Search

During a 2-year study of diarrhea among children in Blantyre, Malawi, greater than 50% of rotavirus strains genotyped by using reverse transcription—polymerase chain reaction possessed previously unrecognized combinations of the neutralization proteins VP7 and VP4. Serotype G8 rotaviruses, which have been identified recently in several African countries, were found to possess P[4] or P[6] VP4 genotype specificity. Two of these short electropherotype rotaviruses were further investigated: these comprised a P[6], G8 representative strain (MW23) and a P[4], G8 representative strain (MW333). The VP7 gene sequences of both strains exhibited greatest homology to human and animal serotype G8 rotaviruses. Sequence analysis of the VP4 gene of MW23 indicated closest identity to the P2A[6], G9 strain US1205 from the United States. The VP4 gene of MW333 was most closely related to the P[4], G12 strain L26 isolated in the Philippines and the Australian P[4], G2 strain RV-5. The NSP4 gene sequences of both strains were classified in NSP4 genetic group I. RNA-RNA hybridization demonstrated that each of these two strains is related to the DS-1 genogroup of human rotaviruses. Subgroup analysis and virus neutralization confirmed complete antigenic characterization of MW23 as subgroup I, P2A[6], G8 and MW333 as subgroup I, P1B[4], G8. The similarity of the VP7 gene sequences of the prototype strains described in this report to bovine serotype G8 rotaviruses suggests that they may represent human/bovine reassortant viruses.

Of five globally important VP7 (G) serotypes (G1–4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.

Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% aa and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8–98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2–98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% aa homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains.

Between 1992 and 1998, serotype G9 human rotavirus (RV) strains have been detected in 10 countries, including Thailand, India, Brazil, Bangladesh, Malawi, Italy, France, the United States, the United Kingdom, and Australia, suggesting the possible emergence of the fifth common serotype worldwide. Unlike the previously characterized reference G9 strains (i.e., WI61 and F45), the recent G9 isolates had a variety of gene combinations, raising questions concerning their origin and evolution. To identify the progenitor strain and examine the on-going evolution of the recent G9 strains, we characterized by genetic and antigenic analyses 16 isolates obtained from children with diarrhea in India, Bangladesh, the United States, and Malawi. Specifically, we sequenced their VP7 and NSP4 genes and compared the nucleotide (nt) and deduced amino acid sequences with the reference G9 strains. To identify reassortment, we examined the products of five gene segments; VP4, VP7, and NSP4 genotypes (genes 4, 9, and 10); subgroups (gene 6); electropherotypes (gene 11); and the genogroup profiles of all of the recent G9 isolates. Sequence analysis of the VP7 gene indicated that the recent U.S. P[6],G9 strains were closely related to the Malawian G9 strains (99% nt identity) but distinct from G9 strains of India ( approximately 97% nt identity), Bangladesh ( approximately 98% nt identity), and the reference strains ( approximately 97% nt identity). Phylogenetic analysis identified a single cluster for the U.S. P[6],G9 strains that may have common progenitors with Malawian P[6],G9 strains whereas separate lineages were defined for the Indian, Bangladeshi, and reference G9 strains. Northern hybridization results indicated that all 11 gene segments of the Malawian P[6],G9 strains hybridized with a probe derived from a U.S. strain of the same genotype and may have the same progenitor, different from the Indian G9 strains, whereas the Bangladesh strains may have evolved from the U.S. G9 progenitors. Overall, our findings suggest that much greater diversity among the newly identified G9 strains has been generated by reassortment between gene segments than through the accumulation of mutations in a single gene.

We recently reported that the culture-adapted neonatal rotavirus strain 116E represented the first P type 11 human rotavirus, based on the close relationship of its VP4 protein to that of the bovine serotype G10P11 strain B223. In this study, we demonstrated by sequence analysis and cross-neutralization studies that the VP7 protein of 116E is closely related to those of the human serotype G9 strains, F45 and WI61, but distinct from B223 and other rotaviruses. Low-level cross-neutralization was also observed between strains 116E and B223, probably because of the antigenic similarity of their VP4 proteins. We have demonstrated by RNA-RNA hybridization that strain 116E is a reassortant between strains from the Wa and bovine (KK3-like) genogroups, deriving at least seven genes from the former and at least one gene from the latter. Together with the recent identification of serotype G10P11 newborn rotavirus strains in Bangalore, India (M. Das et al., Virology, 194, 374-379, 1993), these results are consistent with the hypothesis that reassortment may be an important mechanism for generation of rotavirus strains of newborns.

