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Dispatch
Divergent HIV and Simian Immunodeficiency
Virus Surveillance, Zaire
Amanda Schaefer,* Kenneth E. Robbins,* Eugene Nzila Nzilambi,† Michael
E. St. Louis,* Thomas C. Quinn,‡ Thomas M. Folks,* Marcia L. Kalish,*
and Danuta Pieniazek*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; †Project
SIDA, Kinshasa, Democratic Republic of Congo; and ‡National Institutes
of Health, Bethesda, Maryland, USA
Suggested
citation for this article
Recent HIV infection
or divergent HIV or simian immunodeficiency virus (SIV) strains may
be responsible for Western blot–indeterminate results on 70 serum samples
from Zairian hospital employees that were reactive in an enzyme immunoassay.
Using universal polymerase chain reaction HIV-1, HIV-2, and SIV primers,
we detected 1 (1.4%) HIV-1 sequence. Except for 1 sample, no molecular
evidence for unusual HIV- or SIV-like strains in this sampling was found.
HIV-1 and HIV-2 are believed to be the result of cross-species transmission
from simian immunodeficiency virus (SIV)–infected chimpanzees and sooty
mangabeys, respectively, which represent 2 (SIVcpz and SIVsm) of the 6
major lentiviral phylogenetic lineages (1,2). No evidence
exists that SIV strains from the remaining nonhuman primate lineages have
infected humans, although many grow in human cells in vitro as do SIVcpz
and SIVsm (3). Since humans are exposed to a plethora
of primate lentiviruses through blood or body fluids during hunting, butchering,
eating bushmeat, and keeping primates as pets (3), the
potential exists for zoonotic transmission of diverse primate lentiviruses
in many parts of sub-Saharan Africa, including the Congo River basin (4).
This potential is supported by the identification of a Cameroonian man
whose HIV serologic results were indeterminate but whose serum specimen
reacted strongly and exclusively with an SIVmnd V3 loop peptide (5).
An even more compelling case for cross-species exposure is the recent
finding of a Cameroonian man who may have been exposed to a colobus SIV,
as indicated by a strong humoral (env IDR and V3) and a weak cellular
(gag) immune response (6). Although SIV sequences
were not identified in either case, the findings suggest that humans are
probably exposed to different simian retroviruses that can establish new
infections in humans (3).
Nonhuman primates infected with SIV from the currently recognized lineages
can harbor antibodies that serologically cross-react with some HIV-1 or
HIV-2 antigens (3). In many cases, HIV Western blots
(WBs) with indeterminate profiles of SIV-infected monkeys resemble those
of HIV enzyme immunoassay (EIA)–positive, WB-indeterminate human sera
from Africa. In general, such indeterminate African sera demonstrate a
broad range of reactivity to HIV-1 proteins, in contrast to predominant
p24 reactivity in WB-indeterminate sera from persons in the United States
(7–9). These data suggest that the WB-indeterminate patterns
in HIV EIA-reactive sera from persons living in Africa may reflect more
than just a recent HIV infection or an infection with a highly divergent
HIV-1 strain (10); they may reflect either cross-reactivity
with unknown pathogens of African origin or exposure to new HIV- or SIV-like
strains.
The Study
We investigated the presence of HIV or SIV in 70 HIV EIA-reactive, WB-indeterminate
serum specimens collected in 1984 and 1986 from employees of Mama Yemo
Hospital in Kinshasa, Zaire (currently the Democratic Republic of Congo)
(11). WB bands were identified by using an HIV-1/2 WB
assay (version 2.2, Genelabs Diagnostics, Singapore) and classified as
indeterminate according to criteria for interpreting HIV-1 (http://www.cdc.gov/mmwr/preview/mmwrhtml/00001431.htm).
Briefly, a positive WB result indicates reactivity to at least p24 and
1 of the 3 env (gp41, gp120, or gp160) proteins with banding intensity
at least as strong as that seen in the weak-positive control; a negative
WB result indicates no reactivity and an indeterminate WB result indicates
reactivity to at least 1 protein.
Of the 70 WB-indeterminate serum specimens, 69 showed a broad range of
HIV-1 WB-indeterminate band patterns, and 1 had an HIV-2 WB-indeterminate
pattern with weak reactivity against gp36, p68, and gp80 proteins. Analysis
of HIV-1 WB-indeterminate patterns provided several important observations.
