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

Summary To 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.     

Genetic relatedness among human rotavirus genes coding for VP7, a major neutralization protein, and its application to serotype identification.

Antigenic characterization of human rotaviruses by plaque reduction neutralization assay has revealed four distinct serotypes. The outer capsid protein VP7, coded for by gene 8 or 9, is a major neutralization protein; however, studies of rotaviruses derived from genetic reassortment between two strains have confirmed that another outer capsid protein, VP3, is in some cases equally important in neutralization. In this study, the genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4 was examined by hybridization of their denatured double-stranded genomic RNAs to labeled single-stranded mRNA probes derived from human-animal rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. A high degree of homology was demonstrated between the VP7 genes of strain D and other serotype 1 human rotaviruses, strain DS-1 and other serotype 2 human rotaviruses, strain P and other serotype 3 human rotaviruses, and strain ST3 and other serotype 4 human rotaviruses. Hybrid bands could not be demonstrated between the VP7 gene of D, DS-1, P, or ST3 and the corresponding gene of human rotaviruses belonging to a different serotype. RNA specimens extracted from the stools of 15 Venezuelan children hospitalized with rotavirus diarrhea were hybridized to each of the reassortant probes representing the four human serotypes. All five viruses with short RNA patterns showed homology with the DS-1 strain VP7 gene; two of these were previously adapted to tissue culture and shown to be serotype 2 strains by tissue culture neutralization. Of the remaining 10 viruses with long RNA patterns, 2 hybridized only to the D strain VP7 gene, 6 hybridized only to the P strain VP7 gene, and 2 hybridized only to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternative method for identifying the VP7 serotype of field isolates that would circumvent the need for tissue culture adaptation.     

Isolation, propagation, and characterization of a second equine rotavirus serotype.

A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.     

Isolation and characterization of an equine rotavirus.

A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell culture-adapted strain hemagglutinated erythrocytes of human group O, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests.     

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

Summary Two 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 3 human rotavirus strains with subgroup I specificity.

During an epidemiological study on human rotavirus (HRV) infections in Italy, three subgroup I strains not associated with serotype 2 reactivity were detected. All three strains were serotype 3, each with a distinct RNA pattern showing fast-moving tenth and eleventh segments (long electropherotype). Following successful adaptation to growth in cell cultures, the serotype 3 strains (MZ58, PCP5, and PA710) were further characterized by neutralization and by RNA-RNA (Northern blot) hybridization. Antiserum to reference HRV strain YO (subgroup II, serotype 3), as well as a monoclonal antibody to VP7 of YO neutralized, at comparable titers, the homologous virus, the three unusual HRV strains, and two reference simian strains (SA11 and RRV-2, both subgroup I, serotype 3), whereas SA11 antiserum and a monoclonal antibody to VP7 of SA11 neutralized simian strains more efficiently. However, antiserum to PCP5 neutralized the three unusual isolates and the simian strains at significantly higher titers than it did with reference strain YO. With 32P-labeled RNA from MZ58 as a probe, a high degree of homology was detected by Northern blot hybridization with strains PCP5, PA710, SA11, and UK (bovine rotavirus) at the level of several segments and with strain YO only at the level of genes 7 to 9. Conversely, labeled RNA of strain YO hybridized extensively with Wa (subgroup II, serotype 1 HRV strain) but only at the level of genes 7 to 9 with MZ58, PCP5, PA710, SA11, and UK. Finally, the labeled SA11 probe hybridized at the level of RNA segments 1 to 3 and 6 to 11 to the three unusual strains. These findings suggest that the unusual subgroup I, serotype 3, strains isolated from humans are more likely to be animal rotaviruses rather than natural reassortants between different HRV strains.     

