Recent Progress in Innate Immune Responses to Enterovirus A71 and Viral Evasion Strategies.

Enterovirus A71 (EV-A71) is a major pathogen causing hand, foot, and mouth disease (HFMD) in children worldwide. It can lead to severe gastrointestinal, pulmonary, and neurological complications. The innate immune system, which rapidly detects pathogens via pathogen-associated molecular patterns or pathogen-encoded effectors, serves as the first defensive line against EV-A71 infection. Concurrently, the virus has developed various sophisticated strategies to evade host antiviral responses and establish productive infection. Thus, the virus-host interactions and conflicts, as well as the ability to govern biological events at this first line of defense, contribute significantly to the pathogenesis and outcomes of EV-A71 infection. In this review, we update recent progress on host innate immune responses to EV-A71 infection. In addition, we discuss the underlying strategies employed by EV-A71 to escape host innate immune responses. A better understanding of the interplay between EV-A71 and host innate immunity may unravel potential antiviral targets, as well as strategies that can improve patient outcomes.

Coxsackievirus A7 and Enterovirus A71 Significantly Reduce SARS-CoV-2 Infection in Cell and Animal Models.

In this study, we investigated the features of co-infection with SARS-CoV-2 and the enterovirus vaccine strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-CoV-2 and LEV-8 or EV-A71 in the cell model showed that a competitive inhibitory effect for these viruses was especially significant against SARS-CoV-2. Pre-infection with enteroviruses in the animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tract and more rapid clearance of infectious SARS-CoV-2 from the lower respiratory tract. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection in the animals. Additionally, the histological data illustrated that co-infection with strain LEV8 of coxsackievirus A7 decreased the level of pathological changes induced by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of the antiviral immune response, which is associated with the significant inhibition of SARS-CoV-2 infection. These results demonstrate that there is significant viral interference between the studied strain LEV-8 of coxsackievirus A7 or enterovirus A71 and SARS-CoV-2 in vitro and in vivo.

NLRP3-dependent pyroptosis exacerbates coxsackievirus A16 and coxsackievirus A10-induced inflammatory response and viral replication in SH-SY5Y cells.

Coxsackievirus A16 (CV-A16) and coxsackievirus A10 (CV-A10), more commonly etiological agents of hand, foot and mouth disease (HFMD), are capable of causing severe neurological syndromes with high fatalities, but their neuropathogenesis has rarely been studied. Mounting evidence indicated that pyroptosis is an inflammatory form of cell death that might be widely involved in the pathogenic mechanisms of neurotropic viruses. Our study was designed to examine the effects of NLRP3-mediated pyroptosis in CV-A16- and CV-A10-induced inflammatory neuropathologic formation. In this work, it was showed that SH-SY5Y cells were susceptible to CV-A16 and CV-A10, and meanwhile their infections could result in a decreasing cell viability and an increasing LDH release as well as Caspase1 activation. Moreover, CV-A16 and CV-A10 infections triggered NLRP3-mediated pyroptosis and promoted the release of inflammatory cytokines. Additionally, activated NLRP3 accelerated the pyroptosis formation and aggravated the inflammatory response, but inhibited NLRP3 had a dampening effect on the above situation. Finally, it was further revealed that NLRP3 agonist enhanced the viral replication, but NLRP3 inhibitor suppressed the viral replication, suggesting that NLRP3-driven pyroptosis might support CV-A16 and CV-A10 production in SH-SY5Y cells. Together, our findings demonstrated a mechanism by which CV-A16 and CV-A10 induce inflammatory responses by evoking NLRP3 inflammasome-regulated pyroptosis, which in turn further stimulated the viral replication, providing novel insights into the pathogenesis of CV-A16 and CV-A10 infections.

EV71 5'UTR interacts with 3D protein affecting replication through the AKT-mTOR pathway.

