Pharmacological activation of TLR7 exerts inhibition on the replication of EV-D68 in respiratory cells.

The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.

Clinical application of phage immunoprecipitation sequencing to diagnose enterovirus D68 as the underlying etiology in a case of Gullain-Barré syndrome.

Gullain-Barré syndrome (GBS) is an acute peripheral neuropathy often preceded by respiratory or gastrointestinal infections, though molecular testing of cerebrospinal fluid (CSF) is often inconclusive. In a recent case of severe pediatric GBS in British Columbia, Canada, we detected CSF antibodies against enterovirus D (EV-D) to link GBS with prior EV-D68 respiratory infection.

Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

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.

Real-Time Enterovirus D68 Outbreak Detection through Hospital Surveillance of Severe Acute Respiratory Infection, Senegal, 2023.

In December 2023, we observed through hospital-based surveillance a severe outbreak of enterovirus D68 infection in pediatric inpatients in Dakar, Senegal. Molecular characterization revealed that subclade B3, the dominant lineage in outbreaks worldwide, was responsible for the outbreak. Enhanced surveillance in inpatient settings, including among patients with neurologic illnesses, is needed.

Multimodal Surveillance Model for Enterovirus D68 Respiratory Disease and Acute Flaccid Myelitis among Children in Colorado, USA, 2022.

Surveillance for emerging pathogens is critical for developing early warning systems to guide preparedness efforts for future outbreaks of associated disease. To better define the epidemiology and burden of associated respiratory disease and acute flaccid myelitis (AFM), as well as to provide actionable data for public health interventions, we developed a multimodal surveillance program in Colorado, USA, for enterovirus D68 (EV-D68). Timely local, state, and national public health outreach was possible because prospective syndromic surveillance for AFM and asthma-like respiratory illness, prospective clinical laboratory surveillance for EV-D68 among children hospitalized with respiratory illness, and retrospective wastewater surveillance led to early detection of the 2022 outbreak of EV-D68 among Colorado children. The lessons learned from developing the individual layers of this multimodal surveillance program and how they complemented and informed the other layers of surveillance for EV-D68 and AFM could be applied to other emerging pathogens and their associated diseases.

Respiratory Viruses in Wastewater Compared with Clinical Samples, Leuven, Belgium.

In a 2-year study in Leuven, Belgium, we investigated the use of wastewater sampling to assess community spread of respiratory viruses. Comparison with the number of positive clinical samples demonstrated that wastewater data reflected circulation levels of typical seasonal respiratory viruses, such as influenza, respiratory syncytial virus, and enterovirus D68.

Identification of four neutralizing antigenic sites on the enterovirus D68 capsid.

IMPORTANCE: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 ßB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Telaprevir Treatment Reduces Paralysis in a Mouse Model of Enterovirus D68 Acute Flaccid Myelitis.

In 2014, 2016, and 2018, the United States experienced unprecedented spikes in pediatric cases of acute flaccid myelitis (AFM), which is a poliomyelitis-like paralytic illness. Accumulating clinical, immunological, and epidemiological evidence has identified enterovirus D68 (EV-D68) as a major causative agent of these biennial AFM outbreaks. There are currently no available FDA-approved antivirals that are effective against EV-D68, and the treatment for EV-D68-associated AFM is primarily supportive. Telaprevir is an food and drug administration (FDA)-approved protease inhibitor that irreversibly binds the EV-D68 2A protease and inhibits EV-D68 replication in vitro. Here, we utilize a murine model of EV-D68 associated AFM to show that early telaprevir treatment improves paralysis outcomes in Swiss Webster (SW) mice. Telaprevir reduces both viral titer and apoptotic activity in both muscles and spinal cords at early disease time points, which results in improved AFM outcomes in infected mice. Following intramuscular inoculation in mice, EV-D68 infection results in a stereotypic pattern of weakness that is reflected by the loss of the innervating motor neuron population, in sequential order, of the ipsilateral (injected) hindlimb, the contralateral hindlimb, and then the forelimbs. Telaprevir treatment preserved motor neuron populations and reduced weakness in limbs beyond the injected hindlimb. The effects of telaprevir were not seen when the treatment was delayed, and toxicity limited doses beyond 35 mg/kg. These studies are a proof of principle, provide the first evidence of benefit of an FDA-approved antiviral drug with which to treat AFM, and emphasize both the need to develop better tolerated therapies that remain efficacious when administered after viral infections and the development of clinical symptoms. IMPORTANCE Recent outbreaks of EV-D68 in 2014, 2016, and 2018 have resulted in over 600 cases of a paralytic illness that is known as AFM. AFM is a predominantly pediatric disease with no FDA-approved treatment, and many patients show minimal recovery from limb weakness. Telaprevir is an FDA-approved antiviral that has been shown to inhibit EV-D68 in vitro. Here, we demonstrate that a telaprevir treatment that is given concurrently with an EV-D68 infection improves AFM outcomes in mice by reducing apoptosis and viral titers at early time points. Telaprevir also protected motor neurons and improved paralysis outcomes in limbs beyond the site of viral inoculation. This study improves understanding of EV-D68 pathogenesis in the mouse model of AFM. This study serves as a proof of principle for the first FDA-approved drug that has been shown to improve AFM outcomes and have in vivo efficacy against EV-D68 as well as underlines the importance of the continued development of EV-D68 antivirals.

Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.

IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

Seroprevalence of enterovirus D68 in Yamagata, Japan, between 1976 and 2019.

To clarify the epidemiology of enterovirus D68 (EV-D68), an enterovirus rarely identified in the 20th century, we performed seroepidemiological analysis against EV-D68 using sera collected in 1976, 1985, 1990, 1999, 2009, and 2019, as well as Yamagata isolate (EVD68/Yamagata.JPN/2023-89), in Yamagata, Japan. The neutralizing antibody (Ab)-positive rates for those under 20 years old were 61.0%, 82.5%, 84.3%, 46.7%, 50.5%, and 67.9%, in each year, whereas the rates for those above 20 years old were between 93.4% and 99.1%. Generally, geometric mean titers (GMTs）increased with age among children and the total GMT in each year was 25.4, 49.2, 37.2, 30.8, 29.5, and 33.9, from 1976 to 2019, respectively. The findings in this Yamagata-based study showed that the seroprevalence of EV-D68 over the last four decades has increased with age among children, as a susceptible group, and then reaches a plateau of over approximately 80% among adults. This study clearly revealed that EV-D68 was stably transmitted among children in the 20th century, when EV-D68 detection was quite rare.

Enhanced laboratory surveillance of respiratory infection disclosed the rapid rise of enterovirus D68 cases, northern Italy, August to September 2024.

We report a considerable increase in enterovirus D68 (EV-D68) cases since July 2024, culminating in an ongoing outbreak of acute respiratory infections in northern Italy, accounting for nearly 90% of all enterovirus infections. The outbreak was identified by community- and hospital-based surveillance systems, detecting EV-D68 in individuals with mild-to-severe respiratory infections. These strains belonged to B3 and a divergent A2 lineage. An increase in adult cases was observed. Enhanced surveillance and molecular characterisation of EV-D68 across Europe are needed.

Duration of Enterovirus D68 RNA Shedding in the Upper Respiratory Tract and Transmission among Household Contacts, Colorado, USA.

Enterovirus D68 (EV-D68) causes cyclical outbreaks of respiratory disease and acute flaccid myelitis. EV-D68 is primarily transmitted through the respiratory route, but the duration of shedding in the respiratory tract is unknown. We prospectively enrolled 9 hospitalized children with EV-D68 respiratory infection and 16 household contacts to determine EV-D68 RNA shedding dynamics in the upper respiratory tract through serial midturbinate specimen collections and daily symptom diaries. Five (31.3%) household contacts, including 3 adults, were EV-D68-positive. The median duration of EV-D68 RNA shedding in the upper respiratory tract was 12 (range 7-15) days from symptom onset. The most common symptoms were nasal congestion (100%), cough (92.9%), difficulty breathing (78.6%), and wheezing (57.1%). The median illness duration was 20 (range 11-24) days. Understanding the duration of RNA shedding can inform the expected rate and timing of EV-D68 detection in associated acute flaccid myelitis cases and help guide public health measures.

Trends of Enterovirus D68 Concentrations in Wastewater, California, USA, February 2021-April 2023.

In this retrospective study, we measured enterovirus D68 (EV-D68) genomic RNA in wastewater solids longitudinally at 2 California, USA, wastewater treatment plants twice per week for 26 months. EV-D68 RNA was undetectable except when concentrations increased from mid-July to mid-December 2022, which coincided with a peak in confirmed EV-D68 cases.

