Gold nanoparticles (AuNPs) have been explored for their use in medicine. Here, we report a sustainable, and cost-effective method to produce AuNPs using a bacterial strain such as Pseudomonas mendocina CH50 which is also known to be a polyhydroxyalkanoate (PHA) producer. A cell-free bacterial supernatant, which is typically discarded after PHA extraction, was used to produce spherical AuNPs of 3.5 ± 1.5 nm in size as determined by Transmission Electron Microscopy (TEM) analysis. The AuNPs/PHA composite coating demonstrated antibacterial activity against Staphylococcus aureus 6538P, and antiviral activity, with a 75% reduction in viral infectivity against SARS-CoV-2 pseudotype virus.

There is increasing evidence that endogenous retroviruses (ERVs) play a significant role in central nervous system diseases, including amyotrophic lateral sclerosis (ALS). Studies of ALS have consistently identified retroviral enzyme reverse transcriptase activity in patients. Evidence indicates that ERVs are the cause of reverse transcriptase activity in ALS, but it is currently unclear whether this is due to a specific ERV locus or a family of ERVs. We employed a combination of bioinformatic methods to identify whether specific ERVs or ERV families are associated with ALS. Using the largest post-mortem RNA-sequence datasets available we selectively identified ERVs that closely resembled full-length proviruses. In the discovery dataset there was one ERV locus (HML6_3p21.31c) that showed significant increased expression in post-mortem motor cortex tissue after multiple-testing correction. Using six replication post-mortem datasets we found HML6_3p21.31c was consistently upregulated in ALS in motor cortex and cerebellum tissue. In addition, HML6_3p21.31c showed significant co-expression with cytokine binding and genes involved in EBV, HTLV-1 and HIV type-1 infections. There were no significant differences in ERV family expression between ALS and controls. Our results support the hypothesis that specific ERV loci are involved in ALS pathology.

Over the past two decades a number of studies have demonstrated activity of the retroviral enzyme reverse transcriptase in the serum of patients with sporadic amyotrophic lateral sclerosis (ALS). Known human exogenous retroviruses such as HIV-1 have been eliminated as possible sources of this activity and investigators have therefore considered the possibility that human endogenous retroviruses (HERVs) might be involved. HERV-K (HML-2) is the most recent retroviral candidate to be proposed following the observation of elevated HERV-K expression in cortical and spinal neurons of ALS patients and the demonstration of HERV-K envelope protein neurotoxicity in vitro and in transgenic mice. This retroviral hypothesis is an attractive one, not least because it raises the possibility that ALS might become treatable using antiretroviral drugs. In the present study we have attempted independent confirmation of the observation that HERV-K RNA levels are elevated in ALS brain. Total RNA was extracted from the postmortem premotor cortex of 34 patients with ALS and 23 controls. Quantitative real-time reverse transcription PCR (RT-qPCR) was performed according to the MIQE guidelines using HERV-K gag, pol and env primer sets. Data was analysed by the 2-∆∆Ct method with normalisation against two reference genes, GAPDH and XPNPEP1. Geometric mean HERV-K RNA expression levels in the premotor cortex of ALS patients were not found to be different from the expression levels in non-ALS controls. Our findings do not confirm the recently reported association between elevated cortical HERV-K RNA levels and ALS, and thus raise doubts about the role of this endogenous retrovirus in ALS pathogenesis. The results of this study may have implications for ongoing clinical trials aiming to suppress HERV-K activity with antiretroviral drugs.

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

Single nucleotide polymorphisms (SNPs) in the IL28B gene were shown to have limited utility in predicting response to telaprevir and boceprevir in treatment of chronic HCV infection in clinical trials. Data outside of the clinical trial setting are lacking. We assessed the value of single and combined IL28B SNPs rs12979860 and rs8099917 genotypes in predicting sustained virological response 12 weeks after cessation of triple therapy (SVR12) with telaprevir or boceprevir in a single-centre cohort of treatment-naïve and treatment-experienced patients with genotype 1 HCV mono-infection (n = 105). The overall SVR12 rate was 65.7%. By unadjusted bivariate logistic regression analysis, rs12979860-CC and rs8099917-TT were significantly associated with SVR12 in the subgroup of patients including all naïve patients and all treatment-experienced patients with the exception of partial- and null-responders to previous HCV therapy. The predictive value of rs12979860-CC was stronger than rs8099917-TT and only rs12979860-CC remained significantly predictive of treatment success when the two variants were assessed by adjusted logistic regression analysis in the whole study cohort. In patients presenting the rs12979860-CC variant, the additional determination of rs8099917 genotype had no value. IL28B rs12979860-CC remained significantly associated with SVR12 also in the multivariate analysis including the other baseline characteristics associated to SVR12 in the bivariate analysis (i.e., female gender, HCV genotype 1b, baseline viral load <800,000 IU/mL, advanced liver fibrosis and prior partial- or null-response to HCV therapy). Our study suggests that testing for the IL28B rs12979860 genotype may still be useful in predicting response to triple therapy with boceprevir and telaprevir in naïve patients and treatment-experienced patients other than partial and null-responders.

