Isolation And Identification Of An IHNV Isolate From Rainbow Trout (Oncorhynchus Mykiss)

In 2013, an outbreak emerged in a cultured population of cold water fish of yearling rainbow trout in Pengzhou, China, with fish suffering heavy mortality （young fish=40%, juvenile fish=80%）. Necropsy and histopathological examination were performed. External clinical signs included darkening, a swollen abdomen, ulceration of the snout, petechial hemorrhage on the skin, and hemorrhage at the base of the fins and vent. Internally, visceral mesenteries, adipose tissue, swim bladder, peritoneum, meninges, and pericardium generally exhibited petechiae. The stomach and intestine were devoid of food and contained a yellow mucoid fluid. The liver, kidney, and spleen were pale. Histological examination revealed marked necrosis in the anterior kidney, spleen, liver, pancreas, and digestive tract. Foci of necrosis were present in the hematopoietic tissues, including the kidney and spleen, along with an increased number of macrophages and deteriorating lymphoid cells. Moreover, severe necrosis of the eosinophilic granular cells （EGCs） of the intestinal wall was also observed. Multiple RT-PCR （primers design reference Williams） for imultaneous detection of infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus （IPNV） and viral hemorrhagic septicemia virus （VHSV） were IHNV-positive. a GenBank BLAST search on the resulting sequence revealed the amplification products shared 99.9% identity to that of IHNV nucleoprotein gene.After filtration treatment, the speen and kidey tissue suspension was inoculated to the epithelioma papulosum cyprini cells. The typical cytopathic effect （CPE） was formed after three blind passages in EPC cells. Then, virus suspension was injected intraperitoneally into 20 trouts. Rainbow trouts displayed similar clinical symptoms as fish that were naturally infected. Trouts died acutely in the trial group （cumulative mortality rate=75%）, there was abnormal the control group. Total RNA was extracted from the fish homogenates artificial infected and infected cell culture supernatants for multiplex RT-PCR. A clear single 371 bp target band indicated that target organs and EPC cells were infected with IHNV only. An infectious hematopoietic necrosis virus was isolated from the diseased rainbow trout by using epithelioma papulosum cyprini cells, named LQN131107. In addition. Artificial infection test and multiplex RT-PCR showed that infectious hematopoietic necrosis virus was the causative agents of highly contagious diseases in rainbow trout.The nucleoprotein gene in LQN131107 strain of IHNV was amplified from epithelioma papulosum cyprini cells by RT-PCR （primers design reference KJ441078）. and then was cloned into the vector pMD19-T to construct the recombinant plasmid and sequence. The function and structure of nucleoprotein gene was analyzed by means of bioinformatics software, and then phylogenetic tree was constructed according to the nucleoprotein gene sequences from GenBank. The results showed that the nucleoprotein gene was 1176 bp, encoding a 391 amino acid, protein, the nucleoprotein was rich in leucine （11.80%）, alanine （10.50%）, glutamic acid （9.50%）, glycine （9.20%）. which had a Rhabdo_ncap₂ conserved domain. The protein is hydropathicity with no transmembrane and signal peptide. Prediction of antigenic determinants and B-cell epitopes indicated that the antigenicity of nucleoprotein was good. According to the prediction of modification sites, the nucleoprotein may have one N-glycosylation sites, three O-glycosylation sites and eighteen phosphorylation sites.The partial glycoprotein gene was amplified by RT-PCR （primers design reference Kolodziejek）, and then was cloned into the vector pMD19-T to construct the recombinant plasmid and sequence. Phylogenetic analysis based on the "mid-G" of glycoprotein gene sequences showed that the IHNV-LQN131107 isolate was classified into Asian isolates, belong to the genogroup JRt. Homology analysis of IHNV-LQN131107 with other IHNV isolates showed that the strain has highest identity with the Chinese, Japanese and Korea IHNV strains. More over, Phylogenetic analysis based on the complete nucleoprotein sequences showed that the IHNV-LQN131107 isolate was classified into Chinese isolates,also belong to the genogroup JRt.