Prevalence of SARS-CoV-2 Omicron Sublineages and Spike Protein Mutations Conferring Resistance against Monoclonal Antibodies in a Swedish Cohort during 2022-2023.

Monoclonal antibodies (mAbs) are an important treatment option for COVID-19 caused by SARS-CoV-2, especially in immunosuppressed patients. However, this treatment option can become ineffective due to mutations in the SARS-CoV-2 genome, mainly in the receptor binding domain (RBD) of the spike (S) protein. In the present study, 7950 SARS-CoV-2 positive samples from the Uppsala and Örebro regions of central Sweden, collected between March 2022 and May 2023, were whole-genome sequenced using amplicon-based sequencing methods on Oxford Nanopore GridION, Illumina MiSeq, Illumina HiSeq, or MGI DNBSEQ-G400 instruments. Pango lineages were determined and all single nucleotide polymorphism (SNP) mutations that occurred in these samples were identified. We found that the dominant sublineages changed over time, and mutations conferring resistance to currently available mAbs became common. Notable ones are R346T and K444T mutations in the RBD that confer significant resistance against tixagevimab and cilgavimab mAbs. Further, mutations conferring a high-fold resistance to bebtelovimab, such as the K444T and V445P mutations, were also observed in the samples. This study highlights that resistance mutations have over time rendered currently available mAbs ineffective against SARS-CoV-2 in most patients. Therefore, there is a need for continued surveillance of resistance mutations and the development of new mAbs that target more conserved regions of the RBD.

SARS-CoV-2 variants of concern and spike protein mutational dynamics in a Swedish cohort during 2021, studied by Nanopore sequencing.

BACKGROUND: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. METHODS: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. RESULTS: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. CONCLUSION: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Lastly, we report multiple systematic errors that occurred when following the ARTIC Network SARS-CoV-2 tiled amplicon approach using the V3 primers and Nanopore sequencing, which led to the masking of some of the important signature mutations in the Delta sequences.

Characteristics of hepatitis C virus resistance in an international cohort after a decade of direct-acting antivirals.

Background & Aims: Direct-acting antiviral (DAA) regimens provide a cure in >95% of patients with chronic HCV infection. However, in some patients in whom therapy fails, resistance-associated substitutions (RASs) can develop, limiting retreatment options and risking onward resistant virus transmission. In this study, we evaluated RAS prevalence and distribution, including novel NS5A RASs and clinical factors associated with RAS selection, among patients who experienced DAA treatment failure. Methods: SHARED is an international consortium of clinicians and scientists studying HCV drug resistance. HCV sequence linked metadata from 3,355 patients were collected from 22 countries. NS3, NS5A, and NS5B RASs in virologic failures, including novel NS5A substitutions, were examined. Associations of clinical and demographic characteristics with RAS selection were investigated. Results: The frequency of RASs increased from its natural prevalence following DAA exposure: 37% to 60% in NS3, 29% to 80% in NS5A, 15% to 22% in NS5B for sofosbuvir, and 24% to 37% in NS5B for dasabuvir. Among 730 virologic failures, most were treated with first-generation DAAs, 94% had drug resistance in ≥1 DAA class: 31% single-class resistance, 42% dual-class resistance (predominantly against protease and NS5A inhibitors), and 21% triple-class resistance. Distinct patterns containing ≥2 highly resistant RASs were common. New potential NS5A RASs and adaptive changes were identified in genotypes 1a, 3, and 4. Following DAA failure, RAS selection was more frequent in older people with cirrhosis and those infected with genotypes 1b and 4. Conclusions: Drug resistance in HCV is frequent after DAA treatment failure. Previously unrecognized substitutions continue to emerge and remain uncharacterized. Lay summary: Although direct-acting antiviral medications effectively cure hepatitis C in most patients, sometimes treatment selects for resistant viruses, causing antiviral drugs to be either ineffective or only partially effective. Multidrug resistance is common in patients for whom DAA treatment fails. Older patients and patients with advanced liver diseases are more likely to select drug-resistant viruses. Collective efforts from international communities and governments are needed to develop an optimal approach to managing drug resistance and preventing the transmission of resistant viruses.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

[How did Omicron evolve and why does this SARS-CoV-2 variant spread so fast?] / Hur har omikron uppstått och varför sprider den sig så snabbt?

