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Molecular screening for HIV-1 group N and simian immunodeficiency virus cpz-like virus infections in Cameroon

Fonjungo, Peter N.a; Dash, Bipin C.a; Mpoudi, Eitel N.c; Torimiro, Judith N.c; Alemnji, George A.c; Eno, Laura T.c; Nkengasong, Johnbd; Rayfield, Marka; Folks, Thomas M.a; Pieniazek, Danutaa; Lal, Renu B.a

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aHIV/AIDS and Retrovirology Branch, Division of AIDS, STD and TB Laboratory Research, National Center for Infectious Diseases and bInternational Activity Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS/TB/STD, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA; cHopital Militaire de Yaounde, Yaounde, Cameroon; and dProjet RETRO-CI, Abidjan, Côte d'Ivoire.

Received 7 January 2000; accepted: 14 January 2000.

On the basis of genetic diversity, HIV-1 has been divided into three groups, M (major), O (outlier) and N (nonM-nonO) [1–3]. HIV-1 group M infections are common worldwide, group O infections are restricted to central Africa, whereas group N infections have only been identified in Cameroon [3–6]. It was further shown that HIV-1 group N is a mosaic of divergent HIV-1 and simian immunodeficiency virus (SIV)cpz lineages, indicating possible co-infection and recombination in an ancestral chimpanzee host [7]. These data suggest that zoonotic transmission of lentiviruses plays an important role in the emergence of human retroviruses [7,8]. Given that the conditions favoring human exposure to chimpanzees have increased in Cameroon and other parts of Africa because of commercial logging and hunting practices, it is possible that additional new transmissions of this virus may have occurred. Therefore, more studies are needed to assess the prevalence of HIV-1 group N and SIVcpz viruses to obtain a better understanding of their spread and their continued emergence into the human population.

All civilian applicants to the Cameroon army during September–November 1998 were screened for antibodies to HIV using HIV-1/HIV-2 enzyme immunoassay kits (Murex and Genetic Systems Corporation, Redmond, USA). We obtained randomly selected specimens from 196 of these individuals, including 132 HIV-1-seropositive and 64 seronegative individuals for the present study. The viral RNA extraction and polymerase chain reaction (PCR) were performed essentially as described previously, using the primers in integrase (intM-Z) and gp41 (gpM-Z) regions [9]. The target sizes were 445 base pairs for the gpM-Z primer set and 263 bp for the intM-Z primer set [9]. PCR products were directly sequenced using both forward and reverse nested PCR primers, and phylogenetic analysis was performed in a similar manner to methods described previously [10,11].

PCR analysis of HIV-1 seropositive specimens using primers specific for the env gp41 primers that amplify all known HIV-1 as well as SIVcpz [9], revealed that 123 out of 132 (93.2%) could be amplified (Table 1). Phylogenetic analysis of the gp41 region from 123 seropositive individuals showed that the majority (98%) of viruses identified were group M and the remainder (2%) were group O. Within group M, subtype A viruses accounted for the majority of infections (with more than 70% of the sequences showing close similarity to IbNg-like sequences), whereas the remainder were subtypes D (10%), G (9%), and F (4% in subcluster F2) or were unclassifiable (2%) (Table 1). These data are in accordance with recent studies showing the presence of all known subtypes of HIV-1 and low prevalence of group O viruses in Cameroon [4–6,12]. Among the HIV-1-seropositive specimens, none of the gp41 sequences clustered with the new HIV-1 group N or with the SIVcpz-like viruses (Table 1).

