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We analyzed >4,000 pig samples from slaughterhouses in Cambodia and found higher influenza A seroprevalence (40.0%) and prevalence (1.5%) among pigs from commercial farms than smallholder farms (seroprevalence 8.9%; prevalence 0.6%). Multivariable analyses revealed evidence of transmission after leaving farms. Findings have implications for influenza risk and surveillance in emerging livestock systems.

In July 2023, cases of highly pathogenic avian influenza (HPAI) were reported at 2 shelters for stray cats in Seoul, South Korea. The cause of infection was suspected to be improperly sterilized raw food made from domestic duck meat, which was manufactured in South Korea. All viruses isolated from cats at the shelters and from the raw food belonged to HPAI A(H5N1) clade 2.3.4.4b. The gene constellation of all viruses was most similar to that of viruses isolated in Korea in November 2022. Of note, the viruses isolated from infected cats harbored mutations E627K or D701N in polymerase basic 2, which are indicative of adaptation to mammals. Postmortem examination revealed systemic pathologic lesions and the presence of widespread virus in different tissues. Thus, consumption of raw duck meat contaminated with HPAI virus likely caused systemic symptoms and death in cats, indicating the introduction of mammal-adapted mutations of the virus.

During the active surveillance, we isolated nine H4N6 subtype influenza A viruses from wild birds in China. To reveal the epidemiology and biology characteristics of H4 subtype influenza A virus from wild birds, we investigated H4 subtype viruses available in the public source, and found that the H4 viruses have been detected in at least 37 countries to date, and more than 73.6% of the viruses were from wild Anseriformes. Bayesian phylogeographic analysis showed that Mongolia worked as the important transmission centre for Eurasian lineage H4 viruses spreading. Phylogenetic analysis of HA genes indicated that global H4 influenza A viruses were divided into Eurasian and North American lineage, our nine H4N6 isolates fell into the Eurasian lineage. Recombination analysis suggested that nine H4N6 isolates underwent complex gene recombination with various subtypes of influenza A viruses and formed two genotypes. Notably, nine H4N6 isolates acquired mammalian virulence-increasing residues. Two representative H4N6 viruses possessed dual receptor binding specificity, they could efficiently replicate in MDCK and 293 T cells in vitro infection, also could cross the species barrier to infect mice directly without prior adaption in vivo experiments. These findings emphasize the public health issues represented by H4 viruses, and highlight the need to strengthen the active surveillance of H4 viruses from wild birds.

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.

We detected highly pathogenic avian influenza A(H5N1) virus in wild rats collected from a rural area in Giza, Egypt, near poultry farms, markets, and backyard flocks. Sequence and phylogenetic analyses indicated that the virus from the rats belonged to clade 2.3.4.4b, which has been the predominant virus genotype circulating in Egypt and worldwide since 2021-2022. Active surveillance of avian influenza viruses in wild and domestic mammals is recommended to prevent further spread to mammals and humans.

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.

Influenza A viruses (IAVs) pose a persistent potential threat to human health because of the spillover from avian and swine infections. Extensive surveillance was performed in 12 cities of Guangxi, China, during 2018 and 2023. A total of 2540 samples (including 2353 nasal swabs and 187 lung tissues) were collected from 18 pig farms with outbreaks of respiratory disease. From these, 192 IAV-positive samples and 19 genomic sequences were obtained. We found that the H1 and H3 swine influenza A viruses (swIAVs) of multiple lineages and genotypes have continued to co-circulate during that time in this region. Genomic analysis revealed the Eurasian avian-like H1N1 swIAVs (G4) still remained predominant in pig populations. Strikingly, the novel multiple H3N2 genotypes were found to have been generated through the repeated introduction of the early H3N2 North American triple reassortant viruses (TR H3N2 lineage) that emerged in USA and Canada in 1998 and 2005, respectively. Notably, when the matrix gene segment derived from the H9N2 avian influenza virus was introduced into endemic swIAVs, this produced a novel quadruple reassortant H1N2 swIAV that could pose a potential risk for zoonotic infection.

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.

