Search

The H9N2 avian influenza virus (AIV) is spreading worldwide. Presence of H9N2 virus tends to increase the chances of infection with other pathogens which can lead to more serious economic losses. In a previous study, a regulated delayed lysis Salmonella vector was used to deliver a DNA vaccine named pYL233 encoding M1 protein, mosaic HA protein and chicken GM-CSF adjuvant. To further increase its efficiency, chitosan as a natural adjuvant was applied in this study. The purified plasmid pYL233 was coated with chitosan to form a DNA containing nanoparticles (named CS233) by ionic gel method and immunized by intranasal boost immunization in birds primed by oral administration with Salmonella strain. The CS233 DNA nanoparticle has a particle size of about 150 nm, with an encapsulation efficiency of 93.2 ± 0.12 % which protected the DNA plasmid from DNase I digestion and could be stable for a period of time at 37°. After intranasal boost immunization, the CS233 immunized chickens elicited higher antibody response, elevated CD4 + T cells and CD8 + T cells activation and increased T-lymphocyte proliferation, as well as increased productions of IL-4 and IFN-γ. After challenge, chickens immunized with CS233 resulted in the lowest levels of pulmonary virus titer and viral shedding as compared to the other challenge groups. The results showed that the combination of intranasal immunization with chitosan-coated DNA vaccine and oral immunization with regulatory delayed lytic Salmonella strain could enhance the immune response and able to provide protection against H9N2 challenge., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The H7 subtype avian influenza viruses are circulating widely worldwide, causing significant economic losses to the poultry industry and posing a serious threat to human health. In 2019, H7N2 and H7N9 co-circulated in Chinese poultry, yet the risk of H7N2 remained unclear. We isolated and sequenced four H7N2 viruses from chickens, revealing them as novel reassortants with H7N9-derived HA, M, NS genes and H9N2-derived PB2, PB1, PA,NP, NA genes. To further explore the key segment of pathogenicity, H7N2-H7N9NA and H7N2-H9N2HA single-substitution were constructed. Pathogenicity study showed H7N2 isolates to be highly pathogenic in chickens, with H7N2-H7N9NA slightly weaker than H7N2-Wild type. Transcriptomic analysis suggested that H7N9-derived HA genes primarily drove the high pathogenicity of H7N2 isolates, eliciting a strong inflammatory response. These findings underscored the increased threat posed by reassorted H7N2 viruses to chickens, emphasizing the necessity of long-term monitoring of H7 subtype avian influenza viruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The H9N2 subtype of the avian influenza virus (AIV) is widely prevalent in birds, threatening the poultry industry and providing genetic material for emerging human pathogens. The prevalence and genetic characteristics of H9N2 in Yunnan Province, China, are largely unknown. Samples were collected from live poultry markets (LPMs) and breeding farms in Yunnan Province. H9N2-positive samples were identified by polymerase chain reaction (PCR), with a high positivity rate of 42.86% in tissue samples. The positivity rate of swab samples in the LPMs in Kunming was 3.97% (17/564), but no AIV was detected in samples from poultry farms in Lijiang, Wenshan, and Yuxi. Evolutionary analysis and genotyping were performed for the 17 strains of isolated H9N2 virus. Phylogenetic analysis revealed that all H9N2 viral genes had 91.6%-100% nucleotide homology, belonged to the G57 genotype, and had high homology with H9N2 viruses isolated from Guangdong and Guangxi, suggesting that the H9N2 viruses in Yunnan Province may have been imported by chicks. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to evolve. The surface genes of the H9N2 isolates displayed substantial genetic diversity, highlighting the genetic diversity and complexity of the H9N2-subtype AIVs in Yunnan. Molecular analysis demonstrated that all 17 strains of H9N2 isolates had mutations at H183N, Q226L, L31P, and I268V in hemagglutinin; S31N in matrix protein 2; and no replacements at positions 274 and 292 of the neuraminidase protein. Sixteen strains had the A558V mutation and one strain had the E627V mutation in polymerase basic protein 2. Analysis of these amino acid sites suggests that H9N2 influenza viruses in Yunnan continue to mutate and adapt to mammals and are sensitive to neuraminidase inhibitors but resistant to adamantanes. It is necessary to strengthen surveillance of AIV H9N2 subtypes in poultry and LPMs in Yunnan to further understand their genetic diversity., Competing Interests: DISCLOSURES The authors report no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

