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Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.

Nanoparticles have gained attention as potential antiviral agents, but the effects of graphene oxide nanoparticles (GONPs) on influenza virus remain unclear. In this study, we evaluated the antiviral activity of GONPs against influenza virus strain A/Hunan-Lengshuitan/11197/2013(H9N2). Our results show that GONPs with a diameter of 4 nm exerted an antiviral effect, whereas those with a diameter of 400 nm had no effect. Treatment with 4-nm GONPs reduced viral titers by more than 99% and inhibited viral nucleoprotein expression in a dose-dependent manner. We also confirmed that 4-nm GONPs inhibited the infectivity of H9N2 in MDCK cells. A transmission electron microscopic analysis revealed morphological abnormalities in the GONP-treated virus, including the destruction of the envelope glycoprotein spikes and an irregular shape, suggesting that GONPs cause the destruction of the viral coat proteins. Our results highlight the potential utility of GONPs in the prevention and treatment of viral infections, especially those of emerging and re-emerging viruses., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.)

Objectives: This study sought to detect and characterize influenza A (IAV) and influenza D (IDV) viruses circulating among commercial birds and shop owners in Pakistan's live bird markets., Methods: Oropharyngeal swabs (n = 600; n = 300 pools) collected from poultry and nasopharyngeal swabs (n = 240) collected from poultry workers were studied for molecular evidence of IAV and IDV using real-time and conventional real-time reverse transcription polymerase chain reaction protocols., Results: Nineteen (6.3%) poultry pools were positive for IAV and 73.9% of these were positive for H9N2 subtypes. Two (0.83%) poultry workers had evidence of IAV, and both were also H9N2 subtypes. The poultry and human IAV-positive specimens all clustered phylogenetically by Sanger and next-generation sequencing with previously detected H9N2 poultry isolates. No field specimens were positive for IDV., Conclusion: H9N2 IAV is likely enzootic in Punjab Province Pakistan's live bird markets and may be colonizing the noses of workers and market visitors. Regular monitoring for avian influenza-associated human illness in Punjab seems to be a needed public measure., Competing Interests: Declarations 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 article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Avian influenza outbreaks, including ones caused by highly pathogenic A(H5N1) clade 2.3.4.4b viruses, have devastated animal populations and remain a threat to humans. Risk elements assessed for emerging influenza viruses include their susceptibility to approved antivirals. Here, we screened >20,000 neuraminidase (NA) or polymerase acidic (PA) protein sequences of potentially pandemic A(H5Nx), A(H7Nx), and A(H9N2) viruses that circulated globally in 2010-2023. The frequencies of NA or PA substitutions associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NA inhibitors (NAIs) (oseltamivir, zanamivir) or a cap-dependent endonuclease inhibitor (baloxavir) were low: 0.60% (137/22,713) and 0.62% (126/20,347), respectively. All tested subtypes were susceptible to NAIs and baloxavir at sub-nanomolar concentrations. A(H9N2) viruses were the most susceptible to oseltamivir, with IC 50 s 3- to 4-fold lower than for other subtypes (median IC 50 : 0.18 nM; n = 22). NA-I222M conferred RI of A(H5N1) viruses by oseltamivir (with a 26-fold IC 50 increase), but NA-S246N did not reduce inhibition. PA-E23G, PA-K34R, PA-I38M/T, and the previously unreported PA-A36T caused RI by baloxavir in all subtypes tested. Avian A(H9N2) viruses endemic in Egyptian poultry predominantly acquired PA-I38V, which causes only a <3-fold decrease in the baloxavir EC 50 and fails to meet the RI criteria. PA-E199A/D in A(H7Nx) and A(H9N2) viruses caused a 2- to 4-fold decrease in EC 50 (close to the borderline for RI) and should be closely monitored. Our data indicate antiviral susceptibility is high among avian influenza A viruses with pandemic potential and present novel markers of resistance to existing antiviral interventions., 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 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.)

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.)