Molecular identification of a novel human rotavirus in relation to subgroup and electropherotype of genomic RNA

A total of 41 stool rotavirus specimens collected from children with acute diarrhea at four different locations in Akita Prefecture, Japan, during the peak of the winter diarrhea epidemic in 1988 were analyzed by polyacrylamide gel electrophoresis of viral RNA in conjunction with subgrouping assay. We found that a single strain predominated, with cocirculating strains with less common electropherotypes at a given location, and that two different strains could predominate at geographically close but different locations even during a very limited time of the epidemic season. Furthermore, we isolated a human rotavirus strain (AU125) that was similar to the AU-1 strain in that it possessed a long RNA pattern yet belonged to subgroup I. Genetic analysis by RNA-RNA hybridization assay indicated that the AU125 strain was distinct from two previously identified human rotavirus gene groups (genogroups) represented by the Wa strain (subgroup II with long RNA electropherotype) and the DS-1 strain (subgroup I with short RNA electropherotype), but was very closely related to the AU-1 strain. These data suggest that the genetic diversity of human rotaviruses may be more extensive than was previously thought.     

Isolation and molecular characterization of a naturally occurring non-structural protein 5 (NSP5) gene reassortant of group A rotavirus of serotype G2P[4] with a long RNA pattern

We report here two unusual strains of group A rotavirus, AU85 and AU102, isolated from children with diarrhea. These strains showed an unusual combination of serotype G2 and a long RNA pattern. RNA-RNA hybridization assays showed that these strains are reassortants in which a single genome segment 11 (the NSP5 gene) was derived from a Wa genogroup strain, while other 10 genome segments from a DS-1 genogroup strain. Phylogenetic analysis showed that the NSP5 gene of strain AU85 did not form cluster with Wa strain, while it belonged to the cluster of YM and other porcine strains. Phylogenetic analysis also showed that NSP5 and VP7 genes of AU85 were derived from the rotavirus circulating in the area. Both co-electrophoresis and RNA-RNA hybridization showed that AU85 and AU102 are identical strains. Moreover, the nucleotide sequence comparison between these two strains revealed that they had 100% identical NSP4, NSP5, and VP7 genes. These results suggest that AU85 was a reassortant formed relatively recently between rotaviruses belonging to the Wa and the DS-1 genogroup.     

Molecular identification by RNA-RNA hybridization of a human rotavirus that is closely related to rotaviruses of feline and canine origin

With a few exceptions subgroup I group A human rotavirus strains have short RNA patterns, whereas most animal rotavirus strains belong to subgroup I and have long RNA patterns. Thus, new isolates of subgroup I human rotaviruses with long RNA patterns are considered to have a high likelihood of being animal rotaviruses. A group of human rotaviruses represented by the AU-1 strain has recently been shown to be genetically related to a feline rotavirus (FRV-1) isolated in Japan. A human rotavirus, strain Ro1845, which is similar to the AU-1 strain in its subgroup (I), serotype (3), and electropherotype (long), was compared with various human and animal strains by RNA-RNA hybridization to determine its genogroup, a term proposed to classify rotaviruses based on their gene homology. The Ro1845 strain did not show a significant level of homology with AU-1, FRV-1, or other human strains, indicating that the Ro1845 strain is different in its genogroup not only from the AU-1 strain but also from other human strains. However, the Ro1845 strain showed a high degree of homology with another feline rotavirus (Cat97) isolated previously in Australia, suggesting that the Ro1845 strain might originate from a feline rotavirus that is genetically distinct from the Japanese FRV-1 strain. Furthermore, the Ro1845 strain as well as the Cat97 strain were related genetically to the canine rotavirus RS15 strain. Taken together, these results indicate that at least two genogroups are present in feline rotaviruses, one resembling the AU-1 strain and the other resembling the Ro1845 strain as well as canine rotaviruses.     

Isolation of a bovine rotavirus with a "super-short" RNA electrophoretic pattern from a calf with diarrhea

A rotavirus with a "super-short" RNA electropherotype was isolated from a calf with diarrhea and was designated VMRI strain. Segments 10 and 11 of this rotavirus migrated more slowly than did those of bovine rotavirus strains NCDV, B641, and B223. The electrophoretic pattern of the VMRI strain was similar to that reported for rotaviruses with super-short RNA electropherotypes from humans and rabbits. Northern (RNA) blot hybridization indicated that gene 11 of the VMRI strain was altered and migrated between gene segments 9 and 10. The subgroup of the VMRI strain was shown to be subgroup I. The VMRI strain of bovine rotavirus was neutralized by antisera containing polyclonal antibodies to rotavirus serotype 6 (bovine rotavirus serotype I) strains NCDV and B641 and by ascitic fluid containing monoclonal antibodies directed to VP7 of serotype 6 rotavirus. The VMRI strain was not neutralized by either polyclonal or monoclonal antibodies to strain B223 (bovine rotavirus serotype II). Collective data on the neutralization of the VMRI strain with monoclonal antibodies and polyclonal antibodies suggest that this virus is a member of the NCDV group (serotype 6) of rotaviruses (bovine rotavirus serotype I).     

