Identification of two subtypes of serotype 4 human rotavirus by using VP7-specific neutralizing monoclonal antibodies

Two distinct subtypes of human rotavirus serotype 4 were identified by using neutralizing monoclonal antibodies directed to the major outer capsid glycoprotein, VP7, of strains ST3 (subtype 4A) and VA70 (subtype 4B). Specimens containing serotype 4 rotavirus, obtained from different countries, were examined for subtyping by using solid-phase immune electron microscopy, enzyme-linked immunosorbent assay, and, for cell culture-adapted strains, neutralization assay. All 59 human rotavirus strains identified as serotype 4 by using animal antisera were classified into either subtype by monoclonal antibodies. This suggests that the antigenic difference between the two subtypes is a consequence of critical variations within the immunodominant serotype 4-specific neutralization site of rotavirus VP7. Subtype 4A (ST3-like) strains were predominant and were detected in stools from patients with gastroenteritis, as well as from healthy infants and young children.     

Rotavirus VP7 neutralization epitopes of serotype 3 strains

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 27 human and animal rotavirus strains of serotype 3 in order to examine genetic variation within strains of identical serotype. Comparisons of the deduced amino acid sequences of the VP7s showed overall sequence identities of 85% or higher. A higher degree of overall VP7 sequence similarity was observed among strains from the same animal species when compared to strains from different animal species, suggesting that there are species-specific sequences in the VP7 protein. Alignment of the amino acid sequences demonstrated that amino acid sequence divergence among serotype 3 strains from different species was located primarily in previously established VP7 serotype-specific regions where genetic variation was identified among strains of different serotype. These regions were highly conserved among serotype 3 strains derived from the same species. The varying reactivities of three anti-VP7 monoclonal antibodies with the 27 strains was consistent with the occurrence of antigenic variation among serotype 3 strains. Moreover the reactivity of monoclonal antibodies correlated with the amino acid sequence found in two serotype-specific regions (VR5 and VR8). A computer-derived predicted phylogenetic tree suggests that rotavirus strains from different animal species belonging to serotype 3 are more closely related to each other than to rotavirus strains of different serotypes.     

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.     

Rotavirus vaccines: an overview.

Rotavirus vaccine development has focused on the delivery of live attenuated rotavirus strains by the oral route. The initial "Jennerian" approach involving bovine (RIT4237, WC3) or rhesus (RRV) rotavirus vaccine candidates showed that these vaccines were safe, well tolerated, and immunogenic but induced highly variable rates of protection against rotavirus diarrhea. The goal of a rotavirus vaccine is to prevent severe illness that can lead to dehydration in infants and young children in both developed and developing countries. These studies led to the concept that a multivalent vaccine that represented each of the four epidemiologically important VP7 serotypes might be necessary to induce protection in young infants, the target population for vaccination. Human-animal rotavirus reassortants whose gene encoding VP7 was derived from their human rotavirus parent but whose remaining genes were derived from the animal rotavirus parent were developed as vaccine candidates. The greatest experience with a multivalent vaccine to date has been gained with the quadrivalent preparation containing RRV (VP7 serotype 3) and human-RRV reassortants of VP7 serotype 1, 2, and 4 specificity. Preliminary efficacy trial results in the United States have been promising, whereas a study in Peru has shown only limited protection. Human-bovine reassortant vaccines, including a candidate that contains the VP4 gene of a human rotavirus (VP4 serotype 1A), are also being studied.     

Clinical studies of a quadrivalent rotavirus vaccine in Venezuelan infants.

Phase I studies of an oral quadrivalent rotavirus vaccine were conducted in 130 Venezuelan infants 10 to 20 weeks of age. The vaccine consists of a mixture of equal amounts of rhesus rotavirus (RRV) vaccine (serotype 3 [VP7]) and each of three human rotavirus-RRV reassortant strains: D x RRV (serotype 1 [VP7]), DS1 x RRV (serotype 2 [VP7]), and ST3 x RRV (serotype 4 [VP7]). Three different doses of the quadrivalent vaccine (0.25 x 10(4), 0.5 x 10(4), and 10(4) PFU of each component) were evaluated sequentially for safety and antigenicity in placebo-controlled, double-blind trials. Starting the day after vaccination, the infants were monitored by daily home visits for 7 days. Only minor reactions were observed during this period; these were limited to mild transient febrile episodes which began day 2 or 3 after vaccination and lasted 1 to 2 days in 15 to 30% of the infants. Serological studies demonstrated that 68 to 96% of the infants developed a rotavirus serum immunoglobulin A response following vaccination. However, when tested by plaque reduction neutralization assay against individual human rotavirus serotype 1, 2, 3, or 4, the response rates ranged from 4 to 23% with the low dose, 21 to 33% with the medium dose, and 32 to 58% with the high dose. Most (73 to 79%) infants developed neutralizing antibodies to RRV following administration of each dose schedule. Vaccine virus shedding was analyzed by utilizing tissue culture isolation of virus from stool. All of the infants who received the lower of medium dose and 89% of those fed the high dose shed one or more components of the vaccine. Analyses of rotavirus serotypes isolated from the stool of infants who received the 0.25 x 10(4) -PFU dose revealed that DS1 x RRV was the most commonly shed vaccine component, followed by RRV, D x RRV, and ST3 x RRV in that order.     

