Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia.

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections. The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

The episodic resurgence of highly pathogenic avian influenza H5 virus.

Highly pathogenic avian influenza (HPAI) H5N1 activity has intensified globally since 2021, increasingly causing mass mortality in wild birds and poultry and incidental infections in mammals1-3. However, the ecological and virological properties that underscore future mitigation strategies still remain unclear. Using epidemiological, spatial and genomic approaches, we demonstrate changes in the origins of resurgent HPAI H5 and reveal significant shifts in virus ecology and evolution. Outbreak data show key resurgent events in 2016-2017 and 2020-2021, contributing to the emergence and panzootic spread of H5N1 in 2021-2022. Genomic analysis reveals that the 2016-2017 epizootics originated in Asia, where HPAI H5 reservoirs are endemic. In 2020-2021, 2.3.4.4b H5N8 viruses emerged in African poultry, featuring mutations altering HA structure and receptor binding. In 2021-2022, a new H5N1 virus evolved through reassortment in wild birds in Europe, undergoing further reassortment with low-pathogenic avian influenza in wild and domestic birds during global dissemination. These results highlight a shift in the HPAI H5 epicentre beyond Asia and indicate that increasing persistence of HPAI H5 in wild birds is facilitating geographic and host range expansion, accelerating dispersion velocity and increasing reassortment potential. As earlier outbreaks of H5N1 and H5N8 were caused by more stable genomic constellations, these recent changes reflect adaptation across the domestic-bird-wild-bird interface. Elimination strategies in domestic birds therefore remain a high priority to limit future epizootics.

Global distribution, receptor binding, and cross-species transmission of H6 influenza viruses: risks and implications for humans.

The H6 subtype of avian influenza virus (AIV) is a pervasive subtype that is ubiquitously found in both wild bird and poultry populations across the globe. Recent investigations have unveiled its capacity to infect mammals, thereby expanding its host range beyond that of other subtypes and potentially facilitating its global transmission. This heightened breadth also endows H6 AIVs with the potential to serve as a genetic reservoir for the emergence of highly pathogenic avian influenza strains through genetic reassortment and adaptive mutations. Furthermore, alterations in key amino acid loci within the H6 AIV genome foster the evolution of viral infection mechanisms, which may enable the virus to surmount interspecies barriers and infect mammals, including humans, thus posing a potential threat to human well-being. In this review, we summarize the origins, dissemination patterns, geographical distribution, cross-species transmission dynamics, and genetic attributes of H6 influenza viruses. This study holds implications for the timely detection and surveillance of H6 AIVs.

Continued Circulation of Highly Pathogenic H5 Influenza Viruses in Vietnamese Live Bird Markets in 2018-2021.

We isolated 77 highly pathogenic avian influenza viruses during routine surveillance in live poultry markets in northern provinces of Vietnam from 2018 to 2021. These viruses are of the H5N6 subtype and belong to HA clades 2.3.4.4g and 2.3.4.4h. Interestingly, we did not detect viruses of clade 2.3.4.4b, which in recent years have dominated in different parts of the world. The viruses isolated in this current study do not encode major determinants of mammalian adaptation (e.g., PB2-E627K or PB1-D701N) but possess amino acid substitutions that may affect viral receptor-binding, replication, or the responses to human antiviral factors. Several of the highly pathogenic H5N6 virus samples contained other influenza viruses, providing an opportunity for reassortment. Collectively, our study demonstrates that the highly pathogenic H5 viruses circulating in Vietnam in 2018-2021 were different from those in other parts of the world, and that the Vietnamese H5 viruses continue to evolve through mutations and reassortment.

Highly Pathogenic H5 Influenza Viruses Isolated between 2016 and 2017 in Vietnamese Live Bird Markets.

Routine surveillance in live poultry markets in the northern regions of Vietnam from 2016 to 2017 resulted in the isolation of 27 highly pathogenic avian H5N1 and H5N6 viruses of 3 different clades (2.3.2.1c, 2.3.4.4f, and 2.3.4.4g). Sequence and phylogenetic analysis of these viruses revealed reassortment with various subtypes of low pathogenic avian influenza viruses. Deep-sequencing identified minor viral subpopulations encoding variants that may affect pathogenicity and sensitivity to antiviral drugs. Interestingly, mice infected with two different clade 2.3.2.1c viruses lost body weight rapidly and succumbed to virus infection, whereas mice infected with clade 2.3.4.4f or 2.3.4.4g viruses experienced non-lethal infections.

