Determination of naturally occurring noroviruses in coastal seawater by alkaline elution after acid rinse using negatively charged membrane

Abstract

A new procedure for concentrating viruses from seawater using a negatively charged membrane eluting with alkaline solution (NaOH, pH 10.5) after acid rinse (H2SO4, pH 3.0) was applied to determine naturally occurring enteric viruses in seawater in Tokyo bay. The levels of total coliforms and fecal coliforms ranged from 40 to 68000 (cfu/100ml) and from 2 to 32000 (cfu/100ml), respectively. The F-specific phages were not detected from 5 ml of 53 samples out of 61 tested. The levels of indicator microbes were not found to be related to the tide in Tokyo bay. Enteroviruses were not detected by cell culture RTÐPCR, but detected by direct RTÐPCR from approximately 10% of the samples. Noroviruses were found positive from approx. 31% of the winter samples (n=29), whereas only 3% from the summer samples (n=32). These results of direct RTÐPCR were equivalent to determination of Norwalk viruses occuring in 50 ml of seawater. These results suggested that the levels of noroviruses in Tokyo bay were higher in winter than those of enteroviruses The virus concentration method used was proved to be useful for determination of naturally occurring viruses in seawater, especially when applied prior to PCR detection of nonculturable viruses.