Avian Influenza Virus In Poultry Research Articles

Re-evaluating efficacy of vaccines against highly pathogenic avian influenza virus in poultry: A systematic review and meta-analysis

The global spread of highly pathogenic avian influenza (HPAI) A (H5N1) clade 2.3.4.4b virus since 2021 necessitates a re-evaluation of the role of vaccination in controlling HPAI outbreaks among poultry, which has been controversial because of the concern of silent spread with viral mutation and spillover to human. We systematically reviewed and meta-analyzed all existing data from experimental challenge trials to assess the efficacy of HPAI vaccines against mortality in specific pathogen free (SPF) chickens, with evaluation of the certainty of evidence (CoE) using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Out of 223 screened publications, 46 trials met our eligibility criteria. Inactivated vaccines showed an efficacy of 95% (risk ratio [RR] = 5% [95% CI: 1% to 17%], I2 = 0%, CoE high) against homologous strains and an efficacy of 78% (RR = 22% [95% CI: 14% to 37%], I2 = 18%, CoE high) against heterologous strains (test for subgroup difference p = 0.02). Live recombinant vaccines exhibited the highest efficacy at 97% (RR = 3% [95% CI: 1% to 13%], I2 = 0%, CoE high). Inactivated recombinant vaccines had an overall efficacy of 90% (RR = 10% [95% CI: 6% to 16%], I2 = 47%, CoE high). Commercial vaccines showed an overall efficacy of 91% (RR = 9% [95% CI: 5% to 17%], I2 = 23%, CoE high), with 96% efficacy (RR = 4% [95% CI: 1% to 21%], I2 = 0%, CoE high) against homologous strains and 90% efficacy (RR = 10% [95% CI: 5% to 20%], I2 = 31%, CoE moderate) against heterologous strains. Our systematic review offers an updated and unbiased assessment of vaccine efficacy against HPAI-related mortality, providing timely and crucial information for re-evaluating the role of vaccination in poultry avian influenza control policy amist the global HPAI outbreak post-2021.

Potential use of poultry farms by wild waterfowl in California's Central Valley varies across space, times of day, and species: implications for influenza transmission risk

Interactions between wildlife and livestock can lead to cross‐species disease transmission, which incurs economic costs and threatens wildlife conservation. Wild waterfowl are natural hosts of avian influenza viruses (AIVs), are often abundant near poultry farms, and have been linked to outbreaks of AIVs in poultry. Interspecific and seasonal variation in waterfowl movement and habitat use means that the risk of disease transmission between wild birds and poultry inevitably varies across species, space, and time. Here, we used GPS telemetry data from 10 waterfowl species in and near California's Central Valley, a region where both wild waterfowl and domestic poultry are abundant, to study selection of poultry farms by waterfowl across diel, seasonal, and annual cycles. We found that waterfowl selected for wetlands, open water, protected areas, and croplands, which meant that they generally avoided habitats that were likely to be used for poultry farming. These selection patterns were linked to species' ecology and diel behavioral patterns, such that avoidance of poultry habitats was stronger for local or partial migrants than for long‐distance migrants, and stronger during daytime than at night. We then combined these habitat selection results with data on poultry farm locations to map risk of waterfowl–poultry contact across the Central Valley. Average selection strength at poultry farms was low, suggesting that current placement of poultry farms is generally effective for limiting risk of contact with wild birds. When we combined these habitat selection results with data on species' abundances and AIV infection prevalence, we found dramatic variation in potential AIV transmission risk among species. These results could be used to prioritize surveillance and biosecurity efforts for regions and times of relatively high risk. More generally, these results highlight that fine‐scale movement data can help identify interspecific, seasonal, and diel patterns in animal behaviors that affect wildlife and poultry health.

Sequence Analysis of the Malaysian Low Pathogenic Avian Influenza Virus Strain H5N2 from Duck.

