Avian and Human Influenza A Virus Receptors in Bovine Mammary Gland.

An outbreak of influenza A (H5N1) virus was detected in dairy cows in the United States. We detected influenza A virus sialic acid -α2,3/α2,6-galactose host receptors in bovine mammary glands by lectin histochemistry. Our results provide a rationale for the high levels of H5N1 virus in milk from infected cows.

Pinnipeds and avian influenza: a global timeline and review of research on the impact of highly pathogenic avian influenza on pinniped populations with particular reference to the endangered Caspian seal (Pusa caspica).

This study reviews chronologically the international scientific and health management literature and resources relating to impacts of highly pathogenic avian influenza (HPAI) viruses on pinnipeds in order to reinforce strategies for the conservation of the endangered Caspian seal (Pusa caspica), currently under threat from the HPAI H5N1 subtype transmitted from infected avifauna which share its haul-out habitats. Many cases of mass pinniped deaths globally have occurred from HPAI spill-overs, and are attributed to infected sympatric aquatic avifauna. As the seasonal migrations of Caspian seals provide occasions for contact with viruses from infected migratory aquatic birds in many locations around the Caspian Sea, this poses a great challenge to seal conservation. These are thus critical locations for the surveillance of highly pathogenic influenza A viruses, whose future reassortments may present a pandemic threat to humans.

Exploring Potential Intermediates in the Cross-Species Transmission of Influenza A Virus to Humans.

The influenza A virus (IAV) has been a major cause of several pandemics, underscoring the importance of elucidating its transmission dynamics. This review investigates potential intermediate hosts in the cross-species transmission of IAV to humans, focusing on the factors that facilitate zoonotic events. We evaluate the roles of various animal hosts, including pigs, galliformes, companion animals, minks, marine mammals, and other animals, in the spread of IAV to humans.

Amino acid mutations PB1-V719M and PA-N444D combined with PB2-627K contribute to the pathogenicity of H7N9 in mice.

H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies.

Stop H5N1 influenza in US cattle now.

The relentless march of a highly pathogenic avian influenza virus (HPAIV) strain, known as H5N1, to become an unprecedented panzootic continues unchecked. The leap of H5N1 clade 2.3.4.4b from Eurasia and Africa to North America in 2021 and its further spread to South America and the Antarctic have exposed new avian and mammalian populations to the virus and led to outbreaks on an unrivaled scale. The virus has infected wild birds across vast geographic regions and caused wildlife deaths in some of the world's most biodiverse ecosystems. Hundreds of millions of poultry have died or been culled, affecting global food security in some of the world's poorest regions. Numerous mammalian species, including sea lions and fur animals, have been infected. Outbreaks in dairy cows in the United States have been occurring for months, seemingly unchecked in most affected states. Why is there not a greater sense of urgency to control these infections?

Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Identification of sialic acid receptors for influenza A virus in snakes.

The tissue tropism and the wide host range of influenza A viruses are determined by the presence of sialic acid (SA) α2,3-Gal and SA α2,6-Gal receptors. Recent studies have shown that animals possessing both receptors allow for the rearrangement and emergence of new viral strains of public health importance. This study aimed to evaluate the expression and distribution of human and avian influenza A receptors in nine Neotropical snake species using lectin immunohistochemistry. We selected 17 snakes that were examined post mortem at the Veterinary Pathology Sector of the Universidade Federal de Minas Gerais between 2019 and 2023. Sections of nasal turbinate, trachea, lung, oral mucosa, stomach and intestine were subjected to immunohistochemical analysis using the lectins Maackia amurensis and Sambucus nigra. This research detected, for the first time, co-expression of SA α2,3-Gal and SA α2,6-Gal receptors in the respiratory and digestive tracts of snakes, indicating the possible susceptibility of these species to influenza A virus of avian and human origin. Consequently, snakes can be considered important species for monitoring influenza A in wild, urban and peri-urban environments. More studies should be conducted to investigate the role of snakes in influenza A epidemiology.

A(H2N2) and A(H3N2) influenza pandemics elicited durable cross-reactive and protective antibodies against avian N2 neuraminidases.

Human cases of avian influenza virus (AIV) infections are associated with an age-specific disease burden. As the influenza virus N2 neuraminidase (NA) gene was introduced from avian sources during the 1957 pandemic, we investigate the reactivity of N2 antibodies against A(H9N2) AIVs. Serosurvey of healthy individuals reveal the highest rates of AIV N2 antibodies in individuals aged ≥65 years. Exposure to the 1968 pandemic N2, but not recent N2, protected against A(H9N2) AIV challenge in female mice. In some older adults, infection with contemporary A(H3N2) virus could recall cross-reactive AIV NA antibodies, showing discernable human- or avian-NA type reactivity. Individuals born before 1957 have higher anti-AIV N2 titers compared to those born between 1957 and 1968. The anti-AIV N2 antibodies titers correlate with antibody titers to the 1957 N2, suggesting that exposure to the A(H2N2) virus contribute to this reactivity. These findings underscore the critical role of neuraminidase immunity in zoonotic and pandemic influenza risk assessment.

