Characterization of a new simian immunodeficiency virus strain in a naturally infected Pan troglodytes troglodytes chimpanzee with AIDS related symptoms.

Abstract

Data on the evolution of natural SIV infection in chimpanzees (SIVcpz) and on the impact of SIV on local ape populations are only available for Eastern African chimpanzee subspecies (Pan troglodytes schweinfurthii), and no data exist for Central chimpanzees (Pan troglodytes troglodytes), the natural reservoir of the ancestors of HIV-1 in humans. Here, we report a case of naturally-acquired SIVcpz infection in a P.t.troglodytes chimpanzee with clinical and biological data and analysis of viral evolution over the course of infection.
A male chimpanzee (Cam155), 1.5 years, was seized in southern Cameroon in November 2003 and screened SIV positive during quarantine. Clinical follow-up and biological analyses have been performed for 7 years and showed a significant decline of CD4 counts (1,380 cells/mm³ in 2004 vs 287 in 2009), a severe thrombocytopenia (130,000 cells/mm³ in 2004 vs 5,000 cells/mm³ in 2009), a weight loss of 21.8% from August 2009 to January 2010 (16 to 12.5 kg) and frequent periods of infections with diverse pathogens.DNA from PBMC, leftover from clinical follow-up samples collected in 2004 and 2009, was used to amplify overlapping fragments and sequence two full-length SIVcpzPtt-Cam155 genomes. SIVcpzPtt-Cam155 was phylogenetically related to other SIVcpzPtt from Cameroon (SIVcpzPtt-Cam13) and Gabon (SIVcpzPtt-Gab1). Ten molecular clones 5 years apart, spanning the V1V4 gp120 env region (1,100 bp), were obtained. Analyses of the env region showed positive selection (dN-dS >0), intra-host length variation and extensive amino acid diversity between clones, greater in 2009. Over 5 years, N-glycosylation site frequency significantly increased (p < 0.0001).
Here, we describe for the first time the clinical history and viral evolution of a naturally SIV infected P.t.troglodytes chimpanzee. The findings show an increasing viral diversity over time and suggest clinical progression to an AIDS-like disease, showing that SIVcpz can be pathogenic in its host, as previously described in P.t.schweinfurthii. Although studying the impact of SIV infection in wild apes is difficult, efforts should be made to better characterize the pathogenicity of the ancestors of HIV-1 in their natural host and to find out whether SIV infection also plays a role in ape population decline.

Full text

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RESEARCHOpen Access
Characterization of a new simian
immunodeficiency virus strain in a naturally
infected Pan troglodytes troglodytes chimpanzee
with AIDS related symptoms
Lucie Etienne1, Eric Nerrienet2,3, Matthew LeBreton4, Godwin Tafon Bibila5, Yacouba Foupouapouognigni2,
Dominique Rousset2, Ahmadou Nana4, Cyrille F Djoko4, Ubald Tamoufe4, Avelin F Aghokeng1,6,
Eitel Mpoudi-Ngole6, Eric Delaporte1, Martine Peeters1*, Nathan D Wolfe4,7, Ahidjo Ayouba1
Abstract
Background: Data on the evolution of natural SIV infection in chimpanzees (SIVcpz) and on the impact of SIV on
local ape populations are only available for Eastern African chimpanzee subspecies (Pan troglodytes schweinfurthii),
and no data exist for Central chimpanzees (Pan troglodytes troglodytes), the natural reservoir of the ancestors of
HIV-1 in humans. Here, we report a case of naturally-acquired SIVcpz infection in a P.t.troglodytes chimpanzee with
clinical and biological data and analysis of viral evolution over the course of infection.
Results: A male chimpanzee (Cam155), 1.5 years, was seized in southern Cameroon in November 2003 and
screened SIV positive during quarantine. Clinical follow-up and biological analyses have been performed for 7 years
and showed a significant decline of CD4 counts (1,380 cells/mm3in 2004 vs 287 in 2009), a severe
thrombocytopenia (130,000 cells/mm3in 2004 vs 5,000 cells/mm3in 2009), a weight loss of 21.8% from August
2009 to January 2010 (16 to 12.5 kg) and frequent periods of infections with diverse pathogens.
DNA from PBMC, leftover from clinical follow-up samples collected in 2004 and 2009, was used to amplify
overlapping fragments and sequence two full-length SIVcpzPtt-Cam155 genomes. SIVcpzPtt-Cam155 was
phylogenetically related to other SIVcpzPtt from Cameroon (SIVcpzPtt-Cam13) and Gabon (SIVcpzPtt-Gab1). Ten
molecular clones 5 years apart, spanning the V1V4 gp120 env region (1,100 bp), were obtained. Analyses of the env
region showed positive selection (dN-dS >0), intra-host length variation and extensive amino acid diversity
between clones, greater in 2009. Over 5 years, N-glycosylation site frequency significantly increased (p < 0.0001).
Conclusions: Here, we describe for the first time the clinical history and viral evolution of a naturally SIV infected
P.t.troglodytes chimpanzee. The findings show an increasing viral diversity over time and suggest clinical
progression to an AIDS-like disease, showing that SIVcpz can be pathogenic in its host, as previously described in
P.t.schweinfurthii. Although studying the impact of SIV infection in wild apes is difficult, efforts should be made to
better characterize the pathogenicity of the ancestors of HIV-1 in their natural host and to find out whether SIV
infection also plays a role in ape population decline.
