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Rotavirus A strains obtained from children with acute gastroenteritis in Mozambique, 2012-2013: G and P genotypes and phylogenetic analysis of VP7 and partial VP4 genes.

ABSTRACT: In Mozambique rotavirus (RV) was shown to be the greatest cause of acute diarrhoea in infants from 0 to 11 months, and in 2015, national rotavirus vaccination was introduced. As with other developing countries, there is very limited active strain characterisation. Rotavirus positive clinical specimens, collected between 2012 and 2013, have now provided information on the genotypes circulating in southern Mozambique prior to vaccine introduction. Genotypes G2 (32.4%), G12 (28.0%), P[4] (41.4%) and P[6] (22.9%) (n = 157) strains were commonly detected with G2P[4] (42.3%) RVs being predominant, specifically during 2013. Phylogenetic evaluation of the VP7 and VP8* encoding genes showed, for the majority of the Mozambican strains, that they clustered with other African strains based on genotype. RVA/Human-wt/MOZ/0153/2013/G2P[4], RVA/Human-wt/MOZ/0308/2012/G2P[4] and RVA/Human-wt/MOZ/0288/2012/G12P[8] formed separate clusters from the other Mozambican strains with similar genotypes, suggesting possible reassortment. Amino acid substitutions in selected epitope regions also supported phylogenetic clustering. As expected, the VP7 and VP8* genes from the Mozambican strains differed from both the RotaTeq^® (SC2-9) G2P[5] and Rotarix^® (A41CB052A) G1P[8] genes. This study provides information on the genetic diversity of rotavirus strains prior to vaccine introduction and generates baseline data for future monitoring of any changes in rotavirus strains in response to vaccine pressure.

SUBMITTER: Joao ED

PROVIDER: S-EPMC5756281 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:BackgroundGroup A rotavirus (RVA) is a common causative agent of acute gastroenteritis in infants and young children worldwide. RVA P genotypes, determined by VP4 sequences, have been confirmed to infect humans and animals. However, their codon usage patterns that are essential to obtain insights into the viral evolution, host adaptability, and genetic characterization remained unclear, especially across animal hosts.ResultsWe performed a comprehensive codon usage analysis of eight host-specific RVA P genotypes, including human RVA (P[4] and P[8]), porcine RVA (P[13] and P[23]), and zoonotic RVA (P[1], P[6], P[7] and P[19]), based on 233 VP4 complete coding sequences. Nucleotide composition, relative synonymous codon usage (RSCU), and effective number of codons (ENC) were calculated. Principal component analysis (PCA) based on RSCU values was used to explore the codon usage patterns of different RVA P genotypes. In addition, mutation pressure and natural selection were identified by using ENC-plot, parity rule 2 plot, and neutrality plot analyses. All VP4 sequences preferred using A/U nucleotides (A: 0.354-0.377, U: 0.267-0.314) than G/C nucleotides across genotypes. Similarly, majority of commonly used synonymous codons were likely to end with A/U nucleotides (A: 9/18-12/18, U: 6/18-9/18). In PCA, human, porcine, and zoonotic genotypes clustered separately in terms of RSCU values, indicating the host-specific codon usage patterns; however, porcine and zoonotic genotypes were partly overlapped. Human genotypes, P[4] and P[8], had stronger codon usage bias, as indicated by more over-represented codons and lower ENC, compared to porcine and zoonotic genotypes. Moreover, natural selection was determined to be a predominant driver in shaping the codon usage bias across the eight P genotypes. In addition, mutation pressure contributed to the codon usage bias of human genotypes.ConclusionsOur study identified a strong codon usage bias of human RVA P genotypes attributable to both natural selection and mutation pressure, whereas similar codon usage bias between porcine and zoonotic genotypes predominantly attributable to natural selection. It further suggests possible cross-species transmission. Therefore, it warrants further surveillance of RVA P genotypes for early identification of zoonotic infection.

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Rotavirus A strains obtained from children with acute gastroenteritis in Mozambique, 2012-2013: G and P genotypes and phylogenetic analysis of VP7 and partial VP4 genes.

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