Horizontal propagation research model of grass carp reovirus in grass carp

Abstract

The invention belongs to the technical field of virus biology, and particularly discloses a horizontal propagation research model of grass carp reovirus in grass carp. In a 15L water body with the temperature of 27 + / -1 DEG C, 10 gobiocypris rarus with the length of 3 + / -1cm is used as an infection source, 15 grass carps with the length of 7 + / -1cm are cultured together, the death rate of the grass carps is 40-60%, and the infection rate is 80-100%. Therefore, a horizontal transmission research model of the grass carp reovirus in the grass carp is successfully established, and a technical platform is provided for research on subsequent GCRV-II pathological mechanisms, antiviral drug screening and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of virus biology, and in particular relates to a research model of horizontal transmission of grass carp reovirus in grass carp. Background Art

[0002] Grass carp (Ctenopharyngodon idella) has a history of more than 60 years of farming in my country and is the most farmed freshwater fish in the world. However, frequent outbreaks of grass carp haemorrhagic disease (GCHD) have caused large-scale deaths in grass carp, seriously restricting the development of intensive grass carp farming. Grass carp reovirus (GCRV) is a double-stranded RNA virus belonging to the Reoviridae family and the genus Aquatic Reovirus. It is the pathogen of GCHD and can cause severe hemorrhagic diseases in grass carp fingerlings and juveniles with high mortality. At present, the strains found are divided into three genotypes according to the genome sequence: GCRV-Ⅰ, GCRV-Ⅱ and GCRV-Ⅲ. Some studies have reported that the outbreak of GCHD is mainly related to GCRV-Ⅱ. There are several methods for artificially infecting grass carp with GCRV: injection, immersion and gavage. Currently, no research has reported a fixed and specific operating instruction (such as the titer of the virus stock solution, the size of the grass carp, the size of the water body, etc.), resulting in no fixed range for the mortality rate and infection rate of grass carp. This instability has also brought a lot of uncertainty to subsequent research.

[0003] After being infected by GCRV-Ⅱ, grass carp can be divided into three symptom types according to the differences in symptoms and pathological changes. The first is the red muscle type, which is mainly manifested by obvious muscle bleeding, bright red, and pale gills due to blood loss, which is more common in small grass carp species of 5 to 10 cm; the second is the red fin red gill cover type, which is mainly manifested by severe congestion of the fin base and gill cover, and obvious bleeding spots on the top of the head, eye sockets, and mouth, which is more common in large grass carp species over 10 cm; the last is the enteritis type, which is mainly manifested by severe congestion of the intestine, which is bright red in all or part, different from the purple-red color of bacterial enteritis, punctate bleeding of the internal organs, and bleeding spots can also be seen on the body surface, which can be seen in grass carp species of various sizes. The symptoms of grass carp species of different sizes after being infected by GCRV-Ⅱ show instability. Therefore, the present invention establishes a stable and reliable GCRV-Ⅱ infected grass carp model, which provides a powerful technical means for subsequent research on GCRV-Ⅱ pathological mechanism, antiviral drug screening, etc. Summary of the invention

[0004] Rare gobiocypris rarus is a small carp fish unique to my country. It has a clear genetic background, small body size and short reproductive cycle, and is often used as a model organism. The inventors found that when the diseased rare gobiocypris and healthy rare gobiocypris are cultured in the same water body, the healthy rare gobiocypris can also be infected with GCRV. Therefore, the present invention uses an injection method to infect rare gobiocypris with GCRV, and co-cultivates grass carp with the infected rare gobiocypris, with the aim of establishing a stable and reliable grass carp infection model.

[0005] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0006] The invention provides a research model of horizontal transmission of grass carp reovirus in grass carp. The establishment method of the model comprises the following steps: in a water body at 27±1°C, using 3±1cm long rare minnows injected with GCRV-II virus as infection sources, and culturing 7±1cm long grass carp to obtain grass carp infected with GCRV-II.