* Correspondence: martine.peeters@ird.fr
1UMR145, Institut de Recherche pour le Développement (IRD) and Université
Montpellier 1, Montpellier, France
Full list of author information is available at the end of the article
Etienne et al. Retrovirology 2011, 8:4
http://www.retrovirology.com/content/8/1/4
© 2011 Etienne et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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Background
While non-invasive studies have provided a clear picture
on the prevalence and genetic diversity of simian immu-
nodeficiency virus (SIV) infection in wild apes in Central
Africa and allowed the tracing of the origins of human
immunodeficiency virus type 1 (HIV-1) infection in
humans, there is almost no information on clinical,
immunological, and intra-host viral evolution for natural
SIV infections in chimpanzees and gorillas. Studying
SIV infection over time in apes is not facilitated by their
isolated habitat and endangered status. While non-inva-
sive studies could potentially allow evaluation of viral
evolution over time, they cannot yet provide information
on the clinical history of the animal. Follow-up studies
are thus performed on captive animals, but only a hand-
ful of captive chimpanzees with natural SIVcpz infec-
tions have been identified (Additional file 1: Table S1),
and no captive SIV infected gorilla has been described.
To date, six strains of SIVcpzPtt were characterised in
captive Pan troglodytes troglodytes chimpanzees, but no
virological or clinical follow-up data were available
because the animals died upon arrival, were of a young
age, or were tested retrospectively [1-4]. The clinical
measurements and disease course for one captive SIV
positive Pan troglodytes ellioti (Cam4) were described,
but this animal most likely acquired his infection in cap-
tivity from his naturally infected P. t. troglodytes cage
mate (Cam3); no natural SIVcpz infection has been
identified in wild animals from P. t. ellioti [1,5]. Finally,
a confiscated Pan troglodytes schweinfurthii chimpanzee
[6], rescued following illegal export from Africa to Bel-
gium, is currently the only naturally SIVcpz infected
chimpanzee known to be alive. This animal is infected
with SIVcpzPts-Ant, and had been regularly monitored
over a 7-year period from the ages of 4 to 11 years old.
This observation period has provided unique data on
virological and immunological characteristics of a nat-
ural SIVcpzPts infection [7-9]; the SIVcpzPts-Ant strain
showed an important genetic variability in V1 and V2
env regions and the animal presented no signs of immu-
nodepression with a strong humoral antibody response,
fluctuating plasma viremia, and a strong but transient
neutralizing antibody response [8]. Nevertheless, the ani-
mal’s platelet count eventually dropped to extremely low
values at age 7, leading to a profound and permanent
thrombocytopenia [10], a characteristic that has been
observed and associated with progressive HIV and SIV
infections in humans and pigtail macaques, respectively
[11,12].
Recently, the paradigm that SIVs are non-pathogenic
in their natural hosts has been challenged for chimpan-
zees [13]. It was shown on habituated communities of
wild P. t. schweinfurthii chimpanzees in Gombe,
Tanzania, that SIVcpzPts infection is associated with a
10 to 16 fold increase in age-corrected risk for death,
reduced fertility in SIV positive females in terms of
birth rate and survival of off-spring, and an AIDS
(acquired immune deficiency syndrome)-like syndrome
which correlated with low CD4 counts, revealed by
post-mortem immunohistochemistry. Thus, these obser-
vations suggest that SIVcpz has a similar effect on chim-
panzees as HIV-1 has on humans.
Chimpanzees are also the only animals that can be
experimentally infected with HIV-1; they are readily sus-
ceptible to HIV-1 but, in contrast to humans, the infec-
tion generally does not progress to AIDS. Over 100
chimpanzees have been infected with HIV-1, but only a
few cases of immune deficiency were reported, all
occurring in the Yerkes cohort [14-17]. It has to be
noted that chimpanzees used for the experiments were
almost all from the P. t. verus subspecies from West
Africa, in which no natural SIV infection has been
documented to date.
Today, data on the evolution of natural SIVcpz infec-
tion are only available from a single chimpanzee, and no
data exist for representatives of the P. t. troglodytes sub-
species in which the reservoir of the ancestors of HIV-1
in humans has been identified. Here, we report a new
case of a natural SIVcpzPtt infection in a P. t. troglo-
dytes chimpanzee (Cam155/Ch-Go) from Cameroon; we
characterized the full-length genome and analyzed the
viral diversity and evolution of the SIVcpzPtt-Cam155
strain at a five-year interval. Importantly, the clinical
and biological data recorded on this chimpanzee
following his arrival at the sanctuary suggest progression
to AIDS.
Results
Clinical history and observations of the SIVcpzPtt infected
animal, Cam155
Cam155 (Ch-Go) is a male chimpanzee that arrived in
the sanctuary in Cameroon, in November 2003 at
approximately 1.5 years old. The animal was confiscated
by the Ministry of Environment and Forestry near the
Dja Faunal Reserve in south-central Cameroon, located
within the natural range and habitat of P. t. troglodytes.
Upon arrival, the animal was emaciated, dehydrated, and
had wounds to the groin (Table 1). During his quaran-
tine in December 2003, Ch-Go was screened for SIV
infection and had a positive reaction in HIV screening
and confirmatory tests. Serological tests for other viral
infections, such as hepatitis A, B and C viruses, and
simian T-lymphotropic virus, were all negative, and
there was no evidence of tuberculosis infection.
