Genetic diversity and recombination of bovine enterovirus strains in China.

Bovine enterovirus (BEV) consisting of enterovirus species E (EV-E) and F (EV-F) is the causative agent associated with respiratory and gastrointestinal diseases in cattle. Here, we reported the characterization, genetic diversity, and recombination of novel BEV strains isolated from the major cattle-raising regions in China during 2012-2018. Twenty-seven BEV strains were successfully isolated and characterized. Molecular characterization demonstrated that the majority of these novel BEV strains (24/27) were EV-E, while only few strains (3/27) were EV-F. Sequence analysis revealed the diversity of the circulating BEV strains such as species and subtypes where different species or subtype coinfections were detected in the same regions and even in the same cattle herds. For the EV-E, two novel subtypes, designated as EV-E6 and EV-E7, were revealed in addition to the currently reported EV-E1-EV-E5. Comparative genomic analysis revealed the intraspecies and interspecies genetic exchanges among BEV isolates. The representative strain HeN-B62 was probably from AN12 (EV-F7) and PS-87-Belfast (EV-F3) strains. The interspecies recombination between EV-E and EV-F was also discovered, where the EV-F7-AN12 might be from EV-E5 and EV-F1, and EV-E5-MexKSU/5 may be recombined from EV-F7 and EV-E1. The aforementioned results revealed the genetic diversity and recombination of novel BEV strains and unveiled the different BEV species or subtype infections in the same cattle herd, which will broaden the understanding of enterovirus genetic diversity, recombination, pathogenesis, and prevention of disease outbreaks. IMPORTANCE: Bovine enterovirus (BEV) infection is an emerging disease in China that is characterized by digestive, respiratory, and reproductive disorders. In this study, we first reported two novel EV-E subtypes detected in cattle herds in China, unveiled the coinfection of two enterovirus species (EV-E/EV-F) and different subtypes (EV-E2/EV-E7, EV-E1/EV-E7, and EV-E3/EV-E6) in the same cattle herds, and revealed the enterovirus genetic exchange in intraspecies and interspecies recombination. These results provide an important update of enterovirus prevalence and epidemiological aspects and contribute to a better understanding of enterovirus genetic diversity, evolution, and pathogenesis.

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Emerging porcine Enterovirus G infections, epidemiological, complete genome sequencing, evolutionary and risk factor analysis in India.

The current study reports the in-depth analysis of the epidemiology, risk factors, and molecular characterization of a complete genome of Enterovirus G (EV-G) isolated from Indian pigs. We analysed several genes of EV-G isolates collected from various provinces in India, using phylogenetic analysis, recombination detection, SimPlot, and selection pressure analyses. Our analysis of 534 porcine faecal samples revealed that 11.61% (62/534) of the samples were positive for EV-G. While the G6 genotype was the most predominant, our findings showed that Indian EV-G strains also clustered with EV-G types G1, G6, G8, and G9. Furthermore, Indian EV-G strains exhibited the highest nucleotide similarity with Vietnamese (81.3%) and Chinese EV-G isolates (80.3%). Moreover, we identified a recombinant Indian EV-G strain with a putative origin from a Japanese isolate and South Korean EV-G isolate. In summary, our findings provide significant insights into the epidemiology, genetic diversity, and evolution of EV-G in India.

Distinct transcriptomic profiles in children prior to the appearance of type 1 diabetes-linked islet autoantibodies and following enterovirus infection.

Although the genetic basis and pathogenesis of type 1 diabetes have been studied extensively, how host responses to environmental factors might contribute to autoantibody development remains largely unknown. Here, we use longitudinal blood transcriptome sequencing data to characterize host responses in children within 12 months prior to the appearance of type 1 diabetes-linked islet autoantibodies, as well as matched control children. We report that children who present with insulin-specific autoantibodies first have distinct transcriptional profiles from those who develop GADA autoantibodies first. In particular, gene dosage-driven expression of GSTM1 is associated with GADA autoantibody positivity. Moreover, compared with controls, we observe increased monocyte and decreased B cell proportions 9-12 months prior to autoantibody positivity, especially in children who developed antibodies against insulin first. Lastly, we show that control children present transcriptional signatures consistent with robust immune responses to enterovirus infection, whereas children who later developed islet autoimmunity do not. These findings highlight distinct immune-related transcriptomic differences between case and control children prior to case progression to islet autoimmunity and uncover deficient antiviral response in children who later develop islet autoimmunity.

