Etiologic Study And Development Of Detection Methods For Viral Nervous Necrosis Virus

Betanodavirus infection is responsible for Viral nervous necrosis（VNN） an emerging disease,which was described for the first time at the end of the 1980’s and is now prevalent in all continents except Africa.The infection of Betanodavirus has caused great economic losses of madculture in many countries.The fast diffusion of the infection is due both to the large host range of the virus and extensive translocation of stock wihin and between countries.Betanodavirus are RNA viruses whose genome consists of two molecules:RNA1 encoding for the RNA-dependent RNA-polymerase and RNA2 encoding for the coat protein.According to phylogenetic analysis of the coat protein gene,Betanodavirus can be calssified into four genotypes:SJNNV,TPNNV, BFNNV and RGNNV.The infection of Betanodavirus also caused great loss in Southern China,especially in the larvae of cultured marine fish.Some study about this virus has been carried out, but no strain has ever isolated until 6 strains were isolated with SSN-1 cell line in 2006. In this study,a double antibody sandwich ELISA was developed based on monoclonal antibody and rabit anti-VNNV polyclonal antibody.This ELISA was used to detected fish samples infected with VNNV and other virus than VNNV.The results suggested that the ELISA have good specificity and sensitivity.The ELISA was also used to detected 336 clinical samples,RT-PCR was used as a control method.The results indicated that the ELISA had good specificity（97.6%） and high sensitivity（91.4%）.The agreement ratio between ELISA and RT-PCR was 94.3%.These data suggested that the ELISA could be used as a rapid test for VNNV detection.In this study,a real-time RT-PCR assay for fish nervous necrosis virus was developed.The specific primers and TaqMan probes were designed according to the highly conservative sequence of capsid protein（CP） gene of Viral nervous necrosis virus from Genbank.After the procedure of the real-time RT-PCR assay for fish nervous necrosis virus was optimized,the specificity,sensitivity,reproducibility of the method were estimated.It was found that the specificity of this assay was high without any cross-reactions with SVC、IPN、VHSV、GCHV、IHNV,and so on.A minimum of 1.2 pg/μl total RNA could be detected,indicating a good sensitivity of the assay.The coefficients of variance（CVs） were 0.9%and 1.5%for the intra-assay and inter-assay tests respectively,which indicated good reproducibility.It took only 4 hours to complete the whole course of reaction including extraction of viral RNA and the real-time RT-PCR. Detected by the real-time RT-PCR assay for fish nervous necrosis virus,it was found that forty specimen were positive among 500 clinical samples.Those results confirmed that the real-time RT-PCR assay for VNNV was a rapid,sensitive and specific method for the detection of viral nervous necrosis virus.Six nervous necrosis virus were isolated from cultured yellow grouper in 2006 in shore of china.Total viral RNA genome was extracted.Based on the complete sequences of RNA2 which have been submitted to Genbank,a pair of primers were designed and the coat protein genes from the 6 isolates were amplified by means of reverse-tramcriptase polymerase chain reaction（RT-PCR）.The RT-PCR products were ligated into the PMD18-T vector and transformed into E.coli DH5a.The recombinant plasmid were sequenced respectively.There was more than 96.1%nucleotide identityies and 79.6-99.1%among the deduced amino acid sequences within the six isolates. Phylogentic analysis showed that the isolates shared evolutionary direction with EVNNV,ETNNV and MNNV.It was revealed that these isolates belonged to genotype RGNNV.It was deduced that the strains of RGNNV genotype were dominant and prevalent in spotted grouper cultured in China during the past several years.The cp gene of VNNV 0603 Strain was amplified successfully by reverse-transcription polymerase chain reaction（RT-PCR）,and inserted into plasmid pFastBacI of the Baculovirus expression system Bac-To-Bac to form plasmid pFastBac-cp.Through homologous recombination between pFastBac-cp and bacmid which is genome modified of Autogragha california nuclear polyhedrosis virus（AcNPV）,recombinant shuttle vector named Bacmid-cp was obtained in E.coli DH₁₀Bac.After tranfection with Cellfectin Reagent,recombined Baculovirus（AcNPV-cp） was developed in Sf9 cells.The specific 37kD band was showed by analysis of SDS-PAGE and Western blotting,which indicated that VNNV CP protein had expressed in insect cells sfg.It was noteworthy that many self-assembled virus-like particles（VLPs） were found in CP crude extract and in the infected Sf9 cells by electron microscope.The VLPs were similar to VNNV whole virion, and became a crystalline structure in cytoplasm.