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Purpose: The rapid emergence and spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has sparked concerns of spillover to naïve wildlife populations and it is unclear if wildlife could ultimately serve as a reservoir for the virus. Evidence of SARS-CoV-2 has been detected in captive and wild animals (e.g., mink, tiger, lion). Moreover, the SARS-CoV-2 variants of concern (VOCs) likely have a broader host range, suggested by the susceptibility of laboratory mice (Mus musculus) to the Beta and Gamma variants but not wild-type SARS-CoV-2. Peri-domestic animals (i.e., wild and feral animals living near humans) represent highly relevant species to assess spillover since they likely have a greater chance of exposure to the virus from humans. Thus, monitoring wildlife at the human-animal interface is critical in understanding the epidemiology of SARS-CoV-2. We therefore are investigating the presence of SARS-CoV-2 in key peri-domestic and wild animals across southern Ontario, Canada through a One Health framework. Methods & Materials: In collaboration with the Ministry of Natural Resources and Forestry, oral and rectal swabs are opportunistically collected from two peri-domestic species, raccoons (Procyon lotor) and skunks (Mephitis mephitis), across southern Ontario where high population densities and incidence of SARS-CoV-2 in humans likely provide spillover opportunity. Additionally, oral and rectal swab, and respiratory and intestinal tissues from trapped wild mink (Neovison vison) are collected in collaboration with the Ontario Fur Managers Federation. Samples are analyzed at the Sunnybrook Research Institute via a RT-qPCR for two SARS-CoV-2 gene targets: the 5’ untranslated region (UTR) and the envelope gene (E). Results: From September 2020 to January 2021, 264 oral and 264 rectal swabs were collected from 207 raccoons and 57 skunks. All samples were negative for SARS-CoV-2 RNA. The study is presently ongoing. Conclusion: Based on these preliminary data, there is little evidence to suggest spillover of SARS-CoV-2 to the sampled animals during this period. Notably, these animals were sampled prior to the emergence of SARS-CoV-2 VOCs, and our results do not preclude the possibility of future spillover to wildlife at the human-animal interface. Results from ongoing surveillance efforts post-VOC emergence will also be discussed.

The incorporation of sequencing technologies in frontline and public health healthcare settings was vital in developing virus surveillance programs during the Coronavirus Disease 2019 (COVID-19) pandemic caused by transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, increased data acquisition poses challenges for both rapid and accurate analyses. To overcome these hurdles, we developed the SARS-CoV-2 Illumina GeNome Assembly Line (SIGNAL) for quick bulk analyses of Illumina short-read sequencing data. SIGNAL is a Snakemake workflow that seamlessly manages parallel tasks to process large volumes of sequencing data. A series of outputs are generated, including consensus genomes, variant calls, lineage assessments and identified variants of concern (VOCs). Compared to other existing SARS-CoV-2 sequencing workflows, SIGNAL is one of the fastest-performing analysis tools while maintaining high accuracy. The source code is publicly available (github.com/jaleezyy/covid-19-signal) and is optimized to run on various systems, with software compatibility and resource management all handled within the workflow. Overall, SIGNAL illustrated its capacity for high-volume analyses through several contributions to publicly funded government public health surveillance programs and can be a valuable tool for continuing SARS-CoV-2 Illumina sequencing efforts and will inform the development of similar strategies for rapid viral sequence assessment., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

The COVID-19 pandemic accelerated the development of automated systems for detecting molecular targets for the point-of-care. However, these systems have limited multiplexing capabilities because of the need to alter their hardware to accommodate additional targets and probes. Quantum dot barcodes address this multiplexing obstacle, but their assays have multiple steps that rely on extensive training and laboratory equipment. Here, we built a portable cartridge-and-instrument system that automates the extraction, reverse transcription, amplification, and detection steps of a quantum dot barcode assay. This entire workflow can be completed in 40 minutes. We clinically validated the system with SARS-CoV-2 patient samples ( n = 50, 92% sensitivity, 100% specificity). We then demonstrated multiplexing with 4-barcode respiratory and 5-barcode bloodborne pathogen panels. Our portable system opens quantum dot barcodes for broad use in rapid multiplexed detection of infectious pathogens with the potential for detecting cancer and other genetic diseases.

Background: Essential workers, including those working in healthcare and education, are at higher risk of exposure to communicable diseases, including SARS-CoV-2. Reducing the rates of infection is important for their personal health and for the ongoing safe operation of essential services., Methods: Data from participants in two prospective cohort studies who tested positive for SARS-CoV-2 in 2020 through 2023 were used to determine whether vaccination against SARS-CoV-2 is associated with the severity of symptoms in working-age adults., Results: SARS-CoV-2-positive tests (N = 3757) were reported by 3093 participants (mean: 1.2 per person); 1229 (33%) illnesses did not interfere with regular activities, 1926 (51%) made participants too unwell for regular activities, and 602 (16%) required participant bed rest. Compared with vaccine receipt more than 12 months earlier, receipt within six months of an infection was associated with lower risk ratios for more severe illness (too unwell: 0.69 and bed rest: 0.67) compared with being able to conduct regular activities. More recent vaccination was also associated with lower odds reporting of systemic symptoms (fever, myalgia, arthralgia) and fewer solicited symptoms., Conclusion: Staying current with COVID-19 vaccinations should continue to be recommended since receiving a recent immunization lessened the severity of illness. Also, as symptoms of COVID-19 are now largely similar to other respiratory viruses, practitioners need to use this evidence to inform diagnostic testing and return-to-work policies.

