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International audience; Objectives: Molecular epidemiology is applied to various aspects of HIV transmission analyses. With ultradeep sequencing (UDS), in-depth characterization of transmission episodes involving minority variants is permitted. We explored HIV-1 epidemiological linkage and evaluated characteristics of transmission dynamics and transmitted drug resistance (TDR) detection through the added value of UDS.Design: HIV pol gene fragments were sequenced by UDS and Sanger sequencing on samples of 70 HIV-1-infected, treatment-naive recently diagnosed MSM.Methods: Pairwise genetic distances and maximum likelihood phylogenies were computed. Transmission events were identified as clades with branch support at least 70% and intraclade genetic difference less than 4.5%. TDR mutations were recognized from the TDR consensus list. Transmission directionality, directness and inoculum size were inferred from tree topologies.Results: Both datasets concurred in the identification of seven transmission pairs and one cluster of three patients. With UDS, direction of transmission was inferred in four out of eight chains. Evidence for multiple founder viruses was found in two out of eight chains. No transmission of minority-resistant variants was evidenced. TDR mutations prevalence in protease and reverse transcriptase fragments was 4.3% with Sanger sequencing and 18.6% with UDS.Conclusion: Although Sanger sequencing and UDS identified the same transmission chains, UDS provided additional information on founder viruses, direction of transmission and levels of TDR. Nevertheless, topology of clusters was not always consistent across gene fragments, calling for a cautious interpretation of the data. Moreover, unobserved intermediary links cannot be excluded. Phylogenetic analysis use as a forensic technique for HIV transmission investigations is risky.

Background. The United Kingdom human immunodeficiency virus (HIV) epidemic was historically dominated by HIV subtype B transmission among men who have sex with men (MSM). Now 50% of diagnoses and prevalent infections are among heterosexual individuals and mainly involve non-B subtypes. Between 2002 and 2010, the prevalence of non-B diagnoses among MSM increased from 5.4% to 17%, and this study focused on the drivers of this change. Methods. Growth between 2007 and 2009 in transmission clusters among 14 000 subtype A1, C, D, and G sequences from the United Kingdom HIV Drug Resistance Database was analysed by risk group. Results. Of 1148 clusters containing at least 2 sequences in 2007, >75% were pairs and >90% were heterosexual. Most clusters (71.4%) did not grow during the study period. Growth was significantly lower for small clusters and higher for clusters of ≥7 sequences, with the highest growth observed for clusters comprising sequences from MSM and people who inject drugs (PWID). Risk group (P < .0001), cluster size (P < .0001), and subtype (P < .01) were predictive of growth in a generalized linear model. Discussion. Despite the increase in non-B subtypes associated with heterosexual transmission, MSM and PWID are at risk for non-B infections. Crossover of subtype C from heterosexuals to MSM has led to the expansion of this subtype within the United Kingdom.

Objectives The hepatitis C virus (HCV) epidemic is evolving quickly despite new treatments, and due to behaviour changes increasing at-risk situations. We investigated potential origins and evolution of the HCV-4d French emergence among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM), in Paris in 2003. Methods We analysed all HCV sequences from the initial Paris outbreak with all newly available sequences publicly available, including sampling date and geographical location, resulting in 184, 68, 156, 107, 13 and 2 sequences from France, The Netherlands, other European countries, Africa, the Middle East or Turkey, Americas and Asia, respectively. Phylogenetic reconstruction was performed using maximum likelihood and Bayesian approaches. Results HCV-4d sequences from Europe were strongly separated from non-European sequences. Sequences from the initial Paris outbreak were all included into two well-separated and supported clusters with branch support at 100%, mean genetic distance 3.4 substitutions/100 nucleotides between their common ancestor and the previous node. The largest cluster interleaved French (n = 98) and Dutch (n = 28) sequences, suggesting several translocations between these countries. This cluster included 41 French sequences from Lyon sampled after 2014, highlighting its continuous spread within France since the initial outbreak. The smallest cluster included one Paris sequence with UK sequences (n = 9). Discussion A few previous works have shown HCV-4d transmissions occurring between a few countries. In our work, we suggest a new and large connection between France and The Netherlands MSM communities and highlight a well-separated pan-European transmission network. Large collaborative networks are needed to investigate ongoing transmissions across countries and help specific prevention measures.

Background Transmitted drug resistance (TDR) compromises clinical management and outcomes. Transmitted drug resistance surveillance and identification of growing transmission clusters are needed in the Southeast, the epicenter of the US HIV epidemic. Our study investigated prevalence and transmission dynamics in North Carolina. Methods We analyzed surveillance drug resistance mutations (SDRMs) using partial pol sequences from patients presenting to 2 large HIV outpatient clinics from 1997 to 2014. Transmitted drug resistance prevalence was defined as ≥1 SDRMs among antiretroviral therapy (ART)–naïve patients. Binomial regression was used to characterize prevalence by calendar year, drug class, and demographic and clinical factors. We assessed the transmission networks of patients with TDR with maximum likelihood trees and Bayesian methods including background pol sequences (n = 15 246). Results Among 1658 patients with pretherapy resistance testing, ≥1 SDRMs was identified in 199 patients, with an aggregate TDR prevalence of 12% (95% confidence interval, 10% to 14%) increasing over time (P = .02). Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs; 8%) was common, followed by nucleoside reverse transcriptase inhibitors (4%) and protease inhibitors (2%). Factors associated with TDR were being a man reporting sex with men, white race, young age, higher CD4 cell count, and being a member of a transmission cluster. Transmitted drug resistance was identified in 106 clusters ranging from 2 to 26 members. Cluster resistance was primarily NNRTI and dominated by ART-naïve patients or those with unknown ART initiation. Conclusions Moderate TDR prevalence persists in North Carolina, predominantly driven by NNRTI resistance. Most TDR cases were identified in transmission clusters, signifying multiple local transmission networks and TDR circulation among ART-naïve persons. Transmitted drug resistance surveillance can detect transmission networks and identify patients for enhanced services to promote early treatment.

