Caspase-3 inhibitor inhibits enterovirus D68 production.

Enterovirus D68 (EVD68) is an emerging pathogen that recently caused a large worldwide outbreak of severe respiratory disease in children. However, the relationship between EVD68 and host cells remains unclear. Caspases are involved in cell death, immune response, and even viral production. We found that caspase-3 was activated during EVD68 replication to induce apoptosis. Caspase-3 inhibitor (Z-DEVD-FMK) inhibited viral production, protected host cells from the cytopathic effects of EVD68 infection, and prevented EVD68 from regulating the host cell cycle at G0/G1. Meanwhile, caspase-3 activator (PAC-1) increased EVD68 production. EVD68 infection therefore activates caspase-3 for virus production. This knowledge provides a potential direction for the prevention and treatment of disease related to EVD68.

Bypassing pan-enterovirus host factor PLA2G16.

Enteroviruses are a major cause of human disease. Adipose-specific phospholipase A2 (PLA2G16) was recently identified as a pan-enterovirus host factor and potential drug target. In this study, we identify a possible mechanism of PLA2G16 evasion by employing a dual glycan receptor-binding enterovirus D68 (EV-D68) strain. We previously showed that this strain does not strictly require the canonical EV-D68 receptor sialic acid. Here, we employ a haploid screen to identify sulfated glycosaminoglycans (sGAGs) as its second glycan receptor. Remarkably, engagement of sGAGs enables this virus to bypass PLA2G16. Using cryo-EM analysis, we reveal that, in contrast to sialic acid, sGAGs stimulate genome release from virions via structural changes that enlarge the putative openings for genome egress. Together, we describe an enterovirus that can bypass PLA2G16 and identify additional virion destabilization as a potential mechanism to circumvent PLA2G16.

Susceptibilities of enterovirus D68, enterovirus 71, and rhinovirus 87 strains to various antiviral compounds.

Compounds were evaluated for antiviral activity in rhabdomyosarcoma (RD) cells against a recent 2014 clinical isolate of enterovirus D68 (EV-D68), a 1962 strain of EV-68D, rhinovirus 87 (RV-87, serologically the same as EV-D68), and enterovirus 71 (EV-71). Test substances included known-active antipicornavirus agents (enviroxime, guanidine HCl, pirodavir, pleconaril, and rupintrivir), nucleobase/nucleoside analogs (3-deazaguanine and ribavirin), and three novel epidithiodiketopiperazines (KCN-2,2'-epi-19, KCN-19, and KCN-21). Of these, rupintrivir was the most potent, with 50% inhibition of viral cytopathic effect (EC50) and 90% inhibition (EC90) of virus yield at 0.0022-0.0053 µM against EV-D68. Enviroxime, pleconaril and the KCN compounds showed efficacy at 0.01-0.3 µM; 3-deazaguanine and pirodavir inhibited EV-D68 at 7-13 µM, and guanidine HCl and ribavirin were inhibitory at 80-135 µM. Pirodavir was active against EV-71 (EC50 of 0.78 µM) but not against RV-87 or EV-D68, and all other compounds were less effective against EV-71 than against RV-87 and EV-D68. The most promising compound inhibiting both virus infections at low concentrations was rupintrivir. Antiviral activity was confirmed for the ten compounds in virus yield reduction (VYR) assays in RD cells, and for enviroxime, guanidine HCl, and pirodavir by cytopathic effect (CPE) assays in A549, HeLa-Ohio-1, and RD cells. These studies may serve as a basis for further pre-clinical discovery of anti-enterovirus inhibitors. Furthermore, the antiviral profiles and growth characteristics observed herein support the assertion that EV-D68 should be classified together with RV-87.

Global reemergence of enterovirus D68 as an important pathogen for acute respiratory infections.

We previously detected enterovirus D68 (EV-D68) in children with severe acute respiratory infections in the Philippines in 2008-2009. Since then, the detection frequency of EV-D68 has increased in different parts of the world, and EV-D68 is now recognized as a reemerging pathogen. However, the epidemiological profile and clinical significance of EV-D68 is yet to be defined, and the virological characteristics of EV-D68 are not fully understood. Recent studies have revealed that EV-D68 is detected among patients with acute respiratory infections of differing severities ranging from mild upper respiratory tract infections to severe pneumonia including fatal cases in pediatric and adult patients. In some study sites, the EV-D68 detection rate was higher among patients with lower respiratory tract infections than among those with upper respiratory tract infections, suggesting that EV-D68 infections are more likely to be associated with severe respiratory illnesses. EV-D68 strains circulating in recent years have been divided into three distinct genetic lineages with different antigenicity. However, the association between genetic differences and disease severity, as well as the occurrence of large-scale outbreaks, remains elusive. Previous studies have revealed that EV-D68 is acid sensitive and has an optimal growth temperature of 33 °C. EV-D68 binds to α2,6-linked sialic acids; hence, it is assumed that it has an affinity for the upper respiratory track where these glycans are present. However, the lack of suitable animal model constrains comprehensive understanding of the pathogenesis of EV-D68.

