RNA dependent RNA polymerase (RdRp) as a drug target for SARS-CoV2.

RNA-dependent RNA polymerase (RdRp), also called nsp12, is considered a promising but challenging drug target for inhibiting replication and hence, the growth of various RNA-viruses. In this report, a computational study is performed to offer insights on the binding of Remdesivir and Galidesivir with SARS-CoV2 RdRp with natural substrate, ATP, as the control. It was observed that Remdesivir and Galidesivir exhibited similar binding energies for their best docked poses, -6.6 kcal/mole and -6.2 kcal/mole, respectively. ATP also displayed comparative and strong binding free energy of -6.3 kcal/mole in the catalytic site of RdRp. However, their binding locations within the active site are distinct. Further, the interaction of catalytic site residues (Asp760, Asp761, and Asp618) with Remdesivir and Galidesivir is comprehensively examined. Conformational changes of RdRp and bound molecules are demonstrated using 100 ns explicit solvent simulation of the protein-ligand complex. Simulation suggests that Galidesivir binds at the non-catalytic location and its binding strength is relatively weaker than ATP and Remdesivir. Remdesivir also binds at the catalytic site and showed high potency to inhibit the function of RdRp. Binding of co-factor units nsp7 and nsp8 with RdRp (nsp12) complexed with Remdesivir and Galidesivir was also examined. MMPBSA binding energy for all three complexes has been computed across the 100 ns simulation trajectory. Overall, this study suggests, Remdesivir has anti-RdRp activity via binding at a catalytic site. In contrast, Galidesivir may not have direct anti-RdRp activity but it can induce a conformational change in the RNA polymerase.

Dry eye disease survey among schoolteachers and children using visual display terminals during COVID-19 lockdown-CODE study ( Co vid and d ry e ye study).

Background: Lockdown during COVID-19 led to teachers and children shifting to online classes, using visual display terminals (VDTs) for education, resulting in increased screen time. The present study was done to assess and understand the nature and magnitude of the problem and to suggest preventive or remedial measures. Methods: A questionnaire-based cross-sectional study was conducted. The questionnaire was prepared for an online survey (using Google Forms) and circulated among school children belonging to different schools across India using multiple groups on social media. Results: A total of 3327 participants from 46 schools across India participated in the survey. We found a marked rise in cumulative screen time for both teachers and students before and during the lockdown. There was a threefold increase in the number of participants with a cumulative screen time 6 h or more compared to the pre-COVID era. Teachers (older participants) had worse symptom scores than students. Larger screens, like televisions, were better VDTs compared to smartphones, tablets, or laptops. Conclusions: School administrators and policymakers should pay due attention to institutionalizing the guidelines about class duration, appropriate screens, and stipulating break duration during online classes, which will continue to remain the predominant mode of education for teachers and students alike, at least in the near future.

Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19.

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.

Pharmacophore screening to identify natural origin compounds to target RNA-dependent RNA polymerase (RdRp) of SARS-CoV2.

Several existing drugs have gained initial consideration due to their therapeutic characteristics against COVID-19 (Corona Virus Disease 2019). Hydroxychloroquine (HCQ) was proposed as possible therapy for shortening the duration of COVID-19, but soon after this, it was discarded. Similarly, known antiviral compounds were also proposed and investigated to treat COVID-19. We report a pharmacophore screening using essential chemical groups derived from HCQ and known antivirals to search a natural compound chemical space. Molecular docking of HCQ under physiological condition with spike protein, 3C-like protease (3CLpro), and RNA-dependent RNA polymerase (RdRp) of SARS-CoV2 showed - 8.52 kcal/mole binding score with RdRp, while the other two proteins showed relatively weaker binding affinity. Docked complex of RdRp-HCQ is further examined using 100 ns molecular dynamic simulation. Docking and simulation study confirmed active chemical moieties of HCQ, treated as 6-point pharmacophore to screen ZINC natural compound database. Pharmacophore screening resulted in the identification of potent hit molecule [(3S,3aR,6R,6aS)-3-(5-phenylsulfanyltetrazol-1-yl)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl]N-naphthalen-ylcarbamate from natural compound library. Additionally, a set of antiviral compounds with similar chemical scaffolds are also used to design a separate ligand-based pharmacophore screening. Antiviral pharmacophore screening produced a potent hit 4-[(1,5-dimethyl-3-oxo-2-phenylpyrazol-4-yl)-(2-hydroxyphenyl)methyl]-1,5-dimethyl-2-phenylpyrazol-3-one containing essential moieties that showed affinity towards RdRp. Further, both these screened compounds are docked (- 8.69 and - 8.86 kcal/mol) and simulated with RdRp protein for 100 ns in explicit solvent medium. They bind at the active site of RdRp and form direct/indirect interaction with ASP618, ASP760, and ASP761 catalytic residues of the protein. Successively, their molecular mechanics Poisson Boltzmann surface area (MMPBSA) binding energies are calculated over the simulation trajectory to determine the dynamic atomistic interaction details. Overall, this study proposes two key natural chemical moieties: (a) tetrazol and (b) phenylpyrazol that can be investigated as a potential chemical group to design inhibitors against SARS-CoV2 RdRp.

Synergistic Effects of Natural Compounds Toward Inhibition of SARS-CoV-2 3CL Protease.

The biggest challenge in medical management and control of the COVID-19 pandemic is the nonavailability of the treatment molecules. While vaccines and other biotherapeutic products for managing COVID-19 have reached the market, a small-molecule cure is yet to be developed. This is relevant because the cost of production, storage, and ease of distribution of a small-molecule drug are significantly more favorable than those of biologics. In this paper, we present a multicompound approach, where two drug molecules are administered concurrently to offer an effective therapy for COVID-19. The co-action of the two compounds, each derived from natural origins, has been demonstrated against the 3CL protease, already recognized as a potential drug target for inhibiting SARS-CoV-2. The pair of compounds pursued in this study are flavonoid and naphthalene scaffold. Individually, they offer ∼30 to 35% inhibition at 10 µM. Comprehensive docking and molecular dynamics simulations elucidate that these compounds exhibit excellent binding in the process, which however quickly deteriorates, and the ligand is separated from the binding site. This suggests that while the ligands initially bind with the protease, they are unable to maintain it for an extended period. However, the simulation showed that a simultaneous docked complex of both the compounds together with the protein boosts the stronger binding for a sufficient time. The enzyme assay exhibited 97 and 85% inhibition activity when both compounds were used together at 100 and 50 µM, respectively. Later, a multiconcentration assay was used to determine the coinhibitory activity, and it was observed that the compounds have ∼20 to 30% inhibition activity even at lower concentrations of 0.5 and 1 µM. Surface plasmon resonance was used to measure the binding of the compounds, and when used together, the compounds had a 10-fold greater binding affinity. Thus, the results demonstrate a synergistic mechanism between the two compounds that enhances the inhibition activity against SARS-CoV-2 3CL protease.

Health Perceptions and Adopted Lifestyle Behaviors During the COVID-19 Pandemic: Cross-National Survey.

BACKGROUND: Social isolation measures are requisites to control viral spread during the COVID-19 pandemic. However, if these measures are implemented for a long period of time, they can result in adverse modification of people's health perceptions and lifestyle behaviors. OBJECTIVE: The aim of this cross-national survey was to address the lack of adequate real-time data on the public response to changes in lifestyle behavior during the crisis of the COVID-19 pandemic. METHODS: A cross-national web-based survey was administered using Google Forms during the month of April 2020. The settings were China, Japan, Italy, and India. There were two primary outcomes: (1) response to the health scale, defined as perceived health status, a combined score of health-related survey items; and (2) adoption of healthy lifestyle choices, defined as the engagement of the respondent in any two of three healthy lifestyle choices (healthy eating habits, engagement in physical activity or exercise, and reduced substance use). Statistical associations were assessed with linear and logistic regression analyses. RESULTS: We received 3371 responses; 1342 were from India (39.8%), 983 from China (29.2%), 669 from Italy (19.8%), and 377 (11.2%) from Japan. A differential countrywise response was observed toward perceived health status; the highest scores were obtained for Indian respondents (9.43, SD 2.43), and the lowest were obtained for Japanese respondents (6.81, SD 3.44). Similarly, countrywise differences in the magnitude of the influence of perceptions on health status were observed; perception of interpersonal relationships was most pronounced in the comparatively old Italian and Japanese respondents (ß=.68 and .60, respectively), and the fear response was most pronounced in Chinese respondents (ß=.71). Overall, 78.4% of the respondents adopted at least two healthy lifestyle choices amid the COVID-19 pandemic. Unlike health status, the influence of perception of interpersonal relationships on the adoption of lifestyle choices was not unanimous, and it was absent in the Italian respondents (odds ratio 1.93, 95% CI 0.65-5.79). The influence of perceived health status was a significant predictor of lifestyle change across all the countries, most prominently by approximately 6-fold in China and Italy. CONCLUSIONS: The overall consistent positive influence of increased interpersonal relationships on health perceptions and adopted lifestyle behaviors during the pandemic is the key real-time finding of the survey. Favorable behavioral changes should be bolstered through regular virtual interpersonal interactions, particularly in countries with an overall middle-aged or older population. Further, controlling the fear response of the public through counseling could also help improve health perceptions and lifestyle behavior. However, the observed human behavior needs to be viewed within the purview of cultural disparities, self-perceptions, demographic variances, and the influence of countrywise phase variations of the pandemic. The observations derived from a short lockdown period are preliminary, and real insight could only be obtained from a longer follow-up.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).