Rotavirus strain 116E was isolated from the fecal specimen of a newborn infant from New Delhi who had no symptoms of diarrhea. The strain could not be P typed using the PCR method recently developed in our laboratory so we have sequenced the 4th gene segment that encodes the VP4 protein using viral transcript RNA prepared in vitro. This gene is 2353 base pairs in length and codes for a protein 772 amino acids long which begins with a methionine initiation codon at nucleotides 11 to 13 and ends with a single termination codon at nucleotides 2327 to 2329. Northern blot analysis demonstrates that the VP4 protein of this strain is encoded by genome segment 4. This gene is closely related to the VP4 gene of the bovine B223 strain (P type 11) at both the nucleotide (90.8% identity) and amino acid (92.2% identity) levels, but is unique from those of strains from P types 1 to 10. The close relatedness of the VP4 proteins of strains 116E and B223 is demonstrated by the amino acid composition of the potential trypsin cleavage sites and their flanking sequences, the size of the cleavage fragments, and conservation of most cysteine and proline residues. Comparative amino acid analyses of the variable regions thought to be important in VP4 antigenicity are consistent with the hypothesis that strain 116E may represent the first reported human P type 11 strain.

To further study the molecular characteristics of group (gp) C rotaviruses, we produced, cloned, and sequenced cDNA to gene 5 of the Shintoku strain of bovine gp C rotavirus. The resulting clone was specific for gene 5 and was genetically related to the human and porcine gp C rotaviruses, as demonstrated by Northern blot hybridization analysis. The Shintoku gene 5 is 1352 nucleotides in length and has one open reading frame encoding a polypeptide of 395 amino acids with a predicted molecular mass of 44.5 kDa. Comparative sequence analysis indicated that: (i) the Shintoku gene 5 protein shared 88.4 to 90.6% homology with the VP6 of the human (Bristol and 88–220) and porcine (Cowden) strains of gp C rotaviruses, but only low homology with the VP6 of bovine gp A (RF) and human gp B (ADRV) rotaviruses (41.3 and 16.3%, respectively); (ii) the predicted secondary structure was highly conserved among the gene 5 proteins of the bovine, porcine, and human gp C rotaviruses; and (iii) seven highly conserved regions were identified for the first time in the deduced primary amino acid sequences of gene 5 of gp C and gene 6 of gp A rotaviruses. However, only three of these highly conserved areas were present in the regions of VP6, where the secondary structure was predicted to be similar for the rotavirus strains examined. These three regions may contribute to common epitopes between the two groups of rotaviruses. Our results, in comparison with data for other rotaviruses, indicate that gene 5 of the bovine gp C rotavirus codes for the major inner capsid protein (VP6).

In this report we study the interaction of reovirus type 3 Dearing (RV3) with vertebrate erythrocytes whose membrane glycoconjugates differ in the degree and position of O -acetylation of their sialic acid (NeuAc) residues. Binding to erythrocytes required the presence of NeuAc on cellular glycoconjugates, since pretreatment with sialidase (neuraminidase) abolished hemagglutination by RV3. Furthermore, we found that RV3 binds efficiently to and hemagglutinates all erythrocyte preparations possessing exclusively NeuAc, or a mixture of NeuAc and 4- O -acetyl-NeuAc (4- O -Ac-NeuAc), but poorly to erythrocytes bearing a mixture of 9- O -Ac-NeuAc and NeuAc, suggesting that RV3 binds preferentially to NeuAc-containing glycoconjugates. To gain further evidence for this hypothesis we treated chicken erythrocytes with influenza C virus neuraminate, 9- O -acetylesterase, to convert their 9- O -Ac-NeuAc residues to NeuAc. When hemagglutination assays were carried out on these cells, we observed a 16-fold increase in the hemagglutination titer for RV3 compared to untreated cells. When we treated bovine submaxillary mucin (BSM) with influenza C virus, we observed a dramatic increase in its potency as an inhibitor of RV3 hemagglutination. Concomitant with this, the 9- O -Ac-NeuAc residues on BSM were converted to NeuAc. Taken together and in conjunction with a previous report (A. F. Pacitti and J. R. Gentsch, 1987, J. Virol. 61, 1407–1415) , these results suggest that the virion attachment protein exhibits a strong preference for NeuAc over 9- O -Ac-NeuAc as a receptor component on erythrocytes.

