A Panel of Eight miRNAs Is Deregulated in HTLV-2 Infected PBMCs and BJABGu Cell Line.

Despite human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 being retroviruses closely related at a genomic level, HTLV-2 differs from HTLV-1 in terms of pathogenicity in both single infection and coinfection contexts. Moreover, the HTLV-2 association with clinical outcomes is still debated and several mechanisms underlying HTLV-2 infection remain unexplored as well. Cellular miRNAs are key factors in the post-transcriptional regulation of gene expression and they are known to be potential targets for several pathogens to control the host microenvironment and, in particular, escape immune responses. Here, we identified a HTLV-2-related signature of eight miRNAs (miR-125a-3p, miR-381-3p, miR-502-5p, miR-708-5p, miR-548d-5p, miR-548c-5p, miR-1-3p, and miR-511-5p) in both HTLV-2 infected PBMC and BJABGu cell lines. Altered miRNA expression patterns were correlated with the impairment of Th cell differentiation and signaling pathways driven by cytokines and transcriptional factors such as the Runt-related transcription factor (RUNX) family members. Specifically, we demonstrated that the RUNX2 protein was significantly more expressed in the presence of Tax-2 compared with Tax-1 in an in vitro cell model. To the best of our knowledge, these data represent the first contribution to elucidating the HTLV-2 mediated alteration of host cell miRNA profiles that may impact on HTLV-2 replication and persistent infection.

Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2.

HTLV Tax proteins (Tax-1 and Tax-2) are known to be able to transactivate several host cellular genes involved in complex molecular pathways. Here, we describe a stable and regulated high-level expression model based on Tet-On system, to study the capacity of Tax-2 to transactivate host genes. In particular, the Jurkat Tet-On cell line suitable for evaluating the ability of Tax-2 to stimulate transactivation of a specific host gene, CCL3L1 (C-C motif chemokine ligand 3 like 1 gene), was selected. Then, a plasmid expressing tax-2 gene under control of a tetracycline-response element was constructed. To avoid the production of a fusion protein between the report gene and the inserted gene, a bidirectional plasmid was designed. Maximum expression and fast response time were achieved by using nucleofection technology as transfection method. After developing an optimized protocol for efficiently transferring tax-2 gene in Jurkat Tet-On cellular model and exposing transfected cells to Dox (doxycycline, a tetracycline derivate), a kinetics of tax-2 expression through TaqMan Real-time PCR assay was determined.

A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents.

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

Proviral load determination of HTLV-1 and HTLV-2 in patients' peripheral blood mononuclear cells by real-time PCR.

TaqMan real-time PCR assays were developed to determine the proviral load (PVL) of human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) in peripheral blood mononuclear cells (PBMCs) of infected subjects. In particular, separate single-plex assays for HTLV-1 tax-1, and HTLV-2 tax-2 and pol-2 genes were designed for quantitation of HTLV-1 and HTLV-2 PVLs. The specificity of both tax-2 and pol-2 assays was verified by testing the DNA extracted from C10, a chronically HTLV-1-infected cell line, used as a negative control. As far as HTLV-2 assay, the specificity was checked by testing C344 cells which are stably infected by HTLV-2. Quantitative determination of HTLV PVLs was obtained by performing standard reference curves by a serial dilution of DNA extracted from C10 and C344 cells, assuming one proviral genome per C10 cell and two per C344 cell. The human albumin gene, of which there are 2 copies per cell, was quantified in the same reactions to normalize the results. Intra-assay reproducibility was checked by running 30 replicates of the same sample in a plate (coefficient of variance <6 %), while inter-assay reproducibility was measured by amplifying the same sample in three independent experiments (coefficient of variance <6 %).

HTLV-1/-2 and HIV-1 co-infections: retroviral interference on host immune status.

