Abundant Neutrophils-initiated Acute Myocardial Injury Following Coxsackievirus A6 Infection.

Coxsackievirus (CV) A6 is currently considered as a predominant pathogen of hand, foot, and mouth disease (HFMD), and is occasionally linked to myocardial injury. We first established a mouse model of CVA6-induced myocardial injury. Next, we analyzed the immune cell phenotypes CVA6-infected mice hearts by FACS, and found that CVA6 led to massive neutrophils infiltration, suggesting their potential link with the occurrence of myocardial injury. We further used either αGr-1 or αLy6G antibody to deplete neutrophils, and found that neutrophil-depleted animals showed decreased cardiac enzymes, lower degree pathology in hearts, and reduced inflammatory cytokine production compared to isotype controls. Finally, we confirmed the involvement of neutrophils in myocardial injury of clinical patients with severe HFMD. Overall, our study suggests that excessive neutrophils contribute to myocardial injury caused by CVA6 infection, which provides new insight into myocardial injury during the development of HFMD severity and the outcome of immune cell-mediated therapies.

Mechanism of RNA m6 A methylation modification regulating NLRP3 inflammasome activation for hand, foot, and mouth disease progression.

Some children infected with hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) progressed to severe disease with various neurological complications in the short term, with a poor prognosis and high mortality. Studies had revealed that RNA N6 -methyladenosine (m6 A) modification had a significant impact on EV71 replication, but it was unknown how m6 A modification regulated the host cell's innate immune response brought on by EV71 infection. We used MeRIP-seq (methylation RNA immunoprecipitation sequencing), RNA-seq (RNA sequencing), cell transfection, and other techniques. MeRIP-seq and RNA-seq results showed the m6 A methylation modification map of control and EV71-infected groups of RD cells. And multilevel validation indicated that decreased expression of demethylase FTO (fat mass and obesity-associated protein) was responsible for the elevated total m6 A modification levels in EV71-infected RD cells and that thioredoxin interacting protein (TXNIP) may be a target gene for demethylase FTO action. Further functional experiments showed that demethylase knockdown of FTO promoted TXNIP expression, activation of NLRP3 inflammasome and promoted the release of proinflammatory factors in vitro, and the opposite result occurred with demethylase FTO overexpression. And further tested in an animal model of EV71 infection in vitro, with results consistent with in vitro. Our findings elucidated that depletion of the demethylase FTO during EV71 infection increased the m6 A modification level of TXNIP mRNA 3' untranslated region (UTR), enhancing mRNA stability, and promoting TXNIP expression. Consequently, the NLRP3 inflammasome was stimulated, leading to the release of proinflammatory factors and facilitating HFMD progression.

Local immune dysregulation and subsequent inflammatory response contribute to pulmonary edema caused by Enterovirus infection in mice.

Pulmonary edema that comes on suddenly is the leading cause of mortality in hand-foot-and-mouth disease (HFMD) patients; however, its pathogenesis is still largely unclear. A range of research suggest immunopathogenesis during the occurrence of pulmonary edema in severe HFMD patients. Herein, to investigate the potential mechanism of immune dysregulation in the development of pulmonary edema upon Enterovirus (EV) infection, we established mouse infection models for Enteroviruses (EVs) including Coxsackievirus (CV) A6, Enterovirus A71 (EVA71), and CVA2 exhibiting a high incidence of pulmonary edema. We found that EVs infection induced an immune system disorder by reducing the numbers of pulmonary and circulatory T cells, B cells, macrophages, and monocytes and increasing the numbers of lung neutrophils, myeloid-derived suppressor cells (MDSCs), and activated T cells. In addition, the concentrations of C-X-C motif chemokine ligand 1 (CXCL-1), tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and interleukin 6 were increased in EV-infected lungs. Moreover, we found that EVs replication in mice lungs lead to apoptosis of lung cells and degradation of tight junction proteins. In conclusion, EVs infection likely triggered a complexed immune defense mechanism and caused dysregulation of innate immune cells (MDSCs, neutrophils, monocytes, and macrophages) and adaptive cellular immunity (B cells, T cells). This dysregulation increased the release of cytokines and other inflammatory factors from activated immune-related cells and caused lung barrier damage and pulmonary edema.