Avian influenza virus (AIV) infection and vaccination against live attenuated infectious bronchitis virus (aIBV) are frequent in poultry worldwide. Here, we evaluated the clinical effect of H9N2 subtype AIV and QX genotype aIBV co-infection in specific-pathogen-free (SPF) white leghorn chickens and explored the potential mechanisms underlying the observed effects using by 4D-FastDIA-based proteomics. The results showed that co-infection of H9N2 AIV and QX aIBV increased mortality and suppressed the growth of SPF chickens. In particular, severe lesions in the kidneys and slight respiratory signs similar to the symptoms of virulent QX IBV infection were observed in some co-infected chickens, with no such clinical signs observed in single-infected chickens. The replication of H9N2 AIV was significantly enhanced in both the trachea and kidneys, whereas there was only a slight effect on the replication of the QX aIBV. Proteomics analysis showed that the IL-17 signaling pathway was one of the unique pathways enriched in co-infected chickens compared to single infected-chickens. A series of metabolism and immune response-related pathways linked with co-infection were also significantly enriched. Moreover, co-infection of the two pathogens resulted in the enrichment of the negative regulation of telomerase activity. Collectively, our study supports the synergistic effect of the two pathogens, and pointed out that aIBV vaccines might increased IBV-associated lesions due to pathogenic co-infections. Exacerbation of the pathogenicity and mortality in H9N2 AIV and QX aIBV co-infected chickens possibly occurred because of an increase in H9N2 AIV replication, the regulation of telomerase activity, and the disturbance of cell metabolism and the immune system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The H10 subtype avian influenza virus (AIV) poses an ongoing threat to both birds and humans. Notably, fatal human cases of H10N3 and H10N8 infections have drawn public attention. In 2022, we isolated two H10N3 viruses (A/chicken/Shandong/0101/2022 and A/chicken/Shandong/0603/2022) from diseased chickens in China. Genome analysis revealed that these viruses were genetically associated with human-origin H10N3 virus, with internal genes originating from local H9N2 viruses. Compared to the H10N8 virus (A/chicken/Jiangxi/102/2013), the H10N3 viruses exhibited enhanced thermostability, increased viral release from erythrocytes, and accumulation of hemagglutinin (HA) protein. Additionally, we evaluated the pathogenicity of both H10N3 and H10N8 viruses in mice. We found that viral titers could be detected in the lungs and nasal turbinates of mice infected with the two H10N3 viruses, whereas H10N8 virus titers were detectable in the lungs and brains of mice. Notably, the proportion of double HA Q222R and G228S mutations in H10N3 viruses has increased since 2019. However, the functional roles of the Q222R and G228S double mutations in the HA gene of H10N3 viruses remain unknown and warrant further investigation. Our study highlights the potential public health risk posed by the H10N3 virus. A spillover event of AIV to humans could be a foretaste of a looming pandemic. Therefore, it is imperative to continuously monitor the evolution of the H10N3 influenza virus to ensure targeted prevention and control measures against influenza outbreaks., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Low pathogenic avian influenza (LPAI) subtype H9N2 is a causative agent that has raised increasing concern about its impact on poultry and potential public health threats. Even though H9N2 is endemic in Peninsular Malaysia, it was first reported in Sabah in August 2022, after an outbreak associated with high mortality in broiler chickens. In the present study, based on the hemagglutinin (HA) gene, we report the genetic variations and phylogenetic analysis of a H9N2 virus isolated from broiler chickens in Sabah. The sequence analysis of the HA gene revealed a 98% similarity to the H9N2 virus recently isolated from China in 2018. The amino acids in the HA cleavage site displayed a characteristic LPAI motif (PARSSR/ GLF). Notably, at position 226, the isolate had amino acid Leucine (L) demonstrating its ability to bind to the receptor of mammals, resulting in the potential risk of transmission to humans. In addition, the H9N2 isolate harboured seven potential N-glycosylation sites. The phylogenetic analysis revealed that the isolate belonged to clade h9.4.2.5 in the Y280 lineage, similar to previously reported in Malaysia. However, we observed that the isolate in this study falls in a different cluster compared with previous Malaysian isolates, suggesting different source of H9N2 introduction into the country. This prompts us to propose continuous and thorough surveillance of poultry across the country and the necessity of implementing farm biosecurity to minimize economic losses and potential threats to public health.

The application of recombinant herpesvirus of turkey, expressing the H9 hemagglutinin gene from low pathogenic avian influenza virus (LPAIV) H9N2 and the avian orthoavulavirus-1 (AOAV-1) (commonly known as Newcastle Disease virus (NDV)) fusion protein (F) as an rHVT-H9-F vaccine, is an alternative to currently used classical vaccines. This study investigated H9- and ND-specific humoral and mucosal responses, H9-specific cell-mediated immunity, and protection conferred by the rHVT-H9-F vaccine in specific pathogen-free (SPF) chickens. Vaccination elicited systemic NDV F- and AIV H9-specific antibody response but also local antibodies in eye wash fluid and oropharyngeal swabs. The ex vivo H9-specific stimulation of splenic and pulmonary T cells in the vaccinated group demonstrated the ability of vaccination to induce systemic and local cellular responses. The clinical protection against a challenge using a LPAIV H9N2 strain of the G1 lineage isolated in Morocco in 2016 was associated with a shorter duration of shedding along with reduced viral genome load in the upper respiratory tract and reduced cloacal shedding compared to unvaccinated controls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction ( P < 0.01) in log 2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

This paper aims to explore the effects of chicken interferon-γ (ChIFN-γ) and interleukin-2 (ChIL-2) on type 1 helper (Th1) T lymphocyte differentiation. To be specific, ChIFN-γ and ChIL-2 were first expressed in Escherichia competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine coli competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine in vitro and in vivo . The results of this study can lay a theoretical basis for using ChIFN-γ and ChIL-2 as vaccine adjuvants.

This paper presents genetic data on the full genome analysis of A/chicken/Tajikistan/2379/2018 H9N2 influenza virus isolated in September 2018 from chicken pathological material received from poultry farms of the Republic of Tajikistan and subtyped as H9N2 by serological and molecular methods. According to the results of hemagglutinin gene sequencing, the amino acid sequence of the cleavage site was RSSR/GLF, which is typical for low-virulent avian influenza virus. Phylogenetic analysis of the nucleotide sequence of a hemagglutinin gene fragment (nt 1-1539 of the open reading frame) showed that the A/chicken/Tajikistan/2379/2018 H9N2 isolate belongs to the Y280 genetic group of low-virulent A/H9 influenza virus, which is widespread in Southeast Asia. The complete nucleotide sequence of the viral genome was determined. Comparative analysis of all genomic segments revealed that the A/chicken/Tajikistan/2379/2018 H9N2 virus is closely related to an A/H9 influenza virus isolated in the Far East of the Russian Federation in 2018. Genetic similarity (97.1-99% identity in four out of eight viral genes) was found to isolates of an H7N9 subtype virus recovered in the Inner Mongolia and Hebei regions of China in 2017. According to the analysis of the predicted amino acid sequence of the studied isolate, the positions of some molecular markers indicate possible adaptation of the virus to mammals. Further genetic analysis showed that this virus belongs to genotype G57.

