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The relationship of SARS-CoV-2 with the host translation remains largely unexplored. Using polysome profiling of SARS-CoV-2 infected Caco2 cells, we here demonstrate that the virus induces a strong suppression of global translation by 48 hours of infection. Heavy polysome fractions displayed substantial depletion in the infected cells, indicating the loss of major translational activities in them. Further assessment of the major pathways regulating translation in multiple permissive cell lines revealed strong eIF4E dephosphorylation accompanied by Mnk1 depletion and ERK1/2 dephosphorylations. p38MAPK showed consistent activation and its inhibition lowered viral titers, indicating its importance in viral survival. mTORC1 pathway showed the most profound inhibition, indicating its potential contribution to the suppression of global translation associated with the infection. Pharmacological activation of mTORC1 caused a drop in viral titers while inhibition resulted in higher viral RNA levels, confirming a critical role of mTORC1 in regulating viral replication. Surprisingly, the infection did not cause a general suppression of 5’-TOP translation, as evident from the continued expression of ribosomal proteins. Our results collectively indicate that the differential suppression of mTORC1 might allow SARS-CoV-2 to hijack translational machinery in its favor and specifically target a set of host mRNAs.

SUMMARYSeveral variants of SARS-CoV-2 have been emerging across the globe, continuing to threaten the efforts to end COVID-19 pandemic. Recent data indicate the prevalence of variants with N440K Spike substitution in several parts of India, which is under the second wave of the pandemic. Here, we first analyze the prevalence of N440K variants within the sequences submitted from India and identify a rising trend of its spread across various clusters. We then compare the replicative fitness and infectivity of a prototype of this variant with two other previously prevalent strains. The N440K variant produced ten times higher infectious viral titers than a prevalent A2a strain, and over 1000 folds higher titers than a much less prevalent A3i strain prototype in Caco2 cells. Similar results were detected in Calu-3 cells as well, confirming the increased potency of the N440K variant. Interestingly, A3i strain showed the highest viral RNA levels, but the lowest infectious titers in the culture supernatants, indicating the absence of correlation between the RNA content and the infectivity of the sample. N440K mutation has been reported in several viral sequences across India and based on our results, we predict that the higher infectious titers achieved by N440K variant could possibly lead to its higher rate of transmission. Availability of more sequencing data in the immediate future would help understand the potential spread of this variant in more detail.

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. β-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly causing undesirable outcomes including a potential immune escape by infectious virions, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.

The ongoing pandemic, COVID-19, caused by SARS-CoV-2 has taken the world, and especially the scientific community by storm. While vaccines are being introduced into the market, there is also a pressing need to find potential drugs and therapeutic modules. Remdesivir is one of the antivirals currently being used with a limited window of action. As more drugs are being vetted, passive immunotherapy in the form of neutralizing antibodies can provide immediate action to combat the increasing numbers of COVID-positive cases. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 generate high titers of antibody with a strong virus neutralizing potential. ELISA performed with pooled antisera displayed highest immunoglobulin titer on 42 days post-immunization, at 1:51,200 dilutions. F(ab’)2 immunoglobulin fragments generated from the pools also showed very high, antigen-specific affinity at 1:102,400 dilutions. Finally, in vitro virus neutralization assays confirmed that different pools of F(ab’)2 fragments could successfully neutralize SARS-CoV-2 with titers well above 25,000, indicating the potential of this strategy in treating severe COVID-19 cases with high titers. The F(ab’)2 was able to cross neutralize another SARS-CoV-2 strain, demonstrating its efficacy against the emerging viral variants and the importance of this approach in our efforts of eradication of COVID-19. In conclusion, this study demonstrates that virus-neutralizing antibodies raised in equines can potentially be used as a treatment regimen in the form of effective passive immunotherapy to combat COVID-19.