Modulation of nucleotide metabolism by picornaviruses.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

Emerging concerns of blood-brain barrier dysfunction caused by neurotropic enteroviral infections.

Enteroviruses (EVs), comprise a genus in the Picornaviridae family, which have been shown to be neurotropic and can cause various neurological disorders or long-term neurological condition, placing a huge burden on society and families. The blood-brain barrier (BBB) is a protective barrier that prevents dangerous substances from entering the central nervous system (CNS). Recently, numerous EVs have been demonstrated to have the ability to disrupt BBB, and further lead to severe neurological damage. However, the precise mechanisms of BBB disruption associated with these EVs remain largely unknown. In this Review, we focus on the molecular mechanisms of BBB dysfunction caused by EVs, emphasizing the invasiveness of enterovirus A71 (EVA71), which will provide a research direction for further treatment and prevention of CNS disorders.

Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6).

Hand, foot, and mouth disease (HFMD) is caused by more than 20 pathogenic enteroviruses belonging to the Picornaviridae family and Enterovirus genus. Since the introduction of the enterovirus-71 (EV71) vaccine in 2016, the number of HFMD cases caused by EV71 has decreased. However, cases of infections caused by other enteroviruses, such as coxsackievirus A6 (CA6) and coxsackievirus A10, have been increasing accordingly. In this study, we used a clinical isolate of CA6 to establish an intragastric infection mouse model using 7-day-old mice to mimic the natural transmission route, by which we investigated the differential gene expression profiles associated with virus infection and pathogenicity. After intragastric infection, mice exhibited hind limb paralysis symptoms and weight loss, similar to those reported for EV71 infection in mice. The skeletal muscle was identified as the main site of virus replication, with a peak viral load reaching 2.31 × 107 copies/mg at 5 dpi and increased infiltration of inflammatory cells. RNA sequencing analysis identified differentially expressed genes (DEGs) after CA6 infection. DEGs in the blood, muscle, brain, spleen, and thymus were predominantly enriched in immune system responses, including pathways such as Toll-like receptor signaling and PI3K-Akt signaling. Our study has unveiled the genes involved in the host immune response during CA6 infection, thereby enhancing our comprehension of the pathological mechanism of HFMD.IMPORTANCEThis study holds great significance for the field of hand, foot, and mouth disease (HFMD). It not only delves into the disease's etiology, transmission pathways, and severe complications but also establishes a novel mouse model that mimics the natural coxsackievirus A6 infection process, providing a pivotal platform to delve deeper into virus replication and pathogenic mechanisms. Additionally, utilizing RNA-seq technology, it unveils the dynamic gene expression changes during infection, offering valuable leads for identifying novel therapeutic drug targets. This research has the potential to enhance our understanding of HFMD, offering fresh perspectives for disease prevention and treatment and positively impacting children's health worldwide.

Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.

IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

SIRT-1 is required for release of enveloped enteroviruses.

Enterovirus D68 (EV-D68) is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1's proviral activity is, we demonstrate, mediated through the repression of endoplasmic reticulum stress (ER stress). Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.

HMGB1 is involved in viral replication and the inflammatory response in coxsackievirus A16-infected 16HBE cells via proteomic analysis and identification.

Coxsackievirus A16 (CV-A16) is still an important pathogen that causes hand, foot and mouth disease (HFMD) in young children and infants worldwide. Previous studies indicated that CV-A16 infection is usually mild or self-limiting, but it was also found that CV-A16 infection can trigger severe neurological complications and even death. However, there are currently no vaccines or antiviral compounds available to either prevent or treat CV-A16 infection. Therefore, investigation of the virus‒host interaction and identification of host proteins that play a crucial regulatory role in the pathogenesis of CV-A16 infection may provide a novel strategy to develop antiviral drugs. Here, to increase our understanding of the interaction of CV-A16 with the host cell, we analyzed changes in the proteome of 16HBE cells in response to CV-A16 using tandem mass tag (TMT) in combination with LC‒MS/MS. There were 6615 proteins quantified, and 172 proteins showed a significant alteration during CV-A16 infection. These differentially regulated proteins were involved in fundamental biological processes and signaling pathways, including metabolic processes, cytokine‒cytokine receptor interactions, B-cell receptor signaling pathways, and neuroactive ligand‒receptor interactions. Further bioinformatics analysis revealed the characteristics of the protein domains and subcellular localization of these differentially expressed proteins. Then, to validate the proteomics data, 3 randomly selected proteins exhibited consistent changes in protein expression with the TMT results using Western blotting and immunofluorescence methods. Finally, among these differentially regulated proteins, we primarily focused on HMGB1 based on its potential effects on viral replication and virus infection-induced inflammatory responses. It was demonstrated that overexpression of HMGB1 could decrease viral replication and upregulate the release of inflammatory cytokines, but deletion of HMGB1 increased viral replication and downregulated the release of inflammatory cytokines. In conclusion, the results from this study have helped further elucidate the potential molecular pathogenesis of CV-A16 based on numerous protein changes and the functions of HMGB1 Found to be involved in the processes of viral replication and inflammatory response, which may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.

