A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein and viral entry.

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is the read out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.

Plasmonic Micro-Channel Assisted Photonic Crystal Fiber Based Highly Sensitive Sensor for Multi-Analyte Detection.

A dual-channel propagation controlled photonic crystal fiber (PCF)-based plasmonic sensor was presented to detect multiple analytes simultaneously. Plasmonic micro-channels were placed on the outer surface of the PCF, which facilitates an easy sensing mechanism. The sensor was numerically investigated by the finite element method (FEM) with the perfectly matched layer (PML) boundary conditions. The proposed sensor performances were analyzed based on optimized sensor parameters, such as confinement loss, resonance coupling, resolution, sensitivity, and figure of merit (FOM). The proposed sensor showed a maximum wavelength sensitivity (WS) of 25,000 nm/refractive index unit (RIU) with a maximum sensor resolution (SR) of 4.0 × 10-6 RIU for channel 2 (Ch-2), and WS of 3000 nm/RIU with SR of 3.33 × 10-5 RIU for channel 1 (Ch-1). To the best of our knowledge, the proposed sensor exhibits the highest WS compared with the previously reported multi-analyte based PCF surface plasmon resonance (SPR) sensors. The proposed sensor could detect the unknown analytes within the refractive index (RI) range of 1.32 to 1.39 in the visible to near infrared region (550 to 1300 nm). In addition, the proposed sensor offers the maximum Figure of Merit (FOM) of 150 and 500 RIU-1 with the limit of detection (LOD) of 1.11 × 10-8 RIU2/nm and 1.6 × 10-10 RIU2/nm for Ch-1 and Ch-2, respectively. Due to its highly sensitive nature, the proposed multi-analyte PCF SPR sensor could be a prominent candidate in the field of biosensing to detect biomolecule interactions and chemical sensing.

Repurposing nonnucleoside antivirals against SARS-CoV2 NSP12 (RNA dependent RNA polymerase): In silico-molecular insight.

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.

Polymonoclonal (Not Polyclonal) Antibodies Derived from Convalescent Human B Cell Hybridomas Might Be a Better Therapeutic Option than Single Target Monoclonal Antibodies.

Both human B cell hybridoma technology and convalescent plasma therapy are promising immunological tools for therapeutic interventions. Here we propose using antibody producing B cells from convalescent SARS-CoV2 patients for developing human B cell hybridomas, and a combination of monoclonal antibodies against multiple immunogenic targets of SARS-CoV-2 spike protein might deliver an antibody cocktail for long-lasting therapeutic targeting.

Refining Network Lifetime of Wireless Sensor Network Using Energy-Efficient Clustering and DRL-Based Sleep Scheduling.

Over the recent era, Wireless Sensor Network (WSN) has attracted much attention among industrialists and researchers owing to its contribution to numerous applications including military, environmental monitoring and so on. However, reducing the network delay and improving the network lifetime are always big issues in the domain of WSN. To resolve these downsides, we propose an Energy-Efficient Scheduling using the Deep Reinforcement Learning (DRL) (E2S-DRL) algorithm in WSN. E2S-DRL contributes three phases to prolong network lifetime and to reduce network delay that is: the clustering phase, duty-cycling phase and routing phase. E2S-DRL starts with the clustering phase where we reduce the energy consumption incurred during data aggregation. It is achieved through the Zone-based Clustering (ZbC) scheme. In the ZbC scheme, hybrid Particle Swarm Optimization (PSO) and Affinity Propagation (AP) algorithms are utilized. Duty cycling is adopted in the second phase by executing the DRL algorithm, from which, E2S-DRL reduces the energy consumption of individual sensor nodes effectually. The transmission delay is mitigated in the third (routing) phase using Ant Colony Optimization (ACO) and the Firefly Algorithm (FFA). Our work is modeled in Network Simulator 3.26 (NS3). The results are valuable in provisions of upcoming metrics including network lifetime, energy consumption, throughput and delay. From this evaluation, it is proved that our E2S-DRL reduces energy consumption, reduces delays by up to 40% and enhances throughput and network lifetime up to 35% compared to the existing cTDMA, DRA, LDC and iABC methods.

Analyses of spike protein from first deposited sequences of SARS-CoV2 from West Bengal, India.

India has recently started sequencing SARS-CoV2 genome from clinical isolates. Currently only few sequences are available from three states in India. Kerala was the first state to deposit complete sequence from two isolates followed by one from Gujarat. On April 27, 2020, the first five sequences from the state of West Bengal (Eastern India) were deposited on GISAID, a global initiative for sharing avian flu data. In this study, we have analysed the spike protein sequences from all five isolates and also compared their similarities or differences with other sequences reported in India and with isolates of Wuhan origin. We report one unique mutation at position 723 and another at 1124 in the S2 domain of spike protein of the isolates from West Bengal only.  There was one mutation downstream of the receptor binding domain at position 614 in S1 domain which was common with the sequence from Gujarat (a state of western India).  Mutation in the S2 domain showed changes in the secondary structure of the spike protein at region of the mutation. We also studied molecular dynamics using normal mode analyses and found that this mutation decreases the flexibility of S2 domain.  Since both S1 and S2 are important in receptor binding followed by entry in the host cells, such mutations may define the affinity or avidity of receptor binding.

Insight into the Tropism of Dengue Virus in Humans.

In tropical and subtropical zones, arboviruses are among the major threats to human life, affecting a large number of populations with serious diseases. Worldwide, over three hundred million people are infected with dengue virus (DENV) every year as per the World Health Organization (WHO). DENV-mediated disease severity ranges from a mild fever to hemorrhagic fever and shock syndrome. Patients suffering from severe infection might experience multi-organ failure, cardiomyopathy and even encephalopathy, further complicating the disease pathogenesis. In life-threatening cases, DENV has been reported to affect almost all organs of the human body. In this review, we discuss the organ tropism of DENV in humans in depth as detected in various autopsy studies. Keeping in mind the fact that there is currently no DENV-specific antiviral, it is of utmost importance to achieve a vivid picture of the susceptible cells in humans which might help in designing antivirals against DENV, especially targeting those tissues in which infection might lead to life-threatening conditions.

Hijacking the Host Immune Cells by Dengue Virus: Molecular Interplay of Receptors and Dengue Virus Envelope.

Dengue virus (DENV) is one of the lethal pathogens in the hot climatic regions of the world and has been extensively studied to decipher its mechanism of pathogenesis and the missing links of its life cycle. With respect to the entry of DENV, multiple receptors have been recognized in different cells of the human body. However, scientists still argue whether these identified receptors are the exclusive entry mediators for the virus. Adding to the complexity, DENV has been reported to be infecting multiple organ types in its human host. Also, more than one receptor in a particular cell has been discerned to take part in mediating the ingress of DENV. In this review, we aim to discuss the different cells of the human immune system that support DENV infection and their corresponding receptors that DENV deploy to gain access to the cells.

The Mosquito Immune System and the Life of Dengue Virus: What We Know and Do Not Know.

Flaviviruses are largely transmitted to humans by their arthropod vectors such as mosquitoes or ticks. The dengue virus (DENV) is one of the members of the family Flaviviridae and is the causative agent of dengue fever. In the mosquito vector, DENV enters through viremic blood meal and replicates in the mid-gut. Newly formed virion particles circulate to various mosquito organs and get transmitted to the next host in subsequent bites. Aedes aegypti and Aedes albopictus have intricate immune control to allow DENV production at a sub-pathogenic level. In the mosquito, antimicrobial peptides (AMP) and RNA inference (RNAi) are the two main antiviral strategies used against DENV. Apart from innate immunity, mosquito resident microbes play a significant role in modulating DENV replication. In this review, we discuss different immune mechanisms and preventive strategies that act against DENV in two of its vectors: Aedes aegypti and Aedes albopictus.