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Objective The aim of this study was to investigate introductions and spread of different HIV-1 subtypes in the Netherlands. Design We identified distinct HIV-1 transmission chains in the Netherlands within the global epidemic context through viral phylogenetic analysis of partial HIV-1 polymerase sequences from individuals enrolled in the ATHENA national HIV cohort of all persons in care since 1996, and publicly available international background sequences. Methods Viral lineages circulating in the Netherlands were identified through maximum parsimony phylogeographic analysis. The proportion of HIV-1 infections acquired in-country among heterosexuals and MSM was estimated from phylogenetically observed, national transmission chains using a branching process model that accounts for incomplete sampling. Results As of 1 January 2019, 2589 (24%) of 10 971 (41%) HIV-1 sequenced individuals in ATHENA had non-B subtypes (A1, C, D, F, G) or circulating recombinant forms (CRF01AE, CRF02AG, CRF06-cpx). The 1588 heterosexuals were in 1224, and 536 MSM in 270 phylogenetically observed transmission chains. After adjustments for incomplete sampling, most heterosexual (75%) and MSM (76%) transmission chains were estimated to include only the individual introducing the virus (size = 1). Onward transmission occurred mostly in chains size 2-5 amongst heterosexuals (62%) and in chains size at least 10 amongst MSM (64%). Considering some chains originated in-country from other risk-groups, 40% (95% confidence interval: 36-44) of non-B-infected heterosexuals and 62% (95% confidence interval: 49-73) of MSM-acquired infection in-country. Conclusion Although most HIV-1 non-B introductions showed no or very little onward transmission, a considerable proportion of non-B infections amongst both heterosexuals and MSM in the Netherlands have been acquired in-country.

Background Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. Methods In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. Results These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. Conclusions Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.

Summary Background Pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir disoproxil fumarate is highly effective against acquisition of HIV infection, and only two cases of infection with a multidrug-resistant virus have been reported under adequate long-term adherence, as evidenced by tenofovir diphosphate concentrations in dried blood spots. We report a case of wild-type HIV-1 infection despite consistent use of emtricitabine and tenofovir disoproxil fumarate. Methods The patient participated in the Amsterdam PrEP project, a demonstration project of daily and event-driven PrEP. We did extensive testing for HIV, including plasma HIV RNA and nested PCR on bulk peripheral blood mononuclear cells (PBMCs) and sigmoid biopsies after seroconversion. Findings A 50-year-old man who has sex with men and had been on daily emtricitabine and tenofovir disoproxil fumarate for 8 months presented with fever, urinary tract infection caused by Escherichia coli , anal lymphogranuloma venereum infection, and a positive fourth-generation HIV test. We found an atypical seroconversion pattern, with initially only gp160 antibodies detected in the western blot. HIV RNA could not be detected in plasma, and nested PCR for HIV RNA and DNA on bulk PBMCs and sigmoid biopsies were negative. PrEP was discontinued; 3 weeks later HIV RNA was detected in plasma. No drug-resistant mutations were detected. Tenofovir diphosphate concentrations in dried blood spots were stable and high. Interpretation To our knowledge, this is the first detailed case report suggesting wild-type HIV-1 infection despite good adherence, evidenced by repeatedly high concentrations of tenofovir diphosphate in dried blood spots. PrEP providers need to be aware that infection can occur despite good adherence. Regular HIV testing and awareness of atypical patterns of seroconversion is highly recommended. Funding ZonMw, National Institute for Public Health and the Environment, Internal GGD research funds, Aidsfonds, Stichting AmsterdamDiner Foundation, Gilead Sciences, Janssen Pharmaceutica, M A C AIDS Fund, and ViiV Healthcare.

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.

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.

