Isolation and characterization of a novel single-stranded RNA virus infecting the bloom-forming diatom Rhizosolenia setigera

Abstract

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15 degrees C under a 12-h-12-h light-dark cycle of ca. 110 micro mol of photons m(-2) s(-1) with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.

FIG. 1.

Optical micrographs of an intact cell (A) and an RsRNAV01-infected cell (B) of R. setigera and a transmission micrograph of its frustule (C). Note that frustule pores of R. setigera are 91 ± 6 nm (n = 10; 80 to 98 nm) in length and 73 ± 6 nm (n = 10; 60 to 81 nm) in breadth (C). Bars, 50 μm (A and B) and 100 nm (C).

FIG. 2.

Transmission electron micrographs of R. setigera cultures. (A) Thin section of a healthy cell; (B) thin section of a cell 96 h after addition of the clonal pathogen RsRNAV01; (C) close-up of intracellular virus particles in panel B; (D) negatively stained virus particles in the culture lysate. CH, chloroplast; M, mitochondrion; F, frustule.

FIG. 3.

(A) Nucleic acids isolated from RsRNAV01 (lane 3) and RsRNAV06 (lane 4). RNA molecular size markers are shown in lanes 1 and 5, and Sendai virus RNA (15.8 kb) is shown in lane 3 to estimate the lengths of viral RNAs. A smaller faint band (0.6 kb) is also observed (arrowhead). (B) Nucleic acids extracted from RsRNAV06 without (lane 2) or with (lane 3) DNase I treatment at 37°C for 1 h. No host DNA is found in lane 3 (arrow). RNA molecular size markers are shown in lane 1. Smaller RNA bands (1.5 and 1.2 kb) extracted from RsRNAV particles are also observed.

FIG. 4.

Major structural proteins of RsRNAV. Proteins extracted from RsRNAV01 and RsRNAV06 were loaded in lanes 2 and 3. Molecular mass markers are shown in lane 1.

FIG. 5.

(A) Growth curve of R. setigera S2 used for the one-step growth experiments. (B and C) Changes in density of R. setigera S2 cells with (closed circles) or without (open circles) viral inoculation. (D and E) Changes in viral titer. RsRNAV06 inoculation was performed in the exponential growth phase (A [open arrow], B, and D) and the stationary phase (A [closed arrow], C, and E) at multiplicities of infection of 138 and 156, respectively. The error bars indicate standard deviations (B and C) or 95% confidence limits (D and E).