Omicron has more than twenty new mutations in the S1 domain of the spike gene as compared to the other previously known variants of SARS-CoV-2. Many of these new mutations, especially those located in the receptor binding domain, are likely to improve binding to the ACE2 receptor and to avoid binding to antibodies induced by a previous infection or by vaccination. Today there are several different hypotheses about the origin of Omicron, for example that it would have arisen in an immunosuppressed individual. Alternatively, a SARS-CoV-2 variant could have infected an unknown animal, and re-infection of humans would then have occurred. Furthermore, Omicron may have picked up a piece of a human common cold coronavirus.  The hitherto available data suggest that the rapid spread of Omicron is a combination of properties of the virus replication ability in addition to its ability to avoid pre-existing immune responses.

Global Prevalence of Adaptive and Prolonged Infections' Mutations in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

Several vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called 'adaptive mutations' and 'mutations identified in the prolonged infections', in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections' mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus.

SHARED: An International Collaboration to Unravel Hepatitis C Resistance.

The advent of direct-acting antivirals (DAAs) has transformed the treatment landscape of hepatitis C [...].

Targeting the NS2B-NS3 protease of tick-borne encephalitis virus with pan-flaviviral protease inhibitors.

Tick-borne encephalitis (TBE) is a severe neurological disorder caused by tick-borne encephalitis virus (TBEV), a member of the Flavivirus genus. Currently, two vaccines are available in Europe against TBEV. However, TBE cases have been rising in Sweden for the past twenty years, and thousands of cases are reported in Europe, emphasizing the need for antiviral treatments against this virus. The NS2B-NS3 protease is essential for flaviviral life cycle and has been studied as a target for the design of inhibitors against several well-known flaviviruses, but not TBEV. In the present study, Compound 86, a known tripeptidic inhibitor of dengue (DENV), West Nile (WNV) and Zika (ZIKV) proteases, was predicted to be active against TBEV protease using a combination of in silico techniques. Further, Compound 86 was found to inhibit recombinant TBEV protease with an IC50 = 0.92 µM in the in vitro enzymatic assay. Additionally, two more peptidic analogues were synthetized and they displayed inhibitory activities against both TBEV and ZIKV proteases. In particular, Compound 104 inhibited ZIKV protease with an IC50 = 0.25 µM. These compounds represent the first reported inhibitors of TBEV protease to date and provides valuable information for the further development of TBEV as well as pan-flavivirus protease inhibitors.

Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro.

The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations' health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 µM and 400 µM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine.

Identification of a C2-symmetric diol based human immunodeficiency virus protease inhibitor targeting Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus and infection by ZIKV Asian lineage is known to cause fetal brain anomalies and Guillain-Barrés syndrome. The WHO declared ZIKV a global public health emergency in 2016. However, currently neither vaccines nor antiviral prophylaxis/treatments are available. In this study, we report the identification of a C2-symmetric diol-based Human immunodeficiency virus type-1 (HIV) protease inhibitor active against ZIKV NS2B-NS3 protease. The compound, referred to as 9b, was identified by in silico screening of a library of 6265 protease inhibitors. Molecular dynamics (MD) simulation studies revealed that compound 9b formed a stable complex with ZIKV protease. Interaction analysis of compound 9b's binding pose from the cluster analysis of MD simulations trajectories predicted that 9b mostly interacted with ZIKV NS3. Although designed as an aspartyl protease inhibitor, compound 9b was found to inhibit ZIKV serine protease in vitro with IC50 = 143.25 ± 5.45 µM, in line with the in silico results. Additionally, linear interaction energy method (LIE) was used to estimate binding affinities of compounds 9b and 86 (a known panflavivirus peptide hybrid with IC50 = 1.64 ± 0.015 µM against ZIKV protease). The LIE method correctly predicted the binding affinity of compound 86 to be lower than that of 9b, proving to be superior to the molecular docking methods in scoring and ranking compounds. Since most of the reported ZIKV protease inhibitors are positively charged peptide-hybrids, with our without electrophilic warheads, compound 9b represents a less polar and more drug-like non-peptide hit compound useful for further optimization.Communicated by Ramaswamy Sarma.

Effect of the baseline Y93H resistance-associated substitution in HCV genotype 3 for direct-acting antiviral treatment: real-life experience from a multicenter study in Sweden and Norway.