Background: Drug resistance mutations (DRMs) increasingly jeopardize paediatric HIV programmes in sub-Saharan Africa. As individual monitoring of DRMs and viral loads has limited availability, population data on DRMs are essential to determine first-line susceptibility. Paediatric data from sub-Saharan Africa are scarce and unavailable for Malawi. Objectives: To determine the prevalence of virological failure (VF) and DRMs among ART-naive HIV-infected Malawian children during the first year of first-line ART. Methods: In a prospective cohort of HIV-infected Malawian children, on first-line treatment, children were followed monthly; blood was collected for viral load testing (6 and 12 months) and genotypic resistance testing (12 months). VF was defined as at least one viral load >1000 copies/mL or death after 6 months of ART. DRMs were identified and susceptibility to NRTIs and NNRTIs was scored using the Stanford algorithm and by calculating genotypic susceptibility scores (GSSs). Results: VF occurred in 66% (23/35) of the children during 12 months of follow-up. DRMs were detected in 44% (15/34); all had NNRTI resistance and 12% (4/34) had dual-class NNRTI/NRTI resistance. Reduced susceptibility (DRMs and GSS

We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project, which analyzes complete viral genomes from more than 3000 HIV-1-infected Europeans with high-throughput deep sequencing techniques. The goal of the BEEHIVE project is to determine the contribution of viral genetics to virulence. Recently we performed a pilot experiment with 125 patient plasma samples to identify the method that is most suitable for isolation of HIV-1 viral RNA for subsequent long-amplicon deep sequencing. We reported that manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) provides superior results over robotically extracted RNA. The latter approach used the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics), the QIAcube robotic system (Qiagen), or the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular). Here we present a detailed protocol for the labor-intensive manual extraction method that yielded the best results.

Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5′ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5′ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and the trans -acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein. IMPORTANCE The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.

A central feature of pathogen genomics is that different infectious particles (virions, bacterial cells, etc.) within an infected individual may be genetically distinct, with patterns of relatedness amongst infectious particles being the result of both within-host evolution and transmission from one host to the next. Here we present a new software tool, phyloscanner, which analyses pathogen diversity from multiple infected hosts. phyloscanner provides unprecedented resolution into the transmission process, allowing inference of the direction of transmission from sequence data alone. Multiply infected individuals are also identified, as they harbour subpopulations of infectious particles that are not connected by within-host evolution, except where recombinant types emerge. Low-level contamination is flagged and removed. We illustrate phyloscanner on both viral and bacterial pathogens, namely HIV-1 sequenced on Illumina and Roche 454 platforms, HCV sequenced with the Oxford Nanopore MinION platform, and Streptococcus pneumoniae with sequences from multiple colonies per individual. phyloscanner is available from https://github.com/BDI-pathogens/phyloscanner.

HIV-1 set-point viral load—the approximately stable value of viraemia in the first years of chronic infection—is a strong predictor of clinical outcome and is highly variable across infected individuals. To better understand HIV-1 pathogenesis and the evolution of the viral population, we must quantify the heritability of set-point viral load, which is the fraction of variation in this phenotype attributable to viral genetic variation. However, current estimates of heritability vary widely, from 6% to 59%. Here we used a dataset of 2,028 seroconverters infected between 1985 and 2013 from 5 European countries (Belgium, Switzerland, France, the Netherlands and the United Kingdom) and estimated the heritability of set-point viral load at 31% (CI 15%–43%). Specifically, heritability was measured using models of character evolution describing how viral load evolves on the phylogeny of whole-genome viral sequences. In contrast to previous studies, (i) we measured viral loads using standardized assays on a sample collected in a strict time window of 6 to 24 months after infection, from which the viral genome was also sequenced; (ii) we compared 2 models of character evolution, the classical “Brownian motion” model and another model (“Ornstein–Uhlenbeck”) that includes stabilising selection on viral load; (iii) we controlled for covariates, including age and sex, which may inflate estimates of heritability; and (iv) we developed a goodness of fit test based on the correlation of viral loads in cherries of the phylogenetic tree, showing that both models of character evolution fit the data well. An overall heritability of 31% (CI 15%–43%) is consistent with other studies based on regression of viral load in donor–recipient pairs. Thus, about a third of variation in HIV-1 virulence is attributable to viral genetic variation., Author summary The severity of the outcome of infection by a pathogen depends on many distinct factors. These include the environment and the genetic sequences of both the host and the pathogen, among others. The fraction of variability in disease outcome explained by pathogen genetic factors is termed “heritability”, because these factors are “inherited” by the new host upon infection. Quantifying heritability is key to understanding the development of the disease and the evolution of the virus. Here, we determined heritability of set-point viral load (SPVL) in HIV-1. SPVL is the stable value of viraemia in asymptomatic infection and it is a strong predictor of disease severity. While heritability of SPVL has been estimated using comparisons of viral genome sequences, this has resulted in widely variable estimates of heritability. Using a large dataset of patients living in Europe, standardised viral loads measures, and new methods, we obtain a more definitive estimate of HIV-1 SPVL heritability in Europe at about 30%. Thus, a significant amount of the variation in disease outcome is explained by the genetics of the virus.

