Expression of recombinant dengue virus type 1 non-structural protein 1 in mammalian cells and preliminary assessment of its suitability to detect human IgG antibodies elicited by viral infection.

In recent years dengue has become a rapidly growing public health problem worldwide, however, the availability of accurate and affordable diagnostic immunoassays is limited, partly due to the difficulty of producing large quantities of purified antigen. Non-structural protein 1 (NS1) has shown to be a good candidate for inclusion in diagnostic assays and for serosurveys, particularly in endemic countries as a prerequisite for vaccination. In this work the NS1 antigen derived from dengue virus type-1 (DENV1) was expressed in HEK293-T cells and purified by affinity chromatography. The recombinant protein was recovered properly folded as dimers, highly purified and with good yield (1.5 mg/L). It was applied as a serological probe in an indirect ELISA developed in this work to detect human IgG antibodies. Preliminary comparative performance values of 81.1% sensitivity and 83.0% specificity of the developed and preliminary validated iELISA, relative to a commercial kit were obtained, suggesting that the purified recombinant DENV1 NS1 antigen is suitable to detect IgG antibodies, indicative of past DENV infection.

Targeting SARS-CoV-2 nsp13 Helicase and Assessment of Druggability Pockets: Identification of Two Potent Inhibitors by a Multi-Site In Silico Drug Repurposing Approach.

The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.

Molecular Mechanism of the Non-Covalent Orally Targeted SARS-CoV-2 Mpro Inhibitor S-217622 and Computational Assessment of Its Effectiveness against Mainstream Variants.

Convenient and efficient therapeutic agents are urgently needed to block the continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, the mechanism for the novel orally targeted SARS-CoV-2 main protease (Mpro) inhibitor S-217622 is revealed through a molecular dynamics simulation. The difference in the movement modes of the S-217622-Mpro complex and apo-Mpro suggested S-217622 could inhibit the motility intensity of Mpro, thus maintaining their stable binding. Subsequent energy calculations showed that the P2 pharmacophore possessed the highest energy contribution among the three pharmacophores of S-217622. Additionally, hot-spot residues H41, M165, C145, E166, and H163 have strong interactions with S-217622. To further investigate the resistance of S-217622 to six mainstream variants, the binding modes of S-217622 with these variants were elucidated. The subtle differences in energy compared to that of the wild type implied that the binding patterns of these systems were similar, and S-217622 still inhibited these variants. We hope this work will provide theoretical insights for optimizing novel targeted Mpro drugs.

Inhibition of the hexamerization of SARS-CoV-2 endoribonuclease and modeling of RNA structures bound to the hexamer.

Non-structural protein 15 (Nsp15) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) forms a homo hexamer and functions as an endoribonuclease. Here, we propose that Nsp15 activity may be inhibited by preventing its hexamerization through drug binding. We first explored the stable conformation of the Nsp15 monomer as the global free energy minimum conformation in the free energy landscape using a combination of parallel cascade selection molecular dynamics (PaCS-MD) and the Markov state model (MSM), and found that the Nsp15 monomer forms a more open conformation with larger druggable pockets on the surface. Targeting the pockets with high druggability scores, we conducted ligand docking and identified compounds that tightly bind to the Nsp15 monomer. The top poses with Nsp15 were subjected to binding free energy calculations by dissociation PaCS-MD and MSM (dPaCS-MD/MSM), indicating the stability of the complexes. One of the identified pockets, which is distinctively bound by inosine analogues, may be an alternative binding site to stabilize viral RNA binding and/or an alternative catalytic site. We constructed a stable RNA structure model bound to both UTP and alternative binding sites, providing a reasonable proposed model of the Nsp15/RNA complex.

Assessment of protein-protein interfaces in cryo-EM derived assemblies.

Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we comprehensively assessed protein-protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed Protein Interface-score (PI-score), a density-independent machine learning-based metric, trained using the features of protein-protein interfaces in crystal structures. We evaluated 5873 interfaces in 1053 PDB-deposited cryo-EM models (including SARS-CoV-2 complexes), as well as the models submitted to CASP13 cryo-EM targets and the EM model challenge. We further inspected the interfaces associated with low-scores and found that some of those, especially in intermediate-to-low resolution (worse than 4 Å) structures, were not captured by density-based assessment scores. A combined score incorporating PI-score and fit-to-density score showed discriminatory power, allowing our method to provide a powerful complementary assessment tool for the ever-increasing number of complexes solved by cryo-EM.

