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 Induces TDP-43 Cleavage, Aggregation, and Neurotoxicity.

Enterovirus D68 (EV-D68), which causes severe respiratory diseases and irreversible central nervous system damage, has become a serious public health problem worldwide. However, the mechanisms by which EV-D68 exerts neurotoxicity remain unclear. Thus, we aimed to analyze the effects of EV-D68 infection on the cleavage, subcellular translocation, and pathogenic aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in respiratory or neural cells. The results showed that EV-D68-encoded proteases 2A and 3C induced TDP-43 translocation and cleavage, respectively. Specifically, 3C cleaved residue 327Q of TDP-43. The 3C-mediated cleaved TDP-43 fragments had substantially decreased protein solubility compared with the wild-type TDP-43. Hence, 3C activity promoted TDP-43 aggregation, which exerted cytotoxicity to diverse human cells, including glioblastoma T98G cells. The effects of commercially available antiviral drugs on 3C-mediated TDP-43 cleavage were screened, and the results revealed lopinavir as a potent inhibitor of EV-D68 3C protease. Overall, these results suggested TDP-43 as a conserved host target of EV-D68 3C. This study is the first to provide evidence on the involvement of TDP-43 dysregulation in EV-D68 pathogenesis. IMPORTANCE Over the past decade, the incidence of enterovirus D68 (EV-D68) infection has increased worldwide. EV-D68 infection can cause different respiratory symptoms and severe neurological complications, including acute flaccid myelitis. Thus, elucidating the mechanisms underlying EV-D68 toxicity is important to develop novel methods to prevent EV-D68 infection-associated diseases. This study shows that EV-D68 infection triggers the translocalization, cleavage, and aggregation of TDP-43, an intracellular protein closely related to degenerative neurological disorders. The viral protease 3C decreased TDP-43 solubility, thereby exerting cytotoxicity to host cells, including human glioblastoma cells. Thus, counteracting 3C activity is an effective strategy to relieve EV-D68-triggered cell death. Cytoplasmic aggregation of TDP-43 is a hallmark of degenerative diseases, contributing to neural cell damage and central nervous system (CNS) disorders. The findings of this study on EV-D68-induced TDP-43 formation extend our understanding of virus-mediated cytotoxicity and the potential risks of TDP-43 dysfunction-related cognitive impairment and neurological symptoms in infected patients.

MicroRNA-195 suppresses enterovirus A71-induced pyroptosis in human neuroblastoma cells through targeting NLRX1.

Enterovirus A71 (EV-A71) emerged as a leading cause of virus derived infant encephalitis in most Asian countries. Some recent studies point out the critical role of microRNA (miRNA) in the regulation of pyroptosis. However, the role of miRNAs in the regulation of EV-A71 infection-induced pyroptosis was not previously explored. In this study, we utilized microRNA array and real-time PCR to verify that miR-195 significantly down-regulate in EV-A71-infected SH-SY5Y human neuroblastoma cells. An inverse correlation of NLRX1 with miR-195 expression in EV-A71-infected SH-SY5Y cells was found. Target prediction of miR-195 showed that NLRX1 could directly interact with miR-195. Results from luciferase reporter assays, qRT-PCR and western blotting demonstrated the negative regulation between miR-195 and NLRX1. Silencing NLRX1 expression with small interfering RNAs (siRNAs-NLRX1) and over-expression of miR-195 also attenuate the EV-A71 associated pyroptosis. Our findings provided evidence showed that miR-195 can regulate EV-A71 infection-induced pyroptosis, by directly targeting NLRX1.

A Mouse Model for Infection with Enterovirus A71 in Small Extracellular Vesicles.

Enterovirus A71 (EV-A71) is the major pathogen of hand, foot, and mouth disease (HFMD); in some severe cases, it could develop into central nervous system (CNS) disease such as aseptic meningitis, encephalitis, and neurogenic pulmonary edema in children under 5 years. The EV-A71 pathogenesis which is involved with the CNS is unclear due to the lack of a simple and reliable mouse model thus far. Most clinical EV-A71 isolates could not effectively infect the neonatal mouse, which used to be an EV-A71 infection model. The small extracellular vesicles (sEVs) released from clinical EV-A71 isolate-infected cells were infectious in cell lines and could cause a high viral replication in mice. Neonatal ICR mice were injected intraperitoneally with these infectious sEVs and showed more weight loss and higher mortality than those mice injected with the clinical EV-A71 isolate. By using these sEVs, we provided a simple and effective method by which we can generate a stable and valuable animal model for the studies of EV-A71 pathogenesis and therapy.IMPORTANCE EV-A71 was supposed to infect the CNS through the neural pathway and the circulation of the blood in previous studies. Reverse axon transport had been confirmed as an important pathway for EV-A71 to infect the CNS; however, it is still unknown how EV-A71 infects the CNS through the circulation of the blood. Combined with the infectivity of sEVs secreted from EV-A71-infected cells and the characteristic that sEVs could cross the blood-brain barrier, we considered that sEVs may play a vital role in EV-A71 pathogenesis of the CNS.

Innate Immune Responses in Serum and Cerebrospinal Fluid From Neonates and Infants Infected With Parechovirus-A3 or Enteroviruses.

BACKGROUND: Parechovirus (PeV)-A3 and enteroviruses (EV) are the most common viruses causing sepsis and meningoencephalitis in neonates and young infants. Clinical manifestations of PeV-A3 infection are more severe than those of EV infection, and no pleocytosis with a positive polymerase chain reaction (PCR) result for PeV-A3 in cerebrospinal fluid (CSF) are characteristic findings. We hypothesized that innate immune responses to PeV-A3 and EV are distinct in serum and CSF. METHODS: We evaluated 22 cytokines/chemokines in serum and CSF from PeV-A3- or EV-infected patients younger than 4 months in Niigata, Japan, from 2015 through 2018. Infection was diagnosed with real-time PCR followed by sequencing. Febrile neonates and infants with sepsis-like syndrome who had negative bacterial culture and viral PCR for both PeV-A and EV were also included (non-PeV-A/EV patients). RESULTS: Among 192 febrile patients, we evaluated 16 PeV-A3-infected, 15 EV-infected, and 8 non-PeV-A/EV patients. Serum pro-/anti-inflammatory cytokine/chemokine levels were higher in PeV-A3-infected patients than in EV-infected patients (P < .02). Although most cytokine/chemokine were elevated in CSF from EV-infected patients, levels were low or undetectable in PeV-A3-infected and non-PeV-A/EV patients (P < .001). CONCLUSIONS: Distinct cytokine/chemokine patterns in serum and CSF may explain the different clinical manifestations of PeV-A3-infected and EV-infected neonates and young infants.

Mechanism for the lethal effect of enterovirus A71 intracerebral injection in neonatal mice.

Enterovirus A71 (EV-A71) infection is primarily responsible for fatal hand, foot, and mouth disease (HFMD) cases. Infants and younger children are more likely to suffer central nervous system damage as a result of EV-A71 infection, but this virus mostly does not affect older children and adults. This study investigated the possible mechanism underlying the age-dependent lethal effect of EV-A71 infection by comparing neonatal and adult mouse models of EV-A71 infection. Although viral proliferation is absent in both neonatal and adult mice, we observed that EV-A71, as a stimulus for astrocytes, elevates the levels of cytokines and monoamine neurotransmitters in neonatal mice. Then, we selected IL-6 and adrenaline as targets in a pharmacological approach to further validate the roles of these factors in mediating the mortality of neonatal mice after EV-A71 infection. Intracerebral injection of IL-6 and adrenaline enhanced the severity of EV-A71 infection, while treatment with an anti-IL-6-neutralizing antibody or the adrenergic-antagonist phenoxybenzamine reversed the lethal effect of EV-A71 in neonatal mice. These results suggest that the central nervous system (CNS) damage in neonatal cases of EV-A71 infection might be caused by an activated fetal cerebral immune response to the virus, including the disruption of brainstem function through increased levels of cytokines and neurotransmitters, rather than the typical cytopathic effect (CPE) of viral infection.

Rhinovirus C15 Induces Airway Hyperresponsiveness via Calcium Mobilization in Airway Smooth Muscle.

Rhinovirus (RV) exposure evokes exacerbations of asthma that markedly impact morbidity and mortality worldwide. The mechanisms by which RV induces airway hyperresponsiveness (AHR) or by which specific RV serotypes differentially evoke AHR remain unknown. We posit that RV infection evokes AHR and inflammatory mediator release, which correlate with degrees of RV infection. Furthermore, we posit that rhinovirus C-induced AHR requires paracrine or autocrine mediator release from epithelium that modulates agonist-induced calcium mobilization in human airway smooth muscle. In these studies, we used an ex vivo model to measure bronchoconstriction and mediator release from infected airways in human precision cut lung slices to understand how RV exposure alters airway constriction. We found that rhinovirus C15 (RV-C15) infection augmented carbachol-induced airway narrowing and significantly increased release of IP-10 (IFN-γ-induced protein 10) and MIP-1ß (macrophage inflammatory protein-1ß) but not IL-6. RV-C15 infection of human airway epithelial cells augmented agonist-induced intracellular calcium flux and phosphorylation of myosin light chain in co-cultured human airway smooth muscle to carbachol, but not after histamine stimulation. Our data suggest that RV-C15-induced structural cell inflammatory responses are associated with viral load but that inflammatory responses and alterations in agonist-mediated constriction of human small airways are uncoupled from viral load of the tissue.