While mammals can be infected by influenza A virus either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of the pathogen. So far most of the knowledge on influenza virus dynamics was however gained on mammalian models. In this study, we infected turkeys using a low pathogenic avian influenza virus and determined the infection dynamics with a target-cell limited model. Results showed that turkeys had a different set of infection characteristics, compared with humans and ponies. The viral clearance rates were similar between turkeys and ponies but higher than that in humans. The cell death rates and cell to cell transmission rates were similar between turkeys and humans but higher than those in ponies. Overall, this study indicated the variations of within-host dynamics of influenza infection in avian, humans, and other mammalian systems., 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 sustained circulation of H9N2 avian influenza viruses (AIVs) poses a significant threat for contributing to a new pandemic. Given the temporal and spatial uncertainty in the antigenicity of H9N2 AIVs, the immune protection efficiency of vaccines remains challenging. By developing an antigenicity prediction method for H9N2 AIVs, named PREDAC-H9, the global antigenic landscape of H9N2 AIVs was mapped. PREDAC-H9 utilizes the XGBoost model with 14 well-designed features. The XGBoost model was built and evaluated to predict the antigenic relationship between any two viruses with high values of 81.1 %, 81.4 %, 81.3 %, 81.1 %, and 89.4 % in accuracy, precision, recall, F1 value, and area under curve (AUC), respectively. Then the antigenic correlation network (ACnet) was constructed based on the predicted antigenic relationship for H9N2 AIVs from 1966 to 2022, and ten major antigenic clusters were identified. Of these, four novel clusters were generated in China in the past decade, demonstrating the unique complex situation there. To help tackle this situation, we applied PREDAC-H9 to calculate the cluster-transition determining sites and screen out virus strains with the high cross-protective spectrum, thus providing an in silico reference for vaccine recommendation. The proposed model will reduce the clinical monitoring workload and provide a useful tool for surveillance and control of H9N2 AIVs., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests. There is no financial/personal interest or belief that could affect their objectivity., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Influenza A/H9 viruses circulate worldwide in wild and domestic avian species, continuing to evolve and posing a zoonotic risk. A substantial increase in human infections with A/H9N2 subtype avian influenza viruses (AIVs) and the emergence of novel reassortants carrying A/H9N2-origin internal genes has occurred in recent years. Different names have been used to describe the circulating and emerging A/H9 lineages. To address this issue, an international group of experts from animal and public health laboratories, endorsed by the WOAH/FAO Network of Expertise on Animal Influenza, has created a practical lineage classification and nomenclature system based on the analysis of 10,638 hemagglutinin sequences from A/H9 AIVs sampled worldwide. This system incorporates phylogenetic relationships and epidemiologic characteristics designed to trace emerging and circulating lineages and clades. To aid in lineage and clade assignment, an online tool has been created. This proposed classification enables rapid comprehension of the global spread and evolution of A/H9 AIVs.

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, resulting in significant economic losses and numerous fatalities. Current vaccines, typically administered through injection, provide limited protection due to the frequent antigenic shift and drift of IAV strains. Therefore, the development of alternative broad-spectrum vaccine strategies is imperative. Lactic acid bacteria (LAB) represent promising candidates for vaccine engineering due to their low cost, high safety profile, and suitability for oral administration. In this study, we identified a strain of Lactobacillus plantarum (Lp) that is resistant to acid and bile salts and capable of colonizing the intestines of mice. Subsequently, we employed the RecE/T gene editing system to integrate headless hemagglutinins (mini-HA) into the genome of Lp, generating Lp-mini-HA-SP. Remarkably, immunization with Lp-mini-HA-SP elicited serum IgG antibody responses and conferred immune protection against H9N2 and H1N1 influenza virus challenges. Collectively, our findings offer a novel approach for the development of orally administered IAV vaccines and hold significant potential for future drug development endeavors., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (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.)

The influenza virus neuraminidase (NA) protein is responsible for actively cleaving the sialic acid (SA) bound to the viral hemagglutinin. In the present study, we identified a combination of five novel amino acid substitutions in the NA, conferring increased substrate binding and altered surface characteristics to a low pathogenic avian influenza (LPAI) H9N2 virus strain. The H9N2 strain reported from India, A/Environmental/India/1726265/2017 (H9N2-1726265) showed the combination of amino acid substitutions T149I, R249W, G346A, W403R and G435R, which were in the vicinity of the enzyme active site cavity. The strain A/chicken/India/99321/2009 (H9N2-99321) did not show these substitutions and was used for comparison. Virus elution was studied using turkey red blood cells (tRBCs). NA enzyme kinetics assays were carried out using the MUNANA substrate, which is an SA analogue. Homology modelling and molecular docking were performed to determine alterations in the surface characteristics and substrate binding. H9N2-1726265 showed enhanced elution from tRBCs. Enzyme kinetics revealed a lower K M of H9N2-1726265 (111.5 μM) as compared to H9N2-99321 (135.2 μM), indicating higher substrate binding affinity of H9N2-1726265, due to which the NA enzyme cleaved the SA more efficiently, leading to faster elution. Molecular docking revealed a greater number of binding interactions of H9N2-1726265 to SA as compared to H9N2-99321 corroborating the greater substrate binding affinity. Changes in the surface charge, hydrophobicity, and contour, were observed in H9N2-1726265 NA due to the five substitutions. Thus, the novel combination of five amino acids near the sialic acid binding site of NA, resulted in altered surface characteristics, higher substrate binding affinity, and virus elution., (Copyright © 2024 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 (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475 LRGVRVSK 482 and 528 TITYSSPMMW 537 . Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

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

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs., (© 2024. The Author(s).)

H9N2 avian influenza viruses (AIVs) affect both poultry and humans on a global level, and they are especially prevalent in Egypt. In this study, we sequenced the entire genome of AIV H9N2 isolated from chickens in Egypt in 2021, using next-generation sequencing (NGS) technology. Phylogenetic analysis of the resulting sequences showed that the studied strain was generally monophyletic and grouped within the G1 sublineage of the Eurasian lineage. Four segments (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acidic [PA], and non-structural [NS]) were related to Egyptian genotype II, while the nucleoprotein (NP), neuraminidase (NA), matrix (M), and haemagglutinin (HA) segments were related to Egyptian genotype I. Molecular analysis revealed that HA protein contained amino acid residues (191H and 234L) that suggested a predilection for attaching to human-like receptors. The antigenic sites of HA had two nonsynonymous mutations: V194I at antigenic site A and M40K at antigenic site B. Furthermore, the R403W and S372A mutations, which have been observed in H3N2 and H2N2 strains that caused human pandemics, were found in the NA protein of the detected strain. The internal proteins contained virulence markers: 504V in the PB2 protein, 622G, 436Y, 207K, and 677T in the PB1 protein, 127V, 550L, and 672L in PA protein, and 64F and 69P in the M protein. These results show that the detected strain had undergone intrasubtype reassortment. Furthermore, it contains changes in the viral proteins that make it more likely to be virulent, raising a question about the tendency of AIV H9N2 to become highly pathogenic in the future for both poultry and humans., (© 2024. The Author(s).)