Background: The NS5A resistance-associated substitution (RAS) Y93H is found quite frequently (5-10%) at baseline in direct-acting antiviral agents (DAA) treatment-naïve genotype (GT) 3a patients when studied by the population-sequencing method (cut-off 20%). This RAS may impair HCV DAA treatment response, since it possesses a high fold in vitro resistance to daclatasvir (DCV) and velpatasvir (VEL) in GT 3. We investigated the effect of baseline Y93H in patients with GT 3a infection on treatment outcome, with or without resistance-based DAA-treatment during 2014-2017. Patients/Methods: Treatment in the intervention group (n = 130) was tailored to baseline resistance-findings by population-sequencing method. Detection of baseline Y93H above 20% prompted a prolonged treatment duration of NS5A-inhibitor and sofosbuvir (SOF) and/or addition of ribavirin (RBV). Patients without baseline Y93H in the intervention group and all patients in the control group (n = 78) received recommended standard DAA-treatment. Results: A higher sustained virologic response rate (SVR) in the intervention group was shown compared to the control group at 95.4% (124/130) and 88.5% (69/78), respectively (p = .06). All five patients with baseline Y93H in the intervention group achieved SVR with personalised treatment based on results from resistance testing; either with the addition of RBV or prolonged treatment duration (24w). In the control group, 2/4 patients with Y93H at baseline treated with ledipasvir/SOF/RBV or DCV/SOF without RBV, failed treatment. Conclusion: The results from this real-life study are in accordance with the findings of the randomised controlled trials in 2015 and the EASL-guidelines of 2016, thus, baseline Y93H impacts on DCV and VEL treatment outcome.

Personalized treatment of hepatitis C genotype 1a in Norway and Sweden 2014-2016: a study of treatment outcome in patients with or without resistance-based DAA-therapy.

OBJECTIVES: Resistance-associated substitutions (RASs) may impair treatment response to direct-acting antivirals (DAA) in hepatitis C virus (HCV) treatment. We investigated the effects of baseline NS3-RASs (Q80K and R155K) and clinically relevant NS5A-RASs in patients with HCV genotype (GT) 1a infection on treatment outcome, with or without resistance-based DAA-treatment. This multi-center study was carried out between 2014 and 2016. PATIENTS/METHODS: Treatment in the intervention group (n = 92) was tailored to baseline resistance. Detection of NS3-RAS led to an NS5A-inhibitor-based regimen and detection of NS5A-RAS to a protease-inhibitor regimen. Patients without baseline RAS in the intervention group and all patients in the control group (n = 101) received recommended standard DAA-treatment. RESULTS: The sustained virologic response rates (SVR) in the intervention and control groups were 97.8% (90/92) and 93.1% (94/101), respectively (p = .174). A trend toward higher SVR-rate in cirrhotic patients (p = .058) was noticed in the intervention group compared to the control group with SVR-rates 97.5% (39/40) and 83.3% (35/42), respectively. All patients with baseline NS3 (Q80K/R155K) or NS5A-RASs in the intervention group achieved SVR with personalized resistance-based treatment. In the control group, five patients with Q80K or R155K at baseline were treated with simeprevir + sofosbuvir and treatment failed in two of them. Furthermore, one of three patients who failed ledipasvir + sofosbuvir treatment had NS5A-RASs at baseline. CONCLUSIONS: In line with the findings of the OPTIMIST-2 trial for Q80K and the EASL-guidelines 2016 for NS5A-RASs, baseline RASs appeared to have an impact on treatment outcome albeit a statistical significance was not observed in this low-prevalence population.

Baseline dasabuvir resistance in Hepatitis C virus from the genotypes 1, 2 and 3 and modeling of the NS5B-dasabuvir complex by the in silico approach.

Background: Current combination treatments with direct-acting antiviral agents (DAAs) can cure more than 95% of hepatitis C virus (HCV) infections. However, resistance-associated substitutions (RASs) may emerge and can also be present in treatment-naïve patients. Methods, results and discussion: In this study, a semi-pan-genotypic population sequencing method was developed and used to assess all NS5B amino acid variants between residue positions 310 and 564. Our method successfully sequenced more than 90% of genotype (GT) 1a, 1b, 2b and 3a samples. By using the population sequencing method with a cut-off of 20%, we found the dasabuvir RASs A553V and C445F to be a baseline polymorphism of GT 2b (8 out of 8) and GT 3a (18 out of 18) sequences, respectively. In GT 1a and 1b treatment-naïve subjects (n=25), no high-fold resistance polymorphism/RASs were identified. We further predicted dasabuvir's binding pose with the NS5B polymerase using the in silico methods to elucidate the reasons associated with the resistance of clinically relevant RASs. Dasabuvir was docked at the palm-I site and was found to form hydrogen bonds with the residues S288, I447, Y448, N291 and D318. The RAS positions 316, 414, 448, 553 and 556 were found to constitute the dasabuvir binding pocket.