Around 2.5 million HIV-infected individuals failing first-line therapy qualify for boosted protease inhibitor (bPI)-based second-line therapy globally. Major resistance mutations are rarely present at treatment failure in patients receiving bPI and the determinants of failure in these patients remain unknown. There is evidence that Gag can impact PI susceptibility. Here, we have sequenced Gag-Protease before and following failure in 23 patients in the SARA trial infected with subtypes A, C, and D viruses. Before bPI, significant variation in Protease and Gag was observed at positions previously associated with PI exposure and resistance including Gag mutations L449P, S451N, and L453P and Protease K20I and L63P. Following PI failure, previously described mutations in Protease and Gag were observed, including those at the cleavage sites such as R361K and P453L. However, the emergence of clear genetic determinants of therapy failure across patients was not observed. Larger Gag sequence datasets will be required to comprehensively identify mutational correlates of bPI failure across subtypes.

The current epidemic of Hepatitis C infection in HIV-positive men who have sex with men is associated with increasing use of recreational drugs. Multiple HCV infections have been reported in haemophiliacs and intravenous drug users. Using ultra-deep sequencing analysis, we present the case of an HIV-positive MSM with evidence of three sequential HCV infections, each occurring during the acute phase of the preceding infection, following risk exposures. We observed rapid replacement of the original strain by the incoming genotype at subsequent time points. The impact of HCV super-infection remains unclear and UDS may provide new insights.

In 2012, Plaza et al. [1] reported the prevalence of natural polymorphisms in the HCV NS5A gene associated with resistance to daclatasvir (DCV) in 78 HIV– HCV-coinfected patients and 635 HCV-monoinfected derived NS5A sequences deposited in Los Alamos HCV database. They did not observe NS5A resistanceassociated variants (RAVs) in HCV-1a and HCV-3 NS5A sequences, whereas, major RAVs (Y93H) were detected in 7% and 13% in NS5A sequences from coinfected patients infected with HCV-1b and HCV-4, respectively, with a similar frequency of NS5A RAVs observed in HCV-monoinfected patients for these HCV genotypes (gts) [1]. Additionally, the L31M NS5A variant was observed in 7% of HCV gt-1b patients, irrespective of coinfection status and occurred in >93% of HCV gt-4 monoinfected and coinfected patients [1]. Interestingly, the presence of naturally occurring drug resistance variants in acutely HCV-infected, treatmentnaive HIV patients have been detected by population and deep sequencing in HCV NS3 in a large proportion of subjects [2]. The significance of the threshold at which these RAVs are detectable and whether these will impact on response to therapy with NS3 and NS5A inhibitors in clinical practice is not fully clear. In acute HCV amongst those who are HIV-infected, the role for new HCV direct-acting antivirals (DAAs) has not been established. Treatment with pegylated interferon and ribavirin (PEG-IFN/RBV) early in HCV infection is often successful for most gts [3]. Telaprevir, a firstgeneration protease inhibitor (PI), has been used in a small study of acute HCV infection in HIV, and results suggest sustained virological response (SVR) may be improved using ‘triple therapy’ with shortened treatment durations [4]. It is likely that in the near future DAA-based therapy will be standard of care for both acute and chronic HCV infections, with PIs forming part of this armamentarium [5,6] alongside other DAAs, including NS5B polymerase inhibitors [7] and H-CV NS5A inhibitors [8]. Ultimately, an interferonfree future is heralded, where drug regimens will consist of combinational DAAs, targeting different HCV gene products [9]. It is therefore important to establish the frequency of RAVs in all patients infected with HCV. In this study, we investigated the prevalence of RAVs by population sequencing from three groups of HIV– HCV-coinfected patients: acute HCV infections (n=25), chronic treatment-naive patients (n=20) and chronic treatment-experienced (PEG-IFN/RBV) patients who did not achieve an SVR (n=34) and compared with the prevalence of RAVs in 85 chronic HCV-monoinfected patients. Genomic regions (sites of known RAVs) were amplified from HCV RNA using reverse transcriptase PCR followed by a nested PCR. Typically, amino acids 1–181 of HCV NS3 protease (gt1 only), amino acids 1–213 of domain I of NS5A (for gt-1a, 1b, 2, 3 and 4) and amino acids 219–347 of NS5B (pan-genotypic) were included. Purified PCR amplicons were sequenced using ABI PRISM 3730 genetic analyser (Applied Biosystems, Life Technologies Ltd, Paisley, UK) and consensus sequences aligned against HCV reference sequences. Baseline RAVs were detected in all three cohorts of coinfected patients in NS3 and NS5A but baseline S282T, associated with resistance to sofosbuvir, was not detected in any of our cohorts (Table 1), possibly attributable to the low fitness of this mutation [10]. The Q80K polymorphism was the predominant NS3 variant for gt-1a conferring resistance to firstand secondgeneration PIs [11], and increased in frequency in acute Letter