Current status of hand-foot-and-mouth disease.

Hand-foot-and-mouth disease (HFMD) is a viral illness commonly seen in young children under 5 years of age, characterized by typical manifestations such as oral herpes and rashes on the hands and feet. These symptoms typically resolve spontaneously within a few days without complications. Over the past two decades, our understanding of HFMD has greatly improved and it has received significant attention. A variety of research studies, including epidemiological, animal, and in vitro studies, suggest that the disease may be associated with potentially fatal neurological complications. These findings reveal clinical, epidemiological, pathological, and etiological characteristics that are quite different from initial understandings of the illness. It is important to note that HFMD has been linked to severe cardiopulmonary complications, as well as severe neurological sequelae that can be observed during follow-up. At present, there is no specific pharmaceutical intervention for HFMD. An inactivated Enterovirus A71 (EV-A71) vaccine that has been approved by the China Food and Drug Administration (CFDA) has been shown to provide a high level of protection against EV-A71-related HFMD. However, the simultaneous circulation of multiple pathogens and the evolution of the molecular epidemiology of infectious agents make interventions based solely on a single agent comparatively inadequate. Enteroviruses are highly contagious and have a predilection for the nervous system, particularly in child populations, which contributes to the ongoing outbreak. Given the substantial impact of HFMD around the world, this Review synthesizes the current knowledge of the virology, epidemiology, pathogenesis, therapy, sequelae, and vaccine development of HFMD to improve clinical practices and public health efforts.

Essential Role of Non-Coding RNAs in Enterovirus Infection: From Basic Mechanisms to Clinical Prospects.

Enteroviruses (EVs) are common RNA viruses that can cause various types of human diseases and conditions such as hand, foot, and mouth disease (HFMD), myocarditis, meningitis, sepsis, and respiratory disorders. Although EV infections in most patients are generally mild and self-limiting, a small number of young children can develop serious complications such as encephalitis, acute flaccid paralysis, myocarditis, and cardiorespiratory failure, resulting in fatalities. Established evidence has suggested that certain non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) are involved in the occurrence and progression of many human diseases. Recently, the involvement of ncRNAs in the course of EV infection has been reported. Herein, the authors focus on recent advances in the understanding of ncRNAs in EV infection from basic viral pathogenesis to clinical prospects, providing a reference basis and new ideas for disease prevention and research directions.

The Disruption of the Endothelial Barrier Contributes to Acute Lung Injury Induced by Coxsackievirus A2 Infection in Mice.

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1ß, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

Mast cells contribute to Enterovirus 71 infection-induced pulmonary edema in neonatal mice.

Enterovirus (EV) 71 infection has been widely acknowledged as the leading cause of severe hand, foot and mouth disease (HFMD), which may rapidly lead to fatal pulmonary edema. In this study, we established a mouse model for EV71 infection exhibiting high incidence of severe symptoms with pulmonary edema. Mast cells (MCs) accumulation, activation and allergic inflammation were found in the brains, lungs and skeletal muscle of mice after EV71 infection, especially in the lungs of mice. Levels of histamine, platelet-activating factor (PAF), interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor-α (TNF-α), nitric oxide (NO), endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and noradrenaline (NA) were increased in EV71-infected lungs. In addition, EV71 infection reduced the number of pulmonary T cells, dendritic cells (DCs) and monocytes, and increased the number of lung eosinophils, Tregs and MCs. MCs number and tryptase expression in target organs or tissues posed a trend towards an increase from control to severe mice. There were positive correlations between MCs number in the brains (r = 0.701, P = 0.003), lungs (r = 0.802, P < 0.0001), skeletal muscles (r = 0.737, P = 0.001) and mean clinical score. Thus, our results suggested that MCs contributed to the pulmonary edema during EV71 infection.

Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice.

