MOLECULAR TECHNIQUES FOR THE IDENTIFICATION OF ENTERIC VIRUSES IN MARINE WATERS

The RNAs extracted from 28 high spin pellets of clarified lysates from BGM cells, previously infected by concentrated Adriatic Sea water sam ples, were analyzed by a dot blot test. We used a P-32-labeled cDNA pr obe (BamHI220-670) from the 5' non-coding end of the cloned poliovirus 1 (Mahoney) genome. The probe was selected for its broad range of gen etic specificities. Based on the density of the hybridization signals on the autoradiograph, our dot blot results showed a high degree of re activity of the probe to homologous (poliovirus 1) and heterologous (e chovirus 6 and coxsackievirus B3) reference RNA, as predicted by a com puter analysis of their sequences, and a low reactivity to that of the samples leading us to exclude the presence of enteroviruses like the reference strains. In order to understand if there were other enterovi ral serotypes or not, dot blot tests were supplemented with Northern-b lot hybridization assays. Results from the Northern-blot showed a seri es of fragments ranging from 4.3 to 1.2 kb but no signal corresponding to 7.5 kb (as did the positive control RNAs). These features suggeste d that the tested RNAs might derive from viruses of the Reoviridae fam ily, which includes members with segmented genome. Traditional PAGE an alysis showed clearly the 10-fragments pattern characteristic of reovi ruse. Twenty-three out of the twenty-eight samples tested showed the p resence of viruses, and this confirms the previously noted cytopathic effect (CPF) of the samples on the BGM cells.