Whole genome analyses of African G2, G8, G9, and G12 rotavirus strains using sequence‐independent amplification and 454® pyrosequencing

Abstract

High mortality rates caused by rotaviruses are associated with several strains such as G2, G8, G9, and G12 rotaviruses. Rotaviruses with G9 and G12 genotypes emerged worldwide in the past two decades. G2 and G8 rotaviruses are however also characterized frequently across Africa. To understand the genetic constellation of African G2, G8, G9, and G12 rotavirus strains and their possible origin, sequence-independent cDNA synthesis, amplification, and 454(®) pyrosequencing of the whole genomes of five human African rotavirus strains were performed. RotaC and phylogenetic analysis were used to assign and confirm the genotypes of the strains. Strains RVA/Human-wt/MWI/1473/2001/G8P[4], RVA/Human-wt/ZAF/3203WC/2009/G2P[4], RVA/Human-wt/ZAF/3133WC/2009/G12P[4], RVA/Human-wt/ZAF/3176WC/2009/G12P[6], and RVA/Human-wt/ZAF/GR10924/1999/G9P[6] were assigned G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, G12-P[4]-I1-R1-C1-M1-A1-N1-T1-E1-H1, G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1, and G9-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotypes, respectively. The detection of both Wa- and DS-1-like genotypes in strain RVA/Human-wt/ZAF/3133WC/2009/G12P[4] and Wa-like, DS-1-like and P[6] genotypes in strain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] implies that these two strains were generated through intergenogroup genome reassortment. The close similarity of the genome segments of strain RVA/Human-wt/MWI/1473/2001/G8P[4] to artiodactyl-like, human-bovine reassortant strains and human rotavirus strains suggests that it originated from or shares a common origin with bovine strains. It is therefore possible that this strain might have emerged through interspecies genome reassortment between human and artiodactyl rotaviruses. This study illustrates the swift characterization of all the 11 rotavirus genome segments by using a single set of universal primers for cDNA synthesis followed by 454(®) pyrosequencing and RotaC analysis.

Rotavirus in diarrheal children in rural Burkina Faso: High prevalence of genotype G6P[6]

“…This could be explained by the fact that G and P types in this study were primarily assigned by sequencing all VP7 and VP4 genes from the RVA positive samples, and only with the nested multiplex PCR assay when sequencing PCR was not possible due to low viral load in the samples. As observed previously (Nordgren et al, 2012) the multiplex nested PCR assays could overestimate the amount of mixed infections due to mispriming, hence presence of mixed RVA infections could preferably be determined by deep sequencing (Jere et al, 2011a).…”

Diversity and Relationships of Cocirculating Modern Human Rotaviruses Revealed Using Large-Scale Comparative Genomics

“…Still, a major limitation of this study is in the design of primers to preferentially amplify genotype 1 genes during RT-PCR and sequencing via the established, semiautomated pipeline at the J. Craig Venter Institute. Future studies will rely on sequence-independent amplification and sequencing approaches to address the frequency of gene reassortment between GG-1 and GG-2 strains (22,46). Nonetheless, the fact that all 58 VUMC RVs were GG-1, with genotype 1 internal protein genes, afforded us the unique opportunity to analyze modern RV diversity at the subgenotype level.…”

Identification of porcine rotavirus-like genotype P[6] strains in Taiwanese children

“…However, the various lineages of P[6] strains associated with human infections show marked geographical differences. For example, epidemiologically major P[6] strains, including the abundant African P[6] strains carrying various G types as well as the globally spread G9 and G12 strains, mainly carry a single common P [6] lineage, Ia (Freeman et al, 2009;László et al, 2009;Pietsch & Liebert, 2009;Jere et al, 2011;Le et al, 2011). Based on epidemiological evidence, it is thought that these P[6] strains represent a true human rotavirus VP4 lineage.…”