SIRT-1 is required for release of enveloped enteroviruses.

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

Enterovirus D68 infection upregulates SOCS3 expression to inhibit JAK-STAT3 signaling and antagonize the innate interferon response of the host.

Enterovirus D68 (EV-D68) can cause respiratory diseases and acute flaccid paralysis, posing a great threat to public health. Interferons are cytokines secreted by host cells that have broad-spectrum antiviral effects, inducing the expression of hundreds of interferon-stimulated genes (ISGs). EV-D68 activates ISG expression early in infection, but at a later stage, the virus suppresses ISG expression, a strategy evolved by EV-D68 to antagonize interferons. Here, we explore a host protein, suppressor of cytokine signaling 3 (SOCS3), is upregulated during EV-D68 infection and antagonizes the antiviral effects of type I interferon. We subsequently demonstrate that the structural protein of EV-D68 upregulated the expression of RFX7, a transcriptional regulator of SOCS3, leading to the upregulation of SOCS3 expression. Further exploration revealed that SOCS3 plays its role by inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). The expression of SOCS3 inhibited the expression of ISG, thereby inhibiting the antiviral effect of type I interferon and promoting EV-D68 transcription, protein production, and viral titer. Notably, a truncated SOCS3, generated by deleting the kinase inhibitory region (KIR) domain, failed to promote replication and translation of EV-D68. Based on the above studies, we designed a short peptide named SOCS3 inhibitor, which can specifically bind and inhibit the KIR structural domain of SOCS3, significantly reducing the RNA and protein levels of EV-D68. In summary, our results demonstrated a novel mechanism by which EV-D68 inhibits ISG transcription and antagonizes the antiviral responses of host type I interferon.

Rhinovirus Genotypes Circulating in Bulgaria, 2018-2021.

Rhinoviruses (RV) are one of the most common causative agents of respiratory infections, with significant socioeconomic impact. RV infections are not notifiable in Bulgaria, and little is known about the different RV genotypes circulating in the country. This study aims to investigate the diversity of RV genotypes that were circulating in Bulgaria in the period 2018-2021 in samples from ILI/ARI patients. Genotype assignment was based on sequencing and phylogenetic analysis of the 5' untranslated region and the VP4-VP2 region. Out of a total of 1385 nasopharyngeal swabs tested, 166 were RV-positive (RV detection rate: 11.99% (166/1385)). Those with a cycle threshold <25 were selected for genotyping (n = 63). RV isolates were successfully genotyped and classified into 34 genotypes within Rhinovirus A (RV-A), Rhinovirus B (RV-B) and Rhinovirus C (RV-C) species. Presumptive recombination events between the 5'UTR and VP4-VP2 regions were detected in three of the isolates. RV-A and RV-C were the prevalent RV species, with significantly more frequent detections of RV-A in the years before the COVID-19 pandemic compared to the post-pandemic period, when RV-C prevailed. The present study is the first to determine RV genotypes in Bulgaria and the circulation of RV-C has been described for the first time in the country.

The effects of SCARB2 and SELPLG gene polymorphisms on EV71 infection in hand, foot and mouth disease.

The same viral infection in different hosts may result in varying levels of clinical symptoms, which is related to the genetic background of the host itself. A total of 406 common cases and 452 severe cases of enterovirus 71 (EV71) infection in Yunnan Province were selected as the research subjects, and SNaPshot technology was used to detect genetic polymorphisms for 25 Tag single-nucleotide polymorphisms (TagSNPs) in the selectin P ligand (SELPLG) and scavenger receptor class B member 2 (SCARB2) genes. Our results demonstrate that SCARB2 polymorphisms (rs74719289, rs3733255 and rs17001551) are related to the severity of EV71 infection (A vs G: OR 0.330; 95% CI 0.115 - 0.947; T vs C: OR 0.336; 95% CI 0.118 - 0.958; and A vs G: OR 0.378; 95% CI 0.145 - 0.984). The SELPLG polymorphisms were not significantly different between common cases and severe cases. Therefore, we conclude that the SCARB2 gene has a protective effect on the course of hand, foot and mouth disease caused by EV71 infection and that SCARB2 gene mutations can reduce the severity of the disease.

Rhinovirus-induced epithelial RIG-I inflammasome suppresses antiviral immunity and promotes inflammation in asthma and COVID-19.

Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.

