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A simple method to produce biotinylated probes by utilizing an amine-psoralen to conjugate an ester-biotin to nucleic acid molecules is described. It is simple, rapid, and well suited to label cDNA probes with any PCR-generated amplicon for hybridization assay. Its application to identify or to confirm the G and P genotypes of rotavirus-derived amplicons is described; however, it may be used to label amplicons of any origin. As an alternative or as a complementary test to either PCR-typing assay and/or sequencing, it should reduce considerably the laboratory costs required to genotype fully virus strains in large epidemiological surveys conducted in developing countries.

The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.

A multi-site nested reverse transcription and polymerase chain reaction (RT-PCR) followed by restriction endonuclease analysis (REA) was developed to identify hepatitis E virus (HEV) in clinical specimens. Four sets of primers were selected to amplify regions in the HEV genome supposed to encode the helicase, polymerase, and parts of the viral capsid protein. Digestion of the nested PCR products with HinfI, HaeII, AvaII, BglI, KpnI, SmaI, or EcoRI generated readily recognizable profiles that confirm the HEV sequences and/or distinguish the unique Mexico genotype (our positive control) from all other isolates (Asian genotype). In addition, the hydroxyapatite (HA) adsorption method was compared to other adsorption and extraction methods widely used to purify viral RNA from clinical specimens for RT-PCR. All methods presented the same sensitivity of recovery of HEV RNA, but only the adsorption methods efficiently removed fecal enzymatic inhibitors. The HA method gave the best results and was the most economic in terms of time, cost, manipulations and reagents. The method was validated by screening a small number of serum and fecal specimens available from patients with acute non-A,B,C hepatitis in Nepal. HEV RNA was identified in half (5/11) of the fecal specimens obtained from patients with evidence of recent HEV infection, but in none of the 14 patients without a serological marker for hepatitis E.

A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.

An improved procedure to recover and purify double-stranded RNA (dsRNA) from fecal specimens is described. Guanidine isothiocyanate, hydroxyapatite, and cetyltrimethylammonium bromide were used to extract and purify rotavirus dsRNA from fecal specimens. The method is very efficient and easy to perform, and precludes the use of toxic substances such as phenol, chloroform, and Freon. It yields RNA free of enzymatic inhibitors, permitting its detection by reverse transcription-polymerase chain reaction assays. In addition, it was demonstrated that during initial clarification of the fecal suspension, the pellet must be washed at least twice to avoid massive losses of virus, viral protein, or viral nucleic acid retained in the solid debris.

A combined enzyme immunoassay using nitrocellulose membrane as solid support is described for the detection of rotavirus and adenovirus in faeces from children with gastroenteritis. Its sensitivity and specificity are comparable to those of a previously described assay performed on plastic microplates (Pereira et al. (1985) J. Virol. Methods 10, 21-28). The introduction of nitrocellulose membrane as support for the immune reactions greatly simplifies the multiple washing steps and precludes the need for disposable plastic plates.