for the prevention and control of grass carp hemorrhage disease.
Patent Information
- Application Number
- CN202410602458.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-15
- Publication Date
- 2025-03-18
- Estimated Expiration
- 2044-05-15
AI Technical Summary
The existing methods of artificial infection of viral infection have limitations in grass carp, resulting in instability and accuracy of experimental results, especially in the simulation of natural infection pathways and quantitative infection doses.
Grass carp is infected by mixing virus feed. By mixing the virus with puffed feed, the natural infection pathway is simulated, and quantitative methods and clear judgment standards are used to ensure the standardization of the infection process.
This method effectively avoids accidental damage to fish during infection, ensures the phenotype accuracy of the fish after infection, improves the stability and reliability of experimental results, simulates the natural virus transmission and infection process, and provides a more effective strategy for disease prevention and control.
Smart Images
Figure CN118592401B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of virus biology, and in particular to a method for establishing a grass carp GCRV infection model. Background Art
[0002] Grass carp (Ctenopharyngodon idella) is a traditional high-quality farmed fish in my country with a wide farming area. However, its fingerling stage is susceptible to hemorrhagic disease caused by grass carp reovirus (GCRV), and its survival rate is low. It has always been the biggest disease in the fish fingerling breeding stage. GCRV not only infects grass carp, but also can infect fish such as black carp, causing huge losses to my country's freshwater fisheries. According to the results of the aquatic animal disease monitoring and investigation conducted by the Ministry of Agriculture and Rural Affairs in 2020, grass carp hemorrhagic disease had the highest positive detection rate among the 9 key aquatic animal diseases monitored. Therefore, the prevention and control of hemorrhagic disease and the cultivation of disease-resistant strains have become topics that need to be urgently studied in the current grass carp farming industry.
[0003] Artificial virus infection experiments are an indispensable part of the prevention and control of grass carp hemorrhagic disease, breeding of new disease-resistant strains, and research and development of virus vaccines. Through artificial infection experiments, we can simulate the natural infection environment and observe the pathological changes, immune response, and survival ability of grass carp after virus infection, providing a scientific basis for disease prevention and control.
[0004] However, the currently commonly used artificial virus infection methods all have certain limitations, which have affected the stability and accuracy of the experimental results, especially in the accurate identification of individual phenotypes. The intraperitoneal injection infection method can effectively introduce the virus into the experimental fish, but it may cause greater physiological stress to the experimental fish and lead to accidental death of the experimental fish. The oral gavage method may produce similar results due to improper operation, etc. The hypertonic immersion infection method may cause osmotic pressure imbalance in the experimental fish and have adverse effects on its physiological state. The above methods are not natural infection routes, so there may be problems such as the disease symptoms exhibited by the experimental fish after infection with the virus are quite different from those of natural infection. Summary of the invention
[0005] In view of this, the present invention provides a method for establishing a grass carp GCRV infection model suitable for large-scale virus artificial infection experiments.
[0006] The method of infecting experimental fish with virus-mixed feed adopted by the present invention simulates the natural infection route of the virus, effectively avoids accidental damage to the fish body during the infection process, and avoids death caused by non-viral infection factors during the experiment, thereby ensuring the accuracy of the phenotype of the fish body after infection with the virus. The method of the present invention can better simulate the spread and infection process of the virus in the natural environment, and provide a more effective strategy for the prevention and control of the disease.
[0007] In addition, the present invention also provides a quantitative method for the virus intake dose and a clear judgment standard, thereby achieving standardization of the infection process and making the artificial infection results more stable and reliable.
[0008] The grass carp GCRV infection model provided by the present invention has good reproducibility. When used in experiments such as breeding new disease-resistant strains and developing virus vaccines, the results are highly credible and the cumbersome operations caused by injection, gavage and other methods are reduced, thereby improving experimental efficiency and making large-scale experiments more feasible.
