Medical Microbiology
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Browsing Medical Microbiology by Advisor "Nyaga, Martin"
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Item Open Access Whole genome analysis of rare and/or novel rotavirus strains post-Rotarix® introduction in Zambia(University of the Free State, 2021-06) Maringa, Wairimu Makena; Nyaga, MartinBackground: 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.Item Open Access Whole-genome analyses of rotavirus strains circulating pre- and post- Rotateq™ vaccine introduction in Rwanda(University of the Free State, 2020) Rasebotsa, Sebotsana Paula; Nyaga, Martin; Sabiu, Saheed𝑬𝒏𝒈𝒍𝒊𝒔𝒉 Children living in developing countries are constantly faced with the burden of diarrheal infections that account for over 1.6 million death cases globally. Rotavirus group A (RVA) has been identified as one of the viruses implicated in most viral-induced diarrhoeal infections in children less than five years worldwide. In Rwanda, over 3500 RVA related mortality cases were reported yearly prior to the implementation of the RotaTeq® vaccine in 2012 to overcome this burden, which led to a significant decrease in rotavirus infections. Africa has a huge diversity of rotavirus strains compared to other developed continents especially Europe and North America, thus requiring a deeper understanding of this phenomenon. This study aimed at characterizing all the 11-segments of RVA strains circulating in Rwanda pre- and post-vaccine introduction as part of the World Health Organization (WHO) supported African rotavirus pilot surveillance program. The study was based on 158 rotavirus positive samples that were collected from children presenting symptoms associated with rotavirus infection between 2011 and 2016. The rotavirus double-stranded ribonucleic acid (dsRNA) was extracted from the viral particles and converted into complementary deoxyribonucleic acid (cDNA) prior to library preparation for whole-genome sequencing with an Illumina MiSeq platform. Several bioinformatics tools were utilized to construct phylogenetic trees and the proteins structures. From the sequenced samples, 36 samples were identified as G1P[8] strains, and five samples were reassortant strains. Ten G1P[8] strains were identified pre-vaccine introduction while 26 were identified post-vaccine introduction. Thirty-five of the G1P[8] strains expressed pure Wa-like genome constellations, while one of the strains that was identified in 2012 exhibited a genome constellation typical of a RotaTeq® vaccine strain. On the other hand, the five reassortant strains were identified post-vaccine introduction between 2013-2015. Whole-genome analysis revealed that the G4P[4], G9P[4] and one G12P[8] reassortant strains exhibited both the Wa-like and the DS-1-like genome constellations while two G12P[8] strains had all the three genogroup constellations. Furthermore, the phylogenetic analysis of most of the G1P[8] strains revealed that they segregated according to their vaccination status; strains identified pre-vaccine introduction clustered together while post-vaccine strains also formed a separate cluster. The five reassortant strains were closely related to human RVA strains in all the gene segments and RotaTeq® vaccine strains in the VP1, VP2, NSP2, NSP4, and NSP5 gene segments. Analysis of the neutralization epitopes and cytotoxic T-lymphocytes (CTL) of the G1P[8] strains revealed multiple amino acid substitutions, with some changes influencing the change in polarity thus deemed to be radical in nature. A similar trend was also observed in the reassortant strains, with 27 amino acid substitutions in the VP7 epitope region and only three substitutions in the VP4 epitope region. Changes observed in these epitope regions have the potential of generating vaccine-escape mutants that may undermine the effectiveness of the rotavirus vaccine with time. Whole-genome sequencing has proven to provide information that could have been missed when looking only at the outer capsid proteins. It is thus important to continue conducting rotavirus whole-genome studies to unpack the hidden information behind the huge diversity of rotavirus strains in African countries such as Rwanda. ___________________________________________________________________