HIV-1 subtypes and recombinants in the Republic of Congo
Fabien Roch Niamaa,d, Coumba Toure-Kanea,*, Nicole Vidalb, Pani Obenguic,
Blaise Bikandoud, Marie Yvonne Ndoundou Nkodiad, Ce ´line Montavonb,
Halimatou Diop-Ndiayea, Jean Vivien Momboulid, Etienne Mokondzimobed,
Aı ¨ssatou Gaye Dialloa, Eric Delaporteb, Henri-Joseph Parrad,
Martine Peetersb, Souleymane Mboupa
aLaboratoire de Bacte ´riologie et Virologie, Ho ˆpital Le Dantec, Dakar, Senegal
bUMR 145, Institut de Recherches pour le De ´veloppement (IRD), Montpellier, France
cService Maladies Infectieuses, CHU de Brazzaville, Congo
dNational Laboratory of Public Health of Congo, Brazzaville, Congo
Received 3 October 2005; received in revised form 15 November 2005; accepted 29 December 2005
Available online 13 February 2006
To document the actual genetic diversity of HIV-1 strains in the Republic of Congo, 114 HIV-1 positives persons were sampled in 2003 and
2004 after their informed consent. They were attending the teaching hospital, the reference health center in Makelekele, Brazzaville and the
regional hospital centers inPointe-Noire, Gamboma and Ouesso. A total of 104 samples weregenetically characterized by direct sequencing of the
p24 gag region and 80 were also subtyped in the V3–V5 env region. The genetic subtype distribution of the Congolese strains showed the
predominance of subtype A (36.5% and 32.5% in gag and env, respectively) and G (30.8% and 21.25%), whereas subtype D strains represented
12.5% and 15%. Subtypes C, F, H, J, K and the CRFs-01, -02, -05 -06, and also the recently characterized CRF18 were seen at lower rates. Finally,
4.8% (gag) and 6.25% (env) of the strains could not be classified. Moreover, a high intra-subtype diversity was observed in our study. Among 70
strains which have been characterized in the two genomic regions, 14 (20%) appeared to be unique recombinants. These data show a high genetic
variability in the Republic of Congo, where all the subtypes have been documented together with certain subsubtypes and several CRFs.
# 2006 Elsevier B.V. All rights reserved.
Keywords: HIV; Subtypes; Recombination; Evolution; Congo
One of the major characteristics of HIV is its high
degree of genetic variability and its ability to recombine.
The phylogenetic analysis of many isolates from various parts
of the world revealed three distinct groups of HIV-1: M, N and
O (Simon et al., 1998). Group M is responsible for the global
H, J and K), sub-subtypes (A1/A2/A3, B/D, F1/F2), unique
recombinants and circulating recombinant forms (CRFs)
(Robertson et al., 2000; Peeters and Sharp, 2000). The
distribution of HIV-1 subtypes in the world is very hetero-
geneous; differences have been largely documented between
the different continents, the different countries and within the
countries (Peeters et al., 2000; Vidal et al., 2000). Today,
almost 70% of all HIV-1 infections worldwide are found in
sub-Saharan Africa. Globally, the most predominant subtype
in the world is subtype C, circulating mainly in southern and
eastern Africa and India (Gordon et al., 2003; Kandathil et al.,
2005). In contrast, subtype B circulates mainly in developed
countries; it is the most studied subtype however it represents
less than 5% of all HIV-1 strains. In the western and west-
central part of Africa, CRF02_AG and CRF06_cpx are the
cocirculating in Central Africa suggests that the pandemic
originated in this geographic region (Vidal et al., 2000).
Recombination may play an important role in the dynamic
Infection, Genetics and Evolution 6 (2006) 337–343
* Corresponding author. Laboratoire de Bacte ´riologie-Virologie, CHU A Le
Dantec BP7325, Senegal. Tel.: +221 821 64 20; fax: +221 822 59 19.
E-mail address: firstname.lastname@example.org (C. Toure-Kane).
1567-1348/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
process of genetic diversity. The implications of the genetic
diagnostic assays, ARV treatment and vaccine development
(Parekh et al., 1999; Descamps et al., 1997).
