Detection of Fish Pathogenic Viruses in Seawater Using NegativelyCharged Membranes

摘要

After an outbreak of viral disease in an aquafarm, release of virus (es) from infected fish into environmental seawa-ter has been suspected. In the present study, we utilized a negatively charged membrane (HA type) as an efficient method for concentration and detection of fish pathogenic viruses, specifically, megalocytivirus and viral hemorrhagic septicemia virus (VHSV) present in field-collected seawater samples or inoculated into seawater artificially. Positively charged viruses adsorbed onto thenegatively charged membrane and were eluted with 1 mM NaOH (pH 10.5) following rinsing with 0.5 mM H2S04 (pH 3.0). Megalocytivirus and VHSV particles isolated using an-egatively charged HA membrane from seawater inoculated with each virus at a concentration of 10 viral particles/ mL were of sufficient quantity to show positive results in atwo-step PCR (or RT two-step PCR); however, despite it being negatively charged, a cellulose acetate (CA) membraneshowed negative results.In quantitative PCR, the detection limits of the HA membrane for megalocytivirus and VHSV in seawater were 1.20E+00 viral particles/ mL and 1.22E+01 viralparticles/mL, respectively. The calculated mean recovery yields from 1 L seawater spiked with known concentrations of megalocytivirus and VHSV particles were 28.11% and 23.00%, respectively. The concentrate of a 1-L sample of culturing seawater from the aquatank of flounder suffering from VHSV showed clear positive results in PCR when isolated with an HA, but not a CA, membrane. Thus, viral isolation using an HA membrane is a practical and reliable method for detection of fish pathogenic viruses in seawater.