Skip to main content

A predominance of R5-like HIV genotypes in vaginal secretions is associated with elevated plasma HIV-1 RNA levels and the absence of anti-retroviral therapy


HIV expressed in genital secretions provides the inoculum from which transmitting variants are selected, both in sexual transmission and mother-to-infant transmission during partuition. Characterization of HIV levels and genotypes found in vaginal secretions and the impact of anti-retroviral therapy (ART) on this virus can provide valuable insight for the prevention of HIV transmission. Vaginal HIV was evaluated in a cohort of 43 women attending a New Orleans HIV outpatient clinic. Predominant vaginal genotypes were characterized as R5- or X4-like by heteroduplex tracking analyses of the envelope V3 region. Most women (67.4%) shed R5-like genotypes in vaginal secretions which was associated with elevated plasma HIV levels (≥ 10,000 copies HIV-RNA/mL) and absence of ART. Because R5-like genotypes are more frequently associated with transmission, these observations suggest that the majority of women shedding HIV in genital secretions present a transmission risk. The levels of vaginal virus were similar between both groups, but shedding of X4-like genotypes was associated with lower plasma viral loads and the use of ART, suggesting that ART use may impact the genotypes of virus found in the female genital compartment.


Approximately half of the world's HIV-infected population is female, and the rate of infection is increasing in this population [1]. Mother-to-infant transmission of HIV continues to be a dire issue in endemic areas such as Sub-Saharan Africa and the Caribbean [1, 2]. Currently, heterosexual transmission is the primary route of HIV infection [1], and studies of discordant couples in Sub-Saharan Africa highlight a growing problem of female to male sexual transmission events [3, 4]. Because virus contained in vaginal secretions likely comprises the inoculum from which transmitting variants are selected in mother-to-infant transmission during partuition, as well as through sexual contact, identifying the specific properties and genotypes of virus expressed in this compartment will aid in the design of strategies to prevent HIV transmission.

Studies have described the compartmentalized replication of vaginal HIV as compared to plasma [5, 6]. Viral tropism and the ability of virions to infect cell types that may traffic to specific tissues, such as the vaginal mucosa, are influenced by the virus' envelope sequence, which determines the molecules utilized for entry [7]. The nucleotide sequence of the HIV envelope V3 region determines whether the virion utilizes a CCR5 (R5) or CXCR4 (X4) coreceptor molecule, therefore the V3 genotype can impact compartmentalized replication of virus in the genital mucosa [5, 7, 8].

To evaluate the properties of HIV expressed in the genital tract and the potential impact of ART on genital tract viral genotypes, we measured HIV-1 RNA levels in vaginal secretions and plasma, determined the V3 genotype of predominant variants found in vaginal secretions, and assessed ART use in a cohort of 43 women attending an HIV outpatient clinic in New Orleans. Informed consent was obtained in accordance with Louisiana State University Health Sciences Center and Tulane Medical Center IRB approval. ART use was evaluated by the subject's answer to the question "Are you currently taking ART?" on a survey questionnaire completed at the study visit. ART use was also confirmed by review of medical chart data. Vaginal secretions were collected from the vaginal vault on Dacron swabs and subsequently eluted in phosphate-buffered solution. Supernatant and cell fractions were stored at -80°C within three hours of collection. Samples were not collected during menses, and overtly blood-contaminated samples were excluded. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) anti-coagulant tubes, and plasma aliquots were stored frozen at -80°C. HIV contained in plasma and vaginal secretion supernatant samples was collected by centrifugation and virion-associated HIV-1 RNA copies were quantified using the Roche Amplicor™ Ultra-Sensitive HIV-1 Monitor Assay, according to assay protocol.

Samples from a cohort of 43 women with measurable levels (>50 copies/sample) of vaginal HIV-1 RNA were available for genotypic analysis in this study. To evaluate viral genotypes shed in vaginal secretions, the envelope V3 region was amplified by nested RT-PCR from the remaining isolated RNA and analyzed by a heteroduplex tracking assay (HTA) based on an established protocol [9]. This methodology provides a facile means to evaluate nucleotide changes between a reference sequence and sequences amplified from each vaginal sample. This protocol has been utilized and validated for analyses of HIV genotypes and prediction of coreceptor usage [913]. The genotypic evaluations by heteroduplex analysis in this study were conducted using sample-derived HIV-V3 PCR products, pooled from 2–4 independent PCR amplications. The 334 base pair V3 PCR products were mixed with a single-stranded 32P-radiolabeled probe containing the V3 sequence of an X4-utilizing strain, HIVHXB. Following electrophoresis on a non-denaturing acrylamide gel, mobility ratios of the heteroduplexes formed between sample and probe V3 sequences were analyzed and used to determine the predominant genotype of the patient sample, as described [9, 12, 13]. R5-like genotypes had a reduced mobility on the gel when heteroduplexed with the X4-like sequence probe (mobility ratio < 0.91), while heteroduplexes formed between patient X4-like sequences and the X4 probe had higher mobility ratios (>0.91).

