Modelling the transmission dynamics of H9N2 avian influenza viruses in a live bird market.

H9N2 avian influenza viruses (AIVs) are a major concern for the poultry sector and human health in countries where this subtype is endemic. By fitting a model simulating H9N2 AIV transmission to data from a field experiment, we characterise the epidemiology of the virus in a live bird market in Bangladesh. Many supplied birds arrive already exposed to H9N2 AIVs, resulting in many broiler chickens entering the market as infected, and many indigenous backyard chickens entering with pre-existing immunity. Most susceptible chickens become infected within one day spent at the market, owing to high levels of viral transmission within market and short latent periods, as brief as 5.3 hours. Although H9N2 AIV transmission can be substantially reduced under moderate levels of cleaning and disinfection, effective risk mitigation also requires a range of additional interventions targeting markets and other nodes along the poultry production and distribution network.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

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.

A new nanobody-enzyme fusion protein-linked immunoassay for detecting antibodies against influenza A virus in different species.

Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species.

Molecular and Antigenic Characterization of Avian H9N2 Viruses in Southern China.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10-3 substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 × 10-3 substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

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.

Detection of a Novel Reassortant H9N9 Avian Influenza Virus in Free-Range Ducks in Bangladesh.

Wild aquatic birds are the primary natural reservoir for influenza A viruses (IAVs). In this study, an A(H9N9) influenza A virus (A/duck/Bangladesh/44493/2020) was identified via routine surveillance in free-range domestic ducks in Bangladesh. Phylogenetic analysis of hemagglutinin showed that the H9N9 virus belonged to the Y439-like lineage. The HA gene had the highest nucleotide identity to A/Bean Goose (Anser fabalis)/South Korea/KNU 2019-16/2019 (H9N2). The other seven gene segments clustered within the Eurasian lineage.

Molecular epidemiology and pathogenicity of H5N1 and H9N2 avian influenza viruses in clinically affected chickens on farms in Bangladesh.

Avian influenza virus (AIV) subtypes H5N1 and H9N2 co-circulate in poultry in Bangladesh, causing significant bird morbidity and mortality. Despite their importance to the poultry value chain, the role of farms in spreading and maintaining AIV infections remains poorly understood in most disease-endemic settings. To address this crucial gap, we conducted a cross-sectional study between 2017 and 2019 in the Chattogram Division of Bangladesh in clinically affected and dead chickens in farms with suspected AIV infection. Viral prevalence of each subtype was approximately 10% among farms for which veterinary advice was sought, indicating high levels of virus circulation in chicken farms despite the low number of reported outbreaks. Co-circulation of both subtypes was common in farms, with our findings suggest that in the field, the co-circulation of H5N1 and H9N2 can modulate disease severity, which could facilitate an underestimated level of AIV transmission in the poultry value chain. Finally, using newly generated whole-genome sequences, we investigate the evolutionary history of a small subset of H5N1 and H9N2 viruses. Our analyses revealed that for both subtypes, the sampled viruses were genetically most closely related to other viruses isolated in Bangladesh and represented multiple independent incursions. However, due to lack of longitudinal surveillance in this region, it is difficult to ascertain whether these viruses emerged from endemic strains circulating in Bangladesh or from neighbouring countries. We also show that amino acids at putative antigenic residues underwent a distinct replacement during 2012 which coincides with the use of H5N1 vaccines.

The Impact of the Closure of the Live Poultry Market due to COVID-19 on the Avian Influenza Virus in Nanchang, Jiangxi Province, China.

This article aims to understand the changes in the detection rates of H5, H7, and H9 subtypes of avian influenza viruses (AIVs) in the live poultry markets (LPMs) in Nanchang City, Jiangxi Province, before and after the outbreak of the COVID-19. From 2019 to 2020, we monitored the LPM and collected specimens, using real-time reverse transcription polymerase chain reaction technology to detect the nucleic acid of type A AIV in the samples. The H5, H7, and H9 subtypes of influenza viruses were further classified for positive results. We analyzed 1,959 samples before and after the outbreak and found that the positive rates of avian influenza A virus (39.69%) and H9 subtype (30.66%) after the outbreak were significantly higher than before the outbreak (26.84% and 20.90%, respectively; P < 0.001). In various LPMs, the positive rate of H9 subtypes has increased significantly (P ≤ 0.001). Positive rates of the H9 subtype in duck, fecal, daub, and sewage samples, but not chicken samples, have increased to varying degrees. This study shows that additional measures are needed to strengthen the control of AIVs now that LPMs have reopened after the relaxing of COVID-19-related restrictions.

