Major Group-B Enterovirus populations deleted in the noncoding 5' region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis.

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.

Early plasma interferon-ß levels as a predictive marker of COVID-19 severe clinical events in adult patients.

We assessed relationships between early peripheral blood type I interferons (IFN) levels, clinical new early warning scores (NEWS), and clinical outcomes in hospitalized coronavirus disease-19 (COVID-19) adult patients. Early IFN-ß levels were lower among patients who further required intensive care unit (ICU) admission than those measured in patients who did not require an ICU admission during severe acute respiratory syndrome coronavirus type 2 infection. IFN-ß levels were inversely correlated with NEWS only in the subgroup of patients who further required ICU admission. To assess whether peripheral blood IFN-ß levels could be a potential relevant biomarker to predict further need for ICU admission, we performed receiver operating characteristic (ROC) curve analyses that showed for all study patients an area under ROC curve of 0.77 growing to 0.86 (p = 0.003) when the analysis was restricted to a subset of patients with NEWS ≥5 at the time of hospital admission. Overall, our findings indicated that early peripheral blood IFN-ß levels might be a relevant predictive marker of further need for an ICU admission in hospitalized COVID-19 adult patients, specifically when clinical score (NEWS) was graded as upper than 5 at the time of hospital admission.

Replication Activities of Major 5' Terminally Deleted Group-B Coxsackievirus RNA Forms Decrease PCSK2 mRNA Expression Impairing Insulin Maturation in Pancreatic Beta Cells.

Emergence of 5' terminally deleted coxsackievirus-B RNA forms (CVB-TD) have been associated with the development of human diseases. These CVB-TD RNA forms have been detected in mouse pancreas during acute or persistent experimental infections. To date, the impact of the replication activities of CVB-TD RNA forms on insulin metabolism remains unexplored. Using an immunocompetent mouse model of CVB3/28 infection, acute and persistent infections of major CVB-TD populations were evidenced in the pancreas. The inoculation of mice with homogenized pancreases containing major CVB-TD populations induced acute and chronic pancreatic infections with pancreatitis. In the mouse pancreas, viral capsid protein 1 (VP1) expression colocalized with a decrease in beta cells insulin content. Moreover, in infected mouse pancreases, we showed a decrease in pro-hormone convertase 2 (PCSK2) mRNA, associated with a decrease in insulin plasmatic concentration. Finally, transfection of synthetic CVB-TD50 RNA forms into cultured rodent pancreatic beta cells demonstrated that viral replication with protein synthesis activities decreased the PCSK2 mRNA expression levels, impairing insulin secretion. In conclusion, our results show that the emergence and maintenance of major CVB-TD RNA replicative forms in pancreatic beta cells can play a direct, key role in the pathophysiological mechanisms leading to the development of type 1 diabetes.

Early Emergence of 5' Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues.

Major EV-B populations characterized by 5' terminal deletions (5'TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics of EV-B 5'TD-RNA forms' emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5'TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5'TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5'TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions.

Surfaces and Air Contamination by Severe Acute Respiratory Syndrome Coronavirus 2 Using High-Flow Nasal Oxygenation or Assisted Mechanical Ventilation in Intensive Care Unit Rooms of Patients With Coronavirus Disease 2019.

BACKGROUND: Understanding patterns of environmental contamination by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for infection prevention policies. METHODS: We screened surfaces and air samples from single-bed intensive-care unit rooms of adult patients with coronavirus disease 2019 (COVID-19) for SARS-CoV-2 RNA and viable viruses. RESULTS: We evidenced viral RNA environmental contamination in 76% of 100 surfaces samples and in 30% of 40 air samples without any viable virus detection by cell culture assays. No significant differences of viral RNA levels on surfaces and in ambient air were observed between rooms of patients with assisted mechanical ventilation and those of patients with a high-flow nasal cannula system. Using an original experimental SARS-CoV-2 infection model of surfaces, we determined that infectious viruses may have been present on benches within 15 hours before the time of sampling in patient rooms. CONCLUSIONS: We observed that SARS-CoV-2 environmental contamination around patients with COVID-19 hospitalized in single-bed ICU rooms was extensive and that a high-flow nasal cannula system did not generate more viral aerosolization than a mechanical ventilation system in patients with COVID-19. Despite an absence of SARS-CoV-2 viable particles in study samples, our experimental model confirmed the need to apply strict environmental disinfection procedures and classic standard and droplet precautions in ICU wards.

Coinfection of Parvovirus B19 with Influenza A/H1N1 Causes Fulminant Myocarditis and Pneumonia. An Autopsy Case Report.

Parvovirus-B19 (PVB19) is a frequent causative agent of myocarditis. For unclear reasons, viral reactivation can cause acute myocarditis, a leading cause of sudden death in the young. Influenza A/H1N1(2009) virus (IAV/H1N1) is known for causing flu/pneumonia, but the heart is rarely involved. Co-infections of cardiotropic viruses are rarely reported and the mechanisms of viral interactions remain unknown. A 5-year old girl had a flu-like syndrome, when she suddenly presented with a respiratory distress and cardiac arrest. At autopsy, the lungs were found haemorrhagic. Lungs' histology showed severe bronchiolitis, diffuse haemorrhagic necrosis, and mononuclear inflammation. In the heart, a moderate inflammation was found with no necrosis. IAV/H1N1 was detected in nasal and tracheal swabs, lungs, and the heart. The viral load was high in the lungs, but low in the heart. PVB19 was detected in the heart with a high viral load. Viral co-infection increases the risk of severe outcome but the mechanisms of interaction between viruses are poorly understood. In our case, viral loads suggested a reactivated PVB19-induced acute myocarditis during an IAV/H1N1 pneumonia. Viral interactions may involve an IAV/H1N1-induced cytokine storm, with a fulminant fatal outcome. Clinically, our case shows the importance of investigating inflammatory pathways as therapeutic targets.

Structures and Functions of Viral 5' Non-Coding Genomic RNA Domain-I in Group-B Enterovirus Infections.

Group-B enteroviruses (EV-B) are ubiquitous naked single-stranded positive RNA viral pathogens that are responsible for common acute or persistent human infections. Their genome is composed in the 5' end by a non-coding region, which is crucial for the initiation of the viral replication and translation processes. RNA domain-I secondary structures can interact with viral or cellular proteins to form viral ribonucleoprotein (RNP) complexes regulating viral genomic replication, whereas RNA domains-II to -VII (internal ribosome entry site, IRES) are known to interact with cellular ribosomal subunits to initiate the viral translation process. Natural 5' terminally deleted viral forms lacking some genomic RNA domain-I secondary structures have been described in EV-B induced murine or human infections. Recent in vitro studies have evidenced that the loss of some viral RNP complexes in the RNA domain-I can modulate the viral replication and infectivity levels in EV-B infections. Moreover, the disruption of secondary structures of RNA domain-I could impair viral RNA sensing by RIG-I (Retinoic acid inducible gene I) or MDA5 (melanoma differentiation-associated protein 5) receptors, a way to overcome antiviral innate immune response. Overall, natural 5' terminally deleted viral genomes resulting in the loss of various structures in the RNA domain-I could be major key players of host-cell interactions driving the development of acute or persistent EV-B infections.

Zika Virus-Immune Plasmas from Symptomatic and Asymptomatic Individuals Enhance Zika Pathogenesis in Adult and Pregnant Mice.

Preexisting immunity against dengue virus or West Nile virus was previously reported to mediate antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection in a mouse model. We show here that ZIKV-immune plasma samples from both symptomatic and asymptomatic individuals mediated ZIKV ADE of infection in vitro and in mice. In a lethal infection model with a viral inoculum 10 times higher, both ADE and protection were observed, depending on the amount of infused immune plasma. In a vertical-transmission model, ZIKV-immune plasma infused to timed pregnant mice increased fetal demise and decreased the body weight of surviving fetuses. Depletion of IgG from an immune plasma abolished ADE of infection, and the presence of purified IgG alone mediated ADE of infection. Higher viral loads and proinflammatory cytokines were detected in mice treated with ZIKV-immune plasma samples compared to those receiving control plasma. Together, these data show that passive immunization with homotypic ZIKV antibodies, depending on the concentration, could either worsen or limit a subsequent ZIKV infection.IMPORTANCE Antibody-dependent enhancement (ADE) of virus infection is common to many viruses and is problematic when plasma antibody levels decline to subneutralizing concentrations. ADE of infection is especially important among flaviviruses, many of which are the cause of global health problems. Recently, human plasma samples immune to heterologous flaviviruses were shown to promote Zika virus (ZIKV) infection. Here we showed in immunocompromised mouse models that homologous immune plasma samples protect mice from subsequent infection at high antibody concentrations but that they mediate ADE of infection and increase ZIKV pathogenesis in adult mice and fetal demise during pregnancy at low concentrations.

AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses.

Although a causal relationship between Zika virus (ZIKV) and microcephaly has been established, it remains unclear why ZIKV, but not other pathogenic flaviviruses, causes congenital defects. Here we show that when viruses are produced in mammalian cells, ZIKV, but not the closely related dengue virus (DENV) or West Nile virus (WNV), can efficiently infect key placental barrier cells that directly contact the fetal bloodstream. We show that AXL, a receptor tyrosine kinase, is the primary ZIKV entry cofactor on human umbilical vein endothelial cells (HUVECs), and that ZIKV uses AXL with much greater efficiency than does DENV or WNV. Consistent with this observation, only ZIKV, but not WNV or DENV, bound the AXL ligand Gas6. In comparison, when DENV and WNV were produced in insect cells, they also infected HUVECs in an AXL-dependent manner. Our data suggest that ZIKV, when produced from mammalian cells, infects fetal endothelial cells much more efficiently than other pathogenic flaviviruses because it binds Gas6 more avidly, which in turn facilitates its interaction with AXL.

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis.

RATIONALE: The hallmark of severe influenza virus infection is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. OBJECTIVES: To determine the role of platelets during influenza A virus infections and propose new therapeutics against influenza. METHODS: We used targeted gene deletion approaches and pharmacologic interventions to investigate the role of platelets during influenza virus infection in mice. MEASUREMENTS AND MAIN RESULTS: Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted influenza A virus pathogenesis. Activating protease-activated receptor 4, a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein IIIa protected mice from death caused by influenza viruses, and treating the mice with a specific glycoprotein IIb/IIIa antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other antiplatelet compounds (antagonists of protease-activated receptor 4, MRS 2179, and clopidogrel) were also protected from severe lung injury and lethal infections induced by several influenza strains. CONCLUSIONS: The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and antiplatelet drugs might be explored to develop new antiinflammatory treatment against influenza virus infections.

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication.

UNLABELLED: During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

Switch from protective to adverse inflammation during influenza: viral determinants and hemostasis are caught as culprits.

Influenza viruses cause acute respiratory infections, which are highly contagious and occur as seasonal epidemic and sporadic pandemic outbreaks. Innate immune response is activated shortly after infection with influenza A viruses (IAV), affording effective protection of the host. However, this response should be tightly regulated, as insufficient inflammation may result in virus escape from immunosurveillance. In contrast, excessive inflammation may result in bystander lung tissue damage, loss of respiratory capacity, and deterioration of the clinical outcome of IAV infections. In this review, we give a comprehensive overview of the innate immune response to IAV infection and summarize the most important findings on how the host can inappropriately respond to influenza.

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.

PAR1 contributes to influenza A virus pathogenicity in mice.

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.