Hand-Foot-and-Mouth Disease-Associated Enterovirus and the Development of Multivalent HFMD Vaccines.

Hand-foot-and-mouth disease (HFMD) is an infectious disease of children caused by more than 20 types of enteroviruses, with most cases recovering spontaneously within approximately one week. Severe HFMD in individual children develops rapidly, leading to death, and is associated with other complications such as viral myocarditis and type I diabetes mellitus. The approval and marketing of three inactivated EV-A71 vaccines in China in 2016 have provided a powerful tool to curb the HFMD epidemic but are limited in cross-protecting against other HFMD-associated enteroviruses. This review focuses on the epidemiological analysis of HFMD-associated enteroviruses since the inactivated EV-A71 vaccine has been marketed, collates the progress in the development of multivalent enteroviruses vaccines in different technical routes reported in recent studies, and discusses issues that need to be investigated for safe and effective HFMD multivalent vaccines.

A hSCARB2-transgenic mouse model for Coxsackievirus A16 pathogenesis.

BACKGROUND: Coxsackievirus A16 (CA16) is one of the neurotropic pathogen that has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD), but its pathogenesis is not yet clear. The limited host range of CA16 make the establishment of a suitable animal model that can recapitulate the neurological pathology observed in human HFMD more difficult. Because the human scavenger receptor class B, member 2 (hSCARB2) is a cellular receptor for CA16, we used transgenic mice bearing human SCARB2 and nasally infected them with CA16 to study the pathogenicity of the virus. METHODS: Coxsackievirus A16 was administered by intranasal instillation to groups of hSCARB2 transgenic mice and clinical signs were observed. Sampled at different time-points to document and characterize the mode of viral dissemination, pathological change and immune response of CA16 infection. RESULTS: Weight loss and virus replication in lung and brain were observed in hSCARB2 mice infected with CA16, indicating that these animals could model the neural infection process. Viral antigens were observed in the alveolar epithelia and brainstem cells. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes into the alveolar interstitial in lung and diffuse punctate hemorrhages in the capillaries of the brainstem. In addition, we detected the expression levels of inflammatory cytokines and detected high levels of interleukin IL-1ß, IL-6, IL-18, and IFN-γ in nasal mucosa, lungs and brain tissues. CONCLUSIONS: The hSCARB2-transgenic mice can be productively infected with CA16 via respiratory route and exhibited a clear tropism to lung and brain tissues, which can serve as a model to investigate the pathogenesis of CA16 associated respiratory and neurological disease.

Transcriptome analysis following enterovirus 71 and coxsackievirus A16 infection in respiratory epithelial cells.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.

Suppression of the toll-like receptor 7-dependent type I interferon production pathway by autophagy resulting from enterovirus 71 and coxsackievirus A16 infections facilitates their replication.

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/ß. 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.

An inactivated enterovirus 71 vaccine in healthy children.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children and may be fatal. A vaccine against EV71 is needed. METHODS: We conducted a randomized, double-blind, placebo-controlled phase 3 trial involving healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, China. Two doses of an inactivated EV71 vaccine or placebo were administered intramuscularly, with a 4-week interval between doses, and children were monitored for up to 11 months. The primary end point was protection against hand, foot, and mouth disease caused by EV71. RESULTS: A total of 12,000 children were randomly assigned to receive vaccine or placebo. Serum neutralizing antibodies were assessed in 549 children who received the vaccine. The seroconversion rate was 100% 4 weeks after the two vaccinations, with a geometric mean titer of 170.6. Over the course of two epidemic seasons, the vaccine efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.0) according to the intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.0) according to the per-protocol analysis. Adverse events, such as fever (which occurred in 41.6% of the participants who received vaccine vs. 35.2% of those who received placebo), were significantly more common in the week after vaccination among children who received the vaccine than among those who received placebo. CONCLUSIONS: The inactivated EV71 vaccine elicited EV71-specific immune responses and protection against EV71-associated hand, foot, and mouth disease. (Funded by the National Basic Research Program and others; ClinicalTrials.gov number, NCT01569581.).

Pathogenesis study of enterovirus 71 infection in rhesus monkeys.