BACKGROUND: EV71 is one of the important pathogens of Hand-foot-and-mouth disease (HFMD), which causes serious neurological symptoms. Several studies have speculated that there will be interaction between 5'UTR and 3D protein. However, whether 5'UTR interacts with the 3D protein in regulating virus replication has not been clarified. METHODS: Four 5'UTR mutation sites (nt88C/T, nt90-102-3C, nt157G/A and nt574T/A) and two 3D protein mutation sites (S37N and R142K) were mutated or co-mutated using virulent strains as templates. The replication of these mutant viruses and their effect on autophagy were determined. RESULTS: 5'UTR single-point mutant strains, except for EGFP-EV71(nt90-102-3C), triggered replication attenuation. The replication ability of them was weaker than that of the parent strain the virulent strain SDLY107 which is the fatal strain that can cause severe neurological complications. While the replication level of the co-mutant strains showed different characteristics. 5 co-mutant strains with interaction were screened: EGFP-EV71(S37N-nt88C/T), EGFP-EV71(S37N-nt574T/A), EGFP-EV71(R142K-nt574T/A), EGFP-EV71(R142K-nt88C/T), and EGFP-EV71(R142K-nt157G/A). The results showed that the high replicative strains significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autolysosomes. The low replicative strains had a low ability to regulate the autophagy of host cells. In addition, the high replicative strains also significantly inhibited the phosphorylation of AKT and mTOR. CONCLUSIONS: EV71 5'UTR interacted with the 3D protein during virus replication. The co-mutation of S37N and nt88C/T, S37N and nt574T/ A, R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway AKT-mTOR. The co-mutation of R142K and nt88C/T, and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the AKT-mTOR pathway and reduced the replication ability of the virus.

Mouse Scarb2 Modulates EV-A71 Pathogenicity in Neonatal Mice.

Enterovirus A71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease, which can progress to severe neurological disease. EV-A71 infects humans via the human scavenger receptor B2 (hSCARB2). It can also infect neonatal mice experimentally. Wild-type (WT) EV-A71 strains replicate primarily in the muscle of neonatal mice; however, susceptibility lasts only for a week after birth. Mouse-adapted (MA) strains, which can be obtained by serial passages in neonatal mice, are capable of infecting both muscle and neurons of the central nervous system. It is not clear how the host range and tropism of EV-A71 are regulated and why neonatal mice lose their susceptibility during development. We hypothesized that EV-A71 infection in neonatal mice is mediated by mouse Scarb2 (mScarb2) protein. Rhabdomyosarcoma (RD) cells expressing mScarb2 were prepared. Both WT and MA strains infected mScarb2-expressing cells, but the infection efficiency of the WT strain was much lower than that of the MA strain. Infection by WT and MA strains in vivo was abolished completely in Scarb2-/- mice. Scarb2+/- mice, in which Scarb2 expression was approximately half of that in Scarb2+/+ mice, showed a milder pathology than Scarb2+/+ mice after infection with the WT strain. The Scarb2 expression level in muscle decreased with aging, which was consistent with the reduced susceptibility of aged mice to infection. These results indicated that EV-A71 infection is mediated by mScarb2 and that the severity of the disease, the spread of virus, and the susceptibility period are modulated by mScarb2 expression. IMPORTANCE EV-A71 infects humans naturally but can also infect neonatal mice. The tissue tropism and severity of EV-A71 disease are determined by several factors, among which the virus receptor is thought to be important. We show that EV-A71 can infect neonatal mice using mScarb2. However, the infection efficiency of WT strains via mScarb2 is so low that an elevated virus-receptor interaction associated with mouse adaptation mutation and decrease in mScarb2 expression level during development modulate the severity of the disease, the spread of virus, and the susceptibility period in the artificial neonatal mice model.

PLAC8 promotes EV71 infected inflammatory lesion by disturbing Th-cell-related cytokines release in neonatal mouse.

Enterovirus 71 can cause severe hand, foot, and mouth disease (HFMD) in children. However, little is known about the mechanism of inflammatory disorders caused by EV71 infection and why severe cases are mainly children aged under-three. In current study, using mRNA microarray assay, the differential expression of Placenta-specific 8 (PLAC8) was identified in mice brain. In addition, we found that PLAC8 expression was down-regulated with age in mice lung tissues and human peripheral blood. Then, we further proved that PLAC8 could promote inflammation progress and disturb Th1/Th2/Th17/Treg related cytokines release after EV71 infection using PLAC8 plasmid over-expressed neonatal mouse model. Our data suggest that PLAC8 might play a crucial role in Th cell differentiation and inflammatory damage caused by EV71 infection in infants. Thus, our findings would help understand the causes of severe inflammatory injury in infants during EV71 infection, and provide new insights into the prevention and control of severe HFMD.

Susceptibility and cytokine responses of human neuronal cells to multiple circulating EV-A71 genotypes in India.

