SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.

vSNP: a SNP pipeline for the generation of transparent SNP matrices and phylogenetic trees from whole genome sequencing data sets.

BACKGROUND: Several single nucleotide polymorphism (SNP) pipelines exist, each offering its own advantages. Among them and described here is vSNP that has been developed over the past decade and is specifically tailored to meet the needs of diagnostic laboratories. Laboratories that aim to provide rapid whole genome sequencing results during outbreak investigations face unique challenges. vSNP addresses these challenges by enabling users to verify and validate sequence accuracy with ease- having utility across various pathogens, being fully auditable, and presenting results that are easy to interpret and can be comprehended by individuals with diverse backgrounds. RESULTS: vSNP has proven effective for real-time phylogenetic analysis of disease outbreaks and eradication efforts, including bovine tuberculosis, brucellosis, virulent Newcastle disease, SARS-CoV-2, African swine fever, and highly pathogenic avian influenza. The pipeline produces easy-to-read SNP matrices, sorted for convenience, as well as corresponding phylogenetic trees, making the output easily understandable. Essential data for verifying SNPs is included in the output, and the process has been divided into two steps for ease of use and faster processing times. vSNP requires minimal computational resources to run and can be run in a wide range of environments. Several utilities have been developed to make analysis more accessible for subject matter experts who may not have computational expertise. CONCLUSION: The vSNP pipeline integrates seamlessly into a diagnostic workflow and meets the criteria for quality control accreditation programs, such as 17025 by the International Organization for Standardization. Its versatility and robustness make it suitable for use with a diverse range of organisms, providing detailed, reproducible, and transparent results, making it a valuable tool in various applications, including phylogenetic analysis performed in real time.

Highly Pathogenic Avian Influenza Virus A(H5N1) Clade 2.3.4.4b Infection in Free-Ranging Polar Bear, Alaska, USA.

We report a natural infection with a Eurasian highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a free-ranging juvenile polar bear (Ursus maritimus) found dead in North Slope Borough, Alaska, USA. Continued community and hunter-based participation in wildlife health surveillance is key to detecting emerging pathogens in the Arctic.

Divergent Pathogenesis and Transmission of Highly Pathogenic Avian Influenza A(H5N1) in Swine.

Highly pathogenic avian influenza (HPAI) viruses have potential to cross species barriers and cause pandemics. Since 2022, HPAI A(H5N1) belonging to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing adaptation mutations increase the risk for infection and subsequent reassortment with influenza A viruses endemic in swine. We assessed the susceptibility of swine to avian and mammalian HPAI H5N1 clade 2.3.4.4b strains using a pathogenesis and transmission model. All strains replicated in the lung of pigs and caused lesions consistent with influenza A infection. However, viral replication in the nasal cavity and transmission was only observed with mammalian isolates. Mammalian adaptation and reassortment may increase the risk for incursion and transmission of HPAI viruses in feral, backyard, or commercial swine.

Genome sequences of haemagglutinin cleavage site predict the pathogenicity phenotype of avian influenza virus: statistically validated data for facilitating rapid declarations and reducing reliance on in vivo testing.

Based on the pathogenicity in chickens, most H1-H16 avian influenza viruses (AIV) cause mild diseases, whereas some of the H5 and H7 AI viruses cause severe, systemic disease. The number of basic amino acids in the haemagglutinin (HA) cleavage site of AIV plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (intravenous pathogenicity index [IVPI] and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in the absence of an in vivo study determination. This approach not only expedites the declaration process of highly pathogenic AIV (HPAIV) but also reduces the need for experimental in vivo testing of H5 and H7 viruses.

Lesions and viral antigen distribution in bald eagles, red-tailed hawks, and great horned owls naturally infected with H5N1 clade 2.3.4.4b highly pathogenic avian influenza virus.