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health., Competing Interests: The authors declare no conflict of interest.

The global distribution of avian respiratory viruses highlights the need for effective surveillance programs and international collaboration to monitor viral circulation and implement timely control measures. In the current study, we aim to provide a comprehensive overview of avian respiratory viral infections in the poultry flocks in Jordan, focusing on the major viruses involved, their epidemiology, clinical manifestations, and evolution based on viroinformatics that will be helpful to improve the diagnostic methods, and control strategies including vaccines in the region. In this research, various poultry broiler groups in Jordan experiencing respiratory symptoms were tested for respiratory viral pathogens from January 2021 to February 2022. The mortality rates observed in the examined groups varied between 6% and 40%. The identified strains were authenticated using the RT-qPCR assay. Furthermore, they underwent in-depth characterisation through the sequencing of the complete spike (S1) gene for infectious bronchitis virus (IBV) and the haemagglutinin (HA) gene for avian influenza virus (AIV) subtype H9N2. Co-infection of IBV and AIV H9N2 viruses was detected through molecular analysis. The IBV strains showed affiliation with the variant groups GI-16 (3 strains) and GI-23 (9 strains) and exhibited numerous mutations. Meanwhile, H9N2 avian influenza viruses displayed various changes in amino acids within the HA gene, suggesting the influence of antibody-driven selection pressure. The phylogenetic analysis revealed that the H9N2 viruses identified in this investigation shared close genetic ties with EG3 (3 strains) and the Middle East group (ME1; 8 strains). These strains have been recently found in Jordan and nearby countries in the Middle East. Moreover, their HA genes exhibited similarities to viruses belonging to the G1-like lineage. In conclusion, avian respiratory viral infections remain a significant concern for the poultry industry, requiring constant vigilance and proactive measures to minimise their impact. Continued surveillance, robust diagnostic methods, effective vaccines, and international cooperation are essential components of a comprehensive approach to combat avian respiratory viral infections (AI, IBV, ND and ILT 'viruses) and safeguard avian health and global poultry production., (Copyright © 2024 The Authors. Published by Elsevier Inc. 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.

Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.

Avian H9N2 viruses have wide host range among the influenza A viruses. However, knowledge of H9N2 mammalian adaptation is limited. To explore the molecular basis of the adaptation to mammals, we performed serial lung passaging of the H9N2 strain A/chicken/Hunan/8.27 YYGK3W3-OC/2018 (3W3) in mice and identified six mutations in the hemagglutinin (HA) and polymerase acidic (PA) proteins. Mutations L226Q, T511I, and A528V of HA were responsible for enhanced pathogenicity and viral replication in mice; notably, HA-L226Q was the key determinant. Mutations T97I, I545V, and S594G of PA contributed to enhanced polymerase activity in mammalian cells and increased viral replication levels in vitro and in vivo. PA-T97I increased viral polymerase activity by accelerating the viral polymerase complex assembly. Our findings revealed that the viral replication was affected by the presence of PA-97I and/or PA-545V in combination with a triple-point HA mutation. Furthermore, the double- and triple-point PA mutations demonstrated antagonistic effect on viral replication when combined with HA-226Q. Notably, any combination of PA mutations, along with double-point HA mutations, resulted in antagonistic effect on viral replication. We also observed antagonism in viral replication between PA-545V and PA-97I, as well as between HA-528V and PA-545V. Our findings demonstrated that several antagonistic mutations in HA and PA proteins affect viral replication, which may contribute to the H9N2 virus adaptation to mice and mammalian cells. These findings can potentially contribute to the monitoring of H9N2 field strains for assessing their potential risk in mammals., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

(1) Background: Avian influenza has attracted widespread attention because of its severe effect on the poultry industry and potential threat to human health. The H9N2 subtype of avian influenza viruses was the most prevalent in chickens, and there are several commercial vaccines available for the prevention of the H9N2 subtype of avian influenza viruses. However, due to the prompt antigenic drift and antigenic shift of influenza viruses, outbreaks of H9N2 viruses still continuously occur, so surveillance and vaccine updates for H9N2 subtype avian influenza viruses are particularly important. (2) Methods: In this study, we constructed a stable Chinese hamster ovary cell line (CHO) to express the H9 hemagglutinin (HA) protein of the major prevalent H9N2 strain A/chicken/Daye/DY0602/2017 with genetic engineering technology, and then a subunit H9 avian influenza vaccine was prepared using the purified HA protein with a water-in-oil adjuvant. (3) Results: The results showed that the HI antibodies significantly increased after vaccination with the H9 subunit vaccine in specific-pathogen-free (SPF) chickens with a dose-dependent potency of the immunized HA protein, and the 50 μg or more per dose HA protein could provide complete protection against the H9N2 virus challenge. (4) Conclusions: These results indicate that the CHO expression system could be a platform used to develop the subunit vaccine against H9 influenza viruses in chickens.

