Development of SARS-CoV-2 packaged RNA reference material for nucleic acid testing

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80–8.23% and is stable at 4 °C for 1 week and at −70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.

SARS-CoV-2 variants of concern are dependent on IFITM2 for efficient replication in human lung cells

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several "Variants of Concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant.

ISGylation in Innate Antiviral Immunity and Pathogen Defense Responses: A Review

The interferon-stimulating gene 15 (ISG15) protein is a ubiquitin-like protein induced by interferons or pathogens. ISG15 can exist in free form or covalently bind to the target protein through an enzymatic cascade reaction, which is called ISGylation. ISGylation has been found to play an important role in the innate immune responses induced by type I interferon, and is, thus, critical for the defense of host cells against RNA, DNA, and retroviruses. Through covalent binding with the host and viral target proteins, ISG15 inhibits the release of viral particles, hinder viral replication, and regulates the incubation period of viruses, thereby exerting strong antiviral effects. The SARS-CoV-2 papain-like protease, a virus-encoded deubiquitinating enzyme, has demonstrated activity on both ubiquitin and ISG15 chain conjugations, thus playing a suppressive role against the host antiviral innate immune response. Here we review the recent research progress in understanding ISG15-type ubiquitin-like modifications, with an emphasis on the underlying molecular mechanisms. We provide comprehensive references for further studies on the role of ISG15 in antiviral immunity, which may enable development of new antiviral drugs.

In Silico Drug Discovery of 4-Hyroxypanduratin A, 6-Gingerol and Luteolin Targeting Nipah Virus

Nipah Virus is a zoo tonic virus and has re-emerged again with more deadliness. NiV has infected many animals and humans worldwide and a huge loss to life has been faced. NiV contains a Fusion protein on its outer membrane which helps in the virus entry into the host cell. This fusion protein is a virulent factor and is a major anti-viral target. Many medicinal plants have been used against viral diseases, current research aims towards the potential of three daily dietary food elements that can be used as an anti-viral agent. In-silico studies are performed with 4-hyroxypanduratin A, 6-gingerol and Luteolin against the NiV-F and binding energies were calculated. It was reported that these phyto-compounds have good negative binding energies and they have the promising potential against Nipah Virus. Further in-vitro research can be performed with these phyto-compounds to design a specific drug against Nipah Virus.

Multicomponent reactions in the synthesis of antiviral compounds

Background: Multicomponent reactions are one-pot processes for the synthesis of highly functionalized hetero-cyclic and hetero-acyclic compounds, often endowed with biological activity. Objective: Multicomponent reactions are considered green processes with high atom economy. In addition, they present advantages compared to the classic synthetic methods such as high efficiency and low wastes production. Method: In these reactions two or more reagents are combined together in the same flask to yield a product containing almost all the atoms of the starting materials. Results: The scope of this review is to present an overview of the application of multicomponent reactions in the synthesis of compounds endowed with antiviral activity. The syntheses are classified depending on the viral target. Conclusion: Multicomponent reactions can be applied to all the stages of the drug discovery and development process making them very useful in the search for new agents active against emerging (viral) pathogens.

A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.

Cross Talk between Viruses and Insect Cells Cytoskeleton

Viruses are excellent manipulators of host cellular machinery, behavior, and life cycle, with the host cell cytoskeleton being a primordial viral target. Viruses infecting insects generally enter host cells through clathrin-mediated endocytosis or membrane fusion mechanisms followed by transport of the viral particles to the corresponding replication sites. After viral replication, the viral progeny egresses toward adjacent cells and reaches the different target tissues. Throughout all these steps, actin and tubulin re-arrangements are driven by viruses. The mechanisms used by viruses to manipulate the insect host cytoskeleton are well documented in the case of alphabaculoviruses infecting Lepidoptera hosts and plant viruses infecting Hemiptera vectors, but they are not well studied in case of other insect–virus systems such as arboviruses–mosquito vectors. Here, we summarize the available knowledge on how viruses manipulate the insect host cell cytoskeleton, and we emphasize the primordial role of cytoskeleton components in insect virus motility and the need to expand the study of this interaction.

BCR-ABL Tyrosine Kinase Inhibitors as Candidates for the Treatment of COVID-19: Molecular Docking, Pharmacophore Modeling, ADMET Studies

The novel coronavirus pandemic (COVID-19) caused by SARS-CoV-2 has affected more than 53 million individuals worldwide. Currently, there is a dire need to develop or find potential drugs that can treat SARS-CoV-2 infection. One of the standard methods to accelerate drug discovery and development in pandemics is to screen currently available medications against the critical therapeutic targets to find potential therapeutic agents. The literature has pointed out the 3CLpro and RdRp proteins as the most important proteins involved in viral replications. In the present study, we used an in-silico modeling approach to examine the affinity of six tyrosine kinases inhibitors (TKIs), Imatinib, Ponatinib, Nilotinib, Gefitinib, Erlotinib, and Dasatinibagainst the 3CLpro and RdRp by calculating the energy balance. The six tested TKIs had more than -7 Kcal/mol energy balance values for both viral target proteins. Nilotinib and Ponatinib showed the highest affinity for 3CLpro (-8.32, -8.16, respectively) while Dasatinib, Ponatinib, and Imatinib presented the strongest binding toRdRp(-14.50, -10.57, -9.46, respectively). Based on these findings, we recommend future evaluations of TKIs for SARs-CoV-2 infection in-vitro and further testing in clinical trials.

Host ADP-ribosylation and the SARS-CoV-2 macrodomain

The COVID-19 pandemic has prompted intense research efforts into elucidating mechanisms of coronavirus pathogenesis and to propose antiviral interventions. The interferon (IFN) response is the main antiviral component of human innate immunity and is actively suppressed by several non-structural SARS-CoV-2 proteins, allowing viral replication within human cells. Differences in IFN signalling efficiency and timing have emerged as central determinants of the variability of COVID-19 disease severity between patients, highlighting the need for an improved understanding of host–pathogen interactions that affect the IFN response. ADP-ribosylation is an underexplored post-translational modification catalyzed by ADP-ribosyl transferases collectively termed poly(ADP-ribose) polymerases (PARPs). Several human PARPs are induced by the IFN response and participate in antiviral defences by regulating IFN signalling itself, modulating host processes such as translation and protein trafficking, as well as directly modifying and inhibiting viral target proteins. SARS-CoV-2 and other viruses encode a macrodomain that hydrolyzes ADP-ribose modifications, thus counteracting antiviral PARP activity. This mini-review provides a brief overview of the known targets of IFN-induced ADP-ribosylation and the functions of viral macrodomains, highlighting several open questions in the field.