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Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. The roles of viral proteins in these processes have been studied mostly in vitro , by ectopic overexpression, or by surrogate infection systems, all of which have shortcomings. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2A pro in the virus context. Viruses with an inactive 2A pro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2A pro is essential for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, suppressing the induction of the IFN response, and overcoming IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2A pro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. Thus, 2A pro plays a critical role in subverting antiviral responses and establishing a favorable environment to expedite enterovirus replication.

Enteroviruses are a group of highly prevalent human pathogens responsible for a wide range of illnesses, ranging from common cold symptoms to life-threatening diseases. A deep understanding of enterovirus biology, evolution, and host interaction is required for the development of effective vaccines and antivirals. Recombinant reporter viruses encoding luminescent or fluorescent proteins have been developed to facilitate such investigation. In this work, using coxsackievirus B3 as our model, we analyze how the insertion of fluorescent reporter genes at three distinct sites within the viral polyprotein affects viral fitness, identifying the most tolerant site for reporter insertion. We then describe a set of experimental workflows for measuring viral fitness, sera neutralization, antiviral efficacy, and recombination using fluorescent reporter viruses. The high homology between different enteroviruses suggests these assays can be readily adapted to study additional members of this medically and economically relevant group of viruses.

Background. Scarce information exists about immunity to hand, foot, and mouth disease (HFMD) among household contacts of index cases in Vietnam and what that means for reducing ongoing HFMD transmission in the community. Methods. We analyzed neutralizing antibodies (NT) and the incidence of enterovirus (EVs) infection among household contacts of index cases in a province where HFMD remains endemic. Throat swab and 2 mL blood samples from household contacts were collected at enrollment, during and after 2 weeks follow-up. Results. The incidence of EV-A71 infection among household contacts was 40/84 (47.6%, 95% Cl: 36.9-58.3%), compared with 106/336 (31.5%, 95% Cl: 26.6-36.5%) for CV-A6 and 36/107 (33.6%, 95% Cl: 24.7-42.6%) for CV-A16. The incidence of CV-A6 infection was fairly constant across ages; in contrast, CV-A71 and CV-A16 had some variation across ages. At baseline, higher geometric mean titer (GMT) of EV-A71, CV-A6, and CV-A16 antibody titers was found for 25-34-year groups (range 216.3 to 305.0) compared to the other age groups. There was a statistically significant difference in GMT values of CV-A6 and CV-A16 between those who had an infection or did not have infection among households with an index case of these serotypes. Conclusions. Our results indicated that adults were becoming infected with HFMD and could be contributing to the transmission. There is, therefore, a need for considering the household setting as an additional target for intervention programs for HFMD.

Toll-like receptors (TLRs) act as molecular sentinels, detecting invading viral pathogens and triggering host innate immune responses, including autophagy. However, many viruses have evolved a series of strategies to manipulate autophagy for their own benefit. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16), as the primary agents causing hand, foot and mouth disease (HFMD), can induce autophagy leading to their replication. Therefore, the objective of this study was to investigate whether enhanced viral replication caused by autophagy in EV71 and CA16 infections was associated with a TLR-related signaling pathway. Our results demonstrate that complete autophagy and incomplete autophagy were observed in human bronchial epithelial (16HBE) cells infected with EV71 and CA16. Moreover, suppression of autophagy by the pharmacological modulator 3-MA significantly and clearly decreased the survival rates and viral replication of EV71 and CA16 in 16HBE cells. Inhibition of autophagy also enhanced the expression of molecules related to the TLR7-dependent type I interferon (IFN-I) production pathway, such as TLR7, MyD88, IRF7 and IFN-α/β. Finally, immunofluorescence staining demonstrated that TLR7 endosome marker M6PR levels were clearly reduced in EV71- and CA16-infected cells, while they were markedly elevated in infected cells treated with 3-MA. These findings suggest that increased EV71 and CA16 replication meditated by autophagy in 16HBE cells might promote degradation of the endosome, leading to suppression of the TLR7-mediated IFN-I signaling pathway.

