Identification of two novel subtypes of hepatitis C virus genotype 8 and a potential new genotype successfully treated with direct acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral (DAA) efficacy and global HCV elimination. METHODS: We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences. RESULTS: Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F). CONCLUSIONS: This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.

Real world SOF/VEL/VOX retreatment outcomes and viral resistance analysis for HCV patients with prior failure to DAA therapy.

Sustained viral response (SVR) rates for direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance-associated substitutions (RAS) in a real-world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first-line DAA treatment regimens. Full-length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n = 55, GT3a n = 54) of the cohort. 40% (n = 58) of patients had liver cirrhosis of whom 7% (n = 4) were decompensated, 10% (n = 14) had hepatocellular carcinoma (HCC) and 8% (n = 12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR = 81%, p = .009), cirrhosis (47/58; SVR = 81%, p = .01) and prior treatment with SOF/VEL (12/17; SVR = 71%, p = .02) or SOF+DCV (14/19; SVR = 74%, p = .012) were significantly associated with retreatment failure, but existence of pre-retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non-GT3-infected patients. However, for GT3-infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.

Molecular epidemiology reveals the role of war in the spread of HIV in Ukraine.

Ukraine has one of the largest HIV epidemics in Europe, historically driven by people who inject drugs (PWID). The epidemic showed signs of stabilization in 2012, but the recent war in eastern Ukraine may be reigniting virus spread. We investigated the movement of HIV-infected people within Ukraine before and during the conflict. We analyzed HIV-1 subtype-A pol nucleotide sequences sampled during 2012-2015 from 427 patients of 24 regional AIDS centers and used phylogeographic analysis to reconstruct virus movement among different locations in Ukraine. We then tested for correlations between reported PWID behaviors and reconstructed patterns of virus spread. Our analyses suggest that Donetsk and Lugansk, two cities not controlled by the Ukrainian government in eastern Ukraine, were significant exporters of the virus to the rest of the country. Additional analyses showed that viral dissemination within the country changed after 2013. Spearman correlation analysis showed that incoming virus flow was correlated with the number of HIV-infected internally displaced people. Additionally, there was a correlation between more intensive virus movement and locations with a higher proportion of PWID practicing risky sexual behaviors. Our findings suggest that effective prevention responses should involve internally displaced people and people who frequently travel to war-affected regions. Scale-up of harm reduction services for PWID will be an important factor in preventing new local HIV outbreaks in Ukraine.

Surveillance of HIV-1 transmitted integrase strand transfer inhibitor resistance in the UK.

BACKGROUND: HIV treatment guidelines have traditionally recommended that all HIV-positive individuals are tested for evidence of drug resistance prior to starting ART. Testing for resistance to reverse transcriptase inhibitors and PIs is well established in routine care. However, testing for integrase strand transfer inhibitor (InSTI) resistance is less consistent. OBJECTIVES: To inform treatment guidelines by determining the prevalence of InSTI resistance in a national cohort of recently infected individuals. PATIENTS AND METHODS: Recent (within 4 months) HIV-1 infections were identified using a Recent Infection Testing Algorithm of new HIV-1 diagnoses in the UK. Resistance-associated mutations (RAMs) in integrase, protease and reverse transcriptase were detected by ultradeep sequencing, which allows for the sensitive estimation of the frequency of each resistant variant in a sample. RESULTS: The analysis included 655 randomly selected individuals (median age = 33 years, 95% male, 83% MSM, 78% white) sampled in the period 2014 to 2016 and determined to have a recent infection. These comprised 320, 138 and 197 samples from 2014, 2015 and 2016, respectively. None of the samples had major InSTI RAMs occurring at high variant frequency (≥20%). A subset (25/640, 3.9%) had major InSTI RAMs occurring only as low-frequency variants (2%-20%). In contrast, 47/588 (8.0%) had major reverse transcriptase inhibitor and PI RAMs at high frequency. CONCLUSIONS: Between 2014 and 2016, major InSTI RAMs were uncommon in adults with recent HIV-1 infection, only occurring as low-frequency variants of doubtful clinical significance. Continued surveillance of newly diagnosed patients for evidence of transmitted InSTI resistance is recommended to inform clinical practice.

