Characterisation of rotavirus strains responsible for breakthrough diarrhoeal disease among Zambian children using whole genome sequencing

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dc.contributor.advisorDe Beer, Corenaen_ZA
dc.contributor.authorMwape, Innocenten_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.en_ZA
dc.date.accessioned2024-01-30T02:43:18Zen_ZA
dc.date.accessioned2024-04-26T23:50:31Zen_ZA
dc.date.available2024-01-30T02:43:18Zen_ZA
dc.date.available2024-04-26T23:50:31Zen_ZA
dc.date.issued2024-01en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2024.en_ZA
dc.description.abstractENGLISH ABSTRACT: Background Genetically altered viruses or variants have the potential to increase their virulence, pathogenicity, transmission, and ability to evade both natural and vaccine-induced immune responses leading to diarrhoeal disease in under five-year-old children who have received all recommended doses of Rotavirus (RV) vaccines, also known as breakthrough infections. Studies characterising RV strains responsible for breakthrough infections are rare in resource-limited countries like Zambia where RV-associated diarrhoeal disease is endemic. We aimed to characterise RV strains detected in fully vaccinated under five-year-old children residing in Zambia using next generation sequencing. Methods This was a case study nested under an open label randomised controlled RV vaccine clinical trial that evaluated safety and immune boosting effects of a third dose of Rotarix compared to a two-dose schedule. The Rotaclone kit was used to screen for RV in stool. We performed VP7 and VP4 genotyping on RV positive stool using Sanger sequencing. Whole genome sequencing was done on the Illumina Miseq platform. Genome assembly was done using Geneious software and multiple sequence alignment using Muscle in MEGA version 6. Results A total of 76 diarrhoeal stool specimens were collected and screened for RV of which 4/76 (5.2%) were positive. Genotypes of three of the four cases were identified as G1P[4], G12P[4] and G12P[8] using Sanger sequencing. Whole genome analysis revealed that the RVA/Human-wt/ZMB/CIDRZRV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and RVA/Human-wt/ZMB/CIDRZ-RV2106/ 2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 strains were mostly DS-1-like with mono and multiple reassortant respectively, whilst the RVA/Human-wt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1- M1-A1-N1-T1-E1-H1 was a typical Wa-like strain. Comparison of VP7 antigenic epitope of strains causing breakthrough infections and Rotarix vaccine strains revealed several amino acid differences like G96P and M217E. Comparison of P[4] strains with VP4 of the Rotarix vaccine strain demonstrated two amino acids differences (P114Q and V115T) were P114Q is an immune escape mutation. Discussion and Conclusion Differences observed in amino acids in antigenic epitope suggested its role in the immune evasion of neutralising antibodies elicited by the vaccine. Findings from this study have potential to inform national RV vaccination strategies and the design of highly efficacious universal RV vaccines. Furthermore, there might be need to monitor strains that have escaped vaccine-induced immunity to prevent diarrheal diseases in children under five years of age.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Agtergrond Geneties veranderde virusse of variante het die potensiaal om hul virulensie, patogenisiteit, oordrag en vermoë te verhoog om beide natuurlike infeksie en entstof-geïnduseerde immuunreaksies te ontduik wat lei tot diarree by kinders onder vyf jaar oud wat al die aanbevole dosisse Rotavirus (RV) entstowwe ontvang het, ook bekend as deurbraakinfeksies. Studies wat RV-stamme kenmerk wat vir deurbraakinfeksies verantwoordelik is, is skaars in hulpbronbeperkte lande soos Zambië waar RV-geassosieerde diarreesiekte endemies is. Ons het ten doel gehad om RV-stamme wat opgespoor is in volledig ingeënte kinders onder vyf jaar in Zambië woonagtig is, te karakteriseer met behulp van volgende generasie volgordebepaling. Metodes Hierdie studie is deel van 'n oop-etiket gerandomiseerde kliniese proef oor RV-entstof wat die veiligheid en immuunversterkende effekte van 'n derde dosis Rotarix in vergelyking met 'n tweedosis skedule geëvalueer het. Die Rotaclon-stel is gebruik om stoelgang te sondersoek vir die teenwoordigheid van RV. Ons het VP7 en VP4 genotipering op RV positiewe stoelgang monsters uitgevoer deur gebruik te maak van Sanger volgordebepaling. Heelgenoomvolgordebepaling is op die Illumina Miseq-platform gedoen. Genoomsamestelling is gedoen met behulp van Geneious sagteware en meervoudige volgorde-belyning met behulp van Muscle in MEGA weergawe 6. Resultate 'n Totaal van 76 diarree-stoelgangmonsters is versamel en getoets vir RV waarvan 4/76 (5.2%) positief was. Genotipes van drie van die vier gevalle is geïdentifiseer as G1P[4], G12P[4] en G12P[8] deur gebruik te maak van Sanger-volgordebepaling. Heelgenoomanalise het aan die lig gebring dat die RVA/Human-wt/ZMB/CIDRZ-RV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 en RVA/Human-wt/ZMB/CIDRZ-RV2106/2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 meestal DS-1- agtige stamme was met onderskeidelik mono- en veelvuldige reassortante, terwyl die RVA/Humanwt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 'n tipiese Wa-agtige stam was. Vergelyking van VP7-antigeniese epitoop van stamme wat deurbraakinfeksies veroorsaak, en Rotarix-entstofstamme het verskeie aminosuurverskille soos G96P en M217E aan die lig gebring. Vergelyking van P[4]-stamme met VP4 van die Rotarix-entstofstam het twee aminosureverskille (P114Q en V115T) gedemonstreer, waar P114Q 'n immuunontsnappingsmutasie is. https://scholar.sun.ac.za vi Bespreking en Slot Verskille waargeneem in aminosure in antigeniese epitoop het die rol daarvan voorgestel in die immuunontduiking van neutraliserende teenliggaampies wat deur die entstof ontlok word. Bevindinge van hierdie studie het potensiaal om nasionale RV-inentingstrategieë en die ontwerp van hoogs doeltreffende universele RV-entstowwe in te lig. Verder kan dit nodig wees om stamme te monitor wat entstofgeïnduseerde immuniteit vrygespring het om diarreesiektes by kinders jonger as vyf jaar te voorkom.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxvii, 54 pagesen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130609
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshRotavirus infections -- Zambiaen_ZA
dc.subject.lcshDiarrhea in children -- Zambiaen_ZA
dc.subject.lcshWhole genome sequencing -- Zambiaen_ZA
dc.subject.lcshNucleotide sequence -- Zambiaen_ZA
dc.titleCharacterisation of rotavirus strains responsible for breakthrough diarrhoeal disease among Zambian children using whole genome sequencingen_ZA
dc.typeThesisen_ZA
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