Objective Transmission of HIV-1 involves a bottleneck in which generally a single HIV-1 variant from a diverse viral population in the transmitting partner establishes infection in the new host. It is still unclear to what extent this event is driven by specific properties of the transmitted viruses or the result of a stochastic process. Our study aimed to better characterize this phenomenon and define properties shared by transmitted viruses. Design We compared antigenic and functional properties of envelope glycoproteins of viral variants found during primary infection in 27 patients belonging to eight transmission chains. Methods We generated pseudotyped viruses expressing Env variants of the viral quasispecies infecting each patient and compared their sensitivity to neutralization by eight human monoclonal broadly neutralizing antibodies (HuMoNAbs). We also compared their infectious properties by measuring their infectivity and sensitivity to various entry inhibitors. Results Transmitted viruses from the same transmission chain shared many properties, including similar neutralization profiles, sensitivity to inhibitors, and infectivity, providing evidence that the transmission bottleneck is mainly nonstochastic. Transmitted viruses were CCR5-tropic, sensitive to MVC, and resistant to soluble forms of CD4, irrespective of the cluster to which they belonged. They were also sensitive to HuMoNAbs that target V3, the CD4-binding site, and the MPER region, suggesting that the loss of these epitopes may compromise their capacity to be transmitted. Conclusion Our data suggest that the transmission bottleneck is governed by selective forces. How these forces confer an advantage to the transmitted virus has yet to be determined.

Small ruminant lentiviruses (SRLVs) cause chronic, persistent infections in populations of domestic sheep and goats throughout the world. In this study, we use genomic data to investigate the origins and history of the SRLV pandemic. To explore the hypothesis that SRLV infection disseminated during Neolithic times, we performed a serology and DNA sequencing-based investigation of SRLVs diversity in the Fertile Crescent region, where domestication of sheep and goats is thought to have originally occurred. While we found an elevated level of viral genetic diversity compared to other regions of the world, we did not find unambiguous evidence that the Fertile Crescent region was the centre of the contemporary SRLV pandemic. We therefore examined historical reports to investigate the relationship between contemporary SRLV distribution and diversity and the emergence of SRLV-associated disease. Historical data suggested that the emergence of SRLV-associated disease might be associated with the long-distance export of exotic small ruminant breeds - in particular, karakul sheep from Central Asia - during the late 19thand early 20thcenturies. Phylogeographic analysis could neither confirm nor refute this hypothesis. However, we anticipate that future accumulation of genomic data from SRLV strains found throughout the world may allow for a more definitive assessment. The openly available data and resources assembled in this study will facilitate future investigations in this area.ImportanceViruses that cause chronic, persistent infections have circulated in animals for millions of years. However, many have only emerged as pathogens within the far shorter timeframe of recorded human history. It is important to understand the history of chronic viral infections in domestic animals, so that more effective control and eradication programs can be developed.

Supplemental Digital Content is available in the text, Objective: HIV-1 frequently adapts in response to immune pressure from cytotoxic T-lymphocytes (CTL). Many HIV-2 infected individuals have robust capsid-specific CTL responses associated with viral control. Despite this CTL pressure, adaptive changes in this key immunogenic HIV-2 protein have not previously been described. We sought to compare selective pressure on HIV-1 and HIV-2 capsids and identify HLA-associated viral polymorphisms in HIV-2. Design and methods: Bioinformatic algorithms to identify sites under positive and negative selective pressure and a statistical model of evolution to identify HLA-associated polymorphisms in HIV-2 was applied to sequences from a community cohort in Guinea-Bissau. IFN-γ ELISpots were used to compare T-cell responses to wild-type and variant epitopes. Results: We identified greater purifying selection and less sites under positive selective pressure in HIV-2 compared with HIV-1. Five HIV-2 codons with HLA-associated polymorphisms were detected all within or around known or predicted CTL epitopes. One site was within the HLA-B58 SuperType (ST)-restricted epitope (TSTVEEQIQW), the HIV-2 equivalent of the HIV-1 TW10 epitope. In contrast to HIV-1, where a T→N mutation at position 3 is associated with resulting loss of CTL control, an E→D mutation at position 5 was observed in HIV-2. Robust CTL responses to the variant HIV-2 epitope were seen, suggesting that HIV-2 adaptation may be at the level of T-cell receptor recognition. Conclusion: Greater constraints on evolution may exist in HIV-2, resulting in more purifying selection and different immune adaptation pathways in HIV-1 and HIV-2 capsids. This may allow CTL responses to persist in HIV-2.

Background The large and constantly evolving HIV-1 pandemic has led to an increasingly complex diversity. Because of some taxonomic difficulties among the most diverse HIV-1 subtypes, and taking advantage of the large amount of sequence data generated in the recent years, we investigated novel lineage patterns among the main HIV-1 subtypes. Results All HIV full-length genomes available in public databases were analysed (n = 2017). Maximum likelihood phylogenies and pairwise genetic distance were obtained. Clustering patterns and mean distributions of genetic distances were compared within and across the current groups, subtypes and sub-subtypes of HIV-1 to detect and analyse any divergent lineages within previously defined HIV lineages. The level of genetic similarity observed between most HIV clades was deeply consistent with the current classification. However, both subtypes A and D showed evidence of further intra-subtype diversification not fully described by the nomenclature system at the time and could be divided into several distinct sub-subtypes. Conclusions With this work, we propose an updated nomenclature of sub-types A and D better reflecting their current genetic diversity and evolutionary patterns. Allowing a more accurate nomenclature and classification system is a necessary step for easier subtyping of HIV strains and a better detection or follow-up of viral epidemiology shifts. Electronic supplementary material The online version of this article (10.1186/s12977-018-0461-y) contains supplementary material, which is available to authorized users.