Structural basis for cloverleaf RNA-initiated viral genome replication.

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

Natural Oncolysis of Enterovirus 71 in Antitumor Therapy of Colorectal Cancer.

Colorectal cancer (CRC) is an intestinal malignant tumor with high morbidity and mortality worldwide. Inoperability or resistanance to radiation and chemotherapy occur in the conventional treatments against CRC. Oncolytic viruses (OVs) are one kind of virus that selectively infects and lyses cancer cells, which is considered to be a new anticancer therapy with biological and immune-based approaches. Enterovirus 71 (EV71), belonging to the enterovirus genus in the family Picornaviridae, is a single positive-stranded RNA virus. EV71 is transmitted in a fetal-oral route and infects gastrointestinal tract in infants. Here, EV71 is exploited to be a novel oncolytic virus in colorectal cancer. It is revealed that EV71 infection can selectively cause colorectal cancer cells cytotoxicity but not primary intestinal epithelial cells. Consistently, EV71 injection significantly inhibits tumor growth in nude mice xenografted colorectal cancer cells. In detail, EV71 infects colorectal cancer cells to repress the expression of Ki67 and B-cell leukemia 2 (Bcl-2) leading to the inhibition of cell proliferation, while activating the cleavage of poly-adenosine diphosphatase-ribose polymerase and Caspase-3 protein resulting in the promotion of cell apoptosis. The findings demonstrate the oncolytic feature of EV71 in CRC treatment and may provide a potential clue for clinical anticancer therapy.

Persistent Coxsackievirus B3 Infection in Pancreatic Ductal Cells In Vitro Downregulates Cellular Polyamine Metabolism.

Picornaviruses infect a wide variety of cell types in vitro, with rapid replication kinetics and pronounced cytopathic effect. Coxsackievirus B3 (CVB3) can also establish a persistent infection in vivo that can lead to pathology, including dilated cardiomyopathy and myocarditis. One model system to study persistent infection is the pancreatic ductal cell line PANC-1, which CVB3 infects and is maintained indefinitely. We have characterized this model for CVB3 infection to study persistent infection for over 6 months. We find that CVB3 rapidly replicates within PANC-1 cells without robust cytopathic effect, and after 1 month in culture, titers stabilize. We find that infection does not significantly affect cellular viability. Persistent virus reverts to lytic infection when transferred to Huh7 or Vero cells. We find that persistent CVB3 adapts to PANC-1 cells via mutation of its capsid proteins and, curiously, the viral polymerase (3Dpol) to generate a high-fidelity polymerase. Persistent infection is associated with reduced cleavage of eIF4G, reduced plaque size, and decreasing particle infectivity. We further find that polyamine metabolism is altered in persistently infected cells, with the rate-limiting enzyme ornithine decarboxylase (ODC1) reduced in translation. We further find that targeting polyamine synthesis reduces persistent infection without affecting the viability of the PANC-1 cells. Finally, we find that viral fidelity is essential to maintaining CVB3 infection, and targeting viral fidelity reduces persistent virus infection. Together, these data highlight a novel role for polyamines and fidelity in persistent CVB3 infection and suggest avenues for therapeutic development to target persistent infection. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease, including cardiomyopathies. Viruses like coxsackievirus B3 (CVB3) can cause tissue damage by lytically infecting cells; however, CVB3 can also persistently infect, which has been associated with several pathologies. Studying persistent infection in vitro is challenging, as CVB3 lytically infects most cellular model systems. Here, we show that CVB3 establishes persistent infection in pancreatic ductal cells in vitro, similar to prior studies on other coxsackieviruses. We also show that this infection results in adaptation of the virus to these cells, as well as changes to cellular metabolism of polyamines.

The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D.