Importance: Despite the accumulation of evidence showing that airborne transmissible influenza A virus (IAV) typically has a lower pH threshold for hemagglutinin (HA) fusion activation, the underlying mechanism for such a link remains unclear. In our study, by using a pair of isogenic recombinant A(H9N2) viruses with a phenotypical difference in virus airborne transmission in a ferret model due to an acid-destabilizing mutation (HA1-Y17H) in the HA, we demonstrate that an acid-stable A(H9N2) virus possesses a multitude of advantages over its less stable counterpart, including better fitness in the ferret respiratory tract, more effective aerosol emission from infected animals, and improved host susceptibility. Our study provides supporting evidence for the requirement of acid stability in efficient airborne transmission of IAV and sheds light on fundamental mechanisms for virus airborne transmission., Competing Interests: The authors declare no conflict of interest.

The reassortment between avian H9N2 and Eurasian avian-like (EA) H1N1 viruses may have potentially changed from avian-to-mammals adaptation. This study generated 20 reassortant viruses with the introduction of H1N1/2009 internal genes from EA H1N1 virus into H9N2 virus. 12 of these recovered the replication capability both in the lungs and turbinate samples. 10 of 12 obtained PA gene segments from the ribonucleoprotein (RNP) complexes of the EA H1N1 virus, and 3 exhibited extreme virulence. Specially, the combination of PB2, PA and NP genes could overcome the species-specific restriction in human cells. Analysis of the polymerase activities found that introduction of the PA gene resulted in increased polymerase activity. These findings indicated that RNP complexes from EA H1N1 virus could confer an adaptation advantage and high compatibility to avian H9N2 virus. This raises new concerns for public health due to the possible coexistence of H9N2 and EA H1N1 viruses in dogs., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: The continued emergence of human infections of H9N2 avian influenza virus (AIV) poses a serious threat to public health. The prevalent Y280/G9 lineage of H9N2 AIV in Chinese poultry can directly bind to human receptors, increasing the risk of spillover infections to humans. Since 2013, the number of human cases of H9N2 avian influenza has been increasing continuously, and in 2021, China reported the highest number of human cases, at 25., Methods: In this study, we analyzed the age, geographic, temporal, and sex distributions of humans with H9N2 avian influenza in 2021 using data from the National Influenza Center (Beijing, China). We also conducted evolutionary, gene homology, and molecular characterization analyses of the H9N2 AIVs infecting humans., Results: Our findings show that children under the age of 12 accounted for 80% of human cases in 2021, and females were more frequently affected than males. More cases occurred in winter than in summer, and most cases were concentrated in southern China. Human-infecting H9N2 viruses showed a high level of genetic homology and belonged to the prevalent G57 genotype. Several additional α2,6-SA-binding sites and sites of mammalian adaptation were also identified in the genomes of human-infecting H9N2 viruses., Discussion: Therefore, continuous monitoring of H9N2 AIV and the implementation of further measures to control the H9N2 virus in poultry are essential to reduce the interspecies transmission of the virus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tan, Zeng, Xie, Li, Liu, Yang, Yang and Wang.)

ABSTRACT Live poultry markets (LPMs) are regarded as hubs for avian influenza virus (AIV) transmission in poultry and are a major risk factor in human AIV infections. We performed an AIV surveillance study at a wholesale LPM, where different poultry species were sold in separate stalls, and nine retail LPMs, which received poultry from the wholesale LPM but where different poultry species were sold in one stall, in Guangdong province from 2017 to 2019. A higher AIV isolation rate was observed at the retail LPMs than the wholesale LPM. H9N2 was the dominant AIV subtype and was mainly present in chickens and quails. The genetic diversity of H9N2 viruses was greater at the retail LPMs, where a complex system of two-way transmission between different poultry species had formed. The isolated H9N2 viruses could be classed into four genotypes: G57 and the three novel genotypes, NG164, NG165, and NG166. The H9N2 AIVs isolated from chickens and quails at the wholesale LPM only belonged to the G57 and NG164 genotypes, respectively. However, the G57, NG164, and NG165 genotypes were identified in both chickens and quails at the retail LPMs. We found that the replication and transmission of the NG165 genotype were more adaptive to both poultry and mammalian models than those of its precursor genotype, NG164. Our findings revealed that mixed poultry selling at retail LPMs has increased the genetic diversity of AIVs, which might facilitate the emergence of novel viruses that threaten public health.

We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

Influenza virological surveillance was conducted in Bangladesh from January to December 2021 in live poultry markets (LPMs) and in Tanguar Haor, a wetland region where domestic ducks have frequent contact with migratory birds. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses. Additional LPAIs were found in both LPM (H4N6) and Tanguar Haor wetlands (H7N7). Genetic analyses of these LPAIs strongly suggested long-distance movement of viruses along the Central Asian migratory bird flyway. We also detected a novel clade 2.3.4.4b H5N1 virus from ducks in free-range farms in Tanguar Haor that was similar to viruses first detected in October 2020 in The Netherlands but with a different PB2. Identification of clade 2.3.4.4b HPAI H5N1 viruses in Tanguar Haor provides continued support of the role of migratory birds in transboundary movement of influenza A viruses (IAV), including HPAI viruses. Domestic ducks in free range farm in wetland areas, like Tangua Haor, serve as a conduit for the introduction of LPAI and HPAI viruses into Bangladesh. Clade 2.3.4.4b viruses have dominated in many regions of the world since mid-2021, and it remains to be seen if these viruses will replace the endemic clade 2.3.2.1a H5N1 viruses in Bangladesh.