Since his arrival in the sanctuary, the animal has regu-
larly suffered from bite wounds on hands, feet, ears and
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genitalia from other chimpanzees, as well as from fre-
quent periods of illness. In 2004, various infections with
helminths and protozoans were detected (Balantidium
coli, Entamoeba hartmanni, Trichomonas hominis,
Strongyloides and Ancylostoma), and the animal suffered
from an undiagnosed respiratory illness (coughing)
(Table 1). Plasma viral load was measured with the
commercially available HIV viral load assay in March
2004 (Versant HIV-1 RNA 3.0 (b-DNA), Siemens, Erlan-
gen, Federal Republic of Germany), which revealed a
high viral load of 5.09 log10copies/ml. CD4 and CD8
counts were measured in February and May of the same
year with Dynabeads technology (Invitrogen, Cergy Pon-
toise, France) [18,19] and were of 700 and 1,380 CD4
cells/mm3and 570 and 1,010 CD8 cells/mm3, respec-
tively (Table 1). The mean CD4 counts, measured
between 2002 and 2004 on 15 SIV negative chimpanzees
from the same sanctuary with the same technique, were
1,740 +/- 776, ranging from 540 to 3,460. In 2006, the
animal suffered from another unidentified respiratory ill-
ness (coughing/catarrh) between February and April,
from a swelling in the eyelid in March, from a fungal
skin infection in May, and yet another respiratory illness
with bilateral nasal discharges, coughing, elevated tem-
perature and mouth breathing in November of the same
year. In 2008, oral candidiasis was detected in January
and nose bleeding in March. In August 2009, an eye
infection (inflammation, weeping and pain) was noted;
and, despite treatment attempts, the infection led to cat-
aract and blindness in one eye. Cam155 has experienced
growth retardation, and weighed only 16 kg in August
2009, at 7.3 years old, compared to an average weight of
28 kg for four other seven-year old animals in the same
sanctuary in Cameroon, and approximately 28-30 kg or
more for laboratory raised P. t. versus [20,21]. Between
August 2009 and January 2010, a significant weight loss
(from 16 to 12.5 kg, 21.8%) was observed together with
halitosis, bleeding gums and notable tooth decay.
In August 2009, a blood sample was taken to measure
CD4/CD8 counts and revealed a significant drop in CD4
counts to 287 cells/mm3, CD8 counts were 1,523 cells/
mm3, a total CD3 average of 1,856 cells/mm3and CD4/
CD8 ratio of 0.19 using a Becton Dickinson FACSCount
system (Table 1). By comparison, a healthy seronegative
Table 1 Clinical history of the SIVcpzPtt-Cam155 infected chimpanzee (Ch-Go) since his arrival at the sanctuary in
November 2003
Year Months Symptoms
(Log copies/ml)a
Viral load CD4/CD8 counts
(cells/mm3)b
Platelets
(cells/mm3)
Weight
(kg)
Age
(years)
2003 11 Cachexia, dehydratation, wounds to the groin at
arrival in sanctuary
3.31.5
2004 02 Anorexic CD4 = 700; CD8 = 570
02-04 Respiratory illness
03
Balantidium coli, Entamoeba hartmanni,
Trichomonas hominis
5.09
04
Strongyloides
05CD4 = 1,380; CD8 = 1,010 130,000
06
Balantidium coli, Ancylostoma
2005 10Cestode infection8 3.5
2006 02-04Respiratory illness
03 Swelling in eye lid surgically relieved
05Fungal skin infection
11 Respiratory illness
2007 0214 4.8
2008 01 Oral candidiasis complicated with bacterial infection
03 Nose bleeding
2009 08 An eye infection led to cataract and
blindness in one eye
CD4 = 287; CD8 = 1,523167.3
11 Halitosis, bleeding gums and tooth decay4.04 5,000
2010 01Halitosis, bleeding gums and tooth decay 12.57.7
aViral load was determined with the b-DNA method (Versant HIV-1 RNA 3.0, Siemens, Erlangen, Federal Republic of Germany) in 2004 and with the Abbott
RealTime™ HIV-1 assay (Abbott, Chicago, USA) in August 2009.
bCD4 and CD8 counts were determined with Dynabeads in February and May 2004 and with Flow Cytometry in August 2009.
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chimpanzee of similar age from the same population
had average CD4 counts of 1,256/mm3and normal
chimpanzee values between 800 and 2,000 have also
been reported for other captive HIV/SIV negative,
asymptomatic experimentally HIV-1 infected chimpan-
zees and the naturally SIVcpzPts-Ant infected chimpan-
zee (Ch-No) [22,10].
A commercially available HIV viral load test, Abbott
RealTime™ HIV-1 assay (Abbott, Chicago, USA), was
used to quantify viral load on a blood sample from
November 2009, and plasma viral load was estimated at
10,995 copies/ml (4.04 log10). A full blood count in Jan-
uary 2010 revealed slight anaemia (RBC 4,420,000/mm3
compared to 5,040,000 ± 460,000/mm3; haemoglobin
10.1 g/dl compared with 13.5 ± 1.2 g/dl and haematocrit
30.33% compared to 41.7 ± 4%); slight leukopenia
(8,140/mm3compared to 13,700 ± 4,600/mm3); and
severe thrombocytopenia (5,000/mm3compared with
385,000 ± 77,000/mm3) (Table 1). Normal values
between brackets correspond to data reported on
healthy chimpanzees [23].
Overall, episodes of infections have also been observed
in other animals from the sanctuary, but Ch-Go suffered
from 8 symptomatic episodes: 4 with parasite infections,
2 with fungal infections, and 2 respiratory illnesses
(Table 1). Overall, repeated symptomatic events were
rarely seen in the other chimpanzees, especially oral
candidiasis together with frequent episodes of eye,
respiratory and parasite infections in a single animal was
not seen in the other chimpanzees from the same sanc-
tuary. We evaluated the symptoms and clinical history
observed in Cam155/Ch-Go using the CDC and WHO
classification systems for human HIV infections and
found that the disease stage in this animal corresponds
at least to pre-AIDS, CDC stage B2 and WHO stage III.