The avian influenza viruses (AIV) of the H5 subtype have the ability to mutate from low pathogenic (LPAI) to highly pathogenic (HPAI), which can cause high mortality in poultry. Little is known about the pathogenic switching apart from the mutations at the haemagglutinin cleavage site, which significantly contributes to the virus virulence switching phenomenon. Therefore, this study aimed to compare the molecular markers in the haemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of a locally isolated LPAI AIV strain H5N2 from Malaysia with the reference HPAI strains using bioinformatics approaches, emphasising the pathogenic properties of the viral genes. First, the H5N2 strain A/Duck/Malaysia/8443/2004 was propagated in SPF eggs. The viral presence was verified by haemagglutination assay, RT-PCR, and sequencing. Results showed successful amplifications of HA (1695 bp), NA (1410 bp), and M (1019 bp) genes. The genes were sequenced and the deduced amino acid sequences were analysed computationally using MEGA 11 and NetNGlyc software. Analysis of the HA protein showed the absence of the polybasic cleavage motif, but presence of two amino acid residues that are known to affect pathogenicity. There were also two glycosylation sites (glycosites) compared to the reference HPAI viruses, which had three or more at the HA globular head domain. No NA stalk deletion was detected but the haemadsorbing and active centres of the studied NA protein were relatively similar to the reference HPAI H5N2 isolates of duck but not chicken origins. Six NA glycosites were also identified. Finally, we observed a consistent M1 and M2 amino acid sequences between our LPAI isolate with the other HPAI H5N1 or H5N2 reference proteins. These data demonstrate distinct characteristics of the Malaysian LPAI H5N2, compared to HPAI H5N2 or H5N1 from ducks or chickens, potentially aiding the epidemiological research on genetic dynamics of circulating AIV in poultry.

Multiple Vaccines and Strategies for Pandemic Preparedness of Avian Influenza Virus.

Avian influenza viruses (AIV) are a continuous cause of concern due to their pandemic potential and devasting effects on poultry, birds, and human health. The low pathogenic avian influenza virus has the potential to evolve into a highly pathogenic avian influenza virus, resulting in its rapid spread and significant outbreaks in poultry. Over the years, a wide array of traditional and novel strategies has been implemented to prevent the transmission of AIV in poultry. Mass vaccination is still an economical and effective approach to establish immune protection against clinical virus infection. At present, some AIV vaccines have been licensed for large-scale production and use in the poultry industry; however, other new types of AIV vaccines are currently under research and development. In this review, we assess the recent progress surrounding the various types of AIV vaccines, which are based on the classical and next-generation platforms. Additionally, the delivery systems for nucleic acid vaccines are discussed, since these vaccines have attracted significant attention following their significant role in the fight against COVID-19. We also provide a general introduction to the dendritic targeting strategy, which can be used to enhance the immune efficiency of AIV vaccines. This review may be beneficial for the avian influenza research community, providing ideas for the design and development of new AIV vaccines.

Epidemiology and molecular characterization of avian influenza A viruses H5N1 and H3N8 subtypes in poultry farms and live bird markets in Bangladesh

Avian influenza virus (AIV) remains a global threat, with waterfowl serving as the primary reservoir from which viruses spread to other hosts. Highly pathogenic avian influenza (HPAI) H5 viruses continue to be a devastating threat to the poultry industry and an incipient threat to humans. A cross-sectional study was conducted in seven districts of Bangladesh to estimate the prevalence and subtypes (H3, H5, and H9) of AIV in poultry and identify underlying risk factors and phylogenetic analysis of AIVs subtypes H5N1 and H3N8. Cloacal and oropharyngeal swab samples were collected from 500 birds in live bird markets (LBMs) and poultry farms. Each bird was sampled by cloacal and oropharyngeal swabbing, and swabs were pooled for further analysis. Pooled samples were analyzed for the influenza A virus (IAV) matrix (M) gene, followed by H5 and H9 molecular subtyping using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Non-H5 and Non-H9 influenza A virus positive samples were sequenced to identify possible subtypes. Selected H5 positive samples were subjected to hemagglutinin (HA) and neuraminidase (NA) gene sequencing. Multivariable logistic regression was used for risk factor analysis. We found that IAV M gene prevalence was 40.20% (95% CI 35.98–44.57), with 52.38%, 46.96%, and 31.11% detected in chicken, waterfowl, and turkey, respectively. Prevalence of H5, H3, and H9 reached 22%, 3.4%, and 6.9%, respectively. Waterfowl had a higher risk of having AIV (AOR: 4.75), and H5 (AOR: 5.71) compared to chicken; more virus was detected in the winter season than in the summer season (AOR: 4.93); dead birds had a higher risk of AIVs and H5 detection than healthy birds, and the odds of H5 detection increased in LBM. All six H5N1 viruses sequenced were clade 2.3.2.1a-R1 viruses circulating since 2015 in poultry and wild birds in Bangladesh. The 12 H3N8 viruses in our study formed two genetic groups that had more similarity to influenza viruses from wild birds in Mongolia and China than to previous H3N8 viruses from Bangladesh. The findings of this study may be used to modify guidelines on AIV control and prevention to account for the identified risk factors that impact their spread.

