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G3 rotaviruses rank among the most common rotavirus strains worldwide in humans and animals. However, despite a robust long-term rotavirus surveillance system from 1997 at Queen Elizabeth Central Hospital in Blantyre, Malawi, these strains were only detected from 1997 to 1999 and then disappeared and re-emerged in 2017, five years after the introduction of the Rotarix rotavirus vaccine. Here we analysed representative 27 whole genome sequences (G3P[4], n=20; G3P[6], n=1; and G3P[8], n=6) randomly selected each month between November 2017 and August 2019 to understand how G3 strains re-emerged in Malawi. We found four genotype constellations that were associated with the emergent G3 strains and co-circulated in Malawi post-Rotarix vaccine introduction: G3P[4] and G3P[6] strains with the DS-1-like genetic backbone genes (G3-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) and G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2), G3P[8] strains with the Wa-like genetic backbone genes (G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1), and reassortant G3P[4] strains consisting of the DS-1-like genetic backbone genes and a Wa-like NSP2 (N1) gene (G3-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2). Time-resolved phylogenetic trees demonstrated that the most recent common ancestor for each RNA segment of the emergent G3 strains was between 1996 and 2012, possibly through introductions from outside the country due to the limited genetic similarity with G3 strains which circulated before their disappearance in the late 1990s. Further genomic analysis revealed that the reassortant DS-1-like G3P[4] strains acquired a Wa-like NSP2 genome segment (N1 genotype) through intergenogroup reassortment; an artiodactyl-like VP3 through intergenogroup interspecies reassortment; and VP6, NSP1 and NSP4 segments through intragenogroup reassortment likely before importation into Malawi. Additionally, the emergent G3 strains contain amino acid substitutions within the antigenic regions of the VP4 proteins which could potentially impact the binding of rotavirus vaccine-induced antibodies. Altogether, our findings show that multiple strains with either Wa-like or DS-1-like genotype constellations have driven the re-emergence of G3 strains. The findings also highlight the role of human mobility and genome reassortment events in the cross-border dissemination and evolution of rotavirus strains in Malawi necessitating the need for long-term genomic surveillance of rotavirus in high disease burden settings to inform disease prevention and control.

Strong CD4+ T cell-mediated immune protection following rotavirus infection has been observed in animal models, but its relevance in humans remains unclear. Here, we characterized acute and convalescent CD4+ T cell responses in Rotarix®-vaccinated children who were hospitalized with rotavirus-positive and rotavirus-negative diarrhoea in Blantyre, Malawi. Children presenting with laboratory-confirmed rotavirus infection had higher proportions of effector and central memory T helper 2 cells during acute infection i.e., at disease presentation compared to convalescence, 28 days post-infection defined by a follow-up 28 days after acute infection. However, circulating cytokine-producing (IFN-γ and/or TNF-α) rotavirus-specific VP6-specific CD4+ T cells were rarely detectable in children with rotavirus infection at both acute and convalescent stages. Moreover, following whole blood mitogenic stimulation, the responding CD4+ T cells were predominantly non-cytokine producers of IFN-γ and/or TNF-α. Our findings demonstrate limited induction of anti-viral IFN-γ and/or TNF-α-producing CD4+ T cells in rotavirus-vaccinated Malawian children following the development of laboratory-confirmed rotavirus infection.

Genotype G3 rotaviruses rank among the most common rotavirus strains worldwide in humans and animals. However, despite a robust long-term rotavirus surveillance system from 1997 in Blantyre, Malawi, these strains were only detected from 1997 to 1999 and then disappeared and re-emerged in 2017, five years after the introduction of the Rotarix rotavirus vaccine. Here we analysed 27 whole genome sequences to understand how G3 strains re-emerged in Malawi. We randomly selected samples each month between November 2017 and August 2019 from stool samples of children hospitalised with acute diarrhoea at the Queen Elizabeth Hospital in Blantyre, Malawi. We found three genotypes namely G3P[4] (n=20), G3P[6] (n=1) and G3P[8] (n=6) associated with the re-emergence of G3 strains in Malawi post-Rotarix vaccine introduction. The identified genotypes co-circulated at different time points and were associated with three typical human G3 strains consisting of either a Wa-like or DS-1-like genetic constellation and reassortant strains possessing Wa-like and DS-1-like genetic backbones. Time-resolved phylogenetic trees demonstrated that the most recent common ancestor for each segment of the re-emerged G3 strains emerged between 1996 and 2012, possibly through introductions from outside the country due to the limited genetic similarity with G3 strains which circulated before their disappearance in the late 1990s. Further genomic analysis revealed that the reassortant DS-1-like G3P[4] strains acquired a Wa-like NSP2 genome segment (N1 genotype) through intergenogroup reassortment; an artiodactyl-like VP3 through intragenogroup interspecies reassortment; and VP6, NSP1 and NSP4 segments through intragenogroup reassortment likely before importation into Malawi. Additionally, the re-emerged G3 strains contain amino acid substitutions within the antigenic regions of the VP4 proteins which could potentially impact the binding of rotavirus vaccine-induced antibodies. Altogether, our findings shows that multiple rather than a single genotype have driven the re-emergence of G3 strains likely from other countries highlighting the role of human mobility and genome reassortment events in the dissemination and evolution of rotavirus strains in Malawi necessitating the need for long-term genomic surveillance of rotavirus in high disease burden settings to inform disease prevention and control.