BACKGROUND: Enterovirus (EV) infection has been a serious health issue in Asia-Pacific region. It has been indicated that the occurrence of fatal hand foot and mouth disease (HFMD) cases following EV71 infection is mainly attributed to pulmonary edema. However, the development of pulmonary disorders after EV71 infection remains largely unknown. To establish an EV71-infected animal model and further explore the underlying association of central nervous system (CNS) invasion with pulmonary edema, we isolated a clinical source EV71 strain (ZZ1350) from a severe case in Henan Province. METHODS: We evaluated the cytotoxicity of ZZ1350 strain and the susceptibility in 3-day-old BALB/c mice with intraperitoneal, intracerebral and intramuscular inoculation. Various histopathological and immunohistochemical techniques were applied to determine the target organs or tissue damage after infection. Correlation analysis was used to identify the relationship between CNS injury and pulmonary disorders. RESULTS: Our experimental results suggested that ZZ1350 (C4 subtype) had high cytotoxicity against African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells and neonatal BALB/c mice were highly susceptible to the infection with ZZ1350 through three different inoculation routes (2 × 106 pfu/mouse) exhibiting severe neurological and respiratory symptoms that were similar to clinical observation. Viral replication was found in brain, spinal cord, skeletal muscle, lung, spleen, liver, heart of infected mice and these sections also showed histopathological changes. We found that brain histology score was positive correlated with lung histology score in total experimental mice and mice under the three inoculation routes (P < 0.05). At the same time, there were positive correlations between spinal cord score and lung score in total experimental mice and mice with intracerebral inoculation (P < 0.05). CONCLUSIONS: ZZ1350 strain is effective to establish animal model of EV71 infection with severe neurological and respiratory symptoms. The development of pulmonary disorders after EV71 infection is associated with severity of CNS damage.

Application and Comparison of Laboratory Parameters for Forecasting Severe Hand-Foot-Mouth Disease Using Logistic Regression, Discriminant Analysis and Decision Tree.

BACKGROUND: In recent years, the prevalence of hand-foot-mouth disease (HFMD) in China and some other countries has caused worldwide concern. Mild cases tend to recover within a week, while severe cases may progress rapidly and tend to have bad outcome. Since there is no vaccine for HFMD and anti-inflammatory treatment is not ideal. In this study, we aimed to establish a valid forecasting model for severe HFMD using common laboratory parameters. METHODS: Retrospectively, 77 severe HFMD cases from Zhengzhou Children's hospital in the peaking period between years 2013 to 2015 were collected, with 77 mild HFMD cases in the same area. The study recorded common laboratory parameters to assist in establishment of the severe HFMD model. After screening the important variables using Mann-Whitney U test, the study also matched the logistic regression (LR), discriminant analysis (DA), and decision tree (DT) to make a comparison. RESULTS: Compared with that of the mild group, serum levels of WBC, PLT, PCT, MCV, MCH, LCR, SCR, LCC, GLO, CK-MB, K, S100, and B in the severe group were higher (p < 0.05), while MCR, EOR, BASOR, SCC, MCC, EO, BASO, NA, CL, T, Th, and Th/Ts were lower (p < 0.05). Five indicators including MCR, LCC, Th, CK-MB, and CL were screened out by LR and the same for DA, and five variables including EO, LCC, CL, GLO, and MCC screened out by DT. The area under the curve (AUC) of LR, DA, and DT was 0.805, 0.779 and 0.864, respectively. CONCLUSIONS: The findings were that common laboratory indexes were effectively used to distinguish the mild HFMD cases and severe HFMD cases by LR, DA, and DT, and DT had the best classification effect with an AUC of 0.864.

Epidemiology and etiology of hand, foot, and mouth disease in Zhengzhou, China, from 2009 to 2021.