Despite the low pathogenicity of the H9N2 avian influenza viruses, they can induce severe economic losses in various poultry sectors in conjunction with other factors. In Egypt, low-pathogenic avian influenza (LPAI) H9N2 became endemic in 2011 and has undergone continuous genetic evolution since then. The regular monitoring of the evolution of the virus is necessary to control its spread. During 2017-2020, there were 44 positive samples isolated, and these viruses were genetically sequenced to determine the hemagglutinin (HA) gene circulating in Egypt. The molecular analysis revealed at least nine changes in amino acid residues in comparison with the reference Egyptian strain from the original introduction in 2011 (A/qu/Egypt/113413v/2011), with a similarity of 95%-96%. Amino acid residues 180 and 216 are the most important residues in terms of positive selection pressure. Phylogenetically, the new Egyptian H9N2 viruses in 2017-2020 belonged to a new subcluster related to the strains that had been circulating since 2015. Comparative analysis of the HA gene of LPAI H9N2 viruses in Egypt from 2011 to 2020 supports a continuous evolution through the years with persistent markers., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Korean Society of Veterinary Science.)

H9N2, a low pathogenic avian influenza virus, causes significant economic losses in the poultry industry worldwide. Herein, we describe the construction of an attenuated Salmonella Gallinarum (SG) strain for expression and delivery of H9N2 haemagglutinin (HA) 1 (SG-HA1), HA2 (SG-HA2) and/or the conserved matrix protein 2 ectodomain (SG-M2e). We demonstrated that recombinant SG strains expressing HA1, HA2 and M2e antigens were immunogenic and safe in a chicken model. Chickens ( n  = 8) were vaccinated once orally with SG alone, SG-HA1, SG-HA2, SG-M2e, or mixture of SG-HA1, SG-HA2 and SG-M2e, or vaccinated once intramuscularly with an oil-adjuvant inactivated H9N2 vaccine. Our results demonstrated that vaccination with SG mutants encoding influenza antigens, administered individually or as a mixture, elicited significantly ( P  < 0.05) greater antigen-specific humoral and cell-mediated immune responses in chickens compared with those vaccinated with SG alone. A conventional H9N2 vaccine induced significantly ( P  < 0.05) greater HA1 and HA2 antibody responses than SG-based H9N2 vaccine strains, but significantly ( P  < 0.05) less robust M2e-specific responses. Upon challenge with the virulent H9N2 virus on day 28 post-vaccination, chickens vaccinated with either the SG-based H9N2 or conventional H9N2 vaccines exhibited comparable lung inflammation and viral loads, although both were significantly lower ( P  < 0.05) than in the group vaccinated with SG alone. In conclusion, our results showed that SG-based vaccination stimulated efficient immune responses against virulent H9N2. Further studies are needed to fully develop this approach as a preventive strategy for low pathogenic avian influenza viruses affecting poultry. RESEARCH HIGHLIGHTS S. gallinarum expressing HA1, HA2 and M2e antigens are immunogenic and safe. Salmonella has dual function of acting as a delivery system and as a natural adjuvant. Vaccine constructs elicit specific humoral and cell-mediated immune responses.

In recent years, the H9N2 influenzavirus has been circulating widely in poultry farms causing extensive damage. The hemagglutinin (HA) genes of the two virus isolates of H9N2 subtype in specific pathogen-free chickens were studied to determine the shedding rate in the host&rsquo;s oropharyngeal and cloacal routes and their genetic relationship. The sequence analysis and phylogenetic study of the samples were performed by comparing each isolate with other H9N2 isolates in the gene bank. In the present study, the chickens were inoculated with low pathogenic avian influenza virus (LPAIV) (A/Chicken/Iran/ZMT-101/1998 [H9N2]) through the intranasal route. Oropharyngeal and cloacal swabs were collected from the chickens within 1-10 days after inoculation. The rate of viral shedding was measured within the previous 10 days by the real-time reverse transcriptase polymerase chain reaction molecular technique. No clinical symptoms were observed during the experiment in the chickens. The results obtained from this technique showed that the main route of shedding for LPAIV was oropharyngeal areas (p &lt;0.05). Both isolates had a similar proteolytic R-S-S-R sequence at the cleavage site of the HA gene and contained glutamine (Q) amino acid at position 226 of the HA receptor-binding site, indicating that these isolates were nonpathogenic. Phylogenetic analysis demonstrated that both isolates belonged to the Eurasian clade. The comparison of these isolates with other isolates in the gene bank showed that they had the greatest similarity with the isolates in clade 1 and the least homology with the isolates in clade 4., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

Despite the use of wide-scale vaccination programmes against the H9N2 virus, enzootic outbreaks of H9N2 avian influenza (AI) have often occurred and caused serious nationwide economic losses, particularly in broiler chickens. In this study, the haemagglutinin (HA) and neuraminidase (NA) genes of nine recent H9N2s and a common vaccine strain were fully sequenced and compared with other representative Iranian viruses. Phylogenetic analysis revealed that all Iranian viruses were grouped into the G1 sub-lineage with different clusters in which recent isolates (2014-2017) formed a distinct cluster compared to the vaccine group (1998-2004). All Iranian H9N2s exhibited low pathogenicity AI connecting peptide feature with an R/KSSR motif. Amino acid 226, located in the 220 loop of the receptor binding site, was leucine among the recent Iranian viruses, a characteristic of human influenza viruses. With an overall gradual increase in the genetic diversity of H9N2s, Bayesian skyline plots of Iranian HA and NA genes depicted a fluctuation and a relative stable situation, respectively. It is recommended to apply constant surveillance to assess any increase in viral human adaptation and evolutionary changes in circulating field H9N2s. Moreover, antigenic characterisation of the prevailing H9N2 viruses seems to be necessary for evaluating the possible antigenic drift from the vaccine strain.