Enterovirus-A71 (EV-A71) associated Hand, foot and mouth disease (HFMD) is a highly contagious viral infection affecting children in Asia-Pacific region and has become a major threat to public health. Although several EV-A71 genotypes (C, D, and G) were isolated in India in recent years, no recognizable outbreak of EV-A71 caused HFMD, Acute Flaccid paralysis (AFP) or encephalitis have been reported so far. It is essential to study the pathogenicity or cell tropism of these Indian isolates in order to understand their tendency to cause disease. We investigated the susceptibility and cytokine responses of indigenous EV-A71 genotypes (D and G) isolated from cases of AFP and genotype C viruses isolated from cases of HFMD and encephalitis, in human cells in-vitro. Although all three EV-A71 genotypes could infect and replicate in human muscle and neuronal cells, the genotype D virus showed a delayed response in human neuronal cells. Quantification of cytokine secretion in response to these isolates followed by confirmation with gene expression assays in human neuronal cells revealed significantly higher secretion of pro-inflammatory cytokines TNF-α IL-8, IL-6, IP-10 (p < 0.001) in G genotype infected cells as compared to pathogenic C genotypes whereas the genotype D virus could not induce any of the inflammatory cytokines. These findings will help to better understand the host response to indigenous EV-A71 genotypes for management of future EV-A71 outbreaks in India, if any.

A Novel Attenuated Enterovirus A71 Mutant with VP1-V238A,K244R Exhibits Reduced Efficiency of Cell Entry/Exit and Augmented Binding Affinity to Sulfated Glycans.

Enterovirus A71 (EV-A71) is one of the major etiological agents of hand, foot, and mouth disease (HFMD), and infection occasionally leads to fatal neurological complications in children. However, only inactivated whole-virus vaccines against EV-A71 are commercially available in Mainland China. Furthermore, the mechanisms underlying the infectivity and pathogenesis of EV-A71 remain to be better understood. By adaptation of an EV-A71 B5 strain in monkey Vero cells in the presence of brilliant black BN (E151), an anti-EV-A71 agent, a double mutant with VP1-V238A,K244R emerged whose infection was enhanced by E151. The growth of the reverse genetics (RG) mutant RG/B5-VP1-V238A,K244R (RG/B5-AR) was promoted by E151 in Vero cells but inhibited in other human and murine cells, while its parental wild type, RG/B5-wt, was strongly prevented by E151 from infection in all tested cells. In the absence of E151, RG/B5-AR exhibited defective cell entry/exit, resulting in reduced viral transmission and growth in vitro. It had augmented binding affinity to sulfated glycans, cells, and tissue/organs, which probably functioned as decoys to restrict viral dissemination and infection. RG/B5-AR was also attenuated, with a 355 times higher 50% lethal dose (LD50) and a shorter timing of virus clearance than those of RG/B5-wt in suckling AG129 mice. However, it remained highly immunogenic in adult AG129 mice and protected their suckling mice from lethal EV-A71 challenges through maternal neutralizing antibodies. Overall, discovery of the attenuated mutant RG/B5-AR contributes to better understanding of virulence determinants of EV-A71 and to further development of novel vaccines against EV-A71. IMPORTANCE Enterovirus A71 (EV-A71) is highly contagious in children and has been responsible for thousands of deaths in Asia-Pacific region since the 1990s. Unfortunately, the virulence determinants and pathogenesis of EV-A71 are not fully clear. We discovered that a novel EV-A71 mutant, VP1-V238A,K244R, showed growth attenuation with reduced efficiency of cell entry/exit. In the Vero cell line, which has been approved for manufacturing EV-A71 vaccines, the growth defects of the mutant were compensated by a food dye, brilliant black BN. The mutant also showed augmented binding affinity to sulfated glycans and other cellular components, which probably restricted viral infection and dissemination. Therefore, it was virulence attenuated in a mouse model but still retained its immunogenicity. Our findings suggest the mutant as a promising vaccine candidate against EV-A71 infection.

circRNA expression patterns and circRNA-miRNA-mRNA networks during CV-A16 infection of SH-SY5Y cells.

