ÐIn 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 poly- acrylamide gel electrophoresis. Water Res. 31, 1980±1984] enterovirus strains were isolated from Adria- tic seawater and estuarine water from the Foglia River, by infecting susceptible cells with ultra®ltrated water samples. In the present work we have studied three of those samples, in which routine reverse transcriptase±polymerase chain reaction (RT±PCR) and sequencing analysis had identi®ed the presence of poliovirus type 3 (P3). In order to better estimate the risk to human health of such occurrence in bathing water (having bacteriological standards in line with the EEC directive 76/160), we set up a pro- tocol to distinguish wild from Sabin P3 strains. Three sets of RT±PCR primers were engineered and their predicted products were: 593 nucleotides (nt) in the 5' noncoding (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 (4209±4628) region, which is regarded as the hotspot of recombinant polioviruses. Eight reference ATCC strains, whose sequences were known, were also tested under the same experimental conditions in order to ver- ify the accuracy of the RT±PCR reactions. The amplicons were directly sequenced by Big-dye2 termin- ator sequencing using a capillary automatic sequencer. The latter two regions found the same viral species Polio 3 in all the sample strains, with no meaningful distinction between P3/Leon/37 and P3/ Leon/12a1b, the vaccine strain. The analyses in the 5'NC region were more useful, where genetic re- lationships and the predicted secondary structure suggested that the viruses were of vaccinal sources. Molecular data were con®rmed by in vitro phenotypic marker tests rct/40, where all the examined samples displayed a temperature sensitive phenotype rct/40(ÿ). Our results suggest that the 472U4C transition alone, is not a predictive marker of reversion to neurovirulence. Finally, we conclude that the 220U constantly found in the consensus sequences of the samples can serve as a good predictor of rct/ 40(ÿ) phenotype.
|Autori interni:||CARDUCCI, ANNALAURA|
|Autori:||MUSCILLO M; LA ROSA G; CARDUCCI A; CANTIANI L; MARIANELLI C|
|Titolo:||Molecular and biological characterization of poliovirus 3 strains isolated in adriatic seawater samples|
|Anno del prodotto:||1999|
|Appare nelle tipologie:||1.1 Articolo in rivista|