Next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of rapid between- and within-host evolution may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions.De novoassembly avoids this bias by effectively aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the toolshiverto preprocess reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We useshiverto reconstruct the consensus sequence and minority variant information from paired-end short-read data produced with the Illumina platform, for 65 existing publicly available samples and 50 new samples. We show the systematic superiority of mapping toshiver’s constructed reference over mapping the same reads to the standard reference HXB2: an average of 29 bases per sample are called differently, of which 98.5% are supported by higher coverage. We also provide a practical guide to working with imperfect contigs.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1599-7) contains supplementary material, which is available to authorized users.

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

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.

We aimed to study patterns of HIV transmission among Suriname, The Netherlands Antilles, and The Netherlands. Fragments of env, gag, and pol genes of 55 HIV-infected Surinamese, Antillean, and Dutch heterosexuals living in The Netherlands and 72 HIV-infected heterosexuals living in Suriname and the Antilles were amplified and sequenced. We included 145 pol sequences of HIV-infected Surinamese, Antillean, and Dutch heterosexuals living in The Netherlands from an observational cohort. All sequences were phylogenetically analyzed by neighbor-joining. Additionally, HIV-1 mobility among ethnic groups was estimated. A phylogenetic tree of all pol sequences showed two Surinamese and three Antillean clusters of related strains, but no clustering between ethnic groups. Clusters included sequences of individuals living in Suriname and the Antilles as well as those who have migrated to The Netherlands. Similar clustering patterns were observed in env and gag. Analysis of HIV mobility among ethnic groups showed significantly lower migration between groups than expected under the hypothesis of panmixis, apart from higher HIV migration between Antilleans in The Netherlands and all other groups. Our study shows that HIV transmission mainly occurs within the ethnic group. This suggests that cultural factors could have a larger impact on HIV mobility than geographic distance.

Background Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up. Aim To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue. Methods Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used. Results The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P 3; HBV DNA > 20 000 IU/ml) and inactive hepatitis (histological activity index scorer

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.

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.

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 ( 34) in RT it is important to consider the possibility of a dual HIV-1 infection. 2007 Lippincott Williams & Wilkins AIDS 2007, 21:807‐811

Summary Background Lichen planus (LP) is a common inflammatory skin disease of unknown aetiology. Viral causes have been suggested. Objectives To find candidate viruses associated with LP. Methods Lesional and nonlesional skin samples, peripheral blood mononuclear cells and serum were obtained from patients with LP. Ultrastructural, viral DNA, immunohistochemical and serological analyses were performed, and comparisons were made with psoriatic and normal skin. Results Electron microscopy revealed typical 120–200-nm enveloped particles with a 100-nm nucleus resembling human herpesvirus (HHV) virions both in dermis and in epidermis of lesional LP tissue. HHV-7 DNA was found in 11 of 18 lesional LP samples, as opposed to only one of 11 nonlesional LP samples (P =0·06), two of 11 lesional psoriasis samples (P = 0·05) and none of four normal skin samples. No relation was found between LP skin and DNA of other known HHVs (HHV-1–6 and 8). With immunohistochemistry, significantly more HHV-7+ cells were found in lesional LP epidermis than in normal epidermis. Lesional LP dermis contained significantly more HHV-7+ cells than nonlesional LP, psoriatic or normal dermis. Moreover, LP skin contained overwhelmingly and consistently more plasmacytoid dendritic cells (upregulated in virally induced conditions) than nonlesional LP samples. Conclusions We conclude that HHV-7 replicates in LP lesions, but not in psoriasis, another inflammatory skin condition. HHV-7 is possibly involved in the pathogenesis of LP. These preliminary data make further research on this topic of interest.