Construction of a Vero Cell Line Expressing Human ICAM1 for the Development of Rhinovirus Vaccines.

Human rhinoviruses (HRVs) are small non-enveloped RNA viruses that belong to the Enterovirus genus within the Picornaviridae family and are known for causing the common cold. Though symptoms are generally mild in healthy individuals, the economic burden associated with HRV infection is significant. A vaccine could prevent disease. The Vero-cell-based viral vaccine platform technology was considered for such vaccine development. Unfortunately, most HRV strains are unable to propagate on Vero cells due to a lack of the major receptor of HRV group A and B, intercellular adhesion molecule (ICAM1, also known as CD54). Therefore, stable human ICAM1 expressing Vero cell clones were generated by transfecting the ICAM1 gene in Vero cells and selecting clones that overexpressed ICAM1 on the cell surface. Cell banks were made and expression of ICAM1 was stable for at least 30 passages. The Vero_ICAM1 cells and parental Vero cells were infected with four HRV prototypes, B14, A16, B37 and A57. Replication of all four viruses was detected in Vero_ICAM1, but not in the parental Vero cells. Altogether, Vero cells expressing ICAM1 could efficiently propagate the tested HRV strains. Therefore, ICAM1-expressing cells could be a useful tool for the development and future production of polyvalent HRV vaccines or other viruses that use ICAM1 as a receptor.

Long non-coding RNA ENST00000469812 promotes Enterovirus type 71 replication via targeting the miR-4443/NUPR1 axis in rhabdomyosarcoma cells.

Hand, foot, and mouth disease (HFMD) caused by Enterovirus type 71 (EV71) is a serious threat to children's health. However, the pathogenic mechanism of EV71 is still unclear. Long non-coding RNAs (lncRNAs), some of which bind to miRNA as competitive endogenous RNAs (ceRNA) and weaken the silencing effect on the mRNA of downstream target genes, play a key role in regulating the viral infection process. In this study, through experimental verification, we found miR-4443 to be downregulated in cells infected with EV71. Next, by predicting lncRNAs that potentially regulate miR-4443, we found that EV71 infection induced upregulation of lncRNA ENST00000469812 and then further downregulated miR-4443 expression by direct interaction. We also demonstrated that nuclear protein 1 (NUPR1) is one of the target genes of miR-4443 and is involved in the ENST00000469812/miR-4443/NUPR1 regulatory axis. Finally, the ENST00000469812/miR-4443/NUPR1 regulatory axis exhibited a positive effect on EV71 replication. Here, we lay a foundation for exploring the pathogenic mechanism of EV71 and identify potential targets for HFMD treatment.

Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade.

Enterovirus A (EV-A) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc., revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A. In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity.

Association of TLR3 gene 1377C/T (rs3775290) and TLR7 gene C/G (rs3853839) polymorphism with hand, foot, and mouth disease caused by human enterovirus 71 infection susceptibility and severity in the Chinese Han population: A meta-analysis of case-control studies.

BACKGROUND: Several case-control studies have been conducted on the relationship between rs3775290 C/T and rs3853839 C/G single nucleotide polymorphisms of the Toll-like receptor (TLR) gene and hand, foot, and mouth disease (HFMD) susceptibility and severity. This meta-analysis aimed to offer a systemic review of HFMD susceptibility and severity among the Chinese Han population associated with the C/T (rs3775290) polymorphism of the TLR3 gene or C/G (rs3853839) polymorphism of the TLR7 gene. METHODS: A computer search was conducted using PubMed, Web of Science, Embase, CNKI, CBM, VIP, and WanFang databases. The time ranges were from database establishment to 30/7/2021. Articles selected according to the inclusion and exclusion criteria underwent data extraction and methodological quality evaluation. RevMan 5.4 and Stata 16.0 were adopted for meta-analysis, and the incorporated odds ratio (OR) values and 95% confidence intervals (CIs) were calculated. Sensitivity and publication bias assessments were performed. RESULTS: 8 articles with 9 studies were selected. Among them, there were 858 cases and 577 controls in TLR3 rs3775290 studies as well as 2151 cases and 1554 controls in TLR7 rs3853839 studies. Regarding rs3775290 of TLR3, susceptibilities of the severe type of T-possessing individuals were larger than those of C-possessing individuals [OR = 1.34, 95%CI (1.10, 1.64), P = .004]. The susceptibility of individuals with the severe TT genotype was 1.61 times that of individuals with the CC genotype [95%CI (1.07, 2.43), P=0.02], while susceptibility to HFMD was not influenced by the genotype. In terms of the rs3853839 of the TLR7 gene, C allele carriers have a higher risk of developing HFMD than G allele carriers. The susceptibility to HFMD in CC+CG individuals was 1.24 times than that in GG individuals [95%CI (1.07, 1.43), P = .004]. However, no relationship was found between this polymorphism and severity of the severe type. No significant publication bias was observed in this study. CONCLUSIONS: rs3775290 (C/T) of TLR3 is associated with susceptibility to the severe type, whereas rs3853839 (C/G) of TLR7 is associated with susceptibility to HFMD. However, owing to the limited quantity and quality of the research, the aforementioned conclusions are yet to be justified by more high-quality research.