The human retroviruses HIV-1 and HTLV-1/HTLV-2 share similar routes of transmission but cause significantly different diseases. In this review we have outlined the immune mediated mechanisms by which HTLVs affect HIV-1 disease in co-infected hosts. During co-infection with HIV-1, HTLV-2 modulates the cellular microenvironment favoring its own viability and inhibiting HIV-1 progression. This is achieved when the HTLV-2 proviral load is higher than that of HIV-1, and thanks to the ability of HTLV-2 to: (i) up-regulate viral suppressive CCL3L1 chemokine expression; (ii) overcome HIV-1 capacity to activate the JAK/STAT pathway; (iii) reduce the activation of T and NK cells; (iv) modulate the host miRNA profiles. These alterations of immune functions have been mainly attributed to the effects of the HTLV-2 regulatory protein Tax and suggest that HTLV-2 exerts a protective role against HIV-1 infection. Contrary to HIV-1/HTLV-2, the effect of HIV-1/HTLV-1 co-infection on immunological and pathological conditions is still controversial. There is evidence that indicates a worsening of HIV-1 infection, while other evidence does not show clinically relevant effects in HIV-positive people. Possible differences on innate immune mechanisms and a particularly impact on NK cells are becoming evident. The differences between the two HIV-1/HTLV-1 and HIV-1/HTLV-2 co-infections are highlighted and further discussed.

Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1.

MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4(+) T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients. We proposed that miRNA expression may discriminate between HIV-1-infected and -exposed but negative patients. Analysis of miRNAs expression after exposure of healthy CD4(+) T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro condition revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.

Serum adipokine zinc α2-glycoprotein and lipolysis in cachectic and noncachectic heart failure patients: relationship with neurohormonal and inflammatory biomarkers.

Chronic heart failure is often complicated by the development of cachexia with the loss of fat mass. Zinc α2-glycoprotein (ZAG) is a serum adipokine with lipolytic effects in cancer cachexia. We evaluated in patients with advanced heart failure with (CxHF) or without cachexia (nCxHF) the relationship of ZAG with circulating free fatty acid (FFA), as an index of lipolysis, and with other neurohormonal and inflammatory biomarkers. Two groups, nCxHF (n = 46) and CxHF (n = 18), the latter having a documented, involuntary, edema-free loss of body weight of at least 7.5% in the previous 6 months, underwent plasma determination of FFA, ZAG, norepinephrine (NE), tumor necrosis factor-α, and natriuretic peptide levels (atrial natriuretic, B-type natriuretic peptide). The patients were compared with age-matched healthy controls (CTR) (n = 21). Zinc α2-glycoprotein, atrial natriuretic peptide, B-type natriuretic peptide, and tumor necrosis factor-α circulating levels were similarly greater in CxHF and nCxHF than in CTR. Free fatty acid and NE were higher in CxHF than in nCxHF. A positive correlation between FFA and NE was found in both CxHF (r = 0.73, P < .01) and nCxHF (r = 0.48, P < .01) but only in CxHF between ZAG and FFA (r = 0.54, P = .02) and between ZAG and NE (r = 0.70, P < .01). No correlations between natriuretic peptides and ZAG were found. Serum ZAG levels are increased in advanced heart failure patients compared with CTR, without differences between CxHF and nCxHF. Only in CxHF, ZAG levels are directly correlated to circulating levels of FFA and NE, suggesting a close interaction of ZAG with sympathetic-mediated lipolysis.

Antiretroviral activity of thiosemicarbazone metal complexes.

Thiosemicarbazones display a wide antimicrobial activity by targeting bacteria, fungi, and viruses. Here, we report our studies on the antiviral activity of two thiosemicarbazone metal complexes, [bis(citronellalthiosemicarbazonato)nickel(II)] and [aqua(pyridoxalthiosemicarbazonato)copper(II)] chloride monohydrate, against the retroviruses HIV-1 and HTLV-1/-2. Both compounds exhibit antiviral properties against HIV but not against HTLVs . In particular, the copper complex shows the most potent anti-HIV activity by acting at the post-entry steps of the viral cycle.

Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy.

OBJECTIVE: The continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population. METHODS: The distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995-2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses. RESULTS: 18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.

Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities.

BACKGROUND: Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. METHODOLOGY/PRINCIPAL FINDINGS: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. CONCLUSIONS/SIGNIFICANCE: The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Molecular and cellular interactions of HIV-1/HTLV coinfection and impact on AIDS progression.

