Isolation And Identification Of Micropterus Salmoides Rhabdovirus And Study On Antiviral Activity Of Roc-A

Micropterus salmoides rhabdovirus（MSRV）is a single negative strand RNA virus with a typical bullet-like structure,with a diameter of 85～95 nm and a length of 115～130nm.It is the pathogen of Micropterus salmoides rhabdovirus disease.It has caused larger economic loss to largemouth bass breeding industry.Seedlings between 2-5 cm are the primary targets of Micropterus salmoides rhabdovirus disease,which spreads rapidly and is highly lethal,with a mortality rate of up to 90%within a week.However,there is still a lack of effective prevention and control measures for Micropterus salmoides rhabdovirus disease,so it is urgent to find new effective prevention and control drugs to achieve healthy development of largemouth bass aquaculture.According to the existing research results,the natural compound Rocaglamide（Roc-A）has the activity of inhibiting a variety of viruses,the composition is safe,and it is an ideal compound for drug development.In this study,a strain of MSRV-SG01 virus was isolated from sick largemouth bass in Guangdong Province,and the anti-MSRV effect of Roc-A was evaluated in vitro and in vivo.The specific research content and results are as follows:1.Isolation and identification of a Micropterus salmoides rhabdovirusIn this study,a batch of larvae of sick largemouth bass（0.8-1.0 cm）were collected from a breeding base in Guangdong Province.The whole fish was ground and the virus was isolated from the fish.RT-PCR results showed that bands with a size of about 217 bp could be amplified using MSRV specific primers.（Epithelioma papillosum cyprini cells,EPC）,（Fathead minnow muscle cells,FHM）,（Channel catfish ovary cells,CCO）,（Grouper spleen cells,GS）.It was found that EPC,FHM and CCO cells showed varying degrees of cytopathic effect（CPE）,and the sensitivity was EPC>FHM>CCO and GS are not sensitive.The virus infected EPC cells with a titer of 10^6.5 PFU/m L.The results of the growth kinetics curve showed that 6-24 h was a logarithmic growth phase,and the virus rapidly replicated.24-48 h was the plateau period,the virus prolificated slowly,and the titer was relatively highest at 36 h.Many bullet-like virions with length of 120～170 nm and diameter of 50～65 nm was observed under electron microscope from EPC cells infected with MSRV.After intraperitoneal injection of different challenge doses of largemouth bass,the challenge group showed typical symptoms of Micropterus salmoides rhabdovirus disease,and the mortality rate of the fish in the challenge group reached 100%within one week.Phylogenetic tree analysis showed that MSRV-SG01 was grouped with MSRV-FJ985,MSRV-YH01 and SCRV.Based on the above results,it was speculated that the virus was MSRV isolate and named MSRV-SG01.2.To investigate the anti-MSRV activity of Roc-A at the cell levelFirst,the toxicity of Roc-A on EPC cells was determined by MTT assay,and the concentration was within the safe range（cell viability>90%）,select 10,25,50 n M for follow-up tests.Based on the determination of safe concentration,real-time quantitative PCR（q RT-PCR）was used to evaluate the anti-MSRV effect of Roc-A.The results showed that Roc-A has significant antiviral activity in a dose-dependent manner.In order to explore the anti-MSRV activity of Roc-A in vitro,the antiviral effect of Roc-A was investigated from three aspects of prevention,co-breeding and treatment by plaque test,CPE observation,q RT-PCR and other experimental techniques.The results of the prevention group showed that Roc-A had no preventive effect on EPC cells infected by MSRV.The results of co-breeding group showed the neutralization effect of Roc-A on MSRV.At high dose,the CPE induced by MSRV was significantly reduced,and the virus titer was decreased by about 1 log PFU/m L.The expression of MSRV G gene was significantly decreased by q RT-PCR（p<0.001）.The results of the treatment group showed that Roc-A could significantly inhibit MSRV-induced CPE,significantly reduce virus titer and inhibit the expression of MSRV G gene even at low doses（p<0.001）.3.The anti-MSRV activity of Roc-A was investigated at individual levelIn order to further evaluate the anti-MSRV effect of Roc-A,the safe concentration of Roc-A for injection was first determined.The in vivo anti-MSRV effect of Roc-A was evaluated by survival rate statistics,histopathological observation of liver,kidney and spleen,and viral load measurement.The results showed that Roc-A significantly reduced the mortality rate of largemouth bass infected with MSRV（p<0.01）,increased their survival rate by 37%;Histomathological results showed that the largemouth bass treated with Roc-A had no obvious pathological damage to the liver,kidney and spleen,and significantly reduced the viral load in the liver,kidney and spleen of largemouth bass at day 1,3 and 5 of infection（p<0.001）,indicating that Roc-A has a good therapeutic effect on largemouth bass infected with MSRV.In conclusion,an MSRV-SG01 virus strain was isolated and identified in this study,and it was found that Roc-A had good anti-MSRV activity,and its therapeutic rate for largemouth bass was up to 37%,which is a drug with great potential for development.These results provide a reference for the subsequent research on MSRV.