Background: Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options.Objective: To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B.Study design: Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA.Results: Of 100 samples from 50 patients, >= 2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively.LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available.Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be sub-typed by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold.Conclusion: Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA. (C) 2015 Elsevier B.V. All rights reserved.

The use of triple-therapy, pegylated-interferon, ribavirin and either of the first generation hepatitis C virus (HCV) protease inhibitors telaprevir or boceprevir, is the new standard of care for treating genotype 1 chronic HCV. Clinical trials have shown response rates of around 70-80%, but there is limited data from the use of this combination outside this setting. Through an expanded access programme, we treated 59 patients, treatment naïve and experienced, with triple therapy. Baseline factors predicting treatment response or failure during triple therapy phase were identified in 58 patients. Thirty seven (63.8%) of 58 patients had undetectable HCV RNA 12weeks after the end of treatment. Genotype 1a (p=0.053), null-response to previous treatment (p=0.034), the rate of viral load decline after 12weeks of previous interferon-based treatment (p=0.033) were all associated with triple-therapy failure. The most common cause of on-treatment failure for telaprevir-based regimens was the development of resistance-associated variants (RAVs) at amino acids 36 and/or 155 of HCV protease (p=0.027) whereas in boceprevir-based regimens mutations at amino acid 54 were significant (p=0.015). SVR12 rates approaching 64% were achieved using triple therapy outside the clinical trial setting, in a patient cohort that included cirrhotics.

Objectives: HIV-1 tropism needs to be determined before the use of CCR5 antagonist drugs such as maraviroc (MVC), which are ineffective against CXCR4-using HIV-1. This study assessed how different computational methods for predicting tropism from HIV sequence data performed in a large clinical cohort. The value of adding clinical data to these algorithms was also investigated. Design and methods: PCR amplification and sequence analysis of the HIV-1 gp120 V3 loop region was performed on triple replicates of plasma viral RNA or proviral DNA extracted from peripheral blood monocytes (PBMCs) in 242 patients. Coreceptor usage was predicted from V3 sequences using seven bioinformatics interpretation algorithms, combined with clinical data where appropriate. An intention-to-treat approach was employed for exploring outcomes and performance for different viral subtypes was examined. Results: The frequency of R5 predictions varied by 22.6%, with all seven algorithms agreeing for only 75.3% of tests. The identification of individuals likely to fail was poor for all algorithms. The addition of clinical data improved this, but at the expense of their ability to predict success. The clinical algorithms varied across subtypes, whereas other algorithms were more consistent. Furthermore, individuals with discordant clonal and clinical predictions were more likely to fail MVC treatment. Conclusion: Eligibility for MVC varied depending on the algorithm method used. The addition of clinical parameters alongside sequence data may help predict X4 emergence during treatment. It could be that V3 loop analysis in isolation may not be the best method for selecting individuals for MVC.