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

Bioaccumulation Dynamic by Crassostrea gigas Oysters of Viruses That Are Proposed as Surrogates for Enteric Virus Contamination in Environmental Samples.

Oysters are filter-feeders and retain sewage-derived pathogens in their organs or tissues. Since most enteric viruses involved in outbreaks cannot grow in cell culture, studies using viral surrogate models are essential. Some species are proposed as surrogates for enteric viruses in environmental samples, including in bivalve mollusk samples, such as murine norovirus type 1 (MNV-1) and somatic (as φX) or F-specific coliphages (as MS2) bacteriophages. This study evaluated the tissue distribution of viral surrogates for enteric virus contamination after their bioaccumulation by Crassostrea gigas. Oyster tissues were analyzed for the distribution of viral surrogates (MNV-1, φX-174, and MS2) in digestive tissue (DT), gills (GL), and mantle (MT) after 4, 6, and 24 h of experimental bioaccumulation. MNV-1 had higher counts at 6 h in DT (1.2 × 103 PFU/g), followed by GL and MT (9.5 × 102 and 3.8 × 102 PFU/g, respectively). The bacteriophage φX-174 had a higher concentration in the MT at 4 and 6 h (3.0 × 102 PFU/g, in both) and MS2 in the GL after 24 h (2.2 × 102 PFU/g). The bioaccumulation pattern of MNV-1 by oysters was similar to the other enteric viruses (more in DT), while that of phages followed distinct patterns from these. Since the MNV-1 is bioaccumulated by C. gigas and is adapted to grow in cell culture, it is an important tool for bioaccumulation and viral inactivation tests in oysters. Although bacteriophage bioaccumulation was not similar to enteric viruses, they can be indicated for viral bioaccumulation analysis, analyzing MT and GL, since they do not bioaccumulate in DT.

Anti- IL-22 neutralizing antibodies decrease inflammation lesions and reduce mortality in enterovirus 71-infected mice.

Hand, foot, and mouth disease (HFMD) can cause fatal encephalitis in 0-5-year-old infants and children. There is no effective antiviral drug available to treat HFMD caused by enterovirus 71 (EV71). Our study investigates the relationship between levels of IL-22 expression and the severity of disease after EV71 infection in a mouse model. Anti-IL-22 neutralizing antibodies were tested in EV71-infected mice of different ages. Our results show that anti-IL-22 neutralizing antibodies can effectively reduce mortality in EV71-infected mice. Anti-IL-22 neutralizing antibody effectively reduced various EV71-associated symptoms indicating promising potential of this therapeutic effector in patients with EV71-associated HFMD.

Persistent coxsackievirus B infection and pathogenesis of type 1 diabetes mellitus.

Enteroviruses are believed to trigger or accelerate islet autoimmunity in genetically susceptible individuals, thereby resulting in loss of functional insulin-producing ß-cells and type 1 diabetes mellitus (T1DM). Although enteroviruses are primarily involved in acute and lytic infections in vitro and in vivo, they can also establish a persistent infection. Prospective epidemiological studies have strongly associated the persistence of enteroviruses, especially coxsackievirus B (CVB), with the appearance of islet autoantibodies and an increased risk of T1DM. CVB can persist in pancreatic ductal and ß-cells, which leads to structural or functional alterations of these cells, and to a chronic inflammatory response that promotes recruitment and activation of pre-existing autoreactive T cells and ß-cell autoimmune destruction. CVB persistence in other sites, such as the intestine, blood cells and thymus, has been described; these sites could serve as a reservoir for infection or reinfection of the pancreas, and this persistence could have a role in the disturbance of tolerance to ß-cells. This Review addresses the involvement of persistent enterovirus infection in triggering islet autoimmunity and T1DM, as well as current strategies to control enterovirus infections for preventing or reducing the risk of T1DM onset.

Host Restrictive Factors Are the Emerging Storm Troopers Against Enterovirus: A Mini-Review.

Enterovirus infection continues to be a global health problem. The lack of specific drugs and broad-spectrum vaccines means an urgent need to develop effective strategies against enteroviruses. Host restrictive factors are a class of intrinsic host antiviral factors that have been broadly defined and investigated during HIV infections and have great significance for drug development and treatment design. In recent years, the essential role of host restrictive factors in regulating enteroviral infections has been gradually recognized and investigated. An increasing number of studies have shown that host-restrictive factors regulate multiple steps in the life cycle of enteroviruses. This mini-review discusses the restrictive factors against enteroviruses, their antiviral mechanism, and the arms race between them and enteroviruses. We also summarise the pathways that enteroviruses use to impair host antiviral signals. This mini-review characterizes the essential role of host restriction factors in enterovirus infections, which provides ideas and potential targets for antiviral drug design by regulating host restrictive factors. It also reveals potential future research on the interplay between host restrictive factors and enteroviruses.

