Characterization of human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997.

Serotyping of human rotavirus was conducted in 396 Japanese and 100 Thai rotavirus-positive fecal specimens collected from 1995 to 1997. Serotype G9 was found to be the third most common serotype with frequency of 16.2% in Thailand from 1996 to 1997. It was also detected in Japan with a low frequency (0.7%) in this year. The genetic analyses of VP4 and NSP4 genes of these G9 strains showed that 1 strain from Japan possessed P[8] genotype and NSP4 Wa-group with long electropherotype (e-type). In contrast, 5 strains from Thailand belonged to P[6] and 1 strain belonged to P[4]. All of the Thai strains were in the NSP4 KUN-group with a short e-type. Sequence analysis of their VP7 gene revealed that there was the highest homology among fecal G9 strains (> 96.3%, amino acid identity) and a relatively high degree of homology to standard viruses, F45 from Japan (95.4-96.3%, amino acid identity) and 116E from India (92-92.3%, amino acid identity). However, immunological analysis using G9 specific monoclonal antibodies (Mabs) against VP7 protein showed that the G9 strains isolated from the two countries had different antigenic specificity. It was confirmed further by intraserotypical phylogenetic analysis of VP7 amino acid. These results indicated that the prevalence of G9 rotavirus in 1996-1997 in Thailand was relative to the continuing recent emergence of it on a worldwide basis, while the Japanese G9 strain isolated in this survey was identified to have progenitors common to the F45 strain that was prevalent in 1985 in Japan. Phylogenetic analysis of VP7 amino acid of G1-14 prototype rotavirus showed that the G9 strains were most closely related to the equine G14 rotavirus FI23 strain but G3 strains, interserotypically. These findings suggest that G9 rotaviruses might be divided into two or more subtypes.

Determination of interferon-alpha-producing capacity in whole blood cultures from patients with various diseases and from healthy persons.

To assess the clinical value of determination of the interferon (IFN)-producing capacity of patients, IFN production induced by Sendai virus (HVJ) in vitro was measured in cell cultures of whole blood from patients with various diseases. IFN production in patients with lung cancer, myelodysplastic syndromes, noninsulin-dependent diabetes mellitus, pulmonary tuberculosis, and asymptomatic HIV-1 infection was lower than that in healthy persons. Furthermore, periodic measurements of IFN production revealed decreasing IFN producing capacities in patients with lung cancer with progression of the tumor stage. However, increased IFN-producing capacities were observed in patients with tuberculosis after standard therapy. Further experiments showed that the main type of IFN induced in whole blood cultures was IFN-alpha, and decreased IFN production in patients did not result from a decreased number of leukocytes but rather from an impairment of cellular IFN production. The evaluation of IFN production in whole blood cell cultures may be a feasible method of assessing the impaired immune status.

Propagation of JC virus in human neuroblastoma cell line IMR-32.

JC virus (JCV), the causative agent of a human demyelinating disease, progressive multifocal leukoencephalopathy, has a very narrow host range. Cells permissive for infection by JCV have been essentially limited to primary human fetal glial cells, which are difficult to obtain and maintain. In pilot studies, it was found that JCV can multiply in an established cell line of human neuroblastoma. JCV strains Mad-1 and Tokyo-1 were inoculated, respectively, into two cell lines, IMR-32 (neuroblastoma) and A-172 (glioblastoma). Viral infection with cytopathic effect was observed only in IMR-32 cells, and the most efficient viral proliferation was obtained in cells cultured in medium containing 2% fetal calf serum (FCS). Both Mad-1 and Tokyo-1 strains propagated well, with the former being more efficient than the latter. Viral replication was confirmed by immunofluorescence, electron microscopy, and a hemagglutination assay. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis of the purified virus revealed the characteristic JCV protein profile. Thus, IMR-32 cells have been found to be permissive for JCV, which should provide a useful system for further studies of virus proliferation and viral tissue tropism.

DNA-sequence rearrangement required for the adaptation of JC polyomavirus to growth in a human neuroblastoma cell line (IMR-32).

Infection of a human neuroblastoma cell line (IMR-32) with the JC polyomavirus (JCV) strain Mad-1 with subsequent serial passage results in the generation of a virus adapted to growth in IMR-32 (K. Akatani, M. Imai, M. Kimura, K. Nagashima, and N. Ikegami, J. Med. Virol., in press). To understand the basis of this adaptation, we molecularly cloned JCV DNAs from the adapted virus. The cloned JCV DNAs consisted of essentially three species (M1-IMRa, -IMRb, and -IMRc) with rearranged regulatory regions. Two TATA sequences are present in the regulatory region of the parental virus Mad-1, but one distal from the origin of replication was commonly deleted in M1-IMRa, -IMRb, and -IMRc. We showed that these regulatory regions were required for the efficient growth of JCV in IMR-32. Various JCV strains should be propagated in IMR-32, if their regulatory regions are replaced with those defined in this study. Since it is difficult to propagate JCV in cells other than primary human fetal glial cells, this system may be useful for structural and immunological studies of JCV.

Serotyping of human rotavirus by enzyme immuno-assay with monoclonal antibodies.

Two series of enzyme immunoassays with monoclonal antibodies produced in two laboratories (A and B) were compared in use for serotyping of human rotavirus in stool samples collected in Japan between 1988 and 1991 from patients with gastroenteritis. Of 358 samples, 222 were determined to be the same serotype, while 61 samples could not be serotyped by either assay. A hundred and one and 92 samples were not serotyped by the A and B antibodies, respectively. We believe that the A and B monoclonal antibodies are useful clinically for serotyping rotaviruses at the present time.

