Characterization of neutralization specificities of outer capsid spike protein VP4 of selected murine,lapine, and human rotavirus strains

Neutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine x human, three lapine x human, and two human x human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study.     

VP7 gene polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR

Rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide. Surveillance of rotavirus serotypes/genotypes (both VP7[G] and VP4[P]) is in progress globally in which polymerase chain reaction (PCR) has been the assay of choice. We investigated polymorphism of the VP7 gene of serotype G9 rotavirus strains and its impact on the determination of VP7 gene genotype by PCR assay. By VP7 gene sequence analysis, we and others have previously shown that the G9 rotavirus strains belong to one of three VP7 gene lineages. By PCR assay using three different sets of commonly used primers specific for G1-4, 8 and 9, 23 Brazilian G9 strains and 5 well-characterized prototype G9 strains which collectively represented all three VP7 gene lineages were typed as: (i) G3; (ii) G4; (iii) G9; (iv) G3 and G9; or (v) G9 and G4 depending on a primer pool employed. This phenomenon appeared to be due to: (i) a VP7 gene lineage-specific polymorphism, more specifically mutation(s) in the primer binding region of the VP7 gene of G9 strain; and (ii) the magnitude of difference in nucleotide homology at respective primer binding site between homotypic (G9) and heterotypic (G3 or G4) primers present in a primer pool employed.     

VP7 gene polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR

Rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide. Surveillance of rotavirus serotypes/genotypes (both VP7[G] and VP4[P]) is in progress globally in which polymerase chain reaction (PCR) has been the assay of choice. We investigated polymorphism of the VP7 gene of serotype G9 rotavirus strains and its impact on the determination of VP7 gene genotype by PCR assay. By VP7 gene sequence analysis, we and others have previously shown that the G9 rotavirus strains belong to one of three VP7 gene lineages. By PCR assay using three different sets of commonly used primers specific for G1-4, 8 and 9, 23 Brazilian G9 strains and 5 well-characterized prototype G9 strains which collectively represented all three VP7 gene lineages were typed as: (i) G3; (ii) G4; (iii) G9; (iv) G3 and G9; or (v) G9 and G4 depending on a primer pool employed. This phenomenon appeared to be due to: (i) a VP7 gene lineage-specific polymorphism, more specifically mutation(s) in the primer binding region of the VP7 gene of G9 strain; and (ii) the magnitude of difference in nucleotide homology at respective primer binding site between homotypic (G9) and heterotypic (G3 or G4) primers present in a primer pool employed.     

New P serotype of group A human rotavirus closely related to that of a porcine rotavirus

The VP7 and VP4 genes of two human group A rotavirus strains Mc323 and Mc345 with unique serologic and genomic properties, and isolated in Chiang Mai, Thailand, in 1989 [Urasawa et al. (1992) Journal of Infectious Diseases 166:227-234] were further characterized. The nucleotide and deduced amino acid sequences of the VP7 genes allowed the classification of both strains as serotype G9. The VP4 genes of both strains are 2,359 nucleotides in length and encode a protein of 775 amino acids like in most human rotaviruses. A comparison of the VP4 amino acid sequence of strain Mc323 with those of strain Mc345 and 24 human and animal rotaviruses representing 20 distinct VP4 genotypes reported to date showed that VP4 of Mc323 and Mc345 belong to genotype 19 previously reported for porcine rotavirus [Burke et al. (1994) Journal of General Virology 75:2205-2212]. To investigate the serological type (P serotype) of these VP4s, six reassortant viruses each containing a distinct VP4 gene characteristic of human rotaviruses and the VP7 gene of porcine rotavirus strain Gottfried (G4) were prepared, and antisera to these reassortants produced in rabbits. In neutralization tests, the P serotype of Mc323 was clearly differentiated from the five major P serotypes reported previously for human rotaviruses, suggesting that Mc323 and Mc345 represent a new human rotavirus P serotype tentatively called P11.     

