Genomic characterization of Norwalk-like human caliciviruses (NLVs) originating from outbreaks and sporadic cases of acute gastroenteritis has revealed surprisingly high levels of diversity, even in the RNA polymerase gene, which is anticipated to be highly conserved. Since information on antigenic relationship is limited, due to the lack of a tissue culture system for these viruses, strains mostly are described on the basis of their genetic relatedness. However, the lack of uniformly applied criteria has led to a confusing array of strains with different groups employing different names for similar genetic lineages. Our goal was to conduct a structured analysis of genomic relationships among NLV strains in an attempt to provide an interim framework for genotyping. We assembled a panel of 31 potentially distinct genogroup I (GGI) and genogroup II (GGII) NLVs that reflected the diversity seen in strains detected by our laboratories and in published sequences. Phylogenetic analysis of sequences from regions of the open reading frames (ORF) 1, 2 and 3 was performed in order to investigate genomic relationships. The strains sequenced fell into seven phylogenetic groups in GGI and at least five phylogenetic groups in GGII, based on greater than 80% nucleotide identity in the region of ORF2 encoding the N-terminus of the capsid protein, and consistent clustering with high bootstrap values irrespective of the method used. Analysis of the ORF1 and ORF3 regions supported for most strains the clustering as established for those derived from ORF2. In the ORF1 region, used by most laboratories for diagnostic RT-PCR, clustering was consistent when a putative genotype border was set at 15% nucleotide mismatches for viruses in GGI and at 10% for viruses in GGII. Two strains grouped within different clusters based on ORF1 and ORF2 indicating that recombination may have occurred. We discuss the implications of these observations for the classification and typing of NLVs.
Fifty-two faecal specimens collected in the United Kingdom between 1986 and 1992, which contained small round structured virus (SRSV) particles, were tested by reverse transcriptase polymerase chain reaction assays using two primer pairs derived from sequences of Snow Mountain Agent and Norwalk virus. There was poor correlation between results obtained with each primer pair. Twenty specimens (38%) gave positive bands with SM51/31 primers and 18 (34%) were positive with SM52/32 primers, with a total of 30 specimens (57.7%) giving amplification products of the expected size with one or both primer pairs. Genomic variation was investigated by sequencing a 266 bp region of the RNA polymerase gene from nine strains which had been antigenically typed by solid phase immune electron microscopy (SPIEM). RNA sequence identities ranged from 53 to 99%. Three genomic groups were suggested by phylogenic analysis, the first of which contained Norwalk virus, Southampton virus, and strains typed by SPIEM as SRSV UK2. The second contained Snow Mountain agent and strains typed as either SRSV UK3 or UK4. The third contained strains typed as SRSV UK1 and strains untypeable by SPIEM. Some correlation was demonstrated when antigen typing by SPIEM and phylogenic grouping based on sequence data were compared.
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