Since the early 2000s, the avian influenza virus (AIV) subtype H9N2 has been widely circulating in North African countries, including Libya, Tunisia, and Egypt. For unknown reasons, H9N2 was not detected in Moroccan farms until the end of 2016, and at present, it is endemic in poultry. This study was carried out to evaluate the evolution of H9 AIVs in Morocco from 2017 to 2019. In this study, 16 H9 viruses collected from 2017 to 2019 in Morocco were isolated and sequenced. The genomic signatures and protein sequences of these isolates were analyzed. Moroccan H9 viruses were closely related to viruses isolated from African and Middle Eastern countries, and the hemagglutinin gene resembled those of viruses of G1-like lineage, and multiple mammalian host-associated mutations were detected that favor transmission from avian to mammalian hosts. Other mutations related to virulence were also identified. Our findings suggest that attention be given to the control of H9N2 influenza viruses in chickens and continuous monitoring of the prevalence and pathogenic potential of AIV H9N2 in the poultry population in Morocco.

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1-like M and PB2 genes for the earlier F/98-like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1-like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98-like counterpart, while the G1-like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1-like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses., (© 2019 Blackwell Verlag GmbH.)

To understand the evolution of H9N2 avian influenza virus genotype and its molecular evolution rate, we systematically analyzed 72 H9N2 avian influenza virus sequences isolated from Guangdong province from 2014 to 2018. We found three genotypes (G57, G68, and G118) of the H9N2 avian influenza virus, of which G118 is a newly discovered genotype and G57 is the dominant genotype. The internal gene cassette of the G57 genotype H9N2 avian influenza virus is a stable combination that can easily transport internal genes to other novel avian influenza viruses, and the internal gene cassettes of the G68 and G118 are identical to those of G57.In addition, we estimated the nucleotide substitution rate of the HA and NA genes of the H9N2 influenza virus from 2014 to 2018.The nucleotide substitution rate of HA and NA genes showed an upward trend in 2015 and 2016. In the past two years, H9N2 avian influenza virus recombination has produced genotype G68, which disappeared in 2014 for one year. And very coincidentally, in 2015, there was a new genotype G118. We observed that the emergence of new genotypes was accompanied by a slight increase in overall nucleotide substitution rate. Therefore we hypothesize that the emergence of new genotypes could accelerate the molecular evolution rate of genes. Our research shows that the H9N2 avian influenza virus in Guangdong province has been undergoing intense evolution, demonstrating the need to strengthen influenza surveillance in the region., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The process of avian influenza virus (AIV) evolution in a new host was investigated in the experiment in which ten serial passages of a turkey-derived H9N2 AIV were carried out in specific pathogen free chickens (3 birds/group) inoculated by oculonasal route. Oropharyngeal swabs collected 3 days post infection were used for inoculation of birds in the next passage and subjected to analysis using deep sequencing. In total, eight mutations in the consensus sequence were found in the viral pool derived from the 10th passage: four mutations (2 in PB1 and 2 in HA) were present in the inoculum as minority variants while the other four (2 in NP, 1 in PA and 1 in HA) emerged during the passages in chickens. The detected fluctuations in the genetic heterogeneity of viral pools from consecutive passages were most likely attributed to the selective bottleneck. The genes known for bearing molecular determinants of the AIV host specificity (HA, PB2, PB1, PA) contributed most to the overall virus diversity. In some cases, a fast selection of the novel variant was noticed. For example, the amino-acid substitution N337K in the haemagglutinin (HA) cleavage site region detected in the 6th passage as low frequency variant had undergone rapid selection and became predominant in the 7th passage. Interestingly, detection of identical mutation in the field H9N2 isolates 1-year apart suggests that this substitution might provide the virus with a selective advantage. However, the role of specific mutations and their influence on the virus adaptation or fitness are mostly unknown and require further investigations., (Copyright © 2019. Published by Elsevier B.V.)

H9N2 avian influenza has been prevalent in chicken flocks of China for years. In the first half year of 2018, clinical cases of suspected H9N2 infection were collected from chicken flocks in Shandong province. Nine strains of H9N2 influenza virus were isolated. The pathological changes of the dead chickens were mainly respiratory inflammation, renal swelling, and secondary infection. The microscopic lesions were consistent with the pathogenic characteristics of H9N2 influenza virus. From November 2017 to June 2018, a total of 3,380 serum samples were randomly collected from commercial laying hens in Shandong Province. The H9 antibody levels were tested with the isolated strain (CK/SD/231/17) as the antigen. It showed that the average of antibody titers of H9 avian influenza was 9.24 1og2. Hemagglutination inhibition experiments were conducted on chicken serum with the vaccine virus and the isolated virus (CK/SD/231/17) as the antigens. It was found that the antibody titer measured with the vaccine virus was 1 or 2 titers higher than the isolated strain. It indicated that the antigenicity of H9N2 circulating strain was different from that of vaccine strain. The nucleotide sequences of HA gene of these recent H9N2 avian influenza virus isolates shared homologies from 93.8 to 99.9%. Phylogenetic analysis revealed that the eight gene segments of the viruses were in the same clades with G57 gene reference strain. The amino acid site analysis of influenza resistance showed that the virus was sensitive to neuraminidase inhibitors and resistant to amantadine. Highlights: The protection rate of the H9N2 AIV vaccine almost reached 100% before 2016, but the antibody level of serum samples showed high diversity in this study, which means the poultry were infected. The antigenicity of isolated H9N2 strains was different from that of vaccine strain. Current available vaccines may provide only limited protection., (© 2019 Poultry Science Association Inc.)

Background: Little is known about the antigenic and genetic characteristics of influenza A viruses circulating in poultry in Iraq., Objective: This study describes the genetic and antigenic characteristics of the detected avian influenza H9N2 viruses in Iraq during 2016., Methods: Full genome sequences of two H9N2 viruses isolated from chickens in Iraq during 2016 were assembled. Antigenic analyses of Iraqi H9N2 viruses and contemporary H9N2 isolates from Lebanon and Egypt were performed by hemagglutination inhibition assay., Results: Phylogenetic analysis of surface glycoproteins and internal segments (PB2, PA, NP, M, and NS) indicated that the Iraqi H9N2 viruses were closely related to G1-like lineage of H9N2 viruses isolated from Pakistan and Iran indicating possible epidemiological links. The PB1 segments of the current characterized H9N2 viruses were not related to any of the previously characterized H9N2 viruses and closely similar to H7N7 virus detected in chickens in Germany in 2015. Multiple genetic determinants for virulence and mammalian transmission were characterized in the characterized H9N2 viruses in Iraq. The antigenic analysis showed a close relationship between H9N2 viruses in Iraq and contemporary H9N2 viruses in Egypt and Lebanon. Like H9N2 viruses, Iraqis H9N2 virus bound to human-like receptor rather than avian-like receptor thus represent a public health risk., Conclusion: Active surveillance of avian influenza virus in poultry and migratory birds should be adopted to monitor the genesis and emergence of new viruses in Iraq., Competing Interests: The authors declare that there is no conflict of interest.