Surveillance of Class I Newcastle Disease Virus at Live Bird Markets in China and Identification of Variants with Increased Virulence and Replication Capacity.

In this study, 232 class I Newcastle disease viruses (NDVs) were identified from multiple bird species at nationwide live bird markets (LBMs) from 2017 to 2019 in China. Phylogenetic analysis indicated that all 232 isolates were clustered into genotype 1.1.2 of class I on the basis of the fusion (F) gene sequences, which were distinct from the genotypes identified in other countries. Most of the isolates (212/232) were shown to have the typical F gene molecular characteristics of class I NDVs, while a few (20/232) contained mutations at the site of the conventional start codon of the F gene, which resulted in open reading frames (ORFs) altered in length. The isolates with ACG, CTA, and ATA mutations showed different levels of increased virulence and replication capacity, suggesting that these viruses may be transitional types during the evolution of class I NDVs from avirulent to virulent. Further evaluation of biological characteristics with recombinant viruses obtained by reverse genetics demonstrated that the ATG located at genomic positions 4523 to 4525 was the authentic start codon in the F gene of class I NDV, and the specific ATA mutations which contributed to the expression of F protein on the surface of infected cells were the key determinants of increased replication capacity and virulence. Interestingly, the mutation at the corresponding site of genotype II LaSota of class II had no effects on the virulence and replication capacity in chickens. Our results suggest that the alteration of virulence and replication capacity caused by specific mutations in the F gene could be a specific characteristic of class I NDVs and indicate the possibility of the emergence of virulent NDVs due to the persistent circulation of class I NDVs. IMPORTANCE The available information on the distribution, genetic diversity, evolution, and biological characteristics of class I Newcastle disease viruses (NDVs) in domestic poultry is currently very limited. Here, identification of class I NDVs at nationwide live bird markets (LBMs) in China was performed and representative isolates were characterized. A widespread distribution of genotype 1.1.2 of class I NDVs was found in multiple bird species at LBMs in China. Though most isolates demonstrated typical molecular characteristics of class I NDVs, a few that contained specific mutations at the site of the conventional start codon of the fusion gene with increased virulence and replication capacity were identified for the first time. Our findings indicate that the virulence of class I NDVs could have evolved, and the widespread transmission and circulation of class I NDVs may represent a potential threat for disease outbreaks in poultry.

Phylogenetic and genetic characterization of influenza A H9N2 viruses isolated from backyard poultry in selected farms in Ghana.

INTRODUCTION: Avian influenza viruses (AIV) cause significant economic losses to poultry farmers worldwide. These viruses have the ability to spread rapidly, infect entire poultry flocks, and can pose a threat to human health. The National Influenza Centre (NIC) at the Noguchi Memorial Institute for Medical Research in collaboration with the Ghana Armed forces (GAF) and the U.S. Naval Medical Research Unit No. 3, Ghana Detachment (NAMRU-3) performs biannual surveillance for influenza viruses among poultry at military barracks throughout Ghana. This study presents poultry surveillance data from the years 2017 to 2019. METHODOLOGY: Tracheal and cloacal swabs from sick and healthy poultry were collected from the backyards of GAF personnel living quarters and transported at 4°C to the NIC. Viral ribonucleic acid (RNA) was isolated and analyzed for the presence of influenza viruses using real-time polymerase chain reaction (PCR) assays. Viral nucleic acids extracted from influenza A-positive specimens were sequenced using universal influenza A-specific primers. RESULTS: Influenza A H9N2 virus was detected in 11 avian species out of 2000 samples tested. Phylogenetic analysis of viral haemagglutinin (HA) protein confirms the possibility of importation of viruses from North Africa and Burkina Faso. Although the detected viruses possess molecular markers of virulence and mammalian host adaptation, the HA cleavage site anlaysis confirmed low pathogenicity of the viruses. CONCLUSIONS: These findings confirm the ongoing spread of H9 viruses among poultry in Ghana. Poultry farmers need to be vigilant for sick birds and take the appropriate public health steps to limit the spread to other animals and spillover to humans.