Enterovirus 71 (EV71), a major pathogen that is responsible for causing hand-foot-and-mouth disease (HFMD) worldwide, is a member of the Human Enterovirus species A, family Picornaviridae. HFMD that is caused by EV71 is usually characterized by vesicular lesions on the skin and oral mucosa and high morbidity rates in children; additionally, occasional fatal cases have been reported involving brainstem encephalitis and myelitis associated with cardiopulmonary collapse. Although viral pathogenesis in humans is unclear, previous animal studies have indicated that EV71, inoculated via various routes, is capable of targeting and injuring the central nervous system (CNS). We report here the pathogenic process of systemic EV71 infection in rhesus monkeys after inoculation via intracerebral, intravenous, respiratory and digestive routes. Infection with EV71 via these routes resulted in different rates of targeting to and injury of the CNS. Intracerebral inoculation resulted in pulmonary edema and hemorrhage, along with impairment of neurons. However, intravenous and respiratory inoculations resulted in a direct infection of the CNS, accompanied by obvious inflammation of lung tissue, as shown by impairment of the alveoli structure and massive cellular infiltration around the terminal bronchioles and small vessels. These pathological changes were associated with a peak of viremia and dynamic viral distribution in organs over time in the infected monkeys. Our results suggest that the rhesus monkey model may be used to study not only the basic pathogenesis of EV71 viral infections, but also to examine clinical features, such as neurological lesions, in the CNS and pathological changes in associated organs.

Optimized development of a candidate strain of inactivated EV71 vaccine and analysis of its immunogenicity in rhesus monkeys.

Enterovirus type 71 (EV71) is one of the main etiologic agents responsible for periodic epidemics of hand-foot-and-mouth disease (HFMD). The prevention and control of EV71 epidemics with effective anti-viral agents and vaccines is very important for public health. Because the pathogenesis of EV71 in the human body is not completely clear and genetic variations in the virus during its replication are difficult to control, we have focused on the development of an inactivated whole-virus vaccine. In this study, we screened 16 strains isolated from different areas of China and selected one strain for the development of an inactivated EV71 vaccine. The results of our study suggest that the FY-23K-B strain, which is a candidate strain for an EV71 inactivated vaccine, satisfied the requirements of vaccine production in terms of genetic stability, biological activity, and good immunogenicity. The experimentally inactivated vaccine produced using this strain was capable of inducing an immune response and offered protection to rhesus monkeys against future virus attacks.

[Biological characters of a coxsackievirus B3 variant strain isolated from a hand-foot-mouth disease patient with severe clinical symptoms].

OBJECTIVE: To analyze the genetic and biological characters of a new isolate of coxsackievirus B3 (CoxB3), i.e. FY-19 strain, and investigate its mechanistic role in causing different clinical symptoms of hand-foot-mouth disease (HFMD). METHODS: FY-19 strain, isolated from a patient with severe clinical symptoms from Fuyang, China in 2008, was identified by the serological parameters via the Lim Benyesh-Melnick (LBM) antiserum pools. Its genotype was further characterized by sequencing the whole genome. And its biological characters were also examined by proliferation kinetic and pathogenetic analysis. RESULTS: FY-19 strain was identified as CoxB3 showing 23.0%, 16.5% and 32.1% difference with Nancy strain in 3'-, 5'-noncoding and coding regions respectively. FY-19 also showed a high homology with other HFMD-related CoxB3 isolates in China. But its homology with non-HFMD-related CoxB3 isolates was lower (13.5% and 25.0% difference in 3'-NCR and coding region respectively). The viral replication kinetic analysis suggested that the FY-19 proliferation increased rapidly and peaked at 14 hours post-infection. In pathological analysis, FY-19 strain induced mortal pathology in sucking mice. CONCLUSION: Differences in genetic and biological characters exist between FY-19 and Nancy strains. Further analysis on the pathogenesis of this variant may aid in elucidating the mechanisms of HFMD.

Comparative analysis of putative novel microRNA expression profiles induced by enterovirus 71 and coxsackievirus A16 infections in human umbilical vein endothelial cells using high-throughput sequencing.

Hand, foot and mouth disease (HFMD) is mainly caused by human enterovirus 71 (EV71) and coxsackievirus A16 (CA16), which circulate alternatively or together in epidemic areas. Although the two viruses exhibit genetic homology, their clinical manifestations have some discrepancies. However, the factors underlying these differences remain unclear. Herein, we mainly focused on the alterations and roles of putative novel miRNAs in human umbilical vein endothelial cells (HUVECs) following EV71 and CA16 infections using high-throughput sequencing. The results identified 247 putative novel, differentially expressed miRNAs, of which only 11 miRNAs presented an opposite trend between the EV71- and CA16-infected samples and were used for target prediction. Gene ontology (GO) and pathway enrichment analysis of the predicted targets displayed the top 15 significant biological processes, molecular functions, cell components and pathways. Subsequently, regulatory miRNA-predicted targets and miRNA-GO and miRNA-pathway networks were constructed to further reveal the complex regulatory mechanisms of the miRNAs during infection. Therefore, our data provide useful insights that will help elucidate the different host-pathogen interactions following EV71 and CA16 infections and may offer novel therapeutic targets for these infections.