Since 1998, Iran's poultry industry has faced several outbreaks of low pathogenic avian influenza H9N2. Tissue samples were collected from a broiler flock with respiratory symptoms in autumn 2017. After that, virus isolation and confirmation of H9N2 using RT-PCR, sequencing, and bioinformatic analysis for all eight genes were performed. The phylogenic analysis revealed HA gene of recent Iranian isolate (A/chicken/Mashhad/UT-Barin/2017) which was clustered in G1 sublineage. In addition, all eight genes of the virus were placed with Pakistani isolates of 2015 in separate group. Based on amino acid motif KSSR in HA cleavage site, the UT-Barin is considered as low pathogenic avian influenza with eight HA and seven NA potential N-glycosylated sites. No evidence was detected regarding adamantane and neuraminidase inhibitors' drug's resistance. Multiple point mutations were observed in all genes that were responsible for increasing virulence of the virus for avian host and also increasing affinity to mammalian host cells.

In Morocco in early 2016, a low pathogenic avian influenza virus serotype H9N2 caused large economic losses to the poultry industry, with specific clinical symptoms and high mortality rates on infected farms. Subsequent to the H9N2 outbreak, the causal agent was successfully isolated from chicken flocks with high morbidity and mortality rates, propagated on embryonated eggs, and fully sequenced. The phylogenetic analysis suggested that the Moroccan isolate could have derived from the Middle East isolate A/chicken/Dubai/D2506.A/2015. This study was designed to assess the pathogenicity of the Moroccan isolate H9N2 in experimentally infected broiler and specific-pathogen-free (SPF) chickens. At 22 days of age, one broiler and two SPF chicken groups were inoculated by dropping 0.2 ml of the H9N2 isolate (10 7.5 EID 50 /ml) in both nostrils and eyes. Clinically inoculated chickens with H9N2 displayed mild lesions, low mortality rates, and an absence of clinical signs. The H9N2 virus was more pathogenic in broiler chickens and produced more severe tissue lesions compared to SPF chickens. The viral shedding was detected up to 6 days postinoculation (pi) in oropharyngeal and cloacal swabs in infected birds with a maximum shedding in the oropharynges of the broiler group. All experimental chickens seroconverted and registered high hemagglutination inhibition titers as early as day 7 pi. The present study indicates that the H9N2 virus isolated from a natural outbreak was of low pathogenicity under experimental conditions. However, under field conditions infection with other pathogens might have aggravated the disease.

Newcastle disease virus (NDV) and H9 subtype avian influenza virus (AIV) are two avian pathogens across the globe. Inasmuch as most poultry flocks worldwide are vaccinated with a live low-virulence or attenuated NDV vaccine, we embarked on the development of vaccine prototypes that would have dual specificities and would allow a single immunization against both avian influenza (AI) and Newcastle disease (ND). Therefore, in the present work, a cloned full-length copy of the genome of the lentogenic NDV strain rmNA-1 was selected as a backbone vector to construct three chimeric NDVs that expressed (i) the ORF encoding the HA, (ii) the ectodomain of HA fused with the transmembrane domain and cytoplasmic tail regions derived from the NDV F protein and (iii) the ectodomain of HA fused with a short GS linker and the GCN4 sequences, and designated as rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO), respectively. rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO) stably expressed the modified HA gene for 10 egg passages and the three recombinants were found innocuous to chickens. The insertion of the chimeric HA-F, rather than HA-ECTO or ORF of HA, resulted in a recombinant virus with enhanced incorporation of the HA protein into the viral surface. A single immunization of SPF chickens with the three recombinants induced NDV- and AIV H9-specific antibodies, and protected chickens against a challenge with a lethal dose of velogenic NDV or AIV H9N2. Remarkably, non-shedding of influenza virus and higher levels of H9 subtype HI titers were observed 7 days post challenge (dpc) in rmNA-H9F vaccinated chickens, than other recombinants. Furthermore, a prime-boost vaccination of chickens with rmNA-H9F induced higher levels of NDV- and H9- HI and secretory IgA, as well as reduced viral shedding and virus-induced gross lesions, compared with the commercial vaccine. Therefore, the recombinant rmNA-H9F is a promising bivalent vaccine candidate against NDV and H9 subtype AIV in chickens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Surveillance of H9N2 is currently focused on areas central to the commercial poultry industry. This study determined the prevalence of H9N2 virus in commercial and backyard poultry flocks in Punjab Province, Pakistan. Oral and tracheal swabs were collected from commercial and backyard poultry from January 2015 through June 2016. Antisera against H5, H7, H9, and Newcastle disease viruses were used for virus identification. Molecular confirmation was made by reverse transcription PCR. Avian influenza virus subtypes H5 and H7 were not detected. The H9N2 virus was isolated in 5.7% of 905 tested flocks (5-10 birds/flock). Prevalence in commercial and backyard poultry was 6.7% of 687 flocks and 2.7% of 218 flocks, respectively. Hemagglutinin and neuraminidase-gene-based phylogenetic analysis of commercial and backyard poultry isolates showed 100% homology. Within sublineage B2 of Pakistan, identity among most recent isolates (2015) was 100%, compared to 75%-99% identity with previously isolated viruses (2010-12), indicating continued virus evolution. Most of the previously reported and currently studied viruses were isolated near the Pakistan-India border. Phylogenetic analysis showed that Pakistani and Indian isolates were closely related, indicating that avian influenza virus transmission may occur across this border.