Genetic and antigenic characterization of a serotype G6 human rotavirus isolated in melbourne,Australia

An unusual rotavirus strain, MG6, was isolated from a 16-month-old child admitted to hospital with acute gastroenteritis. The virus could not be serotyped (G-typed) by enzyme immunoassay using standard reagents specific for common serotypes of human Group A rotaviruses. Nucleotide sequencing of cDNA derived from the gene encoding the outer capsid protein, VP7, and deduction of the VP7 amino acid sequence indicated that this strain belonged to serotype G6, a serotype normally associated with viruses causing disease in cattle. This was confirmed by polymerase chain reaction typing and enzyme immunoassay using a G6-specific monoclonal antibody. The VP4 genotype of MG6 was determined by hybridization of its VP4 cDNA to genomic RNA isolated from standard strains of defined P-types. This analysis, confirmed by deduced amino acid sequence analysis, classified MG6 into the novel genotype P13. MG6, therefore, is related to the previously described G6P13 human strain PA169, isolated in Italy. The emergence of strain MG6, the first human G6 rotavirus identified in Australia, provides further evidence of reassortment between human and animal rotaviruses. © Wiley-Liss, Inc.     

Molecular characterization of unusual human G5P[6] rotaviruses identified in China.

BACKGROUND: We found an unusual human rotavirus, LL36755 of G5P[6] genotype, in a stool sample collected in Lulong County, Hebei Province, China. This is the first detection of rotavirus serotype G5 in Asia. OBJECTIVES: To identify and characterize G5 rotaviruses in 988 stool samples collected from children under 5 years old with acute gastroenteritis. STUDY DESIGN: We analyzed 459 rotavirus-positive samples with RT-PCR using G5 genotype-specific primers. The G5 strains were sequenced. RESULTS: Two additional G5-positive samples (LL3354 and LL4260) were identified. VP7, VP4, VP6 and NSP4 genes of LL3354, LL4260 and LL36755 strains were sequenced. The VP4 sequences formed a group with porcine P[6] strains. The VP6 sequences of strains LL3354 and LL36755 were phylogenetically close to the major clusters of SGI and SGII rotaviruses, respectively. The deduced VP6 protein of strain LL4260 had characteristics of both SGI and SGII strains, but best fit with a cluster of atypical SGI viruses. In addition, based on NSP4 sequences, the three G5 strains belonged to genogroup B and were closest to human strain Wa. CONCLUSION: These results indicate a dynamic interaction of human and porcine rotaviruses and suggest that reassortment could result in the stable introduction and successful spread of porcine gene alleles into human rotaviruses.     

Antigenic and genetic characterization of serotype G2 human rotavirus strains from the African continent

Serotype G2 rotavirus strains were isolated in seven countries on the African continent during 1999 and 2000. To investigate the associated DS-1 genogroup characteristics, subgroup (VP6) enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and P genotyping were performed on 10 G2 strains. The antigenic and genetic variation of the gene encoding the major neutralization glycoprotein (VP7) was also investigated by using G2-specific monoclonal antibodies and sequence analysis. Alterations in the characteristic DS-1 genogroup gene constellations were more likely to occur in the VP4 gene, and three genotypes were observed: P[4], P[6], and a dual P[4]-P[6] type. The failure of G2-specific monoclonal antibodies to type African G2 strains was more likely due to improper storage of the original stool, although G2 monotypes were detected. Phylogenetic analyses revealed clusters of serotype G2 strains that were more commonly associated with seasons during which G2 was predominant. No rotavirus vaccine trials have been conducted in an area where G2 strains were the predominant circulating serotype, and the continued surveillance of rotavirus epidemics in Africa will be preparation for future vaccine implementation in an area that clearly needs these preventative medicines.     

Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus: comparison with homologous genes from four strains of human and animal rotaviruses

A full-size cloned cDNA copy of the rotavirus gene encoding the structural neutralization glycoprotein (VP7) of Nebraska calf diarrhea virus (NCDV), a strain recently shown to be effective as a vaccine in children, has been sequenced. Comparison of the deduced amino acid sequence of NCDV (serotype 6) VP7 with that of four other rotavirus strains (human WA serotype 1, human HU-5 serotype 2, simian SA-11 serotype 3, and bovine UK serotype 6) indicates that the degree of amino acid homology among VP7 neutralization proteins of these serotypes ranges from 75 to 86%. Four hydrophilic regions at amino acid residues 174-183, 248-256, 287-294, and 310-317 exhibit significant homology and hence may represent common antigenic determinants, while one hydrophilic area at amino acid residues 83-102 exhibits sufficient divergence to suggest it may be involved in serotype specificity.     

Geographic distribution of human rotavirus VP4 genotypes and VP7 serotypes in five South African regions.