In the last 10 years HIV-1/human T-cell leukemia virus (HIV-1/HTLV) coinfection has emerged as a worldwide health problem. The numbers of HIV-1/HTLV-1 coinfections in South America and Africa are increasing, as well as HIV-1/HTLV-2 coinfections in the USA and Europe. Coinfections by either HTLV-1 or HTLV-2 and HIV-1 frequently occur in persons with a history of injection drug use. Since HTLV-1 preferentially infects CD4+ T-cells and HTLV-2 has a tropism for CD8+ T-cells, the influence of coinfection on HIV-1 disease progression may be different. The effect of HIV-1/HTLV-1 coinfection on HIV-I pathogenesis is controversial as soluble factors produced by HTLV-1 infected cells can either enhance or suppress HIV-1 infection. In HTLV-1/HIV-1 coinfected patients, upregulation of HIV-1 expression was attributed to strong activation of cytokines that promoted HIV infection. The introduction of HAART has dramatically reduced HIV-1 morbidity and mortality, but has given rise to an increased number of inflammatory syndromes. While HAART is successful for controlling HIV disease, it has little impact on HTLV-1/2 genome expression. The consequence of coinfection, even with HAART, may well be the reported increase in neurologic disease. Several epidemiologic and in vitro studies of the influence of HTLV infection on HIV-1 related AIDS progression suggest that HTLV-1 infection can promote HIV-1 replication and accelerate the clinical progression to AIDS. However, other studies have not confirmed these observations. The differences in study outcomes could be related to the occurrence of different HIV-1 phenotypes in clinical disease. In contrast, evidence points to a confirmed protective role of HTLV-2 that is manifested as improved survival and delayed progression to AIDS. The protective effect may be the result of maintaining normal-range levels of CD4 and CD8 counts, lowering HIV replication, and immune activation. As a corollary, the number of long-term nonprogressors for AIDS in the HIV-1/HTLV-2 coinfected group was found to be significantly higher than in HIV-1 monoinfected cases. Investigations of the natural factors induced by HTLV-2 that influence HIV-1 replication show that CCL3L1 (an isoform of CCL3) is preferentially induced in HTLV-2 exposed seronegative HIV individuals and in long-term nonprogressor HTLV-2/HIV-1 coinfected persons. The CCL3L 1 inhibits HIV replication and thus acts as a potent effector against both HIV infection and disease progression. As a complement to upregulation of CCL3L1, other chemokines and cytokines induced by HTLV-2 may contribute to induction of the Th1 response against invading pathogens, in contrast to the dominant Th2 response that appears to favor HIV infection. The number of individuals with either single HIV-1 or HTLV-2 infection, in a cohort of Italian intravenous drug users monitored for 20 years, decreased significantly over time. However, the magnitude of HTLV-2 decrease was significantly less than that of HIV-1, pointing to the need for increased attention to, and control of, HTLV infection. In conclusion, the long-term effects of HIV and HTLV coinfections are poorly understood and the mechanisms of dysregulation of cellular biosynthesis by HTLV that impact HIV disease progression remain elusive.

Dynamics of HIV viral load in blood and semen of patients under HAART: impact of therapy in assisted reproduction procedures.

We examined the efficacy and effect of HAART in HIV-1-infected men confronted with assisted fertilization procedures. We showed that HAART did not always reduce the HIV-1-RNA level in blood and semen compartments, and that a significant upward shift in mitochondrial DNA was observed in spermatozoa from a HAART-treated patient group compared with spermatozoa from HAART-untreated or HIV-1-uninfected groups (P < 0.001). These findings emphasize the negative role of HAART, but not of HIV-1 infection, in determining semen alterations.

Postgenomic up-regulation of CCL3L1 expression in HTLV-2-infected persons curtails HIV-1 replication.

