Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance.

The major arboviruses mainly belong to the Bunyaviridae, Togaviridae, and Flaviviridae families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R2 = 0.99, for DENV-2 and DENV-3; R2 = 0.98, for DENV-4; and CHIKV; R2 = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10-1 copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 101 for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.

Oxidative stress governs mosquito innate immune signalling to reduce chikungunya virus infection in Aedes-derived cells.

Arboviruses such as chikungunya, dengue and zika viruses cause debilitating diseases in humans. The principal vector species that transmits these viruses is the Aedes mosquito. Lack of substantial knowledge of the vector species hinders the advancement of strategies for controlling the spread of arboviruses. To supplement our information on mosquitoes' responses to virus infection, we utilized Aedes aegypti-derived Aag2 cells to study changes at the transcriptional level during infection with chikungunya virus (CHIKV). We observed that genes belonging to the redox pathway were significantly differentially regulated. Upon quantifying reactive oxygen species (ROS) in the cells during viral infection, we further discovered that ROS levels are considerably higher during the early hours of infection; however, as the infection progresses, an increase in antioxidant gene expression suppresses the oxidative stress in cells. Our study also suggests that ROS is a critical regulator of viral replication in cells and inhibits intracellular and extracellular viral replication by promoting the Rel2-mediated Imd immune signalling pathway. In conclusion, our study provides evidence for a regulatory role of oxidative stress in infected Aedes-derived cells.

Cytokine profile, neutralisation potential and viral replication dynamics in sera of chikungunya patients in India: a cross-sectional study.

Background: Chikungunya disease (CHIKD) is a threat to global health, as it impairs the quality of life of an infected individual ranging from months to years. A systematic evaluation of the serological, virological, and immunological aspects of the circulating viruses and their impact on the host response is imperative for better understanding of the evolving disease dynamics. Methods: Serum samples were collected from 196 acute CHIKD patients from ten tertiary care hospitals across India during 2016-2021. Out of 196 patients, paired convalescent samples were collected from 51 patients (one-month post-onset of symptoms). The serum samples were profiled for cytokines and neutralisation capacity. Further, chikungunya virus (CHIKV) was isolated from the acute sera and the replication kinetics of the clinical isolates was evaluated. Findings: Serological analysis indicated that neutralisation could be correlated to seroconversion in the convalescent phase but not found significant in acute phase. In the acute phase samples, there was a correlation between elevated serum levels of IFN-γ, IP-10, MCP-1 and MIG and disease severity. During convalescent phase, pro-inflammatory markers such as IL-6, IL-1ß, IL-9 and IP-10 were found to be elevated with a corresponding decline in the secretion of anti-inflammatory cytokines such as IL-4 and IL-10, which correlated with persistent arthralgia. Analysis of replication of the clinical isolates revealed that 68.4% of viruses were fast-growing in the Vero cells (cytopathic effect [CPE] observed within 24 h post-infection), and their corresponding acute serum samples showed an elevated secretion of IFN-α, IL-1RA, IL-17F, IL-9, MCP-1 and MIP-1α. Interpretation: This study provides an important overview of neutralisation capabilities and cytokine responses along with virus pathogenesis associated with CHIKV infections in India. Funding: Biotechnology Industry Research Assistance Council (BIRAC).

Transcriptome analysis of human macrophages upon chikungunya virus (CHIKV) infection reveals regulation of distinct signaling and metabolic pathways during the early and late stages of infection.

Macrophages are efficient reservoirs for viruses that enable the viruses to survive over a longer period of infection. Alphaviruses such as chikungunya virus (CHIKV) are known to persist in macrophages even after the acute febrile phase. The viral particles replicate in macrophages at a very low level over extended period of time and are localized in tissues that are often less accessible by treatment. Comprehensive experimental studies are thus needed to characterize the CHIKV-induced modulation of host genes in these myeloid lineage cells and in one such pursuit, we obtained global transcriptomes of a human macrophage cell line infected with CHIKV, over its early and late timepoints of infection. We analyzed the pathways, especially immune related, perturbed over these timepoints and observed several host factors to be differentially expressed in infected macrophages in a time-dependent manner. We postulate that these pathways may play crucial roles in the persistence of CHIKV in macrophages.

Chikungunya virus infection in Aedes aegypti is modulated by L-cysteine, taurine, hypotaurine and glutathione metabolism.

BACKGROUND: Blood meal and infections cause redox imbalance and oxidative damage in mosquitoes which triggers the mosquito's system to produce antioxidants in response to increased oxidative stress. Important pathways activated owing to redox imbalance include taurine, hypotaurine and glutathione metabolism. The present study was undertaken to evaluate the role of these pathways during chikungunya virus (CHIKV) infection in Aedes aegypti mosquitoes. METHODOLOGY: Using a dietary L-cysteine supplement system, we upregulated these pathways and evaluated oxidative damage and oxidative stress response upon CHIKV infection using protein carbonylation and GST assays. Further, using a dsRNA based approach, we silenced some of the genes involved in synthesis and transport of taurine and hypotaurine and then evaluated the impact of these genes on CHIKV infection and redox biology in the mosquitoes. CONCLUSIONS: We report that CHIKV infection exerts oxidative stress in the A. aegypti, leading to oxidative damage and as a response, an elevated GST activity was observed. It was also observed that dietary L-cysteine treatment restricted CHIKV infection in A. aegypti mosquitoes. This L-cysteine mediated CHIKV inhibition was coincided by enhanced GST activity that further resulted in reduced oxidative damage during the infection. We also report that silencing of genes involved in synthesis of taurine and hypotaurine modulates CHIKV infection and redox biology of Aedes mosquitoes during the infection.

Characterization of an Aedes ADP-Ribosylation Protein Domain and Role of Post-Translational Modification during Chikungunya Virus Infection.

Poly ADP-ribose polymerases (PARPs) catalyze ADP-ribosylation, a subclass of post-translational modification (PTM). Mono-ADP-ribose (MAR) moieties bind to target molecules such as proteins and nucleic acids, and are added as part of the process which also leads to formation of polymer chains of ADP-ribose. ADP-ribosylation is reversible; its removal is carried out by ribosyl hydrolases such as PARG (poly ADP-ribose glycohydrolase), TARG (terminal ADP-ribose protein glycohydrolase), macrodomain, etc. In this study, the catalytic domain of Aedes aegypti tankyrase was expressed in bacteria and purified. The tankyrase PARP catalytic domain was found to be enzymatically active, as demonstrated by an in vitro poly ADP-ribosylation (PARylation) experiment. Using in vitro ADP-ribosylation assay, we further demonstrate that the chikungunya virus (CHIKV) nsp3 (non-structural protein 3) macrodomain inhibits ADP-ribosylation in a time-dependent way. We have also demonstrated that transfection of the CHIKV nsP3 macrodomain increases the CHIKV viral titer in mosquito cells, suggesting that ADP-ribosylation may play a significant role in viral replication.

Mucin Protein of Aedes aegypti Interacts with Dengue Virus 2 and Influences Viral Infection.

Dengue, caused by dengue virus (DENV), is the most prevalent vector-borne viral disease, posing a serious health concern to 2.5 billion people worldwide. DENV is primarily transmitted among humans by its mosquito vector Aedes aegypti; hence, the identification of a novel dengue virus receptor in mosquitoes is critical for the development of new anti-mosquito measures. In the current study, we have identified peptides which potentially interact with the surface of the virion particles and facilitate virus infection and movement during their life cycle in the mosquito vector. To identify these candidate proteins, we performed phage-display library screening against domain III of the envelope protein (EDIII), which plays an essential role during host cell receptor binding for viral entry. The mucin protein, which shared sequence similarity with the peptide identified in the screening, was cloned, expressed, and purified for in vitro interaction studies. Using in vitro pulldown and virus overlay protein-binding assay (VOPBA), we confirmed the positive interaction of mucin with purified EDIII and whole virion particles. Finally, blocking of mucin protein with anti-mucin antibodies partially reduced DENV titers in infected mosquitos. Moreover, mucin protein was found to be localized in the midgut of Ae. aegypti. IMPORTANCE Identification of interacting protein partners of DENV in the insect vector Aedes aegypti is crucial for designing vector control-based strategies and for understanding the molecular mechanism DENV uses to modulate the host, gain entry, and survive successfully. Similar proteins can be used in generating transmission-blocking vaccines.

Phytochemical rich Himalayan Rhododendron arboreum petals inhibit SARS-CoV-2 infection in vitro.

Phytochemicals with potential to competitively bind to the host receptors or inhibit SARS-CoV-2 replication, may prove to be useful as adjunct therapeutics for COVID-19. We profiled and investigated the phytochemicals of Rhododendron arboreum petals sourced from Himalayan flora, undertook in vitro studies and found it as a promising candidate against SARS-CoV-2. The phytochemicals were reported in various scientific investigations to act against a range of virus in vitro and in vivo, which prompted us to test against SARS-CoV-2. In vitro assays of R. arboreum petals hot aqueous extract confirmed dose dependent reduction in SARS-CoV-2 viral load in infected Vero E6 cells (80% inhibition at 1 mg/ml; IC50 = 173 µg/ml) and phytochemicals profiled were subjected to molecular docking studies against SARS CoV-2 target proteins. The molecules 5-O-Feruloyl-quinic acid, 3-Caffeoyl-quinic acid, 5-O-Coumaroyl-D-quinic acid, Epicatechin and Catechin showed promising binding affinity with SARS-CoV-2 Main protease (MPro; PDB ID: 6LU7; responsible for viral replication) and Human Angiotensin Converting Enzyme-2 (ACE2; PDB ID: 1R4L; mediate viral entry in the host). Molecular dynamics (MD) simulation of 5-O-Feruloyl-quinic acid, an abundant molecule in the extract complexed with the target proteins showed stable interactions. Taken together, the phytochemical profiling, in silico analysis and in vitro anti-viral assay revealed that the petals extract act upon MPro and may be inhibiting SARS-CoV-2 replication. This is the first report highlighting R. arboreum petals as a reservoir of antiviral phytochemicals with potential anti-SARS-CoV-2 activity using an in vitro system.

Vathasura Kudineer, an Andrographis based polyherbal formulation exhibits immunomodulation and inhibits chikungunya virus (CHIKV) under invitro conditions.

ETHNOPHARMACOLOGICAL RELEVANCE: Chikungunya disease (CHIKD) is caused by the alphavirus, chikungunya virus (CHIKV) and is characterized by acute fever and joint inflammation; the inflammation continues even after clearance of the virus from the system, persisting for several months to years. Currently, there are no modern medicines/vaccines available for its treatment and use of over-the-counter anti-inflammatory generic medicines to relieve symptoms is generally practiced. In India, Indian traditional medicines hold a lot of promise to treat this infection and are routinely used during outbreaks. AIM OF THE STUDY: In the present study, we characterized the phytochemical and physicochemical properties of aqueous and ethanol extracts of the Vathasura Kudineer (VSK), a Andrographis based Siddha polyherbal formulation. Additionally, we evaluated its immunomodulatory and antiviral potential using an in vitro system. MATERIALS AND METHODS: Aqueous and ethanolic extracts of VSK were prepared and their physico and phytochemical properties were obtained by biochemical and biophysical assays, HPTLC and FTIR. The aqueous extracts of VSK and several of its ingredients were evaluated for their cytotoxicity in Vero cells and using the maximum non-toxic concentration (MNTC), were processed further for evaluating their ability to inhibit CHIKV infection in Vero cells. We performed the co-treatment assay with ethanol extract of VSK and several of its ingredients to assess the antiviral activity against chikungunya virus on Vero cells and through pre-treatment assay (anti-adhesive effect), co-incubation assay (virucidal effect) and post-treatment assay (post-entry effect) were evaluated. Further, we tested the aqueous extract of VSK along with some of its ingredients for their immunomodulatory properties. We performed antioxidant and anti-inflammatory assays using LPS-simulated RAW 264.7 cells. For antioxidant capacity of extracts, we performed extra-cellular ABTS radical scavenging activity and intra-cellular effects on ROS generation and SOD activity. We assessed the effect on most important inflammatory mediators like Nitric oxide (NO) and Prostaglandin E2 (PGE2) and pro-inflammatory cytokines like interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα). RESULTS: We provided the fingerprint of the phytochemicals of both ethanol and aqueous extracts of VSK that can be used for identification. We observed that ethanol extract was able to inhibit CHIKV infection at MNTC with 48 h of treatment on Vero cells. Its ingredient VSKI-As (Anethum sowa) found to be most effective to show virucidal effect while VSKI-Cs (Clerodendrum serratum) and VSKI-Pn (Pipper nigrum) found to be effective in post-entry effect. VSK was able to show ABTS radical scavenging activity, reduce ROS generation, inhibit the inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (IL-1ß and TNFα) production in LPS-stimulated RAW 264.7 cells. CONCLUSIONS: We provided the evidence that VSK has both immunomodulatory as well as antiviral potential. It shows virucidal as well as post-entry effects on chikungunya virus. VSK can inhibit pro-inflammatory cytokines, IL-1ß and TNFα production by suppressing the inflammatory mediators, NO and PGE2.

SARS-CoV-2 variants: Impact on biological and clinical outcome.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that was first identified in December 2019, in Wuhan, China was found to be the etiological agent for a novel respiratory infection that led to a Coronavirus Induced Disease named COVID-19. The disease spread to pandemic magnitudes within a few weeks and since then we have been dealing with several waves across the world, due to the emergence of variants and novel mutations in this RNA virus. A direct outcome of these variants apart from the spike of cases is the diverse disease presentation and difficulty in employing effective diagnostic tools apart from confusing disease outcomes. Transmissibility rates of the variants, host response, and virus evolution are some of the features found to impact COVID-19 disease management. In this review, we will discuss the emerging variants of SARS-CoV-2, notable mutations in the viral genome, the possible impact of these mutations on detection, disease presentation, and management as well as the recent findings in the mechanisms that underlie virus-host interaction. Our aim is to invigorate a scientific debate on how pathogenic potential of the new pandemic viral strains contributes toward development in the field of virology in general and COVID-19 disease in particular.

Impact of CHIKV Replication on the Global Proteome of Aedes albopictus Cells.

Arboviruses are some of the important causative agents of mosquito-mediated viral diseases. These viruses are transmitted between vector and host during the blood meal. Upon viral entry, host replication machinery is hijacked, supporting new virus particle production and thereby allowing viral survival in the host. In this process, host proteins interact with viral proteins to either facilitate viral replication, or they may provide antiviral defense mechanisms. In this study, we analyzed the impact of chikungunya virus (CHIKV) infection on the global proteome of Dicer active Aedes albopictus cells during the early and late time points of infection. We utilized a bottom-up approach of global proteomics analysis, and we used label-free quantitative mass spectrometry to identify the global protein signatures of Ae. albopictus at two different time points upon CHIKV infection. The mass spectrometry data analysis of the early time point revealed that proteins belonging to pathways such as translation, RNA processing, and cellular metabolic processes were less in abundance, whereas those belonging to pathways such as cellular catabolic process and organic substance transport were significantly abundant. At later time points, proteins belonging to pathways such as cellular metabolic processes, primary metabolic process, organonitrogen compound metabolic process, and organic substance metabolic process were found to be decreased in their presence, whereas those belonging to pathways such as RNA processing, gene expression, macromolecule metabolic processing, and nitrogen compound metabolic processing were found to be abundant during CHIKV infection, indicating that modulation in gene expression favoring cell survival occurs at a later time point, suggesting a survival strategy of Aedes cells to counter prolonged CHIKV infection.

Assessment of dengue virus inactivation in random donor platelets using amotosalen and ultraviolet A illumination.

OBJECTIVES: The study objective was evaluation of amotosalen and ultraviolet A (UVA) illumination-based inactivation of dengue virus (DENV) in blood platelets. MATERIALS AND METHODS: Whole blood was collected from healthy donors and platelet concentrates were prepared at a tertiary care hospital in Gurugram, India. Platelet units collected from five blood group matched individuals were pooled and spiked with DENV. The spiked platelet units were subjected to amotosalen treatment followed by UVA illumination, to evaluate the efficiency of this method for viral inactivation. The treated platelet units were evaluated for the presence of infectious DENV. Amotosalen levels were quantified in the treated samples using high-performance liquid chromatography. RESULTS: The presence of replicating DENV was not observed in spiked platelet units treated with amotosalen and UVA illumination, whereas untreated units contained actively replicating DENV. Amotosalen levels were found to be in the permissible range after photochemical inactivation. CONCLUSIONS: Amotosalen/UVA pathogen inactivation treatment showed efficient inactivation of DENV in platelet components. Therefore, it seems to be a promising method for mitigating the risk of dengue transmission through transfusion of potentially contaminated platelet components in dengue-endemic countries such as India.

Short communication: Virological and B cell profiles of chikungunya and Dengue virus co-infections in Delhi during 2017-2019.

With explosive epidemics of chikungunya in India since 2004, chikungunya virus (CHIKV) now co-circulates in geographical areas where Dengue virus (DENV) is already endemic and thus provides opportunity for the same mosquito to be infected with both viruses. Although there are excellent studies that have addressed the clinical of mono and co-infection, we have little to no knowledge on the current viral sequences that pre-dominate co-infections, and the B cell response elicited. In this study, we analyzed febrile patients that were confirmed to have DENV-CHIKV co-infections and asked the following questions: 1) what is the frequency of co-infections found in a single cycle of transmission; 2) what are the viral sequences associated with them; 3) what does the antibody secreting cell / plasmablast response look like in patients that are co-infected with both viruses. We report those co-infections occur at a frequency of 6.7% in the transmission cycle, and while DENV-3 is now frequently detected, we do not see a serotype bias in the patients that are co-infected with ESCA strain of CHIKV. Moreover, the effector B cell response (plasmablasts) observed are specific to both infecting viruses indicating no overt bias. Further studies to associate whether any of these properties have a bearing on clinical disease manifestation will be both timely and important.

In silico identification and in vitro antiviral validation of potential inhibitors against Chikungunya virus.

The Chikungunya virus (CHIKV) has become endemic in the Africa, Asia and Indian subcontinent, with its continuous re-emergence causing a significant public health crisis. The unavailability of specific antivirals and vaccines against the virus has highlighted an urgent need for novel therapeutics. In the present study, we have identified small molecule inhibitors targeting the envelope proteins of the CHIKV to interfere with the fusion process, eventually inhibiting the cell entry of the virus particles. We employed high throughput computational screening of large datasets against two different binding sites in the E1-E2 dimer to identify potential candidate inhibitors. Among them, four high affinity inhibitors were selected to confirm their anti-CHIKV activity in the in vitro assay. Quercetin derivatives, Taxifolin and Rutin, binds to the E1-E2 dimer at different sites and display inhibition of CHIKV infection with EC50 values 3.6 µM and 87.67 µM, respectively. Another potential inhibitor with ID ChemDiv 8015-3006 binds at both the target sites and shows anti-CHIKV activity at EC50 = 41 µM. The results show dose-dependent inhibitory effects of Taxifolin, Rutin and ChemDiv 8015-3006 against the CHIKV with minimal cytotoxicity. In addition, molecular dynamics studies revealed the structural stability of these inhibitors at their respective binding sites in the E1-E2 protein. In conclusion, our study reports Taxifolin, Rutin and ChemDiv 8015-3006 as potential inhibitors of the CHIKV entry. Also, this study suggests a few potential candidate inhibitors which could serve as a template to design envelope protein specific CHIKV entry inhibitors.

An E3 Ubiquitin Ligase Scaffolding Protein Is Proviral during Chikungunya Virus Infection in Aedes aegypti.