Molecular characterization, receptor binding property, and replication in chickens and mice of H9N2 avian influenza viruses isolated from chickens, peafowls, and wild birds in eastern China.

H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

Development of a Rapid Fluorescent Diagnostic System to Detect Subtype H9 Influenza A Virus in Chicken Feces.

The circulation of the H9N2 virus results in significant economic losses in the poultry industry, and its zoonotic transmission highlights the need for a highly sensitive and rapid diagnostic and detection system for this virus. In this study, the performance of lateral flow test strips for a fluorescent immunochromatographic test (FICT) was optimized for the diagnosis of H9N2 virus-infected animal samples. The novel monoclonal antibodies (McAbs) against influenza A H9 viruses were developed, and two categories of McAbs with linear and conformational epitopes were compared for the performance of rapid diagnostic performance in the presence of feces sample at different time points (2, 4, and 6 days) post-infection (dpi). The limit of detection (LOD) of FICT and Kd values were comparable between linear and conformational epitope McAbs. However, superior performance of linear epitope McAbs pairs were confirmed by two animal studies, showing the better diagnostic performance showing 100% relative sensitivity in fecal samples at 6 dpi although it showed less than 80% sensitivity in early infection. Our results imply that the comparable performance of the linear epitope McAbs can potentially improve the diagnostic performance of FICT for H9N2 detection in feces samples. This highly sensitive rapid diagnostic method can be utilized in field studies of broiler poultry and wild birds.

Surveillance on respiratory diseases reveals enzootic circulation of both H5 and H9 avian influenza viruses in small-scale commercial layer farms of Bangladesh.

Poultry production in Bangladesh has been experiencing H5N1 highly pathogenic avian influenza (HPAI) and H9N2 low pathogenic avian influenza (LPAI) for the last 14 years. Vaccination of chickens against H5 HPAI is in practice since the end of 2012. Subsequently, the official reporting of HPAI outbreaks gradually decreased. However, the true extent of circulation of avian influenza virus (AIV) in commercial poultry production is not clear. To explore this, we conducted active surveillance in 422 small-scale commercial layer farms in 20 villages of Mymensingh and Tangail districts of Bangladesh during 2017 and 2018 for the presence of diseases with respiratory signs. A total of 88 farms with respiratory disease problems were identified and investigated during the surveillance. In addition, 22 small-scale commercial layer farms in the neighbouring areas with respiratory disease problem were also investigated on request from the farmers. Pooled samples of oropharyngeal swabs from live birds or respiratory tissues from dead birds of the farm suffering from respiratory disease problem were tested for molecular detection of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum and Avibacterium paragallinarum. A total of 110 farms (88 in the surveillance site and 22 in the neighbouring region) were investigated, and one or more respiratory pathogens were detected from 89 farms. AIV was detected in 57 farms often concurrently with other pathogens. Among these 57 farms, H5, H9, both H5 and H9 or non-H5 and non-H9 AIV were detected in 28, 9, 13 or 7 farms, respectively. Birds of most of the H5 AIV-positive farms did not present typical clinical signs or high mortality. Twenty such farms were observed longitudinally, which had only 1.05%-5.50% mortality but a marked drop in egg production. This widespread circulation of H5 AIV along with H9 AIV and other pathogens in small-scale commercial layer farms, often with low mortality, reaffirms the enzootic circulation of AIV in Bangladesh, which may escape syndromic surveillance focused on unusual mortality only. To reduce public health risks, strengthening of the control programme with comprehensive vaccination, enhanced biosecurity, improved surveillance and outbreak response is suggested.

Double mutations in the H9N2 avian influenza virus PB2 gene act cooperatively to increase viral host adaptation and replication for human infections.