A Machine Learning Framework Based on Extreme Gradient Boosting to Predict the Occurrence and Development of Infectious Diseases in Laying Hen Farms, Taking H9N2 as an Example.

The H9N2 avian influenza virus has become one of the dominant subtypes of avian influenza virus in poultry and has been significantly harmful to chickens in China, with great economic losses in terms of reduced egg production or high mortality by co-infection with other pathogens. A prediction of H9N2 status based on easily available production data with high accuracy would be important and essential to prevent and control H9N2 outbreaks in advance. This study developed a machine learning framework based on the XGBoost classification algorithm using 3 months' laying rates and mortalities collected from three H9N2-infected laying hen houses with complete onset cycles. A framework was developed to automatically predict the H9N2 status of individual house for future 3 days (H9N2 status + 0, H9N2 status + 1, H9N2 status + 2) with five time frames (day + 0, day - 1, day - 2, day - 3, day - 4). It had been proven that a high accuracy rate > 90%, a recall rate > 90%, a precision rate of >80%, and an area under the curve of the receiver operator characteristic ≥ 0.85 could be achieved with the prediction models. Models with day + 0 and day - 1 were highly recommended to predict H9N2 status + 0 and H9N2 status + 1 for the direct or auxiliary monitoring of its occurrence and development. Such a framework could provide new insights into predicting H9N2 outbreaks, and other practical potential applications to assist in disease monitor were also considerable.

Seroprevalence of Newcastle disease virus and avian influenza virus in poultry and captive wild birds in poultry-dense regions of Pakistan.

Newcastle disease virus (NDV) and avian influenza virus (AIV) are causing contagious diseases in chickens and wild birds worldwide; however, there is a paucity of information on the current status of seropositivity of Newcastle and avian influenza diseases in chickens and wild birds of Pakistan. Therefore, the present study aimed to investigate the serological evidence of both diseases in commercial poultry (broiler, layer chickens), backyard poultry, and captive wild birds in poultry‑dense regions of Punjab, Pakistan. Enzyme‑linked immunosorbent (ELISA) and haemagglutination inhibition (HI) assays were performed for the determination of antibodies against NDV and AIV and their genotyping and subtyping, respectively. Overall, 47.5% and 67.4% seroprevalence of NDV and AIV, respectively, was observed in both poultry and wild birds. Based on bird's category, layer chickens had the highest seroprevalence of NDV (60.8%, 95% CI: 52.95‑68.22, OR: 0.71) followed by backyard poultry (56.8%, 95% CI: 47.92‑65.32, OR: 0.82), broilers (52.7%, 95% CI: 46.84‑58.64), pigeons (41.3%, 95% CI: 30.53‑52.81, OR: 1.59), peafowls (26.1%, 95% CI: 11.09‑48.69, OR: 3.16), ducks (23.8%, 95% CI: 12.59‑39.8, OR: 3.57), turkeys (16.7%, 95% CI: 4.41‑42.27, OR: 5.58), parrots (14.3%, 95% CI: 2.52‑43.85, OR: 6.70) and quails (2.3%, 95% CI: 0.2‑13.51, OR: 4.8). Comparatively, backyard chickens had the highest seroprevalence of AIV (78.8%, 95% CI: 70.64‑85.22, OR: 0.63) followed by ducks (73.8%, 95% CI: 57.68‑85.6, OR: 0.83), layers (73.5%, 95% CI: 65.98‑79.89, OR: 0.84), pigeons (72.5%, 95% CI: 61.2‑81.61, OR: 0.89), broilers (70.1%, 95% CI: 64.44‑75.29), turkeys (55.5%, 95% CI: 31.35‑77.6, OR: 1.87), peafowls (47.8%, 95% CI: 27.42‑68.9, OR: 2.56) and parrots (42.8%, 95% CI: 18.8‑70.3, OR: 3.1). Overall, 40.1%, 34.2%, 31.3%, and 25.1% sera were positive for H9 AIV, G‑VII NDV, H7 AIV, and G‑VI NDV, respectively. The current study revealed a widespread exposure to NDV and AIV in poultry and captive wild birds. Therefore, it is crucial to include captive wild birds in NDV and AIV surveillance programs to further strengthen disease control measures, particularly in endemic regions.

