Isolation and identification of grass carp reovirus strain JX-0902

Abstract

We identified a new strain of reovirus in grass carp from Nanchang city,Jiangxi Province.We collected diseased yearling grass carp,Ctenopharyngodon idellus exhibiting typical hemorrhage symptoms from Nanchang city and observed large numbers of viral cells in ultra-thin sections of the spleen and kidney.The virus had a non-enveloped structure with a diameter of approximately 70 nm,very similar to the characteristics of the grass carp reovirus(GCRV).We isolated samples of the virus by homogenizing the tissue,centrifuging the homogenate,then conducting sterilizing filtration.We were able to replicate the hemorrhaging by artificially infecting grass carp with the purified sample.Artificial infection of grass carp kidney cell line(CIK) revealed no obvious cyto-pathic effects(CPE) after six passages.However,observations under the electron microscope revealed that the virus was propagated in the cytoplasm,and had the same shape as the virus observed in the tissue sections.The newly isolated virus(temporarily named GCRV JX-0902) consisted of 11 segments of dsRNA,a typical charac-teristic of aquareovirus.There were significant differences in the genome banding pattern of GCRV873 and JX-0902,but no difference between HZ08 and JX-0902.We designed PCR primers based on sequence segment 6(GQ896337) of GCRV in GenBank to amplify specific bands using JX-0902 cDNA as the template.The amino acid sequence of JX-0902 S6 had 98% and 99% identity with GCRV-HZ08 and GD108,respectively.Thus,we conclude that the new isolate is a member of the genus of aquareovirus.