Assessment of dengue and COVID-19 antibody rapid diagnostic tests cross-reactivity in Indonesia.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic remains ongoing around the world, including in areas where dengue is endemic. Dengue and COVID-19, to some extent, have similar clinical and laboratory features, which can lead to misdiagnosis, delayed treatment and patient's isolation. The use of rapid diagnostic tests (RDT) is easy and convenient for fast diagnosis, however there may be issues with cross-reactivity with antibodies for other pathogens. METHODS: We assessed the possibility of cross-reactivity between SARS-CoV-2 and dengue antibodies by: (1) testing five brands of COVID-19 IgG / IgM RDTs on 60 RT-PCR-confirmed dengue samples; (2) testing 95 RT-PCR-confirmed COVID-19 samples on dengue RDT; and (3) testing samples positive for COVID-19 IgG and/or IgM on dengue RDT. RESULTS: We observed a high specificity across all five brands of COVID-19 RDTs, ranging from 98.3 to 100%. Out of the confirmed COVID-19 samples, one patient tested positive for dengue IgM only, another tested positive for dengue IgG only. One patient tested positive for dengue IgG, IgM, and NS1, suggesting a co-infection. In COVID-19 IgG and/or IgM samples, 6.3% of COVID-19 IgG-positive samples also tested positive for dengue IgG, while 21.1% of COVID-19 IgM-positive samples also tested positive for dengue IgG. CONCLUSION: Despite the high specificity of the COVID-19 RDT, we observed cross-reactions and false-positive results between dengue and COVID-19. Dengue and COVID-19 co-infection was also found. Health practitioners in dengue endemic areas should be careful when using antibody RDT for the diagnosis of dengue during the COVID-19 pandemic to avoid misdiagnosis.

Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase.

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

Epidemiological survey and screening strategy for dengue virus in blood donors from Yunnan Province.

BACKGROUND: Dengue virus (DENV) infection is increasingly common in southern China and can be transmitted through blood transfusion but is not currently part of donor screening throughout the region. We assessed DENV prevalence among donors at the Xishuangbanna Blood Center, Yunnan, to support development of DENV screening strategies. METHODS: Blood samples were collected randomly between June 2019 and August 2019. These were screened for anti-DENV IgG and IgM using enzyme-linked immunosorbent assay (ELISA). Then, all reactive samples and some randomly-chosen non-reactive samples were used to detect DENV RNAs using real-time polymerase-chain-reaction (RT-PCR) assays. After RT-PCR, samples were further tested for soluble nonstructural protein 1 (NS1) using the colloidal gold method. Donors demographics were also collected and assessed. RESULTS: Over the study period, 2254 donor samples were collected and tested for anti-DENV IgG and IgM by ELISA. This revealed 598 anti-DENV IgG and/or IgM reactive samples, a serological prevalence of 26.53%. Of these, 26 were RT-PCR positive and/or NS1 positive. Significant differences in DENV prevalence were noted by occupation (P = 0.001), education (P < 0.001), and ethnicity (P = 0.026). CONCLUSION: The prevalence of DENV in Xishuangbanna Blood Center was higher than most other blood centers that have implemented DENV donor screening. Our study provides first-hand data about the prevalence of DENV and allows the development of a screening strategy for clinical use.

Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential.

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.

Searching for target-specific and multi-targeting organics for Covid-19 in the Drugbank database with a double scoring approach.