Background: The demand for COVID-19 vaccines has diminished as the pandemic lingers. Understanding vaccine hesitancy among essential workers is important in reducing the impact of future pandemics by providing effective immunization programs delivered expeditiously., Method: Two surveys exploring COVID-19 vaccine acceptance in 2021 and 2022 were conducted in cohorts of health care providers (HCP) and education workers participating in prospective studies of COVID-19 illnesses and vaccine uptake. Demographic factors and opinions about vaccines (monovalent and bivalent) and public health measures were collected in these self-reported surveys. Modified multivariable Poisson regression was used to determine factors associated with hesitancy., Results: In 2021, 3 % of 2061 HCP and 6 % of 3417 education workers reported hesitancy (p < 0.001). In December 2022, 21 % of 868 HCP and 24 % of 1457 education workers reported being hesitant to receive a bivalent vaccine (p = 0.09). Hesitance to be vaccinated with the monovalent vaccines was associated with earlier date of survey completion, later receipt of first COVID-19 vaccine dose, no influenza vaccination, and less worry about becoming ill with COVID-19. Factors associated with hesitance to be vaccinated with a bivalent vaccine that were common to both cohorts were receipt of two or fewer previous COVID-19 doses and lower certainty that the vaccines were safe and effective., Conclusion: Education workers were somewhat more likely than HCP to report being hesitant to receive COVID-19 vaccines but reasons for hesitancy were similar. Hesitancy was associated with non-receipt of previous vaccines (i.e., previous behaviour), less concern about being infected with SARS-CoV-2, and concerns about the safety and effectiveness of vaccines for both cohorts. Maintaining inter-pandemic trust in vaccines, ensuring rapid data generation during pandemics regarding vaccine safety and effectiveness, and effective and transparent communication about these data are all needed to support pandemic vaccination programs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Brenda L Coleman reports financial support was provided by Canadian Institutes of Health Research. Allison McGeer reports financial support was provided by Canadian Institutes of Health Research. Allison McGeer reports financial support was provided by Physicians' Services Inc. Foundation. Dawn Bowdish reports financial support was provided by Weston Family Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The COVID-19 pandemic led to a large global effort to sequence SARS-CoV-2 genomes from patient samples to track viral evolution and inform the public health response. Millions of SARS-CoV-2 genome sequences have been deposited in global public repositories. The Canadian COVID-19 Genomics Network (CanCOGeN - VirusSeq), a consortium tasked with coordinating expanded sequencing of SARS-CoV-2 genomes across Canada early in the pandemic, created the Canadian VirusSeq Data Portal, with associated data pipelines and procedures, to support these efforts. The goal of VirusSeq was to allow open access to Canadian SARS-CoV-2 genomic sequences and enhanced, standardized contextual data that were unavailable in other repositories and that meet FAIR standards (Findable, Accessible, Interoperable and Reusable). In addition, the portal data submission pipeline contains data quality checking procedures and appropriate acknowledgement of data generators that encourages collaboration. From inception to execution, the portal was developed with a conscientious focus on strong data governance principles and practices. Extensive efforts ensured a commitment to Canadian privacy laws, data security standards, and organizational processes. This portal has been coupled with other resources, such as Viral AI, and was further leveraged by the Coronavirus Variants Rapid Response Network (CoVaRR-Net) to produce a suite of continually updated analytical tools and notebooks. Here we highlight this portal (https://virusseq-dataportal.ca/), including its contextual data not available elsewhere, and the Duotang (https://covarr-net.github.io/duotang/duotang.html), a web platform that presents key genomic epidemiology and modelling analyses on circulating and emerging SARS-CoV-2 variants in Canada. Duotang presents dynamic changes in variant composition of SARS-CoV-2 in Canada and by province, estimates variant growth, and displays complementary interactive visualizations, with a text overview of the current situation. The VirusSeq Data Portal and Duotang resources, alongside additional analyses and resources computed from the portal (COVID-MVP, CoVizu), are all open source and freely available. Together, they provide an updated picture of SARS-CoV-2 evolution to spur scientific discussions, inform public discourse, and support communication with and within public health authorities. They also serve as a framework for other jurisdictions interested in open, collaborative sequence data sharing and analyses.

Competing Interests: Intérêts concurrents :: Robert Kozak déclare avoir reçu des subventions gouvernementales ou des bourses universitaires de la part des Instituts de recherche en santé du Canada (IRSC) et de University of Toronto. Le Dr Kozak déclare aussi avoir agi à titre de membre du conseil d’administration du Collège canadien de microbiologie de 2019 à ce jour et de président de la planification scientifique de l’Association canadienne de microbiologie clinique et d’infectiologie. Samira Mubareka déclare avoir reçu une aide des IRSC — Agence de la santé publique du Canada par l’entremise du Programme de chaires de recherche appliquée en santé publique pour la prévention, la préparation, l’intervention et le rétablissement en contexte de pandémie et d’urgences sanitaires. Isaac Bogoch déclare avoir reçu des subventions des IRSC pour la prévention du VIH et le dépistage des maladies tropicales négligées, et des honoraires de consultation du Programme canadien de réduction de la menace liée aux armes d’Affaires mondiales Canada.