In the Republic of Congo, previous studies tried to
understand the genetic diversity and strain distribution of
HIV-1 since 1992, but all of them were based on small sample
sizes (Candotti et al., 1999; Mboudjeka et al., 1999; Bikandou
et al., 2000, 2004; Taniguchi et al., 2002). Therefore we
reconducted recently a new study with a higher sample size in
order tobetter understand the real distributionand the evolution
over time of HIV-1 strains.
2. Materials and methods
2.1. Specimens and DNA isolation
A total of 114 blood samples were collected between 2003
and 2004 from patients with evident signs of AIDS or attending
a voluntary testing center at the teaching hospital center and
Makelekele reference center in Brazzaville, but also from
regional health centers (Albert Cisse in Pointe-Noire,
Gamboma, Ouesso and Pokola). For HIV serology, the
Determine kit (Abbott Minato-Ku, Tokyo Japan) and Gen-
Screen (Bio-Rad, Marnes la coquette, France) were used as
screening test and NewLav Blot II (Bio-Rad, Marnes la
coquette, France) as a confirmation test. ImmunoComb II
(Orgenics Ltd., Yavne, Israel) and Genie II (Bio-Rad, Marne la
coquette, France) were used to discriminate between HIV1 and
HIV2. Plasma and PBMC were separated by Ficoll gradient
centrifugation(Nacalaı ¨,Japan),andwere stored at?20 8C.The
DNA was extracted from uncultured peripheral blood mono-
nuclear cells (PBMC) by using the Qiagen DNA isolation kit
2.2. DNA amplification
Two fragments of approximatively 715 base pairs in the p24
region from thegag gene and 627 bp in the V3–V5 region from
the env gene each, were obtained by previously described
nested PCRs (Sanders-Buell et al., 1995; Delwart et al., 1993;
inner primers for the gag fragment, and ED5/ED12 or env1/
env2 in the first round and ES7/ES8 in the second round for the
env fragment. The polymerase chain reaction (PCR) conditions
were as follows: the first rounds of PCR were carried out in
50 ml of PCR mixture containing 10 ml of genomic DNA,
1.4 mmol/MgCl2, 10 pmol for each primers, 0.2 mM/l each
dNTP, and 2.5 U Taq polymerase; cycling consisted in a first
denaturation step of 3 min at 94 8C, followed by 30 s at 94 8C,
30 s at 55 8C and 1.5 min at 72 8C by 35-fold and a final
extension for 7 min at 72 8C. The second rounds of PCR were
performed in 100 ml with 5 or 7 ml (for samples weakly
concentrated) of the first round product with the same cycling
parameters. One percent of agarose gel for electrophoresis and
ethidium bromide was used to detect the PCR second round
2.3. DNA sequencing and phylogenetic analysis
PCR products were first purified by using the Qiaquik gel
extraction kit (Qiagen, Courtaboeuf, France), and then directly
sequenced with the second round primers. According to the
manufacturer’s instructions, cycle sequencing was performed
by fluorescent dye terminator technology (BDT v3.1, Applied
Biosystems, France). Electrophoresis and data collection were
done on an automated DNA sequencer (Applera ABI Prism
3100 Avant Genetic Analyzer). Sequence plots were corrected
and edited under SeqScape (Applied Biosystems, Courtaboeuf,
France) and the reconstituted fragments were aligned with
CLUSTAL ? 1.83 computer program (Thompson et al., 1994).
Regions that could not be aligned unambiguously, because of
length or sequence variability, were omitted from the analysis.
The phylogenetic relationships of the new sequences from
Congo were investigated with previously reported sequences
representing the different HIV-1 group M reference strains
circulating in West and West Central Africa (http://www.hiv_-
web.lanl.gov/HTML/alignment.html). Phylogenetic trees were
constructed by the neighbour-joining method, and the
reliability of the branching patterns was assessed using the
bootstrap approach (confidence value for individual branch of
the resulting trees evaluated with 100 bootstrap replicates)
(Felsenstein,1985)wereimplementedwithClustal ? Program.
Genetic distances were calculated with the Kimura two-
parameters method (ratio t/T = 2.0). Phylogenetic analysis was
conducted first for each new sequence individually. To clearly
identify whether a sequence belonged to a subgroup
corresponding to a CRF within a certain subtype, all the
HIV-1 variants circulating in West and Central Africa were
considered in each individual tree, including CRFs and unique
recombinants. Subtypes and CRFs were assigned to each new
strain when clusters are supported with high bootstrap values
(>80%). To confirm each cluster, a tree was then constructed
with all the Congolese strains belonging to this cluster; i.e. for
the G subtype, all the strains individually subtyped as G viruses
were taken together against the panel of reference sequences.