[0009] The technical solution of the present invention is achieved in this way:
[0010] In a first aspect, the present invention provides a method for establishing a grass carp GCRV infection model, comprising the following steps:
[0011] S1 collected diseased fish naturally infected with GCRV in the wild, quickly frozen them at -4℃ for later use, took intestinal tissues of diseased fish, extracted RNA, reverse transcribed to obtain cDNA, and used PCR virus typing and identification using specific primers for three subtypes of GCRV;
[0012] S2 collects muscle and visceral tissue samples of the diseased fish, weighs them, adds sterile PBS at a weight ratio of 1:3, and grinds them to obtain a virus suspension, centrifuges and obtains the supernatant to obtain the virus stock solution, performs reverse transcription to obtain cDNA, and then uses qPCR to detect the virus titer in the supernatant. After determining the virus titer, dilute it with PBS to obtain a virus solution, and freeze it at -80°C for later use;
[0013] S3 Select healthy grass carps of the same family weighing 8-10g and with negative GCRV test results, divide them into experimental group and control group, put them into different breeding tanks, and control the water temperature at 26-28℃; artificially train the grass carps for 7-10 days, so that the grass carps can form conditioned reflexes and actively grab food;
[0014] S4: after the virus solution is revived at 28°C for 2 hours, it is mixed evenly with grass carp extruded feed in a weight ratio of 1:1 to prepare a virus-mixed feed.
[0015] Toxic feed;
[0016] The S5 experimental group was fed with virus-mixed feed in the breeding tank, while the control group was fed with PBS-mixed feed in the breeding tank. The dosage of PBS in the PBS-mixed feed was equal to the dosage of the virus liquid in the virus-mixed feed. Each group was fed twice a day, and the number of deaths and symptoms were observed and recorded until no deaths occurred in all groups for 7 consecutive days.
[0017] In a second aspect, the present invention provides an application of a grass carp GCRV infection model, wherein the application includes at least one of the following applications: (1) screening and breeding new disease-resistant strains; (2) evaluating the safety and effectiveness of GCRV vaccines; (3) screening and developing drugs for preventing and treating GCRV infection; (4) developing methods for preventing and treating GCRV infection; and (5) determining the viral activity and immunogenicity of GCRV strains in fish. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0019] Figure 1 It is a virus typing identification diagram of an embodiment of the present invention;
[0020] Figure 2 This is a statistical diagram of artificial infection results according to an embodiment of the present invention;
[0021] Figure 3 The phenotype diagram of the artificial infection group and the control group of the embodiment of the present invention;
[0022] Figure 4 RT-PCR identification diagram of GCRV of the embodiment of the present invention;
[0023] Figure 5 These are electron microscopic images of virus particles in the artificial infection group and the control group of the embodiment of the present invention. DETAILED DESCRIPTION
[0024] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.
[0025] Artificial infection experiments with GCRV are essential links in the prevention and control of grass carp hemorrhagic disease, the screening and breeding of new disease-resistant strains, and the research and development of drugs or vaccines for the prevention and treatment of GCRV infection. However, the commonly used artificial infection methods have technical problems such as the inability to accurately identify individual phenotypes, the lack of clear judgment standards for the quantification of virus doses, and the inability of infection routes to simulate natural infection routes.
[0026] Individual phenotype specifically refers to the phenotypic characteristics exhibited by an individual after infection. Individual phenotype is directly and closely related to the infection dose, infection route and operation process in artificial infection experiments.
[0027] Studies have shown that there are significant differences in clinical symptoms, target organs, tissue pathology and virus distribution between the intraperitoneal injection infection method, which is most commonly used in artificial infection experiments, and naturally diseased fish. These differences are related to the fact that the injection infection method uses artificially enriched viruses and that GCRV does not enter the fish body through water as a medium.
[0028] In view of this, the embodiments of the present application disclose at least the following technical solutions:
[0029] In a first aspect, the present invention provides a method for establishing a grass carp GCRV infection model, comprising the following steps:
[0030] S1 collected diseased fish naturally infected with GCRV in the wild, quickly frozen them at -4℃ for later use, took intestinal tissues of diseased fish, extracted RNA, reverse transcribed to obtain cDNA, and used PCR virus typing and identification using specific primers for three subtypes of GCRV;
[0031] S2 collects muscle and visceral tissue samples of the diseased fish, weighs them, adds sterile PBS at a weight ratio of 1:3, and grinds them to obtain a virus suspension, centrifuges and obtains the supernatant to obtain the virus stock solution, performs reverse transcription to obtain cDNA, and then uses qPCR to detect the virus titer in the supernatant. After determining the virus titer, dilute it with PBS to obtain a virus solution, and freeze it at -80°C for later use;
[0032] S3 Select healthy grass carps of the same family weighing 8-10g and with negative GCRV test results, divide them into experimental group and control group, put them into different breeding tanks, and control the water temperature at 26-28℃; artificially train the grass carps for 7-10 days, so that the grass carps can form conditioned reflexes and actively grab food;
[0033] S4: after the virus solution is revived at 28°C for 2 hours, it is mixed evenly with grass carp extruded feed in a weight ratio of 1:1 to prepare a virus-mixed feed.