No specific antiviral agent against hand foot and mouth disease (HFMD) is available for clinical practice today. To evaluate the efficacy and safety of Jinzhen oral solution in treating uncomplicated HFMD. In this randomized, double-blind, placebo-controlled trial, 399 children aged 1 to 7 years with laboratory confirmed HFMD were randomized to receive Jinzhen oral liquid or placebo 3 times daily for 7 days with a 3-day follow-up. The primary outcomes were time to the first disappearance of oral ulcers and vesicles on hand or foot and time to the first normalization of temperature (fever clearance). There were 199 children enrolling into the Jinzhen group including 79 with fever and 200 into the placebo group including 93 with fever. Jinzhen reduced the time to the first disappearance of oral ulcers and vesicles on hand or foot to 4.9 days (95% CI, 4.6 to 5.2 days), compared with 5.7 days (95% CI, 5.4 to 6.0 days) in the placebo group (P = 0.0036). The median time of fever clearance was shorter in the 79 children who received Jinzhen (43.41 hrs, 95% CI, 37.05 to 49.76) than that in the 93 children who received placebo (54.92 hrs, 95% CI, 48.16 to 61.68) (P = 0.0161). Moreover, Jinzhen reduced the risk of symptoms by 28.5% compared with placebo (HR, 0.7150, 95% CI, 0.5719 to 0.8940, P = 0.0032). More importantly, treatment failure rate was significantly lower in the Jinzhen group (8.04%) compared with that in the placebo group (15.00%) (P = 0.0434). The incidence of serious adverse events did not differ significantly between the two groups (9 in Jinzhen group vs. 18 in placebo, P = 0.075). Children with HFMD may benefit from Jinzhen oral liquid treatment as compared with placebo. Chinese Clinical Trial Registry (http://www.chictr.org/en/) ChiCTR-TRC-10000937.

Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

Nonpolio enteroviruses are diverse and common viruses that can circulate year-round but tend to peak in summer. Although most infections are asymptomatic, they can result in a wide range of neurological and other diseases. Many serotypes circulate every year, and different serotypes predominate in different years, but the drivers of their geographical and temporal dynamics are not understood. We use national enterovirus surveillance data collected by the US Centers for Disease Control and Prevention during 1983−2013, as well as demographic and climatic data for the same period, to study the patterns and drivers of the seasonality of these infections. We find that the seasonal pattern of enterovirus cases is spatially structured in the United States and similar to that observed for historical prevaccination poliomyelitis (1931−1954). We identify latitudinal gradients for the amplitude and the timing of the peak of cases, meaning that those are more regularly distributed all year-round in the south and have a more pronounced peak that arrives later toward the north. The peak is estimated to occur between July and September across the United States, and 1 month earlier than that for historical poliomyelitis. Using mixed-effects models,we find that climate, but not demography, is likely to drive the seasonal pattern of enterovirus cases and that the dew point temperature alone explains ∼30% of the variation in the intensity of transmission. Our study contributes to a better understanding of the epidemiology of enteroviruses, demonstrates important similarities in their circulation dynamics with polioviruses, and identifies potential drivers of their seasonality.