Serological comparison of canine rotavirus with various simian and human rotaviruses by plaque reduction neutralization and hemagglutination inhibition tests.

By the plaque reduction neutralization test, the CU-1 strain of canine rotavirus was similar, if not identical, to three strains (no. 14, no. 15, and P) of the tentatively designated third human rotavirus serotype. In addition, strain CU-1 demonstrated a one-way antigenic relationship with two other strains (M and B) of the third human rotavirus serotype. The CU-1 strain of canine rotavirus hemagglutinated human group O, rhesus monkey, dog, sheep, and guinea pig erythrocytes. A two-way antigenic relationship between canine (CU-1) and simian (MMU 18006 and SA11) rotaviruses demonstrated previously by the plaque reduction neutralization test was confirmed further with two additional isolates (A79-10 and LSU 79C-36) of canine rotavirus by the plaque reduction neutralization test and the hemagglutination inhibition test. The CU-1 strain of canine rotavirus, which is known to be distinct from two well-characterized human rotavirus serotypes (Wa and DS-1), was also found to be distinct from the St. Thomas no. 4 strain, which is a newly defined fourth human rotavirus serotype. Thus, this canine strain, which is related antigenically to one of four human rotavirus serotypes, is another example of an animal rotavirus which shares serotype specificity with a human rotavirus.     

Culture adaptation and characterization of group A rotaviruses causing diarrheal illnesses in Bangladesh from 1985 to 1986.

Group A rotaviruses collected between 1985 and 1986 during comprehensive surveillance of treated diarrheal episodes occurring in a rural Bangladesh population were culture adapted and characterized by electropherotype, serotype, and subgroup. Of 454 episodes of rotavirus-associated diarrhea, rotaviruses were culture adapted from 381 (84%), and 335 contained 11 electrophoretically identical segments in unpassaged and cultured preparations. These 335 comprised 69 different electropherotypes with between 1 (32 isolates) and 79 representatives. The persistence of specific rotavirus strains within the study population, as defined by the detection of viruses with particular electropherotypes, was generally limited to a period of only a few months. All 335 isolates were serotyped by neutralization with hyperimmune antisera to prototype rotavirus strains representative of serotypes 1 to 4, i.e., Wa, DS-1, P, and ST-3. It was found that 80, 48, 119, and 88 isolates belonged to serotypes 1 to 4, respectively. The concentrations of hyperimmune antisera required to neutralize these isolates, however, were at least threefold greater than those needed to neutralize the homologous strains. Therefore, the isolates appeared to have altered neutralization epitopes from their prototype strains. Furthermore, the serotype 4 isolates were consistently shown to be much more closely related to the serotype 4B VA70 strain than the serotype 4A ST-3 strain. All but two isolates identified as serotypes 1, 3, or 4 had long electropherotypes and were subgroup II, and all but one serotype 2 isolate were subgroup I and had short electropherotypes. The three disparate strains appeared to be genetic reassortants. Evidence is presented that dual infections required for reassortant formation were not uncommon. Thus, formation of multiple reassortants may have been a cause for the observed rapid shift in viral strains within the study population.     

