Detection of Enterovirus D68 in Wastewater Samples from the UK between July and November 2021

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68′s known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed.

Strain dependent structural effects and in vivo efficacy of enterovirus-D68 inhibitors

Acute flaccid myelitis (AFM) leads to loss of limb control in young children and is likely due to Enterovirus-D68 (EV-D68), for which there is no current treatment. We have developed a lead isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of the pleconaril-resistant CVB3-Woodruff (IC50 6-20 nM), EV-D68 (IC50 58 nM), and other enteroviruses. A mouse respiratory model of EV-D68 infection, in which pleconaril is inactive, showed decreased viremia of 3 log units as well as statistically significant 1 log reduction in lung titer reduction at day 5 after treatment with 11526092. A cryo-electron microscopy (cryo-EM) structure of EV-D68 in complex with 11526092 suggests that the increased potency may be due to additional hydrophobic interactions. Cryo-EM structures of 11526092 and pleconaril demonstrate destabilization of EV-D68 (MO strain) compared to the previously described stabilization of EV-D68 (Fermon strain) with pleconaril, illustrating clear strain dependent mechanisms of this molecule. 11526092 represents a more potent inhibitor in vitro with in vivo efficacy providing a potential future treatment for EV-D68 and AFM, suggesting an improvement over pleconaril for further optimization.

Enterovirus D68 epidemic, UK, 2018, was caused by subclades B3 and D1, predominantly in children and adults respectively, with both subclades exhibiting extensive genetic diversity

Enterovirus D68 (EV-D68) has been recently identified in biennial epidemics coinciding with diagnoses of non-polio acute flaccid paralysis/myelitis (AFP/AFM). We investigated the prevalence, genetic relatedness and associated clinical features of EV-D68 in 194 known EV positive samples from late 2018, UK. EV-D68 was detected in 83 (58%) of the 143 EV positive samples. Sequencing and phylogenetic analysis revealed an extensive genetic diversity, split between subclades B3 (n=50) and D1 (n=33), suggesting epidemiologically unrelated infections. B3 predominated in children and younger adults, and D1 in older adults and the elderly (p=0.0009). Clinical presentation indicated causation or exacerbation of respiratory distress in 91.4% of EV-D68-positive individuals, principally: cough (75.3%), shortness of breath (56.8%), coryza (48.1%), wheeze (46.9%), supplemental oxygen required (46.9%) and fever (38.9%). Clinical features were not distinguished by subclade. Two cases of AFM were observed, one with EV-D68 detectable in the cerebrospinal fluid, otherwise neurological symptoms were rarely reported (n=4).

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1β, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

High throughput sequencing of whole transcriptome and construct of ceRNA regulatory network in RD cells infected with enterovirus D68

Background With the advancement of sequencing technologies, a plethora of noncoding RNA (ncRNA) species have been widely discovered, including microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs). However, the mechanism of these non-coding RNAs in diseases caused by enterovirus d68 (EV-D68) remains unclear. The goal of this research was to identify significantly altered circRNAs, lncRNAs, miRNAs, and mRNAs pathways in RD cells infected with EV-D68, analyze their target relationships, demonstrate the competing endogenous RNA (ceRNA) regulatory network, and evaluate their biological functions. Methods The total RNAs were sequenced by high-throughput sequencing technology, and differentially expressed genes between control and infection groups were screened using bioinformatics method. We discovered the targeting relationship between three ncRNAs and mRNA using bioinformatics methods, and then built a ceRNA regulatory network centered on miRNA. The biological functions of differentially expressed mRNAs (DEmRNAs) were discovered through GO and KEGG enrichment analysis. Create a protein interaction network (PPI) to seek for hub mRNAs and learn more about protein–protein interactions. The relative expression was verified using RT-qPCR. The effects of Fos and ARRDC3 on virus replication were confirmed using RT-qPCR, virus titer (TCID50/ml), Western blotting. Results 375 lncRNAs (154 upregulated and 221 downregulated), 33 circRNAs (32 upregulated and 1 downregulated), 96 miRNAs (49 upregulated and 47 downregulated), and 239 mRNAs (135 upregulated and 104 downregulated) were identified as differently in infected group compare to no-infected group. A single lncRNA or circRNA can be connected with numerous miRNAs, which subsequently coregulate additional mRNAs, according to the ceRNA regulatory network. The majority of DEmRNAs were shown to be connected to DNA binding, transcription regulation by RNA polymerase II, transcription factor, MAPK signaling pathways, Hippo signal pathway, and apoptosis pathway, according to GO and KEGG pathway enrichment analysis. The hub mRNAs with EGR1, Fos and Jun as the core were screened through PPI interaction network. We preliminarily demonstrated that the Fos and ARRDC3 genes can suppress EV-D68 viral replication in order to further verify the results of full transcriptome sequencing. Conclusion The results of whole transcriptome analysis after EV-D68 infection of RD cells were first reported in this study, and for the first time, a ceRNA regulation network containing miRNA at its center was established for the first time. The Fos and ARRDC3 genes were found to hinder viral in RD cells. This study establishes a novel insight host response during EV-D68 infection and further investigated potential drug targets.

Zinc Influx Restricts Enterovirus D68 Replication

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that causes severe respiratory diseases and neurologic manifestations. Since the 2014 outbreak, EV-D68 has been reported to cause severe complications worldwide. However, there are currently no approved antiviral agents or vaccines for EV-D68. In this study, we found that zinc ions exerted substantial antiviral activity against EV-D68 infection in vitro. Zinc salt treatment potently suppressed EV-D68 RNA replication, protein synthesis, and infectious virion production and inhibited cytopathic effects without producing significant cytotoxicity at virucidal concentrations (EC50=0.033mM). Zinc chloride (ZnCl2) treatment moderately inhibited EV-D68 attachment. Time-dose analysis of EV-D68 structural protein VP1 synthesis showed stronger suppression of VP1 in the culture medium than that in the cell lysates. Furthermore, a zinc ionophore, pyrrolidine dithiocarbamate, which can transport zinc ions into cells, also enhanced the anti-EV-D68 activity of ZnCl2 treatment. Taken together, our results demonstrated that the enhancement of zinc influx could serve as a powerful strategy for the therapeutic treatment of EV-D68 infections.