Determining the Origins of Human Immunodeficiency Virus Type 1 Drug-resistant Minority Variants in People Who Are Recently Infected Using Phylogenetic Reconstruction.

BACKGROUND: Drug-resistant minority variants (DRMinVs) detected in patients who recently acquired human immunodeficiency virus type 1 (HIV-1) can be transmitted, generated de novo through virus replication, or technical errors. The first form is likely to persist and result in treatment failure, while the latter two could be stochastic and transient. METHODS: Ultradeep sequencing of plasma samples from 835 individuals with recent HIV-1 infection in the United Kingdom was performed to detect DRMinVs at a mutation frequency between 2% and 20%. Sequence alignments including >110 000 HIV-1 partial pol consensus sequences from the UK HIV Drug Resistance Database (UK-HDRD), linked to epidemiological and clinical data from the HIV and AIDS Reporting System, were used for transmission cluster analysis. Transmission clusters were identified using Cluster Picker with a clade support of >90% and maximum genetic distances of 4.5% or 1.5%, the latter to limit detection to likely direct transmission events. RESULTS: Drug-resistant majority variants (DRMajVs) were detected in 66 (7.9%) and DRMinVs in 84 (10.1%) of the recently infected individuals. High levels of clustering to sequences in UK-HDRD were observed for both DRMajV (n = 48; 72.7%) and DRMinV (n = 63; 75.0%) sequences. Of these, 43 (65.2%) with DRMajVs were in a transmission cluster with sequences that harbored the same DR mutation compared to only 3 (3.6%) sequences with DRMinVs (P < .00001, Fisher exact test). Evidence of likely direct transmission of DRMajVs was observed for 25/66 (37.9%), whereas none were observed for the DRMinVs (P < .00001). CONCLUSIONS: Using a densely sampled HIV-infected population, we show no evidence of DRMinV transmission among recently infected individuals.

An Innovative Study Design to Assess the Community Effect of Interventions to Mitigate HIV Epidemics Using Transmission-Chain Phylodynamics.

Given globalization and other social phenomena, controlling the spread of infectious diseases has become an imperative public health priority. A plethora of interventions that in theory can mitigate the spread of pathogens have been proposed and applied. Evaluating the effectiveness of such interventions is costly and in many circumstances unrealistic. Most important, the community effect (i.e., the ability of the intervention to minimize the spread of the pathogen from people who received the intervention to other community members) can rarely be evaluated. Here we propose a study design that can build and evaluate evidence in support of the community effect of an intervention. The approach exploits molecular evolutionary dynamics of pathogens in order to track new infections as having arisen from either a control or an intervention group. It enables us to evaluate whether an intervention reduces the number and length of new transmission chains in comparison with a control condition, and thus lets us estimate the relative decrease in new infections in the community due to the intervention. We provide as an example one working scenario of a way the approach can be applied with a simulation study and associated power calculations.

Enhanced surveillance of HIV-1 drug resistance in recently infected MSM in the UK.

