Molecular characterization of a novel adult diarrhoea rotavirus strain J19 isolated in China and its significance for the evolution and origin of group B rotaviruses.

The complete genome of a novel adult diarrhoea rotavirus strain J19 was cloned and sequenced using an improved single-primer sequence-independent method. The complete genome is 17,961 bp and is AU-rich (66.49 %). Northern blot analysis and genomic sequence analysis indicated that segments 1-11 encode 11 viral proteins, respectively. Protein alignments with the corresponding proteins of J19 with B219, and groups A, B and C rotaviruses, produced higher per cent sequence identities to B219. Among groups A, B and C rotaviruses, 10 proteins from group B rotaviruses exhibited slightly higher amino acid sequence identity to the J19 proteins, but proteins of J19 showed low amino acid sequence identity with groups A and C rotaviruses. Construction of unrooted phylogenetic trees using a set of known proteins and representatives of three known rotavirus groups revealed that six structural proteins were positioned close to B219 and the basal nodes of groups A, B and C lineages, although with a preferred association with group B lineages. Phylogenetic analysis of the five non-structural proteins showed a similar trend. The results of the serological analysis, protein sequence analysis and phylogenetic analysis suggested that J19 would be a novel rotavirus strain with great significance to the evolution and origin of group B rotaviruses.

[Cultivation and serial propagation of a new rotavirus causing adult diarrhea in primary human embryo kidney cells].

OBJECTIVE: To investigate the methodology of cultivation of the new rotavirus that causes adult diarrhea. METHODS: 10% suspension of new rotavirus positive stool specimens in DMEM with 100 microgram/ml trypsin was made and centrifuged at the speed of 3 000 rpm for 10 minutes. The supernatant was filtered with 0.45 micrometer-pore-sized membrane filter, and the filtrate was incubated at 37 degrees C for 60 minutes. Primary human embryo kidney (PHEK) cells were prepared and seeded into roller tubes at the concentration of 1 ml odf cell suspension per tube. At 1 hour prior to inoculation, the PHEK cells were rinsed and refed with serum-free DMEM. Immediately before inoculation, the DMEM was decanted, and 200 microliter of prepared filtrate was inoculated into each tube. The tubes were incubated at 37 degrees C for 1 hour. At the end of 1 hour 800 microliter of DMEM containing trypsin (100 microgram per ml) were added to each tube. The tubes were placed in a roller tube apparatus at 37 degrees C for 3 days, removed and frozen-thawed once. 200 microliter of the cell culture fluids (CCF) were used for inoculation of each tube for next virus passage. Detection of new rotavirus from CCF was carried out by PAGE, IF, EM and IEM. RESULTS: New rotavirus was successfully isolated in cultured cells from 1 of 10 specimens. The isolated virus, designated J19, was propagated in PHEK cells for 28 passages. RNA patterns of J19 strain were identical to that of original new rotavirus inoculum and different from that of group A, B, C rotavirus. PHEK cells infected with the J19 strain were detected by IF. Infected cells reacted with convalescent antisera to the new rotavirus, but did not react with convalescent antisera to ADRV and hyperimmune antisera against group A, B rotavirus. Rotavirus particles were detected by EM, IEM in infected CCF of J19 strain. Other viral particles were not observed. The particles were aggregated with convalescent antisera to new rotavirus. CONCLUSION: We were successful in adapting a new rotavirus to serial propagation in PHEK cells. The J19 strain is a kind of new rotavirus different from group A, B, C rotavirus. This is the first report on the cultivation and propagation of the new rotavirus in cultured cells.