Dataset Information

Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus.

ABSTRACT: Rotavirus is the most common cause of acute gastroenteritis among infants and young children throughout the world, but rotavirus cases in developing countries account for nearly all of the approximately 600,000 annual deaths. We studied the epidemiology of rotavirus in 22 rural communities in northern coastal Ecuador over a five-year period. From 250 rotavirus positive stool specimens, the percentage that could not be RT-PCR genotyped for VP4 and VP7 was 77% and 63%, respectively. The possibility of sample degradation was considered but discounted after an experimental examination of rotavirus stability and EM visualization of rotavirus-like particles in several untypeable samples. Finally, alternate primers were used to amplify Ecu534, a sample that was untypeable using most published VP4 and VP7 primers. Characterization of the VP7, VP4, and VP6 full gene segments revealed novel genotypes and nucleotide mismatches with most published primer sequences. When considered with other findings, our results suggest that primer mismatch may be a widespread cause of genotyping failure, and might be particularly problematic in countries with greater rotavirus diversity. The novel sequences described in this study have been given GenBank accession numbers EU805775 (VP7), EU805773 (VP4), EU805774 (VP6) and the RCWG has assigned them novel genotypes G20P[28]I13, respectively.

SUBMITTER: Solberg OD

PROVIDER: S-EPMC2729439 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus.

Solberg Owen D OD Hasing Maria Eloisa ME Trueba Gabriel G Eisenberg Joseph N S JN

Virology 20090108 1

Rotavirus is the most common cause of acute gastroenteritis among infants and young children throughout the world, but rotavirus cases in developing countries account for nearly all of the approximately 600,000 annual deaths. We studied the epidemiology of rotavirus in 22 rural communities in northern coastal Ecuador over a five-year period. From 250 rotavirus positive stool specimens, the percentage that could not be RT-PCR genotyped for VP4 and VP7 was 77% and 63%, respectively. The possibilit ...[more]

PMID: 19131083

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Project description:Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates.IMPORTANCE Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.

Project description:Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

Dataset Information

Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus.

Publications

Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus.

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