Enteric virus detection in Adriatic seawater by cell culture, polymerase chain reaction and polyacrylamide gel electrophoresis

Abstract

Forty samples of sea and estuary water were collected from a 40 km strip along the Adriatic coast of Italy between June 1994 and September 1995. Each sample consisted of 10 l of water. Routine bacteriological analyses were carried out and viral particles concentrated on cross-flow membranes; the concentrated water samples, equally divided into two parts, were used to infect both BGM and Hep-2 cells. Lysates from all cell cultures were further tested for the presence of enteroviruses by reverse-transcribed polymerase chain reaction (RT-PCR) and reoviruses by polyacrylamide gel electrophoresis (PAGE). The results showed widespread viral contamination of the waters tested, particularly in late summer. Under our experimental conditions, BGM cells were more efficient than Hep-2 in recovering viruses. In fact, enteroviruses were detected in up to 33% and reoviruses in 80% of BGM infected with seawater, compared to 8% and 53%, respectively, for the Hep-2 cells. In estuarine samples, enteroviruses were detected in 30% and reovirus in 54% of BGM, compared to 23% and 30% of Hep-2. Twenty nine out of 40 samples showed the presence of infectious particles on the basis of the CPE appearance; after identification of the isolated viruses, only 13 turned out to be specifically contaminated by enteroviruses. Of the latter, five were below the bacteriological standards set by the Italian legislation in line with the EEC Directive 76/160 IEEC for bathing waters.