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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.

An enterovirus 71 (EV71) vaccine for the prevention of hand, foot, and mouth disease (HMFD) is available, but it is not known whether the EV71 vaccine cross-protects against Coxsackievirus (CV) infection. Furthermore, although an inactivated circulating CVA16 Changchun 024 (CC024) strain vaccine candidate is effective in newborn mice, the CC024 strain causes severe lesions in muscle and lung tissues. Therefore, an effective CV vaccine with improved pathogenic safety is needed. The aim of this study was to evaluate the in vivo safety and in vitro replication capability of a noncirculating CVA16 SHZH05 strain. The replication capacity of circulating CVA16 strains CC024, CC045, CC090 and CC163 and the noncirculating SHZH05 strain was evaluated by cytopathic effect in different cell lines. The replication capacity and pathogenicity of the CC024 and SHZH05 strains were also evaluated in a neonatal mouse model. Histopathological and viral load analyses demonstrated that the SHZH05 strain had an in vitro replication capacity comparable to the four CC strains. The CC024, but not the SHZH05 strain, became distributed in a variety of tissues and caused severe lesions and mortality in neonatal mice. The differences in replication capacity and in vivo pathogenicity of the CC024 and SHZH05 strains may result from differences in the nucleotide and amino acid sequences of viral functional polyproteins P1, P2 and P3. Our findings suggest that the noncirculating SHZH05 strain may be a safer CV vaccine candidate than the CC024 strain.

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 β-activated kinase 1 (TAK1), resulting in the inhibition of NF-κB 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-κB response. Thus, our data demonstrate that circulating HFMD-associated 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.

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.

Circulating enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) is a major public health problem in the Asian-Pacific region. An EV71 vaccine for HFMD prevention is currently being developed. However, viral determinants that could influence the vaccine's efficacy have not been well characterized. In this study, we isolated and characterized several EV71 strains that are currently circulating in northern and southern China. We determined that VP1 variation is a major determinant of EV71 immunogenicity. A single amino acid variation in VP1 can lead to significant differences in the breadth and potency of immune responses against primary EV71 isolates as well as the sensitivity of EV71 to heterologous neutralizing antibody responses. We also identified EV71 strains that could induce potent immunogenic and cross-neutralizing antibody responses against diverse EV71 strains. Furthermore, these neutralizing antibodies could protect neonatal mice from lethal dose challenge with various circulating EV71 viruses. Our study provides useful information for EV71 vaccine development and evaluation.