microRNA-4516 Contributes to Different Functions of Epithelial Permeability Barrier by Targeting Poliovirus Receptor Related Protein 1 in Enterovirus 71 and Coxsackievirus A16 Infections.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) remain the predominant etiological agents of hand, foot, and mouth disease (HFMD). The clinical manifestations caused by the two viruses are obviously different. CV-A16 usually triggers a repeated infection, and airway epithelial integrity is often the potential causative factor of respiratory repeated infections. Our previous studies have demonstrated that there were some differentially expressed miRNAs involved in the regulation of adhesion function of epithelial barrier in EV-A71 and CV-A16 infections. In this study, we compared the differences between EV-A71 and CV-A16 infections on the airway epithelial barrier function in human bronchial epithelial (16HBE) cells and further screened the key miRNA which leaded to the formation of these differences. Our results showed that more rapid proliferation, more serious destruction of 16HBE cells permeability, more apoptosis and disruption of intercellular adhesion-associated molecules were found in CV-A16 infection as compared to EV-A71 infection. Furthermore, we also identified that microRNA-4516 (miR-4516), which presented down-regulation in EV-A71 infection and up-regulation in CV-A16 infection was an important regulator of intercellular junctions by targeting Poliovirus receptor related protein 1(PVRL1). The expressions of PVRL1, claudin4, ZO-1 and E-cadherin in CV-A16-infected cells were significantly less than those in EV-A71-infected cells, while the expressions of these proteins were subverted when pre-treated with miR-4516-overexpression plasmid in EV-A71 infected and miR-4516-knockdown plasmid in CV-A16 infected 16HBE cells. Thus, these data suggested that the opposite expression of miR-4516 in EV-A71 and CV-A16 infections might be the initial steps leading to different epithelial impairments of 16HBE cells by destroying intercellular adhesion, which finally resulted in different outcomes of EV-A71 and CV-A16 infections.

miR-1303 regulates BBB permeability and promotes CNS lesions following CA16 infections by directly targeting MMP9.

Coxsackievirus A16 (CA16) is a member of the Picornaviridae family and causes mild and self-limiting hand, foot, and mouth disease (HFMD) in infants and young children. CA16 infection can also progress to central nervous system (CNS) complications; however, the underlying mechanism by which CA16 penetrates the blood-brain barrier (BBB) and then causes CNS damage remains unclear. This study aimed to explore the mechanism of CA16 neurotropic tropism by establishing an in vitro BBB model with CA16 infection and an in vivo CA16 rhesus monkey infant infection model. The results showed that CA16 infection induced increased permeability of the BBB accompanied by upregulation of matrix metalloproteinase 9 (MMP9) expression. Subsequently, high-throughput miRNA sequencing technology and bioinformatics analysis revealed that miR-1303 may regulate BBB permeability by targeting MMP9. Next, we used dual-luciferase, qRT-PCR, and western blot assays to provide evidence of MMP9 targeting by miR-1303. Further experiments revealed that CA16 infection promoted the degradation of junctional complexes (Claudin4, Claudin5, VE-Cadherin, and ZO-1), likely by downregulating miR-1303 and upregulating MMP9. Finally, EGFP-CA16 infection could enter the CNS by facilitating the degradation of junctional complexes, eventually causing neuroinflammation and injury to the CNS, which was confirmed using the in vivo rhesus monkey model. Our results indicate that CA16 might penetrate the BBB and then enter the CNS by downregulating miR-1303, which disrupts junctional complexes by directly regulating MMP9 and ultimately causing pathological CNS changes. These results provide new therapeutic targets in HFMD patients following CA16 infection.

Different microRNA profiles reveal the diverse outcomes induced by EV71 and CA16 infection in human umbilical vein endothelial cells using high-throughput sequencing.

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.

Comparison analysis of microRNAs in response to EV71 and CA16 infection in human bronchial epithelial cells by high-throughput sequencing to reveal differential infective mechanisms.

