In July 2022, an unvaccinated young adult living in Rockland County, New York became paralyzed due to a vaccine-derived poliovirus type 2 (VDPV2) infection that was internationally transmitted. Wastewater surveillance in New York State uncovered VDPV2 in neighboring counties, showing silent community spread. These communities have strong epidemiological links to some vaccine-hesitant communities in Canada, spurring the need to monitor these populations for potential poliovirus importation. At the time, Canada did not have an established poliovirus wastewater method. We initiated this study to apply molecular methods to detect poliovirus in these communities and establish poliovirus wastewater surveillance capabilities in Canada. We sequence confirmed one poliovirus detection on August 30th, 2022 using both viral isolation and direct detection methods. Subsequent retrospective and prospective sampling was initiated, resulting in another sequence confirmed detection of VDPV2 in an overlapping catchment area collected on August 27th, 2022. Both VDPV2 detections were genetically linked to the New York clinical case. No clinical cases of poliomyelitis were detected in Canada during this study. The sporadic detection of VDPV2 in Canadian wastewater supports a travel-related shedding event without community transmission. Furthermore, we show that a direct detection method is sensitive to sequence confirm poliovirus in wastewater samples.

Introduction. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS for rapid identification of risk group 3 (RG3) bacteria is impeded by the following two main limitations: (a) equipment and maintenance costs for instruments placed within containment and (b) lack of a validated inactivation protocol to move RG3 material to a lower containment level. A validated inactivation method would improve operations of public health laboratories by allowing safe triage of potential RG3 agents. Albeit a validated, zero-risk inactivation protocol is unlikely, scientific interrogation of methods to identify and mitigate procedural biosafety risks is vital for institutional risk assessment. Gap. To investigate the effect of a standard MALDI-TOF chemical extraction, hypothesized to alter cells, allowing passage through a filter and maintaining ability to replicate, this study paired visualization using a scanning electron microscope (SEM) with extended viability testing. Aim. This work is intended to support risk assessments for the removal of material from a containment laboratory for MALDI-TOF MS. Methodology. A standard set of Bacillus cereus and Bacillus anthracis vegetative and spore preparations was treated with a formic acid:acetonitrile extraction, with or without filtration, and plated on five types of media to monitor growth over 14 days. SEM images were taken of treated and untreated preparations, prior, during and after filtration across two filters. Reference beads provided accurate pore size measurements. Results. SEM demonstrated no difference in treated and untreated cells but did indicate the ineffectiveness of cellulose filters compared to PVDF filters. Growth was observed in preparations that did not include PVDF filtration, whereas all preparations ( n =60) that included PVDF filtration were 100% non-viable. Although non-viability was observed, an important finding was the passage of 0.262 and 0.173 µm microspheres through the 0.1 µm PVDF filter. Growth of unfiltered preparations was detected between 1 and 7 days. Conclusions. This investigation demonstrates the value of interrogating materials used for bacterial inactivation, highlighting significant issues in the application of filters for exclusion purposes. Visual examination via SEM was key to providing evidence towards a low-risk inactivation method. These findings, with an understanding of limitations identified herein, can be used to inform risk assessments for the removal of RG3 bacteria from containment.

BACKGROUND:Mpox (formerly monkeypox) is an emerging zoonotic disease of public health concern that presents as a rash mimicking other common viral exanthems. Unlike traditional testing algorithms relying on several assays, the BioFire FilmArray meningitis/encephalitis (ME) panel simultaneously detects common viruses causing rashes; however, Biofire ME is only licensed for testing on cerebral spinal fluid.OBJECTIVES:This study evaluated use of the Biofire ME panel for detection and discrimination of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), human herpesviruses type 6 (HHV-6), enteroviruses (EVs), and human paraechoviruses (HPeVs) from a dermal or mucocutaneous swabs collected in universal transport media (UTM).STUDY DESIGN:Results of the BioFire ME panel were compared against methods used during clinical testing. Ten-fold serial dilutions in UTM of cultured viruses were used to compare analytical sensitivity, and analytical specificity was assessed using panels of microorganisms in UTM. Clinical sensitivity and specificity were assessed using 20 positive specimens each for HHV-1, HHV-2, HHV-6, VZV, EVs, and HPeV, as well as 35 known negative specimens that included 15 mpox-positive specimens.RESULTS:Biofire ME was as sensitive as comparator methods, and correctly discriminated all HSV-1, HSV-2, VZV, HHV-6, EVs, and HPeVs from mpox and mpox-mimickers. Cross-reaction between EV and rhinoviruses A, B, and C were noted in the specificity panel.CONCLUSIONS:Swabs in UTM collected for mpox testing are suitable for use on the Biofire ME panel, allowing more streamlined diagnostic testing for viral exanthems in patients under investigation for mpox infection.

Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component ( mlaA ) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions ( ompCF ), and over-expressed efflux pumps ( acrB, qacE, aceI ), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, Δ acrB , Δ ompCF , and transformants pCA24N- aceI and pCA24N- mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (Δ ompCF ) and mlaA alterations (Δ mlaA ; pCA24N- mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH- aceI ) and deleted (Δ acrB ) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. Δ mlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA.

BACKGROUND:Serological assays designed to detect SARS-CoV-2 antibodies are being used in serological surveys and other specialized applications. As a result, and to ensure that the outcomes of serological testing meet high quality standards, evaluations are required to assess the performance of these assays and the proficiency of laboratories performing them.METHODS:A panel of 60 plasma/serum samples from blood donors who had reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections and 21 SARS-CoV-2 negative samples were secured and distributed to interested laboratories within Canada (n = 30) and the United States (n = 1). Participating laboratories were asked to provide details on the diagnostic assays used, the platforms the assays were performed on, and the results obtained for each panel sample. Laboratories were blinded with respect to the expected outcomes.RESULTS:The performance of the different assays evaluated was excellent, with the high-throughput platforms of Roche, Ortho, and Siemens demonstrating 100% sensitivity. Most other high-throughput platforms had sensitivities of >93%, with the exception of the IgG assay using the Abbott ARCHITECT which had an average sensitivity of only 87%. The majority of the high-throughput platforms also demonstrated very good specificities (>97%).CONCLUSION:This proficiency study demonstrates that most of the SARS-CoV-2 serological assays utilized by provincial public health or hospital laboratories in Canada have acceptable sensitivity and excellent specificity.

Journal Article Optimization of CryoEM Sample Preparation: A New Freezing Strategy to Reduce the Time to Structure Loop Get access Ouliana Panova, Ouliana Panova SPT Labtech Ltd., Melbourn Science Park, Hertfordshire, UK Search for other works by this author on: Oxford Academic Google Scholar Herve Vandekerckhove, Herve Vandekerckhove SPT Labtech Ltd., Melbourn Science Park, Hertfordshire, UK Search for other works by this author on: Oxford Academic Google Scholar Nicolas Marro, Nicolas Marro SPT Labtech Ltd., Melbourn Science Park, Hertfordshire, UK Search for other works by this author on: Oxford Academic Google Scholar Stefanie Rajasooriya, Stefanie Rajasooriya SPT Labtech Ltd., Melbourn Science Park, Hertfordshire, UK Search for other works by this author on: Oxford Academic Google Scholar Tim Booth, Tim Booth SPT Labtech Ltd., Melbourn Science Park, Hertfordshire, UK Search for other works by this author on: Oxford Academic Google Scholar Michele C Darrow Michele C Darrow SPT Labtech Ltd., Melbourn Science Park, Hertfordshire, UK Corresponding author: Michele.Darrow@sptlabtech.com Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Page 1236, https://doi.org/10.1017/S143192762200513X Published: 01 August 2022

The period responsible for the current generation of cryogenic electron microscopy (cryo-EM) instrumentation and image processing software is often referred to as the “resolution revolution.” The results of these technological advancements have been achievements in higher quality structures and cryo-EM becoming the go-to method for structural biologists. However, sample preparation is widely recognised as a key unresolved step in the high-resolution cryo electron microscopy (cryoEM) workflow [1]. While atomic to with experimental repetition untraceable

Cationic antimicrobials (CA) such as the therapeutic antibiotic colistin (COL) and antiseptics cetrimide (CET) and chlorhexidine (CHX) all exert their mechanism of action by disrupting bacterial membranes, leading to cell content leakage and death. Resistance to CAs is rapidly increasing and of the many antimicrobial resistance mechanisms that may contribute, the role of outer membrane vesicle (OMV) formation is least understood. Here, we gradually adapted E. coli BW215113 K-12 to COL, CET, and CHX by prolonged exposure in vitro to determine how each CA impacts OMV formation. We assessed OMVs isolated from each CA-adapted strain by nanoparticle tracking analysis (NTA) and cryo-transmission electron microscopy (cryo-TEM), and compared OMV proteomes using liquid chromatography-mass spectroscopy. The results of NTA analysis showed that all three CA-adapted strains had increased OMV formation as compared to unadapted BW25113 (WT), where CA-adapted OMVs were significantly larger. Cryo-TEM analyses revealed that each strain’s OMVs had distinctive morphological alterations, where CET-OMVs looked similar to unadapted WT OMVs but were encapsulated and aggregated, CHX-OMVs were multilamellar and COL-OMVs were large (4-12X) and tubular as compared to WT OMVs. Proteomic analysis highlighted significant increases in EptC abundance in COL-OMVs and decreased abundance of MlaA in CHX-OMVs. Antimicrobial susceptibility testing of E. coli BW25113 K-12 supplemented with purified CA-adapted OMVs demonstrated that CET-OMVs did not alter CET susceptibility, CHX-OMVs enhanced BW25113 tolerance to CHX exposure by 2-fold, and COL-OMV supplementation to BW25113 increased COL susceptibility by 2-fold compared to unadapted WT OMVs. Hence, CA adaptation by E. coli has significant ramifications on OMV production and morphology, and CA-adapted OMV exposure has significant consequences for bacterial antimicrobial susceptibility.

Chlorhexidine (CHX) is an essential medicine used as a topical antiseptic in skin and oral healthcare treatments. The widespread use of CHX has increased concerns regarding the development of antiseptic resistance in Enterobacteria and its potential impact on cross-resistance to other antimicrobials. Similar to other cationic antiseptics, resistance to CHX is believed to be driven by three membrane-based mechanisms: lipid synthesis/transport, altered porin expression, and increased efflux pump activity; however, specific gene and protein alterations associated with CHX resistance remain unclear. Here, we adapted Escherichia coli K-12 BW25113 to increasing concentrations of CHX to determine what phenotypic, morphological, genomic, transcriptomic, and proteomic changes occurred. We found that CHX-adapted E. coli isolates possessed no cross-resistance to any other antimicrobials we tested. Scanning electron microscopy imaging revealed that CHX adaptation significantly altered mean cell widths and lengths. Proteomic analyses identified changes in the abundance of porin OmpF, lipid synthesis/transporter MlaA, and efflux pump MdfA. Proteomic and transcriptomic analyses identified that CHX adaptation altered E. coli transcripts and proteins controlling acid resistance ( gadE, cdaR ) and antimicrobial stress-inducible pathways Mar-Sox-Rob, stringent response systems. Whole genome sequencing analyses revealed that all CHX-resistant isolates had single nucleotide variants in the retrograde lipid transporter gene mlaA as well as the yghQ gene associated with lipid A transport and synthesis. CHX resistant phenotypes were reversible only when complemented with a functional copy of the mlaA gene. Our results highlight the importance of retrograde phospholipid transport and stress response systems in CHX resistance and the consequences of prolonged CHX exposure.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating virulence factor dissemination and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating Escherichia coli mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating Escherichia coli K-12 Δ tolA mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and Δ tolA OMVs, where Δ tolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and Δ tolA OMVs confirmed that Δ tolA enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.