Avian H9N2 influenza viruses in East Asia are genetically diversified and multiple genotypes (A-W) have been established in poultry. Genotype S strains are currently the most prevalent strains, have caused many human infections and pose a public health threat. In this study, human adaptation mutations in the PB2 polymerase in genotype S strains were identified by database screening. Several PB2 double mutations were identified that acted cooperatively to produce higher genotype S virus polymerase activity and replication in human cells than in avian cells and to increase viral growth and virulence in mice. These mutations were chronologically and phylogenetically clustered in a new group within genotype S viruses. Most of the relevant human virus isolates carry the PB2-A588V mutation together with another PB2 mutation (i.e. K526R, E627V or E627K), indicating a host adaptation advantage for these double mutations. The prevalence of PB2 double mutations in human H9N2 virus isolates has also been found in genetically related human H7N9 and H10N8 viruses. These results suggested that PB2 double mutations in viruses in the field acted cooperatively to increase human adaptation of the currently prevalent H9N2 genotype S strains. This may have contributed to the recent surge of H9N2 infections and may be applicable to the human adaptation of several other avian influenza viruses. Our study provides a better understanding of the human adaptation pathways of genetically related H9N2, H7N9 and H10N8 viruses in nature.

Vitamin D receptor and 1α-hydroxylase are highly expressed in lungs of mice infected with H9N2 avian influenza viruses.

The H9N2 avian influenza viruses infect poultry worldwide, and can potentially cause a human pandemic without adaptation. Vitamin D3 (D3) is increasingly being recognized for its extra-skeletal roles, such as the inflammatory and immune responses to infection. The aim of this study was to analyze the changes in vitamin D metabolizing enzymes and vitamin D receptor (VDR) in the lung tissues of mice infected with H9N2. The mice were intranasally inoculated with the appropriate dose of the virus, and various clinical indices were measured on days 3, 7, 14 and 21 post-infection. H9N2 infection significantly increased the expression levels of 1α-hydroxylase mRNA and protein, which is the activating enzyme of 25-hydroxyvitamin D (25(OH)D3), but had no significant effect on the 25(OH)D3 inactivating enzyme 24-hydroxylase, indicating that inactive D3 might be converted to its active form in the H9N2-infected lungs. Furthermore, a significant increase was also observed in the VDR mRNA and protein levels, suggesting enhanced responsiveness of the lung tissues to 1, 25(OH)2D3 post H9N2 infection. In addition, daily 25(OH)D3 injection from day 2-14 post-infection did not affect the clinical signs, virus replication and cytokine (IL-1ß and TNF-α) production in the lungs of the infected mice. Given that the biological effects of D3 rely on its activation, and the binding of 1, 25(OH)2D3 to VDR in specific tissues, our findings provide novel insights into the possible role of vitamin D in the development and progression of influenza.

Detection of novel reassortant H9N2 avian influenza viruses in wild birds in Jiangxi Province, China.

The five avian influenza A/H9N2 viruses isolated from wild birds in Jiangxi, China in 2015 are novel reassortants which most likely evolved from multiple lineages. They shared a high similarity with isolates from poultry, suggesting a frequent contact and continuous viral circulation at the bird-poultry interface. Given the continuous reassortment of H9N2 viruses, it will of substantial importance to implement routine surveillance in wild birds to successfully control avian influenza viruses and better the early warning system of the emerging reassortants with pandemic potential.

Patterns of Avian Influenza A (H5) and A (H9) virus infection in backyard, commercial broiler and layer chicken farms in Bangladesh.