OBJECTIVES: To determine the prevalence of inferred low-frequency HIV-1 transmitted drug resistance (TDR) in MSM in the UK and its predicted effect on first-line therapy. METHODS: The HIV-1 pol gene was amplified from 442 newly diagnosed MSM identified as likely recently infected by serological avidity testing in 2011-13. The PCR products were sequenced by next-generation sequencing with a mutation frequency threshold of >2% and TDR mutations defined according to the 2009 WHO surveillance drug resistance mutations list. RESULTS: The majority (75.6%) were infected with subtype B and 6.6% with rare complex or unique recombinant forms. At a mutation frequency threshold of >20%, 7.2% (95% CI 5.0%-10.1%) of the sequences had TDR and this doubled to 15.8% (95% CI 12.6%-19.6%) at >2% mutation frequency (P < 0.0001). The majority (26/42, 62%) of low-frequency variants were against PIs. The most common mutations detected at >20% and 2%-20% mutation frequency differed for each drug class, these respectively being: L90M (n = 7) and M46IL (n = 10) for PIs; T215rev (n = 9) and D67GN (n = 4) for NRTIs; and K103N (n = 5) and G190E (n = 2) for NNRTIs. Combined TDR was more frequent in subtype B than non-B (OR = 0.38; 95% CI = 0.17-0.88; P = 0.024) and had minimal predicted effect on recommended first-line therapies. CONCLUSIONS: The data suggest differences in the types of low-frequency compared with majority TDR variants that require a better understanding of the origins and clinical significance of low-frequency variants. This will better inform diagnostic and treatment strategies.

Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

HIV-1 drug resistance mutations emerging on darunavir therapy in PI-naive and -experienced patients in the UK.

BACKGROUND: Darunavir is considered to have a high genetic barrier to resistance. Most darunavir-associated drug resistance mutations (DRMs) have been identified through correlation of baseline genotype with virological response in clinical trials. However, there is little information on DRMs that are directly selected by darunavir in clinical settings. OBJECTIVES: We examined darunavir DRMs emerging in clinical practice in the UK. PATIENTS AND METHODS: Baseline and post-exposure protease genotypes were compared for individuals in the UK Collaborative HIV Cohort Study who had received darunavir; analyses were stratified for PI history. A selection analysis was used to compare the evolution of subtype B proteases in darunavir recipients and matched PI-naive controls. RESULTS: Of 6918 people who had received darunavir, 386 had resistance tests pre- and post-exposure. Overall, 2.8% (11/386) of these participants developed emergent darunavir DRMs. The prevalence of baseline DRMs was 1.0% (2/198) among PI-naive participants and 13.8% (26/188) among PI-experienced participants. Emergent DRMs developed in 2.0% of the PI-naive group (4 mutations) and 3.7% of the PI-experienced group (12 mutations). Codon 77 was positively selected in the PI-naive darunavir cases, but not in the control group. CONCLUSIONS: Our findings suggest that although emergent darunavir resistance is rare, it may be more common among PI-experienced patients than those who are PI-naive. Further investigation is required to explore whether codon 77 is a novel site involved in darunavir susceptibility.

Evidence of Self-Sustaining Drug Resistant HIV-1 Lineages Among Untreated Patients in the United Kingdom.

BACKGROUND: About 10% of new diagnoses of subtype B human immunodeficiency virus type 1 (HIV-1) in the United Kingdom are with viruses showing transmitted drug resistance (TDR). However, there is discordance between the mutation patterns observed in HIV-infected patients failing therapy and those seen in TDR. METHODS: We extracted all subtype B HIV-1 pol gene sequences from treatment-naive patients within the United Kingdom HIV Drug Resistance Database sampled between 1997 and 2011 and carrying the most common protease inhibitors, nonnucleoside and nucleotide reverse transcriptase inhibitors TDR mutations, namely, L90M, K103N, and T215Y/F/rev, respectively (n = 1140). Transmission clusters (n ≥ 2 sequences) were identified by maximum-likelihood phylogeny using a genetic distance cutoff of ≤ 1.5%. The time of origin and the basic reproductive number (R0) of clusters were estimated by Bayesian methods. RESULTS: T215rev was present alone in 47% of the sequences (n = 540), K103N in 31% (n = 359), and L90M in 10% (n = 109). The remaining sequences contained T215Y or combinations of L90M, K103N, and T215rev. Fifty-five percent (n = 624) of the sequences formed highly supported transmission clusters (n = 193) containing between 2 and 15 sequences. The time of origin of 10 large clusters (≥ 8 sequences) was estimated to be between 2000 (1999-2002; 95% highest posterior density [HPD]) and 2006 (2005-2007; 95% HPD). The oldest cluster had persisted for nearly 8 years. All 10 clusters had R0s ranging from 1.3 (0.4-2.5; 95% HPD) to 2.8 (0.6-6.5; 95% HPD). CONCLUSIONS: A high proportion of the most common TDR in subtype B infections in the United Kingdom is derived by onward transmission from treatment-naive patients.