An epidemic of highly pathogenic avian influenza (HPAI) began in North America in the winter of 2021. The introduced Eurasian H5N1 clade 2.3.4.4b virus subsequently reassorted with North American avian influenza strains. This postmortem study describes the lesions and influenza A virus antigen distribution in 3 species of raptors, including bald eagles (Haliaeetus leucocephalus, n = 6), red-tailed hawks (Buteo jamaicensis, n = 9), and great horned owls (Bubo virginianus, n = 8), naturally infected with this virus strain based on positive reverse transcriptase polymerase chain reaction and sequencing results from oropharyngeal swabs. The birds presented with severe neurologic signs and either died or were euthanized because of the severity of their clinical signs and suspected influenza virus infection. Gross lesions were uncommon and included forebrain hemorrhages in 2 eagles, myocarditis in 1 hawk, and multifocal pancreatic necrosis in 3 owls. Histological lesions were common and included encephalitis, myocarditis, multifocal pancreas necrosis, multifocal adrenal necrosis, histiocytic splenitis, and anterior uveitis in decreasing frequency. Influenza A viral antigen was detected in brain, heart, pancreas, adrenal gland, kidney, spleen, liver, and eye. In conclusion, bald eagles, red-tailed hawks, and great horned owls infected with the HPAI clade 2.3.4.4b virus strain and showing neurological signs of illness may develop severe or fatal disease with histologically detectable lesions in the brain that are frequently positive for viral antigen.

Outbreak of epizootic hemorrhagic disease in captive reindeer (Rangifer tarandus).

In September 2020, an outbreak of epizootic hemorrhagic disease occurred in captive reindeer (Rangifer tarandus) and was associated with neurological signs and mortality. Four reindeer died or were euthanized after acute illness over a 12-day period. Affected reindeer displayed abnormal behavior, neurologic signs, lethargy, and/or lameness. The most consistent gross finding was dark red streaks throughout the adrenal gland cortices (4/4). One animal had acute hemorrhage involving the subcutis and skeletal muscles over the ventrolateral body wall and back, and abomasal serosa. Histologically, the most common lesions were adrenal gland cortical hemorrhage (4/4) with necrosis (3/4) and lymphoplasmacytic meningoencephalitis with gliosis, glial nodules, satellitosis, and nonsuppurative perivascular cuffing (4/4). The brain lesions were most frequent in the gray matter of the cerebrum, hippocampus, and thalamus but also involved the cerebellum and brainstem. Epizootic hemorrhagic disease virus serotype 6 was detected through PCR and sequencing of the spleen in all cases.

SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus).

Widespread human SARS-CoV-2 infections combined with human-wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted white-tailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have angiotensin-converting enzyme 2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, exhibit social behavior, and can be abundant near urban centers. We evaluated 624 prepandemic and postpandemic serum samples from wild deer from four US states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples tested with a SARS-CoV-2 virus neutralization test showed high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2.

Highly Pathogenic Avian Influenza A(H5N1) Virus Outbreak in New England Seals, United States.

We report the spillover of highly pathogenic avian influenza A(H5N1) into marine mammals in the northeastern United States, coincident with H5N1 in sympatric wild birds. Our data indicate monitoring both wild coastal birds and marine mammals will be critical to determine pandemic potential of influenza A viruses.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Wild Terrestrial Mammals, United States, 2022.

We describe the pathology of natural infection with highly pathogenic avian influenza A(H5N1) virus of Eurasian lineage Goose/Guangdong clade 2.3.4.4b in 67 wild terrestrial mammals throughout the United States during April 1‒July 21, 2022. Affected mammals include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals showed primarily neurologic signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.

Evolution of the North American Lineage H7 Avian Influenza Viruses in Association with H7 Virus's Introduction to Poultry.