Full-length genome sequences of the
SIVcpzPtt-Cam155 strain
DNA extracted from PBMCs obtained from residual
blood samples in May 2004 and five years later, in May
2009, was used to generate full-length sequences of the
SIVcpz strain infecting Cam155. These blood samples
were drawn for clinical purposes at time points when
the health status of the animal deteriorated and medical
intervention was needed. Partially overlapping subge-
nomic fragments (1,100 bp to 3,950 bp in length) were
amplified by PCR to obtain two full-length genome
sequences of 9,899 bp for SIVcpzPtt-04Cam155 and
9,870 bp for SIVcpzPtt-09Cam155 infecting Cam155 in
2004 and 2009, respectively (Figure 1). Inspection of
the deduced protein sequences of both SIVcpzPtt-
Cam155 sequences revealed open reading frames for
gag, pol, vif, vpr, tat, rev, vpu, env and nef genes. To
compare SIVcpzPtt-Cam155 to previously characterized
SIVcpzPtt and SIVcpzPts strains, we performed diversity
plot analyses of concatenated sequences (data not
shown) and phylogenetic tree analyses. The diversity
plot and phylogenetic analyses (Figure 2) revealed that
SIVcpzPtt-Cam155 was a typical SIVcpzPtt virus related
across the entire genome to SIVcpzPtt-Cam13 and
SIVcpzPtt-Gab1, infecting wild-caught chimpanzees
from the south-western part of Cameroon and northern
Gabon, respectively [2,3]. Mitochondrial DNA analysis
on host DNA confirmed that Cam155 belongs to the
P. t. troglodytes subspecies. As expected, the virus
sequences obtained from Cam155 in 2004 and 2009
Figure 1 Amplification of SIVcpzPtt-04Cam155 and SIVcpzPtt-09Cam155 full-length genomes. The two full-length genomes were
amplified as partially overlapping PCR fragments (shaded boxes) and directly sequenced, except for the V1V4 env regions (hatched boxes) where
clonal sequences were necessary. The primers used to amplify each PCR fragment are given in Additional file 2: Table S2. Fragments are drawn
to scale and the nucleotide sequences are numbered from the beginning of the R region in the 5’ LTR (see scale bar).
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were very similar along the genome (Figure 2) with an
average nucleotide similarity of 0.979. pol and vif were
highly conserved after a five-year period, while LTR,
gag, nef, and env regions were under higher selective
pressure reflected by the accumulation of mutations.
The coding region with the highest rate of mutations
was in gp120, between V1 and V4 hypervariable loops.
Analysis of the amino acid sequences in Pol revealed the
absence of naturally present mutations typically asso-
ciated with HIV drug resistance (according to HIVDB,
ANRSV2009.07 and RegaV8.0.2 algorithms). It can also
be noted that there were three and two copies of NF-kB
enhancer in the 5’LTR of SIVcpzPtt-04Cam155 and
SIVcpzPtt-09Cam155, respectively.
Genetic variability of the hypervariable V1V4 env
region over time
To perform in-depth analysis on the variability of the
envelope gene, we amplified a sub-genomic fragment
spanning the V1V4 region (1,105 bp) for both 2004 and
2009 samples with specific primers. The amplified and
gel-purified products were cloned and sequences of ten
V1V4 molecular clones at each time point (2004 and
2009) were analyzed. The phylogenetic tree of V1V4
Figure 2 SIV infecting Cam155/Ch-Go is a SIVcpzPtt closely related to SIVs from Cameroon (SIVcpzPtt-Cam13) and Gabon (SIVcpzPtt-
Gab1) across the genome. SIVcpzPtt-04Cam155 and SIVcpzPtt-09Cam155 Gag, Pol1, Pol2, and Env amino acid (aa) sequences were compared
to previously published SIVgor, SIVcpz and HIV-1 references. 422 amino acids were examined for Gag analysis (up left), 700 aa for Pol1 (up right),
251 aa for Pol2 (below left), and 673 aa for Env (below right). Maximum likelihood and Bayesian analysis trees had the same topology. Here
presented in black and above the branches, the bootstraps > 0.70, and the grey stars below the branches are posterior probabilities > 0.80. Scale
bars represent 0.05 (Gag), 0.05 (Pol1), 0.02 (Pol2) and 0.1 (Env) replacements per site.
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clones shows that strains from 2004 and 2009 form
separate clusters according to their collection date
(Figure 3), illustrating viral adaptation in its natural host
over time. The calculations of dN and dS, with SNAP
[24] of the different V1V4 env clones, showed a positive
selective pressure (dN-dS >0) over the 1,000 bp with an
important increase of non-synonymous substitutions
between 2004 and 2009. Together, with a significant
two-fold increased amino acid and nucleotide diversity
(p < 0.00001) of the V1V4 region over a five-year period
of infection (Table 2), these data confirm the important
selective pressure exerted on SIVcpzPtt-Cam155.
Figure 3 Phylogenetic relationships of SIVcpzPtt-Cam155 V1V4 env clones. Analyses were performed using a codon nucleotide alignment
of 648 bp, once the gaps discarded, of the ten clones of SIVcpzPtt-Cam155 from 2004 and the ten ones from 2009 with previously published
HIV-1/SIVcpz/SIVgor sequences. Phylogenetic analyses were run with both PhyML and Mr Bayes. Here presented in black and above the
branches, the bootstraps > 0.70, and the grey stars below the branches are posterior probabilities > 0.80. Scale bars represent 0.1 replacements
per site. On the right side is a zoom of the clones’ phylogenetic relationships. The clone names 040 to 049 and 090 to 099 stand for SIVcpzPtt-
Cam155 clones from 2004 and 2009, respectively, indicated by 04 and 09 followed by the clone number.
Table 2 Summary of SIVcpzPtt-Cam155 amino acid and nucleotide diversities, sequence length and glycan shield of
V1-V4 env clones in 2004 and 2009
SIVcpzPtt-04Cam155SIVcpzPtt-09Cam155
p values (04 vs 09)
** 8.8 10-06
** 1.2 10-05
0.21
** 3.0 10-04
Diversity (aa) (Min, Max) St Dev
Diversity (nt) (Min, Max) St Dev
Length (aa) (Min, Max)
# N-glyc sites (Min, Max)
0.0471 (0, 0.1035) 0.0364
0.0237 (0.0052, 0.0572) 0.0161
332.6 (330, 338)
17.1 (16, 19)
0.0884 (0, 0.1569) 0.0512
0.0491 (0, 0.0926) 0.0206
331.3 (329, 335)
19.9 (18, 21)
At each time point (2004 and 2009), the mean, minimum (Min), maximum (Max), and standard deviation (St Dev) of the pairwise amino acid (aa) and nucleotide
(nt) diversities are shown. The mean, minimum, and maximum are also shown for V1-V4 amino acid length and the number of putative N-linked glycosylation
sites. The p values were calculated by a Mann-Whitney test to assess the statistical differences between each mean in 2004 vs 2009 (** is highly significant).