[0007] Furthermore, the water volume is 15 L, the number of co-cultured rare goby crucian carp is 10, the number of grass carp is 15, and the injection volume of GCRV-Ⅱ virus is 1.5×10 7 copies; at this time, the mortality rate of grass carp is between 40% and 60%, and the infection rate is between 80% and 100%.

[0008] Furthermore, the horizontal transmission research model of grass carp reovirus in grass carp is specifically established by the following steps:

[0009] S1. Virus extraction: Take the rare crucian carp that died from infection with GCRV-JX02, cut it into pieces, add PBS to grind, and take the supernatant after gradient centrifugation. Filter the supernatant to obtain the virus stock solution;

[0010] S2. Artificial infection and co-cultivation: Put grass carp into a fish tank filled with water, place an isolation net bag in the center of the fish tank, take rare goby and place it in the water for 7 days in advance, then inject the virus stock solution into the rare goby, rinse it in clean water, and then place the rare goby in an isolation net bag. Observe for 14 days, remove the dead grass carp in time, and obtain the mortality rate;

[0011] S3. RNA extraction and reverse transcription: After 14 days of continuous observation, RNA was extracted from each grass carp tissue and reverse transcribed to obtain cDNA;

[0012] S4. Perform PCR and qPCR tests on cDNA to obtain the infection rate.

[0013] Furthermore, in step S1, the gradient centrifugation conditions are 4000, 8000, and 12000 r / min for 10 min each; and the filtration method is filtering with 0.45 and 0.22 μm syringe filters in sequence.

[0014] Furthermore, in step S3, four tissues including liver, intestine, brain and muscle of each grass carp are taken for RNA extraction.

[0015] Furthermore, in step S4, the sequences of the primers used for PCR detection are shown in SEQ ID NOs: 1-2, and the sequences of the primers used for qPCR detection are shown in SEQ ID NOs: 3-4.

[0016] The present invention has the following beneficial effects:

[0017] The present invention adopts the method of "taking rare minnows injected with GCRV-Ⅱ virus as the infection source and co-culturing grass carp", combined with the optimization of co-culture parameters, and successfully establishes a research model for the horizontal transmission of grass carp reovirus in grass carp, which solves the problems of unstable grass carp mortality rate, infection rate and symptom manifestation in the existing methods of artificially infecting grass carp with GCRV and the resulting uncertainty in subsequent research, and provides a good technical platform for subsequent research on the pathological mechanism of GCRV-Ⅱ, antiviral drug screening and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : The co-cultivation model diagram of grass carp and rare minnow crucian carp of the present invention.

[0019] Figure 2 : The survival rate of grass carp cultured in Example 1.

[0020] Figure 3 : The electrophoresis results of PCR detection of co-cultured grass carp infected with GCRV-JX02 in Example 1. In the figure, 1-8. Dead grass carp; M. DL 2000 DNA marker; N. Negative control group; P. Positive control group.

[0021] Figure 4 : The average copy number of GCRV-JX02 in the co-cultured grass carp detected by qPCR in Example 1. DETAILED DESCRIPTION

[0022] The inventors used 10 rare minnows of 3±1 cm in length as the infection source in 15L, 27±1°C water, and co-cultured 15 grass carps of 7±1 cm in length. The specific co-culture parameters are shown in Table 1 below. It was found that the mortality rate of grass carp was between 40% and 60%, the infection rate was between 80% and 100%, and the symptoms of grass carp that died of infection were basically consistent with the red muscle type, manifested as bright red muscle bleeding. Thus, a research model of horizontal transmission of grass carp reovirus in grass carp was successfully established.

[0023] Table 1 Description of co-culture experimental parameters

[0024]

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific examples. The detection method in the following examples refers to GB / T 36190-2018, and the PCR primers are designed based on the M6 ​​gene fragment of GCRV-Ⅱ.

