A Tale of Two Lenses: #Emergency department indoor - #air hybrid-capture #metagenomics complements #wastewater by adding a human-focused respiratory #virus perspective

 

Abstract

Background: 

Continuous, non-invasive viral surveillance is essential to monitor emerging pathogens and guide public health responses. Most environmental surveillance studies use targeted qPCR approaches, and comparisons between wastewater and indoor air surveillance remain limited. We aimed to compare the utility of emergency department indoor air and urban wastewater for tracking circulating viruses and resolving genomic information. 

Methods: 

We conducted a matched-pair study comparing 19 weekly indoor air samples from the central ventilation exhaust shaft of an emergency department and 19 24-hour composite municipal wastewater samples in Leuven, Belgium, from December 2024 to April 2025. Both sample sets were processed using probe-based hybrid-capture viral metagenomics targeting over 3000 viral species, using influenza A as a clinically relevant test case. 

Findings: 

Wastewater captured higher overall viral diversity (233 versus 106 species) and more complete genomes compared to indoor air, showing a relatively stable composition, mainly of enteric and animal-associated viruses. Indoor air demonstrated lower overall diversity but was enriched for respiratory viruses, including influenza A, coronaviruses, metapneumovirus, and respiratory syncytial virus, and more frequently achieved high genome coverage for these pathogens. Although both sample types permitted influenza A subtype characterization, influenza A genomes from wastewater were often less well covered. When coverage thresholds were met, indoor air supported targeted antiviral resistance-site screening for influenza A and RSV-A. 

Interpretation: 

Wastewater and indoor air generate distinct but complementary viromes. Wastewater acts as a diverse, population-level monitor for One-Health applications, whereas indoor air serves as a targeted, human-centric sentinel system facilitating further genomic characterization for respiratory viruses.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

Mustafa Karatas is supported by a Research Foundation Flanders (FWO) fundamental research scholarship (number: 11P7I24N). C.G., L.C., E.H., S.G. and E.A. acknowledge support from the DURABLE project. The DURABLE project has been funded by the European Union, under the EU4Health Programme (EU4H), project no. 101102733. Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The computing power in this work was provided by the VSC (Flemish Supercomputer Centre), financed by the FWO and the Flemish government department EWI.

Source: MedRxIV, https://www.medrxiv.org/

Link: https://www.medrxiv.org/content/10.64898/2026.03.13.26348311v1

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A #clinical #SARS-CoV-2 #Mpro #inhibitor blocks replication of multiple #enteroviruses and confers oral in vivo protection in animal models

 

Abstract

Enteroviruses, which belong to the family Picornaviridae, cause hand, foot, and mouth disease (HFMD), respiratory symptoms, and severe neurological complications in children. Since vaccines cannot provide cross-protection against different serotypes of enteroviruses, the development of broad-spectrum anti-enteroviral drugs is imperative. The viral 3C protease (3Cpro), which is essential for polyprotein processing represents a validated target for therapeutic intervention. Importantly, enterovirus 3Cpro shares conserved structural and catalytic features with coronavirus main protease (Mpro, also known as 3C-like protease, 3CLpro), providing a rationale for cross-target inhibitor repurposing. Through targeted screening of peptidomimetic protease inhibitors, a clinical-stage SARS-CoV-2 Mpro inhibitor was identified as a potent inhibitor of enterovirus A71 (EV71) 3Cpro. Bofutrelvir displayed nanomolar antiviral activity in multiple cell lines and demonstrated broad-spectrum efficacy against several enteroviruses including coxsackievirus B5, coxsackievirus A16 (CA16) and echovirus 11. In EV71 infected neonatal mice, intraperitoneal administration of bofutrelvir markedly reduced viral loads in brain, spinal cord, and muscle, alleviated clinical symptoms, and suppressed tissue inflammation. Oral administration of bofutrelvir also provided therapeutic benefits in neonatal mice models of both EV71 and CA16. Crystallographic analysis revealed that bofutrelvir binds in the conserved substrate-binding cleft of EV71 3Cpro, elucidating its molecular mechanism of inhibition. These findings identify bofutrelvir as a broad-spectrum peptidomimetic 3Cpro inhibitor with strong antiviral efficacy against enteroviruses and highlight its potential for repurposing as a promising antiviral candidate for the treatment of enteroviral infections.

