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Isolation and ultrastructural study of a lytic virus in the small marine phytoflagellate, Micromonas pusilla (Prasinophyceae) Mayer, Jolie Arden

Abstract

The discovery, isolation, and ultrastructural investigation of a lytic virus infection in the naked, marine phytoflagellate, Micromonas pusilla, are described. Infected cells were first seen in April, 1975, during a transmission electron microscope examination of a week old enrichment culture containing nanoplankton collected at the mouth of the Burrard Inlet in Vancouver, British Columbia. Selective lysis occurred in this culture» removing nearly all M. pusilla cells without visibly affecting the other microflagellate populations. Ultrastructural examination of the culture indicated the presence of polyhedral inclusions within cells of M. pusilla. Medium from this infected culture was observed to cause the destruction of unialgal strains of M. pusilla maintained in the Northeast Pacific Culture Collection. Ultrastructural studies of the host organism, M. pusi11a, confirmed the earlier findings of Manton and Parke (1959) and provided additional information on the fine structure of this nanoflagellate. Terminal flagellar hairs, a vestigial, second basal body, and a peripheral system of microtubules are described. The polyhedral virus particles in situ within infected cells, or when isolated and negatively stained with uranyl acetate, appear tail-less and present profiles consistent with their having icosahedral symmetry. The virions have an average diameter of 135 nm. Surface substructures analogous to viral capsomeres were observed on partially disrupted particles. Viral entry takes place within 15 minutes following introduction of inoculum and occurs by localized fusion of adjacent cell and virus surfaces, followed by direct penetration of viral genome. After an eclipse period of approximately three hours in duration progeny virus appear in the perinuclear cytoplasm. Associated with the viral assembly process are cytoplasmic fibrils and clusters of membrane-bound vesicles. Viral egress occurs by localized rupture of the plasmalemma. Viral activity is monitored by an assay system based upon the reduction in the in vivo chlorophyl1 a fluorescence of infected cells. Solutions of virus particles stored at 4°C in millipore-filtered, autoclaved seawater or culture medium retain their infective capacity for nine months. The virus is stable in a pH range between 4 and 10. Thermal inactivation occurs between 55° and 63°C. Host range studies indicate that the lytic properties of the virus are probably specific to M. pusilla. The virus has been designated Micrbmbnas pusilla virus, MPV.

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