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AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 21, Number 1, 2005, pp. 103–109
© Mary Ann Liebert, Inc.
Sequence Note
Characterization of Human Immunodeficiency Virus Type 1
from a Previously Unexplored Region of South Africa with a
High HIV Prevalence
PASCAL OBONG BESSONG,1 CHIKWELU LARRY OBI,1 TONIE CILLIERS,2 ISAAC CHOGE,2
MARY PHOSWA,2 CANDICE PILLAY,2 MARIA PAPATHANASOPOULOS,2 and LYNN MORRIS2
ABSTRACT
HIV prevalence in the Limpopo Province has increased rapidly within the past 10 years, as in other parts of
South Africa. Little is known about the genetic and biological properties of HIV circulating in this region including the baseline drug resistance profiles. We therefore collected blood samples from 42 HIV-1-infected
patients residing in this region for analysis. All samples were shown to belong to HIV-1 subtype C by env and
gag heteroduplex mobility assay (HMA). Viral isolates from 14 of these patients were shown to use the CCR5
coreceptor exclusively and had gp120 V3 loop sequences consistent with this phenotype. Sequence analysis of
both protease and reverse transcriptase genes showed that none of 13 isolates harbored primary resistance
mutations. These data suggest that HIV-1 subtype C is the predominant subtype circulating in the Limpopo
Province, and that viral strains from this region are indistinguishable from those found in other parts of South
Africa.
INTRODUCTION
T
HIV-1 IN SOUTH AFRICA has been
among the fastest in the world (www.unaids.org). An antenatal survey conducted in 2001 showed that 24.5% of pregnant women were HIV infected.1 Prevalence rates varied among
the nine provinces with KwaZulu-Natal (33.5%), Free State
(30.1%), Gauteng (29.8%), and Mpumalanga (29.2%) recording the highest levels. The Limpopo Province has experienced
a sharp rise in HIV seroprevalence over the past 10 years, from
0.64% in 1992 to 14.5% in 2001.1,2 It is one of the poorest
provinces, largely rural, and highly dependent on the migrant
labor system. Its geographic location makes it the portal of entry for surrounding countries of Botswana, Mozambique, and
Zimbabwe, which also have high rates of HIV infection. While
there have been a number of studies on HIV-1 genetic diversity in South Africa, none has specifically addressed the genetic
subtypes of HIV-1 in the Limpopo Province.
HE RATE OF SPREAD OF
The vast majority of the estimated 4.7 million infected South
Africans are infected with HIV-1 subtype C viruses. At an earlier stage of the epidemic, subtype B viruses were identified
among homosexual men who reported contacts in the United
States.3 Subtypes A, D, CRFO1-AE, and other recombinant
viruses have also been identified, although in relatively smaller
numbers.3–7 Thus, while subtype C viruses dominate the epidemic, the exceptionally high prevalence rates in South Africa
could provide significant opportunities for the spread of new
genetic subtypes and/or evolution of current subtypes. Analysis should therefore focus on previously unexplored regions
where the opportunity for the introduction of new variants is
high and where prevalence rates are rising, such as the Limpopo
Province.
Given the high prevalence rates in South Africa, there is a
desperate need for the introduction of antiretroviral (ARV) therapies. While ARV use is currently restricted to mother-to-child
prevention programs, more widespread use is expected in the
1Department
2AIDS
of Microbiology, University of Venda, Thohoyandou, South Africa.
Virus Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa.
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near future as drug prices decrease and the demand for ARV
access in developing countries grows. There is no information
regarding baseline drug resistance patterns from the Limpopo
Province, which would be important to establish prior to the introduction of ARV treatment programs. This study was therefore carried out to identify circulating HIV-1 subtypes and to
analyze genotypic drug resistance profiles in a cross-sectional
drug-naive population residing in the Limpopo Province of
South Africa.
MATERIALS AND METHODS
Study population and sample collection
Whole blood samples in ethylenediaminetetraacetic acid
(EDTA) were collected from 42 HIV-1 seropositive volunteers
who were outpatients at the Bela-Bela Clinic in Bela Bela (formerly Warmbaths) in the Limpopo Province of South Africa.
