Isolation, Identification, Genome Sequencing Of A Novel Ranavirus Isolate In Chinese Giant Salamander (Andrias Davidianus)and Genetic Diversity Of Chinese Giant Salamander

Chinese giant salamander (Andrias davidianus) is a rare protected animal and important species in aquaculture in China. Alongside the enlargement of the cultivation scale, the incease of intensification and the frequent trading of the animal, the diseases have become a great concern in recent years. The Chinese giant salamander iridovirus disease was a new emerged viral disease in farmed Chinese giant salamanders, which has caused huge economic loss and represents a great threat the industry. In this study, an irodovirus, belonging to the genus Ranavirus, family Iridovuridae was isolated and identified from the diseased Chinese giant salamander. The physic-chemical and biological properties of the virus were investigated. Moreover, the complete genome sequence of the Chinese giant salamander iridovrisus was determined and analyzed. The results are as follows:1. Isolation and indentification a Ranavirus pathogen in diseased Chinese giant salamander, Andrias davidianusAn iridovirus was isolated form the diseased larvae and adult Chinese giant salamanders. The typical clinical signs of diseased larvae exhibited jaw and bellies swelling, subcutaneous hemorrhage, and in the diseased adults, the symptoms of included skin hemorrhages, ulceration of the hind limbs, and multiple hemorrhagic spots in the visceral organs. Light microscopy demonstrated tissue degeneration and cytoplasmic inclusions in spleen, liver and kidney, suggestive of a viral infection. The tissue necrosis included splenocytes vacuolar degeneration, necrosis of renal hemopoietic tissue cells and liver sinusoids hypertrophied. The tissue homogenates from the diseased animals were inoculated into EPC (Epithelioma papulosum cyprinid) cells and caused typical cytopathic effect. Electron microscopy observation revealed that the virion was140～180nm in diameter both in the renal tubular epithelial cells of the kidney tissue and in the virus-infected EPC cells. The virus was determined pathogenecity to the healthy animal after experimental infection and resulted in the reproduction of clinical signs as naturnaturally ocurred. The whole major capsid protein (MCP) coding gene of the isolated virus was cloned and sequenced (GenBank:JN615141) and the result of sequence analysis shown a98%～99%sililarity with other ranavirus MCP sequences in GenBank. Combined the histopathology, electron microscopy observation, virus culture in EPC cells, experimental infection and MCP sequence analyzed, the isolated virus was determined to iridovirus belonging to the genus Ranavirus, family Iridovurus and was the pathogen of diseased Chinese giant salamander. The disease was tentatively name Chinese giant salamander iridovirus disease, meanwhile the pathogen named Chinese giant salamander iridovirus (GSIV).2. Physico-chemical and biological properties and detection methods of the Chinese giant salamander iridovirusThe physico-chemical characteristics of GSIV were investigated. The physico-chemical and biological factors included viral titer, growth, sensitivity to temperature, to acid and base, to organic solvent and trypsin. Based on the sequence of MCP gene, five methods were established to detect GSIV. They were conventional PCR, fluorogenic quantitative PCR, polyclone antibody, nested PCR and loop-mediated isothermal amplification assay (LAMP). These methods all can detect GSIV effectively. And the detection sensitivity of the fluorogenic quantitative PCR and LAMP methods can achieve10-9DNA template dilution.3. Sequencing and characterization of GSIV genomeThe complete genome of GSIV was determined by using Illumina HiSeq2000. The length of GSIV genome was104373bp. The results of sequence analysis showed that the genome encoded95predicted open reading frames (ORFs) and GC content was55.2%. The numbers of amino acid encoded proteins were between49and129. The genome had the26core genes of Iridovirus. There were48(CA) repeats in40219～40314nt. The ORF align and dot plot analysis all showed the GSIV closed with common midwife toad ranavirus (CMTV). According the amino acid order of26core genes in GSIV genome,16iridovirus genome sequences were used to construct NJ phylogenetic tree. The results revealed that the sequences clustered into five genera of Iridoviridae, respectively. The GSIV is a member of Ranavirus. In addition, the virus infecting host had the same classification status tended to cluster. The complete genome sequence was submitted to GenBank with the accession KC243313.4. Development of microsatellite markers and genetic diversity analysis of Chinese giant salamanderUsing the AG-enriched genomic library of the Chinese giant salamander, seventy microsatellite markers were developed for the Chinese giant salamander. Ten pairs of these microsatellite markers were evaluated the genetic diversity of cultivation and wild populations. Seven markers were polymorphic in all the samples. The number of alleles ranged from6to9. The polymorphism information content (PIC) of all populations was from0.3750to0.8751, suggesting high polymorphism at these loci. Compared with the wild populations, some alleles in the cultivation population were drifted and the PIC was lower.