Latinos represent a growing proportion of HIV cases in North Carolina (NC). Understanding how immigrants are involved in local HIV transmission is important to guide interventions. We used phylogenetics to characterize Latino involvement in local HIV transmission chains. Transmission clusters were identified from maximum-likelihood phylogenies constructed with HIV pol sequences from 177 Latinos and 1,496 non-Latinos receiving care in NC. Highly supported clusters involving one or more Latinos were characterized. Migration data were obtained from interviews and chart review. Factors associated with cluster membership were identified using log-binomial regression. Most Latinos were male (76%), immigrants (83%), and had HIV-1B (99%). Immigrants were more likely to report heterosexual risk (67% vs. 23%) than U.S.-born Latinos (p

The WHO recommends protease inhibitor (PI)–based antiretroviral therapy (ART) for vertically infected children after failed nevirapine (NVP) prophylaxis. Emergence of PI resistance on the backdrop of preexisting non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance could compromise long-term treatment options in such children. We characterized multiclass drug resistance using single genome sequencing (SGS) in children with viremia while receiving PI-based ART. We applied SGS of HIV-1 protease (PR) and reverse transcriptase to longitudinal samples from a cohort of the Children with HIV Early Antiretroviral Therapy trial with viral loads >1000 copies per milliliter after 40 weeks of early ART. Bulk sequencing revealed NVP-selected resistance in 50% of these children, whereas SGS revealed NVP-selected resistance in 70%. Two children had baseline NRTI and PI mutations, suggesting previous maternal ART. Linked multiclass drug resistance after PI-based ART was detected by SGS in 2 of 10 children. In one child, the majority species contained M184V in reverse transcriptase linked to L10F, M46I/L, I54V, and V82A in PR and a triple-class drug-resistant variant with these mutations linked to the NNRTI mutation V108I. In the second child, the majority species contained M184V and V82A linked within viral genomes. We conclude that when PI-based ART is initiated soon after birth after single dose-NVP prophylaxis, PI and NRTI resistance can occur in the majority species as expected and also be selected on the same genomes as preexisting NNRTI-resistant mutations. These observations highlight a future therapeutic challenge for vertically infected children where antiretroviral drug classes are limited.

Background Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients – once before antiviral treatment and once after viral rebound due to resistance. Results With single-virion sequencing, we obtained 248–8796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently through PacBio SMRT and Illumina Pooled deep sequencing. Conclusions Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance and host adaptation, allows differentiation between soft or hard selective sweeps, and may be useful in the reconstruction of intra-host viral population demographic history. Electronic supplementary material The online version of this article (10.1186/s12864-017-4217-1) contains supplementary material, which is available to authorized users.

Patients who do not disclose their sexuality, including men who do not disclose same-sex behaviour, are difficult to characterise through traditional epidemiological approaches such as interviews. Using a recently developed method to detect large networks of viral sequences from time-resolved trees, we localised non-disclosed men who have sex with men (MSM) in UK transmission networks, gaining crucial insight into the behaviour of this group.For this phylogenetic analysis, we obtained HIV pol sequences from the UK HIV Drug Resistance Database (UKRDB), a central repository for resistance tests done as part of routine clinical care throughout the UK. Sequence data are linked to demographic and clinical data held by the UK Collaborative HIV Cohort study and the national HIV/AIDS reporting system database. Initially, we reconstructed maximum likelihood phylogenies from these sequences, then sequences were selected for time-resolved analysis in BEAST if they were clustered with at least one other sequence at a genetic distance of 4·5% or less with support of at least 90%. We used time-resolved phylogenies to create networks by linking together nodes if sequences shared a common ancestor within the previous 5 years. We identified potential non-disclosed MSM (pnMSM), defined as self-reported heterosexual men who clustered only with men. We measured the network position of pnMSM, including betweenness (a measure of connectedness and importance) and assortativity (the propensity for nodes sharing attributes to link).14 405 individuals were in the network, including 8452 MSM, 1743 heterosexual women and 1341 heterosexual men. 249 pnMSM were identified (18·6% of all clustered heterosexual men) in the network. pnMSM were more likely to be black African (p0·0001), less likely to be infected with subtype B (p=0·006), and were slightly older (p=0·002) than the MSM they clustered with. Mean betweenness centrality was lower for pnMSM than for MSM (1·31, 95% CI 0·48-2·15 in pnMSM vs 2·24, 0·98-3·51 in MSM; p=0·002), indicating that pnMSM were in peripheral positions in MSM clusters. Assortativity by risk group was higher than expected (0·037 vs -0·037, p=0·01) signifying that pnMSM were linked to each other. We found that self-reported heterosexual men were more likely to link MSM and heterosexual women than heterosexual women were to link MSM and heterosexual men (Fisher's exact test p=0·0004; OR 2·24) but the number of such transmission chains was small (only 54 in total vs 32 in women).pnMSM are a subgroup distinct from both MSM and from heterosexual men. They are more likely to choose sexual partners who are also pnMSM and might exhibit lower-risk sexual behaviour than MSM (eg, choosing low-risk partners or consistently using condoms). Heterosexual men are the group most likely to be diagnosed with late-stage disease (ie, low CD4 counts) and non-disclosed MSM might put female partners at higher risk than heterosexual men because non-disclosed MSM have male partners. Hence, pnMSM require specific consideration to ensure they are included in public health interventions.National Institutes of Health.