Comparative genome analysis of Alkhumra hemorrhagic fever virus with Kyasanur forest disease and tick-borne encephalitis viruses by the in silico approach.

Alkhumra hemorrhagic fever virus (AHFV), a relatively new member of the Flaviviruses, was discovered in Saudi Arabia 23 years ago. AHFV is classified in the tick-borne encephalitis virus serocomplex, along with the Kyasanur forest disease virus (KFDV) and tick-borne encephalitis virus (TBEV). Currently, very little is known about the pathologies of AHFV. In this study, using the available genome information of AHFV, KFDV and TBEV, we have predicted and compared the following aspects of these viruses: evolution, nucleotide and protein compositions, recombination, codon frequency, substitution rate, N- and O-glycosylation sites, signal peptide and cleavage site, transmembrane region, secondary structure of 5' and 3' UTRs and RNA-RNA interactions. Additionally, we have modeled the 3D protease and RNA-dependent RNA polymerase structures for AHFV, KFDV and TBEV. Recombination analysis showed no evidence of recombination in the AHFV genome with that of either KFDV or TBEV, although single break point analysis showed that nucleotide position 7399 (in the NS4B) is a breakpoint location. AHFV, KFDV and TBEV are very similar in terms of codon frequency, the number of transmembrane regions, properties of the polyprotein, RNA-RNA interaction sequences, NS3 protease and NS5 polymerase structures and 5' UTR structure. Using genome sequences, we showed the similarities between these closely- related viruses on several different areas.

Worldwide prevalence of baseline resistance-associated polymorphisms and resistance mutations in HCV against current direct-acting antivirals.

BACKGROUND: HCV infections can now be completely cured, thanks to the currently marketed direct-acting antivirals (DAAs). It is known that HCV patients carry viral populations with baseline polymorphisms and/or mutations that make them resistant against some of these DAAs, which can negatively impact the patient's treatment outcome. Using complete HCV coding sequences isolated from 1,306 treatment-naive patients of genotypes (GTs) 1, 2, 3, 4 and 6 from around the globe, we studied the prevalence of baseline resistance-associated polymorphisms (RAPs) and resistance mutations (RMs) against DAAs that are currently on the market or in clinical trials. METHODS: The HCV genome sequences used in this study were retrieved from the NCBI database. RAPs and RMs, with reference to HCV GT1a, were identified using the HCV Geno2pheno web server. RESULTS: Nearly 50% of the total amino acid positions (including NS3 protease, NS5A and NS5B) studied are baseline polymorphisms that differentiated one GT from the rest. A proportion of these baseline polymorphisms and baseline non-polymorphic RMs could confer a significant increase in resistance against DAAs. CONCLUSIONS: In this study, we show the presence and prevalence of RAPs and RMs in DAA treatment-naive patients against currently used DAAs or DAAs in clinical trials. Our study suggests that RAPs and RMs profiling of HCV patients should be performed before the start of the therapy. Our results should be relevant especially in low- and middle-income countries, where the patients have a large variation of GTs and subtypes, and where the generic HCV treatment is now increasingly available.

The effect of the first-generation HCV-protease inhibitors boceprevir and telaprevir and the relation to baseline NS3 resistance mutations in genotype 1: experience from a small Swedish cohort.

BACKGROUND: The clinical experience with protease-inhibitor (PI) triple regimen appears disappointing regarding effect, side effects, high work load, and costs. This real-world study evaluates baseline and emerging resistance-associated substitutions (RASs) and their significance for treatment outcome. METHOD: Thirty-six genotype 1a/b patients treated according to Swedish recommendations during 2011-2013 with triple therapy including pegylated interferon and ribavirin in combination with a protease-inhibitor, either boceprevir (BOC) or telaprevir (TVR), were retrospectively evaluated. Frozen serum samples from the patients were tested for resistance with pan-genotypic population sequencing. RESULTS: Overall, 56% (20/36) of the patients achieved sustained viral response (SVR). The SVR was comparable between BOC (64%; 9/14) and TVR (50%; 11/22) (p = 0.07), and the IL28B type non-CC (48%; 12/25) and CC (46%; 6/13) (p = 0.77). The SVR was higher in patients without cirrhosis (89.5%; 17/19) (p < 0.0005), in treatment-naïve patients (70%; 14/20) (p = 0.02), and those with low viral load (<800,000 IU/mL) (66.7%; 8/12) (p < 0.0002), compared to those with cirrhosis (17.6%; 3/17), treatment-experienced (37.5%; 6/16), and high viral load (>800,000 IU/mL) (50%; 12/24). CONCLUSION: PI triple regimes were highly effective in treatment-naïve patients without cirrhosis, but in this real-world cohort an inferior effect was evident in cirrhotic and treatment-experienced patients. Although tested on a limited sample, the baseline resistance testing seems to have no impact on prediction of therapy outcome. The reason could be that the baseline RASs T54S and V55A have relatively low resistance towards BOC and TVR. Emerging RASs, mainly R155K, with known high resistance to BOC and TVR were frequently found in non-responders.

