E. Kaga

Characterization of human rotavirus genotype P(8)G5 from Brazil by probe-hybridization and sequence

SummaryWe report the molecular characterization of rotavirus genotype P[8]G5 strains found in fecal specimens collected in four different regions of Brazil, using digoxigenin (dig)-labeled oligonucleotide probes, sequence analysis, and RNA-RNA hybridization. The closest sequence relationships of the neutralization antigens of these strains were to the VP4 protein of P1A[8]G1 strain KU (93.3% identity in amino acids 11 to 282) and to the VP7 protein of G serotype 5 strain OSU (87.6% identity in amino acids 8 to 232). Based on VP7 sequence differences, we designed dig-probes that allowed us to discriminate porcine OSU-like strains from G5 strains isolated from Brazilian infants. The genetic relationships of two P[8]G5 isolates to other rotavirus genogroups were analyzed by RNA-RNA hybridization with [32P]-GTP probes representative of serotypes P1A[8]G1 (Wa), P[8]G3 (AU17), and P9[7]G5 (OSU). The Brazilian P[8]G5 strains showed sequence homology with genes of Wa-like and OSU-like strains, suggesting that these two strains were naturally occurring reassortants between members of the Wa and porcine rotavirus genogroups. The identification of these strains in diverse geographic areas of Brazil underscores their stability and demonstrates the emergence of clinically important rotavirus diarrhea strains by reassortment.

Human and bovine serotype G 8 rotaviruses may be derived by reassortment

SummaryThe origin of, and relationship between human and bovine serotype G 8 rotaviruses were investigated by genomic hybridisation. Radiolabelled mRNAs of human G 8 rotaviruses 69 M (isolated in Indonesia) and HAL 1271 (isolated in Finland), and bovine rotaviruses KK 3 (G 10) and NCDV (G 6), were used as probes. The products of liquid hybridisation between the probes and the genomic RNA of human and bovine rotaviruses, including bovine G 8 rotavirus 678 (isolated in Scotland) and two other Finnish human G 8 rotaviruses HAL 1166 and HAL 8590, were examined by separation in polyacrylamide gels. The genomes of Finnish human G 8 rotaviruses were similar to those of bovine G 6 and G 10 rotaviruses. Neither Indonesian human G 8 nor bovine G 8 viruses had high levels of similarity to each other or to other bovine and human rotaviruses. Thus these three epidemiologically distinct G 8 rotaviruses have different origins and may be derived by reassortment with rotaviruses of a third, as yet unknown, host species. The similarity between the Finnish isolates and the bovine isolate NCDV suggests that they have diverged recently and that these human G 8 rotaviruses may be derived from a zoonotic infection, or alternatively, from the live rotavirus vaccine of bovine origin which has been used to vaccinate Finnish children.

The Distribution of G (VP7) and P (VP4) Serotypes among Human Rotaviruses Recovered from Japanese Children with Diarrhea

Both the G (VP7) and P (VP4) serotypes of human rotaviruses collected over a 10‐year period from Japanese children with diarrhea were determined by recently‐developed polymerase chain reaction‐based typing assays. The combination of G1 and P8 was found in 65.2% and the combination of G2 and P4 was found in 15.2%. For the rest of the specimens, only a few other combinations occurred and their relative frequencies were less than 10%. The viruses carrying P9 were always associated with G3 as is the prototype strain AU‐1.

Naturally occurring dual infection with human and bovine rotaviruses as suggested by the recovery of G1P8 and G1P5 rotaviruses from a single patient

SummaryCulture adaptation of rotavirues from an infant with severe diarrhea in Cincinnati, Ohio, yielded not only a virus with the original RNA electropherotype (CJN) but also rotaviruses with other electropherotypes, the most dominant of which was called CJN-M [Ward RL, Knowlton DR, Schiff GM, Hoshino Y, Greenberg HB (1988) in J Virol 62: 1543–1549]. RNA-RNA hybridization and sequencing studies indicated that CJN was a typical G1P8 human rotavirus while CJN-M was a G1P5 strain and contained four gene segments (including segment 4) of a bovine rotavirus. Thus, the infant was apparently dually infected with human and bovine rotaviruses.

Human rotavirus strain with unique VP4 neutralization epitopes as a result of natural reassortment between members of the AU-1 and Wa genogroups

SummaryHuman rotavirus strain K8, which possesses unique VP4 neutralization epitopes, was examined by RNA-RNA hybridization to determine its genogroup. While it possessed four gene segments that formed hybrids with strain Wa (a prototype of the Wa genogroup), strain K8 possessed seven gene segments, including gene segment 4, that formed hybrids with strain AU-1 (a prototype of the AU-1 genogroup) which has been shown to share a unique gene 4 allele with feline rotaviruses. These results suggest that strain K8 is an intergenogroup reassortant formed in nature between a member of the Wa genogroup and a member of the AU-1 genogroup.