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Furthermore, the number of putative N-linked glycosyla-
tion sites (PNGS) in V1V4 env region increased signifi-
cantly from an average of 17.1 to 19.9 between 2004 and
2009 (p < 0.0001) (Table 2). Notably, in SIVcpzPtt-
09Cam155 clones, an additional PNGS was observed in
the V2 loop and in the majority of V1 sequences, and the
glycosylation patterns of the V4 loop were variable accord-
ing to indels (Figure 4). There was no significant difference
in the SIVcpzPtt-Cam155 env amino acid length between
2004 and 2009 (Figure 4). Particularly, the V2 loop
remained stable in length over time, while an extension of
the V2 loop has been associated with a slow disease pro-
gression [25,26], and a cycling pattern in V2 length was
observed in the non-progressor SIVcpzPts-Ant infected
chimpanzee [7].
The different analyzed clones were all from the R5
phenotype, according to V3 net charges and the 11/25
rule [27]. In 2004, the V3 net charge of SIVcpzPtt-
04Cam155 was of 2 (<5). In 2009, an increase of the V3
net charge was observed (net charge = 3), but it was still
inferior to the threshold (<5) associated with a switch
from CCR5 to CXCR4 co-receptor use. An amino acid
modification was observed in the V3 crown with a
switch from GPAM (in 2004), mainly found in HIV-1
group N and its SIVcpzPtt precursors, to a GPGM
motif in 2009, found in the large majority of SIVcpz
Figure 4 Env hypervariable loop amino acid diversity of SIVcpzPtt-Cam155 clonal sequences in 2004 and 2009. The hypervariable loops
V1, V2, V3 and V4 are analyzed. The alignment consensus of all clonal sequences is indicated at the top. The dots stand for gaps, dashes for the
same amino acid as the consensus, the question marks in the consensus reveal no major amino acid in the alignment. On the left side, the
clone names 040 to 049 and 090 to 099 stand for SIVcpzPtt-Cam155 clones from 2004 and 2009, respectively. The glycosylation consensus motifs
(NXT/S) are highlighted in grey, important cysteines in red and the V3 crown in yellow stressing the switch from 2004 to 2009.
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strains, with the exception of SIVcpzPtt ancestors of
HIV-1 group M which harbour a GPGQ/R crown
(Figure 4). This amino acid change in a crucial position
of the envelope and the conserved D to N mutation at
position 29 on the V3 loop (Figure 4) are possibly due
to an adaptation of the virus over time in response to
host immune pressure.
Discussion
To-date, data on the evolution of natural SIVcpz infec-
tion over time and on the impact of SIVcpz on chim-
panzee populations are only available for the P. t.
schweinfurthii subspecies from East-Central Africa and
no data currently exist for representatives of the P. t.
troglodytes subspecies, the natural reservoir of the ances-
tors of HIV-1 in humans. In this study, we describe for
the first time the clinical observations and viral history
over time in a naturally SIV infected P. t. troglodytes
chimpanzee (Cam155/Ch-Go). The low CD4 counts
observed in 2009, together with severe thrombocytope-
nia, weight loss and unusual frequent periods of infec-
tions with diverse pathogens, suggest a progressive SIV
infection similar to HIV infection in humans, confirming
previous observations that SIVcpz can be pathogenic in
its natural host. Although, CD4 counts in 2004 and
2009 were measured with two different techniques, the
observed decline in CD4 counts cannot be explained by
potential different performances of the techniques on
chimpanzee cells only. Moreover in 2009, CD4 counts
of 1,256 CD4 cells/mm3were observed on another SIV
negative chimpanzee from the same sanctuary versus
283 CD4 cells/mm3for Ch-Go with the same technique,
and values reported in the literature for healthy SIV
negative chimpanzees range also between 800 and 2,000
CD4 cells/mm3[23].
When applying the CDC and WHO classification sys-
tems to the clinical and biological data available for
Cam155/Ch-Go and reported in this study, the evolu-
tion of the SIV infection in this P. t. troglodytes chim-
panzee currently corresponds to pre-AIDS in humans,
CDC B2 or WHO stage III. The viral loads fluctuated
between 4 and 5 log10copies/ml. Nevertheless, different
techniques were used to measure viral loads in Cam155/
Ch-Go at the different time points, and it cannot be
excluded that the commercial HIV-1 viral load assays
used in this study underestimated values for SIVcpzPtt.
The viral loads observed in Cam155/Ch-Go are in the
range of the values observed for Ch-No (SIVcpzPts-
Ant), the other naturally SIVcpz infected chimpanzee,
although from the P. t. schweinfurthii subspecies and
with an apparent non-progressive SIV infection [8,28].
In the absence of a specific SIVcpz viral load test, com-
parisons over time or with other experimentally or natu-
rally infected animals are difficult. Moreover, in Ch-No,
viral loads fluctuated over time from 3.4 to 5.8 log10
copies/ml and could differ by more than 1 log according
to the technique used [8]. In addition to PCR, quantita-
tive viral isolations have also been done from different
plasma and PBMC dilutions for Ch-No, and important
fluctuations have been observed over time, although
there was no correlation at all between titres of infec-
tious virus in plasma and viral load measured by PCR
[8]. No other data on viral load observed in natural
SIVcpz infections are readily available for comparison
but, for natural non-pathogenic SIVsm and SIVagm
infections in mangabeys and African green monkeys,
viral loads are also generally high [29,30]. In contrast, in
chimpanzees that were previously experimentally
infected with HIV-1, plasma viral loads were undetect-
able or very low, except for the few animals that pro-
gressed to AIDS 4 to 18 years post-inoculation and for
whom viral loads increased over time and could reach
up to 6 log10copies/ml [16]. CD4 decline, severe throm-
bocytopenia, increased plasma viral loads and occur-
rence of opportunistic infections were also observed in
the HIV-1 experimentally infected chimpanzees that
developed AIDS in the Yerkes Primate Center [31]. The
animals that progressed faster to AIDS underwent
superinfections with 2 or 3 strains, which was the case
for the first animal (C499) that was reported with AIDS,
or were infected with the pathogenic strain of this latter
animal.
