Study On The Co-infection Of Grass Carp Reovirus And Proteomic Analysis Of Cytopathic Effect

Grass carp reovirus (GCRV), also named as Grass carp hemorrhage virus, is themost important pathogen of Grass carp, which causes haemorrhagic disease. The viralgenome is composed of11segments of double stranded (ds) RNA, and encodes7structural proteins and5non-structural proteins. Cell membrane permability, apoptosisand stress granule formation can be induced by GCRV infection.Full-length amplification of cDNA technology was developed for sequencing full-length cDNA of double-stranded RNA virus, and it was also widely used to amplifyany uncharacterized Aquareovirus genomes with accuracy and time-saving. Twodimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry wereused to analyze the proteomic changes and mechanisms during virus infection.FLAC technology, real-time RT-PCR, Two dimensional electrophoresis andMALDI-TOF/TOF mass spectrometry assay were employed in the study. The resultswere as follows:1. Two GCRV strains were isolated from diseased grass carp collected in Jiangxiprovince. Partial of full-length cDNAs were sequenced by FLAC, indicating thatco-infection of two grass carp reovirus, named GCRV-JX01and GCRV-JX02whichshared high homology with GCRV-873and GCRV-GD108, respectively, were revealedin the same diseased sample through Blast method. The results demonstrated that therewere two GCRV strains prevalent in major grass carp farms, and the ratio ofco-infection (55%) was higher than single-infection (30%).2. Infinite dilution method and RT-PCR were used to purify the two virus strains in96-wells. Quantitative real-time PCR assay was carried out to monitor the replicationefficiency of both virus strains in either CIK cells or infected cell supernatant. Theresults demonstrated that, although GCRV-JX02did reduce the cellular replication levelof GCRV-JX01up to10folds during co-infection, there was no significant impact onproductive virus progeny level in supernatant compared to that of cells infected byGCRV-JX01alone. No serologicalcross-reaction or cross-protection occurred betweenGCRV-JX01and JX02in our immunization and challenge tests.3.23differentially expressed protein spots were up-regulated3times or above during GCRV-JX01infection in6h,12h and24h by Two dimensional electrophoresis.27spots were further determined using MALDI-TOF/TOF mass spectrometry and23sequences were finally obtained. These determined spots were clsssified into6groups:cytoskeleton proteins, stress response, signal transduction, ubiquitin proteasomepathway, energy metabolism associated proteins and other unkown predicted proteins.4. NS16, NS31, NS38, VP6and VP7cDNAs of GCRV-JX01were cloned intopEGFP-N1vector and the recombined plasmids were tranfected in CIK cells.Western-blot analysis was conducted to verify the expression of these fusion proteins.The results of subcellular localization showed that NS31fusion protein was located inall cell and was highly expressed in certain organelles; NS16fusion protein was locatedin cytoplasm and was highly expressed in certain organelles; NS38, VP6and VP7fusion proteins were located in cytoplasm. No cytopathic effect were observed bytransfecting either one of the5recombined plasmids.Co-infection of two grass carp reoviruses was revealed in our study for the firsttime. The research provides a new technical method for GCRV co-infection diagnosisand epidemiological investigation. It is not only helpful for designing vaccines targetingthe hemorrhagic disease, but also laied foundations for studing the mechanisms ofinteractions between virus and the host cells, and even the biological functions by itsencoding proteins.