Leukocytes of persons coinfected with HTLV-2 and HIV-1 secrete chemokines that prevent CCR5-dependent (R5) HIV-1 infection of CD4+ T cells and macrophages, with HTLV-2-induced MIP-1alpha as dominant HIV-1 inhibitory molecule. Two nonallelic genes code for CCL3 and CCL3L1 isoforms of MIP-1alpha, and the population-specific copy number of CCL3L1 exerts a profound effect on HIV-1 susceptibility and disease progression. Here, we demonstrate that CCL3L1 is secreted spontaneously by leukocytes of HTLV-2-infected persons and superinduced when cells of HTLV-2/HIV-1 multiply exposed-uninfected seronegative (MEU) persons were stimulated with HIV-1 Env peptides. The CCL3L1 median copy number in MEU, HTLV-2/HIV-1-coinfected long-term nonprogressors (LTNPs) and HIV-1-monoinfected LTNPs were 1, 2, and 3, respectively. An increased CCL3L1/CCL3 mRNA ratio versus PHA-activated healthy leukocytes was observed in both HIV-1-monoinfected LTNPs and in HTLV-2/HIV-1(MEU) subjects. An additional potential correlate of HTLV-2 infection was a rapid and persistent leukocyte secretion of GM-CSF and IFN-gamma, 2 cytokines endowed with CCR5 down-regulation capacity. This study confirms a crucial protective role of CCL3L1 from both HIV infection and disease progression, highlighting a previously not described functional up-regulation of this chemokine variant in both HIV-positive and -negative persons infected with HTLV-2.

Inhibition of human T cell leukemia virus type 2 replication by the suppressive action of class II transactivator and nuclear factor Y.

The master regulator of MHC-II gene transcription, class II transactivator (CIITA), acts as a potent inhibitor of human T cell leukemia virus type 2 (HTLV-2) replication by blocking the activity of the viral Tax-2 transactivator. Here, we show that this inhibitory effect takes place at the nuclear level and maps to the N-terminal 1-321 region of CIITA, where we identified a minimal domain, from positions 64-144, that is strictly required to suppress Tax-2 function. Furthermore, we show that Tax-2 specifically cooperates with cAMP response element binding protein-binding protein (CBP) and p300, but not with p300/CBP-associated factor, to enhance transcription from the viral promoter. This finding represents a unique difference with respect to Tax-1, which uses all three coactivators to transactivate the human T cell leukemia virus type 1 LTR. Direct sequestering of CBP or p300 is not the primary mechanism by which CIITA causes suppression of Tax-2. Interestingly, we found that the transcription factor nuclear factor Y, which interacts with CIITA to increase transcription of MHC-II genes, exerts a negative regulatory action on the Tax-2-mediated HTLV-2 LTR transactivation. Thus, CIITA may inhibit Tax-2 function, at least in part, through nuclear factor Y. These findings demonstrate the dual defensive role of CIITA against pathogens: it increases the antigen-presenting function for viral determinants and suppresses HTLV-2 replication in infected cells.

A killer mimotope with therapeutic activity against AIDS-related opportunistic micro-organisms inhibits ex-vivo HIV-1 replication.

OBJECTIVE: To verify whether a synthetic therapeutic killer decapeptide (KP), a functional mimotope of a yeast killer toxin with wide-spectrum microbicidal activity, inclusive of AIDS-related opportunistic micro-organisms, through interaction with beta-glucan receptors, which has been found to possess sequence homology with critical segments in gp160 V1/V2 and V3 loops, may also be inhibiting HIV-1 replication. METHODS: Primary peripheral blood mononuclear cells (PBMCs) cultures established from HIV-1-infected patients were treated with KP in comparison with zidovudine and supernatants and cells were harvested for analysis of HIV RNA and proviral contents, respectively. Virus production in exogenous in-vitro PBMCs infection with lymphocytotropic and monocytotropic HIV-1 strains was also assessed in presence of KP by enzyme-linked immunosorbent assay HIV p24 gag antigen detection. The binding affinity of KP to CD4, CCR5 and CXCR4 was evaluated on CD4-CCR5 or CD4-CXCR4 transfected astroglioma cell lines. RESULTS: KP was shown to be devoid of cytotoxicity on PBMCs and to inhibit HIV-1 replication in PBMCs of a patient in the acute phase of infection. The antiretroviral activity of KP, which proved to be more potent than zidovudine at micromolar concentrations, is abolished by beta 1,3-glucan but not by beta 1,6-glucan. Down-regulation of CCR5 co-receptor, and/or physical block of the gp120-receptor interaction are possible mechanisms of KP activity. CONCLUSION: KP appears to be the first antibody-derived short peptide displaying an inhibitory activity against HIV-1 and related opportunistic micro-organisms by different mechanisms of action.

