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|Title: ||Sequence diversity of HIV-1 subtype C accessory genes VIF, VPR and VPU|
|Authors: ||Mothapo, K. M.|
|Advisors: ||Mphahlele, M. J.|
Musyoki, M. A.
Selabe, S. G.
|Issue Date: ||2010|
|Publisher: ||University of Limpopo ( Medunsa Campus)|
|Abstract: ||OBJECTIVES: To date there is no effective and safe vaccine to stop the
spread of human immunodeficiency virus (HIV) and provide cross protection
among different subtypes. HIV accessory genes were overlooked for many
years and recently they are becoming candidates for development of new
anti-HIV drugs and vaccines. This is supported by their ability to elicit
cytotoxic T lymphocyte response. To date, there are limited studies on
accessory genes (nef, vif, vpr and vpu) on South African HIV strains. This
study sought to amplify and analyse the sequences of HIV-1 subtype C
accessory genes (vif, vpr and vpu) to assess the genetic diversity as well as
the motifs and residues associated with key biological functions of these
genes. This study further sought to compare the degree of genetic diversity
between the accessory and structural genes.
METHODS: The study was an exploratory study using stored (-70ºC) HIV
positive plasma samples. The study population comprised of 25 HIV positive
plasma samples which were already sequenced in the gag and env genes in
another study. The samples were drawn from the neighbouring townships of
Pretoria: Ga-Rankuwa, Soshanguve, Mamelodi, Laudium, Kalafong, Jubilee
and Mabopane. For the purpose of this study, the same samples were
amplified, sequenced and characterised in the pol and accessory (vif, vpr and
vpu) genes in order to obtain near full length sequences of the HIV isolates
from Pretoria region. Six samples were cloned for accessory genes. Five
clones from each sample were selected. Sequence analysis was performed
for all the PCR amplicons and clones. Base calling for the sequences
generated was performed on Chromas Pro program. Computing of
phylogenetic tree was performed with MEGA 4 program. ClustalW software
was used for sequence alignment and translation of nucleotides to amino
acids was performed with BioEdit. The amino acid alignments were analysed
on graphic view.
RESULTS: All 25 samples were successfully amplified for accessory genes
(vif, vpr and vpu) and pol gene. All the 25 pol PCR amplicons were
successfully sequenced, while all but one accessory PCR amplicons were
successfully sequenced. A number of conserved motifs and residues were
observed in all the four genes (vif, vpr, vpu and pol). Vif and vpr showed to
harbour most of these conserved motifs and residues; 144-SLQYLA-149 and
H71 respectively. In addition, the R77Q mutation associated with long term
non-progressors was observed in the vpr gene of 15 sequences. Drug
resistant mutations were evaluated in both protease and RT regions. Nine
samples had one or two drug resistant mutations i.e T74S, L10I, V179D,
E138A/D, Y318F,Y181C and K108N.
Phylogenetic analysis confirmed the 25 HIV positive samples to be HIV-1
subtype C in both structural and accessory genes. The genetic diversity of
HIV-1 subtype C was compared between accessory (vif, vpr and vpu) and
structural (pol, gag and env) genes. The gag and env sequences were
available from a previous project (Musyoki, 2009). The gag and vif gene
sequences were highly conserved (89% to 96% and 88% to 96%,
respectively), as compared to vpr gene (84% to 94%), the pol gene (79% to
95%), the env gene (83% to 93%) and finally the vpu gene (73% to 92%).
CONCLUSION: This study found that amplification of clones was more
sensitive as compared to direct samples and analysis of clone sequences was
more clear than analysis of direct PCR products. Functional motifs and
residues observed in all accessory genes were highly conserved. Vif was
more conserved, followed by vpr and vpu, respectively. Genetic analysis of
pol gene revealed that there were drug resistant strains in circulation. This
indicates that the patients were infected with drug resistant viruses; this
cannot be verified from the study population. And that most of the strains in
this study had mutations associated with long term non-progressors (LTNP’s).
However, it is not known whether these patients were indeed LTNP’s.
Comparison of genetic diversity between structural and accessory genes
demonstrated that, gag, vif and vpr were more conserved than pol, env and
|Description: ||Thesis (MSc Virology)--University of Limpopo, 2010.|
|Appears in Collections:||Theses and Dissertations (Virology)|
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