Genome sequence analysis of H7N3 subtype avian influenza virus originated from wild birds and its potential infectivity in mice.

In 2021, an H7N3 avian influenza virus (AIV) was isolated from a mallard in Tianhewan Yellow River National Wetland Park, Ningxia Hui Autonomous Region, China. Sequences analysis showed that this strain received its genes from H7, H6, H5, H3, and H1 AIVs of domestic poultry and wild birds in Asia and Europe. It was mild pathogenicity in mice. These results suggest the importance of continued surveillance of the H7N3 virus to better understand the ecology and evolution of the AIVs in poultry and wild birds and the potential threat to humans.

Pathogenicity and Transmissibility of Clade 2.3.4.4h H5N6 Avian Influenza Viruses in Mammals.

Avian influenza viruses (AIVs) have the potential for cross-species transmission and pandemics. In recent years, clade 2.3.4.4 H5N6 AIVs are prevalent in domestic poultry, posing a threat to the domestic poultry industry and public health. In this study, two strains of H5N6 AIVs were isolated from chickens in Hebei, China, in 2019: A/chicken/Hebei/HB1907/2019(H5N6) and A/chicken/Hebei/HB1905/2019(H5N6). Phylogenetic analysis showed that both viral HA genes clustered in the 2.3.4.4h clade. Receptor binding analysis showed that the HB1905 strain preferentially binds to α-2,3-linked sialic acid (SA) receptors, while the HB1907 strain preferentially binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors. During early infection, the HB1907 strain is highly replicable in MDCK cells, more so than the HB1905 strain. Pathogenicity assays in mice showed that both viruses could replicate in the lungs without prior adaptation, with HB1907 being more highly pathogenic in mice than the HB1905 strain. Significantly, both the HB1905 and HB1907 strains can be transmitted through direct contact among guinea pigs, but the transmission efficiency of the HB1907 strain through contact between guinea pigs is much greater than that of the HB1905 strain. These results strengthen the need for ongoing surveillance and early warning of H5N6 AIVs in poultry.

Isolation of Genetically Diverse H5N8 Avian Influenza Viruses in Poultry in Egypt, 2019-2021.

The global spread of avian influenza virus (AIV) of clade 2.3.4.4b since 2016 has caused severe losses in wild birds and poultry and has posed a risk for the infection of mammals including humans. The vaccination of poultry has been used to limit the spread of the virus and mitigate its socioeconomic impact. Here, we describe H5N8 epidemics in chickens, turkeys and ducks from different localities in Egypt from 2019 to 2021. About 41.7% (n = 88/211) flocks were tested positive by RT-qPCR for H5N8 viruses with prevalence rates of 45.1% (n = 65/144) and 34.3% (n = 23/67) in vaccinated and non-vaccinated flocks, respectively. A sequence analysis of the hemagglutinin and neuraminidase genes indicated not only the multiple introduction events of H5N8 viruses in Egypt but also the establishment of endemic viruses in commercial poultry in 2020/2021. The recent H5N8 viruses in poultry in Egypt are genetically distinct from the majority of licensed vaccines used in the field. Together, our findings indicate that poultry in Egypt is an endemic center for clade 2.3.4.4b in the Middle East. The efficiency of current vaccines should be regularly evaluated and updated to fully protect poultry flocks in Egypt against H5N8 viruses.

