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.

Neuroinvasion of SARS-CoV-2 may play a role in the breakdown of the respiratory center of the brain.

The recent outbreak of the novel coronavirus, SARS-CoV-2, has emerged to be highly pathogenic in nature. Although lungs are considered as the primary infected organs by SARS-CoV-2, some of the other organs, including the brain, have also been found to be affected. Here, we have discussed how SARS-CoV-2 might infect the brain. The infection of the respiratory center in the brainstem could be hypothesized to be responsible for the respiratory failure in many COVID-19 patients. The virus might gain entry through the olfactory bulb and invade various parts of the brain, including the brainstem. Alternatively, the entry might also occur from peripheral circulation into the central nervous system by compromising the blood-brain barrier. Finally, yet another possible entry route could be its dispersal from the lungs into the vagus nerve via the pulmonary stretch receptors, eventually reaching the brainstem. Therefore, screening neurological symptoms in COVID-19 patients, especially toward the breakdown of the respiratory center in the brainstem, might help us better understand this disease.

Agricultural labor, COVID-19, and potential implications for food security and air quality in the breadbasket of India.

To contain the COVID-19 pandemic, India imposed a national lockdown at the end of March 2020, a decision that resulted in a massive reverse migration as many workers across economic sectors returned to their home regions. Migrants provide the foundations of the agricultural workforce in the 'breadbasket' states of Punjab and Haryana in Northwest India.There are mounting concerns that near and potentially longer-term reductions in labor availability may jeopardize agricultural production and consequently national food security. The timing of rice transplanting at the beginning of the summer monsoon season has a cascading influence on productivity of the entire rice-wheat cropping system. To assess the potential for COVID-related reductions in the agriculture workforce to disrupt production of the dominant rice-wheat cropping pattern in these states, we use a spatial ex ante modelling framework to evaluate four scenarios representing a range of plausible labor constraints on the timing of rice transplanting. Averaged over both states, results suggest that rice productivity losses under all delay scenarios would be low as compare to those for wheat, with total system productivity loss estimates ranging from 9%, to 21%, equivalent to economic losses of USD $674 m to $1.48 billion. Late rice transplanting and harvesting can also aggravate winter air pollution with concomitant health risks. Technological options such as direct seeded rice, staggered nursery transplanting, and crop diversification away from rice can help address these challenges but require new approaches to policy and incentives for change.