H4 and H9 avian influenza viruses (AIVs) are two of the most prevalent influenza viruses worldwide. The co-existence of H4 and H9 viruses in multiple avian species provides an opportunity for the generation of novel reassortants and for viral evolution. The diversity of the biological characteristics of the reassortants enhances the potential threat to the poultry industry and to public health. To evaluate the reassortment of these viruses and the potential public risk of the reassortants, we co-infected chickens with H4N6 and H9N2 viruses derived from poultry and tested the replication and virulence of the reassortant viruses in mice. A high frequency of reassortment was detected in chickens after co-infection with these two viruses and nine reassortants of six genotypes were purified from the chicken samples. Two H9N2 reassortants containing the PA of the parent H4N6 virus showed higher virulence than the parent H9N2 virus, revealing the significant role of the H4N6 wt virus PA gene in viral reassortment. Analysis of the polymerase activity of the ribonucleoprotein (RNP) complex in vitro suggested that the PA of H4N6 wt origin enhanced polymerase activity. Our results indicate that co-infection of an avian individual with the H4N6 and H9N2 viruses leads to a high frequency of reassortment and generates some reassortants that have higher virulence than the wild-type viruses in mammals. These results highlight the potential public risk of the avian influenza reassortants and the importance of surveillance of the co-existence of the H4N6 and H9N2 viruses in avian species and other animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We identified influenza A(H9N2) virus G1 lineage in poultry in Burkina Faso. Urgent actions are needed to raise awareness about the risk associated with spread of this zoonotic virus subtype in the area and to construct a strategy for effective prevention and control of influenza caused by this virus.

Avian influenza A virus (AIV) subtype H9N2 is the most prevalent subtype found in terrestrial poultry throughout Eurasia and has been isolated from poultry outbreaks worldwide. Tracheal tissue specimens from 100 commercial broiler flocks in Afghanistan were collected between 2016 and 2017. After real-time RT-PCR, AI-positive samples were further characterized. A part of the HA gene was amplified using RT-PCR and sequenced. The results of real-time RT-PCR showed that 40 percent of the flocks were AI positive. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian-lineage G1 AIVs and had a correlation with H9N2 AIV circulating in the poultry population of the neighboring countries over the past decade. Analysis of the amino acid sequence of HA revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity and specificity for the avian-like SAα2,3 receptor, demonstrating their specificity for and adaptation to domestic poultry. The results of the current study provide great insights into H9N2 viruses circulating in Afghanistan's poultry industry and demonstrate the necessity of planning an applied policy aimed at controlling and managing H9N2 infection in Afghan poultry.

Avian influenza A H5N1 and H9N2 viruses have been extensively circulating in various avian species and frequently infect mammals, including humans. The synchronous circulation of both viruses in Egypt provides an opportunity for possible genetic assortment, posing a probable threat to global public health. To assess the potential risk of the IAV reassortants derived from co-circulation of these two AI subtypes, reverse genetics technology was used to generate a set of IAV reassortants carrying single genetic segments of clade 2.2.1.2 virus A/duck/Egypt/Q4596D/2012 (H5N1), a representative of the most prevalent H5N1 clade in Egypt, in the genetic backbone of A/chicken/Egypt/S4456B/2011 (H9N2), a representative of G1-like H9N2 lineage which is widely circulating in Egypt. Furthermore, the genetic compatibility, growth kinetics and virulence were evaluated in vitro in mammalian systems using the MDCK cell line and avian system using SPF embryonated chicken eggs. Pathogenicity and virus shedding were further tested using SPF chickens. Out of the eight desired H9-reassortants, we could rescue only 5 reassortant viruses, either due to difficulty in cloning (PB1 of H5N1 virus) or genetic incompatibility (NP-H5/H9 and NA-H5/H9). Results revealed higher replication rates for the H9N2 virus having the NS segment of H5N1 virus. The lowest survival rate in both SPF eggs and SPF chickens was associated with the H5N1 parent virus infection, followed by the HA-H5/H9 virus. Our findings also suggest that all other reassortant viruses were of lower pathogenicity than the wild type H5N1 virus.

During the surveillance for avian influenza viruses (AIVs) in live poultry markets in Eastern China, in 2016, a novel reassortant H7N6 AIV was isolated from a chicken. Phylogenetic analysis showed that this strain received its genes from H9N2, H7N9 and H5N6 AIVs infecting poultry in China. This strain showed moderate pathogenicity in mice and was able to replicate in mice without prior adaptation. Considering that this novel reassorted H7N6 virus was isolated from poultry in this study, it is possible that chickens play an important role in the generation of novel reassorted H7N6 AIVs.

H9N2 avian influenza virus (AIV) has caused significant losses in chicken flocks throughout china in recent years. There is a limited understanding of the genetic and antigenic characteristics of the H9N2 virus isolated in chickens in southwestern China. In this study a total of 12 field strains were isolated from tissue samples from diseased chickens between 2013 and 2016. Phylogenetic analysis of the Hemagglutinin (HA) and Neuraminidase (NA) nucleotide sequences from the 12 field isolates and other reference strains showed that most of the isolates in the past four years could be clustered into a major branch (HA-branch A and NA-branch I) in the Clade h9.4.2 lineages. These sequences are accompanied by nine and seven new amino acids mutations in the HA and NA proteins, respectively, when compared with those previous to 2013. In addition, four new isolates were grouped into a minor branch (HA-branch B) in the Clade h9.4.2 lineages and two potential N-glycosylation sites were observed due to amino acid mutations in the HA protein. Three antigenic groups (1-3), which had low antigenic relatedness with two commonly used vaccines in China, were identified among the 12 isolates by antigenMap analysis. Immunoprotection testing showed that those two vaccines could efficiently prevent the shedding of branch A viruses but not branch B viruses. In conclusion, these results indicate the genotype of branch B may become epidemic in the next few years and that a new vaccine should be developed for the prevention of H9N2 AIV., Competing Interests: The authors have declared that no competing interests exist.

SD/818 and SD/196 are H9N2 influenza virus strains isolated from chickens from the same farm at different times that exhibited similar genetic evolution. However, strain SD/818 exhibited higher pathogenicity in chickens than strain SD/196 and other H9N2 influenza virus epidemic strains from China. The expression of cytokines is an important host defence mechanism following viral infection and their intensity is a major determinant of viral pathogenicity. To elucidate the mechanism underlying the increased pathogenicity of strain SD/818 from the host's perspective, viral replication and cytokine expression were dynamically studied using real-time quantitative reverse transcription PCR in chickens infected with strain SD/818 compared with chickens infected with strain SD/196 in this study. The results showed that the replication of strain SD/818 and the expressions of IL-1β, IL-6, TNF-α, IFN-α and IFN-β induced by strain SD/818 were higher than those induced by strain SD/196 in the chicken host system. Expression of these cytokines in chickens coincided with or followed virus replication. These results suggested that high-level viral replication and pro-inflammatory cytokine expression (but not decreased type I IFN expression) were associated with the higher pathogenicity of strain SD/818 in chickens.

To determine the role of the potential glycosylation site NA264N, which has been shown to be prevalent in recent Chinese H9N2 isolates, four reverse genetic viruses, rgWS1-NA264N, rgWS1-NA264H, rgBJ-NA264H and rgBJ-NA264N, were rescued. Growth kinetics showed that viruses with NA264H grew faster than viruses with NA264N. Mouse studies revealed that rgBJ-NA264H replicated to a significantly higher titer than rgBJ-NA264N at 3dpi. Notably, in contact chickens, rgBJ-NA264H and rgWS1-NA264H shed significantly more virus than rgBJ-NA264N at 6dpi from the larynx and rgWS1-NA264N at 4dpi from the cloaca, respectively. The present study demonstrates that NA264N affects viral replication of H9N2., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveillance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools) were 5.8% (CI 5.4-6.0). Subtyping was performed on 468 pooled swabs with M gene Ct<26. No influenza H7 was detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interaction between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11 partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 distinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Vietnam are warranted to assess H9 endemicity elsewhere in the country., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Zoonotic infections by avian influenza viruses occur at the human-poultry interface, but the modes of transmission have not been fully investigated. We assessed the potential for airborne and fomite transmission at live poultry markets in Guangzhou city and in Hong Kong Special Administrative Region (SAR), China, during 2014 and 2015. Viral genome and infectious avian influenza A viruses of H5N6, H7N9, and H9N2 subtypes were detected predominantly from particles larger or equal to 1 μm in diameter in the air sampled with cyclone-based bioaerosol samplers at the live poultry markets in Guangzhou. Influenza A(H9N2) viruses were ubiquitously isolated every month during the study period from air and environmental swabs, and different lineages of H9N2 virus were isolated from markets where chickens and minor land-based poultry were sold. The use of de-feathering devices increased the quantity of virus-laden airborne particles while market closure reduced the amount of such particles. The results highlight the possibility of airborne transmission of avian influenza viruses among poultry or from poultry to humans within such settings. This may explain epidemiological observations in which some patients with H7N9 infection reported being in markets but no direct contact with live poultry or poultry stalls., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of The Authors, 2016.)

In this study, respiratory viral pathogens were screened using real-time RT-PCR in 86 broiler chicken flocks suffering from respiratory diseases problems in 4 Egyptian governorates between January 2012 and February 2014. The mortality rates in the investigated flocks ranged from 1 to 47%. Results showed that mixed infection represented 66.3% of the examined flocks. Mixed infectious bronchitis (IBV) and avian influenza (AI)-H9N2 viruses were the most common infection (41.7%). Lack of AI-H9N2 vaccination and high rates of mixed infections in which AI-H9N2 is involved indicate an early AI-H9N2 infection with a potential immunosuppressive effect that predisposes for other viral infections. High pathogenic AI-H5N1 and virulent Newcastle disease virus (vNDV) infections were also detected (26.7% and 8.1%, respectively). Interestingly, co-infection of AI-H9N2 with either AIV-H5N1 or vNDV rarely resulted in high mortality. Partial cell-mediated immunity against similar internal AI genes, as well as virus interference between AI and vNDV, could be an explanation for this. Highly prevalent IBV and AI-H9N2 were isolated and were molecularly characterized based on S1 gene hypervariable region 3 ( HVR3: ) and hemagglutinin gene (HA) sequences, respectively. IBV strains were related to the variant group of IBV with multiple mutations in HVR3. Though AI-H9N2 viruses showed low rate of evolution in comparison to recent strains, few amino acid substitutions indicative of antibody selection pressure were observed in the HA gene. In conclusion, mixed viral infections, especially with IBV and AI-H9N2 viruses, are the predominant etiology of respiratory disease problems in broiler chickens in Egypt. Further investigations of the role of AI, IBV, and ND viruses' co-infections and interference in terms of altering the severity of clinical signs and lesions and/or generating novel reassortants within each virus are needed., (© 2016 Poultry Science Association Inc.)

Avian influenza is a highly contagious respiratory disease of poultry caused by influenza A viruses, family Orthomyxoviridae. H9N2 avian influenza outbreaks are a major problem of the poultry industry in Iran. To determine the genetic differences between field viruses and the vaccine strain, the genomes of four strains isolated in 2011 from vaccinated broiler flocks with a history of respiratory illness were sequenced. Genetic and serological comparisons were made. Sequence analysis of the hemagglutinin (HA) and neuraminidase (NA) genes indicated that the isolated strains shared nucleotide homologies of 91.6-93.9 and 90.2-91.7% with the vaccine strain, respectively. Phylogenetic analyses of HA and NA genes showed that all strains isolated in this study fell into the same group and belonged to the influenza A virus (A)/quail/Hong Kong/G1/97 H9N2 sublineage. Several amino acids have changed at the antigenic sites in HA in the field viruses. Extra potential glycosylation sites were observed in the HA and NA proteins expressed by the current isolates relative to those in the vaccine strain. The deduced amino acid sequence at the cleavage site of HA in recent isolates is the KSSR/GLF motif, whereas it is RSSR/GLF in the vaccine strain. A serological analysis revealed that the currently circulating strains are antigenically distinct from the vaccine strain. These results suggest that the commercial vaccine is insufficiently genetically and antigenically similar to the viruses currently circulating in the region. These findings confirm that it is important to monitor the genetic and antigenic variations in H9N2 influenza viruses when selecting a vaccine strain.