The incursions of H7 subtype low-pathogenicity avian influenza virus (LPAIV) from wild birds into poultry and its mutations to highly pathogenic avian influenza virus (HPAIV) have been an ongoing concern in North America. Since 2000, 10 phylogenetically distinct H7 virus outbreaks from wild birds have been detected in poultry, six of which mutated to HPAIV. To study the molecular evolution of the H7 viruses that occurs when changing hosts from wild birds to poultry, we performed analyses of the North American H7 hemagglutinin (HA) genes to identify amino acid changes as the virus circulated in wild birds from 2000 to 2019. Then, we analyzed recurring HA amino acid changes and gene constellations of the viruses that spread from wild birds to poultry. We found six HA amino acid changes occurring during wild bird circulation and 10 recurring changes after the spread to poultry. Eight of the changes were in and around the HA antigenic sites, three of which were supported by positive selection. Viruses from each H7 outbreak had a unique genotype, with no specific genetic group associated with poultry outbreaks or mutation to HPAIV. However, the genotypes of the H7 viruses in poultry outbreaks tended to contain minor genetic groups less observed in wild bird H7 viruses, suggesting either a biased sampling of wild bird AIVs or a tendency of having reassortment with minor genetic groups prior to the virus's introduction to poultry. IMPORTANCE Wild bird-origin H7 subtype avian influenza viruses are a constant threat to commercial poultry, both directly by the disease they cause and indirectly through trade restrictions that can be imposed when the virus is detected in poultry. It is important to understand the genetic basis of why the North American lineage H7 viruses have repeatedly crossed the species barrier from wild birds to poultry. We examined the amino acid changes in the H7 viruses associated with poultry outbreaks and tried to determine gene reassortment related to poultry adaptation and mutations to HPAIV. The findings in this study increase the understanding of the evolutionary pathways of wild bird AIV before infecting poultry and the HA changes associated with adaptation of the virus in poultry.

Intercontinental Movement of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4 Virus to the United States, 2021.

We detected Eurasian-origin highly pathogenic avian influenza A(H5N1) virus belonging to the Gs/GD lineage, clade 2.3.4.4b, in wild waterfowl in 2 Atlantic coastal states in the United States. Bird banding data showed widespread movement of waterfowl within the Atlantic Flyway and between neighboring flyways and northern breeding grounds.

Agricultural and geographic factors shaped the North American 2015 highly pathogenic avian influenza H5N2 outbreak.

The 2014-2015 highly pathogenic avian influenza (HPAI) H5NX outbreak represents the largest and most expensive HPAI outbreak in the United States to date. Despite extensive traditional and molecular epidemiological studies, factors associated with the spread of HPAI among midwestern poultry premises remain unclear. To better understand the dynamics of this outbreak, 182 full genome HPAI H5N2 sequences isolated from commercial layer chicken and turkey production premises were analyzed using evolutionary models able to accommodate epidemiological and geographic information. Epidemiological compartmental models embedded in a phylogenetic framework provided evidence that poultry type acted as a barrier to the transmission of virus among midwestern poultry farms. Furthermore, after initial introduction, the propagation of HPAI cases was self-sustainable within the commercial poultry industries. Discrete trait diffusion models indicated that within state viral transitions occurred more frequently than inter-state transitions. Distance and sample size were very strongly supported as associated with viral transition between county groups (Bayes Factor > 30.0). Together these findings indicate that the different types of midwestern poultry industries were not a single homogenous population, but rather, the outbreak was shaped by poultry industries and geographic factors.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

SARS-CoV-2 Exposure in Escaped Mink, Utah, USA.

In August 2020, outbreaks of coronavirus disease were confirmed on mink farms in Utah, USA. We surveyed mammals captured on and around farms for evidence of infection or exposure. Free-ranging mink, presumed domestic escapees, exhibited high antibody titers, suggesting a potential severe acute respiratory syndrome coronavirus 2 transmission pathway to native wildlife.

Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs.

Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations.IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

Highly Pathogenic Avian Influenza A(H7N3) Virus in Poultry, United States, 2020.

An outbreak of low-pathogenicity avian influenza A(H7N3) virus of North American wild bird lineage occurred on commercial turkey farms in North Carolina and South Carolina, USA, during March-April 2020. The virus mutated to the highly pathogenic form in 1 house on 1 farm via recombination with host 28S rRNA.

Low Pathogenicity Avian Influenza (H5N2) Viruses, Dominican Republic.

Low pathogenicity avian influenza (H5N2) virus was detected in poultry in the Dominican Republic in 2007 and re-emerged in 2017. Whole-genome sequencing and phylogenetic analysis show introduction of an H5N2 virus lineage from Mexico into poultry in the Dominican Republic, then divergence into 3 distinct genetic subgroups during 2007-2019.

First Reported Cases of SARS-CoV-2 Infection in Companion Animals - New York, March-April 2020.

On April 22, CDC and the U.S. Department of Agriculture (USDA) reported cases of two domestic cats with confirmed infection with SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19). These are the first reported companion animals (including pets and service animals) with SARS-CoV-2 infection in the United States, and among the first findings of SARS-CoV-2 symptomatic companion animals reported worldwide. These feline cases originated from separate households and were epidemiologically linked to suspected or confirmed human COVID-19 cases in their respective households. Notification of presumptive positive animal test results triggered a One Health* investigation by state and federal partners, who determined that no further transmission events to other animals or persons had occurred. Both cats fully recovered. Although there is currently no evidence that animals play a substantial role in spreading COVID-19, CDC advises persons with suspected or confirmed COVID-19 to restrict contact with animals during their illness and to monitor any animals with confirmed SARS-CoV-2 infection and separate them from other persons and animals at home (1).

Investigation of the 2018 thick-billed murre (Uria lomvia) die-off on St. Lawrence Island rules out food shortage as the cause.

Die-offs of seabirds in Alaska have occurred with increased frequency since 2015. In 2018, on St. Lawrence Island, seabirds were reported washing up dead on beaches starting in late May, peaking in June, and continuing until early August. The cause of death was documented to be starvation, leading to the conclusion that a severe food shortage was to blame. We use physiology and colony-based observations to examine whether food shortage is a sufficient explanation for the die-off, or if evidence indicates an alternative cause of starvation such as disease. Specifically, we address what species were most affected, the timing of possible food shortages, and food shortage severity in a historical context. We found that thick-billed murres (Uria lomvia) were most affected by the die-off, making up 61% of all bird carcasses encountered during beach surveys. Thick-billed murre carcasses were proportionately more numerous (26:1) than would be expected based on ratios of thick-billed murres to co-occurring common murres (U. aalge) observed on breeding study plots (7:1). Concentrations of the stress hormone corticosterone, a reliable physiological indicator of nutritional stress, in thick-billed murre feathers grown in the fall indicate that foraging conditions in the northern Bering Sea were poor in the fall of 2017 and comparable in severity to those experienced by murres during the 1976-1977 Bering Sea regime shift. Concentrations of corticosterone in feathers grown during the pre-breeding molt indicate that foraging conditions in late winter 2018 were similar to previous years. The 2018 murre egg harvest in the village of Savoonga (on St. Lawrence Is.) was one-fifth the 1993-2012 average, and residents observed that fewer birds laid eggs in 2018. Exposure of thick-billed murres to nutritional stress in August, however, was no different in 2018 compared to 2016, 2017, and 2019, and was comparable to levels observed on St. George Island in 2003-2017. Prey abundance, measured by the National Oceanic and Atmospheric Administration in bottom-trawl surveys, was also similar in 2018 to 2017 and 2019, supporting the evidence that food was not scarce in the summer of 2018 in the vicinity of St. Lawrence Island. Of two moribund thick-billed murres collected at the end of the mortality event, one tested positive for a novel re-assortment H10 strain of avian influenza with Eurasian components, likely contracted during the non-breeding season. It is not currently known how widely spread infection of murres with the novel virus was, thus insufficient evidence exists to attribute the die-off to an outbreak of avian influenza. We conclude that food shortage alone is not an adequate explanation for the mortality of thick-billed murres in 2018, and highlight the importance of rapid response to mortality events in order to document alternative or confounding causes of mortality.