H5N1 high pathogenicity avian influenza virus has killed thousands of marine mammals in South America since 2022. Here we report epidemiological data and full genome characterization of clade 2.3.4.4b H5N1 HPAI viruses associated with a massive outbreak in southern elephant seals (Mirounga leonina) at Península Valdés, Argentina, in October 2023. We also report on H5N1 viruses in concurrently dead terns. Our genomic analysis shows that viruses from pinnipeds and terns in Argentina form a distinct clade with marine mammal viruses from Peru, Chile, Brazil and Uruguay. Additionally, these marine mammal clade viruses share an identical set of mammalian adaptation mutations which were also present in tern viruses. Our combined ecological and phylogenetic data support mammal-to-mammal transmission and occasional mammal-to-bird spillover and suggest multinational transmission of H5N1 viruses in mammals. We reflect that H5N1 viruses becoming more evolutionary flexible and adapting to mammals in new ways could have global consequences for wildlife, humans, and/or livestock., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The present study aimed to determine the dynamics of influenza A virus (IAV) infection in two endemically infected farms (F1 and F2), where a longitudinal follow-up of piglets was performed from birth to 8-12 weeks of age. During the study, a highly virulent isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was introduced on both farms. This allowed us to examine the impact of such introduction on the patterns of infection, disease, and the antibody response of pigs to IAV infection. The introduction of the new PRRSV strain coincided with a change in the dynamics of IAV infection on both farms. In F1, the cumulative incidence of IAV increased from 20% before the outbreak to 67.5%, together with the existence of animals that tested positive for IAV (RT‒qPCR) in nasal swabs for two or more consecutive samples. In F2, the cumulative incidence of IAV increased from 50% before the PRRSV outbreak to 70%, and the proportion of prolonged IAV shedders increased sharply. Additionally, some animals were infected with the same IAV twice during the observation period. In contrast to previous reports, our study revealed that prolonged shedding was not related to the titres of maternally derived antibodies at the time of infection but was significantly (p < 0.05) related to PRRSV infection status. Notably, both before and after the PRRSV outbreak, a high proportion of IAV-infected piglets did not seroconvert, which was significantly (p < 0.05) related to the hemagglutination inhibition titres against IAV when infected., (© 2024. The Author(s).)

Despite the efforts of practical medicine and virology, influenza viruses remain the most important pathogens affecting human and animal health. Swine are exposed to infection with all types of influenza A, B, C, and D viruses. Influenza viruses have low pathogenicity for swine, but in the case of co-infection with other pathogens, the outcome can be much more serious, even fatal. Having a high zoonotic potential, swine play an important role in the ecology and spread of influenza to humans. In this study, we review the state of the scientific literature on the zoonotic spread of swine influenza A viruses among humans, their circulation in swine populations worldwide, reverse zoonosis from humans to swine, and their role in interspecies transmission. The analysis covers a long period to trace the ecology and evolutionary history of influenza A viruses in swine. The following databases were used to search the literature: Scopus, Web of Science, Google Scholar, and PubMed. In this review, 314 papers are considered: n = 107 from Asia, n = 93 from the U.S., n = 86 from Europe, n = 20 from Africa, and n = 8 from Australia. According to the date of publication, they are conditionally divided into three groups: contemporary, released from 2011 to the present (n = 121); 2000-2010 (n = 108); and 1919-1999 (n = 85).

The rapid geographic spread of the highly pathogenic avian influenza (HPAI) A(H5N1) virus in poultry, wild birds, and other mammalian hosts, including humans, raises significant health concerns globally. The recent emergence of HPAI A(H5N1) in agricultural animals such as cattle and goats indicates the ability of the virus to breach unconventional host interfaces, further expanding the host range. Among the four influenza types-A, B, C, and D, cattle are most susceptible to influenza D infection and serve as a reservoir for this seven-segmented influenza virus. It is generally thought that bovines are not hosts for other types of influenza viruses, including type A. However, this long-standing viewpoint has been challenged by the recent outbreaks of HPAI A(H5N1) in dairy cows in the United States. To date, HPAI A(H5N1) has spread into fourteen states, affecting 299 dairy herds and causing clinical symptoms such as reduced appetite, fever, and a sudden drop in milk production. Infected cows can also transmit the disease through raw milk. This review article describes the current epidemiological landscape of HPAI A(H5N1) in US dairy cows and its interspecies transmission events in other mammalian hosts reported across the globe. The review also discusses the viral determinants of tropism, host range, adaptative mutations of HPAI A(H5N1) in various mammalian hosts with natural and experimental infections, and vaccination strategies. Finally, it summarizes some immediate questions that need to be addressed for a better understanding of the infection biology, transmission, and immune response of HPAI A(H5N1) in bovines.

BackgroundHighly pathogenic avian influenza (HPAI) H5Nx and human H1N1pdm2009 influenza viruses can infect cats. Infections in cats may result in viral adaptations or recombinant viruses, which may facilitate zoonotic transfer.AimWe aimed to investigate the presence of HPAI H5 clade 2.3.4.4 and H1 influenza viruses and antibodies to these viruses in domestic and rural stray cats in the Netherlands and factors associated with exposure.MethodsSera from stray and domestic cats, sampled 2020-2023, were analysed by ELISA and confirmed by hemagglutination inhibition assay (HAI) and pharyngeal swabs and lung tissue for influenza A virus by RT-qPCR.ResultsIn 701 stray cats, 83 (11.8%; 95% confidence interval (CI): 9.5-14.5) sera were positive for HPAI H5 and 65 findings were confirmed. In HAI, two sera were positive for both HPAI H5 and H1. In 871 domestic cats, four (0.46%; 95% CI: 0.13-1.2) sera were HPAI H5 positive and none were confirmed but 40 (4.6%; 95% CI: 3.3-6.2) sera were seropositive for H1 and 26 were confirmed. Stray cats living in nature reserves (odds ratio (OR) = 5.4; 95% CI: 1.5-20.1) and older cats (OR = 3.8; 95% CI: 2.7-7.1) were more likely to be HPAI H5 seropositive. No influenza A virus was detected in 230 cats.ConclusionThe higher HPAI H5 seroprevalence in stray cats compared with domestic cats suggests more frequent viral exposure, most likely due to foraging on wild birds. In contrast, exposure to H1 was more common in domestic cats compared with stray cats.

