Genomic characterization of human astrovirus type 6 Katano virus and the establishment of a rapid and effective reverse transcription-polymerase chain reaction to detect all serotypes of human astrovirus.

We previously reported that human astrovirus type 6 (HAstV T6) was the etiologic agent of a large-scale outbreak of acute gastroenteritis that occurred in 1991 in Katano City, Osaka, Japan [Oishi et al., 1994]. The two representative strains, Katano virus K23 and K24, have been analyzed by sequencing the open reading frame 2 (ORF2) region after amplification by reverse transcription-polymerase chain reaction (RT-PCR). The ORF2 region of HAstV T6 strains, including K23, was found to be about 20 bp smaller than those of other types. There was 94% nucleotide sequence identity and 95% amino acid sequence identity between K23 and K24, with the Oxford strains belonging to HAstV T6. The high homology of the ORF2 region between the Katano and Oxford strains shows intratype genomic stability, irrespective of time and place of virus isolation. Comparing sequences of ORF2 of different HAstV serotypes, we established a rapid and highly sensitive detection system for HAstV types using RT-PCR with the AC230/AC1' primer set designed from the 5'-terminal end region of ORF2. This RT-PCR system seems very useful in detecting at least two different viruses in a single PCR test tube using AC230/AC1' in addition to the NV81/82, SM82 primer sets. Thus, our rapid and effective detection system may contribute to the epidemiologic characterization of astrovirus infections as well as Norwalk-like viruses.

Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection.

The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA tests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WB. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test.

Isolation of group B porcine rotavirus in cell culture.

While group A and C rotaviruses have been grown in cell culture, group B rotavirus has never been cultured. In this study we successfully isolated porcine group B rotavirus in swine kidney cells. Pancreatin treatment is essential for the propagation of group B rotavirus.