To establish the effect of the presence in blood cells of cytomegalovirus (CMV) and human herpesvirus 8 (HHV8) DNA, two herpesviruses that are activated frequently in AIDS patients, were selected from the Amsterdam Cohort Studies on HIV/AIDS 181 PBMC samples from patients with and without Kaposi's sarcoma (KS), and with and without CMV-related disease. The viral loads of both HHV8 and CMV were determined by real-time PCR at the time of diagnosis of AIDS. There was no significant difference in prevalence and load for CMV between the KS and non-KS patients. The variable related most strongly to KS was the presence of HHV8 DNA in PBMCs, whilst CMV DNA was related to the development of CMV disease and shortened survival. The frequency of detection of HHV8 increased when the patient presented with more severe KS symptoms at diagnosis, but detection of HHV8 DNA did not influence survival. Therefore, HHV8 and CMV DNA measured in the blood of AIDS patients, are each related mainly to the associated disease, and have no additional predictive value in these patients. (c) 2005 Wiley-Liss, Inc

Semen samples from a donor who seroconverted for human immunodeficiency virus type 1 (HIV-1) during the period that he was donating at our clinic were stored before and after infection. Semen analysis was done on all of these samples before cryopreservation. Retrospectively, both qualitative and quantitative HIV-1 testing was performed on the cryopreserved semen samples to determine the time of primary HIV-1 infection. After HIV-1 infection, semen volume, sperm motility and the percentage of spermatozoa with normal morphology were reduced compared with the same parameters before HIV-1 infection. HIV-1 RNA was intermittently detectable in semen. HIV-1 infection led to a reduction in semen volume, sperm motility and normal sperm morphology in this donor. However, the clinical significance of these findings is unclear. A longitudinal cohort study on the effects of HIV-1 infection on semen quality is necessary to confirm these findings.

The magnitude and complexity of the HIV-1 genetic diversity are major challenges for vaccine development. Investigation of the genotypes circulating in areas of high incidence, as well as their interactions, will be a milestone in the development of an efficacious vaccine. Because HIV-1 subtype C (HIV-1C) is responsible for most of the 36 million infections worldwide we investigated the HIV-1C strains circulating in Ethiopia in a retrospective, cross-sectional study. Serum samples from HIV-1-positive individuals were collected in seven Ethiopian cities and towns. Nucleotide sequences of the gag, pol, and env genes were analyzed. We performed phylogenetic analysis by the neighbor-joining and maximum-likelihood methods with sequences from 30 isolates, and we determined recombination by the bootscanning method as implemented in the SIMPLOT program. Sequence analyses of a 2600-nucleotide fragment (including the gag gene, the protease, and the 5' half of reverse transcriptase of the pol gene) and the corresponding V1V2/C2V3 envelope regions confirmed that two distinct HIV-1C genotypes (C' and C") are cocirculating in Ethiopia, as shown previously by the analysis of the C2V3 envelope region. We have identified intrasubtype recombination between the two HIV-1C genotypes, C' and C", with 6 of the 30 (20%) analyzed viruses being recombinants. The C' sequences were phylogenetically linked to the fast spreading viruses in India and southern Africa. Furthermore, all the recombinant viruses shared the C' V1V3 region of the envelope, suggesting that the prevalence of viruses with the C' envelope is increasing compared to the C" envelope. The possibility that viruses with a C' envelope have a biological advantage over the viruses with a C" envelope should be further investigated in biological and epidemiological studies.

Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8+cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8+CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.

Item does not contain fulltext Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.