The prevalence of highly pathogenic avian influenza (HPAI) A(H5N1) viruses has increased in wild birds and poultry worldwide, and concomitant outbreaks in mammals have occurred. During 2023, outbreaks of HPAI H5N1 virus infections were reported in cats in South Korea. The H5N1 clade 2.3.4.4b viruses isolated from 2 cats harbored mutations in the polymerase basic protein 2 gene encoding single amino acid substitutions E627K or D701N, which are associated with virus adaptation in mammals. Hence, we analyzed the pathogenicity and transmission of the cat-derived H5N1 viruses in other mammals. Both isolates caused fatal infections in mice and ferrets. We observed contact infections between ferrets, confirming the viruses had high pathogenicity and transmission in mammals. Most HPAI H5N1 virus infections in humans have occurred through direct contact with poultry or a contaminated environment. Therefore, One Health surveillance of mammals, wild birds, and poultry is needed to prevent potential zoonotic threats.

Highly pathogenic avian influenza A(H5N1) virus was detected in dead seals on Tyuleniy Island in eastern Russia, in the Sea of Okhotsk. Viruses isolated from dead northern fur seals belong to clade 2.3.4.4b and are closely related to viruses detected predominantly in the Russian Far East and Japan in 2022-2023.

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 documented 5-12 . 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., (© 2024. The Author(s).)

Relevance: Influenza A virus is characterized by a segmented single-stranded RNA genome. Such organization of the virus genome determines the possibility of reassortment, which can lead to the emergence of new virus variants. The main natural reservoir of most influenza A virus subtypes are wild waterfowl. Seasonal migrations gather waterfowl from all major migration routes to nesting areas near the northern and southern polar circles. This makes intercontinental spread of influenza A viruses possible. Objective ‒ to conduct molecular genetic monitoring and study the phylogenetic relationships of influenza A virus variants circulating in Antarctica in 2023., Materials and Methods: We studied 84 samples of biological material obtained from birds and marine mammals in April‒May 2023 in coastal areas of Antarctica. For 3 samples, sequencing was performed on the Miseq, Illumina platform and phylogenetic analysis of the obtained nucleotide sequences of the influenza A virus genomes was performed., Results: The circulation of avian influenza virus in the Antarctic region was confirmed. Heterogeneity of the pool of circulating variants of the influenza A virus (H3N8, H1N1) was revealed. Full-length genomes of the avian influenza virus were sequenced and posted in the GISAID database (EPI&#95;ISL&#95;19032103, 19174530, 19174467)., Conclusion: The study of the genetic diversity of influenza A viruses circulating in the polar regions of the Earth and the identification of the conditions for the emergence of new genetic variants is a relevant task for the development of measures to prevent biological threats.

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 risk 1-4 . 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., (© 2024. The Author(s).)

The emergence and spread of highly pathogenic avian influenza virus A subtype H5N1 (HP H5N1-IAV), particularly clade H5N1 2.3.4.4b, pose a severe global health threat, affecting various species, including mammals. Historically, cattle have been considered less susceptible to IAV, but recent outbreaks of H5N1-IAV 2.3.4.4b in dairy farms suggest a shift in host tropism, underscoring the urgency of expanded surveillance and the need for adaptable diagnostic tools in outbreak management. This study investigated the presence of anti-nucleoprotein (NP) antibodies in serum and milk and viral RNA in milk on dairy farms affected by outbreaks in Texas, Kansas, and Michigan using a multi-species IAV ELISA and RT-qPCR. The analysis of ELISA results from a Michigan dairy farm outbreak demonstrated a positive correlation between paired serum and milk sample results, confirming the reliability of both specimen types. Our findings also revealed high diagnostic performance during the convalescent phase (up to 96%), further improving sensitivity through serial sampling. Additionally, the evaluation of diagnostic specificity using serum and milk samples from IAV-free farms showed an excellent performance (99.6%). This study underscores the efficacy of the IAV NP-blocking ELISA for detecting and monitoring H5N1-IAV 2.3.4.4b circulation in dairy farms, whose recent emergence raises significant animal welfare and zoonotic concerns, necessitating expanded surveillance efforts.