Bacterial matrix metalloproteases and serine proteases contribute to the extra-host inactivation of enteroviruses in lake water.

Enteroviruses are ubiquitous contaminants of surface waters, yet their fate in presence of microbial congeners is poorly understood. In this work, we investigated the inactivation of Echovirus-11 (E11) and Coxsackievirus-A9 (CVA9) by bacteria isolated from Lake Geneva. Incubation of E11 or CVA9 in biologically active lake water caused inactivation of 2- and 4-log10, respectively, within 48 h. To evaluate the antiviral action of individual bacterial species, we isolated 136 bacterial strains belonging to 31 genera from Lake Geneva. The majority of isolates (92) induced decay of at least 1.5-log10 of CVA9, whereas only 13 isolates induced a comparable inactivation on E11. The most extensive viral decay was induced by bacterial isolates producing matrix metalloproteases (MMPs). Correspondingly, the addition of a specific MMP inhibitor to lake water reduced the extent of inactivation for both viruses. A lesser, though significant protective effect was also observed with inhibitors of chymotrypsin-like or trypsin-like proteases, suggesting involvement of serine proteases in enterovirus inactivation in natural systems. Overall, we demonstrate the direct effect of bacterial proteases on the inactivation of enteroviruses and identify MMPs as effective controls on enteroviruses' environmental persistence.

Homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium.

Interferon-lambda (IFN-λ) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-λ receptor (Ifnlr1), and is associated with IFN-λ production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

Enterovirus Infections in Solid Organ Transplant Recipients: a Clinical Comparison from a Regional University Hospital in the Netherlands.

Enterovirus infections are known to cause a diverse range of illnesses, even in healthy individuals. However, information detailing enterovirus infections and their severity in immunocompromised patients, such as transplant recipients, is limited. We compared enterovirus infections in terms of genotypes, clinical presentation, and severity between transplant and nontransplant patients. A total of 264 patients (38 transplant recipients) with 283 enterovirus infection episodes were identified in our hospital between 2014 and 2018. We explored the following factors associated with enterovirus infections: clinical presentation and diagnosis on discharge, length of hospital stay, symptom persistence, and infection episodes in both children and adults. We observed some differences in genotypes between patients, with enterovirus group C occurring mainly in transplant recipients (P < 0.05). EV-associated gastrointestinal infections were more common in patients with a transplant (children [71%] and adults [46%]), compared to nontransplant patients (P < 0.05). Additionally, nontransplant patients had a higher number of hospital stays (P < 0.05), potentially reflecting more severe disease. However, transplant patients were more likely to have symptom persistence after discharge (P < 0.05). Finally, children and adults with a transplant were more likely to have additional enterovirus infection episodes (P < 0.05). In our cohort, enterovirus infections did not seem to be more severe after transplantation; however, patients tended to present with different clinical symptoms and had genotypes rarely found in nontransplant recipients. IMPORTANCE Despite the high prevalence of enteroviruses in the community and the increasing demand for transplants from an aging population, knowledge on enteroviruses in solid organ transplant recipients is currently limited. Transplant recipients represent a significant patient population and require additional considerations in patient management, particularly as they have an increased risk of disease severity. Enteroviruses are known to cause significant morbidity, with a diverse range of clinical presentation from over 100 different genotypes. In this study, we aimed to provide a more comprehensive overview of enteroviral infections in transplant recipients, compared to nontransplant patients, and to bridge some gaps in our current knowledge. Identifying potential clinical manifestation patterns can help improve patient management following enterovirus infections.

Special Issue "Enteroviruses 2021".

Enteroviruses are a group of clinically relevant RNA viruses that causes human diseases [...].

Conserved Residues Adjacent to ß-Barrel and Loop Intersection among Enterovirus VP1 Affect Viral Replication: Potential Target for Anti-Enteroviral Development.

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate.

Regulation of host factor γ-H2AX level and location by enterovirus A71 for viral replication.

Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent in vitro and in vivo data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.