Finally,in order tovisualize the results, ageneraltreewas made
with all sequences, although the inclusion of all sequences,
significantly decreased bootstrap values for certain subtypes/
CRFs due to the high intra-subtype/CRF diversity. The
clustering of each new sequence should be concordant among
all the trees. The reference sequences used for the Phylogenetic
trees in Fig. 1a and b are: A_KE.Q2317, A_UG.92UG037,
A_SE.SOSE7253, A2_97CD.KTB48, A2_94CY017, A2_ZM.
ZAM184, A3_DDJ369, A3_DDJ360, A3_DDI579, B_FR.
HXB2R, B_US.JRFL, B_US.WEAU160, C_ET.ETH2220,
C_IN.21068, C_BW.96BW0502, D_UG.94UG114, D_ZR.
NDK, D_99TC.MN011, D_99TC.MN012, F1_BR.93BR020,
F1_FI.FIN9363, F1_BE.VI850, F2_96CM.MP255, F2_CM-
53657, F2_96CM.MP257, G_BE.DRCBL, G_SE.SE6165,
G_FI_HH9793, H_BE.VI991, H_BE.VI997, H_CF.90CF056,
J_SE.SE92809, J_SE.SE91733, K_96CM.MP535, K_97CD.
EQTB11, CRF01_AE (AE_TH.93TH253, AE_TH.CM240,
G.IBNG, AG_98SN.MP1211), CRF05_DF (05_VI961, 05_
F.R. Niama et al./Infection, Genetics and Evolution 6 (2006) 337–343338
F.R. Niama et al./Infection, Genetics and Evolution 6 (2006) 337–343339
Fig. 1. Phylogenetic relationships of 483 unambiguously aligned nucleotide sequences representing the p24 region in the gag gene (a) and 388 nucleotides
with the neighbour-joining method implemented with Clustal ? Program. References strains are in black and samples in grey. Bootstrap values above 80% are
indicated with asterisk (*).
X492), CRF06_cpx (06_97SE1078, 06_BFP90, 06_95ML84),
CRF9_cpx (09_95SN1795, 09_96GH2911, 09_95SN7808)
and CRF18_cpx (18_99CU14, 18_99CU76). For the phyloge-
netic analysis of each individual new sequence from Congo, we
included also unique recombinant strains from this region like,
HIV-1 MAL, HIV-1 NOGIL, Z321 as well as representatives of
the tentative new subtype l strains, and the other reported CRFS
(CRF03, CRF04, CRF07, CRF08, CRF10, CRF11 TO CRF16).
2.4. Nucleotide sequence accession number
The new Congolese sequences have been deposited in
EMBL for gag sequences accession numbers are as follows:
AM085004–AM085106 and for env sequences: AM086487–
3.1. Study population
The HIV-1 positives samples were obtained from patients
attending the teaching hospital and Makelekele hospital center
at Brazzaville (n = 102), and the regional reference centers at
Pointe-noire (n = 4), Gamboma (n = 3), and Ouesso (n = 5). All
of these patients were Congolese, but their travel history was
not taken into account in this study. All samples were obtained
from antiretroviral (ARV) treatment naı ¨ve patients, except one
pregnant woman who received a single dose of Nevirapine
during delivery. The demographic characteristics of the
study population were as follows: the mean age was 34 years,
ranging from 1 to 60 years. Sixty percent of the samples were
obtained from women and the main route of transmission was
heterosexual contact. The WHO clinical classification showed
that the majority of these patients (45.9%) were at stage 3,
followed by stage 1 (34.23%), stage 4 (12.61%) and stage 2
3.2. Distribution of genetic subtypes in the
Republic of Congo
A total of 104 HIV-1 positive samples were genetically
characterized in gag (p24) and 80 in env (V3–V5). Table 1
illustrates the subtype/CRF distribution obtained in the gag
(p24) and in the env (V3–V5) genomic regions. Phylogenetic
tree analysis of the gag p24 sequences (Fig. 1a) showed the
predominance of subtypes A and G strains (36.5% and 30.8%,
respectively), followed by subtype D strains (12.5%). Subtype
H represented 3.85% of the strains, and 4.8% of the samples
could not be classified and are identified as U (unclassified).
