Molecular and biological characterization of poliovirus 3 strains isolatedin Adriatic seawater samples

In a previous study [Muscillo, M., Carducci;A., La Rosa, G., Cantiani, L., Marianelli, C. (1997a) Enteric virus detection in adriatic seawater by cell culture, polymerase chain reaction and polyacrylamide gel electrophoresis. Water Res. 31, 1980-1984] enterovirus strains were isolated from Adriatic seawater and estuarine water from the Foglia River, by infecting susceptibl e cells with ultrafiltrated water samples. In the present work we have stud ied three: of those samples, in which routine reverse transcriptase-polymer ase chain reaction (RT-PCR) and sequencing analysis had identified the pres ence of poliovirus type 3 (P3). In order to better estimate the risk to hum an health of such occurrence in bathing water (having bacteriological stand ards in line with the EEC directive 76/160), we set up a protocol to distin guish wild from Sabin P3 strains. Three sets of RT-PCR primers were enginee red and their predicted products were: 593 nucleotides (nt) in the 5' nonco ding (5'NC) region (11-603), 350 nt at the Vp3-Vp1 junction (2438-2787) of the capside protein genes, and 420 nt in the 2C (4309-4628) region, which i s regarded as the hotspot of recombinant polioviruses. Eight reference ATCC strains, whose sequences were known, were also tested under the same exper imental conditions in order to verify the accuracy of the RT-PCR reactions. The amplicons were directly sequenced by Big-dye(TM) terminator sequencing using a capillary automatic sequencer. The latter two regions found the sa me viral species Polio 3 in all the sample strains, with no meaningful dist inction between P3/Leon/37 and P3/ Leon/12alb, the vaccine strain. The anal yses in the 5'NC region were more useful, where genetic relationships and t he predicted secondary structure suggested that the viruses were of vaccina l sources. Molecular data were confirmed by in vitro phenotypic marker test s rct/40, where all the examined samples displayed a temperature sensitive phanotype rct/40(-). Our results suggest that the 472(U-->C) transition alo ne, is not a predictive marker of reversion to neurovirulence. Finally, we conclude that the 220(U) constantly found in the consensus sequences of the samples can serve as a good predictor of rct/ 40(-) phenotype. (C) 1999 El sevier Science Ltd. All rights reserved.