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The timely detection of viremia in HIV-infected patients receiving antiviral treatment is key to ensuring effective therapy and preventing the emergence of drug resistance. In high HIV burden settings, the cost and complexity of diagnostics limit their availability. We have developed a novel complementary metal-oxide semiconductor (CMOS) chip based, pH-mediated, point-of-care HIV-1 viral load monitoring assay that simultaneously amplifies and detects HIV-1 RNA. A novel low-buffer HIV-1 pH-LAMP (loop-mediated isothermal amplification) assay was optimised and incorporated into a pH sensitive CMOS chip. Screening of 991 clinical samples (164 on the chip) yielded a sensitivity of 95% (in vitro) and 88.8% (on-chip) at >1000 RNA copies/reaction across a broad spectrum of HIV-1 viral clades. Median time to detection was 20.8 minutes in samples with >1000 copies RNA. The sensitivity, specificity and reproducibility are close to that required to produce a point-of-care device which would be of benefit in resource poor regions, and could be performed on an USB stick or similar low power device., Competing Interests: C.T., L.S., D.D., Z.L. and E.H. are either currently employed, or have been employees of DNA Electronics Ltd which holds patents on the technology described. R.G., M.M., S.K. and G.C. have no conflicts of interest.

Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4(+) count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.

Objective: Interruption of antiretroviral therapy (ART) in chronic HIV disease is associated with increased mortality, predicted by elevations in interleukin-6 (IL-6) and D-dimer. The effect of ART interruption in primary HIV-1 infection on these biomarkers is unknown., Methods: Plasma samples from 200 HIV seroconverters enrolled in the Short Pulse Anti-Retroviral Therapy At HIV Seroconversion trial of deferred ART (standard of care) - 12 or 48 week ART (ART12 or ART48, respectively) - were analysed for IL-6 and D-dimer at weeks 0, 12, 16, 48, 52, 60 and 108 after randomization. Changes in log10 levels from weeks 0 to 12 were analysed using linear regression, as were changes from baseline to 4 weeks after stopping ART. Areas under the biomarker-time curves (AUC) to week 108 were adjusted for baseline values, and compared across all arms., Results: Median (inter-quartile range) baseline IL-6 and D-dimer were 1.45 (0.88, 2.41) pg/ml and 0.34 (0.20, 0.50) mg/l, respectively. At week 12, D-dimer levels were significantly lower among treated compared to untreated individuals (P < 0.001), whereas IL-6 levels were similar (P = 0.23). Within 4 weeks from stopping ART, IL-6 and D-dimer levels rose by 22 and 18%, reaching pre-ART levels. Over 108-week follow-up, there was no difference between arms in IL-6 AUC (P = 0.53), but D-dimer AUC was significantly lower for ART12 and ART48 compared to standard of care (overall P = 0.008)., Conclusion: Stopping ART in primary HIV-1 infection leads to inflammatory biomarker rebound to pre-treatment levels. However, over 108-week follow-up, we found no evidence that biomarker levels were higher for those interrupting ART, compared to those remaining ART-naïve, and D-dimer levels were significantly lower.

Objective: A minority of HIV-1 positive individuals treated with antiretroviral therapy (ART) in primary HIV-1 infection (PHI) maintain viral suppression on stopping. Whether this is related to ART duration has not been explored., Design: And Methods: Using SPARTAC trial data from individuals recruited within 6 months of seroconversion, we present an observational analysis investigating whether duration of ART was associated with post-treatment viraemic control. Kaplan-Meier estimates, logistic regression and Cox models were used., Results: 165 participants reached plasma viral loads (VL) <400 copies/ml at the time of stopping therapy (ART stop). After ART stop, 159 experienced confirmed VL ≥400 copies/ml during median (IQR) follow-up of 167 (108,199) weeks. Most participants experienced VL rebound within 12 weeks from ART stop, however, there was a suggestion of a higher probability of remaining <400 copies/ml for those on ART >12 weeks compared to ≤12 weeks (p=0.061). Cumulative probabilities of remaining <400 copies/ml at 12, 52 and 104 weeks after ART stop were 21% (95%CI=13,30), 4% (1,9), and 4% (1,9) for ≤12 weeks ART, and 32% (22,42), 14% (7,22), and 5% (2,11) for >12 weeks. In multivariable regression, ART for >12 weeks was independently associated with a lower probability of being ≥400 copies/ml within 12 weeks of ART stop (OR=0.11 (95%CI=0.03,0.34), p<0.001)). In Cox models of time to VL ≥400 after 12 weeks, we only found an association with female sex (OR=0.2, p=0.001)., Conclusion: Longer ART duration in PHI was associated with a higher probability of viral control after ART stop., Trial Registration: Controlled-Trials.com 76742797 http://www.controlled-trials.com/ISRCTN76742797.

CXCR4-tropic (X4) HIV-1 variants are associated with faster disease progression compared with CCR5-tropic variants; however, the mechanism for this is unclear. We measured T-cell activation in 120 individuals with primary HIV-1 infection. X4-utilizing variants, determined genotypically, were present in 8.3% of the participants and were associated with higher levels of CD4 T-cell activation, even after adjusting for other prognostic factors. Increased CD4 T-cell activation may influence the more rapid immunological decline associated with X4 virus.

Background: To combat the pandemic of human immunodeficiency virus 1 (HIV-1), a successful vaccine will need to cope with the variability of transmissible viruses. Human hosts infected with HIV-1 potentially harbour many viral variants but very little is known about viruses that are likely to be transmitted, or even if there are viral characteristics that predict enhanced transmission in vivo. We show for the first time that genetic divergence consistent with a single transmission event in vivo can represent several years of pre-transmission evolution., Results: We describe a highly unusual case consistent with a single donor transmitting highly related but distinct HIV-1 variants to two individuals on the same evening. We confirm that the clustering of viral genetic sequences, present within each recipient, is consistent with the history of a single donor across the viral env, gag and pol genes by maximum likelihood and bayesian Markov Chain Monte Carlo based phylogenetic analyses. Based on an uncorrelated, lognormal relaxed clock of env gene evolution calibrated with other datasets, the time since the most recent common ancestor is estimated as 2.86 years prior to transmission (95% confidence interval 1.28 to 4.54 years)., Conclusion: Our results show that an effective design for a preventative vaccine will need to anticipate extensive HIV-1 diversity within an individual donor as well as diversity at the population level.

Individuals with lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis serovar L2, are commonly co-infected with human immunodeficiency virus type 1 (HIV-1), for reasons that remain unknown. One hypothesis is that a biological synergy exists between the two pathogens. We tested this by characterising for the first time in vitro C. trachomatis L2 replication in the presence of HIV-1. The human epithelial cell-line, MAGI P4R5 was infected with C. trachomatis L2 and HIV-1 (MN strain). Co-infected cultures contained fewer and larger chlamydial inclusions, but the inclusions did not contain morphologically aberrant organisms. C. trachomatis remained infectious in the presence of HIV-1 and showed neither an alteration in genome accumulation, nor in the acumulation of ompA, euo or unprocessed 16S rRNA transcripts. However, omcB was slightly elevated. Taken together, these data indicate that HIV-1 co-infection did not significantly alter C. trachomatis replication and the association between HIV-1 and LGV is likely due to other factors that require further investigation. The fewer, larger inclusions observed in co-infected cultures probably result from the fusion of multiple inclusions in HIV-1 induced syncytia and indicate that C. trachomatis-host-cell interactions continue to function, despite considerable host-cell re-modelling., (Copyright © 2011. Published by Elsevier SAS.)

A xenotropic murine leukemia virus-related virus (XMRV) has recently been reported in association with prostate cancer and chronic fatigue syndrome, with a prevalence of up to 3.7% in the healthy population. We looked for XMRV in 230 patients with human immunodeficiency virus type 1 or hepatitis C infection. XMRV was undetectable in plasma or peripheral blood mononuclear cells by polymerase chain reaction targeting XMRV gag or env. T cell responses to XMRV Gag were undetectable in peripheral blood mononuclear cells by ex vivo gamma interferon enzyme-linked immunospot assay. In our cohorts, XMRV was not enriched in patients with blood-borne or sexually transmitted infections from the United Kingdom and Western Europe.