The recently characterized CRF18 (18_99CU14, 18_99CU76)
represented 3.85% of the gag strains circulating in the country.
Finally, subtypes C and F1 were observed (1.9% each) and one
sample of the following HIV-1 variants: J, K, CRF02_AG and
CRF05_DF were also detected (0.96% each).
Phylogenetic tree analysis of the env V3–V5 sequence
revealed similar subtype/CRF distributions (Fig. 1b). We
observed again the predominance of subtype A (32.5%)
followed by subtype G (21.25%), whereas the subtype D and H
strains accounted, respectively, for 15% and 8.75% of the
variants. Then we could not attribute a known subtype/CRF
designation for 6.25% of the samples which were classified as
(U). The recently characterized CRF18_cpx (Thomson et al.,
1 variants. Finally, 2.5% of the strains belong to CRF06_cpx
form and we detected also one sample of each of the following
variants: C, F1, F2, CRF01_AE and CRF05_DF (1.25% each).
Among the A subtype strains, we identified one A2 and one
A3 subsubtype strain in the V3–V5 env region and one A3
subsubtype strain in the p24 gag region.
3.3. Phylogenetic analysis of HIV-1 strains from Congo
The phylogenetic tree analysis of the env and gag sequences
highlighted a high intra-subtype genetic diversity, with several
F.R. Niama et al./Infection, Genetics and Evolution 6 (2006) 337–343340
Genetic subtypes in the gag p24 region and corresponding genetic subtypes
identified in env V3–V5 based on phylogenetic tree analysis
gag subtypen env subtypen
ND: not done, n = number.
sub-clusters within certain subtypes. For example in the gag
p24 tree (Fig. 1a), several clusters could be seen within subtype
A, with some of them close to known sub-subtypes or CRFs
such as CRF02, CRF06, CRF09 or A3. Within subtype G, two
differentclusterswere present,andwithinthe B–Dclusterthree
Congolese samples branched outside the known B, D or CRF05
clusters. Comparable features were observed in the env V3–V5
tree (Fig. 1b) within subtype A, G and H.
We have also observed that some samples displayed a
divergent subtype designation between individual and
general phylogenetic trees, where they clustered inside the
A subtype. They where noted with an asterisk (*) (Fig. 1a
and b), and are listed below: (1*) ARV53, undetermined
between G and J subtypes in individual trees for gag and env;
(2*) CHU18, CRF02 in env and possibly CRF09 in gag; (3*)
ARV07, CRF09 in gag. The resulting subtype of these strains
was therefore subtype A, as observed in the general trees,
and because the CRFs involved in most of these samples
were located within subtype A. However, these Congolese
strains seem to be atypical and may necessitate more
sequencing to be further characterized. Another strain
seemed to belong to the CRF18 cluster in individual trees
in the two genomic regions; as the clustering was not clear in
the general trees, we preferred to classify it as A in gag and
H in env.
In order to complete the description of the high degree of
the mean intra-subtype genetic distances in the very
informative V3–V5 env region were calculated. Table 2 gives
the genetic distances for the most represented subtypes
documented in our study, i.e. A, G, and D, compared to the
values obtained for the same subtypes in 2002 in the
neighbouring country Democratic
(DRC) (Vidal et al., 2005). Mean intra-subtype genetic
distances in the Republic of Congo were comparable to those
observed in the DRC. With the exception of subtype A, they
were slightly lower, probably because the number of samples
was lower too, particularly for the subtype D strains. The most
striking result concerns subtype A, presenting a higher mean
intra-subtype distance despite a significant lower number of
samples (four-fold lower) than in the DRC survey. The later
result confirms the very high genetic diversity of subtype A
strains circulating in the Republic of Congo, as already
delineated by the phylogenetic analyses.