Coxsackievirus A16 (CV-A16) has caused worldwide epidemics of hand, foot, and mouth disease (HFMD) in infants and preschool children. Circular RNAs (circRNAs), a class of noncoding RNA molecules, participate in the progression of viral infectious diseases. Although the function of circRNAs has been a heavily researched topic, their role in CV-A16 infection is still unclear. In this study, the viral effects of CV-A16 on the cellular circRNA transcriptome were investigated using next-generation sequencing technology. The results showed that a total of 8726, 8611, and 6826 circRNAs were identified at 0, 12, and 24 h postinfection, respectively. Moreover, it was found that 1769 and 1192 circRNAs were differentially expressed in at 12 and 24 h postinfection, respectively. The common differentially expressed circRNAs were used for functional annotation analysis, and it was found that the parent genes of differentially expressed circRNAs might be associated with the viral infection process, especially the "Immune system process" in GO analysis and the "Inflammation mediated by chemokine and cytokine signaling pathway" in KEGG analysis. Subsequently, circRNA-miRNA-mRNA regulatory networks were constructed, and the hsa_circ_0004447/hsa-miR-942-5p/MMP2, hsa_circ_0078617/hsa-miR-6780b-5p/MMP2 and hsa_circ_0078617/hsa-miR-5196-5p/MMP2 regulatory axes were identified by enrichment analysis as important networks during the progression of CV-A16 infection. Finally, six dysregulated circRNAs were selected for validation and were verified to be consistent with the sequencing results. Considering all of these results, to the best of our knowledge, this study is the first to present a comprehensive overview of circRNAs induced by CV-A16 infection, and this research demonstrated that a network of enriched circRNAs and circRNA-associated competitive endogenous RNAs (ceRNAs) is involved in the regulation of CV-A16 infection, thereby helping to elucidate the mechanisms underlying CV-A16-host interactions.

Enterovirus A71 Induces Neurological Diseases and Dynamic Variants in Oral Infection of Human SCARB2-Transgenic Weaned Mice.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae, poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

Adaptation and Virulence of Enterovirus-A71.

Outbreaks of hand, foot, and mouth disease caused by enterovirus-A71 (EV-A71) can result in many deaths, due to central nervous system complications. Outbreaks with many fatalities have occurred sporadically in the Asia-Pacific region and have become a serious public health concern. It is hypothesized that virulent mutations in the EV-A71 genome cause these occasional outbreaks. Analysis of EV-A71 neurovirulence determinants is important, but there are no virulence determinants that are widely accepted among researchers. This is because most studies have been done in artificially infected mouse models and because EV-A71 mutates very quickly to adapt to the artificial host environment. Although EV-A71 uses multiple receptors for infection, it is clear that adaptation-related mutations alter the binding specificity of the receptors and allow the virus to adopt the best entry route for each environment. Such mutations have confused interpretations of virulence in animal models. This article will discuss how environment-adapted mutations in EV-A71 occur, how they affect virulence, and how such mutations can be avoided. We also discuss future perspectives for EV-A71 virulence research.

IKKε isoform switching governs the immune response against EV71 infection.

The reciprocal interactions between pathogens and hosts are complicated and profound. A comprehensive understanding of these interactions is essential for developing effective therapies against infectious diseases. Interferon responses induced upon virus infection are critical for establishing host antiviral innate immunity. Here, we provide a molecular mechanism wherein isoform switching of the host IKKε gene, an interferon-associated molecule, leads to alterations in IFN production during EV71 infection. We found that IKKε isoform 2 (IKKε v2) is upregulated while IKKε v1 is downregulated in EV71 infection. IKKε v2 interacts with IRF7 and promotes IRF7 activation through phosphorylation and translocation of IRF7 in the presence of ubiquitin, by which the expression of IFNß and ISGs is elicited and virus propagation is attenuated. We also identified that IKKε v2 is activated via K63-linked ubiquitination. Our results suggest that host cells induce IKKε isoform switching and result in IFN production against EV71 infection. This finding highlights a gene regulatory mechanism in pathogen-host interactions and provides a potential strategy for establishing host first-line defense against pathogens.

A 10-Day-Old Murine Model of Coxsackievirus A6 Infection for the Evaluation of Vaccines and Antiviral Drugs.

Coxsackievirus A6 (CVA6) is recognized as a major enterovirus type that can cause severe hand, foot, and mouth disease and spread widely among children. Vaccines and antiviral drugs may be developed more effectively based on a stable and easy-to-operate CVA6 mouse infection model. In this study, a wild CVA6-W strain was sub-cultured in newborn mice of different ages (in days), for adaptation. Therefore, a CVA6-A mouse-adapted strain capable of stably infecting the mice was generated, and a fatal model was built. As the result indicated, CVA6-A could infect the 10-day-old mice to generate higher levels of IFN-γ, IL-6, and IL-10. The mice infected with CVA6-A were treated with IFN-α1b at a higher dose, with complete protection. Based on this strain, an animal model with active immunization was built to evaluate antiviral protection by active immunization. The three-day-old mice were pre-immunized with inactivated CVA6 thereby generating IgM and IgG antibodies within 7 days that enabled complete protection of the pre-immunized mice following the CVA6 virus challenge. There were eight mutations in the genome of CVA6-A than in that of CVA6-W, possibly attributed to the virulence of CVA6 in mice. Briefly, the CVA6 infection model of the 10-day-old mice built herein, may serve as an applicable preclinical evaluation model for CVA6 antiviral drugs and vaccine study.