The current outbreak of Covid-19 infection due to SARS-CoV-2, a virus from the coronavirus family, has become a major threat to human healthcare. The virus has already infected more than 44 M people and the number of deaths reported has reached more than 1.1 M which may be attributed to lack of medicine. The traditional drug discovery approach involves many years of rigorous research and development and demands for a huge investment which cannot be adopted for the ongoing pandemic infection. Rather we need a swift and cost-effective approach to inhibit and control the viral infection. With the help of computational screening approaches and by choosing appropriate chemical space, it is possible to identify lead drug-like compounds for Covid-19. In this study, we have used the Drugbank database to screen compounds against the most important viral targets namely 3C-like protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp) and the spike (S) protein. These targets play a major role in the replication/transcription and host cell recognition, therefore, are vital for the viral reproduction and spread of infection. As the structure based computational screening approaches are more reliable, we used the crystal structures for 3C-like main protease and spike protein. For the remaining targets, we used the structures based on homology modeling. Further, we employed two scoring methods based on binding free energies implemented in AutoDock Vina and molecular mechanics-generalized Born surface area approach. Based on these results, we propose drug cocktails active against the three viral targets namely 3CLpro, PLpro and RdRp. Interestingly, one of the identified compounds in this study i.e. Baloxavir marboxil has been under clinical trial for the treatment of Covid-19 infection. In addition, we have identified a few compounds such as Phthalocyanine, Tadalafil, Lonafarnib, Nilotinib, Dihydroergotamine, R-428 which can bind to all three targets simultaneously and can serve as multi-targeting drugs. Our study also included calculation of binding energies for various compounds currently under drug trials. Among these compounds, it is found that Remdesivir binds to targets, 3CLpro and RdRp with high binding affinity. Moreover, Baricitinib and Umifenovir were found to have superior target-specific binding while Darunavir is found to be a potential multi-targeting drug. As far as we know this is the first study where the compounds from the Drugbank database are screened against four vital targets of SARS-CoV-2 and illustrates that the computational screening using a double scoring approach can yield potential drug-like compounds against Covid-19 infection.

Patterns of RNA Editing in Newcastle Disease Virus Infections.

The expression of accessory non-structural proteins V and W in Newcastle disease virus (NDV) infections depends on RNA editing. These proteins are derived from frameshifts of the sequence coding for the P protein via co-transcriptional insertion of one or two guanines in the mRNA. However, a larger number of guanines can be inserted with lower frequencies. We analysed data from deep RNA sequencing of samples from in vitro and in vivo NDV infections to uncover the patterns of mRNA editing in NDV. The distribution of insertions is well described by a simple Markov model of polymerase stuttering, providing strong quantitative confirmation of the molecular process hypothesised by Kolakofsky and collaborators three decades ago. Our results suggest that the probability that the NDV polymerase would stutter is about 0.45 initially, and 0.3 for further subsequent insertions. The latter probability is approximately independent of the number of previous insertions, the host cell, and viral strain. However, in LaSota infections, we also observe deviations from the predicted V/W ratio of about 3:1 according to this model, which could be attributed to deviations from this stuttering model or to further mechanisms downregulating the abundance of W protein.

Two Sides of a Coin: a Zika Virus Mutation Selected in Pregnant Rhesus Macaques Promotes Fetal Infection in Mice but at a Cost of Reduced Fitness in Nonpregnant Macaques and Diminished Transmissibility by Vectors.

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.

SARS-CoV-2 pathophysiology and assessment of coronaviruses in CNS diseases with a focus on therapeutic targets.

The novel Coronavirus disease of 2019 (nCOV-19) is a viral outbreak noted first in Wuhan, China. This disease is caused by Severe Acute Respiratory Syndrome (SARS) Coronavirus (CoV)-2. In the past, other members of the coronavirus family, such as SARS and Middle East Respiratory Syndrome (MERS), have made an impact in China and the Arabian peninsula respectively. Both SARS and COVID-19 share similar symptoms such as fever, cough, and difficulty in breathing that can become fatal in later stages. However, SARS and MERS infections were epidemic diseases constrained to limited regions. By March 2020 the SARS-CoV-2 had spread across the globe and on March 11th, 2020 the World Health Organization (WHO) declared COVID-19 as pandemic disease. In severe SARS-CoV-2 infection, many patients succumbed to pneumonia. Higher rates of deaths were seen in older patients who had co-morbidities such as diabetes mellitus, hypertension, cardiovascular disease (CVD), and dementia. In this review paper, we discuss the effect of SARS-CoV-2 on CNS diseases, such as Alzheimer's-like dementia, and diabetes mellitus. We also focus on the virus genome, pathophysiology, theranostics, and autophagy mechanisms. We will assess the multiorgan failure reported in advanced stages of SARS-CoV-2 infection. Our paper will provide mechanistic clues and therapeutic targets for physicians and investigators to combat COVID-19.

