Isolation and characterization of Cyprinid herpesvirus 2 WH strain

Abstract

By using cell culture and virus infection methods,a virus had been isolated from gibel carp(Carassiusauratus gibelio) suffering with severe hemorrhage,which were cultured in a circulation aquaculture system in Wuhan,and then identified as Cyprinid herpesvirus 2(CyHV-2),designated as CyHV-2 WH strain,by electron microscopy observation,nested-PCR and viral DNA sequencing and sequence alignment analysis.The tissue homogenate of diseased fish could cause typical cytopathic effect in Koi-Fin cells.In artificial infection test,the typical symptoms of hemorrhage could be reproduced as naturally infected fish by infecting both tissue homogenate and cell cultured virus,and the death rate reached 100%.Electron microscopy observation of ultra-thin section samples of spleen and kidney tissues revealed that the viral nucleocapsid was spherical in shape measuring110–120 nm in diameter with a 170–200 nm envelope.The virions replicated and assembled in nucleus.A 357 bp DNA fragment obtained by the nested-PCR assay of both tissue homogenate and cell culture.Sequence alignment analysis of the DNA fragment showed that WH shared so high identity(99.44%) with the published DNA helicase gene sequence of CyHV-2 stains in GenBank,meanwhile,it shared 81.55% identity with CyHV-3 stains.Phylogenetic analysis showed that WH strain formed an independent branch on the phylogenetic tree with other CyHV-2 strains such as JS2012,H.Fukuda and YC110907.