Whole-Genome Analyses Identifies Multiple Reassortant Rotavirus Strains in Rwanda Post-Vaccine Introduction.

Children in low-and middle-income countries, including Rwanda, experience a greater burden of rotavirus disease relative to developed countries. Evolutionary mechanisms leading to multiple reassortant rotavirus strains have been documented over time which influence the diversity and evolutionary dynamics of novel rotaviruses. Comprehensive rotavirus whole-genome analysis was conducted on 158 rotavirus group A (RVA) samples collected pre- and post-vaccine introduction in children less than five years in Rwanda. Of these RVA positive samples, five strains with the genotype constellations G4P[4]-I1-R2-C2-M2-A2-N2-T1-E1-H2 (n = 1), G9P[4]-I1-R2-C2-M2-A1-N1-T1-E1-H1 (n = 1), G12P[8]-I1-R2-C2-M1-A1-N2-T1-E2-H3 (n = 2) and G12P[8]-I1-R1-C1-M1-A2-N2-T2-E1-H1 (n = 1), with double and triple gene reassortant rotavirus strains were identified. Phylogenetic analysis revealed a close relationship between the Rwandan strains and cognate human RVA strains as well as the RotaTeq® vaccine strains in the VP1, VP2, NSP2, NSP4 and NSP5 gene segments. Pairwise analyses revealed multiple differences in amino acid residues of the VP7 and VP4 antigenic regions of the RotaTeq® vaccine strain and representative Rwandan study strains. Although the impact of such amino acid changes on the effectiveness of rotavirus vaccines has not been fully explored, this analysis underlines the potential of rotavirus whole-genome analysis by enhancing knowledge and understanding of intergenogroup reassortant strains circulating in Rwanda post vaccine introduction.

Whole genome and in-silico analyses of G1P[8] rotavirus strains from pre- and post-vaccination periods in Rwanda.

Rwanda was the first low-income African country to introduce RotaTeq vaccine into its Expanded Programme on Immunization in May 2012. To gain insights into the overall genetic make-up and evolution of Rwandan G1P[8] strains pre- and post-vaccine introduction, rotavirus positive fecal samples collected between 2011 and 2016 from children under the age of 5 years as part of ongoing surveillance were genotyped with conventional RT-PCR based methods and whole genome sequenced using the Illumina MiSeq platform. From a pool of samples sequenced (n = 158), 36 were identified as G1P[8] strains (10 pre-vaccine and 26 post-vaccine), of which 35 exhibited a typical Wa-like genome constellation. However, one post vaccine strain, RVA/Human-wt/RWA/UFS-NGS:MRC-DPRU442/2012/G1P[8], exhibited a RotaTeq vaccine strain constellation of G1-P[8]-I2-R2-C2-M2-A3-N2-T6-E2-H3, with most of the gene segments having a close relationship with a vaccine derived reassortant strain, previously reported in USA in 2010 and Australia in 2012. The study strains segregated into two lineages, each containing a paraphyletic pre- and post-vaccine introduction sub-lineages. In addition, the study strains demonstrated close relationship amongst each other when compared with globally selected group A rotavirus (RVA) G1P[8] reference strains. For VP7 neutralization epitopes, amino acid substitutions observed at positions T91A/V, S195D and M217T in relation to the RotaTeq vaccine were radical in nature and resulted in a change in polarity from a polar to non-polar molecule, while for the VP4, amino acid differences at position D195G was radical in nature and resulted in a change in polarity from a polar to non-polar molecule. The polarity change at position T91A/V of the neutralizing antigens might play a role in generating vaccine-escape mutants, while substitutions at positions S195D and M217T may be due to natural fluctuation of the RVA. Surveillance of RVA at whole genome level will enhance further assessment of vaccine impact on circulating strains, the frequency of reassortment events under natural conditions and epidemiological fitness generated by such events.

Uncovering the First Atypical DS-1-like G1P[8] Rotavirus Strains That Circulated during Pre-Rotavirus Vaccine Introduction Era in South Africa.

Emergence of DS-1-like G1P[8] group A rotavirus (RVA) strains during post-rotavirus vaccination period has recently been reported in several countries. This study demonstrates, for the first time, rare atypical DS-1-like G1P[8] RVA strains that circulated in 2008 during pre-vaccine era in South Africa. Rotavirus positive samples were subjected to whole-genome sequencing. Two G1P[8] strains (RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1971/2008/G1P[8] and RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1973/2008/G1P[8]) possessed a DS-1-like genome constellation background (I2-R2-C2-M2-A2-N2-T2-E2-H2). The outer VP4 and VP7 capsid genes of the two South African G1P[8] strains had the highest nucleotide (amino acid) nt (aa) identities of 99.6-99.9% (99.1-100%) with the VP4 and the VP7 genes of a locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU1039/2008/G1P[8]. All the internal backbone genes (VP1-VP3, VP6, and NSP1-NSP5) had the highest nt (aa) identities with cognate internal genes of another locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU2344/2008/G2P[6]. The two study strains emerged through reassortment mechanism involving locally circulating South African strains, as they were distinctly unrelated to other reported atypical G1P[8] strains. The identification of these G1P[8] double-gene reassortants during the pre-vaccination period strongly supports natural RVA evolutionary mechanisms of the RVA genome. There is a need to maintain long-term whole-genome surveillance to monitor such atypical strains.