The H9N2 subtype of the avian influenza virus (AIV) is one of the main subtypes of low pathogenic AIV, and it seriously affects the poultry breeding industry. Currently, vaccination is still one of China's main strategies for controlling H9N2 avian influenza. In this study, we selected MW548848.1 on the current popular main branch h9.4.2.5 as the reference strain, and we optimized the amino acid sequence of HA1 to make it suitable for expression in Bacillus subtilis. The B. subtilis expression vector showed good safety and stress resistance; therefore, this study constructed a recombinant B. subtilis expressing H9N2 HA1 protein and evaluated its immunogenicity in mice. The following results were obtained: the sIgA level of HA1 protein in small intestine fluid and the IgG level of PHT43-HA1/B. subtilis in serum were significantly improved (P < 0.01); PHT43-HA1/B. subtilis can cause a special immune response in mice; and cytokine detection interferon-gamma (IFN-γ) (P < 0.05) and Interleukin 2 (IL-2) (P < 0.01) expressions significantly increased. Additionally, the study found that PHT43-HA1/B. subtilis can alleviate the attack of H9N2 AIV in the spleen, lungs, and small intestine of mice. This study was the first to use an oral recombinant B. subtilis-HA1 vaccine candidate, and it provides theoretical data and technical reference for the creation of a new live vector vaccine against H9N2 AIV., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Influenza A virus is a negative-sense single-stranded RNA virus that belongs to Orthomyxoviridae family. Based on the antigenic characteristics of hemagglutinin (HA) and neuraminidase (NA) influenza viruses are classified into multiple subtypes. H9N2 belongs to the low pathogenic Avian Influenza Viruses (AIVs) and is one of the widely spread viruses in poultry, which can pose a threat to humans by directly infecting or providing internal genes for various zoonotic avian influenza strains. It has the potential to directly or indirectly participate in becoming an AIV that causes a human pandemic. When the virus enters a host, the innate immune system is activated first by pattern recognition receptors. The cytokines produced at the site of infection recruit innate immune cells and antigen-presenting cells and those cells subsequently transmit antigenic signals to adaptive immune cells (i.e. B cells and T cells), to trigger specific humoral and cellular immune responses. As a result, humoral and cellular immunity can clear virus and infected cells via antibody-mediated neutralization and cytotoxicity, respectively. Understanding how chicken immune systems respond to H9N2 is a top priority for effectively controlling the virus's spread and designing vaccines. In this review, we comprehensively discuss the role of the chicken immune system in defending against H9N2, and clarify the current limitations in understanding chicken immune responses to H9N2 virus, thereby providing potential directions for future research as research on the chicken respiratory mucosal immune system has been stagnant for more than 20 years especially on how the mucosal immune system in chicken responds to avian influenza.

Vaccination is a crucial prevention and control measure against H9N2 avian influenza viruses (AIVs) that threaten poultry production and public health. However, H9N2 AIVs in China undergo continuous antigenic drift of hemagglutinin (HA) under antibody pressure, leading to the emergence of immune escape variants. In this study, we investigated the molecular basis of the current widespread antigenic drift of H9N2 AIVs. Specifically, the most prevalent h9.4.2.5-lineage in China was divided into two antigenic branches based on monoclonal antibody (mAb) hemagglutination inhibition (HI) profiling analysis, and 12 antibody escape residues were identified as molecular markers of these two branches. The 12 escape residues were mapped to antigenic sites A, B, and E (H3 was used as the reference). Among these, eight residues primarily increased 3`SLN preference and contributed to antigenicity drift, and four of the eight residues at sites A and B were positively selected. Moreover, the analysis of H9N2 strains over time and space has revealed the emergence of a new antigenic branch in China since 2015, which has replaced the previous branch. However, the old antigenic branch recirculated to several regions after 2018. Collectively, this study provides a theoretical basis for understanding the molecular mechanisms of antigenic drift and for developing vaccine candidates that contest with the current antigenicity of H9N2 AIVs.