Background Rotavirus vaccine (Rotarix [RV1]) has reduced diarrhea-associated hospitalizations and deaths in Malawi. We examined the trends in circulating rotavirus genotypes in Malawi over a 22-year period to assess the impact of RV1 introduction on strain distribution. Methods Data on rotavirus-positive stool specimens among children aged Results A rich rotavirus strain diversity circulated throughout the 22-year period; Shannon (H′) and Simpson diversity (D′) indices did not differ between the pre- and postvaccine periods (H′ P < .149; D′ P < .287). Overall, G1 (n = 268/924 [28.7%]), G2 (n = 308/924 [33.0%]), G3 (n = 72/924 [7.7%]), and G12 (n = 109/924 [11.8%]) were the most prevalent genotypes identified following RV1 introduction. The prevalence of G1P[8] and G2P[4] genotypes declined each successive year following RV1 introduction, and were not detected after 2018. Genotype G3 reemerged and became the predominant genotype from 2017 onward. No evidence of genotype selection was observed 7 years post–RV1 introduction. Conclusions Rotavirus strain diversity and genotype variation in Malawi are likely driven by natural mechanisms rather than vaccine pressure.

BackgroundRotavirus is the major cause of severe gastroenteritis in children aged MethodsStool samples were collected from children hospitalised with acute gastroenteritis from December 2011 – October 2019. Gastroenteritis severity was determined using Ruuska and Vesikari scores. Rotavirus was detected in stool using Enzyme Immunoassay. Rotavirus genotypes were determined using nested RT-PCR. Associations between RV1 vaccination and gastroenteritis severity were investigated using adjusted linear regression.ResultsIn total, 3,159 children were recruited. After adjusting for Mid-Upper Arm Circumference, age, weight, gender and receipt of other vaccines, all-cause gastroenteritis severity scores were 2.21 units lower (95% CI 1.85, 2.56; pn=2,224) compared to RV1-unvaccinated children (n=935); the decrease was comparable between rotavirus-positive and rotavirus-negative cases in all age groups. The reduction in severity score was observed against every rotavirus genotype, although the magnitude was smaller among those infected with G12P[6] compared to the remaining genotypes (p=0.011). Other than RV1 vaccination, age was the only variable associated with gastroenteritis severity. Each one-year increment in age was associated with a decrease of 0.43 severity score (95% CI 0.26, 0.60; pConclusionOur findings provide additional evidence of RV1 impact in a high disease burden, low-income country, lending further support to Malawi’s rotavirus vaccine programme.SummaryIn a long-term hospital-based surveillance study in Malawi, we found evidence of the reduction in gastroenteritis severity among hospitalised RV1-vaccinated children infected with both homotypic and heterotypic rotavirus strains and off-target RV1 vaccine effects against non-rotavirus diarrhoeal severity.

Increasing antimicrobial resistance and limited alternative treatments have led to fluoroquinolone-resistant Shigella strain inclusion on the WHO global priority pathogens list. In this study we characterized multiple Shigella isolates from Malawi with whole genome sequence analysis, identifying the acquirable fluoroquinolone resistance determinant qnrS1.

Identifying risk factors for impaired oral rotavirus vaccine (ORV) efficacy in low-income countries may lead to improvements in vaccine design and delivery. We measured maternal rotavirus antibodies, environmental enteric dysfunction (EED), and bacterial gut microbiota development among infants receiving two doses of Rotarix in India (n = 307), Malawi (n = 119), and the UK (n = 60), using standardised methods across cohorts. ORV shedding and seroconversion rates were significantly lower in Malawi and India than the UK. Maternal rotavirus-specific antibodies in serum and breastmilk were negatively correlated with ORV response in India and Malawi, and this was mediated partly by a reduction in ORV replication. In the UK, ORV replication was not inhibited despite comparable maternal antibody levels. In both India and Malawi, pre-vaccination microbiota diversity was negatively correlated with ORV immunogenicity, suggesting that high early-life microbial exposure may contribute to impaired vaccine efficacy.