Source: 

Link: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014051

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#Genetic Diversity of the Non-Polio #Enteroviruses Detected in Samples of Patients with Aseptic #Meningitis in the #Ural Federal District and Western #Siberia

 

Abstract

Human non-polio enteroviruses (NPEVs) cause a plethora of infections in humans, ranging from mild to severe neurological diseases including aseptic meningitis. NPEVs are the leading cause of aseptic meningitis in both children and adults worldwide. In Russia, reports of NPEV infections have surged, especially in the post-COVID era starting in 2022, with elevated infection rates into 2023. A comprehensive examination of the whole genome is crucial for understanding the evolution of NPEV genes and for predicting potential outbreaks. This study focused on identifying the circulating NPEV strains in the Ural Federal District and Western Siberia, using Sanger sequencing and next-generation sequencing (NGS) methodologies. Biological samples were collected from (n = 225) patients diagnosed with aseptic meningitis. Bioinformatics analysis targeted the nucleotide sequences of the major capsid protein (partial VP1) gene fragment, and the assembly of whole NPEV genomes. A total of 159 NPEVs were characterized, representing 70.7% of the collected samples. The main capsid variants forming the predominant genotypic profile included E30 (n = 39, 24.3%), E6 (n = 31, 19.3%), and CVA9 (n = 25, 15.6%). Using NGS, we successfully assembled 13 whole genomes for E6, E30, EV-B80, CVA9, CVB5, E11, and EV-A71 and 3 partial genomes for E6 and EV-B87. This molecular-genetic analysis provides contemporary insights into the genotypic composition, circulation patterns, and evolutionary dynamics of the dominant NPEV associated with aseptic meningitis in the Ural Federal District and Western Siberia. The laboratory-based monitoring and epidemiological surveillance for genetic changes and evolutionary studies are important for improving prevention and healthcare.

Source: 

Link: https://www.mdpi.com/1999-4915/18/1/121

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#Enterovirus D68 #Sequence #Variations and #Pathogenicity: A Review

Abstract

Enterovirus D68 (EV-D68), a neurotropic respiratory pathogen, poses a considerable clinical threat through its link to pediatric acute flaccid myelitis (AFM) and severe respiratory illness. The possibility of recurrent epidemics, evidenced since the 2014 outbreak, remains a major concern. Genomic determinants of virulence are central to this threat. Sequence variations that affect host–receptor interactions, immune evasion, and replication efficiency serve as critical modifiers of pathogenicity. This article systematically reviews the evidence for specific genomic sites that enhance EV-D68 virulence, focusing on three critical regions: the VP1 receptor-binding site, the 2Apro/TRAF3 cleavage site, and the 3Cpro immunoregulatory region. Mutations in the VP1 receptor-binding site can alter affinity for host receptors such as sialic acid, heparan sulfate, and MFSD6, thereby shaping viral entry and tissue tropism. Alterations in the 2Apro/TRAF3 cleavage site may impair proteolytic cleavage of host TRAF3, attenuating immune evasion and reducing viral pathogenicity. Variations in the 3Cpro region affect its efficiency in cleaving host proteins involved in translation and autophagy, ultimately modulating viral replication and antiviral responses. Finally, we propose that monitoring for mutations in these key virulence determinants, particularly within the surface-exposed VP1, is essential for effective outbreak preparedness.

Source: 

Link: https://www.mdpi.com/1999-4915/18/1/73

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Return of the Biennial #Circulation of #Enterovirus D68 in #Colorado #Children in 2024 Following the Large 2022 #Outbreak