Quaternary ammonium compounds (QACs) benzalkonium (BZK) and cetrimide (CET) are common disinfectants used to inhibit or eradicate Gram-negative bacteria in clinical and agricultural products. QAC tolerance in Escherichia coli and other Enterobacterales species can confer cross-resistance to various clinically used antibiotics, making it important to understand mechanisms of QAC tolerance in greater depth. QAC adaptation by E. coli is hypothesized to alter MarRAB regulated genes that converge on the outer membrane, specifically, lipid A biosynthesis and transport genes, porins, and efflux pump systems. To test this, we performed a ‘multi’-omics and phenotypic characterization of E. coli K-12 adapted to BZK and CET, to assess how QACs alter cell growth, genomics, and proteomics. E. coli adapted to either BZK and CET resulted in strains with stable QAC tolerance when either drug was omitted, elongated and narrower cell morphologies by scanning electron microscopy, and reduced growth fitness when compared to un-adapted E. coli . Antimicrobial susceptibility testing revealed that QAC adaptation increased E. coli tolerance by ≥4-fold to BZK, CET, and other QACs but no antibiotic cross-resistance. Single nucleotide variants identified by whole genome sequencing and differentially accumulated proteins by liquid chromatography-mass spectrometry identified alterations to various QAC-adapted E. coli genes and proteins belonging to: lipid A biosynthesis and transport ( lpxLM, msbA, mla ), the mar-sox-rob regulatory pathway ( marR, rob ), DNA/protein translation ( gyrA, rpsA, rpoB, rapA ). These alterations validate the hypothesis that mar-sox-rob network plays a role in QAC tolerance and identifies additional stress inducible genetic and protein QAC tolerant biomarkers.Importance Bacterial tolerance mechanisms associated with disinfectant QAC adaptation is hypothesized to overlap with the mar-sox-rob multiple antimicrobial resistance pathway but has not been directly shown. Here, we generate QAC tolerant E. coli strains and identify phenotypic changes associated with protein and genetic alterations caused by prolonged QAC exposure. We identified genes that overlap with known antibiotic resistance mechanisms as well as distinct genes and proteins specific to QAC adaptation that are useful for future bacterial disinfectant tolerance mechanism studies. However, these altered genes and proteins implicate MarR and Rob pathways specifically in QAC tolerance but, surprisingly, the involvement of mar-sox-rob pathways did not increase antibiotic cross-resistance. Many altered genes we identified were essential genes in lipid A biosynthesis/transport, DNA and RNA transcription, and protein regulation systems potentially explaining why only QAC cross-tolerance was observed and why we observed greater cell fitness costs despite MarR and Rob pathway involvement.

Twelve isolates recovered from 10 cystic fibrosis/other patient types and a variety of clinical sources, were referred to Canada's National Microbiology Laboratory over 7 years. These were assignable to the genus Pseudoxanthomonas but were unidentifiable to species level. Patients included five males and five females from two geographically separated provinces, ranging in age from 2 months to 84 years. In contrast, most Pseudoxanthomonas species described to date have been derived from water, plants or contaminated soils. By 16S rRNA gene sequencing, the patient strains had ≥99.4 % similarity to each other but only 97.73-98.29 % to their closest relatives, Pseudoxanthomonas spadix or Pseudoxanthomonas helianthi. Bacteria were studied by whole genome sequencing using average nucleotide identity by Blastn, digital DNA-DNA hybridization, average amino acid identity, core genome and single nucleotide variant analyses, MALDI-TOF, biochemical and cellular fatty acid analyses, and by antimicrobial susceptibility testing. Bacterial structures were assessed using scanning and transmission electron microscopy. Strains were strict aerobes, yellowish-pigmented, oxidative, non-motile, Gram-stain-negative bacilli and generally unable to reduce nitrate. Strains were susceptible to most of the antibiotics tested; some resistance was observed towards carbapenems, several cephems and uniformly to nitrofurantoin. The single taxon group observed by 16S rRNA gene sequencing was supported by whole genome sequencing; genomes ranged in size from 4.36 to 4.73 Mb and had an average G+C content of 69.12 mol%. Based on this study we propose the name Pseudoxanthomonas winnipegensis sp. nov. for this cluster. Pseudoxanthomonas spadix DSM 18855T, acquired for this study, was found to be non-motile phenotypically and by electron microscopy; we therefore propose the emendation of Pseudoxanthomonas spadix Young et al. 2007 to document that observation.

Auritidibacter ignavus is a Gram-stain-positive bacillus derived from otorrhea. Four strains derived from ear discharges in Canada and Switzerland, with features consistent with but distinguishable from Auritidibacter ignavus IMMIB L-1656T (accession number FN554542) by 16S rRNA gene sequencing (97.5 % similarity), were thought to represent a novel species of the genus Auritidibacter. Auritidibacter ignavus DSM 45359T (=IMMIB L-1656T) was acquired to compare with Canadian and Swiss strains by whole-genome sequencing (WGS). Unexpectedly, those isolates were observed to be consistent with A. ignavus DSM 45359T by WGS (ANIb scores >98 %), MALDI-TOF (Bruker), cellular fatty acid analysis and biochemically (some differences were observed). A nearly full 16S rRNA gene sequence could not be readily prepared from A. ignavus DSM 45359T, even after multiple attempts. A 16S rRNA gene chimeric consensus sequence created from the genome assembly of A. ignavus DSM 45359T had only 97.5 % similarity to that of A. ignavus IMMIB L-1656T, implying that 16S rRNA sequence accession number FN554542 could not be replicated. We concluded that our isolates of members of the genus Auritidibacter were consistent with A. ignavus DSM 45359T, did not represent a novel species, and that the sequence corresponding to FN554542 was not reproducible. By WGS, A. ignavus DSM 45359T had genome of 2.53×106 bp with a DNA G+C content of 59.34%, while genomes of Canadian and Swiss isolates ranged from 2.47 to 2.59×106 bp with DNA G+C contents of 59.3-59.52 %. A. ignavus NML 100628 (=NCTC 14178=LMG 30897) did not demonstrate a rodcoccus cycle. Emendation of Auritidibacter ignavus was proposed based on these results.

Biocides such as quaternary ammonium compounds (QACs) are potentially important contributors towards bacterial antimicrobial resistance development, however, their contributions are unclear due to a lack of internationally recognized biocide testing standards. Methods to detect QAC tolerance are limited to laborious traditional antimicrobial susceptibility testing (AST) methods. Here, we developed a r apid f luorescent d ye-based m embrane i mpermeant a ssay (RFDMIA) to discriminate QAC susceptibility among Gram-negative Enterobacterales and Pseudomonadales species. RFDMIA uses a membrane impermeant fluorescent dye, propidium iodide, in a 30-min 96-well fluorescent microplate-based assay where cell suspensions are exposed to increasing QAC concentrations. Our results demonstrate that RFDMIA can discriminate between QAC-susceptible and QAC-adapted Escherichia coli tolerant phenotypes and predict benzalkonium and cetrimide tolerance in all species tested except for intrinsically fluorescent Pseudomonas aeruginosa . RFDMIA identified a close association to minimum inhibitory concentration values determined by broth microdilution AST and increasing fluorescent dye emission values. RFDMIA emission values and scanning electron microscopy results also suggest that CET-adapted E. coli isolates have a CET dependence, where cells require sub-inhibitory CET concentrations to maintain bacilliform cell integrity. Overall, this study generates a new, rapid, sensitive fluorescent assay capable of detecting QAC-susceptible Gram-negative bacteria phenotypes and cell membrane perturbations.

Enteroviruses are single-stranded positive-sense RNA viruses that primarily cause self-limiting gastrointestinal or respiratory illness. In some cases, these viruses can invade the central nervous system, causing life-threatening neurological diseases including encephalitis, meningitis and acute flaccid paralysis (AFP). As we near the global eradication of poliovirus, formerly the major cause of AFP, the number of AFP cases have not diminished implying a non-poliovirus etiology. As the number of enteroviruses linked with neurological disease is expanding, of which many had previously little clinical significance, these viruses are becoming increasingly important to public health. Our current understanding of these non-polio enteroviruses is limited, especially with regards to their neurovirulence. Elucidating the molecular pathogenesis of these viruses is paramount for the development of effective therapeutic strategies. This review summarizes the clinical diseases associated with neurotropic enteroviruses and discusses recent advances in the understanding of viral invasion of the central nervous system, cell tropism and molecular pathogenesis as it correlates with host responses.

Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6-a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, 'one-pot' coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe3+ and Zn2+ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures7,8 and are, to our knowledge, unique among designed systems9-13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design.

Objective: To create a scoping review on enterovirus D-68 (EV-D68) that will serve as a useful tool to guide future research with the aim of filling critical information gaps and supporting the development of public health preparedness activities.Introduction: EV-D68 is a non-polio enterovirus, primarily resulting in respiratory illness, with clinical symptoms ranging from mild to severe. Infection has also been associated with severe neurological conditions like acute flaccid myelitis (AFM). EV-D68 was first discovered in 1962, with infrequent case reports until 2014 at which point a widespread multi-national outbreak mostly affecting the pediatric population occurred across North America, Europe, Southeast Asia and Africa. This outbreak was associated with an increase in AFM, with cases being reported in Canada, the United States, Norway, and France. With this new and emerging threat, public health and other organizations were called upon to implement response measures such as establishment of case definitions, surveillance mechanisms, and recommendations for clinical and public health management. The response to the 2014 outbreak in Canada highlighted several important EV-D68 evidence gaps including a lack of risk factor and clinical information available for non-severe cases, and uncertainty around seasonal, cyclical and secular trends. Given the increased reporting of EV-D68 cases associated with severe outcomes, it's critical that public health establishes what is known about EV-D68 in order to support decision-making, education and other preparedness activities and to highlight priority areas for future research to fill critical knowledge gaps. Scoping reviews provide a reproducible and updateable synthesis research methodology to identify and characterise all the literature on a broad topic as a means to highlight where evidence exists and where there are knowledge gaps. In order to systematically characterise the EV-D68 knowledge base, a scoping review was conducted to map the current body of evidence.Methods: A literature search of published and grey literature on EV-D68 was conducted on May 1, 2017. A standardized search algorithm was implemented in four bibliographic databases: Medline, Embase, Global Health and Scopus. Relevant grey literature was sought from a prioriidentified sources: the World Health Organization, United States Centers for Disease Control and Prevention, the Public Health Agency of Canada, the European Centre for Disease Prevention and Control, and thesis registries. Two-level relevance screening (title/abstract followed by full-text) was performed in duplicate by two independent reviewers using pretested screening forms. Conflicts between the reviewers were reconciled following group discussion with the study team. English and French articles were included if they reported on EV-D68 as an outcome. There were no limitations by date, publication type, geography or study design. Conference abstracts were excluded if they did not provide sufficient outcome information to characterize. The articles were then characterized by two independent reviewers using a pretested study characterization form. The descriptive characteristics of each article were extracted and categorized into one of the following broad topic categories: 1) Epidemiology and Public Health, 2) Clinical and Infection Prevention and Control (IPC), 3) Guidance Products, 4) Public Health Surveillance, 5) Laboratory, and 6) Impact. The Epidemiology and Public Health category contained citations describing prevalence, epidemiological distribution, outbreak data and public health mitigation strategies. Clinical and IPC citations included details regarding symptoms of EV-D68 infection, patient outcomes, clinical investigation processes, treatment options and infection prevention and control strategies. The Guidance category included citations that assess risk, provide knowledge translation or provide practice guidelines. Public Health Surveillance citations provided details on surveillance systems. Citations in the laboratory category included studies that assessed the genetic characteristics of circulating EV-D68 (phylogeny, taxonomy) and viral characteristics (proteins, viral properties). Lastly, the Impact category contained citations describing the social, economic and resource burden of EV-D68 infection. Each broad topic category was subsequently characterised further into subtopics.Results: The search yielded a total of 384 citations, of which 300 met the inclusion criteria. Twenty-six of forty-three potentially relevant grey literature sources were also included. Preliminary literature characterization suggests that the majority of the published literature fell under the topic categories of Epidemiology, Clinical, and Laboratory. There were limited published articles on public health guidance, IPC, surveillance systems and the impact of EV-D68. The grey literature primarily consisted of webpages directed towards the public (what EV-D68 is, how to prevent it, what to do if ill, etc.). This scoping review work is presently underway and a summary of the full results will be presented at the 2018 Annual Conference.Conclusions: The body of literature on EV-D68 has increased since the 2014 outbreak, but overall remains small and contains knowledge gaps in some areas. To our knowledge, this scoping review is the first to classify the entirety of literature relating to EV-D68. It will serve as a useful tool to guide future research with the aim of filling critical information gaps, and supporting development of public health preparedness activities.

