Whole genome analyses of G1P[8] rotavirus strains from vaccinated and non-vaccinated South African children presenting with diarrhea

Abstract

Group A rotaviruses (RVAs) are the leading cause of severe gastroenteritis and eventually death among infants and young children worldwide, and disease prevention and management through vaccination is a public health priority. In August 2009, Rotarix™ was introduced in the South African Expanded Programme on Immunisation. As a result, substantial reductions in RVA disease burden have been reported among children younger than 5 years old. Rotavirus strain surveillance post-vaccination is crucial to, inter alia, monitor and study the evolution of vaccine escape strains. Here, full-genome sequence data for the 11 gene segments from 11 South African G1P[8] rotavirus strains were generated, including 5 strains collected from non-vaccinated children during the 2004–2009 rotavirus seasons and 6 strains collected from vaccinated children during the 2010 rotavirus season. These data were analyzed to gain insights into the overall genetic makeup and evolution of South African G1P[8] rotavirus strains and to compare their genetic backbones with those of common human Wa-like RVAs from other countries, as well as with the Rotarix™ and RotaTeq™ G1P[8] vaccine components. All 11 South African G1P[8] strains revealed a complete Wa-like genotype constellation of G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. On the basis of sequence similarities, the South African G1P[8] strains (with the exception of strain RVA/Human-wt/ZAF/1262/2004/G1P[8]) were closely related to each other (96–100% identity in all gene segments). Comparison to the Rotarix™ and RotaTeq™ G1P[8] vaccine components revealed a moderate nucleotide identity of 89–96% and 93–95%, respectively. The results indicated that none of the gene segments of these 11 South African G1P[8] strains were vaccine-derived. This study illustrates that large-scale next generation sequencing will provide crucial information on the influence of the vaccination program on evolution of rotavirus strains. This is the first report to describe full genomic analyses of G1P[8] RVA strains collected from both non-vaccinated and vaccinated children in South Africa.

Whole genome detection of rotavirus mixed infections in human, porcine and bovine samples co-infected with various rotavirus strains collected from sub-Saharan Africa

“…The dsRNA genomes were extracted following previously described methods (Jere et al, 2011; 2012; Nyaga et al, 2013; Potgieter et al, 2009), and RNA sequencing performed as described previously (Magagula et al, 2015; Nyaga et al, 2014). Briefly, 11 one-step RT-PCR reactions (QIAGEN OneStep RT-PCR Kit, QIAGEN, Hilden, Germany) were performed to amplify each full-length genome segment using segment-specific primers as described elsewhere (Magagula et al, 2015; Martinez et al, 2014; Nyaga et al, 2014).…”

“…Briefly, 11 one-step RT-PCR reactions (QIAGEN OneStep RT-PCR Kit, QIAGEN, Hilden, Germany) were performed to amplify each full-length genome segment using segment-specific primers as described elsewhere (Magagula et al, 2015; Martinez et al, 2014; Nyaga et al, 2014). The products were quantitated using a SYBR green dsDNA detection assay (SYBR Green I Nucleic Acid Gel Stain, Thermo Fisher Scientific, Waltham, MA, USA), and all 11 RT-PCR products for each genome were pooled in equimolar amounts.…”

“…The first study reported the sequence of RVA/Human-wt/ZAF/2371WC/2008/G9P[8], which revealed co-infection by at least four different strains (Jere et al, 2011), and the second study sequenced the strain RVA/Simian-tc/ZAF/SA11-x/1958/G3P[2], where x represents two distinct sequences for genome segment 8 (NSP2), corresponding to genotypes N2 and N5 (Mlera et al, 2013). In addition, the number of RV whole genomes reported from Africa still remains relatively low compared to whole genomes from other parts of the world (Dennis et al, 2014; Heylen et al, 2014; Jere et al, 2011, 2012, 2014; Magagula et al, 2015; Nakagomi et al, 2013; Nyaga et al, 2013, 2014). Such data would aid our understanding of viral factors that could possibly assist in understanding the lower immunogenicity and efficacy of RV vaccines observed in developing countries (Armah et al, 2010; Madhi et al, 2010; Nakagomi et al, 2012; Sow et al, 2012; Steele et al, 2012).…”

Multiplexed one-step RT-PCR VP7 and VP4 genotyping assays for rotaviruses using updated primers

“…The described VP7 and VP4 typing assays have been shown to consistently and correctly genotype RVA strains belonging in published lineages and sub-lineages of the six common G- and five common P-genotypes (Esona et al, 2013; Esteban et al, 2010; Le et al, 2011; Magagula et al, 2014; Martella et al, 2011; Mascarenhas et al, 2010; Nyaga et al, 2014; Stupka et al, 2009, 2012). The advantages of these two gel-based multiplexed one-step genotyping RT-PCR protocols are: (1) they do not require specialized equipment; (2) laboratory personnel need only basic skills to follow the protocols; (3) improved differentiation of samples with mixed genotypes; (4) this approach involves less handling of samples, is less labor-intensive, and less prone to sample cross-contamination; and (5) other commercially available one-step RT-PCR kits such as the MyTaq™ one-step RT-PCR kit (Bioline) and the SuperScript ® III one-step RT-PCR System with Platinum ® Taq High Fidelity DNA Polymerase (Life Technologies) can be used in place of the Qiagen one-step RT-PCR kit (Qiagen).…”

“…Four hundred of these sequences were representatives of known lineages and sub-lineages of the five common VP4 genotypes (Donato et al, 2012; Magagula et al, 2014; Nakagomi et al, 2012; Nyaga et al, 2014), while 1400 were representatives of known lineages and sub-lineages of the six common VP7 genotypes (Donato et al, 2012; Esona et al, 2013; Magagula et al, 2014; Nakagomi et al, 2012; Nyaga et al, 2014; Stupka et al, 2009, 2012). The consensus sequences obtained from multiple alignments of the six most common RVA VP7 genotypes (G1, G2, G3, G4, G9 and G12) and five most common VP4 genotypes (P[4], P[6], P[8], P[9] and P[10]) were used to design oligonucleotide primers for specificVP7 and VP4 genotypes, respectively.…”