Chikungunya virus (CHIKV) is a reemerging alphavirus causing chikungunya disease (CHIKD) and is transmitted to humans by Aedes mosquitoes. The virus establishes an intricate balance of cellular interactions that ultimately helps in its replication and dodges cellular immune response. In an attempt to identify cellular host factors required during CHIKV replication in Aag2 cells, we performed global transcriptomics of CHIKV-infected Aag2 cells, and further, we compared this library with the Drosophila RNAi Screening Center (DRSC) database and identified transcripts that were regulated in Aedes aegypti during CHIKV infection. These analyses revealed specific pathways, such as ubiquitin-related pathways, proteolysis pathways, protein catabolic processes, protein modification, and cellular protein metabolic processes, involved during replication of the virus. Loss-of-function assays of selected candidates revealed their proviral or antiviral characteristics upon CHIKV infection in A. aegypti-derived Aag2 cells. Further validations identified that the ubiquitin proteasomal pathway is required for CHIKV infection in A. aegypti and that an important member of this family of proteins, namely, AeCullin-3 (Aedes ortholog of human cullin-3), is a proviral host factor of CHIKV replication in Aag2 cells. IMPORTANCE Arboviruses cause several diseases in humans and livestock. Vector control is the main strategy for controlling diseases transmitted by mosquitoes. In this context, it becomes paramount to understand how the viruses replicate in the vector for designing better transmission blocking strategies. We obtained the global transcriptome signature of A. aegypti cells during CHIKV infection, and in order to obtain the maximum information from these data sets, we further utilized the well-characterized Drosophila system and arrived upon a set of transcripts and their pathways that affect A. aegypti cells during CHIKV infection. These analyses and further validations reveal that important pathways related to protein degradation are actively involved during CHIKV infection in A. aegypti and are mainly proviral. Targeting these molecules may provide novel approaches for blocking CHIKV replication in A. aegypti.

Anti-inflammatory and anti-COVID-19 effect of a novel polyherbal formulation (Imusil) via modulating oxidative stress, inflammatory mediators and cytokine storm.

In the current scenario, most countries are affected by COVID-19, a pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has a massive impact on human health. Previous studies showed that some traditionally used medicinal herbs and their combinations showed synergistic anti-viral and anti-inflammatory activity against SARS-CoV-2 type infections. Therefore, the goal of this study is to demonstrate the anti-viral and anti-inflammatory effects of a novel polyherbal formulation, hereinafter referred to as Imusil, on Vero E6 cell lines and Raw 264.7 murine macrophage cells respectively. The Imusil was subjected to identify its chemical characterisations such as UV-Visible spectrum profile, Fourier transform infrared spectroscopy (FT-IR) and gas chromatography-mass spectroscopic (GC-MS) analysis. FT-IR analysis of Imusil peak values with various functional compounds such as alcohol, esters, aliphatic and carboxylic acids. GC-MS analysis of compounds with totally 87 compounds major chemical compounds were identified, such as 3-(Octanoyloxy) propane-1,2-diyl bis(decanoate), Succinic acid, 2-methylhex-3-yl 2,2,2-trifluoroethyl ester, Neophytadiene, 3,5,9-Trioxa-4-phosphaheneicosan-1-aminium, 4-hydroxy-N,N,N-trimethyl-10-oxo-7-[(1-oxododecyl)oxy]-, hydroxide, inner salt, 4-oxide, (R)-. The anti-viral activity of Imusil against SARS-CoV-2 was assessed using plaque reduction assay and anti-inflammatory study was conducted on lipopolysaccharide (LPS)-induced RAW 264.7 cells. The results obtained from the study reveal that Imusil significantly inhibited SARS-CoV-2 replication in Vero E6 cells and the production of inflammatory mediator's cyclooxygenase-2 and pro-inflammatory cytokines like tumour necrosis factor-α and interleukin- 6 were significantly reduced, along with thwarting the significant oxidative stress by preventing the expression of NOX-2 thereby inhibiting the reactive oxygen species formation. Hence, considering the current study as a novel strategy for mediating the COVID-19 associated aliments, inceptive scientific evidence of Imusil promises its potential therapeutic implications against COVID-19 and inflammatory conditions.

