High Interaction Variability of the Bivalve-Killing Dinoflagellate <i>Heterocapsa circularisquama</i> Strains and Their Single-Stranded RNA Virus HcRNAV Isolates

Abstract

HcRNAV is a single-stranded RNA (ssRNA) virus that specifically infects the bivalve-killing dinoflagellate, Heterocapsa circularisquama. HcRNAV strains are grouped into 2 types (UA and CY), based on intra-species host specificity and the amino acid sequence of the major capsid protein (MCP). In the present study, we report the isolation of novel HcRNAV clones (n=51) lytic to the H. circularisquama strains, HU9433-P, HCLG-1, 05HC05 and 05HC06. HcRNAV34, HcRNAV109, HcRNAV641, and HcRNAV659, which displayed lytic activity against the strains, HU9433-P, HCLG-1, 05HC05, and 05HC06, respectively, were selected as typical virus clones and were intensively examined. The infection intensity of each host-virus combination was analyzed by examining the algicidal activity, detecting the intracellular replication of the viral RNA as well as the appearance of host cells with a morphologically abnormal nucleus post-infection. Interestingly, the strains, 05HC05 and 05HC06, were markedly sensitive to HcRNAV641 and HcRNAV659, respectively. Tertiary structural modeling predicted 4 unique amino acid (aa) substitutions in HcRNAV659-MCP to be exposed to an ambient water environment, which contributed towards determining its infection specificity. Neighbor-joining analysis of MCP aa sequences from HcRNAV clones revealed 3 clades, namely, the CY type and the UA1 and UA2 subtypes. The HcRNAV clones lytic to HCLG-1 (ex. HcRNAV109), HU9433-P and 05HC05 (ex. HcRNAV34), and 05HC06 (ex. HcRNAV659) were categorized into CY type, UA1 and UA2 subtypes, respectively. The present study highlights the complexity of the H. circularisquama-HcRNAV host-virus system, i.e., clonal variation, microbial control, and ecology in a natural algal population.

Multiplex reverse transcription quantitative PCR detection of a single-stranded RNA virus HcRNAV infecting the bloom-forming dinoflagellate Heterocapsa circularisquama

“…This study suggested that a newly developed multiplex RT‐qPCR assay can be used for quantifying HcRNAV abundance in vitro and in seawater. There are several advantages to this method: (1) the high specificity for HcRNAV is realized by the simultaneous detection of two conserved regions in the HcRNAV genome, combined with a highly specific annealing temperature; (2) simultaneous detection of various subtypes of HcRNAV is possible because the primers and probes target conserved regions in the HcRNAV genome (Nakayama et al ); (3) the rapidity of the mRT‐qPCR procedure is advantageous for carrying out real time monitoring of HcRNAV abundance; and (4) the numbers obtained by mRT‐qPCR were close to those measured by direct count, and therefore a highly quantitative measurement is presumably possible for the enumeration of HcRNAV in vitro and in seawater samples.…”

“…A portion of each ORF2 region was amplified via PCR using our original primers, HcRNAV‐ORF2a‐F 5′‐TTT CAC CCT GAG CAC CTT CCG C‐3′ and ORF2a‐R 5′‐CGC CAT GCA ACG CAT TAA GCA GC‐3′ (Nakayama et al ). Amplification was carried out in a 20‐ μ L reaction mixture containing 1 μ L (10 μ M stock) of each primer, 2 μ L (2 mM stock) of dNTPs, 2 μ L of 10× buffer, 0.2 μ L of Blend Taq (Toyobo), and 2 μ L of template cDNA.…”

“…In a previous study, we found that there are at least three distinct HcRNAV subtypes, including CY, UA‐1, and UA‐2, which have different intraspecies host specificities. This suggests that the high complexity of the relationship between HcRNAV and its host affects the occurrence and persistence of algal blooms (Nakayama et al ).…”

“…Considering the high species specificity and replication rate of HcRNAV, its algicidal activity is expected to be a promising tool for preventing H. circularisquama blooms. However, it has not yet been practically applied in the field (Nakayama et al ). This is partially due to the lack of precise validation methods for the impact of HcRNAV on natural blooms.…”

Virologists are “Symbionts” in Microbial Ecology

“…The DNA viruses that would be involved in retreat of red tide algaes (24, 25) and the RNA virus infecting dinoflagellates (26, 35, 38) represent excellent examples of functional environmental viruses in aquatic ecosystems. In addition, although it is not yet justified as true viruses, there are pioneering reports on intracellular genetic elements that are of great interest as a gene transfer agent (9, 10).…”