During an epidemiologic survey of rotavirus infections established to monitor the prevalent G serotypes circulating in the United States, human P[6]G9, subgroup I rotavirus strains causing symptomatic infections were identified as the fourth most common serotype. In this report we describe the molecular and antigenic characterization of one of these P[6]G9 isolates (US1205). Neutralization and sequencing studies have demonstrated that both outer capsid proteins, VP7 and VP4, of US1205 are closely related to but genetically and antigenically distinguishable from those of standard G9 strains (e.g., F45, WI61) and standard P2A[6] strains (e.g., ST3, M37). Thus the complete antigenic type of US1205 is P2A[6]G9, subgroup I. Sequence analysis of the VP6 and NSP4 genes of US1205 indicates that strain US1205 possessed VP6 subgroup I and NSP4A genotype specificities. Finally, Northern hybridization studies suggest that the P[6]G9 strains are closely related to members of the DS-1 genogroup except for their P[6] VP4 gene, which has been commonly identified in strains of both major human genogroups, and their G9 VP7 gene, which may have been derived by reassortment with a Wa genogroup strain. Examination of historic collections and prospective surveillance of strains will be needed to determine whether this strain has been present for some time or if it is emerging to compete with the other common serotypes of rotavirus.

Antigenic sites on the G1 glycoprotein of La Crosse bunyavirus were defined by constructing a panel of neutralizing and nonneutralizing monoclonal antibodies ( F. Gonzalez-Scarano, R. E. Shope, C. H. Calisher, and N. Nathanson (1982) , Virology120, 42–53). To analyze the relationship between the individual epitopes delineated by monoclonal antibodies, 11 neutralizing antibodies were used to select variant viruses. These variant viruses were tested against the panel of anti-G1 protein monoclonal antibodies by neutralization and by ELISA. The neutralization tests assigned the 11 epitopes to five groups, consisting of 6, 2, 1, 1, and 1 epitopes. ELISA tests gave a similar pattern, but also demonstrated interrelationships between four of the five epitope groups, suggesting that there may be a single immunodominant antigenic site on the G1 protein. When eight nonneutralizing anti-G1 monoclonal antibodies were tested in ELISA, they fell into three of the five epitope groups defined by neutralization; there was no evidence of a separate nonneutralizing antigenic site on the G1 protein.

Homologous virus genetic recombination has been demonstrated for snowshoe hare (SSH), La Crosse (LAC), Tahyna (TAH), Lumbo (LUM), and Trivittatus (TVT) viruses, members of the California (CAL) serogroup of bunyaviruses, as well as for Guaroa (GRO) virus, a member of the Bunyamwera serogroup. No heterologous virus genetic recombination has been obtained between GRO and LAC, SSH, TAH, or TVT viruses, although recombination has been demonstrated between TAH and LAC, SSH, or TVT viruses, as well as between LAC and TVT or SSH viruses. Not all the dual temperature sensitive ( ts ) mutant virus coinfections (e.g., the reciprocal crosses to those that gave recombinants), yielded the expected recombinant viruses. This paradox is discussed in relation to the conditions necessary to produce recombinant viruses. Eight new recombinant viruses (TAH/LAC/TAH, TAH/LAC/LAC, LAC/TAH/LAC, LAC/TAH/TAH, TVT/LAC/TVT, TVT/TAH/TVT, SSH/TAH/TAH, and SSH/TAH/SSH) have been characterized. Plaque reduction neutralization tests have been performed with seven of these eight viruses, and four recombinant bunyaviruses obtained previously (i.e. SSH/LAC/LAC, SSH/LAC/SSH, SSH/SSH/LAC, and LAC/LAC/SSH). The results obtained using antisera raised against prototype TAH, TVT, SSH, or LAC viruses indicate that one or both of the bunyavirus M RNA gene products (i.e., their glycoproteins G1 and G2) specify the antigenic determinants recognized in the neutralization test.