Background: Hand, foot, and mouth disease (HFMD) is a common childhood infectious disease caused by a variety of enteroviruses (EVs). To explore the epidemiological characteristics and etiology of HFMD in Zhengzhou, China, we conducted a systematic analysis of HFMD surveillance data from Zhengzhou Center for Disease Control and Prevention from January 2009 to December 2021 (https://wjw.zhengzhou.gov.cn/). Methods: Surveillance data were collected from Zhengzhou Center for Disease Control and Prevention from January 2009 to December 2021 (https://wjw.zhengzhou.gov.cn/). Cases were analyzed according to the time of onset, type of diagnosis, characteristics, viral serotype, and epidemiological trends. Results: We found that the primary causative agent responsible for the HFMD outbreaks in Zhengzhou was Enterovirus A71 (EVA-71) (48.56%) before 2014. After 2015, other EVs gradually became the dominant strains (57.68%). The data revealed that the HFMD epidemics in Zhengzhou displayed marked seasonality, with major peaks occurring from April to June, followed by secondary peaks from October to November, except in 2020. Both the severity and case-fatality ratio of HFMD decreased following the COVID-19 pandemic (severity ‰: 13.46 vs. 0.17; case-fatality ‰: 0.21 vs. 0, respectively). Most severe cases were observed in patients aged 1 year and below, accounting for 45.81%. Conclusions: Overall, the incidence rate of HFMD decreased in Zhengzhou following the introduction of the EVA-71 vaccine in 2016. However, it is crucial to acknowledge that HFMD prevalence continues to exhibit a distinct seasonal pattern and periodicity, and the occurrence of other EV infections poses a new challenge for children's health.

Recent advances in exploiting carrageenans as a versatile functional material for promising biomedical applications.

Carrageenans are a group of biopolymers widely found in red seaweeds. Commercial carrageenans have been traditionally used as emulsifiers, stabilizers, and thickening and gelling agents in food products. Carrageenans are regarded as bioactive polysaccharides with disease-modifying and microbiota-modulating activities. Novel biomedical applications of carrageenans as biocompatible functional materials for fabricating hydrogels and nanostructures, including carbon dots, nanoparticles, and nanofibers, have been increasingly exploited. In this review, we describe the unique structural characteristics of carrageenans and their functional relevance. We summarize salient physicochemical features, including thixotropic and shear-thinning properties, of carrageenans. Recent results from clinical trials in which carrageenans were applied as both antiviral and antitumor agents and functional materials are discussed. We also highlight the most recent advances in the development of carrageenan-based targeted drug delivery systems with various pharmaceutical formulations. Promising applications of carrageenans as a bioink material for 3D printing in tissue engineering and regenerative medicine are systematically evaluated. We envisage some key hurdles and challenges in the commercialization of carrageenans as a versatile material for clinical practice. This comprehensive review of the intimate relationships among the structural features, unique rheological properties, and biofunctionality of carrageenans will provide novel insights into their biomedicine application potential.

Hand, Foot, and Mouth Disease Challenges and Its Antiviral Therapeutics.

Hand, Foot, and Mouth Disease (HFMD) is an infectious disease caused by enteroviruses (EVs) and is extremely contagious and prevalent among infants and children under 5 years old [...].

Arising Concerns of Atypical Manifestations in Patients with Hand, Foot, and Mouth Disease.

Hand, foot, and mouth disease (HFMD) is a mild exanthematous, febrile disease, but it also remains a threat to global public health. HFMD is characterized by a brief febrile illness in children and with a typical skin rash of the hand and foot, with or without mouth ulcers. However, the morphology and distribution of vesicles, as well as accompanying symptoms, are varied among atypical HFMD. An upsurge in atypical presentations of HFMD caused by Coxsackievirus A6 (CVA6), including Gianotti-Crosti-like eruptions, eczema coxsackium, petechial/purpuric eruption, and vesiculobullous exanthema, can be difficult to diagnose clinically as it may mimic other severe skin diseases, such as eczema herpeticum, varicella, disseminated zoster, and erythema multiforme major. The recognition of the distinguishing features of atypical HFMD is vital for an accurate and timely diagnosis, as is initiating appropriate laboratory evaluation and supportive care. Clinicians must identify the wide range of cutaneous and mucosal alterations caused by atypical HFMD. A systemic, high-quality overview of atypical HFMD is needed for advances in better strategies for clinical diagnosis and treatment. Hence, this review is aimed at summarizing the available data on clinical investigations and differential diagnostics to provide a scientific guide for the timely diagnosis of HFMD for preventing serious complications.

TBK1 and IRF3 are potential therapeutic targets in Enterovirus A71-associated diseases.

