The evolution of subtype B HIV-1 tat in the Netherlands during 1985-2012.

For the production of viral genomic RNA, HIV-1 is dependent on an early viral protein, Tat, which is required for high-level transcription. The quantity of viral RNA detectable in blood of HIV-1 infected individuals varies dramatically, and a factor involved could be the efficiency of Tat protein variants to stimulate RNA transcription. HIV-1 virulence, measured by set-point viral load, has been observed to increase over time in the Netherlands and elsewhere. Investigation of tat gene evolution in clinical isolates could discover a role of Tat in this changing virulence. A dataset of 291 Dutch HIV-1 subtype B tat genes, derived from full-length HIV-1 genome sequences from samples obtained between 1985-2012, was used to analyse the evolution of Tat. Twenty-two patient-derived tat genes, and the control TatHXB2 were analysed for their capacity to stimulate expression of an LTR-luciferase reporter gene construct in diverse cell lines, as well as for their ability to complement a tat-defective HIV-1LAI clone. Analysis of 291 historical tat sequences from the Netherlands showed ample amino acid (aa) variation between isolates, although no specific mutations were selected for over time. Of note, however, the encoded protein varied its length over the years through the loss or gain of stop codons in the second exon. In transmission clusters, a selection against the shorter Tat86 ORF was apparent in favour of the more common Tat101 version, likely due to negative selection against Tat86 itself, although random drift, transmission bottlenecks, or linkage to other variants could also explain the observation. There was no correlation between Tat length and set-point viral load; however, the number of non-intermediate variants in our study was small. In addition, variation in the length of Tat did not significantly change its capacity to stimulate transcription. From 1985 till 2012, variation in the length of the HIV-1 subtype B tat gene is increasingly found in the Dutch epidemic. However, as Tat proteins did not differ significantly in their capacity to stimulate transcription elongation in vitro, the increased HIV-1 virulence seen in recent years could not be linked to an evolving viral Tat protein.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

Widespread hepatitis B virus genotype G (HBV-G) infection during the early years of the HIV epidemic in the Netherlands among men who have sex with men.

BACKGROUND: Hepatitis B virus (HBV) variants belong to different genotypes, A-J, whose worldwide distribution is linked with geography, probably because viral spread was associated with ancient human migrations. HBV genotype G (HBV-G) is an aberrant genotype with little sequence divergence, suggesting a recent origin. HBV-G is strongly associated with certain risk groups such as intravenous drug users (IDUs) and men who have sex with men (MSM), but hardly with geography. The origin and epidemiology of HBV-G remain unresolved, as is the disease association. METHODS: To estimate the prevalence and possible time of introduction of HBV-G into the MSM community in Amsterdam, the Netherlands, we have retrospectively analysed 226 blood serum samples from HBsAg positive MSM enrolled in the Amsterdam Cohort Studies (ACS) on HIV infection and AIDS dating from 1984 to 1999 using genotype-specific PCR assays. RESULTS: Of the 226 HBsAg-positive samples, 149 were HBV DNA positive. Of those, 104 were positive for HBV genotype A (HBV-A) and five for HBV-G, and 40 showed a dual infection with both HBV-A and HBV-G. Being HIV-infected was significantly associated with a reduced HBV DNA viral load in blood, but not with the prevalence of HBV-G. Early virus already contained stop codons in the precore region and a 36 bp insertion in the core gene which are the characteristics of HBV-G. CONCLUSIONS: HBV-G was introduced before 1985 into the Amsterdam MSM community. Early isolates show very limited sequence variation, confirming a low evolutionary rate. HBV-G acquisition was independent of HIV infection, but being HIV-infected was significantly associated with a reduced HBV viral load in blood, indicating a beneficial effect of early HIV infection in controlling HBV replication.

The Neutralizing Antibody Response in an Individual with Triple HIV-1 Infection Remains Directed at the First Infecting Subtype.

