Isolation and characterization of a single-stranded RNA virus infecting the marine planktonic diatom Chaetoceros tenuissimus Meunier

Abstract

Diatoms are important components of the biological community and food web in the aquatic environment. Here, we report the characteristics of a single-stranded RNA (ssRNA) virus (CtenRNAV01) that infects the marine diatom Chaetoceros tenuissimus Meunier (Bacillariophyceae). The ca. 31-nm virus particle is icosahedral and lacks a tail. CtenRNAV01 forms crystalline arrays occupying most of the infected host's cytoplasm. By growth experiments, the lytic cycle and the burst size were estimated to be <24 h and approximately 1 x 10(4) infectious units per host cell, respectively. Stationary-phase C. tenuissimus cultures were shown to be more sensitive to CtenRNAV01 than logarithmic-phase cultures. The most noticeable feature of this virus is its exceptionally high yields of approximately 10(10) infectious units ml(-1); this is much higher than those of any other algal viruses previously characterized. CtenRNAV01 has two molecules of ssRNA of approximately 8.9 and 4.3 kb and three major proteins (33.5, 31.5, and 30.0 kDa). Sequencing of the total viral genome has produced only one large contig [9,431 bases excluding the poly(A) tail], suggesting considerable overlapping between the two RNA molecules. The monophyly of CtenRNAV01 compared to another diatom-infecting virus, Rhizosolenia setigera RNA virus, was strongly supported in a maximum likelihood phylogenetic tree constructed based on the concatenated amino acid sequences of the RNA-dependent RNA polymerase domains. Although further analysis is required to determine the detailed classification and nomenclature of this virus, these data strongly suggest the existence of a diatom-infecting ssRNA virus group in natural waters.

FIG. 1.

C. tenuissimus strain 2-10. Shown are optical micrographs of an intact cell (A) and a CtenRNAV01-infected cell (B) and transmission electron micrographs of negatively stained intact cells (C and D).

FIG. 2.

Transmission electron micrographs of C. tenuissimus 2-10 and CtenRNAV01; thin sections of an intact C. tenuissimus cell (A) and a CtenRNAV01-infected cell (B) at 48 h p.i., higher magnification of the CtenRNAV01-infected cell's cytoplasm at 48 h p.i. (C), and negatively stained CtenRNAV01 particles (D). CH, chloroplast; MT, mitochondrion.

FIG. 3.

Nucleic acids of CtenRNAV01 with no treatment (lane 2) and treatment with DNase I (lane 3) or RNase A in low- and high-salt buffers (lanes 4 and 5, respectively). Samples were electrophoresed in a formaldehyde agarose gel with RNA molecular size markers (lane M) and Sendai virus genome RNA (15.3 kb) (lane 1).

FIG. 4.

Major proteins of CtenRNAV01.

FIG. 5.

Changes in cell numbers of C. tenuissimus 2-10 without (closed squares) or with (open squares) virus inoculation and virus titers (closed circles). Virus inoculation (arrows) was performed at day 0 (logarithmic phase) (A) or day 4 (stationary phase) (B), where the MOI was set at 11.6 and 12.1, respectively.

FIG. 6.

ML tree based on deduced amino acid sequences of the RdRp whole domain. ML bootstrap values (percent) from 100 samples are shown at the nodes, followed by bootstrap values (percent) based on the NJ analysis of 100 samples. The ML distance scale bar is shown. CrPV, cricket paralysis virus; DCV, Drosophila C virus; BQCV, black queen cell virus; TrV, Triatoma virus; TSV, Taura syndrome virus; SssRNAV, Schizochytrium single-stranded RNA virus; DWV, deformed wing virus; SBV, Sacbrood virus; BPMV, bean pod mottle virus; CPSMV, cowpea severe mosaic virus; RTSV, rice turgo spherical virus; PYFV, parsnip yellow fleck virus; PV, human poliovirus 1 Mahoney; AiV, Aichi virus; BoCV, bovine enteric calicivirus; NV, Norwalk virus.