Isolation and characterization of a rhabdovirus from snakehead fish(Ophicephalus striatus)

The diseased hybrid snakehead(Channa maculate ♀×Channa argus♂)were collected for analysis of the pathogen both in bacteriology and virology to make the pathologic agent clear.None of pathogenic bacteria was obtained using routine bacteria separation method.One rhabdovirus was isolated using cell culture technique,regression tests,electron microscopy,molecular biology techniques and others,which assigned hybrid snakehead rhabdovirus -C1207(HSHRV-C1207).HSHRV-C1207 could replicate and proliferate in EPC,FHM,GSB,SFC,CIK cells,and cause the significant cytopathic effect(CPE).The regression tests in which separated viruses were injected in the abdominal cavity were all presented with symptoms similar to the natural incidence,and the final mortality was 90%.Electron microscopy observation of ultra-thin section samples of specimen-infected EPC cells revealed that the bullet-shaped virus replicated in cytoplasm with regular arrangement like other fish rhabdovirus,and had a non-enveloped structure with a diameter and length of approximately 60 nm and 145 nm respectively.The OIE recommended primers for Infectious haematopoietic necrosis virus(IHNV),spring viraemia of carp virus(SVCV),viral haemorrhagic septicaemia virus(VHSV)and three pairs of specific primers were designed by G gene conserved sequences of snakehead rhabdovirus(SHRV),siniperca chuatsi rhabdovirus(SCRV)and hirame rhabdovirus(HIRRV)respectively,were applied to the RT-PCR amplification for detecting the possible rhabdovirus in diseased fish and virus-infected cells.A specific ladder product about 350 bp only was amplified with primers of SCRV.Sequencing analysis of the G gene of HSHRV-C1207 showed that this gene shares the sequence identities 93.8% and 20.4% with the respective genes of SCRV and SHRV respectively,and the corresponding deduced amino acid sequence identity 93.7% and 18.3% respectively.Phylogenic relationship with other rhabdoviruses using the corresponding amino acid sequences of G protein indicates that HSHRV C1207 is most closely related to the vesiculovirus genus and shares the highest identity with SCRV,but was far from the snakehead rhabdovirus(SHRV)and the other rhabdovirus.The above results indicated that the causes of hybrid snakehead diseases ceaseless breaking out was the rhabdovirus,and this new isolates HSHRV-C1207 belongs to vesiculovirus genus.It may be a novel variant SCRV isolate or a new member.