Serotyping of cell culture-adapted subgroup 2 human rotavirus strains by neutralization

Nine human rotavirus strains from stools of infants with gastroenteritis were serially propagated in MA-104 cell cultures. All strains were identified as subgroup 2 rotaviruses by RNA gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. The human rotavirus strains were propagated for 15 to 20 passages and then used for immunization of guinea pigs and rabbits. Animal antisera were also raised against a subgroup 1 human strain purified from stools and against the cell culture-adapted Wa strain, a reference subgroup 2 rotavirus of human origin. Cross-neutralization studies revealed the existence of two distinct serotypes within the cell culture-adapted subgroup 2 human rotaviruses: strains related and unrelated to strain Wa were classified as serotypes 1 and 3, respectively. Results with convalescent-phase sera from infants with primary rotavirus infections confirmed the existence of two serotypes within subgroup 2, and the serotypes responsible for primary subgroup 2 infections could be determined on the basis of the neutralizing reactivity of convalescent sera.     

Naturally occurring serotype 2/subgroup II rotavirus reassortants in northern Brazil

Nine serotype 2 human rotavirus strains were isolated in a community-based longitudinal study in Northern Brazil. Five of these strains had a 'long' RNA electrophoretic pattern and all five strains were determined to belong to subgroup II by ELISA assay, in contrast to properties common to serotype 2 human rotaviruses previously characterized. Hybridization studies of one of these unusual strains with 32P-labelled mRNAs derived from the prototype human strains Wa (serotype 1, subgroup II) and S2 (serotype 2, subgroup I) suggested that it was generated by a reassortment event in nature, in which a subgroup II, 'long' electropherotype rotavirus exchanged its serotype-specific gene and gene number 10 for the equivalent genes from a serotype 2, 'short' electropherotype virus.     

Isolation of an avianlike group A rotavirus from a calf with diarrhea.

An atypical group A rotavirus (993/83) was isolated from a 3-day-old German calf with diarrhea. It differed from 35 conventional German bovine rotavirus isolates analyzed previously with respect to subgroup (strain 993/83 was non-subgroup I and non-subgroup II), serotype (strain 993/83 showed a two-way cross-reaction with serotype 7 and a one-way cross-reaction with serotype 3), and electropherotype (strain 993/83 showed comigrating gene segments 10 and 11). Isolate 993/83 reacted with only one of four monoclonal antibodies that recognized a common VP6 epitope(s). In addition, VP6 and VP2 of isolate 993/83 showed one-dimensional peptide maps that differed substantially from the peptide maps of VP6 and VP2 from all bovine rotavirus isolates. By RNA-RNA hybridization, the 993/83 probe failed to react with a panel of mammalian rotavirus strains, including bovine rotaviruses. It hybridized, however, to genomic RNA of an avian rotavirus strain. Isolate 993/83 could thus represent a candidate for a natural interspecies transmission of rotavirus between different classes of vertebrates.     

Rescue and serotypic characterization of noncultivable human rotavirus by gene reassortment.

Thirty-three of 50 noncultivable human rotavirus strains from a variety of locations were successfully rescued by gene reassortment. The serotype of each of the 33 strains was investigated by a qualitative cytopathic effect neutralization assay. Nineteen strains resembled the previously characterized human rotavirus serotype Wa, whereas three strains were serologically related to the DS-1 strain. Eleven strains appeared to be serotypically distinct from the Wa and DS-1 strains and thus apparently represent one or more new human rotavirus serotypes.     

Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup II human rotaviruses.

We have previously found (O. Nakagomi, T. Nakagomi, H. Oyamada, and T. Suto, J. Med. Virol. 17:29-34, 1985), during an epidemiological study in Japan, a novel human rotavirus that belongs to subgroup I but has a long RNA pattern typical of subgroup II human rotaviruses. From the stool specimen containing this virus, we successfully isolated in MA104 cells a rotavirus, designated AU-1, which possesses these novel characteristics. The possibility that strain AU-1 was a laboratory contaminant of an animal rotavirus previously adapted to tissue culture cells was ruled out, and the identity of the AU-1 strain was established. Genetic analysis by RNA-RNA hybridization revealed that the AU-1 strain is not a simple reassortant between subgroup I and II human rotaviruses but that it shares a high level of sequence homology only with the gene encoding VP7 (the major neutralization protein) of serotype 3 human rotaviruses. Weak homology of the genomic RNA segments was also observed between the AU-1 strain and animal rotavirus strains, including rhesus rotavirus strain RRV and bovine rotavirus strain NCDV. These results suggest that the AU-1 strain may be an animal rotavirus that infected a human.