[0034] Toxic feed;
[0035] The S5 experimental group was fed with virus-mixed feed in the breeding tank, while the control group was fed with PBS-mixed feed in the breeding tank. The dosage of PBS in the PBS-mixed feed was equal to the dosage of the virus liquid in the virus-mixed feed. Each group was fed twice a day, and the number of deaths and symptoms were observed and recorded until no deaths occurred in all groups for 7 consecutive days.
[0036] In some preferred embodiments, in step S3, the amount of feed fed each time is gradually adjusted according to the feeding situation of the grass carp, until the feed fed each time is consumed within 5 minutes.
[0037] In this embodiment, in order to avoid the feed being soaked in water for too long to affect the virus infection effect, and the feed being gradually dispersed to cause the water quality to deteriorate, thereby interfering with the accuracy of the experiment, the present invention designs a standard process for artificial feeding to ensure that the feed is completely consumed within an appropriate time. This can be achieved by accurately controlling the feed feeding amount and feeding time to ensure that the feed is completely consumed by the grass carp in a relatively short time, thereby maintaining the stability of the water quality and the accuracy of the experimental results.
[0038] In some preferred embodiments, in step S4, the virus liquid and the puffed feed are mixed evenly and then left to stand for 15 minutes to allow all the virus liquid to be absorbed into the puffed feed.
[0039] In this embodiment, the standing time is precisely controlled to ensure that the virus activity and feed quality are stable and unaffected, and at the same time, the virus liquid is completely absorbed by the puffed feed and evenly distributed therein, thereby improving the infection effect and more accurately simulating the distribution pattern of the virus in the natural infection pathway to ensure that the experimental results are accurate and reliable.
[0040] In some preferred embodiments, in step S5, each group is fed with the virus-mixed feed or PBS-mixed feed for 6 consecutive days, and the total daily amount of the virus-mixed feed or PBS-mixed feed for each group is 0.5% of the fish body weight.
[0041] In some preferred embodiments, in step S5, the diseased fish is dissected and samples are taken for virus isolation and identification.
[0042] In some preferred embodiments, the method further comprises the following steps:
[0043] In step S2, the virus stock solution is diluted into virus solutions with different concentration gradients, so as to obtain virus titers C1, C2...C n of virus solution, where C1 is the virus titer of the virus stock solution, n≥3;
[0044] In step S3, the number of experimental groups is n, the number of grass carps selected from each experimental group and the control group is the same, and each group has at least 120 grass carps;
[0045] In step S4, the virus titers of the virus liquid used in the preparation of the virus-mixed feed of each experimental group correspond to C1, C2...C n ;
[0046] Finally, the minimum value of the virus titer corresponding to the experimental group with a mortality rate ≥ 80% in the statistical results was taken as the virus titer of the experimental group infection, and the grass carp GCRV infection model was established.
[0047] In the GCRV artificial infection experiments for the prevention and control of grass carp hemorrhagic disease, screening and breeding of new disease-resistant strains, and research and development of relevant drugs or vaccines for the prevention and treatment of GCRV infection, the judgment standard of the virus infection dose was determined according to the mortality rate (≥80%) of grass carp after infection with GCRV under natural conditions, and a grass carp GCRV infection model was established on this basis, which can better simulate the virus infection dose under natural conditions and avoid experimental errors caused by artificial enrichment of the virus.
[0048] In some preferred embodiments, the virus titer of the virus solution in step S2 is 5×10 3 copy / μL.
[0049] In some preferred embodiments, the reaction system of PCR in step S1 includes: 2×PCRMix 10μ1, cDNA template 1μ1, forward primer 1μ1, reverse primer 1μ1 and RNase-free H2O 7ul, and the total volume of the reaction system is 20μ1.
[0050] In some preferred embodiments, the primers for PCR in step S1 include:
[0051] The forward primer sequence of type I GCRV is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2;
[0052] The forward primer sequence of type II GCRV is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4;
[0053] The forward primer sequence of type III GCRV is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6.
[0054] In some preferred embodiments, the qPCR reaction system in step S2 includes: 2×SYBR qPCR Mix 10 μl, cDNA template 1 μl, forward primer 0.8 μl, reverse primer 0.8 μl and RNase-free H2O 7.4 ul, and the total volume of the reaction system is 20 μl.