HIV-1 diversity is increasing in North American and European cohorts which may have public health implications. However, little is known about non-B subtype diversity in the southern United States, despite the region being the epicenter of the nation’s epidemic. We characterized HIV-1 diversity and transmission clusters to identify the extent to which non-B strains are transmitted locally. We conducted cross-sectional analyses of HIV-1 partial pol sequences collected from 1997 to 2014 from adults accessing routine clinical care in North Carolina (NC). Subtypes were evaluated using COMET and phylogenetic analysis. Putative transmission clusters were identified using maximum-likelihood trees. Clusters involving non-B strains were confirmed and their dates of origin were estimated using Bayesian phylogenetics. Data were combined with demographic information collected at the time of sample collection and country of origin for a subset of patients. Among 24,972 sequences from 15,246 persons, the non-B subtype prevalence increased from 0% to 3.46% over the study period. Of 325 persons with non-B subtypes, diversity was high with over 15 pure subtypes and recombinants; subtype C (28.9%) and CRF02_AG (24.0%) were most common. While identification of transmission clusters was lower for persons with non-B versus B subtypes, several local transmission clusters (≥3 persons) involving non-B subtypes were identified and all were presumably due to heterosexual transmission. Prevalence of non-B subtype diversity remains low in NC but a statistically significant rise was identified over time which likely reflects multiple importation. However, the combined phylogenetic clustering analysis reveals evidence for local onward transmission. Detection of these non-B clusters suggests heterosexual transmission and may guide diagnostic and prevention interventions.

Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients – once before antiviral treatment and once after viral rebound due to resistance. We obtained 248-8,796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were also able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently on PacBio and Illumina sequencers. Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance, differentiating between soft or hard selective sweeps, and the reconstruction of intra-host viral population demographic history.

The extent of HIV-1 diversity was examined among patients attending a rural district hospital in a coastal area of Kenya. The pol gene was sequenced in samples from 153 patients. Subtypes were designated using the REGA, SCUEAL, and jpHMM programs. The most common subtype was A1, followed by C and D; A2 and G were also detected. However, a large proportion of the samples was found to be recombinants, which clustered within the pure subtype branches. Phylogeographic analysis of Kilifi sequences compared with those from other regions of Africa showed that while many sequences were closely related to sequences from Kenya, others were most closely related to known sequences from other parts of Africa, including West Africa. Overall, these data indicate that there have been multiple introductions of HIV-1 into this small rural town and surroundings with ongoing diversity being generated by recombination.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been amplified from human prostate cancer and chronic fatigue syndrome (CFS) patient samples. Other studies failed to replicate these findings and suggested PCR contamination with a prostate cancer cell line, 22Rv1, as a likely source. MLV-like sequences have also been detected in CFS patients in longitudinal samples 15 years apart. Here, we tested whether sequence data from these samples are consistent with viral evolution. Our phylogenetic analyses strongly reject a model of within-patient evolution and demonstrate that the sequences from the first and second time points represent distinct endogenous murine retroviruses, suggesting contamination.

HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization.

TRIM5α proteins recruit and restrict incoming cytoplasmic retroviruses. Primate TRIM5α sequence diversity underlies species-specific restriction and is likely caused by selective pressure from ancient pathogenic infections. Here we show that TRIM5α from the European brown hare restricts diverse retroviruses. Furthermore, it differs significantly in sequence from TRIM5α from the closely related rabbit, suggesting evolutionary changes in the last 12 million years since these species diverged. We propose that, like primates, lagomorphs have been subject to selective pressure from TRIM5-sensitive viruses, possibly related to the endogenous lentivirus RELIK found in both rabbits and hares.

In a recent ARHR publication by Roy et al., the authors report the identification of 30 codon sites under positive selection in the first exon of the HIV--1 tat gene. Unfortunately, the authors have not considered the presence of overlapping coding sequences in HIV--1, invalidating most of their positive selection analysis.

Background. Most published data on antiretroviral-drug resistance is generated from in vitro or in vivo studies of subtype B virus. However, this subtype is associated with

Objectives: We wished to assess the potential of using HIV-1 pol gene for the identification of transmissions events by phylogenetic means in the era of antiretroviral drug selective pressure.Design: The relatedness of the viruses within a large database of pol sequences generated from HIV-1 infected individuals from the UK was reconstructed by phylogenetic analyses.Methods: A total of 140 pol sequences were selected out of the 2500 database entries, on the basis of a pairwise genetic distance higher than 95%. Neighbour joining and Maximum Likelihood trees were implemented. Trees were reconstructed after exclusion of codon positions associated with drug resistance from the original pol alignment. Trees based on the corresponding env and gag genes were implemented to confirm the linkages.Results: Up to 23 transmission clusters were identified, supported by high bootstrap values (> 99), congruent epidemiological data and/or similar drug resistance motifs. The topology of the tree was consistent after exclusion of the drug resistance associated codons. Identical topologies were obtained in trees implemented from gag and env genes alignments.Conclusions: Despite its genetic conservation, the HIV-1 pol gene holds sufficient variability to permit the phylogenetic reconstruction of transmissions. Identical clusters were obtained whichever of the three principal genes is considered and no bias was induced by the presence of drug resistance mutations. These findings demonstrate the important epidemiological information inherent within routinely collected laboratory data, which can assist in estimating rates of recent HIV-1 transmission within a population. (C) 2004 Lippincott Williams Wilkins.

We have investigated a potential transmission chain of HIV-1 with drug resistance-associated mutations between three individuals over a period of 5 years by use of cloning and sequencing of viral genes, and phenotypic characterization. Viruses containing reverse transcriptase drug resistance-associated mutations were transmitted sequentially between three homosexual men (A, B, and C), and persisted in one individual for at least 4 years, despite intermittent therapy and reduced viral replicative capacity compared with wild-type strains. Clonal analysis of the envelope gene from semen and blood virus showed that the virus transmitted to patient C was more closely related to virus from the semen than the blood of patient B. Our data suggest that HIV variants with drug resistance-associated mutations can persist following primary infection, despite intervening antiretroviral therapy, and subsequently sexually transmitted. We provide "proof of principle" that such mutations can therefore become "fixed" within the circulating virus pool.

The accessory gene vpr , common to all primate lentiviruses, induces potent G 2 /M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G 2 /M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G 2 /M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G 2 /M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G 2 /M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vpr in vivo . IMPORTANCE The Vpr protein of HIV-1 and related viruses is essential for the virus in vivo . The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replication in vivo .