The effect of serial HIV-1 infection on the development of the broadly neutralizing antibody (bNAb) response was studied in an individual, H01-10366, with a serial HIV-1 superinfection (SI), hence triple infection, and compared with the bNAb response in three superinfected as well as 11 monoinfected men who have had sex with men (MSM) from Amsterdam, the Netherlands. Neutralization assays measuring heterologous neutralizing antibody (NAb) titers on a panel of six representative viruses from different HIV-1 subtypes were performed on blood serum samples obtained ∼3 years after primary HIV infection (PHI) and longitudinally for H01-10366. A bNAb response was defined as having a geometric mean neutralization titer (the reciprocal serum dilution giving 50% inhibition of virus infection, inhibitory dilution (ID50)) ≥100 and neutralizing >50% of viruses in the panel with an ID50 titer ≥100. H01-10366 quickly developed a potent NAb response against subtype B viruses before subtype B SI, but no broadening of the response occurred after the second subtype B infection or the third infection with CRF01_AE. When comparing H01-10366 with matched monoinfected (N = 11) and superinfected (N = 3) individuals analyzed 3 years after PHI, we found that 5 of the 15 individuals (4/11 monoinfected, 1/4 SI) developed a bNAb response. However, there was no statistically discernible difference between the bNAb response and HIV-1 SI. Thus, HIV-1 SI was not associated with the breadth and potency of the bNAb response in this small group of Dutch MSM with SI that included a triple HIV-1-infected individual.

High prevalence of hepatitis B virus dual infection with genotypes A and G in HIV-1 infected men in Amsterdam, the Netherlands, during 2000-2011.

BACKGROUND: Hepatitis B virus (HBV) is divided into 8 definite (A-H) and 2 putative (I, J) genotypes that show a geographical distribution. HBV genotype G, however, is an aberrant genotype of unknown origin that demonstrates severe replication deficiencies and very little genetic variation. It is often found in co-infections with another HBV genotype and infection has been associated with certain risk groups such as intravenous drug users and men having sex with men (MSM). We aimed to estimate the prevalence of HBV-G in the Netherlands by analysing samples from HBV-positive patients visiting the Academic Medical Center in Amsterdam. METHODS: Ninety-six HBV-infected patients, genotyped as HBV-A or HBV-G infected, were retrieved from the clinical database. Blood plasma samples were analysed with a newly-developed real-time PCR assay that detects HBV-A and HBV-G. For three patients, the HBV plasma viral load (pVL) of both genotypes was followed longitudinally. In addition, three complete genomes of HBV-G were sequenced to determine their relationship to global HBV-G strains. RESULTS: Ten HBV-G infections were found in the selected Dutch patients. All concerned HIV-1 infected males with HBV-A co-infection. Dutch HBV-G strains were phylogenetically closely related to reference HBV-G strains. CONCLUSIONS: In this study, HBV-G infection in the Netherlands is found exclusively in HIV-1 infected men as co-infection with HBV-A. A considerable percentage (37%) of men infected with HBV and HIV-1 are actually co- infected with two HBV genotypes.

HIV-1 dual infection is associated with faster CD4+ T-cell decline in a cohort of men with primary HIV infection.

BACKGROUND: In vitro, animal, and mathematical models suggest that human immunodeficiency virus (HIV) co- or superinfection would result in increased fitness of the pathogen and, possibly, increased virulence. However, in patients, the impact of dual HIV type 1 (HIV-1) infection on disease progression is unclear, because parameters relevant for disease progression have not been strictly analyzed. The objective of the present study is to analyze the effect of dual HIV-1 infections on disease progression in a well-defined cohort of men who have sex with men. METHODS: Between 2000 and 2009, 37 men who had primary infection with HIV-1 subtype B, no indication for immediate need of combination antiretroviral therapy (cART), and sufficient follow-up were characterized with regard to dual infection or single infection and to coreceptor use. Patients were followed to estimate the effect of these parameters on clinical disease progression, as defined by the rate of CD4(+) T-cell decline and the time to initiation of cART. RESULTS: Four patients presented with HIV-1 coinfection; 6 patients acquired HIV-1 superinfection, on average 8.5 months from their primary infection; and 27 patients remained infected with a single strain. Slopes of longitudinal CD4(+) T-cell counts and time-weighted changes from baseline were significantly steeper for patients with dual infection compared with patients with single infection. Multivariate analysis showed that the most important parameter associated with CD4(+) T-cell decline over time was dual infection (P = .001). Additionally, patients with HIV-1 coinfection had a significantly earlier start of cART (P < .0001). CONCLUSIONS: Dual HIV-1 infection is the main factor associated with CD4(+) T-cell decline in men who have untreated primary infection with HIV-1 subtype B.