[0055] In a second aspect, the present invention discloses an application of a grass carp GCRV infection model, wherein the grass carp GCRV infection model is established according to any one of the above-mentioned methods for establishing a grass carp GCRV infection model, and the application includes at least one of the following applications: (1) screening and breeding new disease-resistant strains; (2) evaluating the safety and effectiveness of GCRV vaccines; (3) screening and developing drugs for preventing and treating GCRV infection; (4) developing methods for preventing and treating GCRV infection; and (5) determining the viral activity and immunogenicity of GCRV strains in fish.
[0056] Example 1
[0057] In the embodiment, firstly, diseased fish samples are collected in the wild, RNA is extracted and virus typing is identified from the samples, then the virus stock solution is isolated from the tissue of the diseased fish samples, the virus titer is measured, and virus solution of different dilutions is prepared; then, suitable experimental fish are selected for grouping and artificial feeding, and virus-mixed feed is prepared, and the virus-mixed feed is used for artificial infection experiments, and the infection results are observed and counted to determine the most suitable infection concentration of the virus.
[0058] The specific experimental steps and experimental results are as follows:
[0059] 1.1 Experimental steps (1) Sample collection and virus identification
[0060] During the outbreak season of grass carp hemorrhagic disease, sick fish with typical hemorrhagic disease symptoms were collected from wild ponds, quickly frozen in an ice box at -4°C, and taken back to the laboratory to obtain intestinal tissue of the sick fish. RNA was extracted using an RNA extraction kit (Nanjing Novozymes Biotech Co., Ltd., RC112-01), and the RNA samples were reverse transcribed using a reverse transcription kit (Nanjing Novozymes Biotech Co., Ltd., R212-01) to obtain cDNA, and PCR virus typing was performed using specific primers for the three subtypes of GCRV. Specific primers were set for the S6 segment coding sequences of the three types of GCRV, and PCR virus typing was performed using specific primers for the three subtypes of GCRV.
[0061] Specific primers were set for the S6 segment coding sequences of three types of GCRV, and the sequences are as follows:
[0062] Type I GCRV forward primer 5'-GTTTACTTCTTATCGGGACTTTATGACCG-3' (as shown in SEQ ID NO.1), reverse primer 5'-CGGTGAGCCTAATCATCAACAGACC-3' (as shown in SEQ ID NO.2);
[0063] Type II GCRV forward primer 5'-GGCGGCTGCTATGATACCAGTAGAC-3' (as shown in SEQ ID NO.3), reverse primer 5'-GTCGCTTCAGATCAATCACCAAATC-3' (as shown in SEQ ID NO.4);
[0064] The forward primer of type III GCRV is 5'-GTTTACTTCTTATCGGGACTTTATGACCG-3' (as shown in SEQ ID NO.5), and the reverse primer is 5'-CGGTGAGCCTAATCATCAACAGACC-3' (as shown in SEQ ID NO.6).
[0065] PCR reaction system: 2×PCR Mix 10μ1, cDNA template 1μ1, forward primer 1μ1, reverse primer 1μ1, RNase-free H2O 7ul, the total volume of the reaction system is 20μ1.
[0066] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 30 s, 32 cycles, and final extension for 5 min.
[0067] (2) Isolation of virus stock solution, determination of titer, and dilution to prepare virus solution
[0068] Muscles and visceral tissues such as liver, intestine, spleen, and kidney of diseased fish were taken, weighed, and then added to sterile PBS in a ratio of 1:3 for homogenization and grinding to obtain a virus suspension. The virus supernatant (i.e., virus stock solution) was further obtained by centrifugation, and reverse transcription was performed using a reverse transcription kit (Nanjing Novozyme Biotechnology Co., Ltd., R212-01) to obtain cDNA, and the virus in the supernatant was detected by qPCR.
[0069] The primer sequences for qPCR were: forward 5'-AGCGCAGCAGGCAATTACTATCT-3' (as shown in SEQ ID NO.7), reverse 5'-ATCTGCTGGTAATGCGGAACG-3 (as shown in SEQ ID NO.8).
[0070] The qPCR reaction system was: 2×SYBR qPCR Mix 10μ1, cDNA template 1μ1, forward primer 0.8μ1, reverse primer 0.8μ1, RNase-free H2O 7.4ul, and the total volume of the reaction system was 20μ1.
