Astroviruses are single-stranded, positive-sense RNA viruses that are associated with gastroenteritis in humans and animals. We describe a reverse transcription-polymerase chain reaction (RT-PCR) assay using primers targeted to a nonstructural protein coding region that allowed sensitive detection and genetic typing of representative strains of seven astrovirus serotypes. Phylogenetic analysis of the nucleotide sequences of PCR products from the reference strains and several wild isolates indicated two distinct genogroups of sequences in open reading frame 1a (ORF 1a). These genogroups correlated with serotype: genogroup A included strains of types 1 to 5, while genogroup B included strains of types 6 and 7. This phylogenetic arrangement differs from the nearly equidistant clustering of serotypes seen when comparing nucleotide sequences from either ORF 1b or ORF 2. It is possible that recombination was responsible for the observed difference in genetic relationships.

In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4,6-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10 8 particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.

Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers. An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables. The protocol included washing the fruit or vegetable surface with 100 mM Tris-HCl, 50 mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50 mM MgCl2 to reduce the decrease in viral infectivity during the process. The viral concentration method was based on polyethylene glycol precipitation. Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction. Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml. Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-microm porosity) and inoculated on cell culture for infectious virus detection. About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces. The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus. By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 x 10(1) 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 x 10(3) RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

DC-related infections are associated with DC longevity. As shown by the 5-day-interval analysis, the incidence of DC-related infections did not increase with DC duration, suggesting that the risk for DC-related infections remained unchanged with time. The characteristics of DC-related infections in ICU patients were comparable to those previously reported for CVC-related infections.

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