A new RT-PCR method for the identification of reoviruses in seawater samples.
The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 101 were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3:3:4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.
Muscillo
M
M
Laboratorio di Igiene Ambientale, Department of Environmental Hygiene, Istituto Superiore di Sanita', Viale Regina Elena 299, Rome, Italy. muscillo@water.iss.infn.it
La Rosa
G
G
Marianelli
C
C
Zaniratti
S
S
Capobianchi
M R
MR
Cantiani
L
L
Carducci
A
A
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004755:Enterobacteriaceae / Q000302:isolation & purification; D004770:Enterovirus / Q000302:isolation & purification; D006386:Hemagglutination Tests; D007558:Italy; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D012087:Reoviridae / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012623:Seawater / Q000821:virology*; D017422:Sequence Analysis, DNA; D014871:Water Microbiology
D017931:DNA Primers; D004279:DNA, Viral; D012330:RNA, Double-Stranded; D012367:RNA, Viral