Background: Mosquito-borne orthobunyaviruses are a growing priority for public and animal health in Canada. It is anticipated that disease incidence will increase due to a warming climate, given that habitats are expanding for reservoir hosts and vectors, particularly in Canada. Little is known about the ecology of primary vectors that perpetuate these orthobunyaviruses, including the viral transmission cycle and the impact of climatic and landscape factors. Methods: A scoping review was conducted to describe the current state of knowledge on the epidemiology of orthobunyaviruses relevant to Canada. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines was used to characterize studies focused on vector species. A literature search was conducted in six databases and gray literature. Eligible studies characterized orthobunyavirus epidemiology related to vector species, including viral competency, geospatial distributions, seasonal trends, and/or risk factors. Results: A total of 1734 unique citations were identified. Screening of these citations revealed 172 relevant studies, from which 87 studies presented primary data related to vectors. The orthobunyaviruses included Cache Valley virus (CVV), Jamestown Canyon virus (JCV), Snowshoe Hare virus (SHV), and La Crosse virus (LACV). Surveillance was the predominant study focus, with most citations representing the United States, specifically, LACV surveillance in Tennessee, followed by CVV and JCV in Connecticut. Orthobunyaviruses were detected in many mosquito species across multiple genera, with high vector specificity only being reported for LACV, which included Aedes triseriatus , Aedes albopictus , and Aedes japonicus. Peridomestic areas were positively associated with infected mosquitoes compared with dense forests. Orthobunyavirus infections, coinfections, and gut microbiota affected mosquito feeding and breeding behavior. Conclusion: Knowledge gaps included Canadian surveillance data, disease modeling, and risk projections. Further research in these areas, especially accounting for climate change, is needed to guide health policy for prevention of orthobunyaviral disease.

Aim: Understanding how COVID-19 impacts the expression of clinically relevant drug metabolizing enzymes and membrane transporters (DMETs) is vital for addressing potential safety and efficacy concerns related to systemic and peripheral drug concentrations. This study investigates the impact of COVID-19 severity on DMETs expression and the underlying mechanisms to inform the design of precise clinical dosing regimens for affected patients., Methods: Transcriptomics analysis of 102 DMETs, 10 inflammatory markers, and 12 xenosensing regulatory genes was conducted on nasopharyngeal swabs from 50 SARS-CoV-2 positive (17 outpatients, 16 non-ICU, and 17 ICU) and 13 SARS-CoV-2 negative individuals, clinically tested through qPCR, in the Greater Toronto area from October 2020 to October 2021., Results: We observed a significant differential gene expression for 42 DMETs, 6 inflammatory markers, and 9 xenosensing regulatory genes. COVID-19 severity was associated with the upregulation of AKR1C1, MGST1, and SULT1E1, and downregulation of ABCC10, CYP3A43, and SLC29A4 expressions. Altogether, SARS-CoV-2-positive patients showed an upregulation in CYP2C9, CYP2C19, AKR1C1, SULT1B1, SULT2B1, and SLCO4A1 and downregulation in FMO5, MGST3, ABCC5, and SLCO4C1 compared with SARS-CoV-2 negative individuals. These dysregulations were associated with significant changes in the expression of inflammatory and xenosensing regulatory genes driven by the disease. GSTM3, PPARA, and AKR1C1 are potential biomarkers of the observed DMETs dysregulation pattern in nasopharyngeal swabs of outpatients, non-ICU, and ICU patients, respectively., Conclusion: The severity of COVID-19 is associated with the dysregulation of DMETs involved in processing commonly prescribed drugs, suggesting potential disease-drug interactions, especially for narrow therapeutic index drugs., (© 2024 The Author(s). British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Randomized trials conducted in low- and middle-income settings demonstrated efficacy of influenza vaccination during pregnancy against influenza infection among infants <6 months of age. However, vaccine effectiveness (VE) estimates from settings with different population characteristics and influenza seasonality remain limited., Methods: We conducted a test-negative study in Ontario, Canada. All influenza virus tests among infants <6 months from 2010 to 2019 were identified and linked with health databases to ascertain information on maternal-infant dyads. VE was estimated from the odds ratio for influenza vaccination during pregnancy among cases versus controls, computed using logistic regression with adjustment for potential confounders., Results: Among 23 806 infants tested for influenza, 1783 (7.5%) were positive and 1708 (7.2%) were born to mothers vaccinated against influenza during pregnancy. VE against laboratory-confirmed infant influenza infection was 64% (95% confidence interval [CI], 50%-74%). VE was similar by trimester of vaccination (first/second, 66% [95% CI, 40%-80%]; third, 63% [95% CI, 46%-74%]), infant age at testing (0 to <2 months, 63% [95% CI, 46%-75%]; 2 to <6 months, 64% [95% CI, 36%-79%]), and gestational age at birth (≥37 weeks, 64% [95% CI, 50%-75%]; < 37 weeks, 61% [95% CI, 4%-86%]). VE against influenza hospitalization was 67% (95% CI, 50%-78%)., Conclusions: Influenza vaccination during pregnancy offers effective protection to infants <6 months, for whom vaccines are not currently available., Competing Interests: Potential conflicts of interest . During the conduct of this work, D. B. F. worked for the University of Ottawa and had academic appointments at the Children's Hospital of Eastern Ontario Research Institute and ICES; she is currently employed by Pfizer. K. W. is a cofounder and Chief Scientific Officer of CANImmunize, Inc; he served on the Independent Data Monitoring Committee for Medicago; and is a member of the Moderna Global Advisory Core Consultancy Group. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Highly pathogenic avian influenza (HPAI) viruses have spread at an unprecedented scale, leading to mass mortalities in birds and mammals. In 2023, a transatlantic incursion of HPAI A(H5N5) viruses into North America was detected, followed shortly thereafter by a mammalian detection. As these A(H5N5) viruses were similar to contemporary viruses described in Eurasia, the transatlantic spread of A(H5N5) viruses was most likely facilitated by pelagic seabirds. Some of the Canadian A(H5N5) viruses from birds and mammals possessed the PB2-E627K substitution known to facilitate adaptation to mammals. Ferrets inoculated with A(H5N5) viruses showed rapid, severe disease onset, with some evidence of direct contact transmission. However, these viruses have maintained receptor binding traits of avian influenza viruses and were susceptible to oseltamivir and zanamivir. Understanding the factors influencing the virulence and transmission of A(H5N5) in migratory birds and mammals is critical to minimize impacts on wildlife and public health., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Competing interests:: Robert Kozak reports receiving government or academic grants from the Canadian Institutes of Health Research (CIHR) and the University of Toronto. Dr. Kozak also reports serving as a member of the executive board of the Canadian College of Microbiology from 2019 to the present, and as scientific planning chair of the Canadian Association of Clinical Microbiology and Infectious Diseases. Samira Mubareka reports receiving support from the CIHR — Public Health Agency of Canada Applied Public Health Chair in Pandemic and Health Emergency Prevention, Preparedness, Response and Recovery. Isaac Bogoch reports receiving grants from CIHR for HIV prevention and screening for neglected tropical diseases, and consulting fees from the Weapons Threat Reduction Program at Global Affairs Canada.

Objective: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals., Results: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection., (© 2024. The Author(s).)

Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Background: Mosquito-borne orthobunyaviruses in Canada are a growing public health concern. Orthobunyaviral diseases are commonly underdiagnosed and in Canada, likely underreported as surveillance is passive. No vaccines or specific treatments exist for these disease agents. Further, climate change is facilitating habitat expansion for relevant reservoirs and vectors, and it is likely that the majority of the Canadian population is susceptible to these viruses. Methods: A scoping review was conducted to describe the current state of knowledge on orthobunyavirus epidemiology in Canada. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guideline was used. Literature searches were conducted in six databases and in gray literature. The epidemiology of orthobunyaviruses was characterized for studies focusing on host species, including spatiotemporal patterns, risk factors, and climate change impact. Results: A total of 172 relevant studies were identified from 1734 citations from which 95 addressed host species, including humans, wildlife, and domestic animals including livestock. The orthobunyaviruses-Cache Valley virus (CVV), Jamestown Canyon virus (JCV), Snowshoe Hare virus (SHV), and La Crosse virus (LACV)-were identified, and prevalence was widespread across vertebrate species. CVV, JCV, and SHV were detected across Canada and the United States. LACV was reported only in the United States, predominantly the Mid-Atlantic and Appalachian regions. Disease varied by orthobunyavirus and was associated with age, environment, preexisting compromised immune systems, or livestock breeding schedule. Conclusion: Knowledge gaps included seroprevalence data in Canada, risk factor analyses, particularly for livestock, and disease projections in the context of climate change. Additional surveillance and mitigation strategies, especially accounting for climate change, are needed to guide future public health efforts to prevent orthobunyavirus exposure and disease.

Forest biomass is an essential resource in relation to the green transition and its assessment is key for the sustainable management of forest resources. Here, we present a forest biomass dataset for Europe based on the best available inventory and satellite data, with a higher level of harmonisation and spatial resolution than other existing data. This database provides statistics and maps of the forest area, biomass stock and their share available for wood supply in the year 2020, and statistics on gross and net volume increment in 2010-2020, for 38 European countries. The statistics of most countries are available at a sub-national scale and are derived from National Forest Inventory data, harmonised using common reference definitions and estimation methodology, and updated to a common year using a modelling approach. For those counties without harmonised statistics, data were derived from the State of Europe's Forest 2020 Report at the national scale. The maps are coherent with the statistics and depict the spatial distribution of the forest variables at 100 m resolution., (© 2024. European Union and The Author(s).)

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)- 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Canada experienced a wave of HPAI H5N1 outbreaks in the spring of 2022 with millions of wild and farmed birds being infected. Seabird mortalities in Canada have been particularly severe on the Atlantic Coast over the summer of 2022. Over 7 million birds have been culled in Canada, and outbreaks continue to profoundly affect commercial bird farms across the world. This new H5N1 virus can and has infected multiple mammalian species, including skunks, foxes, bears, mink, seals, porpoises, sea lions, and dolphins. Viruses with mammalian adaptations such as the mutations PB2-E627K, E627V, and D701N were found in the brain of various carnivores in Europe and Canada. To date this specific clade of H5N1 virus has been identified in less than 10 humans. At the ground level, awareness should be raised among frontline practitioners most likely to encounter patients with HPAI., Competing Interests: S Forgie reports support from AMMI Canada to attend their board meeting in Toronto, Canada, April 2023, and a previous leadership role at AMMI Canada. TF Hatchette reports grants from GSK/Pfizer, Dalhousie Pharmacy Fund, Drug Evaluation Alliance of NS, CIHR/CIRN, NSERC, COVID-19 Immunity Task Force, NIH, Genome Atlantic, Nova Scotia COVID-19 Health Research Coalition Funding Competition, NSHA Research Fund, and an IWK Project Grant; consulting fees from RockDoc; speaker fees from Sanofi; and past leadership roles at AMMI Canada and Canadian Public Health Laboratory Network. D Yamamura reports a previous leadership role at AMMI Canada., (© Association of Medical Microbiology and Infectious Disease Canada (AMMI Canada), 2023.)

Africa experiences frequent emerging disease outbreaks among humans, with bats often proposed as zoonotic pathogen hosts. We comprehensively reviewed virus-bat findings from papers published between 1978 and 2020 to evaluate the evidence that African bats are reservoir and/or bridging hosts for viruses that cause human disease. We present data from 162 papers (of 1322) with original findings on (1) numbers and species of bats sampled across bat families and the continent, (2) how bats were selected for study inclusion, (3) if bats were terminally sampled, (4) what types of ecological data, if any, were recorded and (5) which viruses were detected and with what methodology. We propose a scheme for evaluating presumed virus-host relationships by evidence type and quality, using the contrasting available evidence for Orthoebolavirus versus Orthomarburgvirus as an example. We review the wording in abstracts and discussions of all 162 papers, identifying key framing terms, how these refer to findings, and how they might contribute to people's beliefs about bats. We discuss the impact of scientific research communication on public perception and emphasize the need for strategies that minimize human-bat conflict and support bat conservation. Finally, we make recommendations for best practices that will improve virological study metadata.

White-tailed deer (WTD) are susceptible to SARS-CoV-2 and represent an important species for surveillance. Samples from WTD (n = 258) collected in November 2021 from Québec, Canada were analyzed for SARS-CoV-2 RNA. We employed viral genomics and host transcriptomics to further characterize infection and investigate host response. We detected Delta SARS-CoV-2 (B.1.617.2) in WTD from the Estrie region; sequences clustered with human sequences from October 2021 from Vermont, USA, which borders this region. Mutations in the S-gene and a deletion in ORF8 were detected. Host expression patterns in SARS-CoV-2 infected WTD were associated with the innate immune response, including signaling pathways related to anti-viral, pro- and anti-inflammatory signaling, and host damage. We found limited correlation between genes associated with innate immune response from human and WTD nasal samples, suggesting differences in responses to SARS-CoV-2 infection. Our findings provide preliminary insights into host response to SARS-CoV-2 infection in naturally infected WTD., Competing Interests: The authors declare no conflicts relevant to this article., (© 2023 The Authors.)

Background: Remdesivir (RDV) has been shown to reduce hospitalization and mortality in COVID-19 patients. Resistance mutations caused by RDV are rare and have been predominantly reported in patients who are on prolonged therapy and immunocompromised. We investigate the effects of RDV treatment on intra-host SARS-CoV-2 diversity and low-frequency mutations in moderately ill hospitalized COVID-19 patients and compare them to patients without RDV treatment., Methods: From March 2020 to April 2022, sequential collections of nasopharyngeal and mid-turbinate swabs were obtained from 14 patients with and 30 patients without RDV treatment. Demographic and clinical data on all patients were reviewed. A total of 109 samples were sequenced and mutation analyses were performed., Results: Previously reported drug resistant mutations in nsp12 were not identified during short courses of RDV therapy. In genes encoding and surrounding the replication complex (nsp6-nsp14), low-frequency minority variants were detected in 7/14 (50%) and 18/30 (60%) patients with and without RDV treatment, respectively. We did not detect significant differences in within-host diversity and positive selection between the RDV-treated and untreated groups., Conclusions: Minimal intra-host variability and stochastic low-frequency variants detected in moderately ill patients suggests little selective pressure in patients receiving short courses of RDV. The barrier to RDV resistance is high in patients with moderate disease. Patients undergoing short regimens of RDV therapy should continue to be monitored., (© 2023 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Cache Valley virus (CVV) disease is a mosquito-borne zoonosis endemic to North America. CVV disease is reported most often in sheep, causing lethal congenital deformities. There are limited data on CVV in Ontario, which is the largest sheep producing province in Canada. This study aimed to determine CVV seroprevalence in Ontario sheep flocks and investigate farm management factors associated with CVV exposure. A cross-sectional study was performed including 364 mature ewes across 18 farms selected from the five largest sheep districts in the province. A questionnaire was administered at each farm to determine farm management practices pertinent to the flock and ewes specifically sampled. Mixed multivariable logistic regression with a random effect for farm was conducted to assess associations between CVV seropositivity (outcome variable) and farm management risk factors (predictor variables). CVV seroprevalence was 33.2% in individual ewes (95% CI: 28.4%-38.1%) as determined by a virus neutralization assay with a titre > 4. Sixteen of the eighteen flocks (88.9%) had at least one CVV seropositive ewe. Increased age, smaller flock size, and sheep housing near wetlands, lakes, or ponds were found to be significantly associated with higher odds of CVV seropositivity. These findings are valuable in guiding breeding practices and housing during mosquito season to minimize infection and, ultimately, CVV disease in the flock., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bergevin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Respiratory syncytial virus (RSV) contributes significantly to morbidity in children, placing substantial burdens on health systems, thus RSV vaccine development and program implementation are a public health priority. More data on burden are needed by policymakers to identify priority populations and formulate prevention strategies as vaccines are developed and licensed., Methods: Using health administrative data, we calculated incidence rates of RSV hospitalization in a population-based birth cohort of all children born over a six-year period (May 2009 to June 2015) in Ontario, Canada. Children were followed until their first RSV hospitalization, death, 5th birthday, or the end of the study period (June 2016). RSV hospitalizations were identified using a validated algorithm based on International Classification of Diseases, 10th Revision, and/or laboratory-confirmed outcomes. We calculated hospitalization rates by various characteristics of interest, including calendar month, age groups, sex, comorbidities, and gestational age., Results: The overall RSV hospitalization rate for children <5 years was 4.2 per 1000 person-years (PY) with a wide range across age groups (from 29.6 to 0.52 per 1000 PY in children aged 1 month and 36-59 months, respectively). Rates were higher in children born at a younger gestational age (23.2 per 1000 PY for those born at <28 weeks versus 3.9 per 1000 PY born at ≥37 weeks); this increased risk persisted as age increased. While the majority of children in our study had no comorbidities, rates were higher in children with comorbidities. For all age groups, rates were highest between December and March., Conclusions: Our results confirm the high burden of RSV hospitalization and highlight young infants are at additional risk, namely premature infants. These results can inform prevention efforts., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society.)

Background: Older adults are recommended to receive influenza vaccination annually, and many use statins. Statins have immunomodulatory properties that might modify influenza vaccine effectiveness (VE) and alter influenza infection risk., Methods: Using the test-negative design and linked laboratory and health administrative databases in Ontario, Canada, we estimated VE against laboratory-confirmed influenza among community-dwelling statin users and nonusers aged ≥66 years during the 2010-2011 to 2018-2019 influenza seasons. We also estimated the odds ratio for influenza infection comparing statin users and nonusers by vaccination status., Results: Among persons tested for influenza across the 9 seasons, 54 243 had continuous statin exposure before testing and 48 469 were deemed unexposed. The VE against laboratory-confirmed influenza was similar between statin users and nonusers (17% [95% confidence interval, 13%-20%] and 17% [13%-21%] respectively; test for interaction, P = .87). In both vaccinated and unvaccinated persons, statin users had higher odds of laboratory-confirmed influenza than nonusers (odds ratios for vaccinated and unvaccinated persons 1.15 [95% confidence interval, 1.10-1.21] and 1.15 [1.10-1.20], respectively). These findings were consistent by mean daily dose and statin type. VE did not differ between users and nonusers of other cardiovascular drugs, except for β-blockers. We did not observe that vaccinated and unvaccinated users of these drugs had increased odds of influenza, except for unvaccinated β-blocker users., Conclusions: Influenza VE did not differ between statin users and nonusers. Statin use was associated with increased odds of laboratory-confirmed influenza in vaccinated and unvaccinated persons, but these associations might be affected by residual confounding., Competing Interests: Potential conflicts of interest. H. C., M. A. C., S. A. B., T. K., K. L. S., and J. C. K. report support from the Canadian Institutes of Health Research (CIHR; grant PJT 159516, paid to the institution). J. B. G. has received research grants from GSK and Hoffmann-LaRoche for antiviral resistance studies and from Pfizer to conduct microbiological surveillance of Streptococcus pneumoniae. J. B. G. also reports consulting fees from GIDEON Informatics, as a scientific editor, unrelated to the current work. A. J. M. reports research funds from GSK; grants or contracts from Sanofi, Merck, and Pfizer (paid to the institution); payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from AstraZeneca, Merck, Biogen, and Moderna; and participation on a data safety monitoring board or advisory board for Pfizer, GlaxoSmithKline, Moderna, Medicago, Janssen, AstraZeneca, Novavax, and Sanofi. D. C. R. reports participation on a data safety monitoring board or advisory board for GlaxoSmithKline (advisory board meeting on monoclonal antibodies for coronavirus disease 2019 [COVID-19]). M. S. reports grants or contracts from CIHR, Air Canada (staff and reagent costs for the COVID in International Travelers Study), the Ontario Research Fund (equipment costs for COVID-19 automation), and the Juravinski Research Fund (staff and equipment for COVID-19 diagnostic testing). G. Z. reports support from CIHR (paid to grant administered by J. C. K.) and grants from Genome Atlantic and PHAC. Roche and Abbott contribute unrestricted educational funding to the Eastern Health Regional Health Authority for employees, which include G. Z., to attend virtual meetings and, in specific restricted circumstances, travel to educational, technical, and research meetings. G. Z. is also the previous president of the National Molecular Users Group. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Despite similar infection rates, COVID-19 has resulted in more deaths in men than women. To understand the underlying mechanisms behind this sex-biased difference in disease severity, we infected K18-human angiotensin converting enzyme 2 (ACE2) mice of both sexes with SARS-CoV-2. Our study revealed a unique protein expression profile in the lung microenvironment of female mice. As a result, they were less vulnerable to severe infection, with higher ACE2 expression and a higher estrogen receptor α (ERα)/androgen receptor (AR) ratio that led to increased antiviral factor levels. In male mice, inhaling recombinant ACE2 neutralized the virus and maintained the ERα/AR ratio, thereby protecting the lungs. Our findings suggest that inhaling recombinant ACE2 could serve as a decoy receptor against SARS-CoV-2 and protect male mice by offsetting ERα-associated protective mechanisms. Additionally, our study supports the potential effectiveness of recombinant ACE2 therapy in human lung organoids infected with the Delta variant., Competing Interests: GW is an employee, JP a shareholder, and AS an advisor of Apeiron Biologics AG developing soluble ACE2 as a therapy for COVID-19. The other authors declare no competing interests., (© 2023 The Authors.)

