Isolation And Identification Of Largemouth Bass Virus(LMBV-GD),and Determination And Analysis Of Its Genome Sequence

Largemouth bass virus（LMBV）is an icosahedral double-stranded DNA virus with a diameter of 160-200nm.It can cause a serious disease of largemouth bass,which brings serious economic losses to largemouth bass aquaculture.At present,the known replication mode and pathogenic mechanism of LMBV is little.In this study,LMBV was studied and analyzed,which laid a foundation for its further study.In this study,diseased largemouth bass were collected from a farm in Guangdong province.Virus was isolated from liver,spleen and kidney of diseased largrmouth bass.Fathead minnow（FHM）cells were challenged with the isolated virus.The challenged FHM cells showed obvious cytopathic effect（CPE）,with the titer of TCID₅₀=10^6.7/ml.The challenged FHM cells were sected by electron microscopy.The virus particles with hexagonal cysts and diameter about 160nm were observed.The virus was proliferated and collected in FHM cells for regression infection experiment.The challenged largemouth bass acted like typical symptoms of LMBV infection.On the third day after injection,some largemouth bass died;On eighth day after injection,the death rate was 100%.On the ninth day after soaking infection,some largemouth bass died;On the 12th day after challenge,all largemouth bass died.Based on results of electron microscopy and regression infection,we preliminarily speculated that the virus should be a new LMBV isolate,named LMBV-GD.We acquired nucleotide sequences of mcp（major capsid protein）,mmp（myristylated membrane protein）,dnmts（DNA methyltransferase）and DNApol（DNA polymerase）.Homologous analysis showed that the similarity ratio of the four genes in LMBV-GD with that in LMBV USA isolates was higher than 99.7%.Therefore,we determined that LMBV-GD was a new LMBV isolate,named LMBV-GD.In addition,a detection method of LMBV was established based on genes of mcp and mmp,which is proved to be reliable by verification.The complete genome sequence of LMBV-GD was obtained by high-throughput sequencing.Genemarks were used to annotate gene functions.The annotation results are obtained by comparing with databases of NR（Non-redundant Protein Sequence Database）,NT（Nucleotide Sequence Database）and COG（Cluster of Orthologous Groups of Proteins）.The results showed that LMBV-GD was homologous with LMBV-USA1（Gen Bank accession number:MK681855）,LMBV-USA2（Gen Bank accession number:MK681856）and MRV（Mandarin fish Ranavirus,Gen Bank accession number:MG941005）.The results of linear analysis showed that LMBV-GD was similar to LMBV-USA1 and LMBV-USA2 at the linear gene arrangement,CG%,nucleotide length,and amino acid sequences.The phylogenetic trees showed that LMBV-GD was clustered into one clades with LMBV-USA1 and LMBV-USA2.Furthermore,the linear analysis results showed that the LMBVs had specific genes and gene arrangement,which was different from the four know genomic phenotypes（ATV,CMTV,FV3 and SGIV phenotypes）of ranavirus.Therefore,LMBV should be a new genomic phenotype（LMBV phenotype）.LMBV phenotype is the fifth genomic phenotype of the ranavirus.In conclusion,LMBV-GD was successfully isolated and identified in this study,and it was found that LMBV phenotype was the fifth genomic phenotype of ranovirus.This study enriches the data of largemouth bass virus.