[0071] The qPCR reaction conditions were as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, 40 cycles; fluorescence signal collection at 65°C-95°C.
[0072] Based on the determination of the virus titer, the virus stock solution was further diluted 2-fold, and the dilution gradient was 5. The virus stock solution and the dilution solution were frozen at -80°C for later use.
[0073] (3) Experimental fish grouping and artificial domestication
[0074] The experimental fish used in the present invention were all purchased from Wuhan Huangyouyuan Fishery Company, specifically the same batch of family fry bred by the company on May 6, 2023.
[0075] Grass carp weighing 8-10g were selected and randomly divided into 6 groups, with 120 fish in each group. They were placed in 6 breeding tanks for domestication. The diameter of the breeding tank was 1.2 meters, the volume was 1000L, and the water temperature was controlled between 26-28℃. After artificial feeding for 7-10 days, the grass carp could form conditioned reflexes and actively grab food. According to the feeding situation of the grass carp, the amount of feed fed each time was gradually adjusted until the feed was eaten within 5 minutes each time.
[0076] (4) Preparation of virus-mixed feed
[0077] The virus stock solution and dilution stored at -80°C were thawed at 28°C for 2 hours, and then the virus solution was mixed evenly with grass carp extruded feed (Wuhan Dabeinong Aquatic Technology Co., Ltd., item number: 8930) at a weight ratio of 1:1, and allowed to stand for 15 minutes to allow the virus solution to be completely absorbed into the extruded feed. The PBS dose in the PBS-mixed feed of the control group was equal to the dose of the virus solution in the virus-mixed feed.
[0078] (5) Artificial infection
[0079] Virus-mixed feed was put into the breeding tank of the experimental group, so that the experimental fish were infected with the virus by eating the feed, while the same method was used to put PBS-mixed feed into the breeding tank of the control group.
[0080] All the above groups were fed twice a day, and each group was fed with the virus-mixed feed or PBS-mixed feed for 6 consecutive days. The total amount of virus-mixed feed or PBS-mixed feed per day for each group was 0.5% of the fish body weight.
[0081] All the above groups were continuously observed every day, and the number of individuals who died in each group was recorded every day. The infection experiment was considered to be over when no deaths occurred in all groups for 7 consecutive days. The final mortality rate of each group was calculated and compared.
[0082] 1.2 Experimental Results
[0083] In the above step (1), the result of virus typing identification is as follows Figure 1As shown, the virus that infected the sick fish was type II GCRV (GCRV-II), which is the GCRV strain prevalent in the main grass carp breeding areas in my country.
[0084] Table 1 Death statistics after infection with different concentrations of virus
[0085] Dilution Virus concentration Number of survivors Number of deaths mortality rate Virus stock solution <![CDATA[4×10 4 copy / μL]]> 18 102 85.00％ 2-fold dilution <![CDATA[2×10 4 copy / μL]]> 21 91 82.50％ 4-fold dilution <![CDATA[1×10 4 copy / μL]]> 20 100 83.30％ 8-fold dilution <![CDATA[5×10 3 copy / μL]]> 22 98 81.60％ 16-fold dilution <![CDATA[2.5×10 3 copy / μL]]> 57 63 52.50％ Control group none 120 0 0
[0086] In the above step (2), as shown in Table 1, the virus titer in the virus stock solution was 4×10 4 The results of infection with different concentrations of virus are shown in Table 1. 4 copy / μL) and 2 times (2×10 4 copy / μL), 4 times (1×10 4 copy / μL), 8 times (5×10 3 The mortality rates of the groups infected with 16-fold virus dilution (2.5×10 3 The mortality rate of the group with less than 100 copies / μL was only 52.50%, which was significantly lower than the mortality rate of grass carp infected with GCRV under natural conditions; no dead individuals appeared in the control group during the entire experiment.
[0087] The results showed that 5×10 3 The virus concentration of 0.1 copy / μL is the appropriate infection concentration.
[0088] Example 2
[0089] In the example, 5×10 3 copy / μL of virus liquid; then select suitable experimental fish for grouping and artificial feeding, and then prepare virus-mixed feed, use the virus-mixed feed in artificial infection experiments, observe and count the infection results, dissect the dead individuals, resample for electron microscopy observation, and isolate and identify the virus.