A wide range of viral levels in both the plasma and vaginal compartments was observed in this cohort, 50 to 5 × 105 copies/mL plasma and 50 to 2 × 105 copies/vaginal sample. For statistical analysis, HIV RNA levels in both plasma and vaginal secretions were categorically designated as high-level HIV or low-level HIV for each, as defined in Table 1. The majority of women, 67.4%, had R5-like genotypes in vaginal secretions, while 32.6% shed X4-like genotypes. To identify viral and clinical factors associated with vaginal X4- or R5-like genotypes, we evaluated plasma and vaginal viral levels, ART use, and CD4+ T lymphoctye counts using Chi-square and Fisher's exact tests (Table 1). R5-like vaginal HIV genotypes were associated with high levels of plasma virus and the absence of ART. Conversely, X4-like genotypes in vaginal fluids correlated with lower plasma levels and use of ART. Levels of HIV in vaginal secretions were similar between both groups.

Table 1 Vaginal Genotypic Associations in 43 Women

The presence of predominantly R5-like genotypes in the vaginal compartment has significant implications for HIV transmission. Genotypes that utilize the CCR5 coreceptor are most commonly transmitted in both sexual and mother-to-infant transmission [14]. This collective data suggests the majority of women shedding vaginal HIV present a high transmission risk. Furthermore, vaginal R5-like genotypes were associated with absence of ART use. Reasons for ART use/non-use were not documented for this cohort and are often multifactorial, especially in women. Subjects taking ART (n = 23) and those not taking ART (n = 20) had similar levels of peripheral CD4+ T lymphocytes, indicating that advanced disease was not the main factor driving ART use in this cohort. Additionally, subjects shedding R5-like genotypes as compared to women with X4-like vaginal genotypes had similar levels of peripheral CD4+ T lymphocytes (Table 1). Plasma levels of HIV were lower in ART users as compared to non-users, however ART users represented more than half of the women in this shedding cohort, demonstrating that vaginal HIV shedding occurs in a number of women taking ART. ART use was associated with the presence of predominantly X4-like variants in vaginal secretions, which are less likely to be transmitted [14]. These observations suggest that ART effectively controls the expression of R5-like genotypes in the vaginal compartment.

Due to sample availability, HTA analyses were not conducted on matched plasma samples. Although it is possible to harbor dual-tropic viral variants [9, 13], the low levels of HIV detected in many of the samples only allowed the analysis of the predominant genotypes in genital secretions. Despite this, and the inherent limitations of PCR amplification methodologies, these preliminary findings demonstrate ART's impact on genital HIV genotypes. These data provide a basis for future evaluations of HIV treatment in women to fully examine compartmentalized vaginal expression and the effectiveness of ART on X4-like and R5-like genotypes in vaginal secretions as a means to reduce transmission risk.



Human immunodeficiency virus


Antiretroviral therapy


Chemokine (C-C motif) receptor 5


Chemokine (C-X-C motif) receptor 4


Ribonucleic acid


Polymerase chain reaction


Institutional review board.


  1. UNAIDS: 2007 AIDS Epidemic Update--Global Summary of the HIV and AIDS Epidemic. 2007.

    Google Scholar 

  2. Quinn TC, Overbaugh J: HIV/AIDS in women: an expanding epidemic. Science 2005,308(5728):1582-1583. 10.1126/science.1112489

    Article  CAS  PubMed  Google Scholar 

  3. Fideli US, Allen SA, Musonda R, Trask S, Hahn BH, Weiss H, Mulenga J, Kasolo F, Vermund SH, Aldrovandi GM: Virologic and immunologic determinants of heterosexual transmission of human immunodeficiency virus type 1 in Africa. AIDS Res Hum Retroviruses 2001,17(10):901-910. 10.1089/088922201750290023

    Article  PubMed Central  PubMed  Google Scholar 

  4. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F, Meehan MO, Lutalo T, Gray RH: Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med 2000,342(13):921-929. 10.1056/NEJM200003303421303

