ABSTRACT
AIMS: Faecal material from raw sewage or other sources lacking effective treatment sometimes contaminates water for human consumption. The relevant Italian regulations therefore call for testing drinking and recreational water for the presence of enterovirus. METHODS AND RESULTS: Traditional methods of analysis are based on revealing the typical cytopathic effects of enterovirus on cell cultures. However, the presence in environmental samples of different types of virus may cause interference phenomena that mask such cytopathic effects. The paper reports on an experimental test of this interference hypothesis. Buffalo Green Monkey cell cultures were co-infected via mixed suspensions of the polio type 3 virus and reovirus type 1. Cytopathic effects were then sought and the presence of enterovirus tested for via RT-PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that the normally high sensitivity of tests for the detection of enterovirus in samples is considerably decreased by the simultaneous presence of reovirus.
Subject(s)
Environmental Monitoring/methods , Orthoreovirus, Mammalian/growth & development , Poliovirus/growth & development , Viral Interference , Animals , Cells, Cultured , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Orthoreovirus, Mammalian/isolation & purification , Poliovirus/isolation & purification , Water Supply/standardsABSTRACT
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.
Subject(s)
Reoviridae/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Seawater/virology , Water Microbiology , Base Sequence , Consensus Sequence , DNA Primers/chemistry , DNA, Viral/chemistry , Electrophoresis, Agar Gel , Enterobacteriaceae/isolation & purification , Enterovirus/isolation & purification , Hemagglutination Tests , Italy , Molecular Sequence Data , RNA, Double-Stranded/chemistry , RNA, Viral/chemistry , RNA, Viral/isolation & purification , Reoviridae/chemistry , Reoviridae/genetics , Sequence Analysis, DNAABSTRACT
In order to evaluate possible indicators of viral aerosol contamination in sewage treatment plants, a year-long study was carried out on the relationships between the presence of cytopathogenic viruses and the counts of total bacteria, faecal streptococci and somatic coliphages in samples collected at various distances from the aerosol source (aeration tank). The activated sludge plant studied proved to be a significant source of microbe-bearing aerosol with high levels of viral contamination. When the virus was found in sewage, it was also found in the air, at least in the sites closest to the aeration tank. With regard to the possibility of using the chosen parameters as markers of viral contamination, the total bacteria and faecal streptococci counts were generally positively correlated with viral presence, while coliphage counts yielded no analogous relationship.
