Enterovirus D68 (EV-D68) is a member of the Picornaviridae family. Although EV-D68-associated infection was once considered rare, it has been increasing in recent years. EV-D68 infection is most frequently associated with respiratory illness. However, it has also been implicated in a polio-like neurological disorder, acute flaccid myelitis. Although sialic acid has been implicated in EV-D68 entry, the existence of a protein receptor has yet to be clarified. Here we identify neuron-specific intercellular adhesion molecule 5 (ICAM-5/telencephalin) as a cellular receptor for sialic acid-dependent and -independent EV-D68 viruses. EV-D68 bound specifically and efficiently to ICAM-5, and replication of EV-D68 in diverse cell types was inhibited by soluble ICAM-5 fragments. ICAM-5 silencing attenuated EV-D68 replication in permissive cells, and ICAM-5 expression in non-permissive cells allowed EV-D68 replication. The discovery of a neuron-specific adhesion molecule as an EV-D68 receptor has important implications for EV-D68 pathogenesis and may facilitate the development of novel intervention strategies.

Coxsackievirus A16 (CV-A16), CV-A6, and enterovirus D68 (EV-D68) belong to the Picornaviridae family and are major causes of hand, foot, and mouth disease (HFMD) and pediatric respiratory disease worldwide. The biological characteristics of these viruses, especially their interplay with the host innate immune system, have not been well investigated. In this study, we discovered that the 3C pro proteins from CV-A16, CV-A6, and EV-D68 bind melanoma differentiation-associated gene 5 (MDA5) and inhibit its interaction with MAVS. Consequently, MDA5-triggered type I interferon (IFN) signaling in the retinoic acid-inducible gene I-like receptor (RLR) pathway was blocked by the CV-A16, CV-A6, and EV-D68 3C pro proteins. Furthermore, the CV-A16, CV-A6, and EV-D68 3C pro proteins all cleave transforming growth factor ␤-activated kinase 1 (TAK1), resulting in the inhibition of NF-B activation, a host response also critical for Toll-like receptor (TLR)-mediated signaling. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed novel mechanisms to subvert host innate immune responses by targeting key factors in the RLR and TLR pathways. Blocking the ability of 3C pro proteins from diverse enteroviruses and coxsackieviruses to interfere with type I IFN induction should restore IFN antiviral function, offering a potential novel antiviral strategy.IMPORTANCE CV-A16, CV-A6, and EV-D68 are emerging pathogens associated with hand, foot, and mouth disease and pediatric respiratory disease worldwide. The pathogenic mechanisms of these viruses are largely unknown. Here we demonstrate that the CV-A16, CV-A6, and EV-D68 3C pro proteins block MDA5-triggered type I IFN induction. The 3C pro proteins of these viruses bind MDA5 and inhibit its interaction with MAVS. In addition, the CV-A16, CV-A6, and EV-D68 3C pro proteins cleave TAK1 to inhibit the NF-B response. Thus, our data demonstrate that circulating HFMDassociated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed a mechanism to subvert host innate immune responses by simultaneously targeting key factors in the RLR and TLR pathways. These findings indicate the potential merit of targeting the CV-A16, CV-A6, and EV-D68 3C pro proteins as an antiviral strategy.KEYWORDS MDA5, TAK1, MAVS, 3C protease, CV-A16, CV-A6, EV-D68, HFMD, innate immune response

Circulating coxsackievirus A16 (CA16) is a major cause of hand, foot and mouth disease (HFMD) in South-east Asia. At present, there is no vaccine against CA16. Pathogenic animal models that are sensitive to diverse circulating CA16 viruses would be desirable for vaccine development and evaluation. In this study, we isolated and characterized several circulating CA16 viruses from recent HFMD patients. These CA16 viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analysed the pathogenesis of representative circulating recombinant form CA16 viruses. An inactivated CA16 vaccine candidate, formulated with alum adjuvant and containing submicrogram quantities of viral proteins, protected neonatal mice born to immunized female mice from lethal-dose challenge with a series of CA16 viruses. Further analysis of humoral immunity showed that antibody elicited from both the immunized dams and their pups could neutralize various lethal viruses by a cytopathic effect in vitro. Moreover, viral titres and loads in the tissues of challenged pups in the vaccine group were far lower than those in the control group, and some were undetectable. This lethal-challenge model using pathogenic CA16 viruses and the vaccine candidates that mediated protection in this model could be useful tools for the future development and evaluation of CA16 vaccines.

Recent epidemiological data indicate that outbreaks of hand, foot, and mouth disease (HFMD), which can be categorized according to its clinical symptoms as typical or atypical, have markedly increased worldwide. A primary causative agent for typical HFMD outbreaks, enterovirus 71 (EV71), has been shown to manipulate the cell cycle in S phase for own replication; however, it is not clear whether coxsackievirus (CVA6), the main agent for atypical HFMD, also regulates the host cell cycle. In this study, we demonstrate for the first time that CVA6 infection arrests the host cell cycle in G0/G1-phase. Furthermore, synchronization in G0/G1 phase, but not S phase or G2/M phase, promotes viral production. To investigate the mechanism of cell cycle arrest induced by CVA6 infection, we analyzed cell cycle progression after cell cycle synchronization at G0/G1 or G2/M. Our results demonstrate that CVA6 infection promotes G0/G1 phase entry from G2/M phase, and inhibits G0/G1 exit into S phase. In line with its role to arrest cells in G0/G1 phase, the expression of cyclinD1, CDK4, cyclinE1, CDK2, cyclinB1, CDK1, P53, P21, and P16 is regulated by CVA6. Finally, the non-structural proteins of CVA6, RNA-dependent RNA polymerase 3D and protease 3C , are demonstrated to be responsible for the G0/G1-phase arrest. These findings suggest that CVA6 infection arrested cell cycle in G0/G1-phase via non-structural proteins 3D and 3C, which may provide favorable environments for virus production.

Enterovirus D68 (EV-D68) has emerged as a significant respiratory pathogen that can cause severe respiratory disease and acute neurologic disease. At present, there are no approved antiviral agents or vaccines for EV-D68. In this study, we demonstrate that andrographolide (ADO), an active component of Andrographis paniculata, exerts substantial antiviral activity against EV-D68 infection. ADO treatment dramatically inhibited EV-D68 RNA replication (EC50 = 3.45 μM) and protein synthesis without producing significant cytotoxicity at virucidal concentrations. ADO-treated cells did not show any changes in host immune activation, EV-D68 attachment, or viral 5′ UTR activity. Using a pH-sensitive fluorescent indicator system for endocytosis in living cells, we found that ADO prevented the acidification of endocytic vesicles after receptor-mediated endocytosis. Finally, we showed that ADO inhibited the viral replication of circulating isolated EV-D68 strains. In summary, our results demonstrate that ADO suppresses EV-D68 replication by targeting the maturation of virus-containing endosomes of EV-D68. This mechanism represents a promising strategy for drug development.