Intercontinental dispersal of HIV-1 subtype B associated with transmission among men who have sex with men in Japan.

UNLABELLED: Transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia have recently been identified. Using the Los Alamos HIV sequence database and the UK HIV drug resistance database, we explored possible links between HIV MSM epidemics in East Asia and the rest of the world by using phylogenetic and molecular clock analyses. We found that JP.MSM.B-1, a subtype B MSM variant that accounts for approximately one-third of the infections among Japanese MSM, was detected worldwide, in the United Kingdom (n=13), mainland China (n=3), the United States, Germany, Canada, and Taiwan (n=1 each). Interestingly, 10 United Kingdom samples plus two from Germany and the United States formed a distinct monophyletic subgroup within JP.MSM.B-1. The estimated divergence times of JP.MSM.B-1 and the latter subgroup were ∼1989 and ∼1999, respectively. These dates suggest that JP.MSM.B-1 was circulating for many years in Japan among MSM before disseminating to other countries, most likely through global MSM networks. A significant number of other Asian MSM HIV lineages were also detected in the UK HIV drug resistance database. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages. Further study of these strains is warranted to elucidate viral migration and the interrelationship of HIV epidemics on a global scale. IMPORTANCE: We previously identified several transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia. Using the Los Alamos HIV sequence database and the UK HIV drug resistance database, we explored the possible interplay of HIV MSM epidemics in the different geographic regions and found previously unrecognized interrelationships among the HIV-1 epidemics in East Asia, the United Kingdom, and the rest of the world. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages and highlights the importance of strengthening HIV monitoring efforts and the need for implementing effective control measures to reduce HIV transmission on a global scale.

Contribution of Gag and protease to variation in susceptibility to protease inhibitors between different strains of subtype B human immunodeficiency virus type 1.

Recent reports have shown that human immunodeficiency virus type 1 (HIV-1) Gag can directly affect susceptibility to protease inhibitors (PIs) in the absence of known resistance mutations in protease. Inclusion of co-evolved Gag alongside protease in phenotypic drug susceptibility assays can alter PI susceptibility in comparison with protease with a WT Gag. Using a single-replication-cycle assay encompassing full-length Gag together with protease we demonstrated significant variation in PI susceptibility between a number of PI-naïve subtype B viruses. Six publicly available subtype B molecular clones, namely HXB2, NL4-3, SF2, YU2, JRFL and 89.6, displayed up to nine-fold reduced PI susceptibility in comparison with the assay reference strain. For two molecular clones, YU2 and JRFL, Gag contributed solely to the observed reduction in susceptibility, with the N-terminal region of Gag contributing significantly. Gag and protease from treatment-naïve, patient-derived viruses also demonstrated significant variation in susceptibility, with up to a 17-fold reduction to atazanavir in comparison with the assay reference strain. In contrast to the molecular clones, protease was the main determinant of the reduced susceptibility. Common polymorphisms in protease, including I13V, L63P and A71T, were shown to contribute to this reduction in PI susceptibility, in the absence of major resistance mutations. This study demonstrated significant variation in PI susceptibility of treatment-naïve patient viruses, and provided further evidence of the independent role of Gag, the protease substrate and in particular the N-terminus of Gag in PI susceptibility. It also highlighted the importance of considering co-evolved Gag and protease when assessing PI susceptibility.

Phenotypic characterization of virological failure following lopinavir/ritonavir monotherapy using full-length Gag-protease genes.

