Isolation,identification And Biological Characteristics Research Of A Novel Ranavirus Isolated From Black-spotted Pond Frog (Rana Nigromaculata)

In April 2016,an outbreak occurred in a population of cultured Rana nigromaculata in Shuangliu,Sichuan Province,with suffering 90%mortality.External clinical signs included swollen abdomen,underjaw and abdomen exhibited hemorrhage on body surface of tadpoles,internal signs exhibited massive yellow ascites,hepatomegaly and hepatorrhagia,emptiness intestinal with lots of mucus and splenomegaly.Histological examination showed marked necrosis in the liver,spleen,kidney,intestinal tract and heart,basophilic inclusions appeared in the cytoplasm of hepatocyte and intestinal epithelial cells.The tissue homogenates of diseased tadpoles were inoculated into EPC cells at 25?for 4days and caused typical cytopathic effect,virus titer TCID₅₀ is upto 10⁷/0.1mL by using Reed-Muech method.In pathogenicity tests,tadpoles were immersed in 5%virus fluid for8 h,the symptoms observed were similar to those observed in naturally infected tadpoles and mortality reached 80%,so the virus was determined pathogenecity of this disease.In addition,transmission electron microscopy of EPC cells infected with isolated virus,and it was found that the virus was regular hexagon with capsule,the diagonal diameter was about 135±8nm,and virus particles arrayed in crystalline or free in the cytoplasm.And the sequence of isolates revealed more than 99%similarities to ranavirus based on specific PCR detection and sequence of major capsid protein gene alignment.On the combination of the results,we conclude that ranavirus was the causative agent of this outbreak,and named the virus as Rana nigromaculata ranavirus?RNRV?.The physico-chemica characteristics and phenotypes of RNRV were performed with ordinary method,included viral titer,sensitivity to acid,base,trypsin,organic solvent,temperature and suitable culture cell line,proliferation characteristics.Results showed the similar characteristics with ranavirus,it is sensitive to excess acid?pH?3?,excess base?pH?10?,trypsin and organic solvent,it can be inactivated at 60?for 30 minutes.EPC and GCO lines were the most suitable for cultivation,the optimum temperature was 25?-28?.In addition,under the condition of 25?,the proliferation rate was the highest in the12-24 h after EPC inoculated virus,slowed down after 36 h,and finally reached the platform stage in the 72-96 h.Based on the high-throughput sequencing technology,the complete genome of RNRV Was determined by using Illumina HiSeq 2000.Results showed that the consists of the complete genome were 104286 bp included 104 ORFs with 55 genes of known functions,49 genes of unknown functions.The length of amino acid was between 41-1293 AA and protein molecular weight was between 4.4-140.8 KDa.Genome of GC content was 55.2%.There were 59?CA?repeats in 79069-79186 bp.Dot pot analysis showed that RNRV was related most closely to RGV.Phylogenetic tree base on amino acid sequences which was encoded by 26 core genes was constructed in NJ method,the result showed that RNRV appeared in the FV3 cluster,it was related more closely to RGV and STIV from China.