A novel mucosal bivalent vaccine of EV-A71/EV-D68 adjuvanted with polysaccharides from Ganoderma lucidum protects mice against EV-A71 and EV-D68 lethal challenge.

BACKGROUND: Human enteroviruses A71 (EV-A71) and D68 (EV-D68) are the suspected causative agents of hand-foot-and-mouth disease, aseptic meningitis, encephalitis, acute flaccid myelitis, and acute flaccid paralysis in children. Until now, no cure nor mucosal vaccine existed for EV-A71 and EV-D68. Novel mucosal bivalent vaccines are highly important for preventing EV-A71 and EV-D68 infections. METHODS: In this study, formalin-inactivated EV-A71 and EV-D68 were used as antigens, while PS-G, a polysaccharide from Ganoderma lucidum, was used as an adjuvant. Natural polysaccharides have the characteristics of intrinsic immunomodulation, biocompatibility, low toxicity, and safety. Mice were immunized intranasally with PBS, EV-A71, EV-D68, or EV-A71 + EV-D68, with or without PS-G as an adjuvant. RESULTS: The EV-A71 + EV-D68 bivalent vaccine generated considerable EV-A71- and EV-D68-specific IgG and IgA titres in the sera, nasal washes, saliva, bronchoalveolar lavage fluid, and feces. These antibodies neutralized EV-D68 and EV-A71 infectivity. They also cross-neutralized infections by different EV-D68 and EV-A71 sub-genotypes. Furthermore, compared with the PBS group, EV-A71 + EV-D68 + PS-G-vaccinated mice exhibited an increased number of EV-D68- and EV-A71-specific IgA- and IgG-producing cells. In addition, T-cell proliferative responses, and IFN-γ and IL-17 secretion in the spleen were substantially induced when PS-G was used as an adjuvant with EV-A71 + EV-D68. Finally, in vivo challenge experiments demonstrated that the immune sera induced by EV-A71 + EV-D68 + PS-G conferred protection in neonate mice against lethal EV-A71 and EV-D68 challenges as indicated by the increased survival rate and decreased clinical score and viral RNA tissue expression. Taken together, all EV-A71/EV-D68 + PS-G-immunized mice developed potent specific humoral, mucosal, and cellular immune responses to EV-D68 and EV-A71 and were protected against them. CONCLUSIONS: These findings demonstrated that PS-G can be used as a potential adjuvant for EV-A71 and EV-D68 bivalent mucosal vaccines. Our results provide useful information for the further preclinical and clinical development of a mucosal bivalent enterovirus vaccine against both EV-A71 and EV-D68 infections.

Comparison of acute flaccid myelitis and transverse myelitis in children and evaluation of diagnostic criteria.

BACKGROUND AND PURPOSE: Acute flaccid myelitis (AFM) and transverse myelitis (TM) are serious conditions that may be difficult to differentiate, especially at onset of disease. In this study, we compared clinical features of pediatric AFM and TM and evaluated current diagnostic criteria, aiming to improve early and accurate diagnosis. METHODS: Two cohorts of children with enterovirus D68-associated AFM and clinically diagnosed TM were compared regarding presenting clinical features, additional investigations, and outcome. Current diagnostic criteria for AFM and TM were applied to evaluate their specificity. RESULTS: Children with AFM (n = 21) compared to those with TM (n = 36) were younger (median 3 vs. 10 years), more often had a prodromal illness (100% vs. 39%), predominant proximal weakness (69% vs. 17%), and hyporeflexia (100% vs. 44%), and less often had sensory deficits (0% vs. 81%), bowel and/or bladder dysfunction (12% vs. 69%), and hyperreflexia (0% vs. 44%). On magnetic resonance imaging, brainstem involvement was more common in AFM (74% vs. 21%), whereas supratentorial abnormalities were only seen in TM (0% vs. 40%). When omitting the criterion of a sensory level, 11 of 15 (73%) children with AFM fulfilled the diagnostic criteria for TM. Of children with TM, four of 33 (12%) fulfilled the diagnostic criteria for probable/definite AFM. CONCLUSIONS: Although there is considerable overlap between AFM and TM in children, we found important early differentiating clinical and diagnostic features. Meeting diagnostic criteria for AFM in children with TM and vice versa underlines the importance of thorough clinical examination and early and accurate diagnostic studies.

Seroprevalence of Enterovirus D68 Infection among Humans: A Systematic Review and Meta-Analysis.

INTRODUCTION: Human enterovirus D68 (EV-D68), which belongs to enteroviruses of the small RNA family, is a type of enterovirus that can cause acute respiratory tract infection and central nervous system diseases. This study systematically analysed and summarized EV-D68 antibody studies in databases and identified the seropositivity rates of different regions, ages, and sexes. METHODS: Meta-analysis was performed using STATA 16.0 software. I2 and Q tests were used to analyse the heterogeneity of the included studies. Meta-regression analysis was performed for different groups, and Egger's linear regression analysis was used to evaluate publication bias. RESULTS: The results of multiple studies indicated that the serological prevalence range of EV-D68 antibody was 17.78-96.69%. The results of the meta-analysis showed that the seropositivity rate of EV-D68 antibody was 76% (95% confidence interval [CI]: 67-84%), among which that of the Chinese population was 74% (95% CI: 61-86%) and that of other countries was 79% (95% CI: 65-91%). At the same time, a subgroup analysis was conducted. The seroprevalence of EV-D68 antibody was related to age but not sex or region. CONCLUSION: The seropositivity rate was lower in the below 5-year age group; however, it gradually increased with age. The results of this study showed that EV-D68 infection was widespread in the population, and the current clinical infection situation could not reflect the actual epidemic situation of the virus, among which children under 5 years old were vulnerable to infection, which should be given greater attention for epidemic prevention and control.

Insights into the molecular evolution of enterovirus D68.

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.

Neutralizing activity of intravenous immune globulin products against enterovirus D68 strains isolated in Japan.

BACKGROUND: Enterovirus D68 (EV-D68), belonging to Enterovirus D, is a unique human enterovirus mainly associated with common respiratory diseases. However, EV-D68 can cause severe respiratory diseases, and EV-D68 endemic is epidemiologically linked to current global epidemic of acute flaccid myelitis. METHODS: In this study, we measured neutralizing antibody titers against six clinical EV-D68 isolates in nine intravenous immune globulin (IVIG) products commercially available in Japan to assess their potential as therapeutic options for severe EV-D68 infection. RESULTS: Seven IVIG products manufactured from Japanese donors contained high neutralizing antibody titers (IC50 = 0.22-85.01 µg/mL) against all six EV-D68 strains. Apparent differences in neutralizing titers among the six EV-D68 strains were observed for all IVIG products derived from Japanese and non-Japanese blood donors. CONCLUSIONS: High levels of EV-D68-neutralizing antibodies in IVIG products manufactured from Japanese donors suggest that anti-EV-D68 antibodies are maintained in the Japanese donor population similarly as found in foreign blood donors. Apparent differences in neutralizing antibody titers against the six EV-D68 strains suggest distinct antigenicity among the strains used in this study regardless of the genetic similarity of EV-D68.

Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014-2021.

To determine molecular epidemiology and clinical features of enterovirus D68 (EV-D68) infections, we reviewed EV-D68-associated respiratory cases at a hospital in Barcelona, Spain, during 2014-2021. Respiratory samples were collected from hospitalized patients or outpatients with symptoms of acute respiratory tract infection or suggestive of enterovirus infection. Enterovirus detection was performed by real-time multiplex reverse transcription PCR and characterization by phylogenetic analysis of the partial viral protein 1 coding region sequences. From 184 patients with EV-D68 infection, circulating subclades were B3 (80%), D1 (17%), B2 (1%), and A (<1%); clade proportions shifted over time. EV-D68 was detected mostly in children (86%) and biennially (2016, 2018, 2021). In patients <16 years of age, the most common sign/symptom was lower respiratory tract infection, for which 11.8% required pediatric intensive care unit admission and 2.3% required invasive mechanical ventilation; neurologic complications developed in 1. The potential neurotropism indicates that enterovirus surveillance should be mandatory.

Circulation of Enterovirus D68 during Period of Increased Influenza-Like Illness, Maryland, USA, 2021.

We report enterovirus D68 circulation in Maryland, USA, during September-October 2021, which was associated with a spike in influenza-like illness. The characterized enterovirus D68 genomes clustered within the B3 subclade that circulated in 2018 in Europe and the United States.