Objectives: To describe the frequency and risk factors of non-B HIV-1 subtypes in men who have sex with men (MSM) in the UK., Design: Observational study., Methods: MSM diagnosed with HIV-1 infection from 1980-2007, with HIV genotype held in the UK HIV Drug Resistance Database were identified. Protease and reverse transcriptase sequences were collected and viral clade determined using the REGA algorithm. Associations between demographic variables and subtype were analysed using logistic regression., Results: The prevalence of non-B HIV-1 infection amongst MSM in the UK was 5.4% (437/8058). In the UK this increased with year of diagnosis from pre1996 to 2002, and has subsequently remained relatively stable at around 7-9% after 2002, with a recent increase in 2007 to 13%. Multivariate analysis showed that acquisition of non-B HIV-1 infection was independently associated with later year of HIV diagnosis (P < 0.001), black ethnicity (P < 0.001) and non-European country of birth (P = 0.01). Age was also associated with subtype with individuals aged 25-39 years being less likely to have non-B virus than those aged less than 25 years (P = 0.01). Restricting the analysis to white men born in the UK, the association between subtype and year of diagnosis remained statistically significant (P < 0.001), as did the association with age (P < 0.001)., Discussion: The number of MSM in the UK infected with non-B clade HIV-1 is increasing, suggesting that the sociodemographic boundaries between HIV-1 viral subtypes globally are diminishing. Should viral subtypes be relevant to clinical disease progression or vaccine design, the changing pattern of distribution will need to be taken into account.

The worrying finding that up to 19% of newly diagnosed HIV-1 cases in the UK have genotypic evidence of transmitted drug-resistant HIV-1 (TrDR-HIV-1) does not concur with levels observed in one London centre. A study of the prevalence of resistance in primary HIV infection and newly diagnosed antiretroviral-naive individuals demonstrated significantly lower levels of TrDR-HIV-1 than previously reported. Variations in the prevalence of TrDR-HIV-1 may reflect the heterogeneity of methodologies and definitions used for resistance.

Objectives: The aim of the study was to determine the incidence and persistence of transmitted drug-resistant HIV-1 in an incident cohort between 2000 and 2004, and to investigate the impact of transmitted drug-resistant HIV-1 on the response to antiretroviral therapy (ART)., Methods: A prospective, nonrandomized study was carried out on 140 individuals identified with primary HIV-1 infection (PHI). PHI was defined as an HIV-positive antibody test with an HIV antibody-negative result in the prior 6 months (n = 69); positive HIV DNA in the absence of antibody (n = 30); an evolving titre positive HIV antibody test (n = 23), or an incident 'detuned' assay (B clade viruses only) (n = 18). Genotypic resistance testing was performed at baseline, following ART and annually over a 4-year period., Results: The prevalence of transmitted drug-resistant HIV-1 infection between January 2000 and June 2004 was nine in 140 (6.0%) and the annual incidence was stable. Seven of these nine patients had a single point mutation conferring single-class drug resistance and the other two patients had multiple mutations conferring multiclass drug resistance (MDR). In eight of the nine cases, mutations conferring drug resistance persisted for more than 12 months off therapy. In contrast to transmitted MDR HIV-1, the virological response to initial ART and CD4 decline were comparable in those with wild-type virus, virus with 'polymorphisms' (secondary mutations) and virus with single drug-resistance mutations., Conclusions: The incidence of transmitted drug-resistant HIV remained stable and low over a 4-year period. Although MDR remains rare, its presence significantly affects the response to first-line ART, predisposes towards the accumulation of new resistance mutations and is associated with a more rapid CD4 decline.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences.

Aim: In this paper we compare the transduction efficiency, toxicity, and safety of retroviral vectors [equine infectious anemia virus (EIAV), human immunodeficiency virus-1 (HIV-1), human foamy virus (PFV] and adenovirus (Ad) for potential use in gene therapy of corneal endothelial cells., Method: Murine corneal endothelial cells were transduced with EIAV, HIV-1, PFV, and Ad, resulting in the overexpression of a green fluorescent protein (eGFP) transgene marker. The transduction efficiency was assessed by flow cytometry, while cytotoxicity and apoptosis rate were detected by annexin V/propidium iodide (PI) stain., Results: Ad had the highest transduction efficiency with 99% of the cells expressing the transgene, followed by EIAV (95%), HIV-1 (75%), and PFV (43%). However, the high transduction efficiency of Ad also resulted in the highest apoptosis rate (25%) in the corneal endothelial cells. There was no detectable difference in the toxicity between PFV and HIV-1 (10%). EIAV transduction had the lowest cytotoxicity, with only 3% of the cells being annexin V/PI positive., Conclusion: Compared to other vectors EIAV exhibited high transduction efficiency combined with low toxicity to corneal endothelial cells. Therefore, it is a powerful tool for gene therapy applications in selected corneal endothelial diseases.