Fast Identification of Possible Drug Treatment of Coronavirus Disease-19 (COVID-19) through Computational Drug Repurposing Study.

The recent outbreak of novel coronavirus disease-19 (COVID-19) calls for and welcomes possible treatment strategies using drugs on the market. It is very efficient to apply computer-aided drug design techniques to quickly identify promising drug repurposing candidates, especially after the detailed 3D structures of key viral proteins are resolved. The virus causing COVID-19 is SARS-CoV-2. Taking advantage of a recently released crystal structure of SARS-CoV-2 main protease in complex with a covalently bonded inhibitor, N3 (Liu et al., 10.2210/pdb6LU7/pdb), I conducted virtual docking screening of approved drugs and drug candidates in clinical trials. For the top docking hits, I then performed molecular dynamics simulations followed by binding free energy calculations using an end point method called MM-PBSA-WSAS (molecular mechanics/Poisson-Boltzmann surface area/weighted solvent-accessible surface area; Wang, Chem. Rev. 2019, 119, 9478; Wang, Curr. Comput.-Aided Drug Des. 2006, 2, 287; Wang; ; Hou J. Chem. Inf. Model., 2012, 52, 1199). Several promising known drugs stand out as potential inhibitors of SARS-CoV-2 main protease, including carfilzomib, eravacycline, valrubicin, lopinavir, and elbasvir. Carfilzomib, an approved anticancer drug acting as a proteasome inhibitor, has the best MM-PBSA-WSAS binding free energy, -13.8 kcal/mol. The second-best repurposing drug candidate, eravacycline, is synthetic halogenated tetracycline class antibiotic. Streptomycin, another antibiotic and a charged molecule, also demonstrates some inhibitory effect, even though the predicted binding free energy of the charged form (-3.8 kcal/mol) is not nearly as low as that of the neutral form (-7.9 kcal/mol). One bioactive, PubChem 23727975, has a binding free energy of -12.9 kcal/mol. Detailed receptor-ligand interactions were analyzed and hot spots for the receptor-ligand binding were identified. I found that one hot spot residue, His41, is a conserved residue across many viruses including SARS-CoV, SARS-CoV-2, MERS-CoV, and hepatitis C virus (HCV). The findings of this study can facilitate rational drug design targeting the SARS-CoV-2 main protease.

Assessment of diagnostic and analytic performance of the SD Bioline Dengue Duo test for dengue virus (DENV) infections in an endemic area (Savannakhet province, Lao People's Democratic Republic).

BACKGROUND: Rapid tests detecting both dengue virus (DENV) NS1 antigen and anti-DENV IgM and IgG antibodies facilitate diagnosis of dengue fever (DF) in resource-poor settings. METHODOLOGY/PRINCIPAL FINDINGS: 92 acute phase serum samples from patients with a PCR-confirmed DENV infection collected in Lao People's Democratic Republic (Lao PDR) in 2013 and 2015 were analyzed with the SD Bioline Dengue Duo test. A subset of 74 samples was additionally tested with the Platelia NS1 antigen test, the Panbio DENV µ-capture ELISA and the Panbio DENV IgG ELISA. IgM seroconversion was assayed using follow-up samples of 35 patients collected in the convalescent phase. 57.6%, 22.8% and 44.6% of acute phase serum samples tested positive in the SD Bioline Dengue Duo NS1, IgM, and IgG test, respectively. Diagnostic sensitivity of the SD Bioline Dengue Duo NS1 test strongly correlated with viral load, decreased rapidly over the acute phase of the disease, and was significantly reduced in presence of high anti-DENV IgG antibody titers resulting from secondary DENV infection. While a good concordance (Cohen's kappa 0.78) was found between the SD Bioline Dengue Duo NS1 test and the Platelia NS1 antigen ELISA, both the SD Bioline Dengue Duo IgM and IgG test displayed a significantly lower sensitivity than the corresponding ELISA tests. CONCLUSIONS/SIGNIFICANCE: The SD Bioline Dengue Duo test is a valuable tool for diagnosis of DENV infections especially when analyzing early acute phase samples with high viral load. Nevertheless, in endemic areas, where secondary flavivirus infections are common, diagnostic sensitivity of the NS1 and IgM test components may be compromised.