Chikungunya virus non-structural protein nsP3 interacts with Aedes aegypti DEAD-box helicase RM62F.

The non-structural proteins (nsPs) of the chikungunya virus (CHIKV) form the virus's replication complex. They are known to participate in several functions that allow efficient replication of the virus in diverse host systems. One such function is evading the host defense system such as RNA interference (RNAi). Two nsPs of CHIKV, namely, nsP2 and nsP3, were found to suppress the host/vector RNAi machinery and exhibit RNAi suppressor activity. The present study was undertaken to identify interacting partners of CHIKV-nsP3 in Aedes aegypti. We performed pull-down assays with the mass spectrometry approach and showed the interaction of CHIKV-nsP3 with several Aedes proteins. Further co-immunoprecipitation assays revealed that CHIKV-nsP3 interacts with RM62F, a DEAD-box containing RNA known to play roles in multiple gene regulatory processes such as alternative splicing, RNA release, and also is a component of Ago2-RISC complex. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00734-y.

Chikungunya virus molecular evolution in India since its re-emergence in 2005.

Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is among the most medically significant mosquito-borne viruses, capable of causing major epidemics of febrile disease and severe, chronic arthritis. Identifying viral mutations is crucial for understanding virus evolution and evaluating those genetic determinants that directly impact pathogenesis and transmissibility. The present study was undertaken to expand on past CHIKV evolutionary studies through robust genome-scale phylogenetic analysis to better understand CHIKV genetic diversity and evolutionary dynamics since its reintroduction into India in 2005. We sequenced the complete genomes of fifty clinical isolates collected between 2010 and 2016 from two geographic locations, Delhi and Mumbai. We then analysed them along with 753 genomes available on the Virus Pathogen Database and Analysis Resource sampled over fifteen years (2005-20) from a range of locations across the globe and identified novel genetic variants present in samples from this study. Our analyses show evidence of frequent reintroduction of the virus into India and that the most recent CHIKV outbreak shares a common ancestor as recently as 2006.

Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro.

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.

Clinical, Serological, Whole Genome Sequence Analyses to Confirm SARS-CoV-2 Reinfection in Patients From Mumbai, India.

Background: SARS-CoV-2 infection may not provide long lasting post-infection immunity. While hundreds of reinfections have reported only a few have been confirmed. Whole genome sequencing (WGS) of the viral isolates from the different episodes is mandatory to establish reinfection. Methods: Nasopharyngeal (NP), oropharyngeal (OP) and whole blood (WB) samples were collected from paired samples of four individuals who were suspected of SARS-CoV-2 reinfection based on distinct clinical episodes and RT-PCR tests. Details from their case record files and investigations were documented. RNA was extracted from the NP and OP samples and subjected to WGS, and the nucleotide and amino acid sequences were subjected to genome and protein-based functional annotation analyses. Serial serology was performed for Anti-N IgG, Anti- S1 RBD IgG, and sVNT (surrogate virus neutralizing test). Findings: Three patients were more symptomatic with lower Ct values and longer duration of illness. Seroconversion was detected soon after the second episode in three patients. WGS generated a genome coverage ranging from 80.07 to 99.7%. Phylogenetic analysis revealed sequences belonged to G, GR and "Other" clades. A total of 42mutations were identified in all the samples, consisting of 22 non-synonymous, 17 synonymous, two in upstream, and one in downstream regions of the SARS-CoV-2 genome. Comparative genomic and protein-based annotation analyses revealed differences in the presence and absence of specific mutations in the virus sequences from the two episodes in all four paired samples. Interpretation: Based on the criteria of genome variations identified by whole genome sequencing and supported by clinical presentation, molecular and serological tests, we were able to confirm reinfections in two patients, provide weak evidence of reinfection in the third patient and unable to rule out a prolonged infection in the fourth. This study emphasizes the importance of detailed analyses of clinical and serological information as well as the virus's genomic variations while assessing cases of SARS-CoV-2 reinfection.