H9N2 influenza viruses are globally endemic in birds, and a sharp increase in human infections with H9N2 occurred during 2021 to 2022. In this study, we assess the antigenic and pathogenic impact of 23 hemagglutinin (HA) amino acid mutations. Our study reveals that three specific mutations, labeled R164Q, N166D, and I220T, are responsible for the binding of antibodies with escape mutations. Variants containing R164Q and I220T mutations increase viral replication in avian and mammalian cells. Furthermore, T150A and I220T mutations are found to enhance viral replication in mice, indicating that these mutations may have the potential to adapt mammals. Structure analysis reveals that residues 164 and 220 bearing R164Q and I220T mutations increase interactions with the surrounding residues. Our findings enrich current knowledge about the risk assessment regarding which predominant HA immune-escape mutations of H9N2 viruses may pose the greatest threat to the emergence of pandemics in birds and humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Interpreting real-time reverse transcription-polymerase chain reaction (rRT-PCR) results for human avian influenza A virus (AIV) detection in contaminated settings like live bird markets (LBMs) without serology or viral culture poses a challenge., Methods: During February-March 2012 and November 2012-February 2013, we screened workers at nine LBMs in Dhaka, Bangladesh, to confirm molecular detections of AIV RNA in respiratory specimens with serology. We tested nasopharyngeal (NP) and throat swabs from workers with influenza-like illness (ILI) and NP, throat, and arm swabs from asymptomatic workers for influenza virus by rRT-PCR and sera for seroconversion and antibodies against HPAI A(H5N1) and A(H9N2) viruses., Results: Among 1273 ILI cases, 34 (2.6%) had A(H5), 56 (4%) had A(H9), and six (0.4%) had both A(H5) and A(H9) detected by rRT-PCR. Of 192 asymptomatic workers, A(H5) was detected in eight (4%) NP and 38 (20%) arm swabs. Of 28 ILI cases with A(H5) or A(H9) detected, none had evidence of seroconversion, but one (3.5%) and 12 (43%) were seropositive for A(H5) and A(H9), respectively., Conclusion: Detection of AIV RNA in respiratory specimens from symptomatic and asymptomatic LBM workers without evidence of seroconversion or virus isolation suggests environmental contamination, emphasizing caution in interpreting rRT-PCR results in high viral load settings., Competing Interests: Declarations of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Importance: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species., Competing Interests: The authors declare no conflict of interest.

The occurrence of human infections caused by avian H9N2 influenza viruses has raised concerns regarding the potential for human epidemics and pandemics. The molecular basis of viral adaptation to a new host needs to be further studied. Here, the bases of nucleotides 627 and 701 of PB2 were changed according to the uncoverable purine-to-pyrimidine transversion to block the development of PB2 627K and 701N mutations during serial passaging in mice. The purpose of this experiment was to identify key adaptive mutations in polymerase and NP genes that were obscured by the widely known host range determinants PB2 627K and 701N. Mouse-adapted H9N2 variants were obtained via twelve serial lung-to-lung passages. Sequence analysis showed that the mouse-adapted viruses acquired several mutations within the seven gene segments (PB2, PB1, PA, NP, HA, NA, and NS). One variant isolate with the highest polymerase activity possessed three substitutions, PB2 S155N, PA S49Y and D347G, which contributed to the highly virulent and mouse-adaptative phenotype. Further studies demonstrated that these three mutations resulted in increased polymerase activity, viral transcription and replication in mammalian cells, severe interstitial pneumonia, excessive inflammatory cellular infiltration and increased growth rates in mice. Our results suggest that the substitution of these three amino acid mutations may be an alternative strategy for H9N2 avian influenza viruses to adapt to mammalian hosts. The continued surveillance of zoonotic H9N2 influenza viruses should also include these mammalian adaptation markers as part of our pandemic preparedness efforts., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-β, IL1β, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

BackgroundTwo human cases of avian influenza A (H3N8) virus infection were reported in China in 2022.AimTo characterise H3N8 viruses circulating in China in September 2021-May 2022.MethodsWe sampled poultry and poultry-related environments in 25 Chinese provinces. After isolating H3N8 viruses, whole genome sequences were obtained for molecular and phylogenetic analyses. The specificity of H3N8 viruses towards human or avian receptors was assessed in vitro. Their ability to replicate in chicken and mice, and to transmit between guinea pigs was also investigated.ResultsIn total, 98 H3N8 avian influenza virus isolates were retrieved from 38,639 samples; genetic analysis of 31 representative isolates revealed 17 genotypes. Viruses belonging to 10 of these genotypes had six internal genes originating from influenza A (H9N2) viruses. These reassorted viruses could be found in live poultry markets and comprised the strains responsible for the two human infections. A subset of nine H3N8 viruses (including six reassorted) that replicated efficiently in mice bound to both avian-type and human-type receptors in vitro. Three reassorted viruses were shed by chickens for up to 9 days, replicating efficiently in their upper respiratory tract. Five reassorted viruses tested on guinea pigs were transmissible among these by respiratory droplets.ConclusionAvian H3N8 viruses with H9N2 virus internal genes, causing two human infections, occurred in live poultry markets in China. The low pathogenicity of H3N8 viruses in poultry allows their continuous circulation with potential for reassortment. Careful monitoring of spill-over infections in humans is important to strengthen early-warning systems and maintain influenza pandemic preparedness.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. At present, consensus has been reached that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines in China. This project continues to monitor the evolution characteristics of H9N2 hemagglutinin (HA) genes in China over the past 3 yr. The results showed that the current circling H9N2 viruses were diversified into h9.4.2.5 subclade, which was genetically distant from commonly used commercial vaccine strains. Compared with vaccine strains or 2014 strains, more than 42.1% of the variable antigenic sites in recent 3 yr' strains have shown significant changes and these stacked changes have caused significant differences in antigenicity. We constructed a recombinant vaccine strain rCQY-GHHA, which uses A/Chicken/China/SichuanCQY/2014 as the framework and A/Chicken/China/SichuanGH/2020 strain, which meets the recent viral antigenic characteristics, as the HA gene donor. The recombinant strain was prepared as an oil-adjuvant inactivated vaccine following an industrial process. The results of the immune protection experiment showed that the rCQY-GHHA vaccine was better than the commercial vaccine strain SS in reducing the morbidity, pathological lesion, virus shedding, and viral load. These results provide a reference for the control of H9N2 AIV in China., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