INTRODUCTION:Next-generation sequencing (NGS) is capable of detecting resistance-associated mutations (RAMs) present at frequencies of 1% or below. Several studies have found that baseline low-frequency RAMs are associated with failure to first-line HAART. One major limitation to the expansion of this technology in routine diagnostics is the complexity and laboriousness integral to bioinformatics analysis. DeepChek (ABL, TherapyEdge) is a CE-marked software that allows automated analysis and resistance interpretation of NGS data.OBJECTIVE:To evaluate the use of 454 ultra-deep-sequencing (Roche(®) 454, Life Sciences; 454-UDS) and DeepChek for routine baseline resistance testing in a clinical diagnostic laboratory.METHODS:107 newly diagnosed HIV-1-infected patients (subtypes: A, n=9; B, n=52; C, n=21; D, n=2; F, n=3; G, n=1; CRF01, n=7; CRF02, n=7; CRF06, n=1; CRF07, n=1; CRF10, n=1 and unassigned complex, n=2) with a median plasma viral load of 88,727 copies/mL (range: 1380-2,143,543) were tested by 454-UDS and Sanger sequencing for the detection of protease and reverse transcriptase RAMs. In addition, integrase RAMs were investigated in 57 of them. Sequence analysis and resistance interpretation were performed using DeepChek applying 1% and 20% thresholds for variant detections; filters applied were comparison between Sanger and 454-UDS, and Stanford and IAS list for resistance interpretation.RESULTS:The time elapsed from generation of raw 454 data (between 2,000-5,000 sequences/sample) to elaboration of a resistance report was approximately 10 minutes per sample, equivalent to the time required for the same process using Sanger sequencing. Four patients (3.7%) showed baseline resistance by Sanger and 454-UDS at frequencies above 20%, which affected both NRTIs (n=2) and NNRTIs (n=2). In addition, 12 patients (11.2%) showed transmitted drug resistance (TDR) by 454-UDS at frequencies below 20% affecting NRTIs (n=9), NNRTIs (n=7) and PIs (n=2). Integrase resistance was not detected at baseline by 454-UDS or Sanger sequencing.CONCLUSIONS:DeepChek allowed easy and rapid analysis and interpretation of NGS data, thus facilitating the incorporation of this technology in routine diagnostics. The use of NGS considerably increased the detection rates of TDR to NRTI, NNRTIs and PIs. No transmitted resistance to integrase inhibitors was found in our population by Sanger sequencing or UDS.

In recent years antiretroviral availability and access has become more prevalent in resource poor settings, whilst the availability of access to HIV-1 drug resistance testing remains limited and patients are often started on therapy or switched when failing without a resistance test. Proviral DNA has been proposed as a source of archived resistance mutations. We investigated the feasibility of using proviral DNA to detect previously documented plasma resistance mutations. Amplification and sequencing of pol from proviral DNA derived from buffy coat (BCL), buffy coat dried on filter paper (BCS) or dried blood spots (DBS) was performed using an in-house assay. Only 2/20 samples were amplified from long term stored buffy coat (>1 year) compared to at least one prospective sample from 10/20 patients. Six patients had a plasma viral load (PVL) <40 cp/ml at time of sampling, though 4/6 only become undetectable on the most recent sample, 2/4 had been undetectable for 149 and 819 days respectively. The remaining 6 patients had detectable PVL (median 3.1 log10 cp/ml, range: 1.7–6.2 log10 cp/ml). In contrast, 21/28 (75%) of samples which failed to amplify were <40 cp/ml, 2 had become undetectable in the most recent sample and 19 had been undetectable for a median of 577 days (range: 32–2481 days). 6/10 BCL samples showed none of the previously documented resistance for those patients, 3/10 showed some of the previously known mutations and only 1/10 showed all historic resistance, though this patient had only one documented mutation (K103N). For the BCS samples 3/4 showed none of the documented resistance, 1/4 harboured the previously known mutations (M184V, K103N). 3/6 of the DBS had none of the previously documented resistance, 2/6 showed some of the known mutations and 1/6 showed both known resistance mutations (M41L, T215S). In patients where multiple specimen types were amplified, 3/5 showed differences in mutations detected between sample types, with 2/5 correlating but having no mutations detected in any specimens. In patients with undetectable PVL, particularly those undetectable for long periods, it was difficult to sequence archived DNA. Concordance between different specimen types was variable as was concordance with previously documented mutations. Historic resistance reports remain important in the clinical management of patients on antiretroviral therapy, though proviral DNA testing may be useful in patients where historic reports are not available.

A review of a large number of HIV-1 tropism test requests (n=1148) performed at a London tertiary referral centre was carried out. The aim was to establish whether these were being performed in line with recommendations from published guidelines and whether this represented the most cost-effective use of these tests in informing prescribing decisions of the CCR5 antagonist drug, maraviroc. The cost of these assays within the UK was covered by commercial funding until April 2013 which has subsequently been withdrawn. Furthermore, all healthcare settings are under increasing cost constraints and hence establishing the real utility and appropriate use of these tests is of vital importance.