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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Diet-induced obesity affects influenza disease severity and transmission dynamics in ferrets.

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet, the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a diet-induced obese ferret model and tools to demonstrate that, like humans, obesity resulted in notable changes to the lung microenvironment, leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for a longer period, making them more likely to transmit to contacts. These data suggest that the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission and a key tool for therapeutic and intervention development for this high-risk population.

Influenza D virus infection in China, 2022-2023.

Influenza D virus (IDV) plays an important role in the bovine respiratory disease (BRD) complex. Its potential for the zoonotic transmission is of particular concern. In China, IDV has previously been identified in agricultural animals by molecular surveys with no live virus isolates reported. In this study, live IDVs were successfully isolated from cattle in China, which prompted us to further investigate the national prevalence, antigenic property, and infection biology of the virus. IDV RNA was detected in 11.1% (51/460) of cattle throughout the country in 2022-2023. Moreover, we conducted the first IDV serosurveillance in China, revealing a high seroprevalence (91.4%, 393/430) of IDV in cattle during the 2022-2023 winter season. Notably, all the 16 provinces from which cattle originated possessed seropositive animals, and 3 of them displayed the 100% IDV-seropositivity rate. In contrast, a very low seroprevalence of IDV was observed in pigs (3%, 3/100) and goats (1%, 1/100) during the same period of investigation. Furthermore, besides D/Yama2019 lineage-like IDVs, we discovered the D/660 lineage-like IDV in Chinese cattle, which has not been detected to date in Asia. Finally, the Chinese IDVs replicated robustly in diverse cell lines but less efficiently in the swine cell line. Considering the nationwide distribution, high seroprevalence, and appreciably genetic diversity, further studies are required to fully evaluate the risk of Chinese IDVs for both animal and human health in China, which can be evidently facilitated by IDV isolates reported in this study.

Risk assessment of a highly pathogenic H5N1 influenza virus from mink.

Outbreaks of highly pathogenic H5N1 clade 2.3.4.4b viruses in farmed mink and seals combined with isolated human infections suggest these viruses pose a pandemic threat. To assess this threat, using the ferret model, we show an H5N1 isolate derived from mink transmits by direct contact to 75% of exposed ferrets and, in airborne transmission studies, the virus transmits to 37.5% of contacts. Sequence analyses show no mutations were associated with transmission. The H5N1 virus also has a low infectious dose and remains virulent at low doses. This isolate carries the adaptive mutation, PB2 T271A, and reversing this mutation reduces mortality and airborne transmission. This is the first report of a H5N1 clade 2.3.4.4b virus exhibiting direct contact and airborne transmissibility in ferrets. These data indicate heightened pandemic potential of the panzootic H5N1 viruses and emphasize the need for continued efforts to control outbreaks and monitor viral evolution.

Pigs are highly susceptible to but do not transmit mink-derived highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b.

ABSTRACTRapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammalian species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like adaptations such as PB2-E627 K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.