H9N2 subtype of avian influenza virus (AIV) is primarily a bird virus, which is widespread in clinical avian disease, and reported in cases of human infection. As one of the surface proteins of AIV, the neuraminidase (NA) protein plays an important role mainly in viral budding. However, vaccine development and detection methods for NA of H9N2 AIVs are in urgent clinical need. In this study, a truncated NA gene (205-900 bp) was cloned from the NA sequence of H9N2 strain, and then expressed using pET-28a (+) vector. This purified recombinant NA protein was used to immunize BALB/c mice, and the monoclonal antibodies were screened through the indirect enzyme-linked immunosorbent assay (ELISA). Next, eight prokaryotic expression vectors were constructed for epitope identification. After cell fusion, three hybridoma cell lines producing the antibodies special to NA protein were screened by ELISA, western blotting, and indirect immunofluorescence; these were named 1B10, 2B6, and 5B2, respectively. Epitope scanning techniques were used to identify three B-cell epitopes recognized by these three monoclonal antibodies, 196 KNATASIIYDGMLVD 210 , 210 DSIGSWSKNIL 220 and 221 RTQESECVCI 230 . The subsequent homology analysis revealed the three epitopes were highly conserved in H9N2 AIV strains. The structural predictions of the antigenic epitopes indicated that all three epitopes were located in the catalytic region of NA. These results provide a basis for studying the function of the NA protein of H9N2 AIV and technical support for the development of a universal detection method based on anti-NA monoclonal antibodies.

Global poultry production is still severely affected by H9N2 avian influenza virus (AIV), and the development of a novel universal AIV vaccine is still urgently needed. Neuraminidase (NA) has recently been shown to be an efficient conserved protective antigen. In this study, we fused the extracellular region of the NA gene with a ferritin cassette (pYL281), which resulted in self-assembled 24-mer nanoparticles with the NA protein displayed outside the nanoparticles. In addition, a chicken dendritic cell-targeting nanobody-phage74 was also inserted ahead of the NA protein to yield pYL294. Incubation with chicken bone marrow-derived dendritic cells (chBMDCs) showed that the DC-targeting nanoparticles purified from the pYL294 strain significantly increased the maturation of chBMDCs, as shown by increased levels of CCL5, CCR7, CD83 and CD86 compared with nontargeting proteins. Then, a chicken study was performed using Salmonella oral administration together with intranasal boost with purified proteins. Compared with the other groups, oral immunization with Salmonella harboring pYL294 followed by intranasal boost with purified DC-targeting nanoparticles dramatically increased the humoral IgY and mucosal IgA antibody response, as well as increased the cellular immune response, as shown by elevated splenic lymphocyte proliferation and intracellular mRNA levels of IL-4 and IFN-γ. Finally, sequential immunization with DC-targeting nanoparticles showed increased protection against G57 subtype H9N2 virus challenge compared with other groups, as shown by significantly decreased virus RNA copy numbers in oropharyngeal washes (Days 3, 5 and 7 post challenge) and cloacal washes (Day 7), significantly decreased lung virus titers on Day 5 post challenge and increased body weight gains during the challenge., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Since the 2000s, the Y439 lineage of H9N2 avian influenza virus (AIV) has been the predominant strain circulating in poultry in Korea; however, in 2020, the Y280 lineage emerged and spread rapidly nationwide, causing large economic losses. To prevent further spread and circulation of such viruses, rapid detection and diagnosis through active surveillance programs are crucial. Here, we developed a novel H9 rRT-PCR assay that can detect a broad range of H9Nx viruses in situations in which multiple lineages of H9 AIVs are co-circulating. We then evaluated its efficacy using a large number of clinical samples. The assay, named the Uni Kor-H9 assay, showed high sensitivity for Y280 lineage viruses, as well as for the Y439 lineage originating in Korean poultry and wild birds. In addition, the assay showed no cross-reactivity with other subtypes of AIV or other avian pathogens. Furthermore, the Uni Kor-H9 assay was more sensitive, and had higher detection rates, than reference H9 rRT-PCR methods when tested against a panel of domestically isolated H9 AIVs. In conclusion, the novel Uni Kor-H9 assay enables more rapid and efficient diagnosis than the "traditional" method of virus isolation followed by subtyping RT-PCR. Application of the new H9 rRT-PCR assay to AI active surveillance programs will help to control and manage Korean H9 AIVs more efficiently., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

The infection and replication of avian influenza virus (AIV) in host cells is a complex biological process that involves the transport of viral genes through the host cell's transport systems. Actin, microtubules and vimentin are known to facilitate transport of endosomes to the perinuclear region, but the biological role of Keratin, another intermediate filament, in viral transport during AIV replication is not well understood. In this study, the viral NS2 protein was used as the target protein to identify the potential interacting proteins following GST-Pulldown method and protein mass spectrometry. It was discovered that Keratin10 interacted with NS2. Subsequently, it was found AIV infection did not affect the gene level or protein level of keratin10 in HeLa cells, but when Keratin10 was knocked down, the expressions of viral NP mRNA and protein were reduced, and the generation of offspring virus also was also decreased. Furthermore, in early viral infection, Keratin10 could aggregate and co-localize with NP proteins, suggesting that Keratin10 might be connected to early viral transport. Additionally, it was demonstrated that Keratin10 co-localized with Lamp1 and that AIV particles were trapped in late endosomes/Lysosomes after Keratin10 was knocked down. Finally, it was discovered that the knocking down Keratin10 in HeLa cells led to an increase in the acidic pH of endosomes and lysosomes, which prevented AIV from undergoing fusion and uncoating, and then inhibited the process of the viral infection. Overall, the results suggested that Keratin10 might play the critical role in the release of vRNPs from LEs/Ls and can affect the generation of offspring virus. The study provides the novel insights into the role of Keratin10 in the process of AIV infection and transmission, which may have implications for developing new strategies to against AIV infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

H11N9 viruses in wild birds might have provided the NA gene of human H7N9 virus in early 2013 in China, which evolved with highly pathogenic strains in 2017 and caused severe fatalities. To investigate the prevalence and evolution of the H11N9 influenza viruses, 16,781 samples were collected and analyzed during 2016-2020. As a result, a novel strain of influenza A (H11N9) virus with several characteristics that increase virulence was isolated. This strain had reduced pathogenicity in chicken and mice and was able to replicate in mice without prior adaptation. Phylogenetic analyses showed that it was a sextuple-reassortant virus of H11N9, H3N8, H3N6, H7N9, H9N2, and H6N8 viruses present in China, similar to the H11N9 strains in Japan and Korea during the same period. This was the H11N9 strain isolated from China most recently, which add a record to viruses in wild birds. This study identified a new H11N9 reassortant in a wild bird with key mutation contributing to virulence. Therefore, comprehensive surveillance and enhanced biosecurity precautions are particularly important for the prediction and prevention of potential pandemics resulting from reassortant viruses with continuous evolution and expanding geographic distributions., (© 2023. The Author(s).)

1. The H9N2 subtype avian influenza virus can infect both chickens and humans. Previous studies have reported a role for erythrocytes in immunity. However, the role of H9N2 against chicken erythrocytes and the presence of complement-related genes in erythrocytes has not been studied. This research investigated the effect of H9N2 on complement-associated gene expression in chicken erythrocytes.2. The expression of complement-associated genes ( C1s, C1q, C2, C3, C3ar1, C4, C4a, C5, C5ar1, C7, CD93 and CFD ) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative Real-Time PCR (qRT-PCR) was used to analyse the differential expression of complement-associated genes in chicken erythrocytes at 0 h, 2 h, 6 h and 10 h after the interaction between H9N2 virus and chicken erythrocytes in vitro and 3, 7 and 14 d after H9N2 virus nasal infection of chicks.3. Expression levels of C1q, C4, C1s, C2, C3, C5, C7 and CD93 were significantly up-regulated at 2 h and significantly down-regulated at 10 h. Gene expression levels of C1q, C3ar1, C4a, CFD and C5ar1 were seen to be different at each time point. The expression levels of C1q, C4, C1s, C2, C3, C5, C7, CFD, C3ar1, C4a and C5ar1 were significantly up-regulated at 7 d and the gene expression of levels of C3, CD93 and C5ar1 were seen to be different at each time point.4. The results confirmed that all the complement-associated genes were expressed in chicken erythrocytes and showed the H9N2 virus interaction with chicken erythrocytes and subsequent regulation of chicken erythrocyte complement-associated genes expression. This study reported, for the first time, the relationship between H9N2 and complement system of chicken erythrocytes, which will provide a foundation for further research into the prevention and control of H9N2 infection.

Background: Antibiotics are widely used for prophylactic therapy and for improving the growth performance of chicken. The problem of bacterial drug resistance caused by antibiotic abuse has previously attracted extensive attention; however, the influence of early-day use of prophylactic antibiotics on the gut microflora and on the disease resistance ability in chicks has not been explored. Here, we comprehensively evaluate the growth performance, gut microbial dynamics, level of antibiotic resistance genes (ARGs) in the gut microbial community, and resistance to H9N2 avian influenza virus (AIV) in chickens following long-term and short-term early-day prophylactic antibiotic treatment., Results: Unexpectedly, long-term prophylactic enrofloxacin treatment slowed the growth rate of chickens, whereas short-term antibiotics treatments were found to increase the growth rate, but these changes were not statistically significant. Strikingly, expansions of Escherichia-Shigella populations were observed in early-life prophylactic antibiotics-treated groups of chickens, which is in contrast to the general perception that antibiotics should control their pathogenicity in chicks. The gut microbiota composition of chickens treated long term with antibiotics or received early-day antibiotics treatment tend to be more dramatically disturbed compared to the gut microbiome of chickens treated with antibiotics for a short term at a later date, especially after H9N2 AIV infection., Conclusions: Our data provide evidence that early-day and long-term antibiotic treatments have a more adverse effect on the intestinal microbiome of chickens, compared to short-term late age antibiotic treatment. Furthermore, our metagenomic data reveal that both long-term and short-term antibiotic treatment increase the relative abundance of ARGs. Our findings highlight the adverse effects of prophylactic antibiotic treatment and provide a theoretical basis for the cautious administration of antibiotics in food-producing animal management. Video Abstract., (© 2023. The Author(s).)