Detection of highly pathogenic avian influenza virus in Poultry in Nsukka Agricultural Zone of Southeastern Nigeria using rapid test

Detection of highly pathogenic avian influenza virus in Poultry in Nsukka Agricultural Zone of Southeastern Nigeria using rapid test

Annual Report on surveillance for avian influenza in poultry and wild birds in Member States of the European Union in 2020.

In 2020, Council Directive 2005/94/EC required EU Member States (MSs) to carry out surveillance for avian influenza (AI) in poultry and wild birds and notify the results to the responsible authority. Based on this, MSs, Iceland, Norway, Switzerland and the United Kingdom implemented ongoing surveillance programmes to monitor incursions of AI viruses in poultry and wild birds. EFSA received a mandate from the European Commission to collate, validate, analyse and summarise the data resulting from the avian influenza surveillance programmes in an annual report. This is the second such report produced using data directly submitted to EFSA by MSs. This report summarises the results of the surveillance activities carried out in poultry and wild birds in 2020. Overall, 24,768 poultry establishments (PEs) were sampled, of which 46 were seropositive for H5 virus strains and seven for H7 strains. Seropositive PEs were found in nine MSs (Belgium, Denmark, Finland, France, Italy, the Netherlands, Poland, Spain and Sweden) and the United Kingdom. As per previous years, the highest percentages of seropositive PEs were found in establishments raising waterfowl game birds and breeding geese. Out of the 53 PEs with positive serological tests for H5/H7, seven tested positive in polymerase chain reaction (PCR) or virology for H5/H7 virus strains: six for Low Pathogenic Avian Influenza (LPAI) and one for Highly Pathogenic Avian Influenza (HPAI). In addition, 13 countries also reported PCR results from 748 PEs which did not correspond to the follow‐up testing of a positive serology event (e.g. in some PEs, PCR tests were used for screening). Twenty‐five of these PEs were found positive for AI viral RNA. These positive PEs were located in Bulgaria, Estonia, Germany, Romania and Slovakia. A total of 18,968 wild birds were sampled, with 878 birds testing positive to HPAI virus. Fourteen countries reported HPAI‐positive wild birds, with all HPAI strains identified as H5. Most positive birds were infected with H5N8, with a smaller number of N1, N3, N5 and unidentified NA subtypes. In addition, there were 317 birds testing positive for LPAI H5 or H7 virus and 429 birds testing positive for non‐H5/H7 AI virus, reported by 31 countries. The surveillance findings for poultry and wild birds for 2020 are discussed in relation to the current knowledge of the epidemiology of AI in Europe, in particular the H5N8 epidemic which has been identified late 2020.

Molecular characterization and antigenic analysis of reassortant H9N2 subtype avian influenza viruses in Eastern China in 2016

H9N2 avian influenza viruses (AIVs) can cause respiratory symptoms and decrease the egg production. Additionally, H9N2 AIVs can provide internal genes for reassortment with other subtypes. During the monitoring of live poultry markets in 2016, a total of 32 (32/179, 17.88%) H9N2 AIVs were isolated from poultry in Eastern China, and seven representative strains were selected based on the isolation time, isolation location and sequence homology for further characterization. Phylogenetic analysis of hemagglutinin and neuraminidase showed that these H9N2 AIVs clustered into the Y280 sublineage. And the phylogenetic trees of six internal genes showed that the source of these gene fragments was more abundant, suggesting that extensive reassortment has occurred in these H9N2 viruses. Molecular analysis showed that multiple specific amino acid mutations occurred that increased H9N2 AIVs’ infectivity, transmissibility, and affinity to mammals, including Q226L and Q227M in hemagglutinin, E627K in polymerase basic protein 2 (PB2), L13P in polymerase basic protein 1 (PB1), and A70V and S409N in polymerase acidic protein (PA). Pathogenicity tests in mice showed these H9N2 AIVs could replicate in lungs and exhibited slight to moderate virulence. The continuous circulation of these H9N2 viruses suggests the necessity for persistent surveillance of the H9N2 AIVs in poultry.

Genetic analysis and biological characteristics of novel clade 2.3.4.4 reassortment H5N6 avian influenza viruses from poultry in eastern China in 2016

ObjectivesThe continuous evolution of highly pathogenic H5N6 avian influenza viruses (AIVs) causes outbreaks in wildfowl and poultry, and occasional human infections. The aim of this study was to better understand the genetic relationships between these H5N6 AIVs from eastern China and other AIVs. MethodsIn 2016, 1623 cloacal swabs were sampled from poultry in 18 LPMs in eastern China, and subsequently characterized systematically using gene sequencing, phylogenetic studies, and antigenic analysis. In addition, their pathogenicity in mammals was studied in BALB/c mice, which were inoculated with viruses, with survival rate and body weight recorded daily for 14 days. ResultsIn total, 56 H5N6 AIVs were isolated in eastern China and five representative isolates were selected for further study. In our study, the H5N6 AIVs clustered into clade 2.3.4.4, Group C, and their six internal segments were derived from H6N6 and H9N2 viruses, or both, suggesting extensive reassortant among H5N6 AIVs with other subtypes. These H5N6 viruses could replicate in the lungs without prior adaptation, and exhibited slight-to-moderate virulence in mice. ConclusionsThe continuous circulation of these novel H5N6 viruses suggests the importance of persistent surveillance of H5N6 AIVs in poultry.

Within-host model of respiratory virus shedding and antibody response to H9N2 avian influenza virus vaccination and infection in chickens.

Avian influenza virus (AIV) H9N2 subtype is an infectious pathogen that can affect both the respiratory and gastrointestinal systems in chickens and continues to have an important economic impact on the poultry industry. While the host innate immune response provides control of virus replication in early infection, the adaptive immune response aids to clear infections and prevent future invasion. Modelling virus-innate immune response pathways can improve our understanding of early infection dynamics and help to guide our understanding of virus shedding dynamics that could lead to reduced transmission between hosts. While some countries use vaccines for the prevention of H9N2 AIV in poultry, the virus continues to be endemic in regions of Eurasia and Africa, indicating a need for improved vaccine efficacy or vaccination strategies. Here we explored how three type-I interferon (IFN) pathways affect respiratory virus shedding patterns in infected chickens using a within-host model. Additionally, prime and boost vaccination strategies for a candidate H9N2 AIV vaccine are assessed for the ability to elicit seroprotective antibody titres. The model demonstrates that inclusion of virus sensitivity to intracellular type-I IFN pathways results in a shedding pattern most consistent with virus titres observed in infected chickens, and the inclusion of a cellular latent period does not improve model fit. Furthermore, early administration of a booster dose two weeks after the initial vaccine is administered results in seroprotective titres for the greatest length of time for both broilers and layers. These results demonstrate that type-I IFN intracellular mechanisms are required in a model of respiratory virus shedding in H9N2 AIV infected chickens, and also highlights the need for improved vaccination strategies for laying hens.

Limited onward transmission potential of reassortment genotypes from chickens co-infected with H9N2 and H7N9 avian influenza viruses

ABSTRACT The segmented genome of influenza A virus has conferred significant evolutionary advantages to this virus through genetic reassortment, a mechanism that facilitates the rapid expansion of viral genetic diversity upon influenza co-infections. Therefore, co-infection of genetically diverse avian influenza viruses in poultry may pose a significant public health risk in generating novel reassortants with increased zoonotic potential. This study investigated the reassortment patterns of a Pearl River Delta-lineage avian influenza A(H7N9) virus and four genetically divergent avian influenza A(H9N2) viruses upon co-infection in embryonated chicken eggs and chickens. To characterize “within-host” and “between-host” genetic diversity, we further monitored the viral genotypes that were subsequently transmitted to contact chickens in serial transmission experiments. We observed that co-infection with A(H7N9) and A(H9N2) viruses may lead to the emergence of novel reassortant viruses in ovo and in chickens, albeit with different reassortment patterns. Novel reassortants detected in donor chickens co-infected with different combinations of the same A(H7N9) virus and different A(H9N2) viruses showed distinct onward transmission potential to contact chickens. Sequential transmission of novel reassortant viruses was only observed in one out of four co-infection combinations. Our results demonstrated different patterns by which influenza viruses may acquire genetic diversity through co-infection in ovo, in vivo, and under sequential transmission conditions.

Non-basic amino acids in the hemagglutinin proteolytic cleavage site of a European H9N2 avian influenza virus modulate virulence in turkeys

H9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cell-to-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.

Annual Report on surveillance for Avian Influenza in poultryand wild birds in Member States of the European Union in 2019.

Context. Avian influenza (AI) is an infectious viral disease that affects all species of domestic and wild birds. The viruses causing this disease can be of high (HPAI) or low (LPAI) pathogenicity and represent a continuous threat to poultry in Europe. Council Directive 2005/94/EC requires EU Member States (MSs) to carry out surveillance in poultry and wild birds and notify the results to the responsible authority. Therefore, MSs, Iceland, Norway, Switzerland and the United Kingdom have implemented ongoing surveillance programmes to monitor incursions of AI viruses in poultry and wild birds. EFSA received a mandate from the European Commission to collate, validate, analyse and summarise the data resulting from the avian influenza surveillance programmes in an annual report. Poultry. Overall 24,419 poultry establishments (PEs) were sampled, of which 87 were seropositive for H5 virus strains and 22 for H7 strains. Seropositive PEs were found in eight MSs (Belgium, Bulgaria, Denmark, France, Germany, the Netherlands, Poland, Spain) and the United Kingdom (also a MS at the time of collection). The highest percentage of seropositive PEs was found in establishments raising waterfowl game birds and breeding geese. Out of the 109 PEs with positive serological tests for H5/H7, only two tested positive in PCR and virology for H5/H7 virus strains, both of which were LPAI strains (H5N1 and H7N7, respectively) and were reported by Denmark. In addition, 12 countries also reported PCR results from 653 PEs carried out either as a screening test or subsequent to a negative serological test result. Five of these PEs were found positive for AI viral RNA: four H5N8 HPAI in Bulgaria and one H7N3 LPAI in Italy. Wild birds. A total of 19,661 dead/moribund wild birds were sampled, with one bird testing positive to HPAI virus H5N6, which was reported by Denmark. In addition, there were 84 birds testing positive for LPAI H5 or H7 virus and 848 birds testing positive for non‐H5/H7 AI virus, reported by 30 countries. The surveillance findings for poultry and wild birds for 2019 are discussed in relation to findings from previous years and current knowledge of the epidemiology of AI in Europe.

H7N1 Low Pathogenicity Avian Influenza Viruses in Poultry in the United States During 2018.

Here, we report three detections of H7N1 low pathogenicity avian influenza viruses (LPAIV) from poultry in Missouri (n = 2) and Texas (n = 1) during February and March 2018. Complete genome sequencing and comparative phylogenetic analysis suggest that the H7 LPAIV precursor viruses were circulating in wild birds in North America during the fall and winter of 2017 and spilled over into domestic poultry in Texas and Missouri independently during the spring of 2018.

Avian influenza surveillance at the human-animal interface in Lebanon, 2017.

Avian influenza viruses (AIVs) cause severe diseases in poultry and humans. In Lebanon, AIV H9N2 was detected in 2006 and 2010 and H5N1 was detected in 2016. To evaluate the current circulating AIVs in Lebanon at the human-animal interface. A total of 1000 swabs were collected from poultry from 7 Lebanese governorates between March and June 2017. Swabs were screened for influenza infection. Haemagglutinin and neuraminidase AIV subtypes were determined for positive samples. Gene segments were cloned and sequenced. Blood was collected from 69 exposed individuals. Serological studies were performed to test sera for antibodies against AIV. In chickens, 0.6% were positive for AIV H9N2. Sequences obtained clustered tightly with those of Israeli origin as well as Lebanese H9N2 viruses from 2010. All human samples tested negative. We recommend regular surveillance for AIVs in poultry using a One Health approach.