Dengue immunopathogenesis : a cross talk between host and viral factors leading to disease : part I - Dengue virus tropism, host innate immune responses, and subversion of antiviral responses

Two part articleThis detailed paper addresses the general features of the dengue virus (DENV) infections complex, including the virus structure and genome, epidemiology, and clinical outcomes. This is followed by an updated review of the literature describing the host innate immune strategies as well as the viral mechanisms acting against and in favor of the DENV replication cycle and infection.Canadian Institutes of Health ResearchFondo Mixto CONACYT-Gobierno del Estado de Yucatá

CD300LF polymorphisms of inbred mouse strains confer resistance to murine norovirus infection in a cell type-dependent manner

Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC\u27 loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry

Combinations of Host- and Virus-Targeting Antiviral Drugs Confer Synergistic Suppression of SARS-CoV-2

Three directly acting antivirals (DAAs) demonstrated substantial reduction in COVID-19 hospitalizations and deaths in clinical trials. However, these agents did not completely prevent severe illness and are associated with cases of rebound illness and viral shedding. Combination regimens can enhance antiviral potency, reduce the emergence of drug-resistant variants, and lower the dose of each component in the combination. Concurrently targeting virus entry and virus replication offers opportunities to discover synergistic drug combinations. While combination antiviral drug treatments are standard for chronic RNA virus infections, no antiviral combination therapy has been approved for SARS-CoV-2. Here, we demonstrate that combining host-targeting antivirals (HTAs) that target TMPRSS2 and hence SARS-CoV-2 entry, with the DAA molnupiravir, which targets SARS-CoV-2 replication, synergistically suppresses SARS-CoV-2 infection in Calu-3 lung epithelial cells. Strong synergy was observed when molnupiravir, an oral drug, was combined with three TMPRSS2 (HTA) oral or inhaled inhibitors: camostat, avoralstat, or nafamostat. The combination of camostat plus molnupiravir was also effective against the beta and delta variants of concern. The pyrimidine biosynthesis inhibitor brequinar combined with molnupiravir also conferred robust synergistic inhibition. These HTA+DAA combinations had similar potency to the synergistic all-DAA combination of molnupiravir plus nirmatrelvir, the protease inhibitor found in paxlovid. Pharmacodynamic modeling allowed estimates of antiviral potency at all possible concentrations of each agent within plausible therapeutic ranges, suggesting possible in vivo efficacy. The triple combination of camostat, brequinar, and molnupiravir further increased antiviral potency. These findings support the development of HTA+DAA combinations for pandemic response and preparedness. IMPORTANCE Imagine a future viral pandemic where if you test positive for the new virus, you can quickly take some medicines at home for a few days so that you do not get too sick. To date, only single drugs have been approved for outpatient use against SARS-CoV-2, and we are learning that these have some limitations and may succumb to drug resistance. Here, we show that combinations of two oral drugs are better than the single ones in blocking SARS-CoV-2, and we use mathematical modeling to show that these drug combinations are likely to work in people. We also show that a combination of three oral drugs works even better at eradicating the virus. Our findings therefore bode well for the development of oral drug cocktails for at home use at the first sign of an infection by a coronavirus or other emerging viral pathogens.Peer reviewe

Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease: Part I - Dengue Virus Tropism, Host Innate Immune Responses, and Subversion of Antiviral Responses

Dengue is the most prevalent emerging mosquito-borne viral disease, affecting more than 40% of the human population worldwide. Many symptomatic dengue virus (DENV) infections result in a relatively benign disease course known as dengue fever (DF). However, a small proportion of patients develop severe clinical manifestations, englobed in two main categories known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Secondary infection with any of the four dengue virus serotypes (DENV1, -2, -3, and -4) is a risk factor to develop severe forms of dengue disease. DSS is primarily characterized by sudden and abrupt endothelial dysfunction, resulting in vascular leak and organ impairment, which may progress to hypovolemic shock and death. Severe DENV disease (DHF/DSS) is thought to follow a complex relationship between distinct immunopathogenic processes involving host and viral factors, such as the serotype cross-reactive antibody-dependent enhancement (ADE), the activation of T cells and complement pathways, the phenomenon of the cytokine storm, and the newly described viral toxin activity of the nonstructural protein 1 (NS1), which together play critical roles in inducing vascular leak and virus pathogenesis. In this chapter that is divided in two parts, we will outline the recent advances in our understanding of DENV pathogenesis, highlighting key viral-host interactions and discussing how these interactions may contribute to DENV immunopathology and the development of vascular leak, a hallmark of severe dengue. Part I will address the general features of the DENV complex, including the virus structure and genome, epidemiology, and clinical outcomes, followed by an updated review of the literature describing the host innate immune strategies as well as the viral mechanisms acting against and in favor of the DENV replication cycle and infection

Dengue Immunopathogenesis: A Crosstalk between Host and Viral Factors Leading to Disease: PART II - DENV Infection, Adaptive Immune Responses, and NS1 Pathogenesis

Severe disease is associated with serial infection with DENV of different serotypes. Thus, primary DENV infections normally cause asymptomatic infections, and secondary heterotypic infections with a new DENV serotype potentially increase the risks of developing severe disease. Despite many proposed hypotheses trying to explain it, the exact immunological mechanism leading to severe dengue disease is unknown. In turn, severe manifestations are believed to be a consequence of the combinations of many immunopathogenic mechanisms involving viral and host factors leading to increased pathogenesis and disease. Of these mechanisms, the adaptive immune response has been proposed to play a critical role in the development of severe dengue manifestations. This includes the effect of non-neutralizing but enhancing antibodies produced during primary infections, which results in enhanced-DENV infection of Fc-γ-receptor-expressing cells (e.g. monocytes and macrophages) during DENV heterotypic exposure in a phenomenon called antibody-dependent enhancement (ADE); the increased activation of memory T cells during secondary infections, which has low affinity for the current infecting serotype and high affinity for a past infection with a different serotype known as the original antigenic sin; the unbalanced production of pro-inflammatory cytokines that have a direct effect on vascular endothelial cells resulting in plasma leak in a phenomenon known as cytokine storm; and the excessive activation of the complement system that causes exacerbated inflammatory responses, increasing disease severity. In addition to the adaptive immune responses, a secreted viral factor known as the nonstructural protein 1 (NS1) has been recently proposed as the missing corner piece of the DENV pathogenesis influencing disease. This Part II of the chapter will discuss the interplay between the distinct host adaptive immune responses and viral factors that together contribute to the development of DENV pathogenesis and severe disease

Studying the endothelial glycocalyx in vitro: what is missing?

All human cells are coated by a surface layer of proteoglycans, glycosaminoglycans (GAGs) and plasma proteins, called the glycocalyx. The glycocalyx transmits shear stress to the cytoskeleton of endothelial cells, maintains a selective permeability barrier, and modulates adhesion of blood leukocytes and platelets. Major components of the glycocalyx, including syndecans, heparan sulfate, and hyaluronan, are shed from the endothelial surface layer during conditions including ischaemia and hypoxia, sepsis, atherosclerosis, diabetes, renal disease, and some viral infections. Studying mechanisms of glycocalyx damage in vivo can be challenging due to the complexity of immuno-inflammatory responses which are inextricably involved. Previously, both static as well as perfused in vitro models have studied the glycocalyx, and have reported either imaging data, assessment of barrier function, or interactions of blood components with the endothelial monolayer. To date, no model has simultaneously incorporated all these features at once, however such a model would arguably enhance the study of vasculopathic processes. This review compiles a series of current in vitro models described in the literature that have targeted the glycocalyx layer, their limitations, and potential opportunities for further developments in this field

An intranasal ASO therapeutic targeting SARS-CoV-2

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 “variants of concern” tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics

Insight Into the Interaction Between RNA Polymerase and VPg for Murine Norovirus Replication

Norovirus (NoV) is a leading cause of epidemic acute non-bacterial gastroenteritis, and replicates through virion protein genome-linked (VPg)-primed or de novo RNA synthesis by RNA-dependent RNA polymerase (RdRp). VPg is a multifunctional protein that plays crucial roles in viral protein translation and genome replication. However, the interaction between the RdRp and this multifunctional VPg in NoV replication has been unknown. In this study, VPg derived from murine NoV (MNV) was found to mediate the formation of higher-order multimers or tubular fibrils of MNV RdRp, which led to significantly enhanced polymerase activity in vitro. The replication of MNV mutants containing a VPg-binding defective RdRp, based on the crystal structure of an RdRp-VPg(1-73) complex, was completely blocked in a cell culture system. Our data suggest that the interaction between RdRp and VPg plays a crucial role in the multimerization-mediated RdRp activity in vivo and consequently in MNV replication, which can provide a new target of therapeutic intervention for NoV outbreaks