Objective To reconstruct the epidemiological relationships of the HIV epidemics among injecting drug users (IDU) in western Europe. Methods HIV env V3 sequences of and epidemiological data were obtained from 145 IDU who seroconverted in three sequential periods: 1984-1988, 1989-1992 and 1993-1997. The sequences were phylogenetically analysed and examined for signature patterns characteristic of northern European IDU, including the conserved GGC codon in the V3 loop. Results Subpopulations of genetically related HIV strains were observed in Italy, France, Scotland and Spain, in contrast to the Netherlands, Austria and Switzerland. This difference between the two groups of countries suggests that the HIV epidemics amongst IDU in the latter group was caused by multiple virus introductions. In Edinburgh and the surrounding area, most IDU were infected with the same GGC strain over the 12-year study period. The epidemic among IDU in north-western Europe started with GGC viruses, whereas in south-western Europe non-GGC viruses predominated. This geographical separation has faded during the course of the epidemic, most likely because of virus exchange among IDU populations.

Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.

Others and we have previously shown that subtype C is the predominant HIV-1 subtype and the major cause of AIDS in Ethiopia. The present study shows that subtype C in Ethiopia has a genetic subcluster, designated C', has not increased in frequency, or spread geographically, over the period 1988 (%C' = 23/53) to 1996-1997 (%C' = 26/50). There is no association of the HIV-1 subtype C or subcluster C' with geographic location, time of sample collection, or risk group in Ethiopia. Of 105 randomly collected samples representing 7 different towns in Ethiopia, all but 2 (1 subtype A from Addis Ababa, 1997 and 1 subtype D from Dessie, 1996) belong to subtype C.

We have shown previously that human herpesvirus 8 (HHV8) seroconversion for antibodies to the latency-associated nuclear antigen encoded by ORF73 and/or the lytic capsid antigen (vp19) encoded by ORF65 is associated with orogenital contact and is strongly linked to the development of Kaposi's sarcoma among HIV-infected individuals in the Amsterdam Cohort Studies. Here, we investigate the relationship between seroconversion to these antigens and primary HHV8 infection. Between 1984 and 1997, 215 HHV8 seroconversions to ORF73 (106 cases or 49%) and/or to ORF65 (159 cases or 74%) were recorded in the cohort of homosexual men. The HHV8 seroconversion rate among HIV-infected homosexual men (6.2 per 100 person years) was consistently higher than among HIV-uninfected men (2.6 per 100 person years). In HIV-infected but not in uninfected individuals, seroconversion to ORF73/latency-associated nuclear antigen precedes that to ORF65/vp19. Antibody levels to both ORF65- and ORF73-encoded antigens were higher in HIV-infected than in HIV-uninfected men, and among HIV-seropositives, antibody levels to ORF65/vp19 rise even higher with declining CD4 cell counts and peak with Kaposi's sarcoma development, suggesting continuing and increasing viral replication. In 10.3% of HHV8 seroconversions, transient serum viremia could be demonstrated before or at seroconversion. Together with the previously reported link between unprotected orogenital sex and HHV8 seroconversion, our observations suggest that HHV8 seroconversions result from primary infections.

In addition to development or selection of resistance, failure to continuously suppress HIV-1 production while still using initially effective combination antiretroviral therapy (cART) may result from super-infection with a drug-resistant strain. Both transmission of drug resistant HIV and super-infection have been demonstrated. We analysed HIV pol genes obtained before start of initially successful cART and during failure while still on cART in 101 patients. Difference in precART and cART failure sequences were explained by evolution and not by super-infection.

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4 + T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.

To the Editor: Dual infection with different strains of HIV type 1 (HIV-1) is reported with increasing frequency, attributed mostly to coinfection at the time of the primary infection. However, some patients were superinfected with a second virus after the original seroconversion,1 which generally accelerated disease progression.2 We encountered a case of serial HIV-1 superinfection resulting in a triple infection in a Dutch patient who was originally infected with a subtype B virus. A 35-year-old homosexual man was found to be HIV-1–seropositive in March 2001 and was referred for follow-up. Early in July 2003, the patient presented with acute onset . . .

Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses belonging genetically to distinct subtypes (D. L. Robertson, P. M. Sharp, F. E. McCutchan, and B. H. Hahn, Nature [London] 374:124-126, 1995). To establish the frequency of natural infections with recombinant viruses and to exclude tissue culture artifacts, we analyzed plasma samples from the UNAIDS sample collection. The collection includes samples from 53 individuals infected with subtype A (n = 9), subtype B (n = 15), subtype C (n = 1), subtype D (n = 13), and subtype E (n = 15) on the basis of V3 region analysis. Phylogenetic analysis of the gag gene fragment showed intersubtype recombinant genomes in 23 cases: 3 of 9 (33%) of subtype A, 2 of 15 (13%) of subtype B, 3 of 13 (23%) of subtype D, and all of subtype E. Of the 23 recombinant viruses, 19 had a gag gene from one subtype and env from another (B(env)/C(gag), A(env)/C(gag), D(env)/A(gag), and E(env)/A(gag)). Phylogenetic analysis clustered the A(gag) of subtype E viruses as an outgroup of subtype A, suggesting that these viruses may belong to a distinct A' cluster. The remaining four recombinant viruses (B(env)/B(p17)F(p24), A(env)/A(p17)D(p24), A(env)/A(p17)C(p24), and D(env)/ D(p17)A(p24)) had breakpoint crossover sites in the proximity of the p17-p24 protein processing site. We conclude that recombination in the gag gene is highly frequent among the major env subtypes and that selection of recombinants is apparently based on particularly beneficial combinations of gag and env gene products.

Human immunodeficiency type 1 (HIV-1) DNA in peripheral blood cells of HIV-1 infected individuals may be present as integrated and/or unintegrated DNA. Several reports have indicated that a major proportion of HIV-1 DNA in the asymptomatic phase is linear, full-length, and unintegrated and in the symptomatic phase either circular unintegrated or integrated in the host genome. We developed a quantitative polymerase chain reaction (PCR) technique to detect single-LTR HIV-1 DNA junctions, reflecting the presence of unintegrated single-LTR circles. In vitro infection of a CD4+ T-cell line resulted first in the increase of single-LTR junctions followed by syncytium formation and a rise of p24 antigen production. The number of single-LTR HIV-1 DNA junctions was further studied in two acutely infected individuals and in 21 long-term infected individuals. The number of single-LTR junctions was significantly correlated with CD4+ cell decline, p24 antigen expression, and total HIV-1 DNA content of peripheral blood mononuclear cells (PBMC). Single-LTR HIV-1 DNA junctions were absent from PBMC containing other forms of HIV-1 DNA in four of nine non/slow progressors relative to 2 of 12 rapid progressors/AIDS patients. We conclude from our data that quantitative detection of single-LTR HIV-1 DNA junctions can be used as an early DNA marker of the transition from clinical latency to active replication in the peripheral blood. © 1995 Wiley-Liss, inc.

The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan(®) technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan(®) reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form.

Positively charged amino acid substitutions at positions 11 and 25 within the loop of the third variable region (V3) of HIV-1 subtype B envelope have been shown to be associated with the syncytium-inducing (SI) phenotype of the virus. The present study was designed to examine SI and NSI-associated V3 mutations in HIV-1 subtypes other than B. HIV-1 RNA was isolated from 53 virus stocks and 26 homologous plasma samples from 53 recently infected individuals from Brazil, Rwanda, Thailand, and Uganda. The C2-V3 region of the viral envelope was converted to cDNA, amplified, and sequenced. Of 53 primary virus stock samples 49 were biologically phenotyped through measurement of the syncytium-inducing capacity in MT-2 cells (to differentiate between SI and NSI phenotypes). In addition, after passage of primary isolates through PHA stimulated donor PBMC, the replication capacity was determined in U937-2, CEM, MT-2, and Jurkat-tat cell lines (to differentiate rapid/high and slow/low phenotypes). According to the seq...