Protein backbone flexibility pattern is evolutionarily conserved in the Flaviviridae family: A case of NS3 protease in Flavivirus and Hepacivirus.

Viruses belonging to the Flaviviridae family have been an important health concern for humans, animals and birds alike. No specific treatment is available yet for many of the viral infections caused by the members of this family. Lack of specific drugs against these viruses is mainly due to lack of protein structure information. It has been known that protein backbone fluctuation pattern is highly conserved in protein pairs with similar folds, in spite of the lack of sequence similarity. We hypothesized that this concept should also hold true for proteins (especially enzymes) of viruses included in different genera of the Flaviviridae family, as we know that the sequence similarity between them is low. Using available NS3 protease crystal structures of the Flaviviridae family, our preliminary results have shown that the Cα (i.e. backbone) fluctuation patterns are highly similar between Flaviviruses and a Hepacivirus (i.e. hepatitis C virus, HCV). This has to be validated further experimentally.

Biophysical Studies on HCV 1a NS3/4A Protease and Its Catalytic Triad in Wild Type and Mutants by the In Silico Approach.

The hepatitis C virus (HCV), of the family flaviviridae, is one of the major causes of chronic liver diseases. Until the year 2012, HCV infections were treated using PEG-interferon and ribavirin combinations, which have a low cure rate and severe side effects. Currently, many direct-acting antivirals (DAAs) are available, e.g. protease inhibitors, NS5A and polymerase inhibitors. These drugs have proven to be efficient in interferon-free treatment combinations and capable of enhancing the cure rate to above 90 %. Unlike PEG-interferon and ribavirin combinations, DAAs select for resistance in HCV. The R155K mutation in the HCV was found to resist all the currently available protease inhibitors. Here, we studied biophysical parameters like pocket (cavity) geometries and stabilizing residues of HCV 1a NS3/4A protease in wild type and mutants. We also studied HCV 1a NS3/4A protease's catalytic residues: their accessibility, energy, flexibility and binding to Phase II oral protease inhibitor vedroprevir (GS-9451), and compared these parameters between wild type and mutant(s). All these studies were performed using various bioinformatics tools (e.g. Swiss-PdbViewer and Schrödinger's Maestro) and web servers (e.g. DoGSiteScorer, SRide, ASA-View, WHAT IF, elNémo, CABS-flex, PatchDock and PLIP). From our study, we found that introduction of R155K, A156T or D168A mutation to wild-type NS3/4A protease increases the pocket's volume, surface (in the R155K mutant, surface decreases), lipo surface and depth and decreases the number of stabilizing residues. Additionally, differences in catalytic residues' solvent accessibility, energy, root-mean-square deviation (RMSD) and flexibility between wild type and mutants might explain changes in the protease activity and the resistance to protease inhibitors.

Sequencing of hepatitis C virus for detection of resistance to direct-acting antiviral therapy: A systematic review.

The significance of the clinical impact of direct-acting antiviral (DAA) resistance-associated substitutions (RASs) in hepatitis C virus (HCV) on treatment failure is unclear. No standardized methods or guidelines for detection of DAA RASs in HCV exist. To facilitate further evaluations of the impact of DAA RASs in HCV, we conducted a systematic review of RAS sequencing protocols, compiled a comprehensive public library of sequencing primers, and provided expert guidance on the most appropriate methods to screen and identify RASs. The development of standardized RAS sequencing protocols is complicated due to a high genetic variability and the need for genotype- and subtype-specific protocols for multiple regions. We have identified several limitations of the available methods and have highlighted areas requiring further research and development. The development, validation, and sharing of standardized methods for all genotypes and subtypes should be a priority. (Hepatology Communications 2017;1:379-390).

SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase.

There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.