Objective: HIV-1 subtype B infections are associated with MSM in the UK. Yet, around 13% of subtype B infections are found in those reporting heterosexual contact as transmission route. Using phylogenetics, we explored possible misclassification of sexual exposure among men diagnosed with HIV in the UK. Design: Viral gene sequences linked to patient-derived information were used to identify phylogenetic transmission chains. Methods: A total of 22 481 HIV-1 subtype B pol gene sequences sampled between 1996 and 2008 were analysed. Dated phylogenies were reconstructed and transmission clusters identified as clades of at least two sequences with a maximum genetic distance of 4.5%, a branch support of at least 95% and spanning 5 years. The characteristics of clusters containing at least one heterosexually acquired infection were analysed. Results: Twenty-nine percent of the linked heterosexuals clustered exclusively with MSM. These were more likely to be men than women. Estimated misclassification of homosexually acquired infections ranged between 1 and 11% of the reported male heterosexuals diagnosed with HIV. Black African heterosexual men were more often phylogenetically linked to MSM than other ethnic group, with an estimated misclassification range between 1 and 21%. Conclusion: Overall, a small proportion of self-reported heterosexual men diagnosed with HIV could have been infected homosexually. However, up to one in five black African heterosexual men chose not to disclose sex with men at HIV diagnosis and preferred to be identified as heterosexual. Phylogenetic analyses can enhance surveillance-based risk information and inform national programmes for monitoring and preventing HIV infections.

The HIV-1 Vpu protein is expressed from a bi-cistronic message late in the viral life cycle. It functions during viral assembly to maximise infectious virus release by targeting CD4 for proteosomal degradation and counteracting the antiviral protein tetherin (BST2/CD317). Single genome analysis of vpu repertoires throughout infection in 14 individuals infected with HIV-1 clade B revealed extensive amino acid diversity of the Vpu protein. For the most part, this variation in Vpu increases over the course of infection and is associated with predicted epitopes of the individual's MHC class I haplotype, suggesting CD8+ T cell pressure is the major driver of Vpu sequence diversity within the host. Despite this variability, the Vpu functions of targeting CD4 and counteracting both physical virus restriction and NF-κB activation by tetherin are rigorously maintained throughout HIV-1 infection. Only a minority of circulating alleles bear lesions in either of these activities at any given time, suggesting functional Vpu mutants are heavily selected against even at later stages of infection. Comparison of Vpu proteins defective for one or several functions reveals novel determinants of CD4 downregulation, counteraction of tetherin restriction, and inhibition of NF-κB signalling. These data affirm the importance of Vpu functions for in vivo persistence of HIV-1 within infected individuals, not simply for transmission, and highlight its potential as a target for antiviral therapy., Author Summary The accessory protein Vpu, encoded by HIV-1, performs at least two major roles in the virus life cycle, namely the degradation of newly synthesized CD4 molecules and the counteraction of a host antiviral protein, tetherin. These activities promote the release of infectious viruses from host cells, and recent evidence suggests that Vpu function has been crucial for the cross-species transmission of HIV-1 from chimpanzees, and its subsequent pandemic spread in humans. Here we studied the functional variation in Vpu in infected individuals. We found that the Vpu amino acid sequence can be highly variable within an individual, and that this variation is likely to result from host immune responses targeting antigens derived from Vpu. However, despite this variation, Vpu's major functions are preserved, with only a minority of circulating alleles showing defects throughout the course of infection. These data suggest that defective Vpu proteins are selected against within the infected individual, implying that Vpu functions are critical for HIV-1 replication throughout natural infection, not simply at transmission. Therefore Vpu may represent a novel target for antiviral therapy to augment current treatment strategies for HIV/AIDS.

Background As sequence data sets used for the investigation of pathogen transmission patterns increase in size, automated tools and standardized methods for cluster analysis have become necessary. We have developed an automated Cluster Picker which identifies monophyletic clades meeting user-input criteria for bootstrap support and maximum genetic distance within large phylogenetic trees. A second tool, the Cluster Matcher, automates the process of linking genetic data to epidemiological or clinical data, and matches clusters between runs of the Cluster Picker. Results We explore the effect of different bootstrap and genetic distance thresholds on clusters identified in a data set of publicly available HIV sequences, and compare these results to those of a previously published tool for cluster identification. To demonstrate their utility, we then use the Cluster Picker and Cluster Matcher together to investigate how clusters in the data set changed over time. We find that clusters containing sequences from more than one UK location at the first time point (multiple origin) were significantly more likely to grow than those representing only a single location. Conclusions The Cluster Picker and Cluster Matcher can rapidly process phylogenetic trees containing tens of thousands of sequences. Together these tools will facilitate comparisons of pathogen transmission dynamics between studies and countries.

Here, a protein known as MX2 is shown to be a major effector of interferon-α-mediated resistance to HIV-1 infection: susceptibility of the HIV-1 virus to inhibition by MX2 is dictated by the Capsid region of the viral Gag protein, and inhibition occurs at a late post-entry step of infection. Two groups report in this issue of Nature that the human interferon-induced GTP-binding protein MX2 is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) and a number of other lentiviruses. For some years it had been known that the related protein MX1 can inhibit HIV-1 replication in humans, but MX2 was thought to be devoid of antiviral activity. The anti-HIV-1 action of MX2 is much less dependent on GTPase activity than is the broader antiviral activity of MX1, pointing to possible mechanistic differences between them. Animal cells harbour multiple innate effector mechanisms that inhibit virus replication. For the pathogenic retrovirus human immunodeficiency virus type 1 (HIV-1), these include widely expressed restriction factors1, such as APOBEC3 proteins2, TRIM5-α3, BST2 (refs 4, 5) and SAMHD1 (refs 6, 7), as well as additional factors that are stimulated by type 1 interferon (IFN)8,9,10,11,12,13,14. Here we use both ectopic expression and gene-silencing experiments to define the human dynamin-like, IFN-induced myxovirus resistance 2 (MX2, also known as MXB) protein as a potent inhibitor of HIV-1 infection and as a key effector of IFN-α-mediated resistance to HIV-1 infection. MX2 suppresses infection by all HIV-1 strains tested, has equivalent or reduced effects on divergent simian immunodeficiency viruses, and does not inhibit other retroviruses such as murine leukaemia virus. The Capsid region of the viral Gag protein dictates susceptibility to MX2, and the block to infection occurs at a late post-entry step, with both the nuclear accumulation and chromosomal integration of nascent viral complementary DNA suppressed. Finally, human MX1 (also known as MXA), a closely related protein that has long been recognized as a broadly acting inhibitor of RNA and DNA viruses, including the orthomyxovirus influenza A virus15,16, does not affect HIV-1, whereas MX2 is ineffective against influenza virus. MX2 is therefore a cell-autonomous, anti-HIV-1 resistance factor whose purposeful mobilization may represent a new therapeutic approach for the treatment of HIV/AIDS.