Molecular epidemiology of rotaviruses associated with pediatric diarrhea in Bangkok, Thailand.

Rotavirus diarrhea in 453 pediatric patients (29.8% of 1,518) was studied in greater Bangkok during 1985 to 1987. The disease persisted all year, increasing in incidence from August to January (30 to 50%). Polyacrylamide gel electrophoresis of rotavirus RNA from these patients and from an additional 46 patients of a 1982 to 1983 epidemic revealed 26 electropherotypes, 4 with short (S) and 22 with long (L) RNA profiles. Of the analyzed specimens, 85.5% were L forms. Only one or a few electropherotypes predominated in each epidemic, whereas others appeared sporadically at low frequencies. Shifts in the predominant electropherotypes were observed in every epidemic. Of these, 126 strains were tested for subgroup and serotype by monoclonal antibody enzyme immunoassay. Serotype 4 prevailed from 1982 to 1983, while serotype 1 was encountered more frequently than serotypes 2 and 4 from 1985 to 1987. A complete correlation was found between the electrophoretic migration of segments 10 and 11 and the serologically defined subgroup specificity. Distinct electropherotypes occurred within the same serotype, and strains with the identical electropherotype always showed the same serotype specificity. No specific electropherotype or serotype correlated with patient age. In this study, atypical rotaviruses and mixed infections with different rotaviruses were identified.

Isolation and molecular characterization of a serotype 9 human rotavirus strain.

A human rotavirus strain, designated AU32, that belongs to serotype 9 was isolated and was compared by RNA-RNA hybridization with recently established two serotype 9 strains (WI61 and F45) as well as other prototype human strains. These three strains exhibited a very high degree of homology with one another and shared a high degree of homology with strains belonging to the Wa genogroup but not with strains belonging to either the DS-1 or AU-1 genogroup. These results suggest that genetic constellation of the serotype 9 strains is similar to that of the commonest human rotavirus despite the recent recognition of this serotype.

The finest ultrastructural details of the inner layer of rotavirus revealed by minimal electron doses.

The inner layer of human and calf rotavirus ultrastructure was analyzed using minimal doses in transmission electron microscopy. The morphological unit of spontaneously disrupted virions has the aspect of a flower-like structure, and its petals are hexagonally arranged and have a central pin hole. The similar flower appearance can be observed in complete virions and in rotavirus tube structures produced in aged samples of rotavirus kept for more than a year.

Identification of rotavirus genogroups by RNA-RNA hybridization.

The genetic relatedness of various human rotavirus strains was examined by RNA-RNA hybridization in which 32P-labelled single stranded RNAs produced by in vitro transcription from viral RNAs were used as probes. Denatured genomic double stranded RNAs were hybridized to the probes under highly stringent conditions and the resulting hybrids were fractionated by polyacrylamide gel electrophoresis. Based on the hybridization patterns obtained with probes made from prototype strains Wa (subgroup II, long RNA electropherotype), DS-1 (subgroup I, short RNA electropherotype) and AU-1 (subgroup I, long RNA electropherotype), we have observed that human rotaviruses fall into three distinct gene groups which we have termed 'genogroups'. Identification of genogroups among rotavirus isolates will prove to be a valuable asset for the analysis of naturally occurring reassortants, to trace interspecies transmission of animal rotaviruses to man or vice versa and to identify rotaviruses from environmental sources with regard to their original host species. Furthermore, such an approach will contribute to our understanding of the evolution of rotavirus genes.

Occurrence of changes in human rotavirus serotypes with concurrent changes in genomic RNA electropherotypes.

To investigate the serotypic and genetic diversity of human rotavirus strains, we have tested 513 and 519 fecal rotavirus specimens, respectively, by an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies and by polyacrylamide gel electrophoresis of the segmented RNA genome. Of the 513 specimens, 375 were typed as serotype 1 (47.3%), serotype 2 (2.9%), serotype 3 (2.9%), or serotype 4 (17.7%). In addition, a presumptive new human serotype, tentatively referred to as serotype X in this paper, was found in 1.6% of the specimens tested. The remaining 138 specimens (26.9%) were untypeable. Considerable variation in relative frequency of circulating serotypes was observed with respect to geographic locations and observation periods. Rotavirus RNAs were visualized in 481 of 519 specimens tested. Of these, 415 were typed as 33 electropherotypes, many of which were infrequently detected and were restricted to single epidemics. Analysis of the 291 specimens whose electropherotypes and serotypes were available indicated clearly that a given RNA pattern always corresponded to a particular serotype. Heterogeneity of electropherotypes within a serotype was similarly observed in strains belonging to the four previously established serotypes. The results obtained in this study indicated that antigenic changes on the major neutralization antigen occurred always with concurrent changes of genomic RNA electropherotypes. On the other hand, serotypic changes could not be predicted from the changes in RNA electropherotypes.

Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments.

Both 3'- and 5'-terminal structures of human rotavirus genome double-stranded RNA segments were determined. RNAs were labeled at the 3'-termini with [32P]pCp by incubation with RNA ligase and at the 5'-termini with [32P]phosphate by polynucleotide kinase or, in the case of 5' caps, with 3H by chemical modification with [3H]NaBH4. Examination of radiolabeled termini released by digestion with several base-specific RNases revealed that rotavirus RNA segments are base paired end-to-end and contain the same terminal structures: (formula; see text)