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage.

AIMS/HYPOTHESIS: Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes. METHODS: We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes. RESULTS: We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p<0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.

Enterovirus 71 VP1 promotes 5-HT release by upregulating the expression of ERICH3 and methyltransferase ZC3H13.

BACKGROUND AND AIM: The effect of structural viral protein 1 (VP1) on neurological damage caused by enterovirus 71 (EV71) infection is unclear. This study aimed to explore the transcriptome changes in EV infected patients and the role of VP1 on the cell secretion pathway of neuron cells. METHODS: In our cohort, EV infected patients were enrolled, and RNA-seq analysis was used to evaluate the distinct transcript patterns of cerebrospinal fluid (CSF). The EV71 VP1-overexpressing vector (pEGFP-c3-VP1) was generated and transfected into neuron cells. The relationship between Glutamate Rich 3 (ERICH3) and methyltransferase Zinc Finger CCCH-Type Containing 13 (ZC3H13) and their effect on the serotonin (5-HT) release of neuron cells were explored using small interfering RNA. The expression of ERICH3 and ZC3H13 and concentration of 5-HT were determined using real-time PCR, Western blot, and ELISA, respectively. RESULT: The expression of ERICH3 and ZC3H13 were significantly upregulated in EV infected patients with neurological symptoms compared to those without (P < 0.05). The ERICH3 gene had many N6-methyladenosine (m6A) binding sites that can be regulated by m6A modification. Further, the expression of ERICH3 and ZC3H13 were elevated significantly in EV71-VP1 overexpressing neuron cells (P < 0.05). Moreover, ERICH3 or ZC3H13 deficiency could significantly downregulate the release of 5-HT in VP1-overexpressing cells (P < 0.05). Nonetheless, ERICH3 expression was significantly suppressed when ZC3H13 was silenced in neuron cells and vice versa (P < 0.05). CONCLUSIONS: EV71-VP1 can promote 5-HT release by upregulating the expression of ERICH3 and ZC3H13. 5-HT might be a novel therapeutic target for EV71 infection-induced fatal neuronal damage.

Molecular Analysis of Caprine Enterovirus Circulating in China during 2016-2021: Evolutionary Significance.

Here, we report the characterization of 13 novel caprine/ovine enterovirus strains isolated from different regions in China during 2016-2021. Immunoperoxidase monolayer assay showed that these viral strains shared strong cross-reaction with the previously reported caprine enterovirus CEV-JL14. Alignment analysis of the complete nucleotide sequences revealed 79.2%-87.8% and 75.0%-76.7% sequence identity of these novel caprine enterovirus strains to CEV-JL14 and TB4-OEV, respectively. Phylogenetic analyses clustered these novel strains to EV-G based on the amino acid sequences of P1 and 2C+3CD. Moreover, phylogenetic analysis of these caprine enterovirus strains identified three new EV-G types using VP1 sequences. These results demonstrate the genetic variations and the evolution of caprine enterovirus.

Association of the IRAK4 rs4251545 genetic polymorphism with severity of enterovirus-71 infection in Chinese children.

