Isolation and characterization of a novel single-stranded RNA Virus infectious to a marine fungoid protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)

Abstract

Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 x 10(3) to 6.4 x 10(4) infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms.

FIG. 1.

Optical microphotographs of Schizochytrium sp. strain NIBH N1-27. (A) Intact cells; (B) lysed cells 48 h after inoculation of SssRNAV.

FIG. 2.

Transmission electron microphotographs of Schizochytrium sp. strain NIBH N1-27 infected by SssRNAV. (A) Negatively stained SssRNAV particles in the culture lysate; (B) thin section of a healthy cell of Schizochytrium sp. strain NIBH N1-27; (C) thin section of an SssRNAV-infected cell at 8 h postinoculation (the arrowheads indicate fibrils within vacuoles); (D) crystalline arrays of SssRNAV; (E) unordered aggregation of SssRNAV; (F) virus particles concentrated along the intracellular membranes. N, nucleus; G, Golgi body; Mt, mitochondrion.

FIG. 3.

Formaldehyde-agarose gel electrophoresis of viral nucleic acids. SssRNAV nucleic acids were not treated (lane 2), were treated with RNase A at 37°C under low-salt conditions (lane 3) or high-salt conditions (lane 4), or were treated with DNase I at 37°C (lane 5). Lane 1 contained an RNA molecular marker.

FIG. 4.

SDS-PAGE of SssRNAV structural proteins. The gel was stained with Coomassie brilliant blue.

FIG. 5.

Changes in abundance of Schizochytrium sp. strain N1-27 cells with (▪) or without (□) viral inoculation and the viral titer (○). SssRNAV inoculation was performed in the exponential growth phase of host cultures (arrow). Results for only one of the triplicate experiments are shown. The error bars indicate the 95% confidence limits for the viral titer.