Samples were collected between July and August 2001. All patients provided signed informed consent and each completed a
questionnaire for the provision of demographic data. CD4
counts were performed when possible at the NICD using a
FACSCount (Becton Dickinson, CA). Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll–Hypaque
(Amersham Pharmacia, Sweden) gradient centrifugation and
stored in liquid nitrogen. Plasma was stored at 70°C. Ethical
clearance for this study was obtained from the Research Committee of the University of Venda, South Africa.
Viral RNA extraction and RT-PCR for env and gag
HIV-1 regions
Viral RNA was extracted from 140 l plasma using the
QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA). The
V3–V5 region of env was amplified in a nested polymerase
chain reaction (PCR) as described.8 A region in p24/p7 of gag
was amplified in a nested PCR as described,9 except that the
annealing temperature was increased to 55°C. Reverse transcription (RT) was done with AMV RT (Roche Diagnostics)
at 42°C for 60 min for both PCR reactions. Amplification was
carried out in a GeneAmp 9700 thermocycler (Applied
Biosystems, Perkin Elmer) for 35 cycles, for the first and second PCR. Negative controls were included in all PCR reactions. Reference plasmids A1 (92RW20, Rwanda), B1 (BR20,
Brazil), B2 (TH14, Thailand), C1 (MA959, Malawi), C2
(ZM18, Zambia), D1 (UG21, Uganda), and E1 (TH22, Thailand) were amplified in env and A1 (VI310, Rwanda), B2
(UG280, Uganda), C3 (UG286, Uganda), C4 (ZM145, Zambia), and D1 (K31, Kenya) were amplified in gag. Primers
and plasmids were provided in the HIV-1 env and HIV-1 gag
heteroduplex mobility assay (HMA) subtyping kits obtained
from the National Institutes of Health AIDS Research and Reference Reagent Program.
Viral phenotypes and V3 sequencing
HIV-1 isolation by PBMC coculture was attempted in a subset of patients. Viral phenotype was determined in MT-2 cells
as previously described.10 Coreceptor usage was monitored in
U87.CD4.CCR5 and U.87.CD4.CXCR4 cell lines with a p24
antigen readout (DuPont NEN, Life Sciences, Boston, MA).
Cultures with increasing levels of p24 antigen and evidence of
syncytium formation up to day 12 were considered positive for
viral replication. The amino acid sequences of the env V3 region of cultured viral isolates was determined as described.10
Drug resistance genotyping
RT and PR gene regions were amplified from primary virus
isolates as described for the RT11 and PR (Candice Pillay, National Institute for Communicable Diseases, Johannesburg, South
Africa, unpublished). Sequences were edited using Sequencher
v4.0.5 software and the predicted amino acids were translated using DNasis software. Nucleotide sequences were submitted to the
Stanford database for query (HIV-SEQ and beta test) for the detection of mutations associated with drug resistance and closest
subtype identity (www.hivdb.stanford.edu/hiv).
Phylogenetic analysis
The PR and RT nucleotide sequences were aligned using
ClustalX with HIV-1 subtypes A–J obtained from the Los
Alamos Sequence Database (http://hiv-web.lanl.gov). Additional subtype C PR and RT sequences from Botswana (n 8),
Zimbabwe (n 10), and South Africa (n 20) were included.11–16 Dendograms were created by the neighbor-joining
method with a bootstrapping stringency of 1000. Comparisons of
nucleotide homology among the PR and RT nucleotide sequences
were done according to the Kimura two-parameter model.
RESULTS
Characteristics of study population
Blood samples from 42 HIV-1-infected individuals attending an outpatient clinic in the Limpopo Province were used in
this study. The median age of the group was 34 years (range
20–53). There were 33 females and 9 males; 83% were single
and all except one were unemployed. All the subjects were
South Africans and reported heterosexual activity. One individual believed he had been infected in Zimbabwe (PB01). Individuals presented with fungal skin infections or abscesses
(n 8), pulmonary tuberculosis (n 8), sexually transmitted
infections (n 5), diarrhea (n 2), herpes zoster (n 1), and
either minor or no conditions (n 18). The median CD4 count
for 17 of the 42 (41%) individuals was 334 cells/l (range of
77–1903) with four having a CD4 count below 200 cells/l.