Filoviruses are highly filamentous enveloped animal viruses that can cause severe haemorrhagic fevers. The filovirus ribonucleoprotein forms a highly organized double-layered helical nucleocapsid (NC) containing five different virally encoded proteins. The inner layer consists of NP, the RNA binding protein, complexed with the monopartite linear genome. A distinctive outer layer links individual NP subunits with bridges composed of VP24-VP35 heterodimers, which achieves condensation of the NP-RNA into tight helical coils. There are no vertical connections between the outer helical layers, explaining the flexibility of the NC and its ability to bend into tight curves without breaking the genomic RNA. These properties allow the formation of enveloped virions with varying polymorphisms, including single, linear, continuous, linked, comma-shaped and torroidal forms. Virion length is modular so that just one, or two or more genome copies may be present in each virion, producing polyploid particles. The matrix protein VP40, which drives budding and envelopment, is found in a layer adjacent to the inner cytoplasmic side of viral envelope and is arranged in a 5 nm lattice structure, but its exact symmetry is unclear. There is a constant low density gap between VP40 and the nucleocapsid, so that the latter is held rigidly centred on the long axis of the viral filament. This gap likely contains a region of flexible contacts between VP40 and the NC. The unique morphology of filoviruses may be related to high titre replication, their ease of transmission, and abilities to invade a wide range of host cells and tissues.

Background: In August 2014, a large North American outbreak of enterovirus D68 (EV-D68) was observed in association with severe respiratory illnesses in children as well as a possible association with acute flaccid paralysis. The full spectrum of clinical illness associated with EV-D68 is not well known. Objective: To compare children positive and negative for EV-D68 presenting to a community hospital during the North American outbreak in the fall of 2014. Methods: Consecutive nasopharyngeal swabs obtained from children (<18 years old) presenting to a large community hospital with respiratory symptoms in the fall of 2014 were tested for EV-D68. We conducted a test-negative case-control study between EV-D68 positive and EV-D68 negative children to describe the clinical and outcome characteristics. Controls were limited to children who had a nasopharyngeal swab obtained. EV-D68 indeterminate cases were excluded from the primary analysis. All nasopharyngeal swabs were referred to a reference laboratory for polymerase chain reaction testing and identification of EV-D68 through targeting the 5'NTR region and confirmed by amplifying the VP1 gene for serotype determination. Results: Of the 48 children tested there were 9 positive, 36 negative, and 3 indeterminate for EV-D68. Six of the 9 identified cases of EV-D68 were identified in the first week of testing (September 11–18, 2014). There were 35 children (78%) admitted to hospital for ≥1 day. EV-D68 infected children were significantly older (5.2 versus 2.5 years, p = 0.03). They were more likely to be concurrently taking asthma medications (44% versus 11%, p = 0.04) and receiving treatment with salbutamol (100% versus 58%, p = 0.02). There were no cases of severe respiratory distress or acute flaccid paralysis. Three EV-D68 infected children (33%) had viral co-infections identified. Conclusion: This study of a community hospital experience during the fall of 2014 demonstrates a series of mild illnesses from both EV-D68 positive and negative children. EV-D68 isolation was associated with age and asthmatic treatment in children.