We studied the relationship between the rate of disease progression after HIV-1 seroconversion and the level of IgG antibody response to HIV-1 envelope and core epitopes. This was done by comparing a group of fast-progressing individuals and a group of slow-progressing individuals for serum IgG titers to peptides from the gp120-V3 neutralization domain, to a peptide from the immunodominant gp41 epitope (residues 590 to 607), and to recombinant gp120 and p24. The two groups displayed a large overlap in titers to the envelope epitopes, which precluded their differentiation at most time points after seroconversion. Low responsiveness to envelope antigens was not only found in a few fast-progressors but also in one individual who remained asymptomatic for at least 92 months after seroconversion. The only significant differences between the groups were found in the first months after seroconversion when the responses to the V3 domain and the gp41 epitope were more vigorous in the group of fast-progressors. Furthermore, on evaluating ratios of anti-V3 antibody titers to anti-gp120 antibody titers we found no indication that fast disease progression was associated with a restriction in antibody response to the V3 epitope. We did confirm the finding that fast disease progression is associated with low levels of p24-directed antibodies, both early after seroconversion and at later stages. These data demonstrate that levels of IgG antibodies to envelope epitopes are poor predictors of rapid disease progression and suggest that the role of V3-directed neutralizing antibodies in preventing subversion of the immune system is not decisive in natural HIV-1 infection. (C) 1994 Academic Press, Inc

Over time a single patient can be infected by multiple intraor intersubtype human immunodeficiency viruses type 1 (HIV-1) strains. These so-called dual infections are divided into co-infections and superinfections. HIV-1 co-infection is described as a second infection taking place before measurable HIV-1 antibody production by the immune system (seroconversion) and HIV-1 superinfection is defined as a second infection occurring after seroconversion. Here the focus lays on superinfections, which have implications for HIV-1 transmission, treatment and vaccine development (Gottlieb et al., 2004). Moreover superinfections can give rise to HIV-1 circulating recombinant forms (CRF); this significantly increases the global epidemiology (Gottlieb et al., 2007). Also due to these recombination events, two different drug resistant HIV-1 viruses could lead to multi drug resistance or even to more pathogenic viruses (Gottlieb et al., 2004, Blackard et al., 2004, Fernandez Larrosa et al., 2006). Another important issue that is not extensively surveyed in literature is the clinical relevance of HIV-1 superinfection, as most descriptions rely on case reports and not on controlled cohort studies. The individual cases differ in severity, as several superinfected patients with rapidly progressive HIV-1 disease have been described since 2002, but superinfection cases were also found by coincidence in long-term non-progressors (LTNPs). These patients were able to control HIV-1 disease before and sometimes also after the superinfection event. So it is not resolved yet how superinfection affects disease progression in general and, if any, specific host or viral factors are involved. In this chapter, first an overview will be given of HIV-1 superinfected patients from several studies, with regard to HIV-1 disease progression. Disease progression is indicated by an increase in the HIV-1 plasma viral load, a decrease in the CD4+ T-cell count, acquired immunodeficiency syndrome (AIDS) related events and/or the start of antiretroviral treatment. These outcomes are compared to values of disease progression for single HIV-1 infected patients, resulting in an indication of the clinical relevance of HIV-1 superinfection.

The novel human retrovirus xenotropic murine leukemia virus-related virus (XMRV) is arguably the most controversial virus of this moment. After its original discovery in prostate cancer tissue from North American patients, it was subsequently detected in individuals with chronic fatigue syndrome from the same continent. However, most other research groups, mainly from Europe, reported negative results. The positive results could possibly be attributed to contamination with mouse products in a number of cases, as XMRV is nearly identical in nucleotide sequence to endogenous retroviruses in the mouse genome. But the detection of integrated XMRV proviruses in prostate cancer tissue proves it to be a genuine virus that replicates in human cells, leaving the question: how did XMRV enter the human population? We will discuss two possible routes: either via direct virus transmission from mouse to human, as repeatedly seen for, e.g., Hantaviruses, or via the use of mouse-related products by humans, including vaccines. We hypothesize that mouse cells or human cell lines used for vaccine production could have been contaminated with a replicating variant of the XMRV precursors encoded by the mouse genome.

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.