Development of RNA interference (RNAi) as potential antiviral strategy against enterovirus 70.

Enterovirus 70 (EV70) is recognized as the main causative agent of acute hemorrhagic conjunctivitis (AHC), a highly contagious viral infection of the eye. Currently, there is no available treatment for EV70 infections. In this study, we developed a potential intervention strategy using RNA interference (RNAi) against EV70 infection in an in vitro system. Two synthetic 19-mer siRNAs, si-3D1 and si-3D2, were designed to target the 3Dpol region of the EV70 genome. Significant dosage dependent inhibition of EV70 in rhabdomyosarcoma cell line, as shown by reduction of viral RNA and VP1 production, was observed. Both siRNAs prevented EV70 replication in RD cells when transfected into these cells 48 hr prior to virus infection. Introduction of these siRNAs into RD cells 1-3 hr after infection with EV70 reduced production of viral RNA by approximately 60%. Thus, RNAi is a promising strategy to prevent EV70 infections and may have therapeutic potential.

Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses.

Enterovirus (EV) 68 was originally isolated in California in 1962 from four children with respiratory illness. Since that time, reports of EV68 isolation have been very uncommon. Between 1989 and 2003, 12 additional EV68 clinical isolates were identified and characterized, all of which were obtained from respiratory specimens of patients with respiratory tract illnesses. No EV68 isolates from enteric specimens have been identified from these same laboratories. These recent isolates, as well as the original California strains and human rhinovirus (HRV) 87 (recently shown to be an isolate of EV68 and distinct from the other human rhinoviruses), were compared by partial nucleotide sequencing in three genomic regions (partial sequencing of the 5'-non-translated region and 3D polymerase gene, and complete sequencing of the VP1 capsid gene). The EV68 isolates, including HRV87, were monophyletic in all three regions of the genome. EV68 isolates and HRV87 grew poorly at 37 degrees C relative to growth at 33 degrees C and their titres were reduced by incubation at pH 3.0, whereas the control enterovirus, echovirus 11, grew equally well at 33 and 37 degrees C and its titre was not affected by treatment at pH 3.0. Acid lability and a lower optimum growth temperature are characteristic features of the human rhinoviruses. It is concluded that EV68 is primarily an agent of respiratory disease and that it shares important biological and molecular properties with both the enteroviruses and the rhinoviruses.

Towards an in vitro model for acute hemorrhagic conjunctivitis: cytokine-mediated vascular endothelial cell activation triggered by enterovirus type 70 infection.

BACKGROUND: Little is known of the pathogenetic mechanisms of enterovirus type 70 (EV70), a causative agent of acute hemorrhagic conjunctivitis. However, virus- or cytokine-induced perturbation of vascular endothelial cells are potential triggering events. OBJECTIVE: To determine whether EV70 infection of human umbilical vascular endothelial cells (HUVECs) and human corneal epithelial cells (HCEs) causes the release of vasoactive cytokines, capable of triggering vascular endothelial cell activation. STUDY DESIGN: Susceptibility of cultured HUVECs and HCEs to EV70 was tested by observing the appearance of cytopathic effect or immunoprecipitation of viral protein in infected cells. The culture fluids from the virus-infected cells were tested for their ability to stimulate the expression of intercellular adhesion molecule-1 (ICAM-1) on uninfected HUVECs. Anti-cytokine antibodies were used to identify ICAM-1-activating cytokine(s). RESULTS: Both HUVECs and HCEs were susceptible to EV70 infection. Culture fluids from EV70-infected HUVECs and HCEs stimulated ICAM-l expression on uninfected HUVECs, which was completely blocked by anti-interleukin-1alpha (IL-1alpha) antibody but not by interleukin-1beta (IL-1beta) or anti-tumor necrosis factor alpha (TNFalpha) antibodies. CONCLUSION: This study provides the first evidence of EV70 infection of both HCEs and HUVECs, and furthermore, identifies IL-1alpha as the predominant endothelial cell-activating factor produced by EV70-infected cells. Since endothelial cell activation is often an initiating step towards vascular permeability and/or inflammation, the perturbation of endothelial cell function through EV70 induced IL-1alpha is thus a potential contributory factor in the pathogenesis of EV70-associated hemorrhagic conjunctivitis.