Molecular characterisation of HIV-1 recombinants and non-subtype C viruses in South Africa

Varathan, Olivette (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: HIV/AIDS is a severe health burden, affecting 36.9 million people worldwide by the end of 2017. South Africa has the largest HIV-1 epidemic in the world, estimated at 7.2 million infected individuals by the end of 2017. The HIV-1 epidemic in South Africa is dominated by HIV-1 subtype C, accounting for an estimated 98.2% of the infections in the country, based on the viral sequences in the Los Alamos National Library (LANL) database. To date, to the best of our knowledge, 22 papers have been published on non-subtype C viruses in South Africa. This study aimed to characterise two near full-length genome sequences of non-subtype C viruses in South Africa. The study samples were obtained through the diagnostic services of the National Health Laboratory Services (NHLS), performing routine drug resistance testing within the Division of Medical Virology, Stellenbosch University. All samples received were sequenced in the partial pol region (~1.4kb) of the viral genome by the NHLS. The possible subtype of the virus was identified from the sequences using online subtyping programmes. All sequences and samples that identified as possible non-subtype C viruses were recorded in a separate non-subtype C cohort. In 2011, a possible C, D recombinant virus was first identified in this cohort. By the end of 2015, 30 similar recombinant viruses were observed in the cohort, indicating a possible emergence of this recombinant strain. Two of the samples that identified as possible C, D recombinants were selected for further near full-length genome (NFLG) characterisation. Proviral DNA, from sample EC148, was extracted from PBMCs and viral RNA, from sample WC416, was extracted from plasma. The RNA was reverse transcribed to DNA via cDNA synthesis. Both sample viruses were amplified by PCR in two rounds. The first round targeted the amplification of the HIV-1 NFLG (8978bp) and the second targeted the amplification of two overlapping fragments (5455bp and 4909bp). Positive PCR amplicons were purified and sequenced. The generated sequences were read and analysed before being used in online subtyping programmes. The jumping profile hidden markov model (jpHMM), REGA and recombination identification programmes (RIP) were used to preliminary assign subtypes to both samples. Phylogenetic analyses was inferred to confirm / reject the online subtyping programme results. Online subtyping programmes identified the virus sequences of samples EC148 and WC416 as complex A, C, D recombinants. Phylogenetic analysis confirmed the online subtyping programme results for the sequence of sample WC416 in identifying it as a complex A, C, D recombinant. Phylogenetic analysis indicated that the sequence of sample EC148 is consistent with the results observed from the online subtyping programmes. Each HIV-1 sequence identified as a unique complex recombinant form as the breakpoints between the different subtypes differed. The emergence of new and unique non-subtype C recombinants in South Africa indicates that the epidemic is complex and evolving. It is therefore important to monitor the spread of different HIV subtypes circulating in South Africa.

AFRIKAANSE OPSOMMING: MIV / VIGS is 'n ernstige gesondheidsbelasting, wat teen die einde van 2017 wêreldwyd 36,9 miljoen mense affekteer. Suid-Afrika het die grootste MIV-1-epidemie ter wêreld, met 7,2 miljoen mense geafekteer teen die einde van 2017. Die MIV-1-epidemie in Suid-Afrika word oorheers deur MIV-1 subtipe C, wat verantwoordelik is vir 'n geskatte 98,2% van die infeksies in die land, gebaseer op die virusse in die Los Alamos Nasionale Biblioteek (LANL) databasis. Tot op datum is 22 referate gepubliseer oor nie-subtype C virusse in Suid-Afrika tot die beste van ons wete. Hierdie studie het ten doel om twee naby vollengte-genoomvolgorde van nie-subtype C virusse in SuidAfrika te karakteriseer. Die studiemonsters is verkry deur die diagnostiese dienste van die Nasionale Gesondheids Laboratoriumdienste (NHLS), wat roetine dwelm weerstand toetsing in die Afdeling Mediese Virologie, Universiteit Stellenbosch, uitvoer. Alle monsters wat ontvang is, se genoom volgorde van die gedeeltlike pol streek (~ 1.4kb) was bepaal deur die NHLS. Die moontlike subtipe van die virus is geïdentifiseer deur die volgorde te analiseer met behulp van aanlyn subtipieërings programme. Alle vologordes en monsters wat as moontlike nie-subtype C virusse geïdentifiseer is, is in 'n aparte C-kohort van nie-subtype aangeteken. In 2011 is 'n moontlike C, D rekombinante virus vir die eerste keer geïdentifiseer in hierdie kohort. Teen die einde van 2015 is 30 soort gelyke rekombinante virusse waargeneem in die kohort, wat 'n moontlike opkoms van hierdie rekombinante stam aandui. Twee van die monsters wat as moontlike C, D-rekombinante geïdentifiseer is, is gekies vir naby-vol lengte-genoom (NFLG) karakterisering. Provirale DNA, van monster EC148, is onttrek uit PBMCs en virale RNA. Plasma is onttrek van uit monster WC416. Die RNA was getru getransskribeer na DNA via cDNA sintese. Beide virus monsters het twee rondes PKR ondergaan. Die eerste ronde het die versterking van die MIV-1 NFLG (8978bp) geteiken en die tweede het die versterking van twee oorvleuelende fragmente (5455bp en 4909bp) geteiken. Positiewe PKR amplikone is gesuiwer en volgorde bepaal. Die gegenereerde volgordes is gelees en ontleed voordat dit gebruik was,deur gebruik te maak van aanlyn subtipeërings programme. Die jumping profile hidden markov model (jpHMM), REGA en rekombinasie-identifikasieprogramme (RIP) is gebruik om voorlopige subtipes vir beide monsters te bepaal. Verder is filogenetiese ontledings gebruik om die aanlyn subtipeërings program se resultate te bevestig of te verwerp. Die aanlyn subtipeërings programme het monsters EC148 en WC416 as komplekse A, C, D rekombinante geïdentifiseer. Filogenetiese analise het die aanlyn subtipeërings program se resultate vir die volgorde van monster WC416 bevestig as komplekse A, C, D rekombinante. Filogenetiese analise het aangedui dat die volgorde van monster EC148 in ooreenstemming is met die resultate wat vanaf die aanlyn subtipeërings programme waargeneem word. Elke MIV-1- volgorde wat geïdentifiseer is as 'n unieke komplekse rekombinante vorm omskryf, omrede die breekpunte tussen die verskillende subtipes verskil het. Die opkoms van nuwe en unieke nie-subtipe C rekombinante in Suid-Afrika dui aan dat die epidemie kompleks en ontwikkelend is. Dit is dus belangrik om die verspreiding van verskillende MIV-subtipes wat in Suid-Afrika versprei word, te monitor.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105630
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