Background: Dogs in close contact with humans can serve as a source of potentially dangerous reassortant influenza viruses (IVs) with zoonotic potential. The dog's body can serve as a vessel for the emergence of new IVs. These new viruses can become a source of infection for other animals and humans. The potential for zoonotic transmission of IVs from dogs to humans poses a public health risk., Aim: Study of the circulation of IVs in the dog population in Almaty, Kazakhstan., Methods: Biosamples (oropharyngeal swabs and blood serum) from dogs were collected from veterinary clinics in Almaty in 2023-2024. Samples were screened using RT-PCR, HI assay, and ELISA., Results: RT-PCR analysis of 355 nasopharyngeal swabs showed the presence of influenza A virus (IAV) in 32 samples (9.01% of the total number of samples analyzed). When subtyping IAV H1N1 RNA was detected in 19 swabs (5.35%). IAV subtype could not be determined in 13 PCR-positive samples (3.66%). The genetic material of IAV H3N2, H5, H7, and H9, as well as coronavirus, bocavirus, and adenovirus has not been identified. In a serological analysis of 180 blood sera using ELISA, antibodies to IAV were detected in 5.56% ( n = 10). The results of the HI assay showed the presence of antihemagglutinins to A/H1N1pdm in 6.11% (11 samples), to A/H3N2 in 9.44% (17 samples), and no antibodies to IAV H5, H7, and type B were detected., Conclusion: There is no information about human infection with any canine influenza virus. However, many cases of infection in dogs with human IAVs H1N1, H1N1pdm09, and H3N2 have been described. When dogs are co-infected with different IAVs, new recombinant IAVs may emerge that can infect humans and other animals. Therefore, ongoing global surveillance of animal populations is necessary to monitor the evolution and circulation of viruses dangerous to public health. This is also important for timely preparation for the emergence of a new zoonotic influenza virus that has pandemic potential for humans., Competing Interests: The authors declare that there is no conflict of interest.

Viruses that infect animals regularly spill over into the human population, but individual events may lead to anything from a single case to a novel pandemic. Rapidly gaining an understanding of a spillover event is critical to calibrating a public health response. We here propose a novel method, using likelihood-free rejection sampling, to evaluate the properties of an outbreak of swine-origin influenza A(H1N2)v in the United Kingdom, detected in November 2023. From the limited data available, we generate historical estimates of the probability that the outbreak had died out in the days following the detection of the first case. Our method suggests that the outbreak could have been said to be over with 95% certainty between 19 and 29 days after the first case was detected, depending upon the probability of a case being detected. We further estimate the number of undetected cases conditional upon the outbreak still being live, the epidemiological parameter R 0 , and the date on which the spillover event itself occurred. Our method requires minimal data to be effective. While our calculations were performed after the event, the real-time application of our method has potential value for public health responses to cases of emerging viral infection.

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., (© 2024. The Author(s).)

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

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., (© 2024. The Author(s).)

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.

We detected antibodies to H5 and N1 subtype influenza A viruses in 4/194 (2%) dogs from Washington, USA, that hunted or engaged in hunt tests and training with wild birds. Historical data provided by dog owners showed seropositive dogs had high levels of exposure to waterfowl.

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.

Background: Influenza A virus infections can occur in multiple species. Eurasian avian-like swine influenza A (H1N1) viruses (EAS-H1N1) are predominant in swine and occasionally infect humans. A Eurasian avian-like swine influenza A (H1N1) virus was isolated from a boy who was suffering from fever; this strain was designated A/Shandong-binzhou/01/2021 (H1N1). The aims of this study were to investigate the characteristics of this virus and to draw attention to the need for surveillance of influenza virus infection in swine and humans., Methods: Throat-swab specimens were collected and subjected to real-time fluorescent quantitative polymerase chain reaction (RT‒PCR). Positive clinical specimens were inoculated onto Madin-Darby canine kidney (MDCK) cells to isolate the virus, which was confirmed by a haemagglutination assay. Then, whole-genome sequencing was carried out using an Illumina MiSeq platform, and phylogenetic analysis was performed with MEGA X software., Results: RT‒PCR revealed that the throat-swab specimens were positive for EAS-H1N1, and the virus was subsequently successfully isolated from MDCK cells; this strain was named A/Shandong-binzhou/01/2021 (H1N1). Whole-genome sequencing and phylogenetic analysis revealed that A/Shandong-binzhou/01/2021 (H1N1) is a novel triple-reassortant EAS-H1N1 lineage that contains gene segments from EAS-H1N1 (HA and NA), triple-reassortant swine influenza H1N2 virus (NS) and A(H1N1) pdm09 viruses (PB2, PB1, PA, NP and MP)., Conclusions: The isolation and analysis of the A/Shandong-binzhou/01/2021 (H1N1) virus provide further evidence that EAS-H1N1 poses a threat to human health, and greater attention should be given to the surveillance of influenza virus infections in swine and humans., (© 2024. The Author(s).)