Coxsackievirus A6 (CV-A6), coxsackievirus A16 (CV-A16) and enterovirus 71 (EV-A71) were the major enteroviruses causing nationwide hand, foot and mouth disease (HFMD) epidemics in Singapore in the last decade. We estimated the basic reproduction number (R 0) of these enteroviruses to obtain a better understanding of their transmission dynamics. We merged records of cases from HFMD outbreaks reported between 2007 and 2012 with laboratory results from virological surveillance. R 0 was estimated based on the cumulative number of reported cases in the initial growth phase of each outbreak associated with the particular enterovirus type. A total of 33 HFMD outbreaks were selected based on the inclusion criteria specified for our study, of which five were associated with CV-A6, 13 with CV-A16, and 15 with EV-A71. The median R 0 was estimated to be 5·04 [interquartile range (IQR) 3·57–5·16] for CV-A6, 2·42 (IQR 1·85–3·36) for CV-A16, and 3·50 (IQR 2·36–4·53) for EV-A71. R 0 was not significantly associated with number of infected children (P = 0·86), number of exposed children (P = 0·94), and duration of the outbreak (P = 0·05). These enterovirus-specific R 0 estimates will be helpful in providing insights into the potential growth of future HFMD epidemics and outbreaks for timely implementation of disease control measures, together with disease dynamics such as severity of the cases.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) remain the predominant pathogens in hand, foot, and mouth disease (HFMD), but the factors underlying the pathogenesis of EV71 and CA16 infections have not been elucidated. Recently, the functions of microRNAs (miRNAs) in pathogen-host interactions have been highlighted. In the present study, we performed comprehensive miRNA profiling in EV71- and CA16-infected human umbilical vein endothelial cells (HUVECs) at multiple time points using high-throughput sequencing. The results showed that 135 known miRNAs exhibited remarkable differences in expression. Of these, 30 differentially expressed miRNAs presented opposite trends in EV71- and CA16-infected samples. Subsequently, we mainly focused on the 30 key differentially expressed miRNAs through further screening to predict targets. Gene ontology (GO) and pathway analysis of the predicted targets showed the enrichment of 14 biological processes, 9 molecular functions, 8 cellular components, and 85 pathways. The regulatory networks of these miRNAs with predicted targets, GOs, pathways, and co-expression genes were determined, suggesting that miRNAs display intricate regulatory mechanisms during the infection phase. Consequently, we specifically analyzed the hierarchical GO categories of the predicted targets involved in biological adhesion. The results indicated that the distinct changes induced by EV71 and CA16 infection may be partly linked to the function of the blood-brain barrier. Taken together, this is the first report describing miRNA expression profiles in HUVECs with EV71 and CA16 infections using high-throughput sequencing. Our data provide useful insights that may help to elucidate the different host-pathogen interactions following EV71 and CA16 infection and offer novel therapeutic targets for these infections.

The mechanisms underlying pathological changes in the central nervous system (CNS) following Coxsackievirus A16 (CV-A16) infection have not yet been elucidated. IFN-γ-inducible protein-10 (IP-10) is often used as a predictive factor to monitor early virus infection. It has also been reported that IP-10 plays a pivotal role in neuroinflammation. In this study, we aimed to explore the role of IP-10 in the neuropathogenesis of CV-A16 infection. We observed that the level of IP-10, as well as the TLR3-TRIF-TRAF3-TBK1-NF-κB and RIG-I/MDA5-MAVS-TRAFS-TBK1-NF-κB pathways, which are the upstream of IP-10, were significantly elevated during the course of CV-A16 infection. This increase was accompanied by an increase in a series of inflammatory cytokines at different time-points during CV-A16 infection. To determine whether IP-10 influences BBB integrity, we examined junctional complexes. Our results revealed that the expression levels of Claudin5, Occludin, ZO-1 and VE-Cadherin were notably decreased in CV-A16-infected HUVECs, but these indicators were restored in CV-A16-infected HUVECs with Eldelumab treatment. Nevertheless, IP-10 is only a chemokine that primarily traffics CXCR3-positive immune cells to inflammatory sites or promotes the production of inflammatory cytokines. Therefore, the interactions between IP-10 and inflammatory cytokines were evaluated. Our data revealed that IP-10 mediated the production of TNF-α, which was also observed to change the junctional complexes. Moreover, in a suckling mouse model, IP-10 and TNF-α treatments exacerbated clinical symptoms, mortality and pathological changes in the brain of CV-A16-infected mice, but Anti-IP-10 and Anti-TNF-α treatments alleviated these changes. Our data also revealed that IP-10 may be detected early in CV-A16 infection, whereas TNF-α was detected late in CV-A16 infection, and the production of TNF-α was also found to be positively correlated with IP-10. In addition, IP-10 and TNF-α were observed to reduce junctional complexes and enhance virus entry into the CNS. Taken together, this study provides the first evidence that CV-A16 activates the IP-10/TNF-α regulatory axis to cause BBB damage and accelerate the formation of neuroinflammation in infected hosts, which not only provides a new understanding of the neuropathogenesis caused by CV-A16, but also offers a promising target for the development of CV-A16 antiviral drugs.