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Summary: Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour‐intensive and time‐consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)‐PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot‐and‐mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user‐friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post‐infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT‐PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease ( FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus ( FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR ( RT-ii PCR) assay that can be performed on a commercially available, compact and portable POCKIT™ analyser that automatically analyses data and displays '+' or '−' results. The FMDV RT-ii PCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT- PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. [ABSTRACT FROM AUTHOR]

Porcine respiratory disease complex ( PRDC) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. No simple, single-reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Furthermore, the detection of most of the bacterial pathogens implicated in PRDC currently requires time-consuming culture-based methods that can take several days to obtain results. In this study, a novel prototype automated microarray that integrates and automates all steps of post- PCR microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with PRDC is described along with associated multiplex reverse transcriptase PCR. The user-friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus ( PRRSV), influenza A virus, porcine circovirus type 2, porcine respiratory corona virus], four bacteria ( Mycoplasma hyopneumoniae, Pasteurella multocida, Salmonella enterica serovar Choleraesuis, Streptococcus suis), and further differentiated between type 1 and type 2 PRRSV as well as toxigenic and non-toxigenic P. multocida. The assay accurately identified and typed a panel of 34 strains representing the eight targeted pathogens and was negative when tested with 34 relevant and/or closely related non-target bacterial and viral species. All targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material. [ABSTRACT FROM AUTHOR]

Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus ( BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan- BTV reverse transcription-insulated isothermal polymerase chain reaction ( RT-ii PCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '−' without the need for data interpretation. The RT-ii PCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-ii PCR assay was as sensitive as real-time RT- PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory. [ABSTRACT FROM AUTHOR]

Classical swine fever ( CSF) is an OIE-listed disease that can have a severe impact on the swine industry. User-friendly, sensitive, rapid diagnostic tests that utilize low-cost field-deployable instruments for CSF diagnosis can be useful for disease surveillance and outbreak monitoring. In this study, we describe validation of a new probe-based insulated isothermal reverse transcriptase PCR (ii RT- PCR) assay for rapid detection of classical swine fever virus ( CSFV) on a compact, user-friendly device ( POCKIT™ Nucleic Acid Analyzer) that does not need data interpretation by the user. The assay accurately detected CSFV RNA from a diverse panel of 33 CSFV strains representing all three genotypes plus an additional in vitro-transcribed RNA from cloned sequences representing a vaccine strain. No cross-reactivity was observed with a panel of 18 viruses associated with livestock including eight other pestivirus strains (bovine viral diarrhoea virus type 1 and type 2, border disease virus, HoBi atypical pestivirus), African swine fever virus, swine vesicular disease virus, swine influenza virus, porcine respiratory and reproductive syndrome virus, porcine circovirus 1, porcine circovirus 2, porcine respiratory coronavirus, vesicular exanthema of swine virus, bovine herpes virus type 1 and vesicular stomatitis virus. The ii RT- PCR assay accurately detected CSFV as early as 2 days post-inoculation in RNA extracted from serum samples of experimentally infected pigs, before appearance of clinical signs. The limit of detection ( LOD95%) calculated by probit regression analysis was 23 copies per reaction. The assay has a sample to answer turnaround time of less than an hour using extracted RNA or diluted or low volume of neat serum. The user-friendly, compact device that automatically analyses and displays results could potentially be a useful tool for surveillance and monitoring of CSF in a disease outbreak. [ABSTRACT FROM AUTHOR]

Motivation: State-of-the-art tools for classifying metagenomic sequencing reads provide both rapid and accurate options, although the combination of both in a single tool is a constantly improving area of research. The machine learning-based Naïve Bayes Classifier (NBC) approach provides a theoretical basis for accurate classification of all reads in a sample., Results: We developed the multithreaded Minimizer-based Naïve Bayes Classifier (MNBC) tool to improve the NBC approach by applying minimizers, as well as plurality voting for closely related classification scores. A standard reference- and test-sequence framework using simulated variable-length reads benchmarked MNBC with six other state-of-the-art tools: MetaMaps, Ganon, Kraken2, KrakenUniq, CLARK, and Centrifuge. We also applied MNBC to the "marine" and "strain-madness" short-read metagenomic datasets in the Critical Assessment of Metagenome Interpretation (CAMI) II challenge using a corresponding database from the time. MNBC efficiently identified reads from unknown microorganisms, and exhibited the highest species- and genus-level precision and recall on short reads, as well as the highest species-level precision on long reads. It also achieved the highest accuracy on the "strain-madness" dataset., Availability and Implementation: MNBC is freely available at: https://github.com/ComputationalPathogens/MNBC., (© The Author(s) 2024. Published by Oxford University Press.)