Subgroup I serotype 3 human rotavirus strains with long RNA pattern as a result of naturally occurring reassortment between members of the bovine and AU-1 genogroups.

SummaryTwo human rotavirus strains, PCP 5 and MZ 58, which possessed an unusual combination of subgroup (I), serotype (3) and RNA pattern (long) were examined by RNA-RNA hybridization to determine their genogroup. While these two strains did not belong to either the Wa or the DS-1 genogroup, PCP 5 and MZ 58 possessed seven gene segments that formed hybrids with bovine rotavirus strain NCDV and four gene segments that formed hybrids with human rotavirus strain AU-1. These results suggest that PCP 5 and MZ 58 were intergenogroup reassortants formed in nature between a member of the bovine rotavirus genogroup and a member of the AU-1 genogroup.

Serotype G6 human rotavirus sharing a conserved genetic constellation with natural reassortants between members of the bovine and AU-1 genogroups

SummarySerotype G6 human rotavirus PA151 was examined by RNA-RNA hybridization in comparison with another G6 strain PA169 and two naturally-occurring G3 reassortants PCP5 and MZ58. PA151 possessed three gene segments that formed hybrids with AU-1 and seven gene segments that formed hybrids with bovine rotavirus NCDV. PA151 also possessed eight, 10 and 10 gene segments that formed hybrids with genomic RNAs from PA169, PCP5, and MZ58, respectively. Thus, PA151 was an intergenogroup reassortant formed in nature between members of the bovine and AU-1 genogroups and it shared a genome constellation with PA169, PCP5, and MZ58. These results suggest that naturally-occurring intergenogroup reassortants possessing such a genome constellation were perpetuated in human populations.

Distinctness of NSP1 gene of human rota virus AU-1 from NSP1 gene of other human genogroups

Human rotavirus strain AU-1 has a genomic RNA constellation distinct from that of either the Wa or DS-1 genogroup and thus, represents the third human rotavirus genogroup. Nucleotide sequence analysis of the non-structural protein 1 (NSP1) of AU-1 revealed that it was only 53-57% identical at the amino acid level with strains belonging to either the Wa or the DS-1 genogroup. This result confirmed and extended the earlier observation that the grouping based on the NSP1 sequence similarity is usually in good agreement with classification by genogroup. Phylogenetic analysis placed the AU-1 NSP1 gene on the bovine NSP1 branch, although more than 13% amino acid difference was observed.

Recurrent circulation of single nonstructural gene substitution reassortants among human rotaviruses with a short RNA pattern

SummaryTo determine the relative frequency of intergenogroup reassortants of rotavirus in nature, we analyzed the genetic composition of 22 electrophoretically distinct stool isolates which accounted for 95.2% of stool rotaviruses with a short RNA pattern collected during 10 rotavirus seasons. These strains all showed subgroup I and G2 specificities, but two distinct hybridization patterns were observed when the probes prepared from Wa (a member of the Wa genogroup) and KUN (a member of the DS-1 genogroup) were used. Genomic RNAs from 10 strains (accounting for 64% of the field rotaviruses with short RNA pattern) hybridized exclusively to the KUN probe, and thus belonged to the DS-1 genogroup. On the other hand, genomic RNAs of the remaining 12 strains (accounting for 36% of the field rotaviruses with short RNA pattern) formed one hybrid band with the Wa probe and 10 hybrid bands with the KUN probe. Thus, they were single gene substitution intergenogroup reassortants between members of the Wa and DS-1 genogroups. They had a similar genetic constellation in that a gene segment encoding either NS35 or NS34 from a Wa-like strain was introduced into a DS-1-like genome background.

Thermal degradation of RNA-RNA hybrids during hybridization in solution

Degradation of single-stranded RNA molecules at high temperatures was examined in relation to the kinetics of RNA-RNA hybridization in solution. Eleven species (ranging from 670 bases to 3300 bases) of single-stranded RNAs transcribed from rotavirus genomic RNAs degraded significantly after 16 h of incubation at 65 degrees C. The hybridization of these 11 RNA molecules to the corresponding genomic RNAs, however, was completed within 30 min of incubation. Partially homologous hybrids that were once formed at an early time of incubation gradually degraded in proportion to the length of incubation at 65 degrees C. Thus, the length of hybridization has a critical effect on the final hybridization results. Furthermore, thermal hydrolysis of single-stranded RNA molecules provides a plausible explanation why the percent of nucleotide sequence mismatch allowed to form a stable hybrid in the RNA-RNA hybridization assays for rotavirus genes is much less than that predicted by calculation.