First, most of the specimens (41/69, 59%) showed reactivity to multiple
viral proteins (Figure), whereas the remaining specimens
(28/69, 41%) demonstrated reactivity against single HIV-1 proteins, including
p24 (13/69, 18.8%), p17 (n = 8, 11.6%), gp160w (w = weak) (n = 4, 5.8%),
and p51, p66, and gp41w (n = 1 each; 1.4% each). Second, in all but 3
specimens the WB-indeterminate patterns with reactivity to multiple viral
proteins combined proteins of p24 or p17 gag or both with others, giving
the following 21 profiles: p17/p24 (14/69, 20.3%); p24/p66 and p17/p24/p66
(n = 3 each, 4.3% each); p24/p51, p17/p24/p51, and p17/p24/gp41w (n =
2 each, 2.9% each); p17/p66, p17/gp160, p24/gp160, p24/p51/gp41w, p17/p51/p66,
p17/p31/gp41w, p24/p51/p55, p24/p51/p66, p17/p24/p51/p66, p17/p24/p51/p55,
p17/p24/p55/p66, p24/p31/p51/p55, p31/p66, p51/gp41w, and p31/p66/gp41w
(n = 1 each, 1.4% each). Overall, this analysis indicated reactivity to
8 HIV-1 proteins that occurred as single bands or multiple combinations.
Whereas reactivity to p24 might represent either early infection with
HIV or reaction to nonspecific antigens (8), reactivity
to other HIV-1 proteins (p17, p31, p51, p55, p66, gp41, or gp160) could
reflect at least exposure to HIV-1, HIV-2, or SIV variants as well as
cross-reactivity with yet unidentified HIV- or SIV-like strains (3,10).
To determine whether HIV- and SIV-like RNA could be detected in the 70
WB-indeterminate specimens, we attempted reverse transcription–polymerase
chain reaction (RT-PCR) amplification of HIV-1, HIV-2, and SIV. However,
because of a small volume (<300 mL) of some serum specimens,
a limited number of primers was used. We selected 4 sets of primers for
3 highly conserved gene regions, including env-gp41, pol
protease, and pol integrase, which we previously developed and
extensively tested for the efficient PCR amplification of field specimens.
Briefly, gp41 primers allow the amplification of HIV-1 groups M (subtypes
A–K and U [unclassifiable]) N, and O and SIVcpz with an efficiency of
95% (13); HIV-1 type specific protease primers efficiently
(>95%) detect group M viruses (subtypes A–K and U [unclassifiable]),
and allow successful amplification of some PCR–gp41 negative specimens.
HIV-2 type-specific protease primers allow the amplification of HIV-2
at least subtypes A and B (13), and HIV-2/SIV integrase
primers amplify HIV-2 and at least 5 major SIV lineages including SIVcpz,
SIVsm, SIVagm (African green monkey), SIVmndBK12 (mandrill), and SIVsyk
(Sykes monkey) (14). The integrase primers should also
amplify other SIV lineages including SIVcol, SIVLhoest/SIVsun, SIVgsn,
SIVmus, SIVmon, SIVtal, and SIVdeb (3). This prediction
is based on the fact that genetic diversity of these sequences within
the 3´-end of primer regions was the same as in strains previously
used for testing (15), which was clearly visible through
DNA alignment of the integrase primers with all SIV genomic integrase
sequences deposited in GenBank. Positive controls for PCR amplification
included HIV-1 Mn, HIV-2 ROD, and SIVsm. Using this approach, we amplified
a 297-bp HIV-1 protease (PR) gene from only 1 (1.4%) of the 70 serum specimens.
The remaining 69 specimens were PCR negative for all the primers tested.
Phylogenetic analysis of the PR gene sequence showed a close phylogenetic
relationship with the HIV-1 MAL strain (Figure),
a recombinant virus that contains portions of subtypes A and D and an
unclassifiable region that was identified in 1985 in Zaire (16).
Conclusions
The PR gene sequence was identified in the serum of the person with reactivity
to only the p17 gag band by WB, which suggests that this person
was recently infected and that antibodies to all the HIV-1 antigens had
not yet developed. No information was available on demography, risk, or
clinical status of this person. In the remaining 69 WB-indeterminate specimens,
we could not rule out exposure to SIV from nonhuman primates, from handling
infected animal meat before consuming it, or from keeping monkeys as pets.
Although the limited molecular scope of this study and the quality of
the old serum specimens may not be adequate for molecular confirmation
of such viruses, our findings of complex HIV WB-indeterminate patterns
with reactivity to multiple viral proteins in serum specimens from persons
living in the Democratic Republic of Congo provide comprehensive insights
into HIV WB-indeterminate sera in the mid-1980s.
Ms Schaefer is a
biologist at the Laboratory Branch, Division of HIV/AIDS Prevention,
National Center for HIV, STD, and TB Prevention, Centers for Disease
Control and Prevention. Her current area of interest is investigations
of divergent HIV strains and SIV-like variants of public health importance.
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Suggested citation
for this article:
Schaefer A, Robbins
KE, Nzilambi EN, St. Louis ME, Quinn TC, Folks TM, et al. Divergent HIV
and simian immunodeficiency virus surveillance, Zaire. Emerg Infect Dis
[serial on the Internet]. 2005 Sep [date cited]. Available from
http://www.cdc.gov/ncidod/EID/vol11no09/05-0179.htm
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