Hand, foot, and mouth disease (HFMD) mainly caused by Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) infections which presented significantly different clinical manifestations. Nevertheless, the factors underlying these differences remain unclear. Recently, the functions of microRNAs (miRNAs) in pathogen-host interactions have been highlighted. Here, we performed comprehensive miRNA profiling in EV71- and CA16-infected human bronchial epithelial (16HBE) cells at multiple time points using high-throughput sequencing. The results showed that 154 known and 47 novel miRNAs exhibited remarkable differences in expression. Of these, 65 miRNAs, including 58 known and 7 novel miRNAs, presented opposite trends in EV71- and CA16-infected samples. Subsequently, we mainly focused on the 56 known differentially expressed miRNAs by further screening for targets prediction. GO and pathway analysis of these targets demonstrated that 18 biological processes, 7 molecular functions, 1 cellular component and 123 pathways were enriched. Among these pathways, Cadherin signalling pathway, Wnt signalling pathway and angiogenesis showed significant alterations. The regulatory networks of these miRNAs with predicted targets, GOs, pathways and transcription factors were determined, which suggested that miRNAs displayed intricate regulatory mechanisms during the infection phase. Consequently, we specifically analysed the hierarchical GO categories of the predicted targets involved in adhesion. The results indicated that the distinct changes induced by EV71 and CA16 infection may be partly linked to airway epithelial barrier function. Taken together, our data provide useful insights that help elucidate the different host-pathogen interactions following EV71 and CA16 infection and might offer novel therapeutic targets for these infections.

Dynamic Interaction of Enterovirus 71 and Dendritic Cells in Infected Neonatal Rhesus Macaques.

Enterovirus 71 (EV71) is one of the main pathogens responsible for hand, foot, and mouth disease (HFMD). Infection with EV71 can lead to severe clinical disease via extensive infections of either the respiratory or alimentary tracts in children. Based on the previous pathological study of EV71 infections in neonatal rhesus macaques, our work using this animal model and an EV71 chimera that expresses enhanced green fluorescent protein (EGFP-EV71) primarily explored where EV71 localizes and proliferates, and the subsequent initiation of the pathological process. The chimeric EGFP-EV71 we constructed was similar to the wild-type EV71 (WT-EV71) virus in its biological characteristics. Similar clinical manifestations and histo-pathologic features were equally displayed in neonatal rhesus macaques infected with either WT-EV71 or EGFP-EV71 via the respiratory route. Fluorescent signal tracing in tissues from the animals infected with EGFP-EV71 showed that EV71 proliferated primarily in the respiratory tract epithelium and the associated lymphoid tissues. Immunofluorescence and flow cytometry analyses revealed that EV71 was able to enter a pre-conventional dendritic cell (DC) population at the infection sites. The viremia identified in the macaques infected by WT-EV71 or EGFP-EV71 was present even in the artificial presence of a specific antibody against the virus. Our results suggest that EV71 primarily proliferates in the respiratory tract epithelium followed by subsequent entry into a pre-cDC population of DCs. These cells are then hijacked by the virus and they can potentially transmit the virus from local sites to other organs through the blood circulation during the infection process. Our results suggest that the EV71 infection process in this DC population does not interfere with the induction of an independent immune response against the EV71 infection in the neonatal macaques.

Pathologic and immunologic characteristics of coxsackievirus A16 infection in rhesus macaques.

Coxsackievirus A16 (CV-A16) causes human hand, foot and mouth disease, but its pathogenesis is unclear. In rhesus macaques, CV-A16 infection causes characteristic vesicles in the oral mucosa and limbs as well as viremia and positive viral loads in the tissues, suggesting that these animals reflect the pathologic process of the infection. An immunologic analysis indicated a defective immune response, which included undetectable neutralizing antibodies and IFN-γ-specific memory T-cells in macaques infected with CV-A16. Furthermore, existing neutralizing antibodies in macaques immunized with the inactivated vaccine were surprisingly unable to protect against a viral challenge despite the presence of a positive T-cell memory response against viral antigens. The virus was capable of infecting pre-conventional dendritic cells and replicating within them, which may correlate with the immunological characteristics observed in the animals.