Abstract

Enterovirus D68 (EV-D68) caused large biennial cyclical outbreaks of respiratory disease and cases of acute flaccid myelitis from 2014 to 2018 in the USA. An anticipated outbreak did not occur in 2020, likely due to non-pharmaceutical interventions targeting the COVID-19 pandemic. A large respiratory disease outbreak occurred again in 2022, but uncertainty remained regarding if circulation of EV-D68 would return to the pre-pandemic patterns. We conducted prospective active surveillance of clinical respiratory specimens from Colorado children for EV-D68 in 2023 and 2024. A subset of residual specimens positive for rhinovirus/enterovirus (RV/EV) were tested for EV-D68 via a validated in-house EV-D68 reverse transcription–PCR assay. During epi weeks 18–44 in 2023, 525 residual specimens positive for RV/EV all tested negative for EV-D68. In 2024, during epi weeks 18–44, 10 (1.8%) of the 546 RV/EV-positive specimens were EV-D68-positive. The EV-D68-positive cases were predominantly young children (median age 4.8 years) receiving treatment with asthma medications. Following the 2022 EV-D68 outbreak, an anticipated outbreak did not occur in 2023. While EV-D68 was detected in 2024, the number of cases was not as significant as in prior outbreak years. Continued surveillance for EV-D68 will be important to understand the future dynamics of EV-D68 circulation and prepare for future outbreaks.

Source: Viruses, https://www.mdpi.com/1999-4915/17/5/673

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Sustained circulation of #enterovirus D68 in #Europe in 2023 and continued #evolution of #EVD68 B3-lineages associated with distinct amino acid substitutions in VP1 protein

Highlights

• Enterovirus D68 (EV-D68) was circulating in Europe in 2023

• Most EV-D68 cases were captured through clinical EV surveillance

• Phylogenetic analysis of the VP1 region revealed a distinct B3-derived lineage

• The identified B3 lineage presented a previously undescribed residue change, D554E

Abstract

Background

Enterovirus D68 (EV-D68) causes respiratory disease ranging from mild to severe and in rare cases a paralytic syndrome, called acute flaccid myelitis (AFM). Since the global EV-D68 outbreak in 2014, the virus has mainly circulated in biennial epidemic cycles with peaks detected during even years. However, following the COVID-19 pandemic, the seasonal pattern of EV-D68 has been characterized by large yearly upsurges. Here, we describe the circulation of EV-D68 in Europe in 2023 and track its genetic evolution.

Study design

Data was compiled from members of the European Non-Polio Network (ENPEN). This included monthly data on the total number of EV samples tested, EV positive samples, EV-D68 positive samples and cases, and other EV positive samples detected in 2023. Information on sample types and surveillance system was recorded. Sequence data from the VP1 gene was used for phylogenetic and amino acid sequence analysis.

Results

EV was detected in 13585 out of 203622 diagnostic samples tested (6.7%), of which 402 (3.0%) were determined as EV-D68, representing 386 cases. EV-D68 infections peaked in October 2023 (136/386; 35.2%). 267/386 (69.2%) of EV-D68 cases were captured through clinical EV surveillance, almost all of which (202/204 of positive samples with sample type information) were detected in respiratory specimens. Phylogenetic analysis performed on 99 VP1 sequences revealed a distinct B3-derived lineage with a previously undescribed residue change, D554E, in Europe.

Conclusions

The study documents sustained circulation of EV-D68 in Europe in 2023, the evolution of B3-derived lineages, and appearance of previously undescribed amino acid substitutions in Europe. This stresses the need for continuous EV-D68 surveillance and harmonization of EV-D68 detection practices towards better data comparability across countries.

Source: Journal of Clinical Virology, https://www.sciencedirect.com/science/article/abs/pii/S1386653225000277?dgcid=rss_sd_all

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Evolutionary Studies on the #Coxsackievirus A24 #Variants Causing Acute #Hemorrhagic #Conjunctivitis with Emphasis on the Recent #Outbreak of 2023 in #India

Abstract

Acute Hemorrhagic Conjunctivitis (AHC) is primarily caused by viral infections, with Coxsackievirus A-24v (CV-A24v) being a significant culprit. Enteroviruses, including CV-A24v, are responsible for global AHC outbreaks. Over time, CV-A24v has evolved, and genotype IV (GIV) has become the dominant strain. This study focused on examining the genetic features and evolutionary trends of CV-A24v responsible for the recent AHC outbreak of 2023 in India. Researchers isolated viral strains from ocular swabs and confirmed the presence of CV-A24v using reverse transcriptase quantitative PCR (RT-qPCR) and whole-genome sequencing. Genomic comparisons between isolates of 2023 and those from a previous outbreak in 2009 were conducted. Phylogenetic analysis revealed that the 2023 isolates formed a distinct cluster within GIV-5 and were related to recent strains from China and Pakistan. The older Indian isolates from 2009 grouped with GIV-3. New subclades, GIV-6 and GIV-7, were also identified in this study, indicating the diversification of CV-A24. Molecular clock and phylogeographic analysis traced the virus’s circulation back to the 1960s, with the common ancestor likely to have originated in Singapore in 1968. The 2023 Indian strains probably originated from Thailand around 2014, with subsequent spread to China and Pakistan. This study concluded that the 2023 outbreak was caused by a genetically distinct CV-A24v strain with nine mutations, underlining the virus’s ongoing evolution and adaptations and offering valuable insights for future outbreak control.

