Purification and genetic analysis of a new Prorocentrum minimum infecting virus

Abstract

One of the biggest problems in sequencing the whole genome of a marine algal virus is purifying the virus. Most of the host cultured in laboratory has a concentration of 103`~104 particle/cell and hence when the host is inoculated with virus, the virus titer is not sufficient enough for molecular analysis. There is additional losses during the process of filtration and concentration. Therefore, it is difficult to obtain high concentration of pure viral DNA for molecular analysis. In this study, various purification and concentration methods have been carried out to overcome the problems. Pre-filtration and mini-size hollow fiber ultracentrifugation was conducted to obtain large quantities of Prorocentrum minimum DNA Virus 01 (PminDNAV01). The virus suspension were treated with chloroform, DNaseI and RNaseA to make sure that all bacteria have been removed. Next generation sequencing was carried out with total DNA extrated from PminDNAV01 infected genome and the viral genome sequences were assembled using previously reported algal virus sequences. Although, whole genome sequence of the virus could not been obtained, partial genome of the virus has been successfully recovered. Two contigs, PMV53(52,425bp) and PMV93 (46,021bp), were obtained after assembly of sequences. There are 19 ORFs of PMV53 and 7 ORFs of PMV93 that showed homology with proteins in GenBank database. According to the GenBank database, PminDNAV01 is closely related with nucleo­cytoplasmic large DNA viruses (NCLDVs). The improved virus purification process and whole genome shot-gun sequencing using next generation sequencing could reveal new viral genomes of a novel algal­infecting virus, PminDNAV01.