In order to control Highly Pathogenic Avian Influenza (HPAI) H5N1 and Low Pathogenic Avian Influenza (LPAI) H9N2 virus spread in endemically infected countries, a detailed understanding of infection patterns is required. We conducted cross-sectional studies in Bangladesh in 2016 and 2017, on 144 backyard, 106 broiler and 113 layer chicken farms. Although all sampled birds were negative for H5 virus by RT-PCR, H5 antibodies were detected in unvaccinated birds on all three farming systems. Higher H5 antibody prevalence was observed in ducks raised on backyard farms, 14.2% (95% CI: 10.0%-19.8%), compared to in-contact backyard chickens, 4.2% (95% CI: 2.8%-6.1%). The H5 antibody prevalence was lower in broiler chickens, 1.5% (95% CI: 0.9%-2.5%), compared to layer chickens, 7.8% (95% CI: 6.1%-9.8%). H9 viruses were detected by RT-PCR in 0.5% (95% CI: 0.2%-1.3%) and 0.6% (95% CI: 0.3%-1.5%) of broilers and layers, respectively, and in 0.2% (95% CI: 0.0%-1.2%) of backyard chickens. Backyard chickens and ducks showed similar H9 antibody prevalence, 16.0% (95% CI: 13.2%-19.2%) and 15.7% (95% CI: 11.3%-21.4%), which was higher compared to layers, 5.8% (95% CI: 4.3%-7.6%), and broilers, 1.5% (95% CI: 0.9%-2.5%). Over the course of a production cycle, H5 and H9 antibody prevalence increased with the age of backyard and layer chickens. Usually, multiple ducks within a flock were H5 antibody positive, in contrast to backyard chickens, broilers and layers where only individual birds within flocks developed H5 antibodies. Our findings highlight low virus circulation in healthy chickens of all production systems in Bangladesh, which is in contrast to high virus circulation reported from live bird markets. Data generated in this project can be used to adopt risk-based surveillance approaches in different chicken production systems in Bangladesh and to inform mathematical models exploring HPAI infection dynamics in poultry from the source of production.

Molecular characteristics of H9N2 influenza viruses isolated from farmed raccoon dogs and arctic foxes in China.

In this study, eight H9N2 IAVs were isolated from infected diseased, farmed raccoon dogs and arctic foxes. Eight genes shared 98.6%-100% identity among the isolates possessing a PSRSSR/GL motif at the HA cleavage site, which is same as the motif of G1 and Y280 lineages of H9N2 IAVs. The phylogenetic analysis showed that the HA genes of the eight isolates clustered with Y280-like viruses, whereas the NA genes belonged to F/98-like sublineage. Interestingly, the NS, NP, PB2 and PA genes of the isolates were closely related to H7N9 IAVs. This is the first evidence for isolation of H9N2 IAVs from raccoon dogs and arctic foxes. Raccoon dogs and arctic foxes potentially serve as an intermediate host for influenza viruses with pandemic potential toward other animals due to co-expression of both SA α-2,6-Gal and SA α-2,3-Gal receptors in a wide range of their tissues.

Dominant subtype switch in avian influenza viruses during 2016-2019 in China.

We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014-2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide.

Avian Influenza (H9N2 Subtype) in Iranian Broiler Farms: A Cross-sectional Study.

The present study aimed to determine the seroprevalence of H9N2 influenza in broiler farms at the time of slaughter in Iran. A total of 747 birds were sampled from 74 Farms in 13 provinces within 2013-2016. The obtained sera were investigated using the hemagglutination inhibition (HI) test. Out of 74 sampled farms and 747 birds, 57 farms (77%) and 445 (59.57%) birds were reported to be seropositive. In 2013, 10 farms and 110 birds were sampled out of which three farms (29.6%) and 29 birds (30%) were seropositive. In 2014, 24 farms and 220 birds were sampled out of which 22 farms (91.6%) and 220 birds (86.6%) were positive in six provinces. In 2015, 30 farms and 278 birds were sampled out of which 5 farms (16%) and134 birds (48.2%) were positive in four provinces. Finally, in 2016, 7 farms (70%) out of 10 sampled farms and 62 birds (59%) out of 105 sampled birds were positive for H9N2 in eight provinces. The mean titer of units in 2013 was statistically lower, as compared to that in 2014 (p &lt;0.01). In addition, the proportion of positive serum units in 2013 was statistically lower, as compared to that in 2014 (p &lt;0.001). In general, the prevalence of H9N2 was high indicating the continuous circulation of the virus in Iran. Given the importance and impact of this virus on the poultry industry, people&rsquo;s livelihood, and public health, more epidemiological studies are needed to evaluate the effectiveness of the adopted measures and methods in controlling the H9N2 virus.