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/β) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

Background: With the growing volume and complexity of laboratory repositories, it has become tedious to parse unstructured data into structured and tabulated formats for secondary uses such as decision support, quality assurance, and outcome analysis. However, advances in natural language processing (NLP) approaches have enabled efficient and automated extraction of clinically meaningful medical concepts from unstructured reports., Objective: In this study, we aimed to determine the feasibility of using the NLP model for information extraction as an alternative approach to a time-consuming and operationally resource-intensive handcrafted rule-based tool. Therefore, we sought to develop and evaluate a deep learning-based NLP model to derive knowledge and extract information from text-based laboratory reports sourced from a provincial laboratory repository system., Methods: The NLP model, a hierarchical multilabel classifier, was trained on a corpus of laboratory reports covering testing for 14 different respiratory viruses and viral subtypes. The corpus includes 87,500 unique laboratory reports annotated by 8 subject matter experts (SMEs). The classification task involved assigning the laboratory reports to labels at 2 levels: 24 fine-grained labels in level 1 and 6 coarse-grained labels in level 2. A "label" also refers to the status of a specific virus or strain being tested or detected (eg, influenza A is detected). The model's performance stability and variation were analyzed across all labels in the classification task. Additionally, the model's generalizability was evaluated internally and externally on various test sets., Results: Overall, the NLP model performed well on internal, out-of-time (pre-COVID-19), and external (different laboratories) test sets with microaveraged F 1 -scores >94% across all classes. Higher precision and recall scores with less variability were observed for the internal and pre-COVID-19 test sets. As expected, the model's performance varied across categories and virus types due to the imbalanced nature of the corpus and sample sizes per class. There were intrinsically fewer classes of viruses being detected than those tested; therefore, the model's performance (lowest F 1 -score of 57%) was noticeably lower in the detected cases., Conclusions: We demonstrated that deep learning-based NLP models are promising solutions for information extraction from text-based laboratory reports. These approaches enable scalable, timely, and practical access to high-quality and encoded laboratory data if integrated into laboratory information system repositories., (©Elham Dolatabadi, Branson Chen, Sarah A Buchan, Alex Marchand Austin, Mahmoud Azimaee, Allison McGeer, Samira Mubareka, Jeffrey C Kwong. Originally published in JMIR AI (https://ai.jmir.org), 06.06.2023.)

Among outpatients with coronavirus disease 2019 (COVID-19) due to the severe acute respiratory coronavirus virus 2 (SARS-CoV-2) δ (delta) variant who did and did not receive 2 vaccine doses at 7 days after symptom onset, there was no difference in viral shedding (cycle threshold difference 0.59, 95% CI, -4.68 to 3.50; P = .77) with SARS-CoV-2 cultured from 2 (7%) of 28 and 1 (4%) of 26 outpatients, respectively.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

SARS-CoV-2-mediated interactions with drug metabolizing enzymes and membrane transporters (DMETs) in different tissues, especially lung, the main affected organ may limit the clinical efficacy and safety profile of promising COVID-19 drugs. Herein, we investigated whether SARS-CoV-2 infection could dysregulate the expression of 25 clinically relevant DMETs in Vero E6 cells and postmortem lung tissues from COVID-19 patients. Also, we assessed the role of 2 inflammatory and 4 regulatory proteins in modulating the dysregulation of DMETs in human lung tissues. We showed for the first time that SARS-CoV-2 infection dysregulates CYP3A4 and UGT1A1 at the mRNA level, as well as P-gp and MRP1 at the protein level, in Vero E6 cells and postmortem human lung tissues, respectively. We observed that at the cellular level, DMETs could potentially be dysregulated by SARS-CoV-2-associated inflammatory response and lung injury. We uncovered the pulmonary cellular localization of CYP1A2, CYP2C8, CYP2C9, and CYP2D6, as well as ENT1 and ENT2 in human lung tissues, and observed that the presence of inflammatory cells is the major driving force for the discrepancy in the localization of DMETs between COVID-19 and control human lung tissues. Because alveolar epithelial cells and lymphocytes are both sites of SARS-CoV-2 infection and localization of DMETs, we recommend further investigation of the pulmonary pharmacokinetic profile of current COVID-19 drug dosing regimen to improve clinical outcomes., Competing Interests: CN was a former employee of Gilead Sciences and was involved in the development of remdesivir. CN was employed by OneDrug. MK and MP were employed by Ontario Forensic Pathology Service. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nwabufo, Hoque, Yip, Khara, Mubareka, Pollanen and Bendayan.)

The COVID-19 pandemic caused by SARS-CoV-2 virus is an ongoing global health burden. Severe cases of COVID-19 and the rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are both associated with thrombosis and thrombocytopenia; however, the underlying mechanisms remain inadequately understood. Both infection and vaccination utilize the spike protein receptor-binding domain (RBD) of SARS-CoV-2. We found that intravenous injection of recombinant RBD caused significant platelet clearance in mice. Further investigation revealed the RBD could bind platelets, cause platelet activation, and potentiate platelet aggregation, which was exacerbated in the Delta and Kappa variants. The RBD-platelet interaction was partially dependent on the β3 integrin as binding was significantly reduced in β3 -/- mice. Furthermore, RBD binding to human and mouse platelets was significantly reduced with related αIIbβ3 antagonists and mutation of the RGD (arginine-glycine-aspartate) integrin binding motif to RGE (arginine-glycine-glutamate). We developed anti-RBD polyclonal and several monoclonal antibodies (mAbs) and identified 4F2 and 4H12 for their potent dual inhibition of RBD-induced platelet activation, aggregation, and clearance in vivo, and SARS-CoV-2 infection and replication in Vero E6 cells. Our data show that the RBD can bind platelets partially though αIIbβ3 and induce platelet activation and clearance, which may contribute to thrombosis and thrombocytopenia observed in COVID-19 and VITT. Our newly developed mAbs 4F2 and 4H12 have potential not only for diagnosis of SARS-CoV-2 virus antigen but also importantly for therapy against COVID-19., (Copyright © 2023 Xiaoying Ma et al.)

Transmission of H9N2 avian influenza virus (AIV) can occur in poultry by direct or indirect contact with infected individuals, aerosols, large droplets and fomites. The current study investigated the potential of H9N2 AIV transmission in chickens via a fecal route. Transmission was monitored by exposing naïve chickens to fecal material from H9N2 AIV-infected chickens (model A) and experimentally spiked feces (model B). The control chickens received H9N2 AIV. Results revealed that H9N2 AIV could persist in feces for up to 60-84 h post-exposure (PE). The H9N2 AIV titers in feces were higher at a basic to neutral pH. A higher virus shedding was observed in the exposed chickens of model B compared to model A. We further addressed the efficacy of Toll-like receptor (TLR) ligands to limit transmission in the fecal model. Administration of CpG ODN 2007 or poly(I:C) alone or in combination led to an overall decrease in the virus shedding, with enhanced expression of type I and II interferons (IFNs) and interferon-stimulating genes (ISGs) in different segments of the small intestine. Overall, the study highlighted that the H9N2 AIV can survive in feces and transmit to healthy naïve chickens. Moreover, TLR ligands could be applied to transmission studies to enhance antiviral immunity and reduce H9N2 AIV shedding.

Background: COVID-19 presents with a breadth of symptomatology including a spectrum of clinical severity requiring intensive care unit (ICU) admission. We investigated the mucosal host gene response at the time of gold standard COVID-19 diagnosis using clinical surplus RNA from upper respiratory tract swabs., Methods: Host response was evaluated by RNA-sequencing, and transcriptomic profiles of 44 unvaccinated patients including outpatients and in-patients with varying levels of oxygen supplementation were included. Additionally, chest X-rays were reviewed and scored for patients in each group., Results: Host transcriptomics revealed significant changes in the immune and inflammatory response. Patients destined for the ICU were distinguished by the significant upregulation of immune response pathways and inflammatory chemokines, including cxcl2 which has been linked to monocyte subsets associated with COVID-19 related lung damage. In order to temporally associate gene expression profiles in the upper respiratory tract at diagnosis of COVID-19 with lower respiratory tract sequalae, we correlated our findings with chest radiography scoring, showing nasopharygeal or mid-turbinate sampling can be a relevant surrogate for downstream COVID-19 pneumonia/ICU severity., Conclusions: This study demonstrates the potential and relevance for ongoing study of the mucosal site of infection of SARS-CoV-2 using a single sampling that remains standard of care in hospital settings. We highlight also the archival value of high quality clinical surplus specimens, especially with rapidly evolving COVID-19 variants and changing public health/vaccination measures., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1β, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens.