Sapovirus (SV) causes gastroenteritis in humans and comprises genetically divergent viruses. A nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid-protein-coding region was developed using universal and genogroup-specific primer sets. The universal primers were capable of detecting human SV genogroups I, II, IV and V. Genetic analysis of the amplified products enabled us to phylogenetically determine the genotypes of the viruses. In addition, genogroup-specific primers that amplified different lengths of the amplicon depending on the genogroup were developed. These genogroup-specific primers were also used as inner primers for the nested PCR. These two simple RT-PCR methods are powerful tools for both detection and epidemiological studies of human SV.
An outbreak of gastroenteritis occurred at a kindergarten in Yokote City, Japan, between February 2006 and March 2006. Sapovirus was identified in 19 of 26 stool specimens by reverse transcription-PCR. A high viral shedding pattern was found for this strain, which was shown to be antigenically distinct from other genogroups.
In a survey on the etiology of acute gastroenteritis in infants and young children in Nigeria, group C human rotaviruses were detected in two of 112 rotavirus positive stool specimens collected between 1999 and 2000. The VP7, VP6, and VP4 genes of the two Nigerian human group C rotavirus strains (Jajeri and Moduganari) were sequenced in this study. Comparative sequence analysis with other published human group C rotaviruses showed that the genes encoding the three structural proteins were remarkably conserved in primary structure with few mutations. The VP4 and VP7 genes from the two Nigerian strains were related more closely to each other than to those of other published strains, and formed a separate cluster on the phylogenetic tree. In contrast, it was of note that VP6 gene of strain Moduganari was related more closely to the Brazilian strain Belem than to the other Nigerian strain Jajeri. This is the first report of identification of human group C rotavirus in Nigeria and constitutes the first sequence data of human group C rotaviruses in the African continent.
Human standard astroviruses, serotypes 1 to 7, and 35 Japanese isolates were typed by reverse transcription and polymerase chain reaction (RT-PCR) with serotype-specific primers for the first time. The results were identical with those obtained by enzyme immunoassay with serotype-specific polyclonal antibodies, a method which has already been reported. RT-PCR with serotype-specific primers is useful for epidemiological studies of astroviruses where serotype-specific polyclonal antibodies are not available. Two parts of the capsid region, N terminus and C terminus, were sequenced. Serotypes differed in those regions. The N terminus differed less than the C terminus between serotypes. Both the N terminus and C terminus were similar intraserotypically with the exception of serotype-4 isolates which could be divided into A and B subgroups on the basis of their C terminus sequences, which were not known previously. Human astroviruses are non-enveloped, single-stranded RNA viruses which were first identified in 1975 by the electron microscopy of stool specimens from children with diarrhea (1, 8). Not only sporadic cases but also outbreaks of astroviruses have been reported (9,13,19). Astroviruses can be cultured in LLC-MK2 cells and CaCo-2 cells (5,22). From the cultured viruses, seven human astrovirus serotypes have been identified serologically by using serotype-specific polyclonal antibodies (6). Serotype 8 was recently reported in a database (Accession No. Z66541).The complete genomic sequences of human astrovirus 1 were determined in 1994 (7, 23). After that, the total capsid sequences of serotypes 2, 4 and 6 were reported (3, 6, 11,24). The partial sequence of the capsid region of serotype 3 is also known (12). These results enabled us to detect and sequence astroviruses by reverse transcription and polymerase chain reaction (RT-PCR) (16).We obtained and cultured the standard astroviruses, serotypes 1 to 7. We then analyzed the full length of the capsid regions by RT-PCR and sequencing (preparing for submission). From these results, we designed serotypespecific oligonucleotide primers in the capsid region. We then compared the serotypes as determined by RT-PCR and enzyme immunoassay (EIA) using serotypespecific polyclonal antibodies on Japanese strains of astroviruses. Moreover, we compared the nucleotides and the predicted amino acid sequence homologies in two parts of the capsid region.Our main purpose was to verify that RT-PCR with serotype-specific primers is applicable to the epidemiological study of astrovirus serotypes as compared to serotyping by EIA and sequence analysis because serotype-specific antibodies are commercially unavailable and there are limited epidemiological studies. In addition, the sequence of the capsid region is analyzed in two parts of the relatively conservative portion, N terminus, and relatively variable portion, C terminus, to deter-
Group C rotaviruses have been identified recently from fecal samples of children with diarrhea in the United States. Using reverse transcriptasepolymerase chain reaction and sequence analysis, we sequenced gene 8s encoding VP7 from two U.S. strains (RI-1 and RI-2), and eight other strains isolated from patients on four continents, and compared these with the sequences of four published strains. The gene 8s of the 14 strains were remarkably conserved in size and in predicted primary and secondary structures. When the sequences of the human VP7s were compared with that of the prototype porcine Cowden strain, six regions were found variable in both deduced primary and predicted secondary structures, four of which were predicted to be hydrophilic and might determine serotype specificity. Gene 8 of the human S-1 strain was further characterized by expression in recombinant baculoviruses. The expressed product was immunogenic but failed to elicit neutralizing antibodies. Our sequence analysis indicates that all the human strains characterized to date belong to a single G genotype, which may constitute a single G serotype, pending further antigenic analysis. Whether the human strains and the Cowden strain are the same serotype remains to be determined.
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