The naturally SIV infected P. t. schweinfurthii chim-
panzee, Ch-No is still alive and in good health today,
more than 20 years later, despite the relatively high
plasma viral load and a severe and permanent thrombo-
cytopenia that occurred approximately at age 7 [28].
Thrombocytopenia was also seen in the experimentally
infected chimpanzees with AIDS and is observed in
humans and macaques with AIDS [12,32]. Whether the
asymptomatic period for natural SIVcpz is longer than
for HIV in humans, or whether differences in incubation
periods exist like in humans (i.e. rapid versus long-term
progressors), is not known. Thus, it cannot be excluded
that the SIVcpzPts-Ant infected chimpanzee may still
develop a progressive infection.
Given the young age of Cam155 at seizure (1.5 years
old), the chimpanzee was likely infected through vertical
mother-to-child SIV transmission, as chimpanzees are
not sexually active before the age of 8; however, hori-
zontal transmission by blood contact (e.g. biting injuries)
cannot be entirely excluded. In humans, in utero
infected newborns develop AIDS more rapidly compared
to those infected after birth [33], but survival rates and
disease progressions in vertically HIV-1 infected infants
can be variable [34]. Recent studies on SIV pathogeni-
city in wild East African chimpanzees show a higher
mortality rate among infants born to SIV positive
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mothers [13,35]. The majority of the other known
SIVcpz positive captive chimpanzees were most likely
also infected through mother-to-child transmission,
because they were all less than 3-4 years old at time of
rescue (Additional file 1: Table S1) [1-3,6,36,37].
Although they had no signs of AIDS at the time of diag-
nosis, some had chronic lymphadenopathy like Gab1
and cpz-US, or thrombocytopenia as Ch-No. Some died
suddenly from acute infections (Cam5 and Cam13), as
shown in Additional file 1: Table S1 summarizes the
history of the previously reported SIVcpz positive cap-
tive animals; however, whether this was related to the
SIV infection and an eventual degradation of the
immune system is not known.
Phylogenetic analyses revealed that SIVcpzPtt-Cam155
fell within the radiation of the SIVcpzPtt group of viruses,
as part of a clade including all other SIVcpzPtt strains, as
well as HIV-1 groups M and N. However, SIVcpzPtt-
Cam155 clustered most closely with SIVcpzPtt-Gab1 from
northern Gabon and SIVcpzPtt-Cam13 from southwest
Cameroon. We previously reported phylogeographic clus-
tering of SIVcpzPtt strains in Cameroon, and observed
high genetic diversity within small geographic areas.
Although the geographic origin of this animal is not
precisely known, it most likely originated around the Dja
Reserve in south central Cameroon. The SIVcpzPtt-
Cam155 sequence further illustrates the high genetic
diversity among SIVcpzPtt strains in this area [5].
Our data demonstrate an important diversification and
mutation rate of SIVcpzPtt-Cam155 over time, with
nucleotide and amino acid diversity doubling in 5 years
in the envelope, and an evolution of the putative envel-
ope structure leading to escape mutants. Particularly, V1
and V4 loops were highly variable, as similarly observed
in experimentally SIV infected macaques during progres-
sion to simian AIDS [38]. Moreover, variability in V4
region is associated with modification of CD4 binding
and plays a key role in the swarming nature of gp120
[39]. V3 was modified in its crown and V2 was stable, in
contrast to SIVcpzPts-Ant [7] or slow disease progressors
[25,26]. The progressive diversification of HIV in
untreated infected humans underlies its ability to evade
immunologic selective pressure, but this diversification
may also be responsible for disease progression and
destruction of immune system [40]. Overall the evolu-
tionary rate of HIV-1 slows down over time and seems to
be correlated with the slope of the CD4 cell decline. Con-
sidering two time points, the SIVcpzPtt-Cam155 V1V4
nucleotide diversities five years apart (0.0237 in 2004 and
0.0491 in 2009) fit the trends of viral diversification
across HIV-1 infected humans in diverse studies [41,42],
and the theoretical curve established by Lee et al.
describing the evolution of C2V5 HIV diversity over time
[43]. In chimpanzees experimentally infected with HIV-1
[17], a higher viral diversity was seen in the progressor
chimpanzees vs. the non-progressors; however, it has to
be noted that these data originated from animals inocu-
lated with two distinct HIV-1 strains, and recombination
between the different strains could have biased the over-
all diversity observed over time. We also observed an
increase of putative N-linked glycosylation sites over
time in SIVcpzPtt-Cam155 envelope. Specific genetic
modifications leading to the acquisition of PNGS were
shown to result in an evolving protective glycan shield
[44] and to be a characteristic of escape mutants since it
reduces protein epitope exposure and thus facilitates
viral evasion of antibody neutralization [45].