Mortality in sea lions is associated with the introduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole genome sequencing and phylogenetic analysis.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Characterization of Influenza D Virus Reassortant Strain in Swine from Mixed Pig and Beef Farm, France.

Influenza D virus was isolated from pigs on a mixed pig and beef farm in France. Investigation suggested bull-to-pig transmission and spread among pigs. The swine influenza D virus recovered was a reassortant of D/660 and D/OK lineages. Reported mutations in the receptor binding site might be related to swine host adaptation.

Lateral flow paired with RT-LAMP: A speedy solution for Influenza A virus detection in swine.

Influenza A Virus in swine (IAV-S) is a zoonotic pathogen that is nearly ubiquitous in commercial swine in the USA. Swine possess sialic acid receptors that allow co-infection of human and avian viruses with the potential of pandemic reassortment. We aimed to develop a fast and robust testing method for IAV-S detection on swine farms. Two primers of the RT-LAMP assay were labeled for use in a lateral flow readout. A commercially available lateral flow kit was used to read the amplicon product. With a runtime of ∼ 45 minutes, the limit of detection for the assay is comparable with an RT-qPCR Cq less than 35, with a sensitivity of 83.5 % and a specificity of 89.6 %. This assay allows veterinarians and producers with limited access to diagnostic services to perform and detect Matrix gene amplification on-site with low equipment costs. The time from sample collection to detection is less than one hour, making this method an accessible, convenient, and affordable tool to prevent the spread of zoonotic disease.

The evolution, complexity, and diversity of swine influenza viruses in China: A hidden public health threat.

Swine influenza viruses (SIVs), including H1N1, H1N2, and H3N2, have spread throughout the global pig population. Potential pandemics are a concern with the recent sporadic cross-species transmission of SIVs to humans. We collected 1421 samples from Guangdong, Fujian, Henan, Yunnan and Jiangxi provinces during 2017-2018 and isolated 29 viruses. These included 21H1N1, 5H1N2, and 3H3N2 strains. Genome analysis showed that the domestic epidemic genotypes of H1N1 were mainly G4 and G5 reassortant EA swine H1N1. These genotypes have a clear epidemic advantage. Two strains were Clade 6B.1 pdm/09H1N1, suggesting a possible pig-to-human transmission route. Notably, three new H1N2 genotypes were identified using the genomic backbones of G4 or G5 viruses for recombination. The identification of various subtypes and genotypes highlight the complexity and diversity of SIVs in China and need for continuous monitoring of SIV evolution to assess the risks and prepare for potential influenza pandemics.

Risk assessment of influenza transmission between workers and pigs on US indoor hog growing units.

On pig farms ample opportunity exists for pig-to-human and human-to-pig (cross-species) influenza transmission. The purpose of this study was to assess the risks of cross-species influenza transmission within an indoor pig grower unit in the United States and to prioritize data gaps. Using the World Organization for Animal Health risk assessment framework we evaluated influenza transmission across two risk pathways: 1. What is the likelihood that based on current conditions on a single typical hog grower-finisher facility in the Midwest (US), during a single production cycle, at least one hog becomes infected with an influenza virus associated with swine (either H1N1, H3N2, or H1N2) [step 1a] and that at least one worker becomes infected as a result [step 1b] and that the worker develops symptoms [step 1c]? And 2. What is the likelihood that, based on current conditions on a single typical hog grower-finisher facility in the Midwest (US), during a single production cycle, at least one worker becomes infected with an influenza virus associated with people (either H1N1, H3N2, or H1N2) [step 2a] and that at least one pig becomes infected as a result [step 2b] and that the pig(s) develop(s) symptoms [step 2c]? Semi-quantitative probability and uncertainty assessments were based on literature review including passive and active influenza surveillance data. We assumed a typical pig-grower farm has capacity for 4,000 pigs, two workers, and minimal influenza control measures. Probability and uncertainty categories were assessed for each risk step and the combined risk pathway. The combined risk assessment for risk pathway one was estimated to be Very Low for H1N1 and H1N2 with an overall High level of uncertainty. The combined risk assessment for risk pathway two was estimated to be Extremely Low for H1N1 and H3N2 with a High degree of uncertainty. Scenario analyses in which influenza control measures were assumed to be implemented separately (implementing vaccinating sows, mass vaccinating incoming pigs or improved personal protective equipment adherence) showed no reduction in the combined risk category. When implementing three influenza control methods altogether, the combined risk could be reduced to Extremely Low for risk pathway one and remained Extremely Low for risk pathway two. This work highlights that multiple influenza control methods are needed to reduce the risks of inter-species influenza transmission on swine farms.

Evaluation of nasal swab and nasal wipe for detection of Influenza A in swine using Bayesian latent class analysis.

Influenza A virus (IAV) is an important pathogen in Brazilian swine herds, and monitoring the viral circulation is essential to control and reduce the transmission. Surveillance programs for IAV are often based on individual piglets level sampling, making the evaluation of the available diagnostic tools crucial to assessing IAV circulation in herds. Thus, two sample collection methodologies were compared in pig herds in southern Brazil to detect IAV by RT-qPCR: nasal swab (NS) and nasal wipe (NW). A Bayesian latent class model (BLCM) was set for two tests and two populations. The NW and NS used are more specific (higher than 95 % for both) than sensitive. The sensitivity for NW was lower than the NS, 84.14 % (70 % - 95 %; posterior probability interval (PPI): 95 %) and 87.15 % (73 % - 97 %; PPI: 95 %), respectively, and the specificity was 95 % (90 % - 99 %; PPI: 95 %) and 99 % (96 % - 100 %; PPI: 95 %), respectively. Although the wipe sample collection loses both sensitivity and specificity compared with nasal swab, differences in test performance were very limited and PPIs largely overlapped. Therefore NW can also be considered a valuable tool. The decision about the use of both techniques should be based on the trade-off between their performance limitations and feasibility in routine monitoring.

Characterization of a novel functional porcine CD3+CD4lowCD8α+CD8ß+ T-helper/memory lymphocyte subset in the respiratory tract lymphoid tissues of swine influenza A virus vaccinated pigs.

The pig is emerging as a physiologically relevant biomedical large animal model. Delineating the functional roles of porcine adaptive T-lymphocyte subsets in health and disease is of critical significance, which facilitates mechanistic understanding of antigen-specific immune memory responses. We identified a novel T-helper/memory lymphocyte subset in pigs and performed phenotypic and functional characterization of these cells under steady state and following vaccination and infection with swine influenza A virus (SwIAV). A novel subset of CD3+CD4lowCD8α+CD8ß+ memory T-helper cells was identified in the blood of healthy adult pigs under homeostatic conditions. To understand the possible functional role/s of these cells, we characterized the antigen-specific T cell memory responses by multi-color flow cytometry in pigs vaccinated with a whole inactivated SwIAV vaccine, formulated with a phytoglycogen nanoparticle/STING agonist (ADU-S100) adjuvant (NanoS100-SwIAV). As a control, a commercial SwIAV vaccine was included in a heterologous challenge infection trial. The frequencies of antigen-specific IL-17A and IFNγ secreting CD3+CD4lowCD8α+CD8ß+ memory T-helper cells were significantly increased in the lung draining tracheobronchial lymph nodes (TBLN) of intradermal, intramuscular and intranasal inoculated NanoS100-SwIAV vaccine and commercial vaccine administered animals. While the frequencies of antigen-specific, IFNγ secreting CD3+CD4lowCD8α+CD8ß+ memory T-helper cells were significantly enhanced in the blood of intranasal and intramuscular vaccinates. These observations suggest that the CD3+CD4lowCD8α+CD8ß+ T-helper/memory cells in pigs may have a protective and/or regulatory role/s in immune responses against SwIAV infection. These observations highlight the heterogeneity and plasticity of porcine CD4+ T-helper/memory cells in response to respiratory viral infection in pigs. Comprehensive systems immunology studies are needed to further decipher the cellular lineages and functional role/s of this porcine T helper/memory cell subset.

Sialic Acid Receptor Specificity in Mammary Gland of Dairy Cattle Infected with Highly Pathogenic Avian Influenza A(H5N1) Virus.

In March 2024, the US Department of Agriculture's Animal and Plant Health Inspection Service reported detection of highly pathogenic avian influenza (HPAI) A(H5N1) virus in dairy cattle in the United States for the first time. One factor that determines susceptibility to HPAI H5N1 infection is the presence of specific virus receptors on host cells; however, little is known about the distribution of the sialic acid (SA) receptors in dairy cattle, particularly in mammary glands. We compared the distribution of SA receptors in the respiratory tract and mammary gland of dairy cattle naturally infected with HPAI H5N1. The respiratory and mammary glands of HPAI H5N1-infected dairy cattle are rich in SA, particularly avian influenza virus-specific SA α2,3-gal. Mammary gland tissues co-stained with sialic acids and influenza A virus nucleoprotein showed predominant co-localization with the virus and SA α2,3-gal. HPAI H5N1 exhibited epitheliotropism within the mammary gland, and we observed rare immunolabeling within macrophages.

Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020-2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

Rapid mortality in captive bush dogs (Speothos venaticus) caused by influenza A of avian origin (H5N1) at a wildlife collection in the United Kingdom.

Europe has suffered unprecedented epizootics of high pathogenicity avian influenza (HPAI) clade 2.3.4.4b H5N1 since Autumn 2021. As well as impacting upon commercial and wild avian species, the virus has also infected mammalian species more than ever observed previously. Mammalian species involved in spill over events have primarily been scavenging terrestrial carnivores and farmed mammalian species although marine mammals have also been affected. Alongside reports of detections of mammalian species found dead through different surveillance schemes, several mass mortality events have been reported in farmed and wild animals. In November 2022, an unusual mortality event was reported in captive bush dogs (Speothos venaticus) with clade 2.3.4.4b H5N1 HPAIV of avian origin being the causative agent. The event involved an enclosure of 15 bush dogs, 10 of which succumbed during a nine-day period with some dogs exhibiting neurological disease. Ingestion of infected meat is proposed as the most likely infection route.