BACKGROUND: Enterovirus A71 (EV-A71) is an important causative agent of hand-foot-and-mouth disease (HFMD) associated with enormous healthcare and socioeconomic burden. Although a range of studies about EV-A71 pathogenesis have been well described, the underlying molecular mechanism in terms of innate immune response is still not fully understood, especially the roles of TANK-binding kinase 1 (TBK1) and interferon-regulatory factor 3 (IRF3). METHODOLOGY/PRINCIPAL FINDINGS: Here, we applied TBK1 inhibitor and IRF3 agonist, for the first time, to evaluate the antiviral activities of TBK1 and IRF3 in vivo. We found that, through regulating EV-A71-induced type I interferon (IFN) response, IRF3 agonist effectively alleviated EV-A71-induced illness, while TBK1 inhibitor aggravated disease progression. In addition, EV-A71 replication was suppressed in EVA-71-infected mice administrated with IRF3 agonist. On the other hand, more severe pathological alterations of neuronal degeneration, muscle fiber breaks, fractured or fused alveolar walls, and diffuse congestion occurred in EVA-71-infected mice treated with TBK1 inhibitor administration. Furthermore, we determined the concentrations of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), IL-1ß, monocyte chemotactic protein-1 (MCP-1), and IL-10 in both lungs and brains of mice and found that TBK1 inhibitor promoted EV-A71-induced inflammatory response, while IRF3 agonist alleviated it, which was consistent with clinical manifestations and pathological alterations. CONCLUSIONS: Collectively, our findings suggest that TBK1 and IRF3 are potential therapeutic targets in EV-A71-induced illness.

A mouse model and pathogenesis study for CVA19 first isolated from hand, foot, and mouth disease.

ABSTRACTCoxsackievirus A19 (CVA19) is a member of Enterovirus (EV) C group in the Picornaviridae family. Recently, we reported a case of CVA19-infected hand, foot, and mouth disease (HFMD) for the first time. However, the current body of knowledge on the CVA19 infection, particularly the pathogenesis of encephalomyelitis and diarrhoea is still very limited, due to the lack of suitable animal models. Here, we successfully established a CVA19 mouse model via oral route based on 7-day-old ICR mice. Our results found the virus strain could directly infect the neurons, astrocytes of brain, and motor neurons of spinal cord causing neurological complications, such as acute flaccid paralysis. Importantly, viruses isolated from the spinal cords of infected mice caused severe illness in suckling mice, fulfilling Koch's postulates to some extent. CVA19 infection led to diarrhoea with typical pathological features of shortened intestinal villi, increased number of secretory cells and apoptotic intestinal cells, and inflammatory cell infiltration. Much higher concentrations of serum cytokines and more peripheral blood inflammatory cells in CVA19-infected mice indicated a systematic inflammatory response induced by CVA19 infection. Finally, we found ribavirin and CVA19 VP1 monoclonal antibody could not prevent the disease progression, but higher concentrations of antisera and interferon alpha 2 (IFN-α2) could provide protective effects against CVA19. In conclusion, this study shows that a natural mouse-adapted CVA19 strain leads to diarrhoea and encephalomyelitis in a mouse model via oral infection, which provides a useful tool for studying CVA19 pathogenesis and evaluating the efficacy of vaccines and antivirals.

Active inoculation with an inactivated Coxsackievirus A2 vaccine induces neutralizing antibodies and protects mice against lethal infection.

Coxsackievirus A2 (CVA2) is one of the causative agents of hand-foot-and-mouth disease (HFMD), which poses a great challenge for global public health. However, presently, there are no available commercial vaccines or antivirals to prevent CVA2 infection. Here, we present an inactivated Vero cell-based whole CVA2 vaccine candidate and evaluate its safety and efficacy in this study. Neonatal BALB/c mice were vaccinated at 5 and 7 days old, respectively, and then challenged with either homologous or heterologous strain of CVA2 at a lethal dose at 10 days old. The inactivated whole CVA2 vaccine candidate showed a high protective efficacy. Additionally, our inactivated vaccine stimulated the production of CVA2-specific IgG1 and IgG2a antibodies in vivo and high titers of neutralization antibodies (NtAbs) in the serum of immunized mice. Maternal immunization with the inactivated CVA2 vaccine provided full protection to pups against lethal infection. Compared with mice inoculated with only alum, the viral loads were decreased, and pathological changes were relieved in tissue samples of immunized mice. Moreover, the transcription levels of some genes related to cytokines (IFN-γ and TNF-α, MCP-1, IL-6, CXCL-10 etc.) were significantly reduced. The number of immune cells and levels of cytokines in peripheral blood of mice inoculated with only alum were higher than that of immunized mice. It is noteworthy that this vaccine showed a good cross-immunity efficacy against Enterovirus A71 (EVA71) challenge. In conclusion, our findings suggest that this experimental inactivated CVA2 vaccine is a promising component of polyvalent vaccines related to HFMD in the near future.