The rotavirus outer capsid proteins elicit the production of neutralizing antibodies and are known to play a role in inducing resistance to disease. In this study, cDNA probes directed at the six most common human rotavirus VP7 serotypes (G1 to G4, G8, and G9) and five human rotavirus VP4 genotypes (P4, P6, P8, P9, and P10) were utilized. Hybridization analysis of 572 human rotavirus strains collected from five regions in South Africa was performed to determine the distribution of the VP7 serotypes and VP4 genotypes in nature. VP7 serotype G1 was identified most frequently, occurring in 51% of the rotavirus strains tested. VP7 serotypes G2 and G4 occurred in similar numbers, although their distribution varied regionally. Few serotype G3 strains and no G8 or G9 strains were identified. The P8 VP4 genotype occurred most frequently overall (66%), and the P4 genotype was detected next most frequently. The P6 genotype was identified in 28 symptomatically infected neonates and in 8 symptomatic infants. Few P9 strains were identified. The potential for reassortment events was demonstrated by dual infections with different viruses.     

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.     

人轮状病毒G2和G3型主要中和抗原VP7基因在重组腺病毒中的表达

目的 研究我国G2和G3型轮状病毒主要中和抗原VP7基因在重组腺病毒中的表达。方法 在前期成功表达Gl型VP7的基础上，选用我国G2和G3型主要流行株97S43和97S48 VP7基因，用非复制型腺病毒载体对上述基因进行表达。结果 获得了表达我国G2型和G3型人轮状病毒流行株VP7基因的非复制型重组腺病毒rvAdG2VP7和rvAdG3VP7，应用PCR及Southem blot技术证实在重组腺病毒中整合有轮状病毒G2型VP7和G3型VP7基因，RT-PCR证明重组腺病毒在感染的293细胞内均能有效地转录插入基因，Western blot检测到轮状病毒VP7基因的表达。结论 这一工作为进一步进行动物实验，发展多价轮状病毒疫苗打下了基础。     

Electrophoretic typing of nosocomial rotavirus infection in a general paediatric unit showing the continual introduction of community strains

During 1989 stool specimens from hospitalised children with gastroenteritis at Ga-Rankuwa Hospital in South Africa were examined for the presence of rotaviruses. Overall 16% of the children were positive for rotavirus. However, 43% of the rotavirus positive patients were infected in the hospital. Further characterisation of the rotavirus strains was performed by electrophoresis of the RNA genome and hybridisation analysis of the VP7 and VP4 genes present. The strains associated with nosocomial infection were similar to those strains acquired in the community. The majority of the strains, both community- or hospital- acquired, were associated with a serotype 1 strain with a long electrophoretype and bearing the Wa-like VP4 gene. Three minor rotavirus strains with a long electrophoretype were also observed to be circulating bearing serotype 1 or 4 VP7 genes and the Wa-like VP4 gene. Interestingly, a serotype 4 strain bearing the M37-like VP4 gene was identified to occur almost exclusively in neonates although the gene was associated with diarrhoea in these cases. Two strains with differing short RNA electrophoretypes were also observed, members of which hybridised to VP7 serotype 2 and VP4 DS-1 type probes. © 1993 Wiley-Liss, Inc.     

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.     

Analysis by RNA-RNA hybridization assay of intertypic rotaviruses suggests that gene reassortment occurs in vivo

Antigenic characterization of human and animal rotaviruses by the plaque reduction neutralization assay has shown the existence of naturally occurring intertypes. Antiserum to M37, a rotavirus strain isolated from an asymptomatic neonate, neutralizes both Wa and ST3 strains, which are classified as serotype 1 and serotype 4 human rotaviruses, respectively. Likewise, antiserum to SB-1A, a porcine rotavirus, neutralizes rotavirus strains belonging to serotype 4 or 5. Plaque reduction neutralization assay of reassortant rotaviruses produced in vitro from these intertypes indicates that these viruses share one antigenically related outer capsid protein, VP3, with one serotype and another antigenically related outer capsid protein, VP7, with the other serotype. Thus, M37 is related to ST3 on the basis of its fourth-gene product, VP3, and to Wa on the basis of its ninth-gene product, VP7, whereas SB-1A is related to Gottfried (serotype 4 porcine rotavirus) via VP7 and to OSU (serotype 5 porcine rotavirus) via VP3. RNA-RNA hybridization studies revealed a high degree of homology between the VP3 or VP7 gene segments responsible for shared serotype specificity. Thus, the fourth gene segments of M37 and ST3 were highly homologous, while M37 and Wa had homology between their ninth gene segments. SB-1A and Gottfried were homologous not only with respect to the ninth gene but had complete homology in all other genes except the fourth gene. The fourth gene of SB-1A was highly homologous with the fourth gene of OSU. These observations suggested that SB-1A was a naturally occurring reassortant between Gottfried-like and OSU-like porcine rotavirus strains. Our observations also suggested that intertypes may result from genetic reassortment in nature.     

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.