Table 1
Table 1
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All seronegative specimens were tested for the presence of highly divergent HIV-1 group N as well as SIVcpz-like viruses using recently developed molecular detection assays [9–11]. Although both primers amplified the viral RNA from the cultured supernatant of HIV-1 group N isolate (YBF-30) as well as RNA from the plasma of a chimpanzee infected with the SIVcpzANT and SIVcpzUS virus, none of the 64 seronegative samples gave a positive amplification (Table 1). The majority of the specimens in the present investigation were from persons residing in the central–south Cameroon, a region with dense equatorial forest vegetation and where bush meat hunting and household chimpanzee pets are commonly found. Despite our sampling, which was concentrated in the central and western parts of the country, we did not find evidence of chimpanzee-like viruses in this study population. Several reasons may explain the apparent lack of SIVcpz-like viruses, including the possibility that the introduction of such viruses into the human population may be occurring rarely or may require a long duration or additional adaptive events in their new host before spreading. Despite the use of sensitive molecular screening analyses, we thus found no evidence of infection with HIV-1 group N and SIVcpz-like viruses in this limited survey in Cameroon. These results are in agreement with our recent report [13], in which a population-based surveillance in the United States of persons born in central Africa who were at high risk for group O infections did not reveal the presence of group N infections. Further studies examining seronegative individuals with AIDS-defining illness or persons with atypical serologies will be needed to study the emergence of HIV variants. An analysis of a handful of HIV-1-infected individuals from the USA [14], with AIDS-defining illness but persistently HIV-1 seronegative, have identified all to be infected with HIV-1 subtype B virus. Additional molecular epidemiological studies are needed to assess the prevalence of group N viruses and the mechanisms influencing primate lentivirus zoonosis in order to understand their spread and impact on the epidemic.

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Acknowledgement

The authors are grateful to Dr Martine Peeters for providing F2 and F3 sequences as well as helpful comments on the manuscript.

Peter N. Fonjungoa

Bipin C. Dasha

Eitel N. Mpoudic

Judith N. Torimiroc

George A. Alemnjic

Laura T. Enoc

John Nkengasongbd

Mark Rayfielda

Thomas M. Folksa

Danuta Pieniazeka

Renu B. Lala

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References

1. UNAIDS/WHO. Reports on the Global HIV/AIDS Epidemic, June 1998. Geneva, Switzerland: World Health Organization (http://www.who.int/emc-hiv/global report/indexx.html).

2. Carr JK, Foley BT, Leitner T, Salminen M, Korber B, McCutchan F. Reference sequences representing the principal genetic diversity of HIV-1 in the pandemic. In:Human retrovirus and AIDS. Korber B, Kuiken C, Foley B, et al. (editors). Los Alamos, New Mexico: Los Alamos, National Laboratory; 1998. pp. III10–III19.

3. Simon F, Mauclere P, Roques P. et al. Identification of a new human immunodeficiency virus type 1 distinct from group M and group O. Nat Med 1998, 4: 1032–1037.

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7. Gao F, Bailes E, Robertson DL. et al. Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 1999, 397: 436–441.

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9. Yang C, Dash B, Simon F, et al. Detection of diverse variants of HIV-1 groups M, N and O and simian immunodeficiency viruses from chimpanzee using genericpolandenvprimers. J Infect Dis 2000, in press.

10. Pieniazek D, Yang C, Lal RB. Phylogenetic analysis of gp41 envelope of HIV-1 group M, N, and O strains provides an alternate region for subtype determination. In:Human retrovirus and AIDS. Korber B, Hahn BH, Foley B, et al. (editors). Los Alamos, New Mexico: Los Alamos National Laboratory; 1998. pp. III112–III117.

11. Yang C, Pieniazek D, Owen SM. et al. Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma using highly sensitive and specific generic primers. J Clin Microbiol 1999, 37: 2581–2586.

12. Triques K, Bourgeois A, Saragosti S. et al. High diversity of HIV-1 subtype F strains in Central Africa. Virology 1999, 259: 99–109.

13. Sullivan P, Do A, Ellenberger D, et al. HIV subtype surveillance of African-born persons at risk for group O and group N HIV infection in the United States.J Infect Dis 2000, in press.

14. Ellenberger D, Sullivan P, Dorn J, Schable C, Spira TJ, Folks TM, Lal RB. Viral and immunologic examination of human immunodeficiency virus type 1-infected, persistently seronegative persons. J Infect Dis 1999, 180: 1033–1042.

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