Coinfection with HIV-1 and human T-Cell lymphotropic virus type II in intravenous drug users is associated with delayed progression to AIDS.

Human T-cell lymphotropic virus (HTLV) type II has spread among intravenous drug users (IDUs), many of whom are coinfected with HIV-1. We have investigated the rate of HTLV-II infection in 3574 Italian IDUs screened for HIV-1, HTLV-I, and HTLV-II from 1986 to the present. HTLV-II proviral load was determined by a real-time polymerase chain reaction specifically designed for tax amplification. The frequency of HTLV-II infection was 6.7% among HIV-1-positive subjects and 1.1% among HIV-1-negative subjects (P < 0.0001). For examination of AIDS progression, a group of 437 HIV-1-monoinfected subjects and another group of 96 HIV-1/HTLV-II-coinfected subjects were monitored. Enrollees were matched at entry by CD4 cell counts and followed for an average of 13 years. HIV-1/HTLV-II coinfection was associated with older age (P < 0.0001) and higher CD4 (P < 0.0001) and CD8 (P < 0.001) cell counts compared with monoinfected IDUs. The number of long-term nonprogressors for AIDS was significantly higher (P < 0.0001) among coinfected patients (13 [13.5%] of 96 patients) than HIV monoinfected patients (5 [1.1%] of 437 patients), showing that HTLV-II exerts a protective role. An increased incidence of liver disease and hepatitis C virus positivity among coinfected IDUs was observed. Five coinfected subjects undergoing antiretroviral therapy showed a significant (P < 0.05) increase in HTLV-II proviral load concomitant to a decrease in HIV-1 viremia, suggesting that the treatment is ineffective against HTLV-II infection.

Copper(II) and cobalt(III) pyridoxal thiosemicarbazone complexes with nitroprusside as counterion: syntheses, electronic properties, and antileukemic activity.

In this paper the syntheses of new pyridoxal thiosemicarbazone copper(II) and cobalt(III) complexes with nitroprusside as a counterion and tests on the antileukemic activity of three of these complexes toward human cell lines U937 and CEM are reported. Nitroprusside was chosen in order to test if its ability to release NO can increase the biological activity already shown by these complexes. The compounds were characterized by spectroscopic and magnetic methods and by single-crystal X-ray diffraction.

Screening of HIV-1 isolates by reverse heteroduplex mobility assay and identification of non-B subtypes in Italy.

OBJECTIVE: The increasing prevalence of HIV-1 transmission through heterosexual contacts and the growing number of immigrants from non-Western countries, where non-B subtypes and recombinant forms are prevalent, suggest the possible emergence in Italy of a new epidemic wave of HIV-1 non-B subtypes as well as recombinant forms. METHODS: The distribution of HIV-1 subtypes has been evaluated in 63 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the last 5 years. A modified heteroduplex mobility assay (HMA) strategy, reverse HMA (rHMA), has been developed in our laboratory, allowing rapid identification of divergent-from-B-subtype isolates, which have been subsequently characterized by detailed molecular and phylogenetic analyses. RESULTS: Five samples show, on rHMA, an electrophoretic pattern compatible with a non-B subtype classification. Their phylogenetic analysis, performed on both env and gag regions, confirms the rHMA subtyping prediction, given that 3 samples fall into the "A-family" subtype and 2 into the G subtype. The 5 non-B-subtype HIV-1 isolates have been identified among 23 variants (prevalence, 21.74%) isolated during the 2000 to 2001 period in heterosexuals. In parallel, B-subtype isolates show high levels of intrasubtype nucleotide divergence, compatible with a constant HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes, and the data indicate that rHMA represents a powerful tool for HIV-1 biomolecular screening in epidemics characterized by a mono-/dual-subtype predominance.