Bridging the Local Persistence and Long-Range Dispersal of Highly Pathogenic Avian Influenza Virus (HPAIv): A Case Study of HPAIv-Infected Sedentary and Migratory Wildfowls Inhabiting Infected Premises.

The past two decades have seen the emergence of highly pathogenic avian influenza (HPAI) infections that are characterized as extremely contagious, with a high fatality rate in chickens, and humans; this has sparked considerable concerns for global health. Generally, the new variant of the HPAI virus crossed into various countries through wild bird migration, and persisted in the local environment through the interactions between wild and farmed birds. Nevertheless, no studies have found informative cases associated with connecting local persistence and long-range dispersal. During the 2016-2017 HPAI H5N6 epidemic in South Korea, we observed several waterfowls with avian influenza infection under telemetric monitoring. Based on the telemetry records and surveillance data, we conducted a case study to test hypotheses related to the transmission pathway between wild birds and poultry. One sedentary wildfowl naturally infected with HPAI H5N6, which overlapped with the home range of one migratory bird with H5-specific antibody-positive, showed itself to be phylogenetically close to the isolates from a chicken farm located within its habitat. Our study is the first observational study that provides scientific evidence supporting the hypothesis that the HPAI spillover into poultry farms is caused by local persistence in sedentary birds, in addition to its long-range dispersal by sympatric migratory birds.

Strength in numbers: Avian influenza A virus transmission to poultry from a flocking passerine.

The effects of flock size of European starlings (Sturnus vulgaris) was experimentally manipulated to assess the potential of influenza A virus (IAV; H4N6) transmission from a flocking passerine to bobwhite quail (Colinus virginianus) through shared food and water resources to mimic starling intrusions into free-range and backyard poultry operations. Of the three starling flock sizes tested (n = 30, n = 20 and n = 10), all successfully transmitted the virus to all or most of the quail in each animal room (6/6, 6/6 and 5/6) by the end of the experimental period, as determined by seroconversion and/or viral RNA shedding. Although starlings have been shown to be inconsistent shedders of IAVs and when they do replicate and subsequently shed virus they typically do so at low to moderate levels, this study has provided evidence that relatively small flocks (i.e., 10 or possibly a smaller number) of this species can collectively transmit the virus to a highly susceptible gallinaceous bird species. Future work should assess if starlings can transmit IAVs to additional poultry species commonly found in backyard or free-range settings.

Emergence, prevalence, and evolution of H5N8 avian influenza viruses in central China, 2020.

Highly pathogenic influenza A(H5N8) viruses have caused several worldwide outbreaks in birds and are able cross the species barrier to infect humans, posing a substantial threat to public health. After the first detection of H5N8 viruses in deceased swans in Inner Mongolia, we performed early warning and active monitoring along swan migration routes in central China. We isolated and sequenced 42 avian influenza viruses, including 40 H5N8 viruses, 1 H5N2 virus, and 1 H9N2 virus, in central China. Our H5N8 viruses isolated in swan stopover sites and wintering grounds showed high nucleotide homologies in the whole genome, revealing a common evolutionary source. Phylogenetic analysis revealed that the H5 viruses of clade 2.3.4.4b prevalent in 2020 have further diverged into two sub-clades: b1 and b2. The phylogeographic analysis also showed that the viruses of sub-clade b2 most likely originated from poultry in Russia. Notably, whooper swans were found to be responsible for the introduction of sub-clade b2 viruses in central China; whooper and tundra swans play a role in viral spread in the Yellow River Basin and the Yangtze River Basin, respectively. Our findings highlight swans as an indicator species for transborder spreading and monitoring of the H5N8 virus.

Dynamics of inter-farm transmission of highly pathogenic avian influenza H5N6 integrating vehicle movements and phylogenetic information.

