Electron microscopic observation of infected CCB and whole genome analysis of Koi herpesvirus isolate GY01

Abstract

Background：Cyprinid herpesvirus-3 (CyHV-3), commonly known as Koi herpesvirus (KHV) can induce infectious and acute viremia in common/koi carp (Cyprinus carpio). In an earlier study in this laboratory a KHV isolate GY1506 (KHV GY01 in GeneBank ) was isolated from diseased common carp, replicated on CCB cells and identified by PCR targeted on and phylogenetic analysis of thymidine kinase (TK) gene.Electron microscopy examination of GY01 infected CCB cell line and whole genome analysis was studied for further characteristics and epidemiological features of this strain.Results：Electron microscopy examination of CCB infected with KHV GY01 strain revealed destruct of infected cells including incomplete nuclear membranes, deformed nucleus, marginalized nuclear chromosome, and the virus of different development stages and morphologies in the cytoplasm and nucleus, which resembles the herpesvirus. MEGA X and phyML were used for multiple alignment and phylogenetic analysis of whole genomes of GY01 and other 21 KHV strains available in GeneBank. analysis showed that GY01 was more close to E and KHV-I and was predicted it originated from the same ancestor as the E and KHV-I. Pairwise alignment of strain GY01 and strain E by Geneious software and YASS online version revealed that two strains had high identity(99.1%) at the nucleotide sequence level although variations and disagreement existed. The number and structure arrangement of open reading frames (orfs) or protein-encoding genes of GY01 is very similar to KHV E, and also to KHV-U but different from KHV-I. The characteristics and function of each orf need further study in the future. Conclusions:Pathogenic changes of infected CCB cells and morphologies of KHV GY01 resembles the herpesvirus. Pairwise, multiple alignment and phylogenetic analysis of whole genomes of GY01 and other 21 KHV strains available in GeneBank demonstrated that the GY01 is closely related to strain KHV E and KHV-I and suggested it originated from the same ancestor as the E and KHV-I. Keywords:Koi herpesvirus(KHV), Cyprinid herpesvirus 3 (CyHV-3),KHV isolate GY01, Electron microscopy examination, pairwise alignment, multiple alignment, phylogenetic analysis，common carp brain cell line(CCB)