OBJECTIVES: Major protease mutations are rarely observed following first-line failure with PIs and interpretation of genotyping results in this context may be difficult. We performed extensive phenotyping of viruses from five patients failing lopinavir/ritonavir monotherapy in the MONARK study without major PI mutations by standard genotyping. METHODS: Phenotypic susceptibility testing and viral infectivity assessments were performed using a single-cycle assay and fold changes (FC) relative to a lopinavir-susceptible reference strain were calculated. RESULTS: >10-fold reduced baseline susceptibility to lopinavir occurred in two of five patients and >5-fold in another two. Four of five patients exhibited phylogenetic evidence of a limited viral evolution between baseline and failure, with amino acid changes at drug resistance-associated positions in one: T81A emerged in Gag with M36I in the protease gene, correlating with a reduction in lopinavir susceptibility from FC 7 (95% CI 6-8.35) to FC 13 (95% CI 8.11-17.8). Reductions in darunavir susceptibility (>5 FC) occurred in three individuals. DISCUSSION: This study suggests both baseline reduced susceptibility and evolution of resistance could be contributing factors to PI failure, despite the absence of classical PI resistance mutations by standard testing methods. Use of phenotyping also reveals lower darunavir susceptibility, warranting further study as this agent is commonly used following lopinavir failure.

Low frequency of genotypic resistance in HIV-1-infected patients failing an atazanavir-containing regimen: a clinical cohort study.

OBJECTIVES: To determine protease mutations that develop at viral failure for protease inhibitor (PI)-naive patients on a regimen containing the PI atazanavir. METHODS: Resistance tests on patients failing atazanavir, conducted as part of routine clinical care in a multicentre observational study, were randomly matched by subtype to resistance tests from PI-naive controls to account for natural polymorphisms. Mutations from the consensus B sequence across the protease region were analysed for association and defined using the IAS-USA 2011 classification list. RESULTS: Four hundred and five of 2528 (16%) patients failed therapy containing atazanavir as a first PI over a median (IQR) follow-up of 1.76 (0.84-3.15) years and 322 resistance tests were available for analysis. Recognized major atazanavir mutations were found in six atazanavir-experienced patients (P < 0.001), including I50L and N88S. The minor mutations most strongly associated with atazanavir experience were M36I, M46I, F53L, A71V, V82T and I85V (P < 0.05). Multiple novel mutations, I15S, L19T, K43T, L63P/V, K70Q, V77I and L89I/T/V, were also associated with atazanavir experience. CONCLUSIONS: Viral failure on atazanavir-containing regimens was not common and major resistance mutations were rare, suggesting that adherence may be a major contributor to viral failure. Novel mutations were described that have not been previously documented.

Clinical evaluation of a Hepatitis C Virus whole-genome sequencing pipeline for genotyping and resistance testing.

OBJECTIVES: We sought to evaluate clinically a hepatitis C virus (HCV) whole-genome, next-generation sequencing (NGS) pipeline that is agnostic to viral genotype. METHODS: Performance of the NGS pipeline was assessed through comparison of results with Sanger sequencing (SS) of partial HCV genomes. RESULTS: There was 98.7% (376/381) concordance for viral subtype between SS and NGS. The positive and negative per cent agreements for determination of resistance-associated substitutions were 97.8% (95% CI 92.5-99.4%) and 99.9% (95% CI 99.5-100.0%), respectively. The NGS pipeline was also able to detect novel subtypes, mixtures, recombinants, transiently occurring resistance mutations and distinguish re-infection with the same subtype from relapse. DISCUSSION: Particular scenarios where NGS may be used include settings without universal access to pan-genotypic antiviral regimens, those infected with a 'rare' subtype or who have been failed by first-line therapy, and in cases of suspected re-infection.

The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains.

BACKGROUND: The Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring. RESULTS: Single-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain. CONCLUSIONS: We demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

Molecular and epidemiological characterization of HIV-1 infection networks involving transmitted drug resistance mutations in Northern Greece.

OBJECTIVES: To determine the contribution of transmission clusters to transmitted drug resistance (TDR) in newly diagnosed antiretroviral-naive HIV-1-infected patients in Northern Greece during 2000-07. METHODS: The prevalence of TDR was estimated in 369 individuals who were diagnosed with HIV-1 infection in the period 2000-07 at the National AIDS Reference Laboratory of Northern Greece. Phylogenetic analysis was performed using a maximum likelihood method on partial pol sequences. TDR was defined in accordance with the surveillance drug resistance mutation list (2009 update). RESULTS: The overall prevalence of TDR in our population was 12.5% [46/369, 95% confidence interval (CI) 9.1%-15.8%], comprising 7.6% (28/369) resistant to nucleoside reverse transcriptase inhibitors, 5.4% (20/369) resistant to non-nucleoside reverse transcriptase inhibitors and 3.3% (12/369) resistant to protease inhibitors. Dual class resistance was identified in 3.8% (14/369). Infection with subtype A was the sole predictor associated with TDR in multivariate analysis (odds ratio 2.15, 95% CI 1.10-4.19, P = 0.025). Phylogenetic analyses revealed three statistically robust transmission clusters involving drug-resistant strains, including one cluster of 12 patients, 10 of whom were infected with a strain carrying both T215 revertants and Y181C mutations. CONCLUSIONS: Our findings underline the substantial impact of transmission networks on TDR in our population.

APOBEC3F and APOBEC3G inhibit HIV-1 DNA integration by different mechanisms.

APOBEC3F (A3F) and APBOBEC3G (A3G) both are host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination, which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome (J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, A. Engelman, and V. K. Pathak, J. Virol. 81:7099-7110, 2007). Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3' long terminal repeat (3'-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3' processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3'-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.

Intrapatient variation of the respiratory syncytial virus attachment protein gene.

Intrapatient variability of the attachment (G) protein gene of respiratory syncytial virus (RSV) was examined using both population and single-genome sequencing. Samples from three patients infected with a group B virus variant which has a 60-nucleotide duplication in the G protein gene were examined. These samples were chosen because occasional mixed sequence bases were observed. In a minority of RSV genomes from these patients considerable variability was found, including point mutations, insertions, and deletions. Of particular note, the deletion of the exact portion of the gene which had been duplicated in some isolates was observed in viral RNAs from two patients.

Coevolved Multidrug-Resistant HIV-1 Protease and Reverse Transcriptase Influences Integrase Drug Susceptibility and Replication Fitness.

Integrase strand transfer inhibitors (InSTIs) are recommended agents in first-line combination antiretroviral therapy (cART). We examined the evolution of drug resistance mutations throughout HIV-1 pol and the effects on InSTI susceptibility and viral fitness. We performed single-genome sequencing of full-length HIV-1 pol in a highly treatment-experienced patient, and determined drug susceptibility of patient-derived HIV-1 genomes using a phenotypic assay encompassing full-length pol gene. We show the genetic linkage of multiple InSTI-resistant haplotypes containing major resistance mutations at Y143, Q148 and N155 to protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) resistance mutations. Phenotypic analysis of viruses expressing patient-derived IN genes with eight different InSTI-resistant haplotypes alone or in combination with coevolved protease (PR) and RT genes exhibited similar levels of InSTI susceptibility, except for three haplotypes that showed up to 3-fold increases in InSTI susceptibility (p ≤ 0.032). The replicative fitness of most viruses expressing patient-derived IN only significantly decreased, ranging from 8% to 56% (p ≤ 0.01). Interestingly, the addition of coevolved PR + RT significantly increased the replicative fitness of some haplotypes by up to 73% (p ≤ 0.024). Coevolved PR + RT contributes to the susceptibility and viral fitness of patient-derived IN viruses. Maintaining patients on failing cART promotes the selection of fitter resistant strains, and thereby limits future therapy options.