Equine influenza is a viral disease caused by the equine influenza virus (EIV), and according to the WOAH, it is mandatory to report these infections. In Latin America and Colombia, EIV risk factors have not been analyzed. The objective of this research is to perform an epidemiological and molecular analysis of the EIV in horses with respiratory symptoms from 2020 to 2023 in Colombia. Molecular EIV detection was performed using RT-qPCR and nanopore sequencing. A risk analysis was also performed via the GEE method. A total of 188 equines with EIV respiratory symptoms were recruited. The positivity rate was 33.5%. The descriptive analysis showed that only 12.8% of the horses were vaccinated, and measures such as the quarantine and isolation of symptomatic animals accounted for 91.5% and 88.8%, respectively. The variables associated with the EIV were the non-isolation of positive individuals (OR = 8.16, 95% CI (1.52-43.67), p = 0.014) and sharing space with poultry (OR = 2.16, 95% CI (1.09-4.26), p = 0.027). In conclusion, this is the first EIV investigation in symptomatic horses in Colombia, highlighting the presence of the virus in the country and the need to improve preventive and control measures.

Dogs are known to be susceptible to influenza A viruses, although information on influenza D virus (IDV) is limited. We investigated the seroprevalence of IDV in 426 dogs in the Apulia region of Italy during 2016 and 2023. A total of 14 samples were positive for IDV antibodies, suggesting exposure to IDV in dogs.

We describe the evolution of the outbreak of Highly Pathogenic Avian Influenza (HPAI) A(H5N1) in sea lions (Otaria flavescens) of South America. At least 24,000 sea lions died in Peru, Chile, Argentina, Uruguay, and Brazil between January-October 2023. The most plausible route of infection is cohabiting with or foraging on infected birds. However, we urge a detailed evaluation of the sea lions actual source of infection given that the concomitant massive wild bird mortalities registered in the Pacific Ocean did not occur in the Atlantic Ocean., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this commentary., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inactivated influenza A virus (IAV) vaccines help reduce clinical disease in suckling piglets, although endemic infections still exist. The objective of this study was to evaluate the detection of IAV in suckling and nursery piglets from IAV-vaccinated sows from farms with endemic IAV infections. Eight nasal swab collections were obtained from 135 two-week-old suckling piglets from four farms every other week from March to September 2013. Oral fluid samples were collected from the same group of nursery piglets. IAV RNA was detected in 1.64% and 31.01% of individual nasal swabs and oral fluids, respectively. H1N2 was detected most often, with sporadic detection of H1N1 and H3N2. Whole-genome sequences of IAV isolated from suckling piglets revealed an H1 hemagglutinin (HA) from the 1B.2.2.2 clade and N2 neuraminidase (NA) from the 2002A clade. The internal gene constellation of the endemic H1N2 was TTTTPT with a pandemic lineage matrix. The HA gene had 97.59% and 97.52% nucleotide and amino acid identities, respectively, to the H1 1B.2.2.2 used in the farm-specific vaccine. A similar H1 1B.2.2.2 was detected in the downstream nursery. These data demonstrate the low frequency of IAV detection in suckling piglets and downstream nurseries from farms with endemic infections in spite of using farm-specific IAV vaccines in sows.

Highly pathogenic avian influenza (HPAI) has persisted as a One Health threat whose current circulation and impact are addressed in the companion Currents in One Health by Puryear and Runstadler, JAVMA, May 2024. Highly pathogenic avian influenza emerged as a by-product of agricultural practices and adapted to endemic circulation in wild bird species. Over more than 20 years, continued evolution in a complex ecology involving multiple hosts has produced a lineage that expanded globally over the last 2 years. Understanding the continued evolution and movement of HPAI relies on understanding how the virus is infecting different hosts in different contexts. This includes understanding the environmental factors and the natural ecology of viral transmission that impact host exposure and ultimately evolutionary trajectories. Particularly with the rapid host expansion, increased spillover to mammalian hosts, and novel clinical phenotypes in infected hosts, despite progress in understanding the impact of specific mutations to HPAI viruses that are associated with spillover potential, the threat to public health is poorly understood. Active research is focusing on new approaches to understanding the relationship of viral genotype to phenotype and the implementation of research and surveillance pipelines to make sense of the enormous potential for diverse HPAI viruses to emerge from wild reservoirs amid global circulation.