[0090] The specific experimental steps and experimental results are as follows:
[0091] 2.1 Experimental procedures
[0092] (1) Same as described in experimental procedure 1.1(1).
[0093] (2) The difference from step 1.1(2) is that in this step, only 5×10 3 The virus solution was 100 copies / μL, and the rest was the same.
[0094] (3) The difference from experimental step 1.1(3) is that in this step, grass carp weighing 8-10 g were selected and randomly divided into two groups, with 120 fish in each group, and placed in two breeding tanks for domestication, while the rest were the same.
[0095] (4) Same as described in experimental step 1.1(4).
[0096] (5) Other procedures are the same as described in Experimental Step 1.1(5), with the only difference being that the number of individuals who died in each group each day was recorded and a mortality curve was drawn, the clinical symptoms of the deceased individuals were detected, and 10 intestinal, kidney and other tissues of the deceased individuals in the virus-infected group were randomly selected for RT-PCR identification (the reverse transcription and PCR identification steps were both referred to type II GCRV in Example 1) and electron microscopy observation.
[0097] 2.2 Experimental Results
[0098] like Figure 2 As shown, grass carp fed with virus-containing feed began to die on the 7th day after infection, reaching a peak of mortality on the 10th day, with a final mortality rate of 82.50%. No diseased or dead individuals appeared in the control group during this period.
[0099] like Figure 3 As shown, most of the dead individuals showed bleeding on the body surface, muscles, oral cavity and other parts, which are typical symptoms of grass carp hemorrhagic disease.
[0100] like Figure 4 As shown, 10 dead individuals in the virus-infected group were randomly selected for testing, and the results showed that specific bands of GCRV virus were detected in all of them, while no such specific bands were found in the control individuals.
[0101] like Figure 5 As shown, further electron microscopic observation revealed that a large number of GCRV virus particles were found in the kidney tissues of the deceased individuals in the virus infection group, while no GCRV virus particles were found in the control group.
[0102] The above results show that during the whole course of the disease, most of the dead grass carp individuals had obvious symptoms. After testing, all the dead individuals were infected with GCRV and died.
[0103] Comparative Example 1
[0104] The grass carp used in this comparative example had the same source, specifications, and feed conditions as above, and were divided into 5 groups, with 120 fish in each group. The virus titer was 5×10 3 The virus liquid with a concentration of copy / μL was used to carry out virus infection experiments on grass carp through different artificial infection methods.
[0105] The specific experimental steps and experimental methods are as follows:
[0106] 3.1 Experimental procedures
[0107] (1) Virus-mixed feed method: The details are the same as in Example 1.
[0108] (2) Injection method: The virus solution was injected into the fish by intraperitoneal injection, with an injection dose of 20 μL / g fish weight.
[0109] (3) Oral gavage: The virus solution is injected into the fish through the mouth, with an oral dose of 20 μL / g fish weight.
[0110] (4) Hypertonic immersion method: The experimental fish were immersed in 8% NaCl solution for 2 minutes, and then immersed in 10-fold diluted virus solution for 30 minutes. After the immersion, the experimental fish were placed in a tank for breeding.
[0111] (5) Control group: No treatment was given and the mice were raised under the same conditions as the infected group.
[0112] All the above groups were continuously observed every day, and the number of individuals who died in each group was recorded every day. The infection experiment ended when no deaths occurred in all groups for 7 consecutive days. The final mortality rate of each group was calculated and compared.
[0113] 3.2 Experimental Results
[0114] As shown in Table 2, the mortality statistics showed that the final mortality rates of the four virus-infected groups all exceeded 80%, namely 81.67% (feeding method with virus-mixed feed), 84.17% (injection method), 84.17% (gastric gavage method), and 85.00% (hypertonic immersion method), which are all close to the mortality rate of grass carp infected with GCRV under natural conditions (≥80%), indicating the effectiveness of the virus concentration used; no dead individuals occurred in the control group during the entire experiment.
[0115] Table 2 Statistics of deaths after infection with different viral infection methods
[0116]
[0117] Further analysis of the death data for each of the above groups showed that the groups infected with the virus by injection, gavage, and hypertonic immersion had 2, 4, and 3 deaths, respectively, during the incubation period (within 7 days after infection), while the group infected by feeding the virus-mixed feed had no deaths during the incubation period.