[0026] Example 1

[0027] 1. Experimental Materials

[0028] The rare goby used in the experiment was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences; grass carp was purchased from a farm in Foshan, Guangdong; the GCRV-Ⅱ experimental strain was the GCRV-JX02 isolated and preserved in our laboratory (isolated from grass carp with obvious bleeding symptoms using conventional virus isolation methods, identified as the GCRV-Ⅱ strain through sequence analysis and phylogenetic tree construction, and preserved in the National Aquatic Animal Pathogen Bank). TRIzol was purchased from Invitrogen (USA), and the cDNA synthesis reagent PrimeScript TM Ⅱ1 st Strand cDNA Synthesis Kit (6210A), High Specificity qPCR Reagent TB Premix Ex Taq TM Ⅱ(RR820A), Premix Taq TM (TaKaRa Taq TM Version 2.0 plus dye) (RR901A) was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. All PCR and qPCR primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0029] 2. Experimental Methods

[0030] 2.1 Virus extraction and virus titer determination

[0031] GCRV-JX02 was intraperitoneally injected into rare Gobiocypris carp, and after death, it was chopped up, ground in 1× phosphate buffer (PBS), and centrifuged at 4000, 8000, and 12000 r / min for 10 min each. After gradient centrifugation, the supernatant was filtered with 0.45 and 0.22 μm syringe filters to obtain the virus stock solution, and the virus copy number was determined to be 3×10 5 copies / μL.

[0032] Note: Conventional cell adsorption infection is not applicable to the present invention, because GCRV-JX02 does not produce CPE after infecting cells, that is, it cannot infect cells, but can only infect fish. Based on this, the present invention causes rare goby to be infected and killed by injecting GCRV-JX02. At this time, the virus has replicated and amplified in the infected rare goby, and the advantage is that it can stably spread the virus.

[0033] 2.2 Artificial infection and co-culture

[0034] Fifteen grass carps with a body length of 7±1 cm were placed in a fish tank filled with 15L of water, the water temperature was controlled at 27±1℃, and an isolation net bag was placed in the center of the fish tank. Ten rare goby with a body length of 3±1 cm were placed in 27±1℃ water for 7 days in advance, and then 50μL of the virus stock solution was injected intraperitoneally into each goby. After rinsing in clean water, the rare goby was placed in an isolation net bag and observed for 14 consecutive days. The dead grass carp were promptly removed for subsequent experiments.

[0035] Note: The setting of isolation nets is mainly based on the following two considerations: 1) Horizontal transmission mainly uses water as a medium to spread the virus. If no isolation nets are set up, the rare minnow and grass carp will have physical contact, and the virus may be spread through physical contact; 2) Setting up isolation nets can also prevent grass carp from harming the rare minnow.

[0036] 2.3 RNA extraction and reverse transcription

[0037] After 14 days of continuous observation, the liver, intestine, brain, and muscle of each grass carp, the four main target organ tissues of GCRV-JX02, were collected and mixed for RNA extraction. The steps are as follows:

[0038] (1) Add 1 mL of TRIzol to a 1.5 mL centrifuge tube containing tissue, grind and centrifuge, and transfer 500 μL to a centrifuge tube;

[0039] (2) Add 200 μL of chloroform, mix thoroughly, and let stand for 10 min;

[0040] (3) Centrifugation at 12000 rpm for 10 min;

[0041] (4) Take the upper layer of liquid and add 200 μL of pre-cooled isopropanol, mix thoroughly and let stand for 10 min;

[0042] (5) Centrifugation at 12000 rpm for 10 min;

[0043] (6) Discard the supernatant and add 1 mL of 75% enzyme-free ethanol;

[0044] (7) Centrifugation at 12000 rpm for 10 min;

[0045] (8) Discard the supernatant, dry thoroughly, and add 50 μL RNase-free dH2O to dissolve;

[0046] (9) The concentration of nucleic acid protein is detected by nucleic acid protein detector.

