Whole genome analysis of rare and/or novel rotavirus strains post-Rotarix® introduction in Zambia
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Date
2021-06
Authors
Maringa, Wairimu Makena
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
Background: Group A rotaviruses (RVA) cause acute diarrhoea in children under the age of five years. In sub-Saharan Africa, limited studies have been conducted on RVA at whole genome level, particularly post-vaccine implementation. Even though strain oscillation has been documented in Zambia since the countrywide rollout of Rotarix® vaccine in 2013, the approach primarily utilised conventional methods to characterise the outer capsid proteins (VP7 and VP4) of RVA strains. However, analysing the remaining genome-encoded proteins contributes to a better understanding of mechanisms driving genetic diversity in rotaviruses. This study undertook whole genome analysis of the rare and/or novel reassortant strains from Zambia during the post-vaccine era. Methods: Archived samples selected from a WHO-Regional Office for Africa surveillance project (n=133) were sent to the Next Generation Sequencing unit, University of the Free State (Bloemfontein, South Africa). The surveillance project aimed to characterise RVA at a whole genome level in Zambia. These samples had been conventionally genotyped at the WHO-Regional Reference Laboratory located in the Diarrhoeal Pathogens Research Unit at the Sefako Makgatho University (Pretoria, South Africa). The transfer was facilitated by a Material Transfer Agreement (MTA:NGS Unit, UFS(1)). Viral RNA was extracted from the samples, followed by cDNA synthesis and DNA library preparation. Whole genome sequencing was done on the Illumina® MiSeq to generate 300 bp x 2 paired end reads. FASTQ reads were obtained from the MiSeq, de novo assembled on Geneious® Prime and subjected to a quality trim before the genotype constellations were determined using the Virus Pathogen Database and Analysis Resource (ViPR). Strains from the post vaccine-period (2013-2016) that exhibited any form of atypical characteristics were selected for further investigation. The genotypes of the strains (n=5) were confirmed using BLAST, followed by pairwise alignments and bioinformatic analysis on various tools and software. Results: A rare reassortant porcine-like human strain (RVA/Human-wt/ZMB/UFS-NGS-MRC DPRU4724/2014/G5P[6]) with the constellation G5-P[6]-I1-R1-C1-M1-A8-N1-E1-H1 typically found in porcine strains was identified in a sample collected from a child with gastroenteritis who resided in Kasama, Zambia. All the genes of this strain were seen to cluster only among porcine and putative porcine- like human strains on a phylogenetic level. The sequence identities (95.7%-98.0%) were consistent with the phylogenetic relationships observed. Moreover, reassortment analysis demonstrated the genetic similarity between the Zambian G5P[6] strain and other porcine-like human strains, thus acknowledged that the strain may have arisen due to animal-human interactions. Furthermore, four reassortant strains were identified in samples taken from four children who resided in different areas of Ndola and Lusaka, Zambia. The children experienced moderate to severe gastroenteritis. Two strains had the constellation G1-P[8]-I1-R1-C1-M1-A1-N2-T1-E1-H1, while the other two strains had the constellations G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and G2-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2. One of the strains (RVA/Human-wt/ZMB/UFS-NGS-MRC-DPRU4749/2014/G2P[8]) was divergent from other global reference strains in the VP4 and VP1 encoding genes on both nucleotide and amino acid level. Moreover, several amino acid changes were observed in the antigenic sites of the VP4 of the divergent Zambian strain in relation to Rotarix® and global reference strains. Conclusion: Our findings add to the growing global evidence of strains generated through reassortment and/or zoonotic transmission. Further, current rotavirus vaccines do not contain genotypes such as the G5 and P[6], and such animal-like strains may have an impact on vaccines. These findings emphasise the need for continuous active surveillance and analysis of circulating RVA in Zambia at whole genome level.
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Keywords
Dissertation (M.Sc. (Medical Microbiology and Virology))--University of the Free State, 2021, Genome-encoded proteins, Rotavirus vaccines, Rotaviruses (RVA)