Different microRNA alterations contribute to diverse outcomes following EV71 and CA16 infections: Insights from high-throughput sequencing in rhesus monkey peripheral blood mononuclear cells.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the predominant pathogens of hand, foot, and mouth disease (HFMD). Although these viruses exhibit genetic homology, the clinical manifestations caused by the two viruses have some discrepancies. In addition, the underlying mechanisms leading to these differences remain unclear. microRNAs (miRNAs) participate in numerous biological or pathological processes, including host responses to viral infections. Here, we focused on differences in miRNA expression patterns in rhesus monkey peripheral blood mononuclear cells (PBMCs) infected with EV71 and CA16 at various time points using high-throughput sequencing. The results demonstrated that 106 known and 13 novel miRNAs exhibited significant differences, and 32 key miRNAs among them for target prediction presented opposite trends in the EV71- and CA16-infected samples. GO and pathway analysis of the predicted targets showed enrichment in 14 biological processes, 10 molecular functions, 8 cellular components and 104 pathways. Subsequently, regulatory networks of miRNA-transcription factors, miRNA-predicted targets, miRNA-GOs and miRNA-pathways were constructed to reveal the complex regulatory mechanisms of miRNAs during the infection phase. Ultimately, we analysed hierarchical GO categories of the predicted targets involved in immune system processes, which indicated that the innate and adaptive immunity following EV71 and CA16 infections may be remarkably distinct. In conclusion, this report is the first describing miRNA expression profiles in PBMCs with EV71 and CA16 infections using high-throughput sequencing. Our findings could provide a valuable basis for further studies on the regulatory roles of miRNAs related to the different immune responses caused by EV71 and CA16 infections.

Clinical and Associated Immunological Manifestations of HFMD Caused by Different Viral Infections in Children.

Hand, foot, and mouth disease (HFMD), with vesiculae on the hands, feet and mouth, is an infectious disease caused by many viral pathogens. However, the differences of immune response induced by these pathogens are unclear. We compared the clinical manifestations and the levels of immunologic indicators from 60 HFMD patients caused by different viral pathogens to analyze the differences in the immune response. It was shown that Th2 cytokines (IL-4 and IL-10) increased significantly in EV71-infected children; Th1 cytokines (IL-2 and IFN-γ) rose in CA16-infected children; both Th1 and Th2 cytokines elevated in non-EVG-infected children; only individual cytokines (such as IL-10) went up in EVG-infected children. Meanwhile, the antibodies induced by viral infection could not cross-interfere between the different pathogens. These differences might be due to variations in the immune response induced by the individual pathogens or to the pathogenesis of the infections by the individual pathogens.

[Expression of Type-Ι Interferon Production Pathway-Related Genes Induced by Infection Due to Enterovirus 71 or Coxsackievirus A16 in Normal Human Airway Epithelial Cells].

Hand, foot, and mouth disease(HFMD)is caused by mainly enterovirus 71(EV-A71)and coxsackievirus A16(CV-A16),and is a serious healthcare problem worldwide.EV-A71 infection is thought to progress readily to serious complications whereas CV-A16 infection, in general, results in mild symptoms and presents repeatedly. However, the underlying mechanisms leading to these differences are not known. We compared changes in expression of type-I interferon(IFN-I)-related genes in normal human bronchial epithelial(16HBE) cells. Gene-expression levels of TLR3,MAVS,MDA5,MyD88,IRF7,IFNαand IFNßwere elevated significantly after EVA71 infection.MDA5expression was increased markedly, and that of TLR3 and IRF3was decreased obviously after CV-A16 infection, but that of MAVS,MyD88,IFNαand IFNßdid not show significant differences. Viral copy number and viral titers suggested that CV-A16 replicates more efficiently than EV-A71 in 16HBE.These results suggest that IFN-I production pathway-related genes in response to infection by EV-A71 and CV-A16 have notable discrepancies. Such information could shine a light on the different manifestations caused by EV-A71 and CV-A16,and the mechanism of repeat infection by CV-A16.

The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis.

The pathological manifestations of fatal cases of human hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) are characterized by inflammatory damage to the central nervous system (CNS). Here, the dynamic distribution of EV71 in the CNS and the subsequent pathological characteristics within different regions of neonatal rhesus macaque brain tissue were studied using a chimeric EV71 expressing green fluorescence protein. The results were compared with brain tissue obtained from the autopsies of deceased EV71-infected HFMD patients. These observations suggested that the virus was prevalent in areas around the blood vessels and nerve nuclei in the brain stem and showed a preference for astrocytes in the CNS. Interestingly, infected astrocytes within the in vivo and in vitro human and macaque systems exhibited increased expression of excitatory neurotransmitters and cytokines that also stimulated the neuronal secretion of the excitatory neurotransmitters noradrenalin and adrenalin, and this process most likely plays a role in the pathophysiological events that occur during EV71 infection.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus.