Enterovirus D68 (EV-D68) is a significant global health threat, responsible for severe respiratory and neurological complications, with no FDA-approved antiviral treatments currently available. The 3C protease of EV-D68, an essential enzyme involved in viral replication, represents a key target for therapeutic development. In this study, we employed a comprehensive computational approach, including molecular docking, pharmacokinetic predictions, and molecular dynamics simulations, to identify potential natural inhibitors of the EV-D68 3C protease. Screening a library of natural compounds, Withaferin-A (CID: 265,237) and Baicalin (CID: 64,982) emerged as top candidates due to their favorable pharmacokinetic profiles, high binding affinities (-10.7 kcal/mol for Withaferin-A and -9.5 kcal/mol for Baicalin), and interactions with key residues in the protease's active site. The molecular dynamics simulations demonstrated the stability of the protein-ligand complexes, with low root mean square deviation (RMSD) and fluctuation (RMSF) values over a 100-ns trajectory. Free energy calculations further supported the superior binding efficiency of Withaferin-A. These findings suggest that Withaferin-A and Baicalin have significant potential as natural inhibitors of EV-D68 3C protease, offering a promising foundation for future experimental validation and the development of targeted antiviral therapies against EV-D68.

Enterovirus D68 (EV-D68) is a leading non-polio enterovirus that causes severe respiratory diseases and poliomyelitis-like illness in children. Viral entry represents a potential multifaceted target for antiviral intervention; however, there are no approved inhibitors to block EV-D68. Here, we identify the functionally undescribed membrane protein major facilitator superfamily-domain-containing protein 6 (MFSD6) as an EV-D68 entry factor amenable to therapeutic intervention. Specifically, MFSD6 expression is crucial for EV-D68 replication. MFSD6 binds to EV-D68 particles and is necessary for virus attachment to cells. The second extracellular domain of the MFSD6 molecule is involved in the recognition of EV-D68. On the basis of these findings, we engineered a recombinant protein complex comprising the MFSD6 ectodomain fused to Fc (MFSD6-Fc(CH3)), which potently inhibited EV-D68 uptake. MFSD6-Fc(CH3) effectively blocked EV-D68 infection in vitro and prevented lethality in newborn mice. In conclusion, our study not only identifies MFSD6 as an EV-D68 entry factor but also reveals a potential antiviral target and therapeutic agent.

We report the epidemiology, treatment and outcomes of acute flaccid myelitis (AFM), including the first two cases of enterovirus (EV) D68 myelitis, in Singapore.

Retrospective observational study from a paediatric hospital in Singapore, from January 2012 to December 2022. Clinical, laboratory, neuroimaging and outcome data were analysed.

Eight patients were identified (all male). Median age at presentation was 3 (interquartile range (IQR) 2.2-10.0) years. Seven (87.5%) patients were clustered in two time periods (July 2018-January 2019 and April-August 2022). Presenting features were a preceding febrile respiratory illness (n = 6) or hand-foot-and-mouth disease (n = 2), upper limb weakness (n = 5, 62.5%) and neurogenic bladder (3, 37.5%), while the spinal cord lesions were predominantly in the cervical region (7, 87.5%). Five (62.5%) and six (75.0%) patients had elevated cerebrospinal fluid (CSF) white cells (median 7.5/mm3 (IQR 2.8-40.3)) and protein (median 0.6 g/L (IQR 0.4-0.7)), respectively. CSF was negative for pathogens. Two (50%) of four patients with EV detected in respiratory/rectal swabs had EVD68 (2022 cluster). All received intravenous methylprednisolone, six (75%) had additional intravenous immunoglobulin and either plasma exchange therapy (n = 1) or intravenous tocilizumab (n = 1). Median modified Rankin Scale (mRS) at acute illness was 4 (IQR grades 3-5), with an improvement (median 2 (IQR 1.8-2.3) mRS grades) on follow-up (median duration 3.7 (IQR 1.4-4.1) years). One patient (12.5%) had a full recovery and seven (87.5%) have moderate disability (mRS 2-3).

Disability risk in AFM is high despite aggressive immunotherapy. We report the first two confirmed cases of EV D68 AFM in Singapore and Southeast Asia.

This review explores diverse infectious etiologies of peripheral nervous system (PNS) dysfunction, spanning sensory and motor neurons, nerves, and associated structures. Progress in viral and bacterial infections reveals multifaceted mechanisms underlying neuropathies, including viral neurotoxicity and immune-mediated responses. Latest diagnostic advances facilitate early PNS complication detection, with ongoing research offering promising treatment avenues. Emerging pathogens like severe acute respiratory syndrome coronavirus 2, Zika virus, and EV-D68 highlight the evolving infectious neuropathy paradigm. Recognizing characteristic patterns and integrating clinical factors are pivotal for precise diagnosis and tailored intervention. Challenges persist in assessment and management due to varied pathogenic mechanisms. Advancements in understanding pathogenesis have improved targeted therapies, yet gaps remain in effective treatments. Ongoing research is crucial for optimizing approaches and improving patient outcomes.

The development of antivirals for respiratory viruses has advanced markedly in response to the growing threat of pathogens such as Influenzavirus (IAV), respiratory syncytial virus (RSV), and SARS-CoV-2. This article reviews the advances and challenges in this field, highlighting therapeutic strategies that target critical stages of the viral replication cycle, including inhibitors of viral entry, replication, and assembly. In addition, innovative approaches such as inhibiting host cellular proteins to reduce viral resistance and repurposing existing drugs are explored, using advanced bioinformatics tools that optimize the identification of antiviral candidates. The analysis also covers emerging technologies such as nanomedicine and CRISPR gene editing, which promise to improve the stability and efficacy of treatments. While current antivirals offer valuable options, they face challenges such as viral evolution and the need for accessible treatments for vulnerable populations. This article underscores the importance of continued innovation in biotechnology to overcome these limitations and provide safe and effective treatments. Combining traditional and advanced approaches in developing antivirals is essential in order to address respiratory viral diseases that affect global health.

Enterovirus D68 (EV-D68) has had several outbreaks worldwide, yet no FDA-approved antiviral is available for treating this viral infection. EV-D68 infection typically leads to respiratory illnesses and, in severe cases, can cause neurological complications and even death, particularly in children. This study identified a small molecule, A-967079, as an EV-D68 antiviral through phenotypical screening. A-967079 has shown potent and broad-spectrum antiviral activity with a high selectivity index against multiple strains of EV-D68. Pharmacological characterization of the mechanism of action involving time-of-addition, resistance selection, and differential scanning fluorimetry assays suggests that viral 2C protein is the drug target. Overall, A-967079 represents a promising candidate for further development as an EV-D68 antiviral.

Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases in western countries. However, the prevalence and clinical characteristics of EV-D68-associated pneumonia in China remain understudied.

Between January 2022 and January 2024, 28 patients with EV-D68-associated pneumonia were enrolled. We described the prevalence, demographic, and clinical characteristics of patients with EV-D68-associated pneumonia.

Among the 28 enrolled patients, the male-to-female ratio was 1.5:1, and the average age at onset was 4.6 ± 2.7 years. Four (14.3%) required intensive care support. Monoinfection occurred in 11 cases (39.3%), while coinfections were seen in 17 cases (60.7%). 82.1% of patients had a history of one or more atopic diseases. The primary symptoms of EV-D68-associated pneumonia included cough (100%), wheezing (53.6%), and fever (53.6%). Radiologically, patchy opacity was the predominant feature, observed in 72.7% of cases. No statistically significant differences were found in symptoms, laboratory tests, or imaging findings between the monoinfection and coinfection groups. Except for one case who developed quadriplegia sequelae, all patients had a favorable prognosis.

EV-D68 is not a common pathogen for community-acquired pneumonia in China. It mainly affects young children, particularly those with atopic constitution. The overall prognosis is favorable, although neurological complications are rare and may lead to severe sequelae. This study is the first investigation into the prevalence and clinical characteristics of EV-D68-associated pneumonia in China.

A method that has rapidly evolved for detection of viral pathogens are loop-mediated isothermal amplification (LAMP) assays. The available LAMP assays usually target the most common viral strains, including enteroviruses, but for the atypical enterovirus D68 strain VR-1197 this method has not yet been developed. Enterovirus D68 are known for severe respiratory distress in children, and atypical strains are less likely to be detected by traditional methods. This study targets the atypical EVD68 strain VR-1197 and have developed a rapid detection method saving time when differentiating enterovirus strains. This study present method development and review the sensitivity and specificity compared to traditional RT-qPCR, and wet lab cross reactivity with other airway pathogens. The EVD68 VR-1197 assay can be a rapid POC (Point of care) test for atypical EVD68 VR-1197 and have the potential as reliable detection method with minimal technological requirements.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages.

Enterovirus D68 (EV-D68), a respiratory RNA virus in the family Picornaviridae, is implicated as a potential etiological agent for acute flaccid myelitis in preteen adolescents. The absence of a specific therapeutic intervention necessitates the development of an effective animal model for EV-D68. The AG129 mouse strain, characterized by the double knockout of IFN-α/β and IFN-γ receptors on the 129 genetic background, has been proposed as a suitable model for EV-D68. The goals of this study were to assess the effect of a nonmouse-adapted EV-D68 strain (US/MO/14-18947, NR-49129) in AG129 (IFN-α/β and IFN-γ receptors null), A129 (IFN-α/β receptor null), G129 (IFN-γ receptor null), and the 129 background strain (129S2/SvPasCrl) when infected intraperitoneally at 10 d of age. Both AG129 and A129 strains demonstrated similar clinical signs (paralysis, paresis, lethargy, dyspnea [characterized by prominent abdominal respiration], and morbidity requiring euthanasia) induced by EV-D68. While G129 and 129S2 strains also exhibited susceptibility to EV-D68, the severity of clinical signs was less than in AG129 and A129 strains, and many survived to the experimental endpoint. Histopathological and immunohistochemical data confirmed EV-D68 tropism for the skeletal muscle and spinal cord and suggest that the dyspnea observed in infected mice could be attributed, in part, to lesions in the diaphragmatic skeletal muscles. These findings contribute valuable insights into the pathogenesis of EV-D68 infection in this mouse model and provide investigators with key information on virus dose and mouse strain selection when using this mouse model to evaluate candidate EV-D68 therapeutics.

Enterovirus D68 (EV-D68) is responsible for millions of infections. In the last decade, there has been an increase in the number of children requiring hospital or critical care admission due to severe respiratory illness. Nevertheless, the epidemiological and clinical importance of EV-D68 infections remains unclear.

We aimed to determine the local prevalence of EV-D68 infection in pediatric patients and to characterize its clinical presentation and disease burden compared with non-EV-D68 enterovirus and human rhinovirus (RV) infections.

We performed a retrospective single-center study of children presenting with respiratory symptoms and positive respiratory panel polymerase chain reaction for EV/RV from November 2018 to December 2019. We tested EV/RV positive specimens with an EV-D68-specific polymerase chain reaction to discriminate EV-D68, non-EV-D68 and RV and compared their respective clinical presentation, outcomes and treatment.