None of the individuals had received antiretroviral therapy.
Genetic subtypes
Heteroduplex formation and mobility analyses
HMAs for env and gag were performed as described.6,8,9
DNA heteroduplexes were resolved on a 5% acrylamide gel for
2.5 hr (env) or 2 hr (gag) at 250 V.
Plasma samples from all 42 individuals were used in an env
and gag PCR. Amplification products were obtained for 39
(93%) samples in env and 36 (85%) samples in gag. Two samples (01PB14ZA and 01PB23ZA) did not amplify in env and
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SOUTH AFRICAN HIV-1 SUBTYPE C
five samples (01PB11ZA, 01PB20ZA, 01PB37ZA, 01PB38ZA,
and 01PB40ZA) did not amplify in gag. For one sample
(01PB19ZA), amplification was not successful for either gene.
HMA profiles indicated that all samples belonged to HIV-1 subtype C. No ambiguous or indeterminate profiles were obtained
and no other subtype or intersubtype recombinants were identified. All profiles were unique, indicating no evidence for contamination. For the gag HMA, the heteroduplexes migrated
closer to the homoduplexes compared to the env HMA, confirming the lower level of genetic diversity in gag. In some
cases there was insufficient discrimination between references
A and C and it was therefore considered important to include
more than one subtype C reference for the gag HMA. An example is shown in Figure 1 (01PB17ZA), where heteroduplexes
with references C4 and A1 migrated similarly but reference C3
clearly distinguished this sample as a subtype C.
MT-2 coreceptor usage, and V3 loop sequences
Virus isolation from PBMC was successful for 14 of 32
(44%) of individuals. All viruses were nonsyncytium inducing
(NSI) in an MT-2 cell assay suggestive of CCR5 usage. Indeed
all viruses caused extensive syncytia in the U87.CD4.CCR5 cell
line with high p24 antigen levels (4.3–250 ng/ml) (Table 1). No
syncytia formation was observed in the U87.CD4.CXCR4 cell
line and cultures remained p24 antigen negative after 12 days.
Analysis of the V3 loop predicted amino acid sequences of 10
105
isolates (sequence data of 01PB12ZA, 01PB13ZA, 01PB22ZA,
and 01PB23ZA could not be reliably interpreted) revealed conservation of the GPGQ motif, characteristic of subtype C
viruses. All were 35 amino acids in length with a neutral amino
acid at position 11 and a negative or neutral amino acid at position 25 and an overall charge of between 2 and 5, typical
of R5 isolates (Fig. 2).
Drug resistance mutations
The PR and RT genes of the 14 viral isolates were analyzed
to determine if any harbored drug resistance mutations. One
isolate did not amplify in PR (01PB12ZA) and one did not amplify in RT (01PB15ZA). The Stanford sequence database listed
all viruses as subtype C in both PR and RT regions. The predicted amino acid PR sequences relative to a subtype C consensus are shown in Figure 3A. There were no primary resistance mutations. Polymorphisms occurred at sites associated
with secondary drug resistance and included K20R (6/13), I36L
(1/13), L63P/T/V (7/13), V77I (2/13), V82I (1/13), and L93I
(1/13). M36I and I93L, which are known secondary drug resistance mutations in subtype B infections, occurred in 12 of
13 (93%) of these subtype C isolates. The predicted amino acid
sequences of 13 RT genes are shown in Figure 3B. No primary
resistance mutations were detected, but polymorphisms at drug
resistance sites included A98S (2/13), V118I (3/13), V179I
(3/13), and L210V (1/13).