Novel H1N2 and H3N2 swine influenza A viruses (IAVs) have recently been identified in Chile. The objective of this study was to evaluate their zoonotic potential. We perform phylogenetic analyses to determine the genetic origin and evolution of these viruses, and a serological analysis to determine the level of cross-protective antibodies in the human population. Eight genotypes were identified, all with pandemic H1N1 2009-like internal genes. H1N1 and H1N2 were the subtypes more commonly detected. Swine H1N2 and H3N2 IAVs had hemagglutinin and neuraminidase lineages genetically divergent from IAVs reported worldwide, including human vaccine strains. These genes originated from human seasonal viruses were introduced into the swine population since the mid-1980s. Serological data indicate that the general population is susceptible to the H3N2 virus and that elderly and young children also lack protective antibodies against the H1N2 strains, suggesting that these viruses could be potential zoonotic threats. Continuous IAV surveillance and monitoring of the swine and human populations is strongly recommended.IMPORTANCEIn the global context, where swine serve as crucial intermediate hosts for influenza A viruses (IAVs), this study addresses the pressing concern of the zoonotic potential of novel reassortant strains. Conducted on a large scale in Chile, it presents a comprehensive account of swine influenza A virus diversity, covering 93.8% of the country's industrialized swine farms. The findings reveal eight distinct swine IAV genotypes, all carrying a complete internal gene cassette of pandemic H1N1 2009 origin, emphasizing potential increased replication and transmission fitness. Genetic divergence of H1N2 and H3N2 IAVs from globally reported strains raises alarms, with evidence suggesting introductions from human seasonal viruses since the mid-1980s. A detailed serological analysis underscores the zoonotic threat, indicating susceptibility in the general population to swine H3N2 and a lack of protective antibodies in vulnerable demographics. These data highlight the importance of continuous surveillance, providing crucial insights for global health organizations., Competing Interests: The authors declare no conflict of interest.

Interspecies transmission of influenza A viruses (IAV) from pigs to humans is a concerning event as porcine IAV represent a reservoir of potentially pandemic IAV. We conducted a comprehensive analysis of two porcine A(H1N1)v viruses isolated from human cases by evaluating their genetic, antigenic and virological characteristics. The HA genes of those human isolates belonged to clades 1C.2.1 and 1C.2.2, respectively, of the A(H1N1) Eurasian avian-like swine influenza lineage. Antigenic profiling revealed substantial cross-reactivity between the two zoonotic H1N1 viruses and human A(H1N1)pdm09 virus and some swine viruses, but did not reveal cross-reactivity to H1N2 and earlier human seasonal A(H1N1) viruses. The solid-phase direct receptor binding assay analysis of both A(H1N1)v showed a predominant binding to α2-6-sialylated glycans similar to human-adapted IAV. Investigation of the replicative potential revealed that both A(H1N1)v viruses grow in human bronchial epithelial cells to similar high titers as the human A(H1N1)pdm09 virus. Cytokine induction was studied in human alveolar epithelial cells A549 and showed that both swine viruses isolated from human cases induced higher amounts of type I and type III IFN, as well as IL6 compared to a seasonal A(H1N1) or a A(H1N1)pdm09 virus. In summary, we demonstrate a remarkable adaptation of both zoonotic viruses to propagate in human cells. Our data emphasize the needs for continuous monitoring of people and regions at increased risk of such trans-species transmissions, as well as systematic studies to quantify the frequency of these events and to identify viral molecular determinants enhancing the zoonotic potential of porcine IAV., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Transmissibility, the ability to spread within host populations, is a prerequisite for a pathogen to have epidemic or pandemic potential. Here, we estimate the phylogenies of human infectivity and transmissibility using 1,408 genome sequences from 743 distinct RNA virus species/types in 59 genera. By repeating this analysis using data sets censored by virus discovery date, we explore how temporal changes in the known diversity of RNA viruses-especially recent increases in recognized nonhuman viruses-have altered these phylogenies. Over time, we find significant increases in the proportion of RNA virus genera estimated to have a nonhuman-infective ancestral state, in the fraction of distinct human virus lineages that are purely human-transmissible or strictly zoonotic (compared to mixed lineages), and in the number of human viruses with nearest relatives known not to infect humans. Our results are consistent with viruses that are capable of spreading in human populations commonly emerging from a nonhuman reservoir. This is more likely in lineages that already contain human-transmissible viruses but is rare in lineages that contain only strictly zoonotic viruses., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Influenza D virus (IDV) infections have been observed in animals worldwide, confirmed through both serological and molecular tests, as well as virus isolation. IDV possesses unique properties that distinguish it from other influenza viruses, primarily attributed to the hemagglutinin-esterase fusion (HEF) surface glycoprotein, which determines the virus' tropism and wide host range. Cattle are postulated to be the reservoir of IDV, and the virus is identified as one of the causative agents of bovine respiratory disease (BRD) syndrome. Animals associated with humans and susceptible to IDV infection include camels, pigs, small ruminants, and horses. Notably, high seroprevalence towards IDV, apart from cattle, is also observed in camels, potentially constituting a reservoir of the virus. Among wild and captive animals, IDV infections have been confirmed in feral pigs, wild boars, deer, hedgehogs, giraffes, wildebeests, kangaroos, wallabies, and llamas. The transmission potential and host range of IDV may contribute to future viral differentiation. It has been confirmed that influenza D may pose a threat to humans as a zoonosis, with seroprevalence noted in people with professional contact with cattle.