[0118] Since the incubation period after GCRV infection is about 7 days, the individuals in the above groups that died during the incubation period may have been caused by accidental damage to the fish during the artificial infection process, that is, death caused by non-viral factors. This result shows that the virus-mixed feed feeding method can effectively avoid accidental damage to the fish during artificial infection, exclude deaths caused by non-viral infection factors during the experiment, and ensure the accuracy of the fish phenotype.
[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for establishing a grass carp GCRV infection model, characterized in that: The steps include: S1 collected diseased fish naturally infected with GCRV in the wild, quickly frozen them at -4℃ for later use, and took intestinal tissues of diseased fish to extract RNA. Reverse transcription was performed to obtain cDNA, and PCR virus typing was performed using specific primers for the three subtypes of GCRV; S2 collects muscle and visceral tissue samples of the diseased fish, weighs them, adds sterile PBS at a weight ratio of 1:3, and grinds them to obtain a virus suspension. Centrifuges and obtains the supernatant to obtain the virus stock solution, reverse transcribes and obtains cDNA, and then uses qPCR to detect the virus titer in the supernatant. After determining the virus titer, dilute it with PBS to obtain virus solutions with different concentration gradients, thereby obtaining virus titers of C1, C2...C n The virus solution is prepared by freezing at -80°C for future use; S3 Select healthy grass carps of the same family weighing 8-10g and with negative GCRV test results, and divide them into an experimental group and a control group, the number of the experimental group is n, the number of grass carps selected in each experimental group and the control group is the same, and each group has at least 120 grass carps; put them into different breeding tanks, and control the water temperature at 26-28℃; artificially train the grass carps for 7-10 days, so that the grass carps can form conditioned reflexes and actively grab food; gradually adjust the amount of feed fed each time according to the feeding situation of the grass carps, until the feed fed each time is eaten within 5 minutes; S4: after the virus liquid is revived at 28°C for 2 hours, it is mixed evenly with grass carp puffed feed in a weight ratio of 1:1 to prepare virus-mixed feed; after the virus liquid and puffed feed are evenly mixed, they are allowed to stand for 15 minutes to allow the virus liquid to be completely absorbed into the puffed feed; wherein, in the preparation of the virus-mixed feed of each experimental group, the virus titers of the virus liquid used correspond to C1, C2...C n ; The culture tank of the S5 experimental group was filled with virus-mixed feed, while the culture tank of the control group was filled with PBS-mixed feed, wherein the dosage of PBS in the PBS-mixed feed was equal to the dosage of the virus liquid in the virus-mixed feed; each group was fed twice a day, and each group was fed with the virus-mixed feed or PBS-mixed feed for 6 consecutive days, and the total daily feeding amount of the virus-mixed feed or PBS-mixed feed for each group was 0.5% of the fish body weight; the number of deaths and symptoms were observed and recorded until no death individuals appeared in all groups for 7 consecutive days; finally, the minimum value of the virus titer corresponding to the experimental group with a mortality rate ≥80% in the statistical results was used as the virus titer of the experimental group infection, and the grass carp GCRV infection model was established.
2. The method for establishing a grass carp GCRV infection model according to claim 1, characterized in that: The PCR reaction system in step S1 includes: 2×PCR Mix 10 μl, cDNA template 1 μl, forward primer 1 μl, reverse primer 1 μl and RNase-free H2O 7ul, and the total volume of the reaction system is 20 μl.
3. The method for establishing a grass carp GCRV infection model according to claim 2, characterized in that: The primers for PCR in step S1 include: The forward primer sequence of type I GCRV is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; The forward primer sequence of type II GCRV is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4; The forward primer sequence of type III GCRV is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.
6.
4. The method for establishing a grass carp GCRV infection model according to claim 2, characterized in that: The qPCR reaction system in step S2 includes: 2×SYBR qPCR Mix 10 μl, cDNA template 1 μl, forward primer 0.8 μl, reverse primer 0.8 μl and RNase-free H2O 7.4 ul, and the total volume of the reaction system is 20 μl.
5. Application of the grass carp GCRV infection model, characterized in that: The grass carp GCRV infection model is established according to the method for establishing a grass carp GCRV infection model according to any one of claims 1 to 4, and the application includes at least one of the following applications: (1) screening and breeding new disease-resistant strains; (2) evaluating the safety and effectiveness of GCRV vaccines; (3) screening and developing drugs for preventing and treating GCRV infection; (4) developing methods for preventing and treating GCRV infection; and (5) determining the viral activity and immunogenicity of GCRV strains in fish.