Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization

Abstract

Application of reverse transcription (RT)-PCR to detect small round-structured viruses (SRSVs) from fecal specimens of patients with gastroenteritis has been insensitive because of the tremendous sequence heterogeneity between strains. We have designed two RT-PCR primer sets (G-1 and G-2) based on the nucleotide sequence diversity in the RNA polymerase gene of SRSVs belonging to two distinct genogroups represented by Norwalk virus (primers G-1) and Snow Mountain agent (primers G-2). All 22 SRSV strains examined that had been classified previously by solid-phase immune electron microscopy into four antigenic types (UK1, UK2, UK3, and UK4) could be detected by RT-PCR with these two primer sets. The G-1 primer set detected 6 UK2 strains, and the G-2 primers detected 16 strains, including 7 UK1, 5 UK3, and 4 UK4 strains. On the basis of nucleotide sequences of 81-bp fragments of the RT-PCR products from 13 strains determined in this study, together with those previously reported for 17 SRSV strains, we designed four sets of internal oligonucleotide probes (P1-A, P1-B, P2-A, and P2-B) for Southern hybridization, using chemiluminescent detection. The P1-A probe hybridized with PCR products from the UK2 strains; the P1-B probe, with products from two of the seven UK1 strains; the P2-A probe, with four of the remaining five UK1 strains; and the P2-B probe, with products from both UK3 and UK4 strains, as well as with one strain originally typed as UK1 which showed cross-reactivity with UK4 upon retesting by solid-phase immune electron microscopy. RT-PCR with both the G-1 and the G-2 primer sets can increase the detection rate of the many antigenically distinct SRSVs and, when combined with Southern hybridization, may predict the antigenic type of the SRSV associated with infection.

Identification of group B rotavirus as an etiological agent in the gastroenteritis outbreak in Maharashtra, India

“…All patients were examined for fever, number of episodes, and duration of vomiting and diarrhea, extent of dehydration, and treatment for the assessment of disease severity score (DSS). 7 According to the scores obtained, the disease condition of each of the patients was categorized as mild (scores 0-5), moderate (scores 6-10), severe (scores [11][12][13][14][15], and very severe (scores [16][17][18][19][20]. The mean vesikary score between the two groups was compared using t-test.…”

Evaluation of the NucliSens® Basic Kit assay for detection of Norwalk virus RNA in stool specimens

“…Reactions were carried out with the RT-PCR Access System kit (Promega Corporation, Madison, WI) according to manufacturer's protocol and as previously reported (Moe et al, 1994). The stool specimens from the human challenge studies and the NLV outbreaks were tested by RT-PCR using the G1 (SR33/SR48) and G2 primers, that produce a 123-bp product between nucleotides 4754Á/4876, also within the RNA polymerase gene (Ando et al, 1995). The G1 and G2 primers are more broadly reactive than the NV3/ NV51 pair and were routinely used to screen volunteer and outbreak specimens for the presence of any NLV infection.…”

Nonculturable agents of viral gastroenteritis

“…Discussions, views and recommendations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors. strains into different genogroups based on sequence analysis of the RNA polymerase region has not always agreed with the results obtained by SPIEM; for example, different strains with the same SPIEM type have been found to belong to different genogroups (15). The reason(s) for these differences is not yet known.…”

“…Three distinct genogroups of HuCV have been identified based on the analysis of the partial or complete nucleotide and predicted amino acid sequences of at least 50 different HuCVs (10)(11)(12)(13)(14)(15)(16)…”

“…At present, RT-PCR is the most sensitive method for the detection of infection with HuCVs. Most RT-PCR assays use primer pairs that amplify the RNAdependent, RNA polymerase region of the genome (11)(12)(13)(14)(15)(38)(39)(40)(41), although other areas of the genome also have been targeted (12,14,42). The genetic diversity of the HuCVs has prevented the development of a universal primer pair to detect all HuCVs.…”