Influenza A viruses (IAV) cause annual epidemics and occasional pandemics in humans. The most recent pandemic outbreak occurred in 2009 with H1N1pdm09. This virus, which most likely reassorted in swine before its transmission to humans, was reintroduced into the swine population and continues circulating ever since. In order to assess its potential to cause reassortants on a cellular level, human origin H1N1pdm09 and a recent Eurasian avian-like H1N1 swine IAV were (co-)passaged in the newly generated swine lung cell line C22. Co-infection with both viruses gave rise to numerous reassortants that additionally carry different mutations which can partially be found in nature as well. Reassortment most frequently affected the PB1, PA and NA segments with the swine IAV as recipient. These reassortants reached higher titers in swine lung cells and were able to replicate in genuine human lung tissue explants ex vivo, suggesting a possible zoonotic potential. Interestingly, reassortment and mutations in the viral ribonucleoprotein complex influence the viral polymerase activity in a cell type and species-specific manner. In summary, we demonstrate reassortment promiscuity of these viruses in a novel swine lung cell model and indicate a possible zoonotic potential of the reassortants.

Respiratory disease is a significant economic issue in pig farming, with a complex aetiology that includes swine influenza A viruses (swIAV), which are common in European domestic pig populations. The most recent human influenza pandemic in 2009 showed swIAV's zoonotic potential. Monitoring pathogens and disease control are critical from a preventive standpoint, and are based on quick, sensitive, and specific diagnostic assays capable of detecting and distinguishing currently circulating swIAV in clinical samples. For passive surveillance, a set of multiplex quantitative reverse transcription real-time PCRs (mRT-qPCR) and MinION-directed sequencing was updated and deployed. Several lineages and genotypes of swIAV were shown to be dynamically developing, including novel reassortants between human pandemic H1N1 and the avian-derived H1 lineage of swIAV. Despite this, nearly 70% (842/1216) of individual samples from pigs with respiratory symptoms were swIAV-negative, hinting to different aetiologies. The complex and synergistic interactions of swIAV infections with other viral and bacterial infectious agents contribute to the aggravation of pig respiratory diseases. Using a newly developed mRT-qPCR for the combined detection of swIAV and the recently described porcine respirovirus 1 (PRV1) and swine orthopneumovirus (SOV) widespread co-circulation of PRV1 (19.6%, 238/1216 samples) and SOV (14.2%, 173/1216 samples) was evident. Because of the high incidence of PRV1 and SOV infections in pigs with respiratory disease, these viruses may emerge as new allies in the porcine respiratory disease syndrome.

During an investigation in October 2018, two people with diarrhoea, mild abdominal pain, and mild arthralgia symptoms in Guangxi, China, were identified as infected by H9N2 avian influenza virus (AIV). Four H9N2 AIVs were isolated from one of two patients, a pet cat, and a dead chicken (two respective isolates from its lung and kidney tissues) bred by the patients at a backyard farm. Epidemiological investigation indicated that the newly bought chicken died first, and clinical syndromes appeared subsequently in the two owners and one cat. Furthermore, the two individuals possessed high H9N2-specific hemagglutination inhibition and microneutralization antibodies. Shared nucleotide sequence identity (99.9% - 100%) for all genes was detected in the four H9N2 isolates, and hemagglutinin (HA) T138A located on the receptor binding domain (RBD), resulted from nucleotide polymorphisms that were exclusively found in the isolate from the female patient. Moreover, HA K137N on the RBD was found in isolates from these three host species. Importantly, these four H9N2 isolates presented an exclusive binding preference for the human-type receptor (α2-6-SA), and could replicate and cause pathological changes in mice. Phylogenetic analyses showed that these four isolates clustered together and belonged to clade C1.2, lineage Y280. In addition, H9N2 viruses of human origin are genetically divergent and interspersed with the widespread poultry-origin H9N2 AIVs. All these results indicate a high risk of H9N2 AIVs in public health, and effective prevention and control measures against H9N2 AIVs should be considered and performed for both animal and human health.

Competing Interests: Sajid Umar: Formal analysis; investigation; methodology. Mai‐Juan Ma: Conceptualization; data curation; funding acquisition; methodology; project administration; validation; writing—review and editing. Gregory C. Gray: Conceptualization; data curation; funding acquisition; methodology; project administration; supervision; validation; visualization; writing—review and editing.The authors report no conflicts of interest.

