Rotavirus genotypes in children under five years hospitalized with diarrhea in low and middle-income countries: Results from the WHO-coordinated Global Rotavirus Surveillance Network.

Rotavirus is the most common pathogen causing pediatric diarrhea and an important cause of morbidity and mortality in low- and middle-income countries. Previous evidence suggests that the introduction of rotavirus vaccines in national immunization schedules resulted in dramatic declines in disease burden but may also be changing the rotavirus genetic landscape and driving the emergence of new genotypes. We report genotype data of more than 16,000 rotavirus isolates from 40 countries participating in the Global Rotavirus Surveillance Network. Data from a convenience sample of children under five years of age hospitalized with acute watery diarrhea who tested positive for rotavirus were included. Country results were weighted by their estimated rotavirus disease burden to estimate regional genotype distributions. Globally, the most frequent genotypes identified after weighting were G1P[8] (31%), G1P[6] (8%) and G3P[8] (8%). Genotypes varied across WHO Regions and between countries that had and had not introduced rotavirus vaccine. G1P[8] was less frequent among African (36 vs 20%) and European (33 vs 8%) countries that had introduced rotavirus vaccines as compared to countries that had not introduced. Our results describe differences in the distribution of the most common rotavirus genotypes in children with diarrhea in low- and middle-income countries. G1P[8] was less frequent in countries that had introduced the rotavirus vaccine while different strains are emerging or re-emerging in different regions.

Genomic Analysis of G2P[4] Group A Rotaviruses in Zambia Reveals Positive Selection in Amino Acid Site 7 of Viral Protein 3.

The G2P[4] genotype is among the rotavirus strains that circulate commonly in humans. Several countries have reported its immediate upsurge after the introduction of rotavirus vaccination, raising concern about sub-optimal vaccine effectiveness against this genotype in the long term. This study aimed to gain insight into the evolution of post-vaccine Zambian G2P[4] group A rotavirus (RVA) strains and their overall genetic make-up by analysis of sequence alignments at the amino acid (AA) level. Twenty-nine Zambian G2P[4] rotavirus strains were subjected to whole-genome sequencing using the Illumina MiSeq® platform. All the strains exhibited the typical DS-1-like genotype constellation, and the nucleotide sequences of the 11 genome segments showed high nucleotide similarities (>97%). Phylogenetic analyses together with representative global G2P[4] RVA showed that Zambian strains clustered into human lineages IV (for VP2, VP4, VP7, NSP1, and NSP5), V (for VP1, VP3, VP6, NSP2, and NSP3), and XXIII (for NSP4). The AA differences between the lineages where the study strains clustered and lineages of global reference strains were identified and analyzed. Selection pressure analysis revealed that AA site seven in the Viral Protein 3 (VP3) genome segment was under positive selection. This site occurs in the region of intrinsic disorder in the VP3 protein, and Zambian G2P[4] strains could potentially be utilizing this intrinsically disordered region to survive immune pressure. The Zambian G2P[4] strains from 2012 to 2016 comprised the G2P[4] strains that have been circulating globally since the early 2000s, highlighting the epidemiological fitness of these contemporary G2P[4] strains. Continuous whole-genome surveillance of G2P[4] strains remains imperative to understand their evolution during the post-vaccination period.

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.

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

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.