3.4. Recombinant strains in the Republic of Congo
Table 1 summarizes the details of the subtype designations
in env as compared to the gag subtype. Among 70 samples that
were characterized in both genomic regions, 14 samples
displayed different subtype/CRF designations between the two
genomic regions, suggesting that at least 20% of the HIV-1
strains in the Republic of Congo were recombinant. Eleven
different recombination profiles were seen: A/CRF01 (n = 1),
A/H (n = 2), A/J (n = 2), A/U (n = 2), A3/CRF06 (n = 1), G/A
(n = 1), G/H (n = 1), CRF18/A (n = 1), U/A (n = 1), U/C (n 1),
U/CRF06 (n = 1), in gag/env, respectively. Overall, more than
one quarter of the gag subtype A viruses were recombinant
between env and gag; subtypes D or B–D viruses, as well as the
Hstrains, were concordant between gag and env;among 18 gag
subtype G strains, only 2 had a different subtype/CRF
designation in env, whereas 3 of the strains that could not be
classified in gag (U) were very likely recombinant viruses,
because they were classified as A, C or CRF06-CPX in env.
3.5. Comparison with the HIV-1 subtypes patterns
previously reported in the Republic of Congo
Several studies have been performed to describe the HIV-1
variants circulating in the Republic of Congo, with samples
collectedin the beginninguntil the end of the nineties (Candotti
et al., 1999; Mboudjeka et al., 1999; Bikandou et al., 2000,
2004). In order to better compare the different subtype patterns
in the env region, from previous studies with those from our
study collected after 2000, we have re-analyzed the phyloge-
netic relationshipof these older HIV-1 strains, 68 forenvand 48
for gag, by including all new subtypes and CRFs not available
at the time that they were reported. Globally, the HIV-1
epidemicin Congo (Brazzaville) has been drivenby subtypes A
and G, but strains belonging to subtypes C, D, F, H, J and K
subtypes were also present as well as strains that could not be
classified. We have also found that some rare strains were
probably representative of the CRF01 (EKE), CRF02
(96CG39), CRF06 (96CG38) and CRF18 (98CG896) circulat-
ing recombinant forms. Finally, representatives of subsubtype
A2 (98CG860 in gag) and F2 (98CG893 in gag and env) were
also detected in previous surveys.
The main goal of this study was to document the diversity of
HIV-1 strains and recombinants in the Republic of Congo. A
total of 104 samples have been directly sequenced in the p24
gag gene and 80 samples in the V3–V5 env gene. The
phylogenetic analyses showed an important genetic diversity
among HIV-1 strain from Congo. Eight subtypes from A to K
and five circulating recombinant forms (CRFs): CRF01_AE,
CRF02_AG, CRF05_DF, CRF06_cpx and CRF18_cpx were
Subtype A appeared to be dominant in the Republic of
Congo (36.5% in gag and 32.5% in env), immediately followed
by the subtype G (30.8% in gag and 21.25% in env). The high
F.R. Niama et al./Infection, Genetics and Evolution 6 (2006) 337–343341
V3–V5 env mean intra-subtype genetic distances of HIV-1 strains from the
Republic of Congo, as compared to intra-subtype genetic distances of HIV-1
strains in the neighbouring Democratic Republic of Congo (DRC)
SubtypesRepublic of Congo DRC (2002)
(Vidal et al., 2005)
21.37% (14.3–38.8), n = 24
14.53% (12.6–23.8), n = 12
17.46% (12.0–38.5), n = 17
19.8% (13.–27.2), n = 104
19.03% (14.0–24.1), n = 32
20.3% (15.0–28.9), n = 28
Genetic distances were calculated with Kimura-2 parameter method. Range of
lowest to highest distances within a subtype are between parentheses. In each
subtype, the number of samples included in the calculations are indicated by n.
prevalence of subtype A in our study is concordant with
previous data from the Republic of Congo, where A strains
represented about 53% of 17 samples analyzed in 1992, 39% of
strainssampledin1998.Thesevariations couldberelated tothe
low sample size or to a different population sample in the
previous studies. The prevalence of A subtype strains is slightly
lower with those seen in other Central African countries were
they represent about 50% of the circulating strains like in Chad
(Vidal et al., 2003), in Equatorial Guinea (Ortiz et al., 2001),
and in the Democratic Republic of Congo (Vidal et al., 2000,
2005); only in the Central African Republic, subtype A is
largely predominant, representing 89.7% of HIV-1 strains in
Bangui (Muller-Trutwin et al., 1999).
this study represents a unique feature in Central Africa. The
importance of these strains in Congo was previously high-
lighted (Mboudjeka et al., 1999; Bikandou et al., 2000, 2004).