Cost-Effectiveness Analysis of Baseline Testing for Resistance-Associated Polymorphisms to Optimize Treatment Outcome in Genotype 1 Noncirrhotic Treatment-Naïve Patients With Chronic Hepatitis C Virus.

OBJECTIVES: Direct-acting antivirals containing nonstructural protein 5A (NS5A) inhibitors administered over 8 to 12 weeks are effective in ∼95% of patients with hepatitis C virus. Nevertheless, patients resistant to NS5A inhibitors have lower cure rates over 8 weeks (<85%); for these patients, 12 weeks of treatment produces cure rates greater than 95%. We evaluated the lifetime cost-effectiveness of testing for NS5A resistance at baseline and optimizing treatment duration accordingly in genotype 1 noncirrhotic treatment-naïve patients from the perspective of the UK National Health Service. METHODS: A decision-analytic model compared (1) standard 12-week treatment (no testing), (2) shortened 8-week treatment (no testing), and (3) baseline testing with 12-/8-week treatment for those with/without NS5A polymorphisms. Patients who failed first-line therapy were retreated for 12 weeks. Model inputs were derived from published studies. Costs, quality-adjusted life-years, and the probability of cost-effectiveness were calculated. RESULTS: Baseline testing had an incremental net monetary benefit (INMB) of £11 838 versus standard 12 weeks of therapy (no testing) and low probability (31%) of being the most cost-effective, assuming £30 000 willingness to pay. Shortened 8 weeks of treatment (no testing) had an INMB of £12 294 and the highest probability (69%) of being most cost-effective. Scenario analyses showed baseline testing generally had the highest INMB and probability of being most cost-effective if first- and second-line drug prices were low (<£20k). CONCLUSIONS: Optimizing treatment duration based on NS5A polymorphisms for genotype 1 noncirrhotic treatment-naive patients in the United Kingdom is not cost-effective if the drug costs are high; the strategy is generally most cost-effective when drug prices are low (<£20k).

Comparative assessment of commercial enzyme-linked immunosorbent assay & rapid diagnostic tests used for dengue diagnosis in India.

Background & objectives: Dengue diagnosis is routinely carried out by detection of dengue virus (DENV) antigen NS1 and/or anti-DENV IgM antibodies using enzyme-linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs). This study was aimed at evaluation of quality of diagnostic assays currently in use in India for the identification of DENV infection. Methods: During 2016 dengue season (July-November) in Pune, India, comparative assessment of a few immunoassays was undertaken using (i) WHO-approved Panbio-Dengue-Early-(NS1)-ELISA and Panbio-Dengue-IgM-Capture-ELISA as reference tests, and (ii) Bayesian latent class analysis (BLCA) which assumes that no test is perfect. The assays included J.Mitra-Dengue-NS1-Ag-MICROLISA (JME-NS1), J.Mitra-Dengue-IgM-MICROLISA (JME-IgM), and two RDTs, namely, J.Mitra-Dengue-Day-1-Test (JM-RDT) and SD-BIOLINE-Dengue-Duo (SDB-RDT). Serum samples from patients seeking dengue diagnosis (n=809) were tested using the diagnostic kits. The presence of NS1 and/or IgM was taken as evidence for dengue-positive diagnosis. Results: Panbio-NS1/IgM-ELISAs identified 38.6 per cent patients as dengue positive. With Panbio-ELISA as reference, all the tests were less sensitive for IgM detection, while for NS1, JM-RDT was less sensitive. For combined diagnosis (both markers), sensitivity of all the tests was low (55.7-76.6%). According to BLCA, Panbio-ELISA was 84 per cent sensitive for NS1, 86 per cent specific for IgM and 87 per cent specific for combined diagnosis. Accordingly, performance of the other tests was substantially improved with BLCA; however, sensitivity of both the RDTs for IgM detection remained unacceptable. The NS1 ELISAs and RDTs detected all four DENV serotypes, JME being most efficient. All IgM tests exhibited higher sensitivity in secondary infections. Interpretation & conclusions: These results confirmed superiority of ELISAs, and testing for both NS1 and IgM markers for dengue diagnosis, and emphasized on improvement in sensitivity of RDTs.

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.