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Characterization of HIV-1 subtype B near full-length genome sequences identified at the start of HIV epidemic in South Africa

Obasa, Adetayo Emmanuel Adegbenga (2017-03)

Thesis (MMedSc)--Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY: South Africa is home to approximately 20.0% of the global Human Immunodeficiency Virus (HIV) infected population. The first reported cases of HIV-1 in the country were described in 1982 amongst the homosexual male population. This was attributed to HIV-1 subtypes B (HIV-1B) and D (HIV-1D). Since the late 1980s HIV-1 subtype C (HIV-1C), spread mainly through heterosexual contact, has been the driving force of the epidemic. To date, only six HIV-1B near full-length genome (NFLG) sequences from South Africa are available in the Los Alamos National Laboratory database (LANL). During this study we retrieved five HIV-1B positive samples from homosexual and bi-sexual males, stored for up to 30 years, from the early 1980s, for further characterization. The NFLG amplification reactions were performed using a modern Polymerase Chain Reaction (PCR) protocol designed to target two overlapping proviral DNA HIV genome fragments, 5.5 kb and 3.7 kb in size, respectively. All positive PCR products were sequenced to characterize the viruses. The sequences were checked and edited manually using Sequencher V5. Multiple sequence alignments were created using Clustal W and Maft V7. The sequences were subtyped using the REGA V3.0, RIP V3.0 and jumping profile Hidden Markov Model (jpHMM) online subtyping programmes. Maximum likelihood phylogenetic trees were drawn using MEGA V6. Four of the five HIV-1 patient sequences were subtyped as pure HIV-1B. One sequence, ZA|85|R605, was characterized as a novel HIV-1 BD recombinant. This is the first NFLG HIV-1 BD recombinant ever described and indicates that recombination events were most likely already happening at the early stage of the South African epidemic. Two patient sequences, ZA|87|R1296 and ZA|87|R459, clusters with HIV-1B sequences from the United States of America (USA). The sequence from patient ZA|87|R68 clusters with a HIV-1B sequence from France and the sequence of ZA|87|R526 clusters with another South African HIV-1B sequence. Homosexual flight stewards, international tourists and migrants from the European and North American countries were most likely responsible for the introduction of the HIV-1B epidemic into South Africa. The findings of this study provides valuable insights from the beginning of the HIV-1 epidemic in South Africa. We highlight the importance of characterizing complete viral genomes from early archival specimens to give a more detailed picture of landmarks of the HIV/AIDS pandemic. We show that NFLG sequencing is an important tool for the identification of recombinant viral strains. This study can form the basis for continued research in our attempt to reconstruct the epidemiology and evolutionary history of HIV in South Africa. The HIV-1 epidemic is dynamic in nature and is constantly changing.

AFRIKAANSE OPSOMMING: Suid-Afrika is die tuiste van ongeveer 20,0% van die wêreld se menslike immuniteitsgebreksvirus (MIV)-besmette bevolking. Die eerste gerapporteerde gevalle van MIV-1 in die land is beskryf in 1982 onder homoseksuele mans. Dit was toegeskryf aan MIV-1 subtipes B (MIV-1B) en D (MIV-1D). Sedert die laat 1980's oorheers MIV-1 subtipe C (HIV-1 C), hoofsaaklik versprei deur heteroseksuele kontak. Tot hete is slegs ses MIV-1B naby vollengte genoom (NFLG) rye van Suid-Afrika beskikbaar in die LANL MIV databasis. Tydens hierdie studie het ons vyf MIV-1B positiewe monsters van homoseksuele en biseksuele mans, wat gestoor was vir tot 30 jaar, vanaf die vroeë 1980's, verder gekarakteriseer. Die NFLG amplifisering reaksies is uitgevoer met behulp van 'n moderne polimerase-kettingreaksie (PKR) protokol ontwerp om twee oorvleuelende provirale DNS MIV genoom fragmente, 5.5 kb en 3.7 kb in grootte, te teiken. Alle positiewe PKR produkte se DNS volgordes is bepaal om die virusse volledig te karakteriseer. Die volgorde is nagegaan en met die hand geredigeer deur gebruik te maak Sequencher V5. Veelvuldige volgorde roetes is geskep met behulp van CLUSTAL W en Maft V7. Die MIV-1 subtipes is met die aanlyn programme REGA V5, RIP V3.0 en jpHMM bepaal. Die subtipes is bevestig duer maksimum waarskynlikheid filogenetiese bome te trek met behulp van MEGA V6. Vier van die vyf MIV-1 pasiënt volgordes is as suiwer MIV-1B gekarakteriseer. Een volgorde, ZA|85|R605, is gekenmerk as ’n unieke MIV-1 BD rekombinant. Tot ons kennis is dit die eerste en enigste NFLG MIV-1 BD rekombinant ooit beskryf en dui aan dat rekombinasie gebeure reeds in die vroeë jare van die Suid-Afrikaanse epidemie plaasgevind het. Twee pasiënt volgordes, ZA|87|R1296 en ZA|87|R459, is na verwant aan MIV-1B volgordes van die Verenigde State van Amerika (VSA). Die volgorde van pasiënt ZA|87|R68 is na verwant met 'n MIV-1B volgorde van Frankryk en die volgorde van ZA|87|R526 is na verwant aan 'n ander Suid-Afrikaanse MIV-1B volgorde. Homoseksuele vlug bedienaars, internasionale toeriste en immigrante uit die Europese en Noord-Amerikaanse lande was waarskynlik verantwoordelik vir die bekendstelling van die MIV-1B epidemie in Suid-Afrika. Die bevindinge van die studie gee insigte van die begin van die MIV-1-epidemie in Suid-Afrika. Ons beklemtoon die belangrikheid om argiewe MIV-stamme met NFLG volgorde bepalings te karakteriseer aangesien dit vir ons ‘’n meer volledige prentjie van die pandemie kan skep, veral uit die begin jare. Ons wys dat NFLG volgorde bepaling 'n belangrike instrument is vir die identifisering van rekombinante MIV-stamme. Hierdie studie kan die grondslag lê vir voortgesette navorsing om die epidemiologie en evolusionêre geskiedenis van MIV in Suid-Afrika te rekonstrueer.

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