Highly pathogenic avian influenza (HPAI) in poultry holdings commonly spreads through animal trade, and poultry production and health-associated vehicle (PPHaV) movement. To effectively control the spread of disease, it is essential that the contact structure via those movements among farms is thoroughly explored. However, few attempts have been made to scrutinize PPHaV movement compared to poultry trade. Therefore, our study aimed to elucidate the role of PPHaV movement on HPAI transmission. We performed network analysis using PPHaV movement data based on a global positioning system, with phylogenetic information of the isolates during the 2016-2017 HPAI H5N6 epidemic in the Republic of Korea. Moreover, the contribution of PPHaV movement to the spread of HPAI was estimated by Bayesian modeling. The network analysis revealed that there was the relationship between phylogenetic clusters and the contact network via PPHaV movement. Furthermore, the similarity of farm poultry species and the shared integrators between inter-linked infected premises (IPs) were associated with ties within the same phylogenetic clusters. Additionally, PPHaV movement among phylogenetically clustered IPs was estimated to contribute to approximately 30% of HPAI H5N6 infections in IPs on average. This study provides insight into how HPAI spread via PPHaV movement and scientific basis for control strategies.

Survey of low pathogenic avian influenza viruses in live poultry markets in Guangxi Province, Southern China, 2016-2019.

Low pathogenic avian influenza viruses (LPAIVs) have been widespread in poultry and wild birds throughout the world for many decades. LPAIV infections are usually asymptomatic or cause subclinical symptoms. However, the genetic reassortment of LPAIVs may generate novel viruses with increased virulence and cross-species transmission, posing potential risks to public health. To evaluate the epidemic potential and infection landscape of LPAIVs in Guangxi Province, China, we collected and analyzed throat and cloacal swab samples from chickens, ducks and geese from the live poultry markets on a regular basis from 2016 to 2019. Among the 7,567 samples, 974 (12.87%) were LPAIVs-positive, with 890 single and 84 mixed infections. Higher yearly isolation rates were observed in 2017 and 2018. Additionally, geese had the highest isolation rate, followed by ducks and chickens. Seasonally, spring had the highest isolation rate. Subtype H3, H4, H6 and H9 viruses were detected over prolonged periods, while H1 and H11 viruses were detected transiently. The predominant subtypes in chickens, ducks and geese were H9, H3, and H6, respectively. The 84 mixed infection samples contained 22 combinations. Most mixed infections involved two subtypes, with H3 + H4 as the most common combination. Our study provides important epidemiological data regarding the isolation rates, distributions of prevalent subtypes and mixed infections of LPAIVs. These results will improve our knowledge and ability to control epidemics, guide disease management strategies and provide early awareness of newly emerged AIV reassortants with pandemic potential.

Frequency and patterns of exposure to live poultry and the potential risk of avian influenza transmission to humans in urban Bangladesh.

Avian influenza is endemic in Bangladesh, where greater than 90% of poultry are marketed through live poultry markets (LPMs). We conducted a population-based cross-sectional mobile telephone survey in urban Dhaka, Bangladesh to investigate the frequency and patterns of human exposure to live poultry in LPMs and at home. Among 1047 urban residents surveyed, 74.2% (95% CI 70.9-77.2) reported exposure to live poultry in the past year, with the majority of exposure occurring on a weekly basis. While visiting LPMs was less common amongst females (40.3%, 95% CI 35.0-45.8) than males (58.9%, 95% CI 54.0-63.5), females reported greater poultry exposure through food preparation, including defeathering (13.2%, 95% CI 9.5-17.9) and eviscerating (14.8%, 95% CI 11.2-19.4) (p < 0.001). A large proportion of the urban population is frequently exposed to live poultry in a setting where avian influenza viruses are endemic in LPMs. There is thus not only ample opportunity for spillover of avian influenza infections into humans in Dhaka, Bangladesh, but also greater potential for viral reassortment which could generate novel strains with pandemic potential.

Two-stage algorithms for visually exploring spatio-temporal clustering of avian influenza virus outbreaks in poultry farms.

