Rapid evolution leads to extensive genetic diversification of cattle flu Influenza D virus.

Influenza D virus (IDV), the cattle flu virus, is a novel multi-host RNA virus, circulating silently worldwide, with widespread seropositivity among US cattle, reaching up to 80% in some areas raising a potential threat of cattle-to-human transmission. Currently, five genetic lineages of IDV have been described, but their evolutionary dynamics have not been studied. Although IDV was first identified in 2011, our comprehensive analysis of all known IDV genomes suggests that the earliest ancestors of IDV likely to have evolved towards the end of the 20th century and D/OK lineage appears to have emerged in 2005. We confirmed a significantly higher substitution rate in IDV than in Influenza C virus, which is consistent with their global distribution and multi-host tropism. We identified multiple sub-populations within the D/OK lineage, highlighting extensive diversification and dissemination. Other findings are evidence for potential reassortment among IDV strains in the USA and transboundary circulation of IDV in Europe with introductions into Danish cattle, some of which potentially originated from France. IDV, an emerging virus with a higher rate of evolution and uncontrolled circulation, could facilitate its adaptation to humans. Our findings underscore the importance of targeted surveillance for IDV in humans and at-risk animal populations.

Ventilation does not affect close-range transmission of influenza virus in a ferret playpen setup.

Sustained community spread of influenza viruses relies on efficient person-to-person transmission. Current experimental transmission systems do not mimic environmental conditions (e.g., air exchange rates, flow patterns), host behaviors, or exposure durations relevant to real-world settings. Therefore, results from these traditional systems may not be representative of influenza virus transmission in humans. To address this pitfall, we developed a close-range transmission setup that implements a play-based scenario and used it to investigate the impact of ventilation rates on transmission. In this setup, four immunologically naive recipient ferrets were exposed to a donor ferret infected with a genetically barcoded 2009 H1N1 virus (H1N1pdm09) for 4 h. The ferrets interacted in a shared space that included toys, similar to a childcare setting. Transmission efficiency was assessed under low and high ventilation, with air exchange rates of ~1.3 h-1 and 23 h-1, respectively. Transmission efficiencies observed in three independent replicate studies were similar between ventilation conditions. The presence of infectious virus or viral RNA on surfaces and in air throughout the exposure area was also not impacted by the ventilation rate. While high viral genetic diversity in donor ferret nasal washes was maintained during infection, recipient ferret nasal washes displayed low diversity, revealing a narrow transmission bottleneck regardless of ventilation rate. Examining the frequency and duration of ferret physical touches revealed no link between these interactions and a successful transmission event. Our findings indicate that exposures characterized by frequent, close-range interactions and the presence of fomites can overcome the benefits of increased ventilation.

Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket.

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket.

Spatial patterns of influenza A virus spread across compartments in commercial swine farms in the United States.

Multiple genetic variants of H1 and H3 influenza A viruses (IAVs) circulate concurrently in US swine farms. Understanding the spatial transmission patterns of IAVs among these farms is crucial for developing effective control strategies and mitigating the emergence of novel IAVs. In this study, we analysed 1909 IAV genomic sequences from 785 US swine farms, representing 33 farming systems across 12 states, primarily in the Midwest from 2004 to 2023. Bayesian phylogeographic analyses were performed to identify the dispersal patterns of both H1 and H3 virus genetic lineages and to elucidate their spatial migration patterns within and between different systems. Our results showed that both intra-system and inter-system migrations occurred between the swine farms, with intra-system migrations being more frequent. However, migration rates for H1 and H3 IAVs were similar between intra-system and inter-system migration events. Spatial migration patterns aligned with expected pig movement across different compartments of swine farming systems. Sow-Farms were identified as key sources of viruses, with bi-directional migration observed between these farms and other parts of the system, including Wean-to-Finish and Gilt-Development-Units. High intra-system migration was detected across farms in the same region, while spread to geographically distant intra- and inter-system farms was less frequent. These findings suggest that prioritizing resources towards systems frequently confronting influenza problems and targeting pivotal source farms, such as sow farms, could be an effective strategy for controlling influenza in US commercial swine operations.

Nonvirulent Infectious Salmon Anemia Virus (ISAV-HPR0) Not Detectable in Eggs or Progeny of Infected Captive Atlantic Salmon Brood.

The potential for infectious salmon anemia virus (ISAV)-an internationally regulated pathogen of salmon-to transmit vertically from parent to offspring is currently unclear. While the highly virulent ISAV phenotype known as ISAV-HPRΔ has been observed intra-ova, evidence for vertical transmission of the avirulent ISAV phenotype known as ISAV-HPR0 is lacking. In this study, we identified ISAV-HPR0-infected Atlantic salmon broodstock during spawning within a government research recirculating aquaculture facility using qPCR. Eggs and milt from infected brood were used to initiate 16 unique family dam-sire crosses from which 29-60 fertilized eggs per cross were screened for ISAV using qPCR (limit of detection ~100 virus genome copies/egg). A portion of eggs (~300) from one family cross was hatched and further reared in biosecure containment and periodically screened for ISAV by gill clipping over a 2-year period. ISAV was not detected in any of the 781 eggs screened from 16 family crosses generated by infected brood, nor in 870 gill clips periodically sampled from the single-family cohort raised for 2 years in biocontainment. Based on these findings, we conclude that ISAV-HPR0 has a limited likelihood for vertical parent-to-offspring transmission in cultured Atlantic salmon.

Machine learning approaches for influenza A virus risk assessment identifies predictive correlates using ferret model in vivo data.

In vivo assessments of influenza A virus (IAV) pathogenicity and transmissibility in ferrets represent a crucial component of many pandemic risk assessment rubrics, but few systematic efforts to identify which data from in vivo experimentation are most useful for predicting pathogenesis and transmission outcomes have been conducted. To this aim, we aggregated viral and molecular data from 125 contemporary IAV (H1, H2, H3, H5, H7, and H9 subtypes) evaluated in ferrets under a consistent protocol. Three overarching predictive classification outcomes (lethality, morbidity, transmissibility) were constructed using machine learning (ML) techniques, employing datasets emphasizing virological and clinical parameters from inoculated ferrets, limited to viral sequence-based information, or combining both data types. Among 11 different ML algorithms tested and assessed, gradient boosting machines and random forest algorithms yielded the highest performance, with models for lethality and transmission consistently better performing than models predicting morbidity. Comparisons of feature selection among models was performed, and highest performing models were validated with results from external risk assessment studies. Our findings show that ML algorithms can be used to summarize complex in vivo experimental work into succinct summaries that inform and enhance risk assessment criteria for pandemic preparedness that take in vivo data into account.

Adaptation potential of H3N8 canine influenza virus in human respiratory cells.

In 2004, the equine-origin H3N8 canine influenza virus (CIV) first caused an outbreak with lethal cases in racing greyhounds in Florida, USA, and then spread to domestic dogs nationwide. Although transmission of this canine virus to humans has not been reported, it is important to evaluate its zoonotic potential because of the high contact opportunities between companion dogs and humans. To gain insight into the interspecies transmissibility of H3N8 CIV, we tested its adaptability to human respiratory A549 cells through successive passages. We found that CIV acquired high growth properties in these cells mainly through mutations in surface glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). Our reverse genetics approach revealed that HA2-K82E, HA2-R163K, and NA-S18L mutations were responsible for the increased growth of CIV in human cells. Molecular analyses revealed that both HA2 mutations altered the optimum pH for HA membrane fusion activity and that the NA mutation changed the HA-NA functional balance. These findings suggest that H3N8 CIV could evolve into a human pathogen with pandemic potential through a small number of mutations, thereby posing a threat to public health in the future.

Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle.

The highly pathogenic avian influenza (HPAI) H5N1 virus clade 2.3.4.4b has caused the death of millions of domestic birds and thousands of wild birds in the USA since January 2022 (refs. 1-4). Throughout this outbreak, spillovers to mammals have been frequently documented5-12. Here we report spillover of the HPAI H5N1 virus to dairy cattle across several states in the USA. The affected cows displayed clinical signs encompassing decreased feed intake, altered faecal consistency, respiratory distress and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of the HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the ability of the virus to cross species barriers.

Pathogenicity and transmissibility of bovine H5N1 influenza virus.

Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the USA, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1-4. Here we characterize an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species, however, this tropism was also observed for an older HPAI H5N1 virus isolate. Bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.

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.

Multiple transatlantic incursions of highly pathogenic avian influenza clade 2.3.4.4b A(H5N5) virus into North America and spillover to mammals.

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.

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.

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.

Potential pandemic risk of circulating swine H1N2 influenza viruses.

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans due to a potential lack of population level immunity. Here we describe a pipeline to characterize and triage influenza viruses for their pandemic risk and examine the pandemic potential of two widespread swine origin viruses. Our analysis reveals that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against a α-H1 clade strain (α-swH1N2) but do against a γ-H1 clade strain. The α-swH1N2 virus replicates efficiently in human airway cultures and exhibits phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, α-swH1N2 is capable of efficient airborne transmission to both naïve ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity show reduced α-swH1N2 viral shedding and less severe disease signs. Despite this, H1N1pdm09-immune ferrets that became infected via the air can still onward transmit α-swH1N2 with an efficiency of 50%. These results indicate that this α-swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness and pathology in animals with prior immunity.

Infection dynamics of subtype H9N2 low pathogenic avian influenza a virus in turkeys.

While mammals can be infected by influenza A virus either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of the pathogen. So far most of the knowledge on influenza virus dynamics was however gained on mammalian models. In this study, we infected turkeys using a low pathogenic avian influenza virus and determined the infection dynamics with a target-cell limited model. Results showed that turkeys had a different set of infection characteristics, compared with humans and ponies. The viral clearance rates were similar between turkeys and ponies but higher than that in humans. The cell death rates and cell to cell transmission rates were similar between turkeys and humans but higher than those in ponies. Overall, this study indicated the variations of within-host dynamics of influenza infection in avian, humans, and other mammalian systems.

Persistence of Influenza H5N1 and H1N1 Viruses in Unpasteurized Milk on Milking Unit Surfaces.

Examining the persistence of highly pathogenic avian influenza A(H5N1) from cattle and human influenza A(H1N1)pdm09 pandemic viruses in unpasteurized milk revealed that both remain infectious on milking equipment materials for several hours. Those findings highlight the risk for H5N1 virus transmission to humans from contaminated surfaces during the milking process.