Outbreaks of pandemic H1N1 2009 (pH1N1) in turkeys have been reported in several countries. Co-infection of pH1N1 and avian H9N2 influenza viruses in turkeys provide the opportunity for their reassortment, and novel reassortant viruses might further be transmitted to other avian species. However, virulence and transmission of those reassortant viruses in poultry remain unclear. In the present study, we generated 16 single-gene reassortant influenza viruses including eight reassortants on the pH1N1 background by individual replacement with a corresponding gene segment from H9N2 and eight reassortants on the H9N2 background replaced individually with corresponding gene from pH1N1, and characterized reassortants viruses in turkeys and chickens. We found that the pH1N1 virus dramatically increased its infectivity and transmissibility in turkeys and chickens after introducing any gene (except for PB2) from H9N2 virus, and H9N2 virus acquired single gene (except for HA) of pH1N1 almost did not influence its replication and transmission in turkeys and chickens. Additionally, 13 reassortant viruses transmitted from turkeys to chickens. Our results indicate that turkeys and chickens are susceptible to pH1N1-H9N2 reassortant viruses, and mixing breeding of different avian species would facilitate the transmission of these reassortant viruses.

Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus.

Phylogenetic analysis of influenza viruses collected during December 2009-February 2010 from chickens in live poultry retail shops in Lahore, Pakistan, showed influenza A(H9N2) lineage polymerase and nonstructural genes generate through inter- and intrasubtypic reassortments. Many amino acid signatures observed were characteristic of human isolates; hence, their circulation could enhance inter- or intrasubtypic reassortment.

To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity of rPR8-NS(0028) might be associated with the lower virus titer and IFN-β level in the lungs of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028. In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous monitoring of their mammalian pathogenicity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant's internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010-2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSR↓GLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSR↓GLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.

Since H9N2 avian influenza virus (AIV) was first isolated in Guangdong province of China, the virus has been circulating in chicken flocks in mainland China. However, a systematic phylogenetic analysis of H9N2 AIV from chickens in Shandong of China has not been conducted. Based on hemagglutinin (HA) gene sequences of H9N2 AIVs isolated from chickens in Shandong of China between 1998 and 2013, genetic evolution of 35 HA gene sequences was systematically analyzed in this study. Our findings showed that the majority of H9N2 AIVs (21 out of 35) belonged to the lineage h9.4.2.5. Most of isolates (33 out of 35) had a PSRSSR↓GLF motif in HA cleavage site. Importantly, 29 out of these 35 isolates had an amino acid exchange (Q226L) in the receptor-binding site. The substitution showed that H9N2 AIVs had the potential affinity to bind to human-like receptor. The currently prevalent H9N2 AIVs in Shandong belonged to the lineage h9.4.2.5 which are different from the vaccine strain SS/94 clade h9.4.2.3. Therefore, the long-term surveillance of H9N2 AIVs is of significance to combat the possible H9N2 AIV outbreaks.

Avian influenza viruses of subtype H9N2 are widely prevalent in poultry in many Asian countries, and the segmented nature of the viral genome results in multiple distinct genotypes via reassortment. In this study, genetic evolution of H9N2 viruses circulating in eastern China during 2007-2013 was analyzed. The results showed that the diversity of the gene constellations generated six distinct genotypes, in which a novel genotype (S) bearing the backbone of A/chicken/Shanghai/F/98-like viruses by acquiring A/quail/Hong Kong/G1/97-like polymerase basic subunit 2 and matrix genes has gradually established its ecological niche and been consistently prevalent in chicken flocks in eastern China since its first detection in 2007. Furthermore, genotype S possessed the peculiarity to donate most of its gene segments to other emerging influenza A viruses in China, including the novel reassortant highly pathogenic avian influenza H5N2, the 2013 novel H7N7, H7N9 and the latest reassortant H10N8 viruses, with potential threat to poultry industry and human health., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Three H9N2 avian influenza viruses were isolated from the Dongting Lake wetland, among which one was from fresh egret feces, the other two were from chicken cloacal swabs in poultry markets. Phylogenetic analyses suggested that eight genes of the egret-derived H9N2 virus might come from Korean-like or American-like lineages. The two poultry-derived H9N2 viruses were reassortants between the CK/BJ/94-like and G1-like viruses. Except the PB1 genes (90.6%), the nucleotide sequence of other internal genes of the two viruses exhibited high homology (>95%). In addition, they also exhibited high homology (96-98.3%) with some genes of the H7N9 virus that caused an epidemic in China in 2013. Nucleotide sequence of the poultry-derived and egret-derived H9N2 viruses shared low homology. Infection studies showed that the egret-derived H9N2 virus was non-pathogenic to both mice and chickens, and the virus was unable to infect chickens even through 8 passages continuously in the lung. On the other hand, the chickens infected by poultry-derived viruses showed obvious clinical symptoms and even died; the infected mice showed no noticeable clinical symptoms and weight loss, but viruses could be detected in their lungs. In conclusion, for the egret-derived H9N2 virus, it would take a long adaptation process to achieve cross-species transmission in poultry and mammals. H9N2 viruses isolated at different times from the same host species in the same geographical region presented different evolutionary status, and virus isolated from different hosts in the same geographical region exhibited genetic diversity. Therefore, it is important to continue the H9N2 virus surveillance for understanding their evolutionary trends so as to provide guidance for disease control and prevention.

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.