Since 2002, β coronaviruses (CoVs) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the recent outbreak of SARS-CoV-2 late in 2019 (also named as COVID-19 or novel coronavirus 2019 or nCoV2019). Spike (S) protein, one of the structural proteins of this virus plays key role in receptor (ACE2) binding and thus virus entry. Thus, this protein has attracted scientists for detailed study and therapeutic targeting. As the nCoV2019 takes its course throughout the world, more and more sequence analyses are being done and genome sequences are being deposited in various databases. From India, two clinical isolates have been sequenced and the full genome has been deposited in GenBank. We have performed sequence analyses of the Spike protein of the Indian isolates and compared with that of the Wuhan, China (where the outbreak was first reported). While all the sequences of Wuhan isolates are identical, we found point mutations in the Indian isolates. Out of the two isolates, one was found to harbor a mutation in its receptor-binding domain (RBD) at position 407. At this site, arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic amino acid that is also a C-β branched amino acid). This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor binding of the virus. Although this finding needs further validation and more sequencing, the information might be useful in rational drug designing and vaccine engineering.

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.

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.

1. The open reading frame (ORF) 1ab of SARS-CoV2 encodes non-structural proteins involved in viral RNA functions like translation and replication including nsp1-4; 3C like proteinase; nsp6-10; RNA dependent RNA polymerase (RdRp); helicase and 3’-5’ exonuclease. Sequence analyses of ORF1ab unravelled emergence of mutations especially in the viral RdRp and helicase at specific positions, both of which are important in mediating viral RNA replication. Since proteins are dynamic in nature and their functions are governed by the molecular motions, we performed normal mode analyses of the SARS-CoV2 wild type and mutant RdRp and helicases to understand the effect of mutations on their structure, conformation, dynamics and thus function. Structural analyses revealed that mutation of RdRp (at position 4715 in the context of the polyprotein/ at position 323 of RdRp) leads to rigidification of structure and that mutation in the helicase (at position 5828 of polyprotein/ position 504) leads to destabilization increasing the flexibility of the protein structure. Such structural modifications and protein dynamics alterations might alter unwinding of complex RNA stem loop structures, the affinity/ avidity of polymerase RNA interactions and in turn the viral RNA replication. The mutation analyses of proteins of the SARS-CoV2 RNA replication complex would help targeting RdRp better for therapeutic intervention.

Dengue virus NS1 protein is a major pathogenic protein. In this study, we examined the role of NS1 in coagulopathy associated with Dengue infection, a common feature of Dengue virus pathogenesis. Since most coagulation factors are produced by hepatocytes and liver is key organ affected during infection, we conducted transcriptomics using total-RNA extracted from Huh7 cells overexpressing NS1 protein. Coagulation factors 1, 5, 10, and 13 were downregulated and was confirmed using quantitative real-time polymerase chain reaction (RT-PCR) and western blot assays in both adherent and non-adherent cell culture systems across all four serotypes of Dengue. We also determined that downregulation of coagulation factors is a result of reduced expression of transcription activator HNF4α. Furthermore, we demonstrated that phosphorylation of extracellular signal-regulated kinase (ERK) leads to HNF4α downregulation and subsequent downregulation of coagulation factors. The downregulation of HNF4α and the downregulation of subsequent coagulation factors were validated in BALB/c mice by hydrodynamic tail vein injection of NS1 expression plasmids. Western blot assays using plasma from Dengue patients indicated that at least two coagulation factors of the common pathway of coagulation cascade are downregulated during the febrile phase, with levels improving toward the convalescent phase. NS1-mediated downregulation of coagulation factors was observed for both intracellular and secreted NS1. The hypothesis was also validated using virus infection assays. Overall, our study highlights the role of NS1 in mediating coagulopathy by modulating the expression of coagulation factors through transcriptional suppression of HNF4α by elevated phosphorylated ERK. This signaling cascade could be targeted for therapeutic intervention against virus-related coagulopathies. IMPORTANCE: Thrombocytopenia has been linked to coagulopathy of Dengue infection, and Dengue patients with coagulopathies are often administered platelet transfusion. For coagulopathies without thrombocytopenia, platelet transfusion might not help. We demonstrated the role of NS1 in coagulopathy by downregulating coagulation factors themselves. When thrombocytopenia does not exist or when thrombocytopenia as well as reduced levels of coagulation factors are the causative factors for coagulopathies, only platelet transfusion might not be effective. Alternative strategies, like administration of coagulation factor cocktails or platelet transfusion along with coagulation factor cocktail, might be promising. Our work also leads to a signaling pathway of NS1-mediated downregulation of coagulation factors via phosphorylated ERK and HNF4α. HNF4α is a transcription regulator for many other liver-based metabolic factors and pathways like lipid metabolism, carbohydrate metabolism, etc, and thus, therapeutic targeting of NS1-based downregulation of HNF4α can lead to designing therapeutic candidates for managing other Dengue-based liver dysfunction.