INTRODUCTION: This study aimed to explore the association between the IRAK4 polymorphism rs4251545 and the severity of enterovirus 71 (EV71) infection in Chinese children. METHODS: We analyzed the IRAK4 polymorphism rs4251545 in 617 EV71-infected patients and 410 controls using the improved multiplex ligation detection reaction. IRAK4 mRNA expression was tested by qRT-PCR. Serum concentrations of IL-6 and NF-κB were detected using ELISA. RESULTS: The frequencies of the GA + AA genotype and A allele in the mild EV71 infection group and in the severe EV71 infection group were significantly higher than those in the normal control group. The frequency of the GA + AA genotype and A allele in severely infected EV71 patients was markedly higher than that in mildly infected EV71 patients. IRAK4 mRNA expression in mildly infected EV71 patients and severely infected patients was significantly higher than that in the control group. IRAK4 mRNA expression in GA + AA genotypes in both mild and severe EV71 infection groups was significantly higher than that in patients with the GG genotype. IL-6 concentration and the ratio of IL-6/NF-κB in severe EV71 cases were significantly lower in patients with the GA + AA genotype than in those with the GG genotype. The ratio of IL-6/NF-κB was distinctly higher in severely infected EV71 patients than in mildly infected and control subjects. CONCLUSIONS: The IRAK4 polymorphism rs4251545 was associated with the susceptibility and severity of EV71 infection. The A allele is a susceptible factor in the development of severe EV71 infection in Chinese children.

Protein Kinase B2 (PKB2/AKT2) Is Essential for Host Protection in CVB3-Induced Acute Viral Myocarditis.

Protein kinase B2 (AKT2) is involved in various cardiomyocyte signaling processes, including those important for survival and metabolism. Coxsackievirus B3 (CVB3) is one of the most common pathogens that cause myocarditis in humans. The role of AKT2 in CVB3 infection is not yet well understood. We used a cardiac-specific AKT2 knockout (KO) mouse to determine the role of AKT2 in CVB3-mediated myocarditis. CVB3 was injected intraperitoneally into wild-type (WT) and KO mice. The mice's survival rate was recorded: survival in KO mice was significantly decreased compared with WT mice (WT vs. KO: 73.3 vs. 27.1%). Myocardial damage and inflammation were significantly increased in the hearts of KO mice compared with those of WT mice. Moreover, from surface ECG, AKT2 KO mice showed a prolonged atria and ventricle conduction time (PR interval, WT vs. KO: 47.27 ± 1.17 vs. 64.79 ± 7.17 ms). AKT2 deletion induced severe myocarditis and cardiac dysfunction due to CVB3 infection. According to real-time PCR, the mRNA level of IL-1, IL-6, and TNF-α decreased significantly in KO mice compared with WT mice on Days 5 after infection. In addition, innate immune response antiviral effectors, Type I interferon (interferon-α and ß), and p62, were dramatically suppressed in the heart of KO mice. In particular, the adult cardiac myocytes isolated from the heart showed high induction of TLR4 protein in KO mice in comparison with WT. AKT2 deletion suppressed the activation of Type I interferon and p62 transcription in CVB3 infection. In cardiac myocytes, AKT2 is a key signaling molecule for the heart from damage through the activation of innate immunity during acute myocarditis.

Life-Threatening Enterovirus 71 Encephalitis in Unrelated Children with Autosomal Dominant TLR3 Deficiency.

PURPOSE: Enterovirus A71 (EV71) causes a broad spectrum of childhood diseases, ranging from asymptomatic infection or self-limited hand-foot-and-mouth disease (HFMD) to life-threatening encephalitis. The molecular mechanisms underlying these different clinical presentations remain unknown. We hypothesized that EV71 encephalitis in children might reflect an intrinsic host single-gene defect of antiviral immunity. We searched for mutations in the toll-like receptor 3 (TLR3) gene. Such mutations have already been identified in children with herpes simplex virus encephalitis (HSE). METHODS: We sequenced TLR3 and assessed the impact of the mutations identified. We tested dermal fibroblasts from a patient with EV71 encephalitis and a TLR3 mutation and other patients with known genetic defects of TLR3 or related genes, assessing the response of these cells to TLR3 agonist poly(I:C) stimulation and EV71 infection. RESULTS: Three children with EV71 encephalitis were heterozygous for rare mutations-TLR3 W769X, E211K, and R867Q-all of which were shown to affect TLR3 function. Furthermore, fibroblasts from the patient heterozygous for the W769X mutation displayed an impaired, but not abolished, response to poly(I:C). We found that TLR3-deficient and TLR3-heterozygous W769X fibroblasts were highly susceptible to EV71 infection. CONCLUSIONS: Autosomal dominant TLR3 deficiency may underlie severe EV71 infection with encephalitis. Human TLR3 immunity is essential to protect the central nervous system against HSV-1 and EV71. Children with severe EV71 infections, such as encephalitis in particular, should be tested for inborn errors of TLR3 immunity.

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

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