Conclusion
Our study provides additional evidence that SIVcpz
infection is associated with clinical disease in chimpan-
zees and that it affects both Eastern and Central African
chimpanzee subspecies. We also showed SIVcpzPtt viral
diversification and adaptation in its natural host. Only a
future detailed and regular clinical, immunological and
virological follow-up on naturally infected animals over
time will allow us to determine to what extent SIVcpz
infection resembles that of HIV-1 in humans. Given the
poor health status of the animal described in this study,
the administration of antiretroviral therapy may be
necessary in the near future in order to avoid further
progression to AIDS and to ensure the lengthened survi-
val of this chimpanzee. Although studying the impact of
SIV infection in wild chimpanzees is difficult because
they live in isolated forest regions, efforts should be
made to monitor health status in ape populations to
find out whether SIV infection plays a role in population
decline, in addition to habitat destruction, poaching and
other disease pressures, such as Ebola virus. No data are
currently available on the pathogenicity of SIV in goril-
las. Nevertheless, as gorillas are infected with SIVgor,
most likely through cross-species transmission of SIVcpz
from chimpanzees, it is probable that SIVgor also has a
negative impact on the health of gorillas in the wild.
The fact that chimpanzees naturally infected with SIV
have been rescued (7 individuals between 1988 and
2008) further indicates that humans hunting apes are
exposed to SIVs and are at risk for cross-species trans-
mission of SIVcpz. Such cross-species transmissions pre-
sent a risk of potential emergence of new strains in the
human population, which could make HIV treatment
and vaccine development more difficult.
Methods
Serological testing of the SIVcpzPtt-Cam155 infected
chimpanzee
In November 2003, a 1.5 year old male chimpanzee
(Cam155) was seized by the Ministry of Environment
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and Forestry from the area around the Dja reserve,
south central Cameroon. During his quarantine in
December 2003, the animal was screened for SIV infec-
tion with HIV screening tests, i.e. a rapid test (Multispot
HIV-1/HIV-2 Rapid Test (Bio-Rad, Marnes-la-Coquette,
France)), an indirect ELISA (HIV-1 and HIV-2 GenEla-
via Mixt (BioRad)), and a competitive Elisa (Wellcozyme
rec HIV-1 (Murex/Abbott, Dartford, Kent, UK)) [46].
These reactions were confirmed by western-blot analysis
(New Lav Blot HIV-1/2, Bio-Rad). Serological tests for
other viral infections were also performed: hepatitis A
virus (HAV) by Monolisa™ anti HAV IgM EIA
(BioRad), HBV using Monolisa™ Ag HBS plus (BioRad),
and HCV using Monolisa™ anti-HCV plus, version 2
(BioRad), and simian T-lymphotropic virus using Platel-
lia™, HTLV-1/2 (BioRad).
Full-length sequence of the SIVcpzPtt-Cam155 strain and
envelope clones
Total DNA was extracted from leftover buffy coat or
PBMCs using the QIAamp blood kit (Qiagen, Courta-
boeuf, France). Full-length sequences of the SIVcpz
strains infecting Cam155 in 2004 (9,899 bp) and 2009
(9,870 bp) were generated by amplifying partially over-
lapping subgenomic fragments (1,100 bp to 3,950 bp
in length) using SIVcpz/HIV-1 consensus primers and
SIVcpzPtt-Cam155 specific primers (Additional file 2:
Table S2, Figure 1). All PCR reactions were performed
with the Expand Long Template PCR system (Roche
Diagnostics, Indianapolis, IN) and PCR conditions
were as previously described [5]. The resulting ampli-
fication products were gel purified (Geneclean Turbo
Kit, Qbiogene, Carlsbad, CA) and directly sequenced
on an automated sequencer (3130xl Genetic Analyser,
Applied Biosystems, Foster City, CA), except for the
V1V4 env region for which clonal sequences were
necessary since chromatograms from direct sequence
analysis could not be resolved. We amplified a frag-
ment spanning the V1V4 env region (1,105 bp) of
SIVcpzPtt-Cam155 from 2004 and 2009 with specific
primers (Additional file 2: Table S2, Figure 1). The
amplified and gel purified products were cloned fol-
lowing the manufacturer’s instructions (pGEM-T easy
vector system II, Promega, Madison, WI) and ten SIV
clones for each time point were sequenced to analyse
the viral envelope diversity. SIVclone041 and SIV-
clone091 were arbitrarily selected to cover the V1V4
env region in the full-length sequences of SIVcpzPtt-
04Cam155 and SIVcpzPtt-09Cam155 respectively.
Phylogenetic and genetic diversity analyses
Phylogenetic analyses were performed for each main
gene of the SIV genome, but the Pol region was divided
in two fragments according to the recombination point
observed for other SIVcpz and HIV-1 N viruses [4].
SIVcpzPtt-04Cam155 and SIVcpzPtt-09Cam155 Gag,
Pol1, Pol2, and Env amino acid (aa) sequences were com-
pared to previously published SIVgor, SIVcpz and HIV-1
references. Sequences were aligned using Mega4 [47] and
where necessary, minor manual adjustments were per-
formed. Sites that could not be unambiguously aligned or
contained a gap in any sequence were excluded from the
analyses. In the end, 422 amino acids were examined for
Gag analysis, 700 aa for Pol1, 251 aa for Pol2, and 673 aa
for Env. Maximum likelihood (ML) trees were con-
structed using PhyML http://www.atgc-montpellier.fr/
with 1,000 bootstrap replicates [48]. Phylogenies were
also inferred by the Bayesian method [49], implemented
in MrBayes version 3.1 [50], run for 3,000,000 genera-
tions, and trees sampled every 100 generations, the first
25% being discarded as burn-in. Parameters were exam-
ined with the Tracer program http://tree.bio.ed.ac.uk/
software/tracer/. For the Gag, Pol2, and Env regions, the
Jones, Taylor and Thornton (JTT) model for protein
evolution [51] with a gamma distribution [52] across sites
was the most appropriate model according to TOPALI
[53] and Bayesian estimation [50]. Nevertheless, for Pol1
analysis, the RtREV model [54] was found to be the best
model. Phylogenetic analyses were also performed for the
V1V4 region using a codon nucleotide alignment of 648
bp, once the gaps discarded. The best evolution model
was the general time-reversible (GTR) model with a
gamma distribution across sites. The phylogenetic ana-
lyses were run with both PhyML and Mr Bayes with the
same characteristics as shown above.