Source: Viruses, https://www.mdpi.com/1999-4915/17/3/371

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Unexpected #Pediatric #Cluster of #Enterovirus C105, #Verona, #Italy

Abstract

In an epidemiologic investigation of Enterovirus (EV) infections in a Verona hospital, September 2022–September 2024, we detected EV-C105 in six pediatric patients with upper respiratory symptoms between March and May 2023. The primary objective was to describe the local incidence of EV cases. The secondary objective was to perform Sanger’s genomic characterization and the whole-genome sequencing (WGS) of EV-C105. The proportion of positive EV results was calculated based on routine molecular method testing. An available cohort of 114 underwent Sanger sequencing, and the six EV-C105 were characterized with WGS. Overall, 96% EV results were from the upper respiratory tract. The total proportion of positives in children was 83%. Out of the typed 114, 90% were Rhinoviruses and 9%, EVs. Notably, six pediatric cases were EV-C105, placing together in a unique cluster with 99% of nucleotides belonging to the European lineage with the highest Average Nucleotide Identity, including EV-C104, EV-C109, and EV-C118. Our data describes the first cluster indicating that EV-C105 incidence may be higher than previously estimated. However, a limitation for affirming this hypothesis is the lack of a more in-depth epidemiological investigation on a larger case series with the possibility of including data from coordinated laboratories.

Source: Viruses, https://www.mdpi.com/1999-4915/17/2/255

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The Emergence of #Coxsackievirus A16 Subgenotype B1c: A Key Driver of the #HFMD #Epidemic in #Guangdong, #China

Abstract

Background: 

In 2024, mainland China witnessed a significant upsurge in Hand, Foot, and Mouth Disease (HFMD) cases. Coxsackievirus A16 (CVA16) is one of the primary causative agents of HFMD. Long-term monitoring of theCVA16 infection rate and genotype changes is crucial for the prevention and control of HFMD. 

Methods: 

A total of 40,673 clinical specimens were collected from suspected HFMD cases in Guangdong province from 2018 to 2024, including rectal swabs (n = 27,954), throat swabs (n = 6791), stool (n = 5923), cerebrospinal fluid (n = 3), and herpes fluid (n = 2). A total of 24,410 samples were detected as EV-positive and further typed by RT-PCR. A total of 872 CVA16-positive samples were isolated and further sequenced to obtain the full-length VP1 sequence. Phylogenetic analysis was performed based on viral protein 1 gene (VP1). 

Results: 

In the first 25 weeks of 2024, reported cases of HFMD were 1.36 times higher than the mean rates of 2023. In 2024, CVA16 predominated at 75.42%, contrasting with the past etiological pattern in which the CVA6 was predominant with the detection rate ranging from 32.85 to 77.61% from 2019 to 2023. Phylogenetic analysis based on the VP1 gene revealed that the B1a and B1b subtypes co-circulated in Guangdong from 2018 to 2022. The B1c outbreak clade, detected in Guangdong in 2023, constituted 68.24% of the 148 strains of CVA16 collected in 2024, suggesting a subtype shift in the CVA16 virus. There were three specific amino acid variations (P3S, I235V, and T240A) in the VP1 sequence of B1c. 

Conclusions: 

The new emergence of the CVA16 B1c outbreak clade in Guangdong during 2023–2024 highlights the necessity for the enhanced surveillance of the virus evolution epidemiological dynamic in this region. Furthermore, it is imperative to closely monitor the etiological pattern changes in Hand, Foot, and Mouth Disease (HFMD) in other regions as well. Such vigilance will be instrumental in guiding future vaccination strategies for HFMD.

Source: Viruses, https://www.mdpi.com/1999-4915/17/2/219

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