Screening,purification And Immunogenicity Analysis Of A Candidate Of Live-attenuated Vaccine For Siniperca Chuatsi Rhabdovirus Disease

Siniperca chuatsi rhabdovirus(SCRV)is a kind of important pathogen for Chinese perch(Siniperca chuatsi)cultured in China.In recent years,highly frequent outbreaks of SCRV disease with high motality have resulted in the serious losses for Chinese aquaculture industry.The epidemic period of SCRV disease is long and it can infect different sizes fish.Therefore,it is significantly necessary to develop effective vaccine for SCRV disease.Although the adult fish can be immunized by intraperitoneal injection,for fries the immersion is the best immunization method.Live vaccines possess many advantages.It can be immunized by injection vaccination and immersion and has good protective effect as well as low immune dose.Therefore,development of effective live vaccines is the key to the prevention of SCRV disease.In our study Chinese perch was used for the study model and an avirulent virus strain,designed as Micropterus salmoides rhabdovirus Sanshui(MSRV-SS),was selected from six fish rhabdovirus isolates by fish challenge assay.Then,the MSRV-SS was purified by plaque clone assays,and a clonal strain designed as MSRV-SS-7 was obtained.The immune safty and efficacy of the clonal strain were investigated and the results suggested that the MSRV-SS-7 could be a candidate of live vaccine for Siniperca chuatsi rhabdovirus(SCRV)disease.The main results are as follows.1.Screening of avirulent virus strainChinese perch(siniperca chuatsi)was used for the study model and an avirulent virus strain,MSRV-SS,was selected from six fish rhabdovirus isolates by fish challenge assay and viral titers comparison.In order to analyze the protective efficacy of MSRV-SS,Chinese perch were immunized by intraperitoneal injection with MSRV-SS.Then the vaccinated fish were challenged with virulent Siniperca chuatsi rhabdovirus Gaoming(SCRV-GM).The result indicated that the relative percentage survival(RPS)of Chinese perch injected with MSRV-SS was 100%.Therefore,MSRV-SS can protect Chinese perch against virulent SCRV infection.2.Purification of parent MSRV-SS and the biological characteristics study of clonal MSRV-SS-7Parent MSRV-SS was purified by plaque clone assays,and 11 clonal strains were obtained.The clonal strain MSRV-SS-7 was picked out from 11 clonal strains based on the comparison analysis of viral titer and sequence alignment of G protein gene.The biological characteristics of MSRV-SS-7 were investigated.The clonal MSRV-SS-7 has a typical bullet shape and the growth characteristic of MSRV-SS-7 in CPB cells was similar to MSRV-SS.The complete genomes of MSRV-SS and MSRV-SS-7 comprise 11548 nucleotides and the length of five structure genes N,P,M,G,L is 1456,971,946,1661 and 6366 nucleotides,respectively.The leader region has 71 nucleotides and the trailer region has 61 nucleotides,respectively.The sequence of intergenic region is CTTT.The clonal strain MSRV-SS-7 had a good immunogenicity and could protect Chinese perch and largemouth bass against virulent SCRV infection.3.Evaluation of the immune safty and efficacy of MSRV-SS-7The MSRV-SS-7 distribution and change regularity in different tissues of Chinese perch were investigated.The results showed that MSRV-SS-7 could replicate in different tissues and had certain organ targeting in Chinese perch.MSRV-SS-7 was rapidly proliferated in the first 3 days post injection,but sharply decreased on the 4th day post injection.The safety tests for different kinds of fish and of administration of overdose were performed.The results indicated that MSRV-SS-7 was reliablely safe and had no pathogenicity for Chinese perch,snakehead fish(Channa maculate),marbled sand goby(Oxyeleotris marmoratus)and Largemouth bass(Micropterus salmoides).The result of blind passage in fish indicated that the MSRV-SS-7 couldn't be reversed to the virulent strain in the process of continuous infection.The immune efficacy tests of different immunization methods suggested that MSRV-SS-7 could provide protection for Chinese perch by immersion and injection immunization against virulent SCRV infection.Based on the results of challenge test,the minimum vaccination dose was 103.67TCID50.The storage life of lyophilized vaccine powder was 7 months at 4? by the viral titer surveillance.The immune genes expressions results showed that MSRV-SS-7 could not enhance the type?IFN-dependent genes expression,but motivate the Ig M gene expression.Above all,in this study Chinese perch was used as the study model and an avirulent clonal strain MSRV-SS-7 was screened out and purified from 6 fish rhabdovirus isolates.The study of biological characteristics of MSRV-SS-7 suggested that this clonal strain could be a candidate of live vaccine against SCRV infecion.And these results will provide a theoretical basis and reference for development of live-attenuated vaccine against other fish rhabdoviruses.