Sequencing of the complete hepatitis B virus (HBV) genomes from Vietnam, China and Laos led to the identification of a complex recombinant, referred to initially as an aberrant genotype and later proposed to be a new genotype, I. However, epidemiological data regarding this new genotype are lacking. A cross-sectional study was carried out to investigate the epidemiology of HBV candidate genotype I in Guangxi, China using stratified, random cluster sampling. Four thousand five hundred thirteen subjects were recruited from five counties within Guangxi. Three genotypes, B, C, and I, were identified with a prevalence of 32.6% (114/350), 64% (224/350), and 3.4% (12/350), respectively. All the genotype I isolates belong to candidate subgenotype I1 and were found in Bing Yang (15.3%, 9/59) and Na Po (5.0%, 3/60) counties only. The prevalence of this subgenotype is significantly higher in males (5.1%, 10/195) than in females (1.3%, 2/155; X(2) = 3.959, P

Background The combination of phylogenetic analyses of HIV sequences with patients’ demographic data allows us to understand local HIV transmission, a necessary knowledge for designing prevention strategies. The Community of Madrid represents a challenge for the control of HIV epidemic in Spain given its high HIV prevalence and increasing proportion of immigrant people among HIV-infected population. Methods We applied maximum likelihood methods and Bayesian Markov chain Monte Carlo (MCMC) inference using the program BEAST to a set of HIV-1 pol sequences from 1293 patients diagnosed in 1995–2010 in Madrid, Spain. Results Two-hundred and thirty six patients (18.2% of the cohort) were included in 100 transmission chains using phylogenetic criteria, 67 (67%) belonging to HIV-1 subtype B and 33 (33%) to 11 different non-B strains, especially BG and BF recombinants. Most networks involved transmission between MSM (48/100). Half of non-B clusters (15/33) included at least one Spaniard. Sub-Saharan African patients presented a low linkage rate (9%) in contrast to Spanish (21%) and Latin American (25%) patients. Three clusters involving treatment-independent transmission of drug-resistance mutations were found. Conclusions One out of five HIV-infected patients in our cohort in Madrid was epidemically linked, mainly by transmission pairs. The inclusion in transmission networks was more likely for MSM, Spaniards and patients from Latin America. We found no evidence of self-sustained non-B epidemics due to the absence of large transmission chains with the exception of Cuban BG recombinants and CRF47_BF. However, the differences in transmission across variants are probably determined by the patient profile, especially the infection route.

OBJECTIVES Despite prevention efforts, new HIV diagnoses continue in the southern United States, where the epidemic is characterized by significant racial/ethnic disparities. We integrated phylogenetic analyses with clinical data to reveal trends in local HIV transmission. DESIGN Cross-sectional analysis of 1671 HIV-infected individuals each with one B-subtype pol sequence obtained during chronic (82%; UNC Center for AIDS Research Clinical Cohort) or acute/recent (18%; Duke/UNC Acute HIV Consortium) infection. METHODS Phylogenies were inferred using neighbor joining to select related sequences then confirmed with Bayesian methods. We characterized transmission clusters (clades n ≥ 3 sequences supported by posterior probabilities = 1) by factors including race/ethnicity and transmission risk. Factors associated with cluster membership were evaluated for newly diagnosed patients. RESULTS Overall, 72% were men, 59% black and 39% men who have sex with men (MSM). A total of 557 (33%) sequences grouped in either 108 pairs (n = 216) or 67 clusters (n = 341). Clusters ranged from three to 36 (median 4) members. Composition was delineated primarily by race, with 28% exclusively black, and to a lesser extent by risk group. Both MSM and heterosexuals formed discrete clusters, although substantial mixing was observed. In multivariable analysis, patients with age 30 years or less (P = 0.009), acute infection (P = 0.02), local residence (P = 0.002) and transmitted drug resistance (P = 0.02) were more likely to be cluster members, whereas Latinos were less likely (P < 0.001). CONCLUSION Integration of molecular, clinical and demographic data offers a unique view into the structure of local transmission networks. Clustering by black race, youth and transmitted drug resistance and inability to identify Latino clusters will inform prevention, testing and linkage to care strategies.

Background A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans. Results Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression. Conclusions Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.