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved., (Copyright 2003 Wiley-Liss, Inc.)

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01&#95;AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.

A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS--PCR) --- a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant--wild-type population. Gene sequencing was carried out simultaneously for comparison. MS--PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS--PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS--PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS--PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.

HIV-1 is characterised by extensive genetic variability encompassing at least 10 different phylogenetically related clades within the major group of HIV-1 subtypes. Most commercially available HIV-1 RNA plasma viral load assays have been optimised with clade B viruses and may yield misleadingly low RNA levels for nonclade B viruses that are increasingly found in Europe. In this study we compare the most recent versions of the Roche Amplicor HIV Monitor and the Chiron Quantiplex for ability to detect viraemia in a population of patients infected with a range of HIV-1 subtypes. EDTA-treated plasma was obtained from 206 patients. The Amplicor and Quantiplex assays were carried out in accordance with manufacturers' instructions. Results from 53/206 (25.7%) samples differed by >0.4 log between Amplicor 1.5 and Quantiplex 3.0. A >0.5 log and 1.0 log difference was detected between Amplicor 1.5 and Quantiplex 3.0 in 37/206 (17.9%) and 7/206 (3.4%) of samples, respectively. Overall, Amplicor 1.5 gave a median value of 0.22 log higher than Quantiplex 3.0. Discordant results were detected in 53 out of 206 (25.7%) samples. Of these 22 out of 123 (17.9%) samples were of UK origin, 18 out of 43 (41.9%) African, 1 out of 8 (12.5%) South American, 1 out of 6 (16.7%) North American, 4 out of 9 (44.4%) North European, 3 out of 11 (23.7%) South European and 3 out of 7 (42.3%) Asian samples, respectively. Serotyping revealed that discordant viral load results between Amplicor 1.5 and Quantiplex 3.0 occurred within samples from all subtypes (A-E). Despite the improvements made to both the Roche Amplicor and the Chiron Quantiplex assays discordant results were detected between the two assays in 25.7% of cases. In a substantial minority of patients there were major discrepancies between the two assays that were not explained by HIV subtype differences., (Copyright 2000 Wiley-Liss, Inc.)

The prevention of mother to child transmission of HIV-1 by zidovudine monotherapy is well known, but increasingly combination anti-retroviral therapy is prescribed during pregnancy. In this prospective study, 19 pregnant women with human immunodeficiency virus-1 (HIV-1) infection who elected to take anti-retroviral therapy during the second and third trimesters were treated with zidovudine or zidovudine plus lamivudine. Fourteen women treated with zidovudine monotherapy had a mean 0.3 log(10) reduction in viral load and a mean 52 x 10(6)/L (17%) increase in CD4+ lymphocytes at delivery compared with pre-treatment samples. Genotypic mutations associated with decreased susceptibility to zidovudine were detected in 2 of 10 women at delivery. Five women with more advanced HIV-1 infection were treated with zidovudine plus lamivudine and a mean 1.5 log(10) reduction in viral load together with a mean 30 x 10(6)/L (33%) increase in CD4+ lymphocytes was observed in this group. However, four of five women in the dual therapy arm had the M184V mutation in the reverse transcriptase gene associated with decreased susceptibility to lamivudine at delivery. We conclude that zidovudine plus lamivudine reduced HIV-1 plasma viraemia to low levels in pregnant women with advanced HIV-1 disease but the rapid development of genotypic resistance to lamivudine indicates that additional therapy is required both for the long-term benefit of the mothers and to prevent the development of resistant virus that may be transmitted to the infant.

A proposed hemopoietic stem cell gene therapy for treatment for HIV infection would involve transduction of CD34+ hemopoietic stem cells with vectors encoding anti-HIV constructs. Peripheral blood has proved to be a useful source of these hemopoietic stem cells and this study exploits this finding. Small quantities of peripheral blood were obtained from HIV-negative patients and HIV-positive patients who were and were not receiving hemopoietic growth factors (HGFs). CD34+ cells were obtained from these samples using a simple technique and scored for frequency of colony type. This demonstrated that HIV-negative patients had the highest frequency of colony-forming units (CFUs). HIV-positive patients not treated with HGFs had a lower frequency of CFUs, but the same colony type distribution as HIV-negative patients. HIV-positive patients treated with HGFs had the lowest frequency of CFUs, but their colony type distribution demonstrated that they had responded to treatment. CD34+ cells selected in this way were also transduced with the murine retroviral MFG vector using a technique that demonstrated transduction efficiencies ranging from 2% to 16% (median, 11.5%). This study simplifies the experimental requirements for development of a hemopoietic stem cell gene therapy for HIV infection and offers the possibility that longitudinal studies could be performed on peripheral blood CD34+ cells from HIV-positive or HIV-negative patients without the need for granulocyte colony-stimulating factor mobilization.