Sexual transmission of hepatitis C virus in human immunodeficiency virus-negative men who have sex with men: a series of case reports.

Hepatitis C Virus (HCV) has recently emerged as sexual transmitted infection among (human immunodeficiency virus) HIV-positive but not HIV-negative men who have sex with men (MSM). We present 4 case reports showing that HIV-infection is not an absolute prerequisite for sexual HCV transmission in MSM. HIV-negative MSM with ulcerative sexual transmitted infection, those who engage in rough sexual practices or report a HCV-positive sexual partner, should be regularly screened for HCV.

Unusual cluster of HIV type 1 dual infections in Groningen, The Netherlands.

In 2007, 14 Dutch men having sex with men (MSM) filed a criminal case against three other men, accusing them of administering sedative drugs, sexual abuse, and deliberate subcutaneous injections with HIV-1-infected blood. Medical files showed that 9 of 17 men presented with an acute HIV-1 infection syndrome during 2006-2007. Two men were not infected with HIV. Analysis of viral strains in the 12 MSM and the three alleged donors showed that one donor and six recipients were double infected with two distinct HIV-1 subtype B strains, while another five recipients and one donor were single infected with either strain. Two men were infected with unrelated strains. The finding of multiple double infections with very similar HIV-1 strains is without precedent.

Generation of representative primary virus isolates from blood plasma after isolation of HIV-1 with CD44 MicroBeads.

Infection of cell cultures with cell-free virus isolated from HIV-infected patients is notoriously difficult and results in a loss of viral variation. Here, we describe viral sequences from PBMC, U87.CD4.CCR5 and U87.CD4.CXCR4 cell cultures and compare them to those from blood plasma from 12 patients from whom virus particles were isolated using CD44 MicroBeads. In both PBMC and U87.CD4.CCR5 cultures, 66% of the plasma viral strains were retrieved after culturing. In addition, coreceptor use was predicted based on the env-V3 sequence and tested in U87.CD4 cells expressing either CCR5 or CXCR4. Recovery was lower for the CXCR4-using viruses. Only 50% of the virus clusters predicted to use CXCR4 could be retrieved from cell cultures, while 71% of CCR5-using strains were found in U87.CCR5 cultures. Therefore, isolation of primary viruses with CD44 MicroBeads results in a good representation in cell culture of the in vivo divergence.

Lack of detection of XMRV in seminal plasma from HIV-1 infected men in The Netherlands.

BACKGROUND: Xenotropic murine leukaemia virus-related virus (XMRV) is a recently discovered human gammaretrovirus with yet unknown prevalence and transmission route(s). Its presence in prostate stromal fibroblasts and prostatic secretions suggests that XMRV might be sexually transmitted. We chose to study a compartment closely connected to the prostate, a location where XMRV was detected in independent studies. Seminal plasma samples from HIV-1 infected men were examined as they have an increased probability of acquiring sexually transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: We studied the prevalence of XMRV in 93 seminal plasma samples of 54 HIV-1 infected men living in The Netherlands with a nested PCR amplification specifically targeting the XMRV gag gene. As a control for the presence and integrity of retrovirus particles, HIV-1 was amplified from the same samples with a PCR amplification targeting the env gene of the virus, or HIV-1 was quantified with a real-time PCR amplifying part of the pol gene. CONCLUSIONS/SIGNIFICANCE: Although HIV-1 was amplified from 25% of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in the seminal plasma.

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness.

BACKGROUND: Two HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans et al., JAIDS 2008, 47:69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection. RESULTS: Biological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the tat gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus. CONCLUSIONS: These preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.