    Article  CAS  PubMed  Google Scholar 

  5. Kemal KS, Foley B, Burger H, Anastos K, Minkoff H, Kitchen C, Philpott SM, Gao W, Robison E, Holman S, Dehner C, Beck S, Meyer WA 3rd, Landay A, Kovacs A, Bremer J, Weiser B: HIV-1 in genital tract and plasma of women: compartmentalization of viral sequences, coreceptor usage, and glycosylation. Proc Natl Acad Sci U S A 2003,100(22):12972-12977. 10.1073/pnas.2134064100

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Sullivan ST, Mandava U, Evans-Strickfaden T, Lennox JL, Ellerbrock TV, Hart CE: Diversity, divergence, and evolution of cell-free human immunodeficiency virus type 1 in vaginal secretions and blood of chronically infected women: associations with immune status. J Virol 2005,79(15):9799-9809. 10.1128/JVI.79.15.9799-9809.2005

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Berger EA, Murphy PM, Farber JM: Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 1999, 17: 657-700. 10.1146/annurev.immunol.17.1.657

    Article  CAS  PubMed  Google Scholar 

  8. Xiao L, Owen SM, Goldman I, Lal AA, deJong JJ, Goudsmit J, Lal RB: CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage. Virology 1998,240(1):83-92. 10.1006/viro.1997.8924

    Article  CAS  PubMed  Google Scholar 

  9. Nelson JA, Fiscus SA, Swanstrom R: Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay. J Virol 1997,71(11):8750-8758.

    PubMed Central  CAS  PubMed  Google Scholar 

  10. Delwart EL, Gordon CJ: Tracking changes in HIV-1 envelope quasispecies using DNA heteroduplex analysis. Methods 1997,12(4):348-354. 10.1006/meth.1997.0489

    Article  CAS  PubMed  Google Scholar 

  11. Freel SA, Fiscus SA, Pilcher CD, Menezes P, Giner J, Patrick E, Lennox JL, Hicks CB, Eron JJ Jr., Shugars DC: Envelope diversity, coreceptor usage and syncytium-inducing phenotype of HIV-1 variants in saliva and blood during primary infection. Aids 2003,17(14):2025-2033. 10.1097/00002030-200309260-00003

    Article  PubMed  Google Scholar 

  12. Freel SA, Williams JM, Nelson JA, Patton LL, Fiscus SA, Swanstrom R, Shugars DC: Characterization of human immunodeficiency virus type 1 in saliva and blood plasma by V3-specific heteroduplex tracking assay and genotype analyses. J Virol 2001,75(10):4936-4940. 10.1128/JVI.75.10.4936-4940.2001

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Nelson JA, Baribaud F, Edwards T, Swanstrom R: Patterns of changes in human immunodeficiency virus type 1 V3 sequence populations late in infection. J Virol 2000,74(18):8494-8501. 10.1128/JVI.74.18.8494-8501.2000

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. van't Wout AB, Kootstra NA, Mulder-Kampinga GA, Albrecht-van Lent N, Scherpbier HJ, Veenstra J, Boer K, Coutinho RA, Miedema F, Schuitemaker H: Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission. J Clin Invest 1994,94(5):2060-2067. 10.1172/JCI117560

    Article  PubMed Central  PubMed  Google Scholar 

Download references


We wish to thank Nurse Practitioners Jeanne Dumestre and Louann Wenthold for their assistance with this study. We would also like to acknowledge and thank the staff and patients of the New Orleans HIV Outpatient Program. This study was supported by grants from the Louisiana Board of Regents Health Excellence Fund, the Centers for Disease Control and Prevention RA1/CCR622272, and the Gulf South STI/Topical Microbicide Cooperative Research Center NIAID 1U19 AI61972-01.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Angela M Amedee.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

TR conducted HTA analyses, participated in PCR sample preparation, carried out data analysis, and drafted the manuscript. PK participated in the study design and conducted the statistical analyses. RC participated in the design and coordination of the study and facilitated the collection of clinical samples. NL processed clinical samples and conducted PCR and viral load analyses. AA participated in the coordination of the study, directed sample and data analysis, and assisted in drafting the manuscript.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Randolph, T.C., Kissinger, P.J., Clark, R.A. et al. A predominance of R5-like HIV genotypes in vaginal secretions is associated with elevated plasma HIV-1 RNA levels and the absence of anti-retroviral therapy. Virol J 5, 87 (2008).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: