Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy.

Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.

Impact of drug resistance-associated amino acid changes in HIV-1 subtype C on susceptibility to newer nonnucleoside reverse transcriptase inhibitors.

The objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in an in vitro single-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases, <1%. The more common EFV/NVP resistance-associated substitutions, such as K103N, V106M, and G190A, had no major impact on ETR or RPV susceptibility. The low-level resistance to RPV and ETR conferred by E138K was not significantly enhanced in the presence of M184V/I, unlike for EFV and NVP. Among patient samples, 97% were resistant to EFV and/or NVP, while only 24% and 16% were resistant to ETR and RPV, respectively. Overall, only a few, relatively rare NNRTI resistance-associated amino acid substitutions caused resistance to ETR and/or RPV in an HIV-1 subtype C background, suggesting that these newer NNRTIs would be effective in NVP/EFV-experienced HIV-1 subtype C-infected patients.

Analysis of the history and spread of HIV-1 in Uganda using phylodynamics.

HIV prevalence has decreased in Uganda since the 1990s, but remains substantial within high-risk groups. Here, we reconstruct the history and spread of HIV subtypes A1 and D in Uganda and explore the transmission dynamics in high-risk populations. We analysed HIV pol sequences from female sex workers in Kampala (n = 42), Lake Victoria fisher-folk (n = 46) and a rural clinical cohort (n = 74), together with publicly available sequences from adjacent regions in Uganda (n = 412) and newly generated sequences from samples taken in Kampala in 1986 (n = 12). Of the sequences from the three Ugandan populations, 60 (37.1 %) were classified as subtype D, 54 (33.3 %) as subtype A1, 31 (19.1 %) as A1/D recombinants, six (3.7 %) as subtype C, one (0.6 %) as subtype G and 10 (6.2 %) as other recombinants. Among the A1/D recombinants we identified a new candidate circulating recombinant form. Phylodynamic and phylogeographic analyses using BEAST indicated that the Ugandan epidemics originated in 1960 (1950-1968) for subtype A1 and 1973 (1970-1977) for D, in rural south-western Uganda with subsequent spread to Kampala. They also showed extensive interconnection with adjacent countries. The sequence analysis shows both epidemics grew exponentially during the 1970s-1980s and decreased from 1992, which agrees with HIV prevalence reports in Uganda. Inclusion of sequences from the 1980s indicated the origin of both epidemics was more recent than expected and substantially narrowed the confidence intervals in comparison to previous estimates. We identified three transmission clusters and ten pairs, none of them including patients from different populations, suggesting active transmission within a structured transmission network.

Human immunodeficiency virus Tat associates with a specific set of cellular RNAs.

BACKGROUND: Human Immunodeficiency Virus 1 (HIV-1) exhibits a wide range of interactions with the host cell but whether viral proteins interact with cellular RNA is not clear. A candidate interacting factor is the trans-activator of transcription (Tat) protein. Tat is required for expression of virus genes but activates transcription through an unusual mechanism; binding to an RNA stem-loop, the transactivation response element (TAR), with the host elongation factor P-TEFb. HIV-1 Tat has also been shown to alter the expression of host genes during infection, contributing to viral pathogenesis but, whether Tat also interacts with cellular RNAs is unknown. RESULTS: Using RNA immunoprecipitation coupled with microarray analysis, we have discovered that HIV-1 Tat is associated with a specific set of human mRNAs in T cells. mRNAs bound by Tat share a stem-loop structural element and encode proteins with common biological roles. In contrast, we do not find evidence that Tat associates with microRNAs or the RNA-induced silencing complex (RISC). The interaction of Tat with cellular RNA requires an intact RNA binding domain and Tat RNA binding is linked to an increase in RNA abundance in cell lines and during infection of primary CD4+ T cells by HIV. CONCLUSIONS: We conclude that Tat interacts with a specific set of human mRNAs in T cells, many of which show changes in abundance in response to Tat and HIV infection. This work uncovers a previously unrecognised interaction between HIV and its host that may contribute to viral alteration of the host cellular environment.

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.

Host-pathogen interaction in invasive Salmonellosis.

Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by S. enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a bacteremic illness but whose clinical features differ from other Gram-negative bacteremias. Non-typhoidal Salmonella (NTS) serovars cause self-limiting diarrhea with occasional secondary bacteremia. Primary NTS bacteremia can occur in the immunocompromised host and infants in sub-Saharan Africa. Recent studies on host-pathogen interactions in Salmonellosis using genome sequencing, murine models, and patient studies have provided new insights. The full genome sequences of numerous S. enterica serovars have been determined. The S. Typhi genome, compared to that of S. Typhimurium, harbors many inactivated or disrupted genes. This can partly explain the different immune responses both serovars induce upon entering their host. Similar genome degradation is also observed in the ST313 S. Typhimurium strain implicated in invasive infection in sub-Saharan Africa. Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized. Genes for these factors are commonly carried on Salmonella Pathogenicity Islands (SPIs). Plasmids also carry putative virulence-associated genes as well as those responsible for antimicrobial resistance. The interaction of Salmonella pathogen-associated molecular patterns (PAMPs) with Toll-like receptors (TLRs) and NOD-like receptors (NLRs) leads to inflammasome formation, activation, and recruitment of neutrophils and macrophages and the production of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN)-γ. The gut microbiome may be an important modulator of this immune response. S. Typhimurium usually causes a local intestinal immune response, whereas S. Typhi, by preventing neutrophil attraction resulting from activation of TLRs, evades the local response and causes systemic infection. Potential new therapeutic strategies may lead from an increased understanding of infection pathogenesis.

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.

Assessing the Virologic Impact of Archived Resistance in the Dolutegravir/Lamivudine 2-Drug Regimen HIV-1 Switch Study TANGO through Week 144.

The TANGO study (ClinicalTrials.gov, NCT03446573) demonstrated that switching to dolutegravir/lamivudine (DTG/3TC) was non-inferior to continuing tenofovir alafenamide-based regimens (TBR) through week 144. Retrospective baseline proviral DNA genotypes were performed for 734 participants (post-hoc analysis) to assess the impact of archived, pre-existing drug resistance on 144-week virologic outcomes by last on-treatment viral load (VL) and Snapshot. A total of 320 (86%) participants on DTG/3TC and 318 (85%) on TBR had both proviral genotype data and ≥1 on-treatment post-baseline VL results and were defined as the proviral DNA resistance analysis population. Archived International AIDS Society-USA major nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, protease inhibitor, and integrase strand transfer inhibitor resistance-associated mutations (RAMs) were observed in 42 (7%), 90 (14%), 42 (7%), and 11 (2%) participants, respectively, across both groups; 469 (74%) had no major RAMs at baseline. M184V/I (1%), K65N/R (<1%), and thymidine analogue mutations (2%) were infrequent. Through week 144, >99% of participants on DTG/3TC and 99% on TBR were virologically suppressed (last on-treatment VL <50 copies/mL) regardless of the presence of major RAMs. Results from the sensitivity analysis by Snapshot were consistent with the last available on-treatment VL. In TANGO, archived, pre-existing major RAMs did not impact virologic outcomes through week 144.

Virologic Response to Dolutegravir Plus Lamivudine in People With Suppressed Human Immunodeficiency Virus Type 1 and Historical M184V/I: A Systematic Literature Review and Meta-analysis.

Background: To investigate the impact of the M184V/I mutation on virologic response to dolutegravir plus lamivudine (DTG + 3TC) in suppressed-switch populations, a meta-analysis was performed using virologic outcomes from people with human immunodeficiency virus type 1 (PWH) with and without M184V/I before DTG + 3TC switch in real-world studies identified via systematic literature review. Sensitivity analyses were performed using data from PWH with M184V/I in interventional studies identified via targeted literature review. Methods: Single-arm meta-analyses using common- and random-effects models were used to estimate proportions of PWH with virologic failure (VF) among real-world populations with and without M184V/I and interventional study participants with M184V/I at 24, 48, and 96 weeks. Results: Literature reviews identified 5 real-world studies from 3907 publications and 51 abstracts meeting inclusion criteria and 5 interventional studies from 1789 publications and 3 abstracts. All time points had low VF incidence in PWH with M184V/I (real-world: 1.43%-3.81%; interventional: 0.00%) and without (real-world: 0.73%-2.37%). Meta-analysis-estimated proportions (95% confidence interval) with VF were low at weeks 24, 48, and 96, respectively, for PWH with M184V/I (real-world: 0.01 [.00-.04], 0.03 [.01-.06], and 0.04 [.01-.07]; interventional: 0.00 [.00-.02], 0.00 [.00-.01], and 0.00 [.00-.03]) and without (real-world: 0.00 [.00-.02], 0.02 [.01-.04], and 0.02 [.00-.05]). One real-world study (n = 712) reported treatment-emergent M184V at VF in 1 of 652 (0.15%) PWH without prior M184V/I. Conclusions: Results suggest that prior M184V/I has minimal impact on virologic suppression after switching to DTG + 3TC and provide reassurance when considering switching regimens in virologically suppressed PWH with incomplete treatment history or limited treatment options.

Isotopic Radiolabeling of the Antiretroviral Drug [18F]Dolutegravir for Pharmacokinetic PET Imaging.

Deciphering the drug/virus/host interactions at infected cell reservoirs is a key leading to HIV-1 remission for which positron emission tomography (PET) imaging using radiolabeled antiretroviral (ARV) drugs is a powerful asset. Dolutegravir (DTG) is one of the preferred therapeutic options to treat HIV and can be isotopically labeled with fluorine-18. [18F]DTG was synthesized via a three-step approach of radiofluorination/nitrile reduction/peptide coupling with optimization for each step. Radiofluorination was performed on 2-fluoro-4-nitrobenzonitrile in 90% conversion followed by nitrile reduction using sodium borohydride and aqueous nickel(II) chloride with 72% conversion. Final peptide coupling reaction followed by HPLC purification and formulation afforded ready-to-inject [18F]DTG in 5.1 ± 0.8% (n = 10) decay-corrected radiochemical yield within 95 min. The whole process was automatized using a TRACERlab® FX NPro module, and quality control performed by analytical HPLC showed that [18F]DTG was suitable for in vivo injection with >99% chemical and radiochemical purity and a molar activity of 83 ± 18 GBq/µmol (n = 10). Whole-body distribution of [18F]DTG was performed by PET imaging on a healthy macaque and highlighted the elimination routes of the tracer. This study demonstrated the feasibility of in vivo [18F]DTG PET imaging and paved the way to explore drug/virus/tissues interactions in animals and humans.

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.

Three residues in HIV-1 matrix contribute to protease inhibitor susceptibility and replication capacity.

Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC50). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.

Impact of the N348I mutation in HIV-1 reverse transcriptase on nonnucleoside reverse transcriptase inhibitor resistance in non-subtype B HIV-1.

We investigated the effect of N348I alone and with M184V on nonnucleoside reverse transcriptase inhibitor (NNRTI) drug susceptibility and replicative capacity in B and non-B HIV-1 isolates. N348I reduced the susceptibility to all NNRTI drugs across subtypes. The replication capacity of all viruses in a variety of cell lines was impaired by N348I. Interestingly, the N348I and M184V double mutation compensated for the reduced NNRTI drug susceptibility observed in the N348I single mutant and marginally improved viral replicative capacity.

Prolonged permissive hypotensive resuscitation is associated with poor outcome in primary blast injury with controlled hemorrhage.

OBJECTIVE: To determine the effects of primary blast injury (PBI) on survival and the physiological response to resuscitation after hemorrhagic shock. BACKGROUND: Air-blast injury is a significant clinical problem that can reduce blood oxygenation and modify the response to hemorrhage. PBI has specific physiological effects that have not been fully accounted for in resuscitation strategies. Permissive hypotension is a widely adopted strategy in trauma resuscitation. However, the choice of resuscitation strategy requires a full understanding of the mechanisms of injury and their physiological consequences. METHODS: Terminally anesthetized pigs were divided into 4 groups and subjected to either air-blast injury (B) or no blast (S). All received a controlled hemorrhage of 30% blood volume and resuscitation with 0.9% saline to a normotensive (Normot, systolic blood pressure 110 mm Hg) or hypotensive (Hypot, 80 mm Hg) end point for up to 8 hours. (n = 6 in each B and n = 8 in each S subgroup). RESULTS: Survival time was significantly shorter with Hypot (P < 0.0001 Peto log rank). The effect was in the animals subjected to B (P = 0.0005) (mean survival time [95% CI]; BNormot 422 [313-531] vs. BHypot137 [94-181] min), but not those given S (P = 0.06) (SNormot 480 [all survived] vs. SHypot 352 [210-494] min). PBI exacerbated a profound metabolic acidosis during Hypot, possibly due to an overwhelming compromise in tissue oxygen delivery. CONCLUSIONS: Prolonged hypotensive resuscitation is not compatible with survival after primary blast. Casualties most likely to be in this category are those injured by blast in confined spaces or by enhanced blast weapons. The risk of rebleeding associated with normotensive resuscitation needs to be balanced with the metabolic derangement associated with hypotensive resuscitation.

Dexamethasone in Vietnamese adolescents and adults with bacterial meningitis.

BACKGROUND: It is uncertain whether all adults with bacterial meningitis benefit from treatment with adjunctive dexamethasone. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of dexamethasone in 435 patients over the age of 14 years who had suspected bacterial meningitis. The goal was to determine whether dexamethasone reduced the risk of death at 1 month and the risk of death or disability at 6 months. RESULTS: A total of 217 patients were assigned to the dexamethasone group, and 218 to the placebo group. Bacterial meningitis was confirmed in 300 patients (69.0%), probable meningitis was diagnosed in 123 patients (28.3%), and an alternative diagnosis was made in 12 patients (2.8%). An intention-to-treat analysis of all the patients showed that dexamethasone was not associated with a significant reduction in the risk of death at 1 month (relative risk, 0.79; 95% confidence interval [CI], 0.45 to 1.39) or the risk of death or disability at 6 months (odds ratio, 0.74; 95% CI, 0.47 to 1.17). In patients with confirmed bacterial meningitis, however, there was a significant reduction in the risk of death at 1 month (relative risk, 0.43; 95% CI, 0.20 to 0.94) and in the risk of death or disability at 6 months (odds ratio, 0.56; 95% CI, 0.32 to 0.98). These effects were not found in patients with probable bacterial meningitis. Results of multivariate analysis indicated that dexamethasone treatment for patients with probable bacterial meningitis was significantly associated with an increased risk of death at 1 month, an observation that may be explained by cases of tuberculous meningitis in the treatment group. CONCLUSIONS: Dexamethasone does not improve the outcome in all adolescents and adults with suspected bacterial meningitis; a beneficial effect appears to be confined to patients with microbiologically proven disease, including those who have received prior treatment with antibiotics. (Current Controlled Trials number, ISRCTN42986828 [controlled-trials.com] .).

Gag determinants of fitness and drug susceptibility in protease inhibitor-resistant human immunodeficiency virus type 1.

Mutations can accumulate in the protease and gag genes of human immunodeficiency virus in patients who fail therapy with protease inhibitor drugs. Mutations within protease, the drug target, have been extensively studied. Mutations in gag have been less well studied, mostly concentrating on cleavage sites. A retroviral vector system has been adapted to study full-length gag, protease, and reverse transcriptase genes from patient-derived viruses. Patient plasma-derived mutant full-length gag, protease, and gag-protease from a multidrug-resistant virus were studied. Mutant protease alone led to a 95% drop in replication capacity that was completely rescued by coexpressing the full-length coevolved mutant gag gene. Cleavage site mutations have been shown to improve the replication capacity of mutated protease. Strikingly, in this study, the matrix region and part of the capsid region from the coevolved mutant gag gene were sufficient to achieve full recovery of replication capacity due to the mutant protease, without cleavage site mutations. The same region of gag from a second, unrelated, multidrug-resistant clinical isolate also rescued the replication capacity of the original mutant protease, suggesting a common mechanism that evolves with resistance to protease inhibitors. Mutant gag alone conferred reduced susceptibility to all protease inhibitors and acted synergistically when linked to mutant protease. The matrix region and partial capsid region of gag sufficient to rescue replication capacity also conferred resistance to protease inhibitors. Thus, the amino terminus of Gag has a previously unidentified and important function in protease inhibitor susceptibility and replication capacity.

Activation of coagulation and endothelium with concurrent impairment of anticoagulant mechanisms in patients with typhoid fever.

OBJECTIVES: Typhoid fever caused by Salmonella Typhi remains a major burden worldwide. Gastrointestinal bleeding can be seen in up to 10 percent of patients and may be fatal. The coagulopathy, which may be the driver of this severe complication in patients with typhoid fever, however is ill defined. The aim of this study was to evaluate the activation of coagulation, anticoagulation, and fibrinolysis in patients with acute typhoid fever. METHODS: Parameters of coagulation and fibrinolysis were measured in 28 hospitalized patients with culture-confirmed or PCR-confirmed typhoid fever and compared to 38 age- and sex-matched healthy volunteers. RESULTS: Patients demonstrated activation of the coagulation system, as reflected by elevated in vitro thrombin generation and high plasma levels of fibrinogen, D-dimer and prothrombin fragment F1 + 2 in concert with consumption of coagulation factors resulting in a prolonged prothrombin-time and activated-partial-thromboplastin-time. Concurrently, the anticoagulant proteins, protein C and antithrombin, were significantly lower in comparison to healthy controls. Patients also demonstrated evidence of activation and inhibition of fibrinolysis and a marked activation of endothelial cells. The extent of coagulation activation was associated with the course of the disease, repeated testing during convalescence showed a return toward normal values. CONCLUSIONS: Activation of coagulation is an important clinical feature of typhoid fever and is associated with severity of disease.

HIV-1 transmission networks in high risk fishing communities on the shores of Lake Victoria in Uganda: A phylogenetic and epidemiological approach.

BACKGROUND: Fishing communities around Lake Victoria in sub-Saharan Africa have been characterised as a population at high risk of HIV-infection. METHODS: Using data from a cohort of HIV-positive individuals aged 13-49 years, enrolled from 5 fishing communities on Lake Victoria between 2009-2011, we sought to identify factors contributing to the epidemic and to understand the underlying structure of HIV transmission networks. Clinical and socio-demographic data were combined with HIV-1 phylogenetic analyses. HIV-1 gag-p24 and env-gp-41 sub-genomic fragments were amplified and sequenced from 283 HIV-1-infected participants. Phylogenetic clusters with ≥2 highly related sequences were defined as transmission clusters. Logistic regression models were used to determine factors associated with clustering. RESULTS: Altogether, 24% (n = 67/283) of HIV positive individuals with sequences fell within 34 phylogenetically distinct clusters in at least one gene region (either gag or env). Of these, 83% occurred either within households or within community; 8/34 (24%) occurred within household partnerships, and 20/34 (59%) within community. 7/12 couples (58%) within households clustered together. Individuals in clusters with potential recent transmission (11/34) were more likely to be younger 71% (15/21) versus 46% (21/46) in un-clustered individuals and had recently become resident in the community 67% (14/21) vs 48% (22/46). Four of 11 (36%) potential transmission clusters included incident-incident transmissions. Independently, clustering was less likely in HIV subtype D (adjusted Odds Ratio, aOR = 0.51 [95% CI 0.26-1.00]) than A and more likely in those living with an HIV-infected individual in the household (aOR = 6.30 [95% CI 3.40-11.68]). CONCLUSIONS: A large proportion of HIV sexual transmissions occur within house-holds and within communities even in this key mobile population. The findings suggest localized HIV transmissions and hence a potential benefit for the test and treat approach even at a community level, coupled with intensified HIV counselling to identify early infections.

Correction: Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial.

[This corrects the article DOI: 10.1371/journal.pone.0137834.].

Intrapatient Evolutionary Dynamics of Human Immunodeficiency Virus Type 1 in Individuals Undergoing Alternative Treatment Strategies with Reverse Transcriptase Inhibitors.

Structured treatment interruption (STI) has been trialed as an alternative to lifelong antiretroviral therapy (ART). We retrospectively performed single genome sequencing of the HIV-1 pol region from three patients representing different scenarios. They were either failing on continuous therapy (CT-F), failing STI (STI-F), or suppressing on STI (STI-S). Over 460 genomes were generated from three to five different time points over a 2-year period. We found multiple-linked-resistant mutations in both treatment failures. However, the CT-F patient showed a stepwise accumulation of diverse, linked mutations whereas the STI-F patient had lineage turnover between treatment periods with recirculation of wild-type and resistant variants from reservoirs. The STI-F patient showed a 7-fold increase in the third codon position substitution rate relative to the first and second positions compared to a 2-fold increase for CT-F and increased purifying selection in the pol gene (62 vs. 22 sites, respectively). An understanding of intrapatient viral dynamics could guide the future direction of treatment interruption strategies.