Diversity plots were made using a sliding window of
300 nucleotides and moved in steps of 50 residues. The
cumulative number of non-synonymous and synon-
ymous nucleotide substitutions (dN and dS) was
estimated using SNAP [24]. Viral diversity of SIVcpzPtt-
Cam155 V1V4 env region in 2004 and 2009 was deter-
mined by calculating pairwise nucleotide and amino
acid distances between V1V4 env clones with Mega4
[47] with the Tamura and Nei method [55] and the
Gamma distance method, respectively. At each time
point (2004 and 2009), the mean, minimum, maximum,
and standard deviation of amino acid and nucleotide
diversities were calculated. Amino acid sequence length
and putative N-linked glycosylation diversity, which
were five years apart, were compared. P values were
estimated by a Mann-Whitney test to assess the statisti-
cal differences between 2004 vs 2009 viral diversity,
length variation and PNGS.
Accession numbers
GenBank accession numbers for complete genome
sequences used in comparative analyses are as follows:
SIVcpzPts: ANT (U42720), TAN1 (AF447763), TAN2
(DQ374657), TAN3 (DQ374658); SIVcpzPtt: MB897
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(EF535994), LB7 (DQ373064), MB66 (DQ373063), EK505
(DQ373065), CAM5 (AJ271369), DP943 (EF535993),
CAM3 (AF115393), US (AF103818), GAB1 (X52154),
CAM13 (AY169968); SIVgor: CP684 (FJ424871), CP2135
(FJ424863); HIV-1 group M: subtype A U455 (M62320),
subtype B HXB2 (K03455); HIV-1 group N: YBF106
(AJ271370), YBF30 (AJ006022); HIV-1 group O: MVP5180
(L20571), ANT70 (L20587); HIV-1 group P: RBF168
(GQ328744).
The two complete genomes SIVcpzPtt-04Cam155 and
SIVcpzPtt-09Cam155 are available under accession
numbers [EMBL:FR686510-1], and the 20 envelope
clones spanning the V1V4 env region are under
accession numbers [EMBL:FR686512-31].
Additional material
Additional file 1: Table S1 Naturally SIVcpz infected captive
chimpanzees reported in the literature. Table summarizing the history
of the 9 SIVcpz positive captive chimpanzees, with details on their
capture, their SIV strain, their current status and remarks of interest.
Additional file 2: Table S2 Primer sets used to amplify SIVcpzPtt-
04Cam155 and SIVcpzPtt-09Cam155 PCR fragments.
Acknowledgements
We would like to thank the Cameroon Ministry of Scientific Research and
Innovation, the Ministry of Health and the Ministry of Forestry and Wildlife,
which provided the necessary permits for this work. We thank the staff from
PRESICA and the Centre Pasteur for logistical support in Cameroon. Financial
support for this study was obtained by grants from the National Institutes of
Health (R01 AI50529), the Agence Nationale de Recherche sur le SIDA,
France (ANRS 12182) and the Institut de Recherche pour le Développement
(IRD). Lucie Etienne is supported by a grant from Sidaction. Nathan D Wolfe
is supported by the NIH Director’s Pioneer Award (DP1-OD000370), Global
Viral Forecasting Initiative is supported by http://google.org, the Skoll
Foundation, the Henry M. Jackson Foundation for the Advancement of
Military Medicine, and the Global Emerging Infections Surveillance and
Response System (GEIS) - a Division of the United States Armed Forces
Health Surveillance Center. Additional support was provided to GVFI by the
United States Agency for International Development (USAID) Emerging
Pandemic Threats Program, PREDICT project, under the terms of Cooperative
Agreement Number GHN-A-OO-09-00010-00. We also thank the US Embassy
in Cameroon for their support. Additional thanks to the management and
staff of Mfou National Park and Ape Action Africa and in particular to Mr Avi
Sivan, Director of the Cameroon Wild Aid Fund, and Rachel Hogan, Mfou
Park Manager, for their continued support. We thank Joseph Fair and Nancy
Ortiz from GVFI for critical reading of the manuscript. We thank Leo
Heyndrickx for providing the sequential envelope sequences from Ch-No.
Author details
1UMR145, Institut de Recherche pour le Développement (IRD) and Université
Montpellier 1, Montpellier, France.2HIV laboratory, Centre Pasteur du
Cameroun, Yaounde, Cameroon.3HIV/Hepatitis Laboratory, Pasteur Institute,
Phnom Penh, Kingdom of Cambodia.4Global Viral Forecasting (GVF), San
Francisco, USA.5Apes Action Africa, Yaoundé, Cameroon.6Virology
Laboratory CRESAR/IMPM/IRD, Yaoundé, Cameroon.7Stanford University,
Program in Human Biology, Stanford, California, USA.
Authors’ contributions
LE performed all molecular, phylogenetic and diversity analyses with the
contribution of AA. EN, NW, ML, and UT initiated and coordinated collaboration
between Yaoundé Zoo/Sanctuary and the laboratory of virology of Centre
Pasteur du Cameroun (CPC). EN supervised all activities related to initial
diagnosis, virological and immunological monitoring with YF, AA and DR. AFA,
ML, AN, CD and EM continued clinical and laboratory monitoring. GTB rescued
the animal and is involved in daily care of Cam155/Ch-Go. ED and EM provide
advice on clinical monitoring. LE, AA, ML and MP wrote the manuscript. AA, ML,
NW and MP coordinated the study.
Competing interests
The authors declare that they have no competing interests.
Received: 18 October 2010 Accepted: 13 January 2011
Published: 13 January 2011
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doi:10.1186/1742-4690-8-4
Cite this article as: Etienne et al.: Characterization of a new simian
immunodeficiency virus strain in a naturally infected Pan troglodytes
troglodytes chimpanzee with AIDS related symptoms. Retrovirology 2011
8:4.
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