Multiple transmissions of a stable human leucocyte antigen-B27 cytotoxic T-cell-escape strain of HIV-1 in The Netherlands.

OBJECTIVE: The evolution of HIV-1 is largely shaped by the cytotoxic T-cell (CTL) response of the host as encoded by the human leucocyte antigen (HLA) genes. Certain HLA-B alleles can delay disease progression, but it is uncertain whether this protection will sustain or whether the virus is in the process of adaptation. In The Netherlands, HLA-B27 is moderately prevalent (approximately 8-16% of HLA-B alleles). If adaptation to HLA-B alleles is in progress, virus strains carrying escape mutations to HLA-B27 should appear in the epidemic by now. DESIGN: A subtype B HIV-1 strain carrying a HLA-B27 CTL-escape mutation in the main Gag-p24 KK10 epitope, R264G, together with a compensatory mutation outside this epitope, E260D, was detected in four patients from Amsterdam, The Netherlands, by sequence analysis of the gag gene. The patients were a drug user and three men who have sex with men, and were infected with HIV-1 between 2002 and 2008. METHODS: Characterization and evolutionary analysis of the HIV-1 CTL-escape strain was done by sequence analysis of serial blood plasma samples. RESULTS: The mutations involved were stable during follow-up and after transmission, also in two individuals lacking HLA-B27. CONCLUSION: The finding that a stable HLA-B27 CTL-escape strain is circulating in The Netherlands has important implications for the understanding of virus-host interactions and vaccine design alike. Vaccines targeted at inducing a CTL response might easily be circumvented by the virus. Also, patients carrying protective HLA alleles might not be protected anymore from disease progression in the future.

Incidence of human immunodeficiency virus type 1 dual infections in Amsterdam, The Netherlands, during 2003-2007.

BACKGROUND: The occurrence of human immunodeficiency virus type 1 (HIV-1) dual infections in Amsterdam, The Netherlands, was examined during 2003-2007 to investigate whether the number of HIV-1 dual infections increased as the number of HIV-1 infected individuals increased during the same period. METHODS: All first HIV-1 genotyping sequences obtained from 2003 through 2007 were retrieved and examined for the number of degenerate base codes in the reverse-transcriptase fragment. A total of 72 patients had >or=34 degenerate base codes; for these patients, a fragment of the V3-V4 region of the env gene was amplified, cloned, and sequenced to verify the presence of an HIV-1 dual infection. The number of dual infections were counted for each year investigated. RESULTS: No significant change in the incidence of dual infections was observed in our population of patients, who were selected on the basis of the number of degenerate base codes in each patient's first HIV-1 sequence obtained from 2003 through 2007. The frequency of HIV-1 dual infections varied between 1.0% and 2.4% each year, with no significant trend over time (P = .49). Patients with HIV-1 dual infections were similar to patients with single HIV-1 infections in The Netherlands with regard to distribution of risk group, sex, and HIV subtype. CONCLUSION: The proportion of HIV-1 dual infections in The Netherlands did not increase from 2003 through 2007, although the HIV-1-infected population expanded in this period.

Severe anaemia is not associated with HIV-1 env gene characteristics in Malawian children.