In our study, the prevalence of G is higher than previously
reported in the gag region and was perfectly concordant in the
env region. Subtype G is significantly less prevalent in
bordering countries such as Gabon (2.43%) and Cameroon
(3.3%) (Pandrea et al., 2002; Ndembi et al., 2004). However,
subtype G has been found to represent 37.5% of the strains
among female sex workers in Kinshasa, DRC, in 2002 (Yang
et al., 2001).
Subtype D isthe third most important subtype (12.5% in gag
and 15% in env). Its prevalence is in accordancewith the results
obtained in the neighbouring DRC (9.8% in 1997 and 13.9% in
2002 in the env region in Kinshasa) (Vidal et al., 2000, 2005),
and in Gabon (12.9% in 2002) (Pandrea et al., 2002). In
Cameroon (11% in env) (Ndembi et al., 2004), the majority of
the D subtype strains correspond to a particular cluster within
subtype D also described in Chad, where it represents nearly
20% of the circulating HIV-1 viruses (Vidal et al., 2003).
Subtype H is well represented in the env gene (8.75%)
detected at lower rates, as well as five CRFs. Concerning the F
subtypes, strains belonging to the F1 butalso the F2 subsubtype
have been observed in the Republic of Congo. It was surprising
to find an F2 strain, since such variants were up to now only
described in Cameroon. However, the re-analysis of the
previously reported Congolese strains revealed one of them to
be an F2 virus (Bikandou et al., 2004), and one strain was also
documented in DRC (Vidal et al., 2000).
Among the subtype A strains, sub-subtypes A2 and A3 were
documented forthe first time in the country.This isalsothe first
reportforthe presence ofCRFs suchasCRF02, CRF05, CRF06
and the newly defined CRF18. Again, the re-analysis of the
strains from the previous surveys revealed the existence of A2,
CRF02, CRF06 and CRF18 strains, circulating as anecdotical
variants in the Republic of Congo.
Finally, some Congolese strains remained unclassified since
they do not cluster with known subtypes/CRFs or unique
viruses already described elsewhere; they represent 4.8% of the
samples in the gag gene, and 6.25% in the env gene. These
results are concordant with the prevalence of such strains in the
DRC (6.5% in 1997 and 7.6% in 2002 in Kinshasa in the env
region) (Vidal et al., 2000, 2005).
In order to estimate the proportion of recombinant strains in
the Republic of Congo, 70 samples have been sequenced in the
two genomic regions. Eleven different recombination profiles
have been found, in which four involved one circulating
recombinant form: A/CRF01, A3/CRF06, CRF18/A and U/
CRF06. A total of 14 samples displayed different subtype
the strains circulating in Congo were recombinant. This picture
was previously highlighted by other studies in Congo
(Bikandou et al., 2000, 2004; Taniguchi et al., 2002). Indeed,
gag subtyping strategy in Congo Brazzaville (Bikandou et al.,
2004). In the surrounding countries, like DRC (Democratic
Republic of Congo) the proportion of recombinant strains may
be more than 59.3% in certain regions of the country like in
study reported 16% of recombinant strains in the rural North
Western part of the country (Burda et al., 2004), whereas in
Gabon and Angola, the prevalence was respectively 35.15%
and 38% (Pandrea et al., 2002; Abecasis et al., 2005).
Our study reported a high genetic variability in the Republic
of Congo, where all the subtypes cocirculate together with
certain sub-subtypes and CRFs. The presence of recombinants
such as CRF05_DF episodically reported in central Africa and
the newly characterized CRF18_cpx found among the previous
published sequences from Congo, translated the dynamic of the
HIV-1 molecular epidemic in the Republic of Congo. These
results highlighted the need to include as much as possible in
the phylogenetic analyses the different HIV-1 variants already
described, including CRFs and subclusters, especially when
working on HIV-1 strains from Central African countries.
antiretroviral treatment, and the high level of recombinant
viruses involving subtypes G and A in Congo, it would be
important to study more in detail their in vitro and in vivo
responses to antiretroviral drugs.
Fabien Roch Niama has a doctoral fellowship from the AUF
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