FIG. 1. A representative profile of gag (A) and env (B) HMA analyses. Mobilities of heteroduplexes formed between the amplified gag product of 01PB17ZA and reference strains. The fastest mobility is exhibited by the heteroduplex between the unknown 01PB17ZA and the reference strains C3 (Uganda). Lane S is the sample hybridized unto itself. (B) The fastest heteroduplex is equally shown between sample 01PB17ZA and a reference strain C2 (ZM18, Zambia). Sample 01PB17ZA was hence
designated HIV-1 subtype C based on the gag and env regions.
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TABLE 1.
CORECEPTOR USAGE
AND
MT-2 PHENOTYPES
OF ISOLATES
p24 values (ng/ml) and syncytium formation on day 8
Isolate
U.87.CD4.CCR5
Syncytia
U.87.CD4.CXCR4
Syncytia
MT-2
Biotype
12.4
25.3
218.0
53.0
12.0
251.0
179.0
17.3
75.4
4.3
11.6
91.8
5.6
147.2
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.21
0.05
0.05
0.05
NSIa
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
NSI
R5
R5
R5
R5
R5
R5
R5
R5
R5
R5
R5
R5
R5
R5
01PB01ZA
01PB02ZA
01PB03ZA
01PB04ZA
01PB08ZA
01PB12ZA
01PB13ZA
01PB15ZA
01PB17ZA
01PB18ZAb
01PB21ZA
01PB22ZA
01PB23ZA
01PB27ZA
aNSI,
bData
nonsyncytium inducing.
are from day 12 cultures.
Phylogenetic analysis
Phylogenetic analysis of the RT nucleotide sequences with
reference sequences from different subtypes confirmed that all
isolates clustered with subtype C (Fig. 4). A similar phylogenetic relationship was obtained with the PR sequences (data not
shown). Pairwise nucleotide distance analysis indicated a
2.1–10.2% variation for the PR and 2.4–7.2% for the RT regions. Comparison of RT sequences with those from other geographic locations in southern Africa including Botswana (n 8), Zimbabwe (n 10), and South Africa (10 from Durban and
10 from Johannesburg) revealed intermixing of these subtype
C sequences. This suggests that viruses from the Limpopo
Province are indistinguishable from viruses circulating in other
parts of southern Africa.
DISCUSSION
Most of the work performed to date on genetic diversity of
HIV in South Africa has focused on urban centers or highly
populated regions.3,6,17,18 The Limpopo Province is the poorest and most rural and one of the least studied of the nine
provinces of South Africa. In selecting an area for study, we
chose Bela-Bela, which is a holiday resort with luxury hotels
and a thriving sex industry. All 42 individuals residing in this
region were infected with HIV-1 subtype C strains that were
indistinguishable from other sequences from the southern
African region.
In determining the HIV-1 subtypes we employed the heteroduplex mobility assay. This assay has been shown to be a
FIG. 2. V3 loop amino acid sequences, MT-2 phenotypes, and coreceptor biotypes of 10 South African HIV-1 subtype C isolates. The sequences were all 35 amino acids in length, with overall charges between 2 and 5. The tetrapeptide motif GPGQ
(shown in the box) characteristic of nonsyncytium-inducing HIV-1 subtype C viruses was conserved in all the isolates. Positions
11 and 25, known to determine coreceptor phenotype, are shaded in gray.
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107
A
B
FIG. 3. Predicted amino acid sequences of protease (A) and reverse transcriptase (B) of 13 drug-naive South African individuals. The sequences were aligned against a consensus generated from the sequences. There were no deletions or insertions. The
dots indicate identical amino acids and changed amino acids are shown. Shaded regions represent drug-resistance-related loci.
