Abstract

The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P[8] VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006–2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers.

Highlights

  • Diarrhoea is one of the leading causes of childhood mortality and was responsible for nearly 500, 000 childhood deaths in 2015 [1]

  • Reverse transcription polymerase chain reaction genotyping methods used in rotavirus surveillance studies, have been useful in monitoring circulating strains and identifying rare and emerging strains worldwide

  • Genetic analysis of Ghanaian G1P[8] and G9P[8] rotavirus A strains primer and the yellow highlight indicates the region of variability of the primer binding site

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Summary

Introduction

Diarrhoea is one of the leading causes of childhood mortality and was responsible for nearly 500, 000 childhood deaths in 2015 [1]. Rotavirus A (RVA), a member of the genus Rotavirus and family Reoviridae [2], is the leading cause of viral diarrhoea in children under five years. Prior to the introduction of the monovalent rotavirus vaccine—Rotarix into Ghana’s Expanded Programme on Immunisation (EPI) in May 2012, rotaviruses killed about 2,090 children below five years of age accounting for 3.6% of annual diarrhoeal disease deaths in Ghana (https://path.azureedge.net/ media/documents/VAD_rotavirus_ghana_fs.pdf). Rotaviruses are non-enveloped viruses which possess a triple-layered capsid surrounding a genome of 11 segments of double-stranded RNA (dsRNA). The G12 genotype, first detected in the Philippines in 1987 [9], emerged as a significant cause of rotavirus diarrhoea in children and have since persisted worldwide [10,11,12,13,14,15,16,17,18,19]

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