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a tick-borne, risk group 4 pathogen that often causes a severe haemorrhagic disease in humans (CCHF) with high case fatality rates. The virus is believed to be maintained in a tick-vertebrate-tick ecological cycle involving numerous wild and domestic animal species; however the biology of CCHFV infection in these animals remains poorly understood. Here, we experimentally infect domestic sheep with CCHFV Kosovo Hoti, a clinical isolate representing high pathogenicity to humans and increasingly utilized in current research. In the absence of prominent clinical signs, the infection leads to an acute viremia and coinciding viral shedding, fever and markers for potential impairment in liver and kidney functions. A number of host responses distinguish the subclinical infection in sheep versus fatal infection in humans. These include an early reduction of neutrophil recruitment and its chemoattractant, IL-8, in the blood stream of infected sheep, whereas neutrophil infiltration and elevated IL-8 are features of fatal CCHFV infections reported in immunodeficient mice and humans. Several inflammatory cytokines that correlate with poor disease outcomes in humans and have potential to cause vascular dysfunction, a primary hallmark of severe CCHF, are down-regulated or restricted from increasing in sheep. Of particular interest, the detection of CCHFV RNA (including full-length genome) in a variety of sheep tissues long after the acute phase of infection indicates a widespread viral dissemination in the host and suggests a potentially long-term persisting impact of CCHFV infection. These findings reveal previously unrecognized aspects of CCHFV biology in animals.

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners., Importance: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners., Competing Interests: The authors declare no conflict of interest.

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.)

We describe an unusual mortality event caused by a highly pathogenic avian influenza (HPAI) A(H5N1) virus clade 2.3.4.4b involving harbor (Phoca vitulina) and gray (Halichoerus grypus) seals in the St. Lawrence Estuary, Quebec, Canada, in 2022. Fifteen (56%) of the seals submitted for necropsy were considered to be fatally infected by HPAI H5N1 containing fully Eurasian or Eurasian/North American genome constellations. Concurrently, presence of large numbers of bird carcasses infected with HPAI H5N1 at seal haul-out sites most likely contributed to the spillover of infection to the seals. Histologic changes included meningoencephalitis (100%), fibrinosuppurative alveolitis, and multiorgan acute necrotizing inflammation. This report of fatal HPAI H5N1 infection in pinnipeds in Canada raises concerns about the expanding host of this virus, the potential for the establishment of a marine mammal reservoir, and the public health risks associated with spillover to mammals.Nous décrivons un événement de mortalité inhabituelle causé par un virus de l'influenza aviaire hautement pathogène A(H5N1) clade 2.3.4.4b chez des phoques communs (Phoca vitulina) et gris (Halichoerus grypus) dans l'estuaire du Saint-Laurent au Québec, Canada, en 2022. Quinze (56%) des phoques soumis pour nécropsie ont été considérés comme étant fatalement infectés par le virus H5N1 de lignées eurasiennes ou de réassortiment eurasiennes/nord-américaines. Un grand nombre simultané de carcasses d'oiseaux infectés par le H5N1 sur les sites d'échouement a probablement contribué à la contamination de ces phoques. Les changements histologiques associés à cette infection incluaient : méningo-encéphalite (100%), alvéolite fibrinosuppurée et inflammation nécrosante aiguë multi-organique. Cette documentation soulève des préoccupations quant à l'émergence de virus mortels, à la possibilité d'établissement de réservoirs chez les mammifères marins, et aux risques pour la santé publique associés aux propagations du virus chez les mammifères.

Two cat nasal swabs from Canada's earliest confirmed SARS-CoV-2 positive domestic cats were sequenced to over 99% SARS-CoV-2 genome coverage. One cat had lineage A.23.1 SARS-CoV-2 not reported before in animals. Both sequences have multiple spike gene mutations and clustered closely with human-derived sequences in the global SARS-CoV-2 phylogenetic tree., Competing Interests: The authors declare no conflict of interest.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 10 5 TCID 50 /pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 10 3 TCID 50 /pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

We conducted an in silico analysis to better understand the potential factors impacting host adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer, humans, and mink due to the strong evidence of sustained transmission within these hosts. Classification models trained on single nucleotide and amino acid differences between samples effectively identified white-tailed deer-, human-, and mink-derived SARS-CoV-2. For example, the balanced accuracy score of Extremely Randomized Trees classifiers was 0.984 ± 0.006. Eighty-eight commonly identified predictive mutations are found at sites under strong positive and negative selective pressure. A large fraction of sites under selection (86.9%) or identified by machine learning (87.1%) are found in genes other than the spike. Some locations encoded by these gene regions are predicted to be B- and T-cell epitopes or are implicated in modulating the immune response suggesting that host adaptation may involve the evasion of the host immune system, modulation of the class-I major-histocompatibility complex, and the diminished recognition of immune epitopes by CD8+ T cells. Our selection and machine learning analysis also identified that silent mutations, such as C7303T and C9430T, play an important role in discriminating deer-derived samples across multiple clades. Finally, our investigation into the origin of the B.1.641 lineage from white-tailed deer in Canada discovered an additional human sequence from Michigan related to the B.1.641 lineage sampled near the emergence of this lineage. These findings demonstrate that machine-learning approaches can be used in combination with evolutionary genomics to identify factors possibly involved in the cross-species transmission of viruses and the emergence of novel viral lineages.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus capable of infecting and establishing itself in human and wildlife populations, such as white-tailed deer. This fact highlights the importance of developing novel ways to identify genetic factors that contribute to its spread and adaptation to new host species. This is especially important since these populations can serve as reservoirs that potentially facilitate the re-introduction of new variants into human populations. In this study, we apply machine learning and phylogenetic methods to uncover biomarkers of SARS-CoV-2 adaptation in mink and white-tailed deer. We find evidence demonstrating that both non-synonymous and silent mutations can be used to differentiate animal-derived sequences from human-derived ones and each other. This evidence also suggests that host adaptation involves the evasion of the immune system and the suppression of antigen presentation. Finally, the methods developed here are general and can be used to investigate host adaptation in viruses other than SARS-CoV-2., Competing Interests: The authors declare no conflict of interest.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

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.)

Wholly Eurasian highly pathogenic avian influenza H5N1 clade 2.3.4.4b virus was isolated from 2 free-ranging black bears with meningoencephalitis in Quebec, Canada. We found that isolates from both animals had the D701N mutation in the polymerase basic 2 gene, previously known to promote adaptation of H5N1 viruses to mammal hosts.

In December 2022 and January 2023, we isolated clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) viruses from six American crows ( Corvus brachyrhynchos ) from Prince Edward Island and a red fox ( Vulpes vulpes ) from Newfoundland, Canada. Using full-genome sequencing and phylogenetic analysis, these viruses were found to fall into two distinct phylogenetic clusters: one group containing H5N1 viruses that had been circulating in North and South America since late 2021, and the other one containing European H5N1 viruses reported in late 2022. The transatlantic re-introduction for the second time by pelagic/Icelandic bird migration via the same route used during the 2021 incursion of Eurasian origin H5N1 viruses into North America demonstrates that migratory birds continue to be the driving force for transcontinental dissemination of the virus. This new detection further demonstrates the continual long-term threat of H5N1 viruses for poultry and mammals and the subsequent impact on various wild bird populations wherever these viruses emerge. The continual emergence of clade 2.3.4.4b H5Nx viruses requires vigilant surveillance in wild birds, particularly in areas of the Americas, which lie within the migratory corridors for long-distance migratory birds originating from Europe and Asia. Although H5Nx viruses have been detected at higher rates in North America since 2021, a bidirectional flow of H5Nx genes of American origin viruses to Europe has never been reported. In the future, coordinated and systematic surveillance programs for HPAI viruses need to be launched between European and North American agencies.