Avian-origin H3N2 canine influenza A viruses (CIVs) have become enzootic in China and Korea and have sporadically transmitted to North America, causing multiple epidemics. We isolated six CIVs in Korea from CIV-infected patients during 2014-2017 and conducted whole genome sequencing and phylogenetic analyses. Results revealed that CIVs have circulated and evolved in Korea since the early 2000s and then diversified into a new clade, probably contributing to multiple epidemics in China, the USA, and Canada. Our findings bridge an evolutionary gap for understanding the global transmission of CIVs, emphasizing the significance of continuous monitoring of CIVs., Competing Interests: The author declares no conflicts of interest., (© 2023 The Korean Society of Veterinary Science.)

Equine influenza virus (EIV) causes acute respiratory disease in horses and belongs to the influenza A virus family Orthomyxoviridae , genus Orthomyxovirus . This virus may have severe financial implications for the horse industry owing to its highly contagious nature and rapid transmission. In the Republic of Korea, vaccination against EIV has been practiced with the active involvement of the Korea Racing Authority since 1974. In this study, we monitored the viral RNA for EIV using PCR, as well as the antibody levels against 'A/equine/South Africa/4/03 (H3N8, clade 1)', from 2020 to 2022. EIV was not detected using RT-PCR. The seropositivity rates detected using a hemagglutination inhibition assay were 90.3% in 2020, 96.7% in 2021, and 91.8% in 2022. The geometric mean of antibody titer (GMT) was 83.4 in 2020, 135.7 in 2021, and 95.6 in 2022. Yearlings and two-year-olds in training exhibited lower positive rates (59.1% in 2020, 38.9% in 2021, and 44.1% in 2022) than the average. These younger horses may require more attention for vaccination and vaccine responses against EIV. Continuous surveillance of EIV should be performed to monitor the prevalence and spread of this disease.

Bovine respiratory disease (BRD) complex is a multifactorial respiratory disease of cattle. Seven-segmented influenza C (ICV) and D (IDV) viruses have been identified in cattle with BRD, however, molecular epidemiology and prevalence of IDV and ICV in the diseased population remain poorly characterized. Here, we conducted a molecular screening of 208 lung samples of bovine pneumonia cases for the presence of IDV and ICV. Our results demonstrated that both viruses were prevalent in BRD cases and the overall positivity rates of IDV and ICV were 20.88% and 5.99% respectively. Further analysis of three IDV strains isolated from lungs of cattle with BRD showed that these lung-tropic strains belonged to D/Michigan/2019 clade and diverged antigenically from the circulating dominant IDV clades D/OK and D/660. Our results reveal that IDV and ICV are associated with BRD complex and support a role for IDV and ICV in the etiology of BRD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Swine influenza is a respiratory disease that affects the pork industry and is a public health threat. It is caused by type A influenza virus (FLUAV), which continuously undergoes genetic and antigenic variations. A large amount of information regarding FLUAV in pigs is available worldwide, but it is limited in Latin America. The HA sequences of H1 subtype FLUAV-positive samples obtained from pigs in Colombia between 2008-2021 were analyzed using sequence-based antigenic cartography and N-Glycosylation analyses. Of the 12 predicted global antigenic groups, Colombia contained five: four corresponding to pandemic strains and one to the classical swine H1N1 clade. Circulation of these clusters was observed in some regions during specific years. Ca2 was the immunodominant epitope among Colombian viruses. The counts of N-Glycosylation motifs were associated with the antigenic cluster ranging from three to five. The results show for the first time the existence of antigenic diversity of FLUAV in Colombia and highlight the impact of spatial and temporal factors on this diversity. This study provides information about FLUAV variability in pigs under natural conditions in the absence of vaccination and emphasizes the need for surveillance of its phylogenetic and antigenic characteristics.

Influenza A viruses (IAV) are still a cause of concern for public health and veterinary services worldwide. With (-) RNA-segmented genome architecture, influenza viruses are prone to reassortment and can generate a great variety of strains, some capable of crossing interspecies barriers. Seasonal IAV strains continuously spread from humans to pigs, leading to multiple reassortation events with strains endemic to swine. Due to its high adaptability to humans, a reassortant strain based on "human-like" genes could potentially be a carrier of avian origin segments responsible for high virulence, and hence become the next pandemic strain with unseen pathogenicity. The rapid evolution of sequencing methods has provided a fast and cost-efficient way to assess the genetic diversity of IAV. In this study, we investigated the genetic diversity of swine influenza viruses (swIAVs) collected from Polish farms. A total of 376 samples were collected from 11 farms. The infection was confirmed in 112 cases. The isolates were subjected to next-generation sequencing (NGS), resulting in 93 full genome sequences. Phylogenetic analysis classified 59 isolates as genotype T (H1avN2g) and 34 isolates as genotype P (H1pdmN1pdm), all of which had an internal gene cassette (IGC) derived from the H1N1pdm09-like strain. These data are consistent with evolutionary trends in European swIAVs. The applied methodology proved to be useful in monitoring the genetic diversity of IAV at the human-animal interface.

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.