Laboratory Indicators for Identifying Hand, Foot, and Mouth Disease Severity: A Systematic Review and Meta-Analysis.

OBJECTIVE: The purpose of this study is to study laboratory indicators for the identification of hand, foot, and mouth disease (HFMD) severity. METHODS: We searched PubMed, Embase, and the Web of Science for literature that was published before May 2022. The main results are presented as forest plots. Subgroup analyses, sensitivity analyses, and publication bias were also performed. RESULTS: Our study indicated that white blood cells (WBC) (95%CI: 0.205-0.778), blood glucose (95%CI: 0.505-0.778), lymphocytes (95%CI: 0.072-0.239), creatinine (95%CI: 0.024-0.228), interleukin (IL)-2 (95%CI: 0.192-1.642), IL-6 (95%CI: 0.289-0.776), IL-8 (95%CI: 0.499-0.867), IL-10 (95%CI: 0.226-0.930), interferon-γ (IFN-γ) (95%CI: 0.193-2.584), tumor necrosis factor-α (TNF-α) (95%CI: 1.078-2.715), and creatine kinase MB isoenzyme (CK-MB) (95%CI: 0.571-1.459) were associated with an increased risk of HFMD severity, and the results of the sensitivity analysis of these indicators were stable and free of publication bias. CONCLUSIONS: Our results suggest that various deleterious immune and metabolic changes can increase the risk of HFMD severity, which can provide a basis for predicting the prognosis and useful evidence for clinicians to manage patients efficiently.

The CXCL10/CXCR3 Axis Promotes Disease Pathogenesis in Mice upon CVA2 Infection.

Coxsackievirus A2 (CVA2) is an emerging pathogen that results in hand-foot-and-mouth disease (HFMD) outbreaks. Systemic inflammatory response and central nervous system inflammation are the main pathological features of fatal HFMD. However, the immunopathogenesis of CVA2 infection is poorly understood. We first detected the transcriptional levels of 81 inflammation-related genes in neonatal mice with CVA2 infection. Remarkably, CVA2 induced higher expression of chemokine (C-X-C motif) ligand 10 (CXCL10) in multiple organs and tissues. CXCL10 acts through its cognate receptor chemokine (C-X-C motif) receptor 3 (CXCR3) and regulates immune responses. CXCL10/CXCR3 activation contributes to the pathogenesis of many inflammatory diseases. Next, we found CXCL10 and CXCR3 expression to be significantly elevated in the organs and tissues from CVA2-infected mice at 5 days postinfection (dpi) using immunohistochemistry (IHC). To further explore the role of CXCL10/CXCR3 in CVA2 pathogenesis, an anti-CXCR3 neutralizing antibody (αCXCR3) or IgG isotype control antibody was used to treat CVA2-infected mice on the same day as infection and every 24 h until 5 dpi. Our results showed that αCXCR3 therapy relieved the clinical manifestations and pathological damage and improved the survival rate of CVA2-infected mice. Additionally, αCXCR3 treatment reduced viral loads and reversed the proinflammatory cytokine (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α], and IL-1ß) expression, apoptosis, and inflammatory cell infiltration induced by CVA2. Collectively, our study presents evidence for the involvement of the CXCL10/CXCR3 axis in CVA2 pathogenesis. The activation of CXCL10/CXCR3 contributes to CVA2 pathogenesis by inducing apoptosis, proinflammatory cytokine expression, and inflammatory cell infiltration, which can be reversed by αCXCR3 therapy. This study provides new insight into the pathogenesis of HFMD, which has an important guiding significance for the treatment of HFMD. IMPORTANCE Systemic inflammatory response and central nervous system inflammation are the main pathological features of fatal HFMD cases. We detected the expression of 81 inflammation-related genes and found higher expression of CXCL10 in CVA2-infected mice. Next, we confirmed CXCL10/CXCR3 activation using immunohistochemistry and found that anti-CXCR3 neutralizing antibody (αCXCR3) therapy could relieve the clinical manifestations and pathological damage and improve the survival rate of CVA2-infected mice. Additionally, αCXCR3 treatment reduced viral loads and reversed the proinflammatory cytokine (IL-6, TNF-α, and IL-1ß) expression, apoptosis, and inflammatory cell infiltration induced by CVA2. Collectively, our study presents the first evidence for the involvement of the CXCL10/CXCR3 axis in CVA2 pathogenesis. The activation of CXCL10/CXCR3 contributes to CVA2 pathogenesis via inducing apoptosis, proinflammatory cytokine expression, and inflammatory cell infiltration, which can be reversed by αCXCR3 therapy. This study provides new insight into the pathogenesis of HFMD, which has an important guiding significance for the treatment of HFMD.

