The importance of the genetic diversity of the HcRNAV ssRNA virus in the viral-based bloom control of the dinoflagellate Heterocapsa circularisquama

Abstract

The HcRNAV virus has been used to control the blooms of the harmful dinoflagellate Heterocapsa circularisquama through the spraying of autochthonous sediment containing HcRNAV from prior blooms. When using natural substances for harmful algal bloom (HAB) control, the effects on local ecosystems must be ascertained and minimized. Specifically, HcRNAV is a single-stranded RNA virus which replicates with an extremely high mutation rate, leading to significant genetic diversity. Here we demonstrate the genetic diversity of HcRNAV in natural sediments analyzed through the use of the clone library approach and Next Generation Sequencing (NGS) analysis based on the major capsid protein (MCP) sequence of HcRNAV. The sediment samples were collected over four developmental phases, including low- and high- density phases of H. circularisquama blooms in the saltwater Lake Kamo, Niigata, and Mikawa Bay, Aichi, Japan. The HcRNAV sequences obtained from both methods were divided into only two groups (UA2 and CY groups), with most of the sequences classified into the UA2 group. Haplotype network analysis, which is used to reveal the genealogical relationships among haplotypes, has indicated that the HcRNAV sequences varied according to time and location. This analysis suggested that HcRNAV does not randomly diversify, thus indicating that the diversity of HcRNAV might be undergoing a process of natural selection. Furthermore, the present cluster in the haplotype network was consistent with the cross-reactivity tests infecting the H. circularisquama strains with isolated HcRNAV clones. These results suggest that HcRNAV consists of several clusters that are evolving as genetically diverse populations, termed quasispecies. Hence, our findings indicate that for effective utilization, it is necessary to collect and pool sediments containing HcRNAV from multiple local sites and periods to provide more diversity in the viral populations used for bloom control.