Characterization of genotype G8 strains from Malawi, Kenya, and South Africa

Reviews of the global distribution of rotavirus genotypes have revealed the continuous circulation of G8 strains in Africa, often responsible for more cases of rotavirus disease than the more common G1-G4 rotavirus strains. During the study, genotype G8 strains from Malawi, Kenya, and South Africa were detected and the VP7 and VP4 genes of selected specimens were sequenced. Results indicated that G8 strains appeared to reassort frequently and were associated with P[6], P[4], and P[8] specificity. Phylogenetic analysis suggested that G8 strains occurred in a North/South African phylogenetic divide. In addition, G8 strains appear to be able to infect non-human primates and strains with close phylogenetic relationships were detected in the same year on two continents. Any rotavirus vaccine introduced into African environments will need to demonstrate protective efficacy against unusual genotype combinations, new serotypes, and animal strains. Therefore, continuous monitoring of rotavirus strains in human and animal populations in Africa is a necessity.     

Preparation and characterization of a neutralizing monoclonal antibody directed to VP4 of rotavirus strain K8 which has unique VP4 neutralization epitopes

Summary For selecting the neutralizing monoclonal antibodies (N-MAbs) directed to VP4 of rotavirus strain K8, which has unique VP4 neutralization epitopes, we prepared several reassortant viruses by mixed infection of two different strains K8 (serotype 1) and P (serotype 3) in vitro: three reassortant clones having VP4 of K8 and VP7 of P and four clones having VP4 of P and VP7 of K8. By using these reassortants in screening hybridomas, a N-MAb (K8-2C12) directed to strain K8-specific VP4 was obtained. The MAb K8-2C12 neutralized only K8 when tested against numerous strains of different serotypes, while in enzyme-linked immunosorbent assay this MAb reacted also with simian rotavirus SA11 (serotype 3), bovine rotavirus NCDV (serotype 6), and human rotavirus (HRV) strain 69M (serotype 8). Neutralization-resistant mutants of K8 were selected by the K8-2C12 antibody and VP4 amino acid sequences of the mutants were determined. Single amino acid substitution was detected in the three mutant clones at position 394, which is included in the major cross-reactive neutralization region identified in other rotaviruses.     

Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses

During a limited epidemiological study, the serotype specificities of several isolates of bovine rotavirus, exhibiting identical electropherotypes, from a single cattle farm near Bangalore, India, could not be determined using a panel of serotyping monoclonal antibodies (MAbs) specific for G serotypes 1-6 and 10. To determine the genotypes of these isolates, the nucleotide sequences of the genes encoding the outer capsid proteins VP4 and VP7 of two representative isolates, Hg18 and Hg23, were determined. The corresponding gene sequences from the two isolates were identical, indicating that these isolates represented a single strain of bovine rotavirus. Comparison of the VP4 nucleotide (nt) and the deduced amino acid (aa) sequences with those of several human and animal rotavirus strains representing all of the currently recognized 20 different VP4 (P) genotypes revealed low nt and aa sequence identities of 61.0 to 74.2% and 57.9 to 78.2% for VP4. The percentages of amino acid homology for the VP8* and VP5* regions of VP4 were 37.7 to 67.9 and 68.1 to 84.2%, respectively. The nt and aa sequences of the VP7 gene were also distinct from those of human and animal strains belonging to the previously established 14 VP7(G) serotypes (65.9 to 75.5% nt and 59.5 to 77.6% aa identities). These findings suggest the classification of the VP4 and VP7 genes of the bovine isolates represented by Hg18 as new P and G genotypes and provide further evidence for the vast genetic/antigenic diversity of group A rotaviruses.     

Molecular characterization of the VP4, VP6, VP7, and NSP4 genes of lapine rotaviruses identified in italy: emergence of a novel VP4 genotype

The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87-96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90-95%) among each other, but showed low aa identity (34-77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].     

Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the United States

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.     

Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus.

The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.     

Antigenic and Genomic Diversity of Human Rotavirus VP4 in Two Consecutive Epidemic Seasons in Mexico

In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.     

Prevalence of human rotavirus genotypes in Wuhan, China, during 2008-2011: changing trend of predominant genotypes and emergence of strains with the P[8]b subtype of the VP4 gene

Hospital-based surveillance of rotavirus genotypes was conducted in Wuhan, China, between March 2008 and May 2011. The detection rates of group A rotavirus were 24.6% (458/1859) and 12.1% (96/795) in children and adults, respectively, with diarrhea. Among the 554 positive specimens, the most frequent genotype was G3P[8] (57.9%), followed by G1P[8] (29.4%). Compared with previous studies in Wuhan (2000-2008), the relative frequency of G3P[8] has been decreasing year by year, while the predominant genotype G3 shifted to G1 in 2011. In the present study, a rare P[8]b subtype of the VP4 gene (OP354-like P[8]) was identified in nine strains. Full-length sequences of VP7, VP4, VP6 and NSP4 genes of two G9P[8]b strains (RVA/Human-wt/CHN/E1545/2009/G9P[8]b and RVA/Human-wt/CHN/Z1108/2008/G9P[8]b) were determined for phylogenetic analysis. The four genes of these strains were closely related to one another, and the G9-VP7 genes of these strains belonged to lineage III, which contains globally spreading G9 rotaviruses. The full-length sequence of VP4 gene segments of the P[8]b strains in Wuhan clustered with those of P[8]b strains in Vietnam, Russia and Belgium, while they were distinct from those of the OP354 strain from Malawi and Bangladeshi strains. The VP6 and NSP4 genes of two P[8]b strains belonged to the I1 and E1 genotype, respectively, and clustered with those of strains belonging to Wa-like human rotaviruses from various Asian countries. These findings indicate the changing epidemiologic trend of rotavirus genotypes in Wuhan, i.e., the shift of the predominant type from G3 to G1 and the emergence of P[8]b strains genetically related to those distributed in other Asian countries.     

Distribution of human group a rotavirus VP7 and VP4 types circulating in Seoul,Korea between 1998 and 2000

Three hundred forty-eight fecal specimens collected from young children with acute diarrhea in Seoul, Korea between January 1998 and February 2000 were examined for G and P types. Of these, 205 samples (59%) were confirmed as group A rotavirus by ELISA for the detection of VP6 antigen. Confirmed rotavirus isolates were characterized using G serotyping ELISA and RT-PCR methodologies for G and P genotyping of the outer capsid proteins VP7 and VP4, respectively. Serotyping of the outer capsid protein, VP7, revealed G4 as the dominant circulating serotype (41%) followed by G1 (28%) and quite a high incidence of mixed infection (14%). Genotyping of the VP4 protein was carried out on 55 of the rotavirus isolates with the dominant type being P[8] (46%). Of interest were a number of unusual G and P type combinations detected in Korea for the first time, especially the P[4] genotype associated with non-G2 serotypes. There were also a number of P[6] isolates identified including one G2P[6] isolate.     

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains

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.     

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain

Summary.  The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1–4, and 6–11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype. Received September 2, 1997 Accepted January 8, 1998     

Sequence analysis of the gene encoding the serotype-specific glycoprotein (VP7) of two new human rotavirus serotypes

Human rotavirus strains 69M and WI61 are distinct from human rotavirus serotypes 1, 2, 3, and 4 and from each other by plaque reduction neutralization and have been proposed as new human rotavirus serotypes (serotype 8 and serotype 9, respectively). The nucleotide sequence of the gene encoding the serotype-specific capsid glycoprotein, VP7, of strains 69M and WI61 was determined. In addition, the sequence of the VP7 gene of strain F45 (serotypically indistinguishable from WI61) was determined. Comparative analyses of the nucleotide and deduced amino acid sequences with those of reference strains from serotypes 1,2,3,4,5, and 6 demonstrated that WI61 and F45 share a high degree of sequence similarity with each other and that strains 69M, WI61, and F45 are distinct from established serotypes 1,2,3,4,5, and 6 in nine defined regions of the VP7 which are variable across rotavirus serotypes.