We identified 224 patients (median age 21 months), of which 16 (7%) were EV-D68 positive. They presented with cough (88%), wheezing (62%) and dyspnea (75%). EV-D68 infection had an odds ratio regarding pediatric respiratory severity-score of 11.6 relative to non-EV-D68 [confidence intervals (CI): 3.51-41.14], and of 9.9 (CI: 3.75-27.95) relative to RV. The fitted logistic regression showed that the odds of intensive care were 5 times more likely with EV-D68 than RV infection (CI: 1.32-19.28; P = 0.001). Patients with EV-D68 infections were more likely to receive medical support in the form of supplementary oxygen, antibiotics and steroids.

EV-D68 infection is associated with higher morbidity and a higher likelihood of intensive care treatment than non-EV-D68 and RV infections.

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68.

Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.

Spinal infections are a diverse group of diseases affecting different compartments of the spine with variable clinical and imaging presentations. Diagnosis of spinal infections is based on a combination of clinical features, laboratory markers, and imaging studies. Imaging plays a pivotal role in the diagnosis and management of spinal infections. The characteristic imaging manifestations of bacterial and viral infections in the spine are discussed with key teaching points emphasized.

Enteroviruses are a type of RNA-strained virus with more than 100 different genotypes. Infection can be asymptomatic, and, if any, symptoms can range from mild to severe. Some patients can develop neurological involvement, such as aseptic meningitis, encephalitis, or even cardiorespiratory failure. However, in children, the risk factors for developing severe neurological involvement are not well understood. The aim of this retrospective study was to analyze some characteristics associated with severe neurological involvement in children hospitalized for neurological disease after enterovirus infection.

retrospective observational study analyzing clinical, microbiological and radiological data of 174 children hospitalized from 2009 to 2019 in our hospital. Patients were classified according to the World Health Organization case definition for neurological complications in hand, foot and mouth disease.

Our findings showed that, in children between 6 months old and 2 years of age, the appearance of neurological symptoms within the first 12h from infection onset-especially if associated with skin rash-was a significant risk factor for severe neurological involvement. Detection of enterovirus in cerebrospinal fluid was more likely in patients with aseptic meningitis. By contrast, other biological samples (e.g., feces or nasopharyngeal fluids) were necessary to detect enterovirus in patients with encephalitis. The genotype most commonly associated with the most severe neurological conditions was EV-A71. E-30 was mostly associated with aseptic meningitis.

Awareness of the risk factors associated with worse neurological outcomes could help clinicians to better manage these patients to avoid unnecessary admissions and/or ancillary tests.

Enterovirus D68 (EV-D68), a pathogen that causes respiratory symptoms, mainly in children, has been implicated in acute flaccid myelitis, which is a poliomyelitis-like paralysis. Currently, there are no licensed vaccines or treatments for EV-D68 infections. Here, we investigated the optimal viral inactivation reagents, vaccine adjuvants, and route of vaccination in mice to optimize an inactivated whole-virion (WV) vaccine against EV-D68. We used formalin, β-propiolactone (BPL), and hydrogen peroxide as viral inactivation reagents and compared their effects on antibody responses. Use of any of these three viral inactivation reagents effectively induced neutralizing antibodies. Moreover, the antibody response induced by the BPL-inactivated WV vaccine was enhanced when adjuvanted with cytosine phosphoguanine oligodeoxynucleotide (CpG ODN) or AddaVax (MF59-like adjuvant), but not with aluminum hydroxide (alum). Consistent with the antibody response results, the protective effect of the inactivated WV vaccine against the EV-D68 challenge was enhanced when adjuvanted with CpG ODN or AddaVax, but not with alum. Further, while the intranasal inactivated WV vaccine induced EV-D68-specific IgA antibodies in the respiratory tract, it was less protective against EV-D68 challenge than the injectable vaccine. Thus, an injectable inactivated EV-D68 WV vaccine prepared with appropriate viral inactivation reagents and an optimal adjuvant is a promising EV-D68 vaccine.

Enteroviruses (EVs), comprise a genus in the Picornaviridae family, which have been shown to be neurotropic and can cause various neurological disorders or long-term neurological condition, placing a huge burden on society and families. The blood-brain barrier (BBB) is a protective barrier that prevents dangerous substances from entering the central nervous system (CNS). Recently, numerous EVs have been demonstrated to have the ability to disrupt BBB, and further lead to severe neurological damage. However, the precise mechanisms of BBB disruption associated with these EVs remain largely unknown. In this Review, we focus on the molecular mechanisms of BBB dysfunction caused by EVs, emphasizing the invasiveness of enterovirus A71 (EVA71), which will provide a research direction for further treatment and prevention of CNS disorders.

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.

The past decade has seen the global reemergence and rapid spread of enterovirus D68 (EV-D68), a respiratory pathogen that causes severe respiratory illness and paralysis in children. EV-D68 was first isolated in 1962 from children with pneumonia. Sporadic cases and small outbreaks have been reported since then with a major respiratory disease outbreak in 2014 associated with an increased number of children diagnosed with polio-like paralysis. From 2014-2018, major outbreaks have been reported every other year in a biennial pattern with > 90% of the cases occurring in children under the age of 16. With the outbreak of SARS-CoV-2 and the subsequent COVID-19 pandemic, there was a significant decrease in the prevalence EV-D68 cases along with other respiratory diseases. However, since the relaxation of pandemic social distancing protocols and masking mandates the number of EV-D68 cases have begun to rise again - culminating in another outbreak in 2022. Here we review the virology, pathogenesis, and the immune response to EV-D68, and discuss the epidemiology of EV-D68 infections and the divergence of contemporary strains from historical strains. Finally, we highlight some of the key challenges in the field that remain to be addressed.

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

Enterovirus D68 (EV-D68), belonging to the genus Enterovirus of the Picornavirus family, is an emerging pathogen that can cause neurological and respiratory diseases in children. However, there is little understanding of the pathogenesis of EV-D68, and no effective vaccine or drug for the prevention or treatment of the diseases caused by this virus is available. Autophagy is a cellular process that targets cytoplasmic proteins or organelles to the lysosomes for degradation. Enteroviruses strategically harness the host autophagy pathway to facilitate the completion of their life cycle. Therefore, we selected an autophagy compound library to screen for autophagy-related compounds that may affect viral growth. By using the neutralization screening assay, we identified a compound, 'licochalcone A' that significantly inhibited EV-D68 replication. To investigate the mechanism by which licochalcone A inhibits EV-D68 replication and to identify the viral life cycle stage it inhibits, the time-of-addition, viral attachment, viral entry, and dual-luciferase reporter assays were performed. The results of the time-of-addition assay showed that licochalcone A, a characteristic chalcone found in liquorice roots and widely used in traditional Chinese medicine, inhibits EV-D68 replication during the early stages of the viral life cycle, while those of the dual-luciferase reporter assay showed that licochalcone A does not regulate viral attachment and entry, but inhibits EV-D68 IRES-dependent translation. Licochalcone A also inhibited enterovirus A71 and coxsackievirus B3 but did not significantly inhibit dengue virus 2 or human coronavirus 229E replication. Licochalcone A regulates IRES translation to inhibit EV-D68 viral replication.

While infections caused by rhinoviruses and enteroviruses are common among children, the entirety of their clinical impact remains elusive. We compared the clinical outcomes of children with rhinovirus/enterovirus infections to other common respiratory viruses in outpatient settings.

We conducted a retrospective analysis of nasopharyngeal samples singly positive for human rhinovirus/enterovirus (HRV/ENT), influenza A/B (FLU) or respiratory syncytial virus (RSV) from patients ≤17 years submitted for clinical testing via multiplex polymerase chain reaction between 2016 and 2019. We evaluated the following outpatient outcomes: days of respiratory symptoms before testing; visits for respiratory symptoms; receipt of a breathing treatment; receipt of antibiotics and hospital admission. Statistical analyses were conducted controlling for age and comorbid conditions.

There were 1355 positive samples included in this analysis (HRV/ENT: n = 743, FLU: n = 303 and RSV: n = 309). Compared to HRV/ENT, children with FLU had 28% fewer days of respiratory symptoms (β: -0.32; 95% confidence interval: -0.46 to -0.18; P < 0.001), fewer visits for respiratory symptoms, and significantly decreased odds of receiving a breathing treatment or antibiotics, and admission to the hospital. Children with RSV had a similar number of days of respiratory symptoms, outpatient visits and odds of hospital admission, but significantly increased odds of receiving a breathing treatment and antibiotics compared to those with HRV/ENT.

Clinicians should have a high level of vigilance when managing children with positive respiratory viral testing for HRV/ENT given the potential for clinical outcomes similar to and, in some instances, worse than known highly pathogenic viruses.

Enterovirus D68 (EV-D68) is one of hundreds of non-polio enteroviruses that typically cause cold-like respiratory illness. The first EV-D68 outbreak in the United States in 2014 aroused widespread concern among the public and health authorities. The infection was found to be associated with increased surveillance of acute flaccid myelitis, a neurological condition that causes limb paralysis in conjunction with spinal cord inflammation. In vitro studies utilising two-dimensional (2D) and three-dimensional (3D) culture systems have been employed to elucidate the pathogenic mechanism of EV-D68. Various animal models have also been developed to investigate viral tropism and distribution, pathogenesis, and immune responses during EV-D68 infection. EV-D68 infections have primarily been investigated in respiratory, intestinal and neural cell lines/tissues, as well as in small-size immunocompetent rodent models that were limited to a young age. Some studies have implemented strategies to overcome the barriers by using immunodeficient mice or virus adaptation. Although the existing models may not fully recapitulate both respiratory and neurological disease observed in human EV-D68 infection, they have been valuable for studying pathogenesis and evaluating potential vaccine or therapeutic candidates. In this review, we summarise the methodologies and findings from each experimental model and discuss their applications and limitations.

Enterovirus D68 (EV-D68) is a respiratory virus that primarily affects children and has been associated with sporadic outbreaks of respiratory illness worldwide. In the present study, temporal spreading and molecular evolution of EV-D68 clades (A1, A2, B, B1, B2, B3, and C) were evaluated. Bayesian coalescent analysis was performed to study viral evolution. Data from 976 whole-genome sequences (WGSs) collected between 1977 and 2022 were evaluated. For A1, the most recent common ancestor was dated to 2005-04-17 in the USA; for A2 it was 2003-12-23 in China; for B, it was 2003-07-06 in China; for B1, it was 2010-03-21 in Vietnam; for B2, it was 2006-11-25 in Vietnam; for B3, it was 2011-01-15 in China; and for C, it was 2000-06-27 in the USA. The molecular origin of EV-D68 was in Canada in 1995, and later it was disseminated in France in 1997, the USA in 1999, Asia in 2008, the Netherlands in 2009, New Zealand in 2010, Mexico in 2014, Kenya in 2015, Sweden in 2016, Switzerland in 2018, Spain in 2018, Belgium in 2018, Australia in 2018, and Denmark in 2019. In 2022, this virus circulated in the USA. In conclusion, EV-D68 originated in Canada in the 1990s and spread to Europe, Asia, Oceania, Latin America, and Africa.

Enteroviruses (EVs) are classified into 15 species according to their sequence diversity. They include four human EV (A, B, C, and D) and three rhinoviruses (A, B, and C), and cause diseases in millions of people worldwide. Generally, individuals with enteroviral infections have mild clinical symptoms, including respiratory illness, vomiting, diarrhea, dizziness, and fever. More importantly, some members of the human EV family are neurotropic pathogens that may cause a wide range of clinical diseases, such as aseptic meningitis and encephalitis. Previously, the EV that caused the most severe neurotropic symptoms was poliovirus (PV), a member of the EV C group. Poliovirus has been eliminated in most countries through a global vaccination campaign. Non-PV EVs infect the central nervous system (CNS) and are the major EVs causing neurological diseases. These human non-PV EVs include EV A (e.g., EV-A71, CVA6, and CVA16), B (e.g., CVA9 and CVB3, CVB5, echovirus 11 [E11], E30, and E7), C (e.g., CVA24), and D (e.g., EV-D68). Here, we review the relationship between EV infection and CNS diseases and advance in the use of cellular receptors and host immune responses during viral infection.

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

RNA viruses continue to remain a clear and present threat for potential pandemics due to their rapid evolution. To mitigate their impact, we urgently require antiviral agents that can inhibit multiple families of disease-causing viruses, such as arthropod-borne and respiratory pathogens. Potentiating host antiviral pathways can prevent or limit viral infections before escalating into a major outbreak. Therefore, it is critical to identify broad-spectrum antiviral agents. We have tested a small library of innate immune agonists targeting pathogen recognition receptors, including TLRs, STING, NOD, Dectin and cytosolic DNA or RNA sensors. We observed that TLR3, STING, TLR8 and Dectin-1 ligands inhibited arboviruses, Chikungunya virus (CHIKV), West Nile virus (WNV) and Zika virus, to varying degrees. Cyclic dinucleotide (CDN) STING agonists, such as cAIMP, diABZI, and 2',3'-cGAMP, and Dectin-1 agonist scleroglucan, demonstrated the most potent, broad-spectrum antiviral function. Comparative transcriptome analysis revealed that CHIKV-infected cells had larger number of differentially expressed genes than of WNV and ZIKV. Furthermore, gene expression analysis showed that cAIMP treatment rescued cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provided protection against CHIKV in a CHIKV-arthritis mouse model. Cardioprotective effects of synthetic STING ligands against CHIKV, WNV, SARS-CoV-2 and enterovirus D68 (EV-D68) infections were demonstrated using human cardiomyocytes. Interestingly, the direct-acting antiviral drug remdesivir, a nucleoside analogue, was not effective against CHIKV and WNV, but exhibited potent antiviral effects against SARS-CoV-2, RSV (respiratory syncytial virus), and EV-D68. Our study identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses, which can be rapidly deployed to prevent or mitigate future pandemics.

In patients with central nervous system (CNS) infections identification of the causative pathogen is important for treatment. Metagenomic next-generation sequencing techniques are increasingly being applied to identify causes of CNS infections, as they can detect any pathogen nucleic acid sequences present. Viromic techniques that enrich samples for virus particles prior to sequencing may simultaneously enrich ribosomes from bacterial pathogens, which are similar in size to small viruses.

We studied the performance of a viromic library preparation technique (VIDISCA) combined with low-depth IonTorrent sequencing (median ~ 25,000 reads per sample) for detection of ribosomal RNA from common pathogens, analyzing 89 cerebrospinal fluid samples from patients with culture proven bacterial meningitis.

Sensitivity and specificity to Streptococcus pneumoniae (n = 24) before and after optimizing threshold parameters were 79% and 52%, then 88% and 90%. Corresponding values for Neisseria meningitidis (n = 22) were 73% and 93%, then 67% and 100%, Listeria monocytogenes (n = 24) 21% and 100%, then 27% and 100%, and Haemophilus influenzae (n = 18) 56% and 100%, then 71% and 100%. A higher total sequencing depth, no antibiotic treatment prior to lumbar puncture, increased disease severity, and higher c-reactive protein levels were associated with pathogen detection.

We provide proof of principle that a viromic approach can be used to correctly identify bacterial ribosomal RNA in patients with bacterial meningitis. Further work should focus on increasing assay sensitivity, especially for problematic species (e.g. L. monocytogenes), as well as profiling additional pathogens. The technique is most suited to research settings and examination of idiopathic cases, rather than an acute clinical setting.