MicroRNAs miR-18a and miR-452 regulate the replication of enterovirus 71 by targeting the gene encoding VP3.

MicroRNAs (miRNAs) are crucial in the process of host-pathogen interaction. In this study, we established a screening system for miRNAs of target genes to detect the effect of miRNAs on Enterovirus 71 (EV71) replication in rhabdomyosarcoma (RD) cells. A 3'-untranslated region (UTR) dual-luciferase assay was performed to confirm putative miRNA targets in EV71 genome. Firstly, 13 fragments of EV71 genome were inserted into the vector pMIR, and luciferase activities were analyzed to identify the putative miRNAs of target genes. The expression of the reporter protein was significantly downregulated in cells transfected with the vector containing gene VP3. Then we screened for miRNAs that might target to VP3 through online analysis software. In addition, Western blot, real-time PCR, virus titration, and morphological changes were considered to examine the effects of miRNAs on virus replication. The results suggested that miR-18a and miR-452 repress the reproduction of EV71 virus by binding to VP3. Moreover, EV71 infection also affected the expression of endogenous miR-18a and miR-452. In addition, no significant cytotoxic effects were observed. The results from this study suggest that the intracellular miRNAs may play vital roles in the host-virus interaction.

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.

Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus.

Enteroviruses, such as EV-A71 and CVA16, mainly infect the human gastrointestinal tract. Human coronaviruses, including SARS-CoV and SARS-CoV-2, have been variably associated with gastrointestinal symptoms. We aimed to optimize the human intestinal organoids and hypothesize that these optimized intestinal organoids can recapitulate enteric infections of enterovirus and coronavirus. We demonstrate that the optimized human intestinal organoids enable better simulation of the native human intestinal epithelium, and that they are significantly more susceptible to EV-A71 than CVA16. Higher replication of EV-A71 than CVA16 in the intestinal organoids triggers a more vigorous cellular response. However, SARS-CoV and SARS-CoV-2 exhibit distinct dynamics of virus-host interaction; more robust propagation of SARS-CoV triggers minimal cellular response, whereas, SARS-CoV-2 exhibits lower replication capacity but elicits a moderate cellular response. Taken together, the disparate profile of the virus-host interaction of enteroviruses and coronaviruses in human intestinal organoids may unravel the cellular basis of the distinct pathogenicity of these viral pathogens.

Prevalence of recessive infection of pathogens of hand, foot, and mouth disease in healthy people in China: A meta-analysis.

BACKGROUND: To analyze the prevalence of latent infection of pathogens of hand, foot, and mouth disease (HFMD) in Chinese healthy population and its influencing factors, so as to provide reference for the prevention and control of HFMD. METHODS: A systematic literature searching about the incidence of latent infection of HFMD was conducted in Chinese and English databases. The inclusion and exclusion criteria of the retrieved literature were established. The qualified literatures were screened and the data were extracted. The pooled rate and its 95% confidence interval was used to assess the latent infection rate of HFMD pathogens in healthy Chinese population, and subgroup analysis was conducted based on gender and age. All statistical analyses were performed using the STATA version 12.0 software. RESULTS: A total of 31 literatures were included in this meta-analysis. The recessive infection rate of HFMD pathogens reported in the literature of Chinese healthy people ranged from 4.59% to 44.12%. The results of meta-analysis showed that the latent infection rate of human enteroviruses (HEVs) in healthy Chinese population was 17.5% (14.9-20.1%), among which, the latent infection rates of EV-A71, CV-A16, and other HEVs were 3.3% (2.2-4.4%), 1.7% (1.0-2.5%), and 15.1% (11.1-17.1%), respectively. The latent infection rates of HEVs in healthy men and women in China were 16.7% (12.9-20.4%) and 14.4% (10.8-18.0%), respectively. The latent infection rates of HEVs in the healthy population aged 0 to 5 years and over 5 years were 24.4% (20.4-28.5%) and 9.4% (6.5-12.2%), respectively. Meta regression showed that the factors affecting the latent infection rate of HEVs in Chinese healthy population included sampling period, sampling area, and study population. CONCLUSION: The latent infection rate of HEVs is high in healthy people in China, but it is mainly caused by other enteroviruses. The latent infection rate of HEVs in male was higher than that of female and was greater in people aged 0 to 5 than that of aged over 5 years. Limited by the quantity and quality of the included studies, more high-quality studies are needed for further verification in the future.

Sox4 represses host innate immunity to facilitate pathogen infection by hijacking the TLR signaling networks.

Toll-like receptors (TLRs) are essential for the protection of the host from pathogen infections by initiating the integration of contextual cues to regulate inflammation and immunity. However, without tightly controlled immune responses, the host will be subjected to detrimental outcomes. Therefore, it is important to balance the positive and negative regulations of TLRs to eliminate pathogen infection, yet avert harmful immunological consequences. This study revealed a distinct mechanism underlying the regulation of the TLR network. The expression of sex-determining region Y-box 4 (Sox4) is induced by virus infection in viral infected patients and cultured cells, which subsequently represses the TLR signaling network to facilitate viral replication at multiple levels by a distinct mechanism. Briefly, Sox4 inhibits the production of myeloid differentiation primary response gene 88 (MyD88) and most of the TLRs by binding to their promoters to attenuate gene transcription. In addition, Sox4 blocks the activities of the TLR/MyD88/IRAK4/TAK1 and TLR/TRIF/TRAF3/TBK1 pathways by repressing their key components. Moreover, Sox4 represses the activation of the nuclear factor kappa-B (NF-κB) through interacting with IKKα/α, and attenuates NF-kB and IFN regulatory factors 3/7 (IRF3/7) abundances by promoting protein degradation. All these contributed to the down-regulation of interferons (IFNs) and IFN-stimulated gene (ISG) expression, leading to facilitate the viral replications. Therefore, we reveal a distinct mechanism by which viral pathogens evade host innate immunity and discover a key regulator in host defense.

Sulfonated azo dyes enhance the genome release of enterovirus A71 VP1-98K variants by preventing the virions from being trapped by sulfated glycosaminoglycans at acidic pH.

Enterovirus A71 (EV-A71) is a causative agent of hand, foot and mouth disease and occasionally causes death in children. Its infectivity and pathogenesis, however, remain to be better understood. Three sulfonated azo dyes, including acid red 88 (Ar88), were identified to enhance the infectivity of EV-A71, especially isolates with VP1-98K, 145E (-KE), by mainly promoting viral genome release in vitro. Enzymatic removal of sulfated glycosaminoglycans (GAGs) or knockout of xylosyltransferase II (XT2) responsible for biosynthesis of sulfated GAGs weakened the Ar88 enhanced EV-A71 infection. Ar88 is proposed to prevent the -KE variants from being trapped by sulfated GAGs at acidic pH and to facilitate the viral interaction with uncoating factors for genome release in endosomes. The results suggest dual roles of sulfated GAGs as attachment factors and as decoys during host interaction of EV-A71 and caution that these artificial dyes in our environment can enhance viral infection.

Molecular determinants and heterogeneity underlying host response to EV-A71 infection at single-cell resolution.

The pathogenic human enterovirus EV-A71 has raised serious public health concerns. A hallmark of EV-A71 infection is the distortion of host transcriptomes in favour of viral replication. While high-throughput approaches have been exploited to dissect these gene dysregulations, they do not fully capture molecular perturbations at the single-cell level and in a physiologically relevant context. In this study, we applied a single-cell RNA sequencing approach on infected differentiated enterocyte cells (C2BBe1), which model the gastrointestinal epithelium targeted initially by EV-A71. Our single-cell analysis of EV-A71-infected culture provided several lines of illuminating observations: 1) This systems approach demonstrated extensive cell-to-cell variation in a single culture upon viral infection and delineated transcriptomic differences between the EV-A71-infected and bystander cells. 2) By analysing expression profiles of known EV-A71 receptors and entry facilitation factors, we found that ANXA2 was closely correlated in expression with the viral RNA in the infected population, supporting its role in EV-A71 entry in the enteric cells. 3) We further catalogued dysregulated lncRNAs elicited by EV-A71 infection and demonstrated the functional implication of lncRNA CYTOR in promoting EV-A71 replication. Viewed together, our single-cell transcriptomic analysis illustrated at the single-cell resolution the heterogeneity of host susceptibility to EV-A71 and revealed the involvement of lncRNAs in host antiviral response.