The development of visual tools for the timely identification of spatio-temporal clusters will assist in implementing control measures to prevent further damage. From January 2015 to June 2020, a total number of 1463 avian influenza outbreak farms were detected in Taiwan and further confirmed to be affected by highly pathogenic avian influenza subtype H5Nx. In this study, we adopted two common concepts of spatio-temporal clustering methods, the Knox test and scan statistics, with visual tools to explore the dynamic changes of clustering patterns. Since most (68.6%) of the outbreak farms were detected in 2015, only the data from 2015 was used in this study. The first two-stage algorithm performs the Knox test, which established a threshold of 7 days and identified 11 major clusters in the six counties of southwestern Taiwan, followed by the standard deviational ellipse (SDE) method implemented on each cluster to reveal the transmission direction. The second algorithm applies scan likelihood ratio statistics followed by AGC index to visualize the dynamic changes of the local aggregation pattern of disease clusters at the regional level. Compared to the one-stage aggregation approach, Knox-based and AGC mapping were more sensitive in small-scale spatio-temporal clustering.

A 10-Year Retrospective Study of Inclusion Body Hepatitis in Meat-Type Chickens in Spain (2011-2021).

A surge in fowl adenovirus (FAdV) causing inclusion body hepatitis (IBH) outbreaks has occurred in several countries in the last two decades. In Spain, a sharp increase in case numbers in broilers and broiler breeder pullets arose since 2011, which prompted the vaccination of breeders in some regions. Our retrospective study of IBH cases in Spain from 2011 to 2021 revealed that most cases were reported in broilers (92.21%) and were caused by serotypes FAdV-8b and -11, while cases in broiler breeder pullets were caused by serotypes FAdV-2, -11, and -8b. Vertical transmission was the main route of infection, although horizontal transmission likely happened in some broiler cases. Despite the inconsistent and heterogeneous use of vaccines among regions and over time, the number of cases mirrored the use of vaccines in the country. While IBH outbreaks were recorded year-long, significantly more cases occurred during the cooler and rainier months. The geographic distribution suggested a widespread incidence of IBH and revealed the importance of a highly integrated system. Our findings contribute to a better understanding of FAdV infection dynamics under field conditions and reiterate the importance of surveillance, serological monitoring of breeders, and vaccination of breeders against circulating serotypes to protect progenies.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018.

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia-Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China's H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.

Peering into Avian Influenza A(H5N8) for a Framework towards Pandemic Preparedness.

2014 marked the first emergence of avian influenza A(H5N8) in Jeonbuk Province, South Korea, which then quickly spread worldwide. In the midst of the 2020-2021 H5N8 outbreak, it spread to domestic poultry and wild waterfowl shorebirds, leading to the first human infection in Astrakhan Oblast, Russia. Despite being clinically asymptomatic and without direct human-to-human transmission, the World Health Organization stressed the need for continued risk assessment given the nature of Influenza to reassort and generate novel strains. Given its promiscuity and easy cross to humans, the urgency to understand the mechanisms of possible species jumping to avert disastrous pandemics is increasing. Addressing the epidemiology of H5N8, its mechanisms of species jumping and its implications, mutational and reassortment libraries can potentially be built, allowing them to be tested on various models complemented with deep-sequencing and automation. With knowledge on mutational patterns, cellular pathways, drug resistance mechanisms and effects of host proteins, we can be better prepared against H5N8 and other influenza A viruses.

Airborne Transmission of Avian Origin H9N2 Influenza A Viruses in Mammals.

Influenza A viruses (IAV) are widespread viruses affecting avian and mammalian species worldwide. IAVs from avian species can be transmitted to mammals including humans and, thus, they are of inherent pandemic concern. Most of the efforts to understand the pathogenicity and transmission of avian origin IAVs have been focused on H5 and H7 subtypes due to their highly pathogenic phenotype in poultry. However, IAV of the H9 subtype, which circulate endemically in poultry flocks in some regions of the world, have also been associated with cases of zoonotic infections. In this review, we discuss the mammalian transmission of H9N2 and the molecular factors that are thought relevant for this spillover, focusing on the HA segment. Additionally, we discuss factors that have been associated with the ability of these viruses to transmit through the respiratory route in mammalian species. The summarized information shows that minimal amino acid changes in the HA and/or the combination of H9N2 surface genes with internal genes of human influenza viruses are enough for the generation of H9N2 viruses with the ability to transmit via aerosol.