Polymorphisms at these positions are shown in boldface.
rapid, inexpensive, and reliable technique for screening HIV-1
genetic subtypes, especially when both gag and env HMA are
used.6,8,19,20 Using this approach we found that all samples in
this cohort were HIV-1 subtype C, with no other subtype or intersubtype identified. Phylogenetic analyses of the PR and RT
nucleotide sequences provided additional information on their
subtype C designation. It is therefore unlikely that these viruses
were intersubtype recombinants, although this possibility cannot be excluded based on this subgenomic analysis. All the
viruses tested made use of the CCR5 coreceptor and did not
cause syncytia in the MT-2 cell line, irrespective of CD4 count
or the presence of an AIDS-defining illness. This is in agreement with previous reports in which subtype C viruses were
shown to preferentially use CCR5 and rarely induce syncytia.10,21 Our findings on subtype distribution are consistent with
those from studies conducted in other regions and show that
HIV-1 subtype C is the major subtype in the expanding HIV-1
epidemic in South Africa.6,22
Examination of the protease and reverse transcriptase genes did
not reveal any primary resistance mutations to any RT or PR in-
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FIG. 4. Phylogenetic relationship of RT sequences. RT sequences from the Limpopo Province (in bold) do not form a separate cluster, but intermingle with subtype C sequences from Johannesburg (00ZA.AC01–00ZA.AC12), Durban (ZA004p01–
ZA130p01), Botswana (AF110967, AF110980, AF443115–AF443110), and Zimbabwe (U83614–U83605).
hibitors. However, polymorphisms associated with secondary
drug resistance among subtype B viruses were detected in subtype C PR sequences. M36I and I93L associated with resistance
to each of the PIs occurred in 12 of 13 (92%) sequences while
K20R associated with resistance to indinavir, ritonavir, and
lopinavir occurred in 6 of 13 (46%) of the patients studied. V82I,
which confers low level resistance to nelfinavir, was found in 1
of 13 (8%) sequences. Among the RT sequences, 3 of 13 (23%)
viruses had V118I, which confers resistance to nucleoside RTIs
when present with E44A/D. Two viruses (15%) had the A98S
mutation, while V179I was detected in three viruses (23%). The
frequencies of A98S, V118I, and V179I in this cohort were marginally higher than that reported by Pillay et al.,11 in which 1
(0.03%), 3 (0.08%), and 2 (0.05%) of A98S, V118I, and V179I,
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respectively, were observed among 37 pregnant women attending antenatal clinics in Gauteng Province, South Africa. Based on
these genotypic profiles, it is expected these viruses would be susceptible to the currently available PIs and RTIs. However, the impact of secondary resistance mutations and polymorphisms on the
propensity to develop resistance once drug pressure is applied is
unknown. There is therefore a need for continuous surveillance
among patients failing antiretroviral therapies in South Africa to
determine the resistance patterns among subtype C viruses.
This study, to the best of our knowledge, is the first on the
molecular epidemiology and genotypic resistance profiles of
HIV-1 in the Limpopo Province of South Africa. The results on
subtyping may have relevance for the development and evaluation of candidate AIDS vaccines for South Africa. The drug resistance data may be useful in the formulation and implementation of drug delivery policies and programs in the Limpopo
Province of South Africa.
SEQUENCE DATA
The sequence data for the protease, reverse transcriptase, and
envelope V3 loop gene regions obtained in this study have been
submitted to GenBank under the following accession numbers:
AY510031–AY510043, AY510044–AY510056, and AY510057–
AY510066, respectively.
109
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9.
10.
11.
12.
13.
14.
15.
ACKNOWLEDGMENTS
16.
This study received funding from the Fogarty International
Centre (TWO-0231) and the National Research Foundation,
South Africa. We thank Ms. Rebecca Baloyi of the Bela-Bela
Clinic for recruitment of volunteers and sample collection and
Ms. Mia Coetzer and Mr. Shayne Loubser of NICD for assistance with HMA and phylogenetic analyses, respectively. L.M.
is a Wellcome Trust Overseas Senior Research Fellow in Biomedical Sciences. Research done by P.O. Bessong was in partial fulfillment for a Ph.D. degree in the Department of Microbiology, University of Venda, South Africa.
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Address reprint requests to:
Lynn Morris
AIDS Virus Research Unit
National Institute for Communicable Diseases
Private Bag X4
Sandringham 2131
Johannesburg, South Africa
E-mail: [email protected]