The human immunodeficiency virus type-1 (HIV-1) Rev protein regulates the nuclear export of intron-containing viral RNAs by recruiting the CRM1 nuclear export receptor. Here, we employed a combination of functional and phylogenetic analyses to identify and characterize a species-specific determinant within human CRM1 (hCRM1) that largely overcomes established defects in murine cells to the post-transcriptional stages of the HIV-1 life cycle. hCRM1 expression in murine cells promotes the cytoplasmic accumulation of intron-containing viral RNAs, resulting in a substantial stimulation of the net production of infectious HIV-1 particles. These stimulatory effects require a novel surface-exposed element within HEAT repeats 9A and 10A, discrete from the binding cleft previously shown to engage Rev's leucine-rich nuclear export signal. Moreover, we show that this element is a unique feature of higher primate CRM1 proteins, and discuss how this sequence has evolved from a non-functional, ancestral sequence., Author Summary HIV-1 requires multiple cellular co-factors to replicate, and non-human cells often carry species-specific variations in the genes encoding these co-factors that can prevent efficient replication. Here, the basis for murine cell-specific deficiencies in the late steps of HIV-1 replication is addressed. We show that differences between the mouse and human forms of the essential host protein CRM1, a protein required for the transport of macromolecules from the nucleus to the cytoplasm, underlie this problem. More precisely, murine CRM1, unlike its human counterpart, fails to fully support the function of the HIV-1 Rev protein, a factor necessary to transport viral RNAs to the cytoplasm. Key amino acid differences between the mouse/human CRM1 proteins are identified and computational analyses of divergent animal CRM1 proteins reveal a unique motif in higher primates likely acquired in response to ancient evolutionary pressures. This CRM1 element may represent a novel pathogen interaction site that evolved to evade prior infections, but is now contributing to the susceptibility of humans to HIV-1.

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment., Author Summary During infection HIV-1 enters the nucleus by crossing the nuclear membrane and incorporating itself into the host DNA by a process called integration. Here we show that the viral capsid protein gets tethered to a cyclophilin protein called Nup358, a component of the nuclear membrane gateways that allow transport between the cytoplasm and the nucleus. Altering the capsid protein so that it cannot use Nup358 prevents viral replication in macrophages, a natural target cell type for HIV-1. Intriguingly, these viral mutants are not less infectious in certain immortalised cell lines suggesting that in these cells nuclear entry is regulated differently. In this case similar to wild type virus, the mutant viruses integrate into host chromosomes but they integrate into different regions suggesting that the pathway into the nucleus dictates where the virus ends up in the host chromatin. We also show that another cyclophilin, the cytoplasmic protein cyclophilin A, influences the engagement of Nup358 as well as other proteins involved in HIV-1 nuclear entry. We hypothesise that HIV-1 has evolved to use cyclophilins so that it can access a particular pathway into the nucleus because alternative pathways lead to defects in integration targeting and viral replication in human macrophages.

Background Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies. Results Real-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution. Conclusion The lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.

BACKGROUND: The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. METHODOLOGY/PRINCIPAL FINDINGS: Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. CONCLUSIONS: We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.

Background Xenotropic murine leukaemia viruses (MLV-X) are endogenous gammaretroviruses that infect cells from many species, including humans. Xenotropic murine leukaemia virus-related virus (XMRV) is a retrovirus that has been the subject of intense debate since its detection in samples from humans with prostate cancer (PC) and chronic fatigue syndrome (CFS). Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls. Results Here we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission. Conclusions We provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.

Recently, a complex (X/C) hepatitis B virus (HBV) recombinant, first reported in 2000, was proposed as a new genotype; although this was refuted immediately because the strains differ by less than 8 % in nucleotide distance from genotype C. Over 13.5 % (38/281) of HBV isolates from the Long An cohort in China were not assigned to a specific genotype, using current genotyping tools to analyse surface ORF sequences, and these have about 98 % similarity to the X/C recombinants. To determine whether this close identity extends to the full-length sequences and to investigate the evolutionary history of the Long An X/C recombinants, 17 complete genome sequences were determined. They are highly similar (96-99 %) to the Vietnamese strains and, although some reach or exceed 8 % nucleotide sequence difference from all known genotypes, they cluster together in the same clade, separating in a phylogenetic tree from the genotype C branch. Analysis of recombination reveals that all but one of the Long An isolates resembles the Vietnamese isolates in that they result from apparent recombination between genotype C and a parent of unknown genotype (X), which shows similarity in part to genotype G. The exception, isolate QL523, has a greater proportion of genotype C parent. Phylogeographic analysis reveals that these recombinants probably arose in southern China and spread later to Vietnam and Laos.

Background Detailed local HIV-1 sequence data are essential for monitoring the HIV epidemic, for maintaining sensitive sequence-based diagnostics, and to aid in designing vaccines. Results Reported here are full envelope sequences derived from 38 randomly selected HIV-1 infections identified at a Gambian clinic between 1991 and 2009. Special care was taken to generate sequences from circulating viral RNA as uncloned products, either by limiting dilution or single genome amplification polymerase chain reaction (PCR). Within these 38 isolates, eight were subtyped as A and 18 as CRF02_AG. A small number of subtype B, C, D viruses were identified. Surprising, however, was the identification of six isolates with subtype J-like envelopes, a subtype found normally in Central Africa and the Democratic Republic of the Congo (DRC), with gag p24 regions that clustered with subtype A sequences. Near full-length sequence from three of these isolates confirmed that these represent a novel circulating recombinant form of HIV-1, now named CRF49_cpx. Conclusions This study expands the HIV-1 sequence database from the Gambia and will provide important data for HIV diagnostics, patient care, and vaccine development.

TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5α but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations., Author Summary Retroviruses have constantly been infecting mammals throughout their evolution, causing them to evolve defensive mechanisms to protect themselves. One of these mechanisms utilises intracellular antiviral molecules referred to as restriction factors. Restriction factor sequences have changed through primate evolution, suggesting an ongoing battle between retroviruses and their hosts as described by the Red Queen hypothesis. TRIM5 is an important restriction factor able to protect some monkeys, but not humans, from HIV infection. Certain monkeys have modified their TRIM5 genes by swapping the virus binding B30.2 domain with a cyclophilin A domain inserted into the TRIM5 locus by retrotransposition. This leads to expression of a TRIMCyp protein with antiviral activity against viruses, such as HIV-1, that recruit cyclophilins. It appears that cyclophilin makes a particularly flexible virus-binding domain able to restrict divergent lentiviruses from primates as well as cats. Here we characterise the molecular details of Cyclophilin-Capsid interactions focusing on TRIMCyp proteins from Macaca Fascicularis. Using a structure/function approach we can show the molecular details of how adaptive changes in the TRIMCyp sequence switch specificity between members of different primate lentiviral lineages. Mapping these changes onto the macaque phylogeny reveals a history of TRIMCyp evolution that directs restriction to a variety of diverse lentiviruses.