[0047] The reverse transcription steps are as follows:

[0048] (1) Prepare 10 μL of reaction solution, the system is as shown in Table 2:

[0049] Table 2 Reverse transcription 10 μL reaction system

[0050]

[0051] (2) Keep at 65°C for 5 min and then cool rapidly on ice;

[0052] (3) Prepare 20 μL of reaction solution, the system is as shown in Table 3:

[0053] Table 3 Reverse transcription 20 μL reaction system

[0054]

[0055] (4) After thorough mixing, centrifuge for 15 seconds, incubate at 30°C for 10 minutes, incubate at 42°C for 60 minutes, and incubate at 70°C for 15 minutes, and then quickly cool on ice.

[0056] 2.4 PCR detection

[0057] (1) Prepare 25 μL of reaction solution, the system is as shown in Table 4:

[0058] Table 4 PCR reaction system

[0059]

[0060] Common PCR primers are shown in Table 5:

[0061] Table 5 PCR primer sequences

[0062]

[0063] (2) After thorough mixing, centrifuge for 15 seconds and place in a PCR instrument. The reaction program is 94°C pre-denaturation for 4 minutes, 94°C for 40 seconds, 55°C for 40 seconds, 72°C for 1 minute, 35 cycles; 72°C extension for 8 minutes, and storage at 4°C.

[0064] (3) After the PCR reaction was completed, the PCR product was detected using 1.5% agarose gel electrophoresis.

[0065] 2.5qPCR detection

[0066] (1) Prepare 13 μL of reaction solution, the system is as shown in Table 6:

[0067] Table 6 qPCR reaction system

[0068]

[0069] qPCR primers are shown in Table 7:

[0070] Table 7 qPCR primer sequences

[0071]

[0072] (2) Centrifuge for 3 min and place in a fluorescence quantitative PCR instrument. The reaction program is: 95°C pre-denaturation for 30 s; 95°C for 5 s, 58°C for 30 s, 39 cycles; 95°C for 10 s, 65°C for 5 s, 95°C.

[0073] (3) At the same time, a negative control group was set up to perform the same operation. A standard curve was also established. The standard curve was established by constructing the GCRV-Ⅱ virus plasmid, calculating the concentration of the plasmid using a formula, and then performing a gradient dilution of the plasmid for qPCR detection to establish a standard curve between the GCRV-Ⅱ virus concentration and the CT value.

[0074] 3. Results

[0075] The co-culture observation results are shown in Table 8 and Figure 2 As shown, the survival rate of the co-cultured grass carp was 47%. In the dead grass carps numbered 1-8, the infection symptoms were all manifested as bright red flesh, but some were darker and some were lighter. In the surviving grass carps numbered 9-15, no symptoms appeared.

[0076] Table 8 Death of co-cultured grass carp

[0077]

[0078] PCR electrophoresis results are as follows Figure 3As shown, among the dead grass carps numbered 1-8, except for number 6, the target bands can be seen in all other numbers, deep or shallow target bands can be seen. Among the surviving grass carps numbered 9-15, the target bands can not be seen, and the positive control has a lighter band. The absence of the target band in the sample does not mean that the grass carp is not infected, but may be due to a small amount of virus infection, so it is necessary to make an accurate judgment based on the qPCR test results.

[0079] qPCR test results Figure 4 As shown in the figure, the copy number is obtained by substituting the CT value obtained by fluorescent quantitative PCR into the standard curve. When the CT value obtained by fluorescent quantitative PCR is less than 35 (the negative control group of this experiment shows no CT value, when the CT value is higher than 35, slight operating errors or fluctuations in reaction conditions will have a greater impact on the results, that is, the given CT value is distorted and meaningless), that is, the copy number is greater than 10 2.936 When it occurs, it may indicate that grass carp is infected. Figure 4 The results showed that the copy numbers of grass carp samples 1-15 were all greater than 10 2.936 , indicating that all grass carp, dead or alive, were infected, that is, the infection rate was 100%.