Pathological Features of Enterovirus 71-Associated Brain and Lung Damage in Mice Based on Quantitative Proteomic Analysis.

The outbreaks of enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) have emerged as an emergency of global health due to its association with fatal encephalitis and subsequent neurogenic pulmonary edema; however, the molecular characteristics and pathological features underlying EV71-associated encephalitis and pulmonary edema remain largely unknown. In this study, we performed a proteomic analysis of fresh brain and lung tissues from EV71-infected mice at 7 days post infection. We detected a perturbed expression of 148 proteins in the brain and 78 proteins in the lung after EV71 expression. Further analysis showed that the dysregulated proteins in the brain are involved in a variety of fundamental biological pathways, including complement and coagulation cascades, innate and adaptive immune responses, platelet activation, and nitrogen metabolism, and those proteins in the lung participate in innate and adaptive immune responses, phagosome, arginine biosynthesis, and hypoxia-inducible factor 1 signaling pathway. Our results suggested that immune activation, complement and coagulation dysfunction, platelet activation, imbalance of nitrogen metabolism, and hypoxia could be involved in the pathogenesis of EV71, which explains the major clinical manifestation of hyperinflammatory status of severe HFMD cases. Our study provides further understanding of the molecular basis of EV71 pathogenesis.

Pathogenesis Study of Enterovirus 71 Using a Novel Human SCARB2 Knock-In Mouse Model.

Enterovirus 71 (EV71) can cause a severe hand-foot-mouth disease in children. However, the precise mechanism of EV71-associated disease, particularly the neuropathogenesis and pulmonary disorder, is still not fully understood because no suitable animal models are available. The human scavenger receptor class B, member 2 (hSCARB2), is a cellular receptor for EV71. Here, we generated a novel knock-in (KI) mouse model using the CRISPR/Cas9 system to insert the hSCARB2 gene into the mouse Rosa26 locus to study the pathogenesis of EV71. The hSCARB2 KI mice infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia, paralysis, and death. Viral replication was detected in mainly astrocytes and a limited number of neurons and microglia, accompanied by gliosis. Vascular leakage and alveoli filled with erythrocytes were detected, suggesting that edema and hemorrhage, which are observed in human patients, also occurred in EV71-infected KI mice. In addition, proinflammatory cytokines and chemokines were significantly increased in the serum of infected KI mice. These pathological features of the KI mice after infection resembled those of EV71 encephalomyelitis in humans. Therefore, our KI mouse model is suitable to study the pathogenesis of EV71 and is of great significance for development of antiviral drugs and vaccines to treat or prevent EV71 infection.IMPORTANCE Enterovirus 71 (EV71) is associated with severe hand-foot-mouth disease. Recently, outbreaks of EV71 infection with high mortality have been reported in the Asia-Pacific region, posing a great challenge for global public health. To date, the precise mechanism of EV71-induced disease, particularly the neuropathogenesis and respiratory disorders, is still not fully understood because no suitable animal models are available. Human scavenger receptor class B, member 2 (hSCARB2), has been identified as a cellular receptor for EV71. Here, we introduce a novel CRISPR/Cas9-mediated hSCARB2 knock-in (KI) mouse model for the study of EV71 pathogenesis, which is of great significance for the development of antiviral drugs and vaccines.