Summary: The calibrated population resistance (CPR) tool is a web-accessible program for performing standardized genotypic estimation of transmitted HIV-1 drug resistance. The program is linked to the Stanford HIV drug resistance database and can additionally perform viral genotyping and algorithmic estimation of resistance to specific antiretroviral drugs. Availability: http://cpr.stanford.edu/cpr/index.html Contact: robjgiff@gmail.com

HLA-B*5703 is associated with effective immune control in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an escape mutation within the immunodominant HLA-B*5703-restricted epitope in chronic HIV-1 infection, KAFSPEVIPMF (Gag 162-172), and demonstrate that this mutation reduces viral replicative capacity. Reversion of this mutation following transmission to HLA-B*5703-negative recipients was delayed by the compensatory mutation S165N within the same epitope. These data may help explain the observed association between HLA-B*5703 and long-term control of viremia.

The recent identification of antiretroviral tripartite motif-bearing restriction factors that protect against retroviral infection has revealed a novel branch of innate immunity. The factors target the retroviral capsid and inhibit infectivity soon after the capsid has entered the cytoplasm by an incompletely characterized mechanism. Restriction is species specific. For example, TRIM5α from Old World monkeys, but not humans, restricts human immunodeficiency virus type 1 infection. Here, we identify an antiviral TRIM5 molecule in rabbits that is closely related to antiviral TRIM5 of both primates and cattle. We demonstrate that the rabbit TRIM5 protein is active against divergent retroviruses and leads to a strong block to viral DNA synthesis and infectivity. Furthermore, we show that antiviral activity is directed against the viral capsid and that human TRIM5 proteins are dominant negative to restriction in rabbit cells. We propose that the sequence and restriction characteristics conserved between restriction factors from primates, cattle, and rabbits indicate that these factors have evolved from a common ancestor with antiretroviral properties.

Antigenic variation inherent in human immunodeficiency virus type 1 (HIV-1) virions that successfully instigate new infections transferred by sex has not been well defined. Yet this is the viral “challenge” which any vaccine-induced immunity must deal with. Closely timed comparisons of the virus circulating in the “donor” and that which initiates new infection are difficult to carry out rigorously, as suitable samples are very hard to get in the face of ethical hurdles. Here we investigate HIV-1 variation in four homosexual couples where we sampled blood from both parties within several weeks of the estimated transmission event. We analyzed variation within highly immunogenic HIV-1 internal proteins encoding epitopes recognized by cytotoxic Tlymphocytes (CTLs). These responses are believed to be crucial as a means of containing viral replication. In the donors we detected virions capable of evading host CTL recognition at several linked epitopes of distinct HLA class I restriction. When a donor transmitted escape variants to a recipient with whom he had HLA class I molecules in common, the recipient's CTL response to those epitopes was prevented, thus impeding adequate viral control. In addition, we show that even when HLA class I alleles are disparate in the transmitting couple, a single polymorphism can abolish CTL recognition of an overlapping epitope of distinct restriction and so confer immune escape properties to the recipient's seroconversion virus. In donors who are themselves controlling an early, acute infection, the precise timing of onward transmission is a crucial determinant of the viral variants available to compose the inoculum.

We explored the epidemic history of HIV-1 subtype B in the United Kingdom by using statistical methods that infer the population history of pathogens from sampled gene sequence data. Phylogenetic analysis of HIV-1 pol gene sequences from Britain showed at least six large transmission chains, indicating a genetically variable, but epidemiologically homogeneous, epidemic among men having sex with men. Through coalescent-based analysis, we showed that these chains arose through separate introductions of subtype B strains into the United Kingdom in the early to mid-1980s. After an initial period of exponential growth, the rate of spread generally slowed in the early 1990s, which is more likely to correlate with behavior change than with reduced infectiousness resulting from highly active antiretroviral therapy. Our results provide insights into the complexity of HIV-1 epidemics that must be considered when developing HIV monitoring and prevention initiatives.

The evolutionary relationships of 26 sequenced members of the poxvirus family have been investigated by comparing their genome organization and gene content and by using DNA and protein sequences for phylogenetic analyses. The central region of the genome of chordopoxviruses (ChPVs) is highly conserved in gene content and arrangement, except for some gene inversions in Fowlpox virus (FPV) and species-specific gene insertions in FPV and Molluscum contagiosum virus (MCV). In the central region 90 genes are conserved in all ChPVs, but no gene from near the termini is conserved throughout the subfamily. Inclusion of two entomopoxvirus (EnPV) sequences reduces the number of conserved genes to 49. The EnPVs are divergent from ChPVs and between themselves. Relationships between ChPV genera were evaluated by comparing the genome size, number of unique genes, gene arrangement and phylogenetic analyses of protein sequences. Overall, genus Avipoxvirus is the most divergent. The next most divergent ChPV genus is Molluscipoxvirus, whose sole member, MCV, infects only man. The Suipoxvirus, Capripoxvirus, Leporipoxvirus and Yatapoxvirus genera cluster together, with Suipoxvirus and Capripoxvirus sharing a common ancestor, and are distinct from the genus Orthopoxvirus (OPV). Within the OPV genus, Monkeypox virus, Ectromelia virus and Cowpox virus strain Brighton Red (BR) do not group closely with any other OPV, Variola virus and Camelpox virus form a subgroup, and Vaccinia virus is most closely related to CPV-GRI-90. This suggests that CPV-BR and GRI-90 should be separate species.