We compared the adsorption kinetics of Heterosigma akashiwo virus clones 01 (HaV01) and 53 (HaV53) to several other H. akashiwo strains with differing viral susceptibility spectra. When a HaV strain was inoculated into its suitable host strain, only infectious virus particles (3 to 4% of direct count estimates) adsorbed to the host cells, and the adsorption coefficient was estimated to be 4.02 to 5.44 × 10 -8 ml min -1; in contrast, virus adsorption to an unsuitable host strain was not detected. This suggests that virus adsorption is an important first step in determining the sensitivity or resistance of H. akashiwo strains to viral infection, and that it determines the strain-specific host specificity of HaV.
KEY WORDS: Algal virus · Heterosigma akashiwo · Host range · Intraspecies host specificity · Viral adsorptionResale or republication not permitted without written consent of the publisher Aquat Microb Ecol 42: 209-213, 2006
MATERIALS AND METHODSWe used 2 HaV clones and 4 Heterosigma akashiwo strains. HaV01 was isolated from Unoshima Fishing Port (Fukuoka Prefecture, Japan), and HaV53 was isolated from Itsukaichi Fishing Port (northern Hiroshima Bay, Japan). Axenic strains of H. akashiwo (strains H93616, H94608, H98518-4 and H98527-6) were isolated from Itsukaichi Fishing Port. These were selected because they display differing viral susceptibilities to HaV01 and HaV53 (Table 1; Nagasaki & Yamaguchi 1998a, Tarutani et al. 2000. Algal cultures were grown in modified SWM3 medium enriched with 2 nM Na 2 SeO 3 (Chen et al. 1969, Itoh & Imai 1987, Imai et al. 1996, and incubated at 20°C using a 12:12 h light:dark cycle (light provided by cool white fluorescent illumination FL40S D EDL D65, Toshiba, at ~50 µmol photons m -2 s -1). Aliquots of HaV were inoculated into 25 ml exponentially growing cultures of Heterosigma akashiwo at a multiplicity of infection of ~0.1. This corresponds to a total virus to host ratio of ~3, because 3 to 4% of the HaV particles stainable with DAPI (4'-6-diamidino-2-phenylindole) were considered to be infectious (see 'Results and discussion'). As a control, an algal culture without viral inoculation was passed through a glass fiber filter (Whatman GF/F) and the same volume as the HaV suspension (25 ml) was inoculated. Each experiment was conducted in triplicate. Samples were taken immediately after gentle mixing, and then at 30 min intervals over a period of 120 min; they were then diluted 10-fold with SWM3 medium and centrifuged at 3000 rpm (800 × g), at 4°C for 3 min to remove algal cells with adsorbed viruses. An aliquot of the resulting supernatant was fixed with glutaraldehyde at a final concentration of 1%. The unadsorbed virus particles were directly counted using epifluorescence microscopy following the protocol of Weinbauer & Suttle (1997) with partial modifications. Briefly, DAPI solution -15 µg ml -1 DAPI in Tris buffer (10 mM TrisHCl, 10 mM EDTA-Na, 100 mM NaCl, 10 mM 2-mercaptoethylamine hydrochloride, pH 7.4; Hamada & Fujita 1983) -was added to each fixed sa...