[0080] The above experiments show that rare minnows infected with GCRV-JX02 can infect grass carp horizontally through water bodies, causing the grass carp to develop symptoms of hemorrhagic disease and die or become a virus carrier.

[0081] Comparative Example 1

[0082] In this comparative example, the virus was directly injected into grass carp for infection, rather than using the method of infecting grass carp with rare minnows in Example 1. As a result, some grass carp died due to severe stress or improper injection operation within a short period of time after the injection. These deaths were not caused by virus infection, and the model establishment failed.

[0083] Direct virus injection into grass carp will cause the following problems: 1) For grass carp of different sizes, the concentration and dosage of the injected virus are uncertain. 2) Grass carp may die due to improper injection operation. 3) The injection wound is susceptible to infection with bacteria and other diseases. 4) It causes severe stress response in grass carp, which will have an adverse effect on the detection of various physiological indicators of grass carp in subsequent research. The present invention can infect grass carp with virus without directly harming the grass carp by co-culturing with rare minnows and crucian carp, so the infection effect is more stable and controllable.

[0084] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A research model for horizontal transmission of grass carp reovirus in grass carp, characterized in that: The establishment method is as follows: in water at 27±1℃, 3±1cm long rare minnows injected with GCRV-Ⅱ virus are used as the infection source, and 7±1cm long grass carp are cultured to obtain grass carp infected with GCRV-Ⅱ.

2. The horizontal transmission research model of grass carp reovirus in grass carp according to claim 1, characterized in that: The volume of the water body is 15 L, the number of the co-cultured rare goby crucian carp is 10, the number of the co-cultured grass carp is 15, and the injection volume of the GCRV-Ⅱ virus is 1.5×10 7 copies; at this time, the mortality rate of grass carp is between 40% and 60%, and the infection rate is between 80% and 100%.

3. The horizontal transmission research model of grass carp reovirus in grass carp according to claim 2, characterized in that: Specifically, it is established through the following steps: S1. Virus extraction: Take the rare crucian carp that died from infection with GCRV-JX02, cut it into pieces, add PBS to grind, and take the supernatant after gradient centrifugation. Filter the supernatant to obtain the virus stock solution; S2. Artificial infection and co-cultivation: Put grass carp into a fish tank filled with water, place an isolation net bag in the center of the fish tank, take rare goby and place it in the water for 7 days in advance, then inject the virus stock solution into the rare goby, rinse it in clean water, and then place the rare goby in an isolation net bag. Observe for 14 days, remove the dead grass carp in time, and obtain the mortality rate; S3. RNA extraction and reverse transcription: After 14 days of continuous observation, RNA was extracted from each grass carp tissue and reverse transcribed to obtain cDNA; S4. Perform PCR and qPCR tests on cDNA to obtain the infection rate.

4. The horizontal transmission research model of grass carp reovirus in grass carp according to claim 3, characterized in that: In step S1, the gradient centrifugation conditions are 4000, 8000, and 12000 r / min for 10 min each; the filtration method is filtering with 0.45 and 0.22 μm syringe filters in sequence.

5. The horizontal transmission research model of grass carp reovirus in grass carp according to claim 3, characterized in that: In step S3, four tissues including liver, intestine, brain and muscle of each grass carp are taken for RNA extraction.

6. The research model of horizontal transmission of grass carp reovirus in grass carp according to claim 3, characterized in that: In step S4, the sequences of the primers used for PCR detection are shown in SEQ ID NOs: 1 to 2. The sequences of the primers used for qPCR detection are shown in SEQ ID NOs: 3-4.

7. Application of the horizontal transmission research model of grass carp reovirus in grass carp as described in any one of claims 1 to 6 in the research on the pathological mechanism of GCRV-Ⅱ and antiviral drug screening.

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