BACKGROUND: Anaemia is the most common haematological complication of HIV and associated with a high morbidity and a poor prognosis. The pathogenesis of HIV-associated anaemia is poorly understood and may include a direct effect of HIV on erythropoiesis. In vitro studies have suggested that specific HIV strains, like X4 that uses the CXCR4 co-receptor present on erythroid precursors, are associated with diminished erythropoiesis. This co-receptor affinity is determined by changes in the hypervariable loop of the HIV-1 envelope genome. In a previous case-control study we observed an association between HIV and severe anaemia in Malawian children that could not be fully explained by secondary infections and micronutrient deficiencies alone. We therefore explored the possibility that alterations in the V1-V2-V3 fragment of HIV-1 were associated with severe anaemia. METHODS: Using peripheral blood nucleic acid isolates of HIV-infected children identified in the previous studied we assessed if variability of the V1-V2-V3 region of HIV and the occurrence of X4 strains were more common in HIV-infected children with (cases, n = 29) and without severe anaemia (controls, n = 30). For 15 cases bone marrow isolates were available to compare against peripheral blood. All children were followed for 18 months after recruitment. RESULTS: Phylogenetic analysis showed that HIV-1 subtype C was present in all but one child. All V1-V2-V3 characteristics tested: V3 charge, V1-V2 length and potential glycosylation sites, were not found to be different between cases and controls. Using a computer model (C-PSSM) four children (7.8%) were identified to have an X4 strain. This prevalence was not different between study groups (p = 1.00). The V3 loop characteristics for bone marrow and peripheral blood isolates in the case group were identical. None of the children identified as having an X4 strain developed a (new) episode of severe anaemia during follow up. CONCLUSION: The prevalence of X4 strains in these young HIV-1-subtype-C-infected children that were most likely vertically infected and naïve to anti-retroviral therapy can be considered high compared to previous results from Malawi. It is unlikely that V1-V2-V3 fragment characteristics and HIV co-receptor affinity is an important feature in the development of severe anaemia in Malawian children.

A sudden rise in viral load is infrequently associated with HIV-1 superinfection.

OBJECTIVE: To investigate the association between an unexpected increase in the blood plasma HIV-1 viral load in chronically untreated HIV-infected patients and the occurrence of an HIV superinfection, we analyzed the HIV-1 quasispecies in plasma samples before and at peak level in 14 patients. RESULTS: Phylogenetic analysis of HIV-1 env-V3 fragments showed that in 2 patients a superinfection had occurred: their dominant V3 population at the peak level clustered separately from the V3 sequences in a sample predating the peak level. The rapid rise in viral load could be attributed to upper respiratory tract infections or a vaccination in 4 patients, suggesting that even minor health problems can result in significantly increased HIV-1 replication. In most other patients, no minor or major medical condition accompanied the rise in HIV-1 viral load, implying that in these patients the viral load increase was probably associated with disease progression. CONCLUSION: This study suggests that an unexpected rapid rise in the plasma HIV-1 viral load of untreated patients can infrequently be ascribed to an HIV-1 superinfection.

HIV-1 sequence evolution in vivo after superinfection with three viral strains.

With millions of people infected worldwide, the evolution of HIV-1 in vivo has been the subject of much research. Although recombinant viruses were detected early in the epidemic, evidence that HIV-1 dual infections really occurred came much later. Dual infected patients, consisting of coinfected (second infection before seroconversion) and superinfected (second infection after seroconversion) individuals, opened up a new area of HIV-1 evolution studies. Here, we describe the in-depth analysis of HIV-1 over time in a patient twice superinfected with HIV-1, first with a subtype B (B2) strain and then with CRF01_AE after initial infection with a subtype B (B1) strain. The nucleotide evolution of gag and env-V3 of the three strains followed a similar pattern: a very low substitution rate in the first 2-3 years of infection, with an increase in synonymous substitutions thereafter. Convergent evolution at the protein level was rare: only a single amino acid in a gag p24 epitope showed convergence in the subtype B strains. Reversal of CTL-epitope mutations were also rare, and did not converge. Recombinant viruses were observed between the two subtype B strains. Luciferase-assays suggested that the CRF01_AE long terminal repeat (LTR) constituted the strongest promoter, but this was not reflected in the plasma viral load. Specific real-time PCR assays based upon the env gene showed that strain B2 and CRF01_AE RNA was present in equal amounts, while levels of strain B1 were 100-fold lower. All three strains were detected in seminal plasma, suggesting that simultaneous transmission is possible.

Lichen planus remission is associated with a decrease of human herpes virus type 7 protein expression in plasmacytoid dendritic cells.