Parechovirus and enterovirus infections in neonates.

BACKGROUND: Parechovirus and enterovirus belong to a family of Picornaviridae, non- enveloped, small-sized RNA viruses, responsible for multiple human diseases. Recent introduction of molecular tests enabled the identi cation of parechovirus and enterovirus infections. Our aim was a retrospective analysis of signs and symptoms associated with confirmed parechovirus or enterovirus infections among children treated in the Department of Neonatology, St. Louis Regional Children's Hospital in Kraków, Poland. METHODS: Based on laboratory records, we identified all cases of parecho- or enterovirus infections confirmed by identification of viral RNA in nasal swab or cerebrospinal fluid samples. Hospital records and laboratory tests results of selected patients were then analyzed, and selected data were summarized, with emphasis on clinical and laboratory findings at admission. RESULTS: We identified 11 cases of parechovirus and three of enterovirus infections. All cases were neonates admitted to hospital with fever and irritability. Except for leukopenia in 50% of patients, no significant abnormalities were noted in blood counts and serum biochemistry, including low C-reactive protein and procalcitonin. In nine cases, cerebrospinal fluid was collected, the fluid protein concentrations and cell counts were moderately increased. Final diagnosis was meningitis in 12 children, and other viral infections in two. CONCLUSIONS: Viral infection, including parecho- and enteroviruses, should be considered in the etiology of fever and meningitis in neonates. The available molecular tests allow for detection of viral genetic material even in a scant biological specimen collected from neonates.

Cerebrospinal Fluid Neopterin in Children With Enterovirus-Related Brainstem Encephalitis.

BACKGROUND: Enterovirus-A71 causes outbreaks of brainstem encephalitis, ranging from self-limited disease to acute flaccid paralysis. The aim of this study was to assess the role of cerebrospinal fluid (CSF) neopterin as a biomarker of disease severity in children with enterovirus-related brainstem encephalitis. METHODS: A descriptive, prospective cohort study was conducted from April 2016 to March 2017 in a tertiary hospital. Pediatric patients with a diagnosis of brainstem encephalitis with or without myelitis due to enterovirus infection were enrolled. The final study group comprised a convenience sample including all patients with sufficient CSF volume for neopterin determination. The major variables considered in estimating the severity were the diagnosis of encephalomyelitis, the presence of lesions and extensive lesions on brain and spinal magnetic resonance imaging (MRI), hospital stay length greater than seven days, and sequelae at day 30. RESULTS: Of 60 patients, CSF neopterin could be measured in 36. Median age was 26 months (interquartile range: 19 to 32). Thirty-three were diagnosed with brainstem encephalitis and three with encephalomyelitis. Enterovirus-A71 was the only identified genotype (25 of 25). CSF neopterin levels were elevated (>61 nmol/L) in 33 of 36 (92%), with a median of 347 nmol/L (interquartile range: 204 to 525). CSF neopterin was useful to distinguish patients with lesions on MRI (area under the receiver operating characteristic curve = 0.76; P = 0.02) and extensive lesions (area under the receiver operating characteristic curve = 0.76; P = 0.04). CONCLUSIONS: This study suggests an association between CSF neopterin levels and the presence of inflammatory lesions on MRI.

Efficacy of Long-term Selenium Supplementation in the Treatment of Chronic Keshan Disease with Congestive Heart Failure.

Few effective treatments for chronic Keshan disease have been available till now. The efficacy of long-term selenium supplementation in the treatment of chronic Keshan disease with congestive heart failure is inconclusive. This study aimed to determine whether selenium supplementation is associated with a decreased risk of cardiac death in chronic Keshan disease with congestive heart failure by ten years of follow-up. A retrospective long-term follow-up analysis was performed on a monitored cohort consisting of 302 chronic Keshan disease patients with a mean age of 40.8±11.4 years. Of the 302 chronic Keshan disease patients, 170 (56.3%) were given selenium supplementation until the end point of follow-up. Cox proportional hazards regression models were used to identify the independent predictors of cardiac events. Our results showed that during the follow-up, there were 101 deaths of patients with chronic Keshan disease in the selenium supplementation group (101/170, 59.4%) and 98 in non-selenium supplementation group (98/132, 74.2%). Multivariate analyses suggested that selenium supplementation was associated with a decreased risk of cardiac death (HR 0.39, 95% CI 0.28-0.53) after adjustment for baseline age, sex, cigarette smoking, family history of Keshan disease, body mass index (BMI), heart rate, electrocardiogram (ECG) abnormalities, blood pressure, initial cardiothoracic ratio, left ventricular ejection fractions (LVEF) and whole-blood selenium concentration. Our ten-year follow-up analysis indicated that selenium supplementation, specifically combined with the use of angiotensin-converting enzyme inhibitor and beta blocker therapy, improved the survival of patients with chronic Keshan disease with congestive heart failure. BMI, selenium deficiency, male, combined ECG abnormalities, LVEF, and fast heart rate increased the risk of cardiac events.

Clinical and laboratory characteristics of enteroviral meningitis in children, including qRT-PCR and sequencing analysis.

AIMS: Enteroviruses (EVs) are the most common agents of aseptic meningitis. Some serotypes can cause serious neuroinfection leading to death. The aim of this study was to determine the representation of EVs in the etiology of aseptic meningitis in children and to analyze the demographic, clinical, laboratory, and epidemiological characteristics of patients with EV meningitis. PATIENTS AND METHODS: This was a prospective study including 147 patients in three groups: EV meningitis, tick-borne encephalitis, and aseptic meningitis with unidentified agent. RESULTS: Boys with EV meningitis predominated over girls. The average patient age was 11 years. Compared to the control group, these patients suffered more from stiff back (P=0.010), vomiting and nausea (P=0.009). They had shorter symptom duration (P<0.001), higher C-reactive protein in blood (P<0.001), higher predominance of polynuclears (P=0.026), and greater lactate (P=0.003) in cerebrospinal fluid (CSF). The serotype seen most frequently (68%) was ECHO virus (ECV) 30. CONCLUSIONS: Enteroviruses play the most important role in the differential diagnosis of aseptic meningitis. Short symptom duration, slightly higher inflammatory parameters in blood, predominance of polynuclears, and elevated CSF lactate have predictive value in diagnosing this disease. ECV 30 (frequently the agent of epidemics in the Czech Republic) was the aseptic meningitis agent most often seen.

Longitudinal survey of Teschovirus A, Sapelovirus A, and Enterovirus G fecal excretion in suckling and weaned pigs.

Fecal samples from 27 pigs were longitudinally analyzed for Teschovirus A (TV-A), Sapelovirus A (SV-A), and Enterovirus G (EV-G) RNA presence. Suckling piglet fecal samples were negative for the three enteric picornaviruses. However, these picornaviruses were detected in 22/27 weaned pig fecal samples. This study provides new data on TV-A, SV-A, and EV-G infection dynamics.

Enterovirus and parechovirus infections in children: differences in clinical presentation, mechanisms for meningitis without pleocytosis and mechanisms involved in the neurological outcome.

Enterovirus (EV) and Parechovirus (HPeV) are a frequent cause of infection in children. This review gives an overview of possible causes for differences in clinical presentation. EV and HPeV can cause a meningitis with or without pleocytosis. Different possible mechanisms for meningitis without pleocytosis are given. Little is known about the prognosis and long-term effects of EV and HPeV meningitis in children. Only some studies with a small number of children with EV or HPeV meningitis are reported. The different possible mechanisms involved in the neurological outcome after EV or HPeV meningitis will be discussed.

Evaluation of antiviral therapies in respiratory and neurological disease models of Enterovirus D68 infection in mice.

Enterovirus D68 (EV-D68) is unique among enteroviruses because of the ability to cause severe respiratory disease as well as neurological disease. We developed separate models of respiratory and neurological disease following EV-D68 infection in AG129 mice that respond to antiviral treatment with guanidine. In four-week-old mice infected intranasally, EV-D68 replicates to high titers in lung tissue increasing the proinflammatory cytokines MCP-1 and IL-6. The respiratory infection also produces an acute viremia. In 10-day-old mice infected intraperitoneally, EV-D68 causes a neurological disease with weight-loss, paralysis, and mortality. In our respiratory model, treatment with guanidine provides a two-log reduction in lung virus titers, reduces MCP-1 and IL-6, and prevents histological lesions in the lungs. Importantly, viremia is prevented by early treatment with guanidine. In our neurological model, guanidine treatment protects mice from weight-loss, paralysis, and mortality. These results demonstrate the utility of these models for evaluation of antiviral therapies for EV-D68 infection.

Estimating the Severity Profile of Enterovirus A71 Infections in Children: A Bayesian Synthesis Framework.

Enterovirus A71 (EV-A71) is responsible for the majority of severe cases of hand, foot, and mouth disease, but little evidence is available on the severity profile of EV-A71 infections. We formulated a hierarchical Bayesian model that synthesized data on diseases/events associated with EV-A71 and EV-A71 antibody responses to infection among unvaccinated children from large clinical trials of EV-A71 vaccination, which were conducted in Jiangsu and Beijing during 2012 and 2013, to reconstruct the severity profile in a unified framework. On average, 15.1% of the children aged 6-35 months were infected by EV-A71 during 1-year follow-up in a mild epidemic season. We estimated that 9.7%, 2.2%, and 0.6% of children infected with EV-A71 were diagnosed with EV-A71-associated diseases, were hospitalized, and showed severe complications, respectively. We estimated on average 1 death per 10,000 EV-A71 infections for children aged 6-35 months. Approximately 70% of children had ≥4-fold rises in antibody titers after infection. Most EV-A71 infections in young children are mild, and overall 2.2% of the infected patients were hospitalized in the 2 trials. There remain several uncertainties about the immune response after infection and the duration of immunity against EV-A71 reinfection.

Urinary ß2-microglobulin as an early marker of infantile enterovirus and human parechovirus infections.

Enterovirus and human parechovirus (HPeV) are RNA viruses belonging to the family Picornaviridae that frequently infect infants. These infections show a wide variety of clinical manifestations, from mild to severe. However, there are no known early clinical markers for diagnosis and prediction of disease severity. The aim of this study was to examine the clinical utility of urinary beta 2-microglobulin (ß2MG) for the early detection and prognosis of infantile enterovirus and HPeV infections.This retrospective study included 108 full-term infants younger than 60 days of age, including 15 with enterovirus or HPeV-3 (enterovirus/HPeV-3), 22 with respiratory syncytial virus (RSV), and 24 with bacterial infections. Laboratory data and clinical characteristics were compared among these 3 groups. Of the 15 patients with enterovirus/HPeV-3, 6 were treated with intravenous immunoglobulin (IVIG subgroup) because of severe clinical conditions.Urinary ß2MG to creatinine ratio (ß2MG/Cr) was significantly higher in the enterovirus/HPeV-3 group compared to bacterial and RSV infection groups (both P < .001). In the enterovirus/HPeV-3 group, mean peak urinary ß2MG/Cr was observed on day 1 or 2. Urinary ß2MG/Cr values were significantly higher in the IVIG subgroup than the non-IVIG subgroup (P < .001).Increased urinary ß2MG/Cr in early-stage infection may be a useful clinical marker for the detection and prediction of infantile enterovirus and HPeV infection severity.

Acute flaccid myelitis associated with enterovirus D68 infection: A case report.

RATIONALE: We present the first case of enterovirus (EV) D68, lineage B3 infection, associated with acute flaccid myelitis (AFM) in Taiwan. AFM caused by EV D68 is relatively rare. This report highlights the importance of clinical recognition of the disease and discusses treatments that can benefit such patients. PATIENT CONCERNS: A 5-year-old boy experienced sudden onset of acute flaccid paralysis (AFP) involving left arm after fever and respiratory symptoms for 3 days. DIAGNOSES: Magnetic resonance imaging (MRI) of the spinal cord revealed signal changes over segments C1 to T5 on a T2-weighted image (T2WI), compatible with the diagnosis of AFM. The EV D68 strain, cultured from the throat of the patient was identified. INTERVENTIONS: We administered intravenous immunoglobulin (IVIG, 1g/kg, twice), pulse steroid therapy (methylprednisolone, 30 mg/kg, twice) and oral prednisolone (1mg/kg/day). Rehabilitation was also arranged. OUTCOMES: The patient still had mild muscle atrophy over left arm after following-up for 1 year. LESSONS: Early diagnosis and prompt management are essential for managing this kind of patient. IVIG, pulse therapy, and oral prednisolone may play crucial roles in controlling its clinical course.