This study investigated the effects of a combination antiretroviral drug regimen (indinavir and two nucleoside analogs or ritonavir and saquinavir) on the levels of CD34+ colony-forming units (CFU-Cs) in the peripheral blood of HIV-1+ patients. Ten patients who were receiving combination antiretroviral drug therapy were studied and their peripheral blood CD34+ CFU-Cs were measured prior to, 1 month after, and 4 to 6 months after the commencement of therapy. The levels of CD4+ T cells increased significantly in these patients (paired t test, p = 0.0027) and plasma viral load became undetectable in all but one patient studied. Measurements of the CFU-Cs showed that their levels tended to increase on the commencement of therapy, and these levels became significantly higher than baseline by 4-6 months (paired t test, p = 0.0293). Analysis of the different colony phenotype demonstrated that the main contributor to this increase consisted of burst-forming unit erythroid (BFU-E) cells. These data also demonstrated that there was an inverse correlation between the rise in CFU-Cs at 4-6 months compared with CD4+ cell, CD8+ cell, and neutrophil counts, and hemoglobin concentration, at baseline. The demonstrated increase in the levels of CD34+ CFU-Cs suggests that HIV-1 may have an inhibitory effect on these cells in vivo, and that this inhibition may be abrogated by suppression of viral replication.

The majority of vertical infections with human immunodeficiency virus type 1 (HIV-1) occur at or near delivery, strongly suggesting a mucosal route of transmission. The frequency and level of intrapartum mucosal exposure to HIV-1 of 22 infants born to infected mothers was investigated. Maternal plasma HIV-1 RNA and CD4 cell count were measured at delivery. Infant oropharyngeal aspirates obtained at birth were examined for HIV-1 RNA by reverse transcription-polymerase chain reaction and qualitative nucleic acid sequence-based amplification. Nine infants (41%) had detectable levels of HIV-1 RNA, 3 of which were quantifiable (mean, 3000 copies/mL). This mucosal exposure to HIV-1 during delivery did not lead to infection of any infant. Cesarian delivery did not reduce mucosal exposure to HIV-1. Mucosal exposure did not correlate with maternal CD4 cell count but did correlate with maternal plasma virus load and was reduced by antiretroviral therapy.

In common with oncoviruses but unlike the lentivirus human immunodeficiency virus type 1, foamy (spuma) viruses require host cell proliferation for productive infection. We show that human immunodeficiency virus type 1 replicates in RD-CD4 cells regardless of the growth arrest condition of the cells, while murine leukemia virus is unable to infect growth-arrested RD-CD4 cells or cells progressing through a partial cell cycle that includes S phase but not mitosis. Human foamy virus, like murine leukemia virus, does not productively infect G1/S or G2 growth-arrested cells. Two other foamy viruses, simian foamy virus type 1, isolated from a macaque, and simian foamy virus type 6, isolated from a chimpanzee, also fail to establish productive infection in G1/S-arrested cells.

Sulfated polysaccharides have been shown to inhibit human immunodeficiency virus (HIV) infection in vitro. Dextrin sulfate, fucoidan, and dextran sulfate fail to neutralize virions directly, but interact with target cells to inhibit virus entry. Ionic interactions of sulfated polyanions with oppositely charged cell surface components, including CD4, have been assumed to be the inhibitory mechanism. It is shown that the sulfated polysaccharides inhibit infection of both CD4+ and CD4- cell lines by HIV and also that they inhibit HTLV-1 and, to a lesser extent, the simian retrovirus, MPMV, which use receptors other than CD4. One binding site for radiolabeled fucoidan on the surface of human T cells is an 18 kD protein, but its significance is not yet clear.

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.

The susceptibility of common marmosets and cotton-top tamarins to infection by HIV-2 in vivo was tested. One year and 19 months, respectively, post-inoculation, sera taken from three of four animals from each species are reactive for HIV-2 antibodies and HIV-specific nucleotide sequences were demonstrated in short-term cultures of PBL from two cotton-top tamarins. The animals remain in good health.