The cause of lichen planus is still unknown. Previously we showed human herpes virus 7 (HHV-7) DNA and proteins in lesional lichen planus skin, and significantly less in non-lesional lichen planus, psoriasis or healthy skin. Remarkably, lesional lichen planus skin was infiltrated with plasmacytoid dendritic cells. If HHV-7 is associated with lichen planus, then HHV-7 replication would reduce upon lichen planus remission. HHV-7 DNA detection was performed by nested PCR and HHV-7 protein by immunohistochemistry on lesional skin biopsies from lichen planus patients before treatment and after remission. Biopsies were obtained from lichen planus lesions before treatment (n = 18 patients) and after remission (n = 13). Before treatment 61% biopsies contained HHV-7 DNA versus 8% after remission (P = 0.01). HHV-7-protein positive cell numbers diminished significantly after remission in both dermis and epidermis. Expression of HHV-7 was mainly detected in BDCA-2 positive plasmacytoid dendritic cells rather than CD-3 positive lymphocytes. HHV-7 replicates in plasmacytoid dendritic cells in lesional lichen planus skin and diminishes after remission. This study further supports our hypothesis that HHV-7 is associated with lichen planus pathogenesis.

Routine HIV-1 genotyping as a tool to identify dual infections.

OBJECTIVES: The incidence of HIV-1 dual infections is generally thought to be low, but as dual infections have been associated with accelerated disease progression, its recognition is clinically important. Methods to identify HIV-1 dual infections are time consuming and are not routinely performed. DESIGN: Genotyping of the HIV-1 protease and reverse transcriptase (prot/RT) genes is commonly performed in the western world to detect drug-resistance mutations in clinical isolates. In our hospital, prot/RT baseline sequencing is part of the patient care for all newly infected patients in the Amsterdam region since 2003. We reasoned that degenerate base codes in this sequence could indicate either extensive viral evolution or infection with multiple HIV-1 strains. METHODS: We amplified, cloned and sequenced multiple HIV-1 envelope (env)-V3 and gag sequences from patients with 34 or more (range 34-99) degenerate base codes in the ViroSeq genotyping RT sequence (37 out of 1661 available records) to estimate the number of HIV-1 dual infections in this group. RESULTS: Of the 37 patients included in this study, 16 (43.2%, equal to 1% of the 1661 total records) had an HIV-1 dual infection based on phylogenetic analysis of env-V3/gag sequences. If only sequences with 45 or more degenerate base codes were taken into account, 73.3% of patients showed evidence of a dual infection. CONCLUSION: We describe an additional use of routinely performed HIV-1 genotyping. In patients with a high number of degenerate bases (> or = 34) in RT it is important to consider the possibility of a dual HIV-1 infection.

Sialoadhesin (CD169) expression in CD14+ cells is upregulated early after HIV-1 infection and increases during disease progression.

BACKGROUND: Sialoadhesin (CD169, siglec-1 or Sn) is an activation marker seen on macrophages in chronic inflammatory diseases and in tumours, and on subsets of tissue macrophages. CD169 is highly expressed by macrophages present in AIDS-related Kaposi's sarcoma lesions. It is also increased on blood monocytes of HIV-1 infected patients with a high viral load despite antiretroviral treatment. METHODOLOGY/PRINCIPAL FINDINGS: We investigated expression of sialoadhesin in untreated HIV-1 and HHV-8 infected patients, by real-time PCR and FACS analysis to establish its expression in relation to infection and disease progression. Patients analysed were either HIV-1 seroconverters (n = 7), in the chronic phase of HIV-1 infection (n = 21), or in the AIDS stage (n = 58). Controls were HHV-8 infected, but otherwise healthy individuals (n = 20), and uninfected men having sex with men (n = 24). Sialoadhesin mRNA was significantly elevated after HIV-1, but not HHV-8 infection, and a further increase was seen in AIDS patients. Samples obtained around HIV-1 seroconversion indicated that sialoadhesin levels go up early in infection. FACS analysis of PBMCs showed that sialoadhesin protein was expressed at high levels by approximately 90% of CD14(+) and CD14(+)CD16(+)cells of HIV-1(+) patients with a concomitant 10-fold increase in sialoadhesin protein/cell compared with uninfected controls. CONCLUSIONS/SIGNIFICANCE: We have shown that sialoadhesin is induced to high levels on CD14(+) cells early after HIV-1 infection in vivo. The phenotype of the cells is maintained during disease progression, suggesting that it could serve as a marker for infection and probably contributes to the severe dysregulation of the immune system seen in AIDS.