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

Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-μl samples (concentration: 100 pg/ml-100 ng/ml) were needed to form the antigen-antibody complexes during 20-30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses., (© 2022. The Author(s).)

The enzootic and zoonotic nature of H9N2 avian influenza viruses poses a persistent threat to the global poultry industry and public health. In particular, the emerging sublineage h9.4.2.5 of H9N2 viruses has drawn great attention. In this study, we determined the effects of the flexibility at residues 226 and 227 in the hemagglutinin on the receptor avidity and immune evasion of H9N2 viruses. The solid-phase direct binding assay showed that residue 226 plays a core role in the receptor preference of H9N2 viruses, while residue 227 affects the preference of the virus for a receptor. Consequently, each of these two successive residues can modulate the receptor avidity of H9N2 viruses and influence their potential of zoonotic infection. The antigenic map based on the cross-hemagglutination inhibition (HI) titers revealed that amino acid substitutions at positions 226 or 227 appear to be involved in antigenic drift, potentially resulting in the emergence of H9N2 immune evasion mutants. Further analysis suggested that increased receptor avidity facilitated by residue 226Q or 227M resulted in a reduction in the HI titer. Among the four naturally-occurring amino acid combinations comprising QQ, MQ, LQ, and LM, the number of viruses with LM accounted for 79.64% of the sublineage h9.4.2.5 and the rescued virus with LM exhibited absolute advantages of in vitro and in vivo replication and transmission. Collectively, these data demonstrate that residues 226 and 227 are under selective pressure and their synergistic regulation of receptor avidity and antigenicity is related to the evolution of circulating H9N2 viruses.

Influenza A H9N2 virus causes economic loss to the poultry industry and has likely contributed to the genesis of H5N1 and H7N9 viruses. The neuraminidase (NA) of H9N2 virus, like haemagglutinin, is under antibody selective pressure and may undergo antigenic change; however, its antigenic structure remains to be elucidated. In this study, we used monoclonal antibodies (mAbs) to probe the H9N2 viral NA residues that are key for antibody binding/inhibition. These mAbs fell into three groups based on their binding/inhibition of the NA of H9N2 viruses isolated during 1999-2019: group I only bounded the NA of the early 2000 H9N2 viruses but possessed no neutralizing ability, group II bounded and inhibited the NA of H9N2 viruses isolated before 2012, and group III reacted with most or all tested H9N2 viruses. We showed that NA residue 356 is key for the recognition by group I mAbs, residues 344, 368, 369, and 400 are key for the binding/inhibition of NA by group II antibodies, whereas residues 248, 253, and the 125/296 combination are key for neutralizing antibodies in group III. Our findings highlighted NA antigenic change of the circulating H9N2 viruses, and provided data for a more complete picture of the antigenic structure of H9N2 viral NA.

Sub-Saharan Africa was historically considered an animal influenza cold spot, with only sporadic highly pathogenic H5 outbreaks detected over the last 20 years. However, in 2017, low pathogenic avian influenza A(H9N2) viruses were detected in poultry in Sub-Saharan Africa. Molecular, phylogenetic, and antigenic characterization of isolates from Benin, Togo, and Uganda showed that they belonged to the G1 lineage. Isolates from Benin and Togo clustered with viruses previously described in Western Africa, whereas viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Viruses from Benin exhibited decreased cross-reactivity with those from Togo and Uganda, suggesting antigenic drift associated with reduced replication in Calu-3 cells. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2). Therefore, viral genetic and antigenic surveillance in Africa is of paramount importance to detect further evolution or emergence of new zoonotic strains.

Pandemic influenza, typically caused by the reassortment of human and avian influenza viruses, can result in severe or fatal infections in humans. Timely identification of potential pandemic viruses must be a priority in influenza virus surveillance. However, the range of host species responsible for the generation of novel pandemic influenza viruses remains unclear. In this study, we conducted serological surveys for avian and human influenza virus infections in farmed mink and determined the susceptibility of mink to prevailing avian and human virus subtypes. The results showed that farmed mink were commonly infected with human (H3N2 and H1N1/pdm) and avian (H7N9, H5N6, and H9N2) influenza A viruses. Correlational analysis indicated that transmission of human influenza viruses occurred from humans to mink, and that feed source was a probable route of avian influenza virus transmission to farmed mink. Animal experiments showed that mink were susceptible and permissive to circulating avian and human influenza viruses, and that human influenza viruses (H3N2 and H1N1/pdm), but not avian viruses, were capable of aerosol transmission among mink. These results indicate that farmed mink could be highly permissive "mixing vessels" for the reassortment of circulating human and avian influenza viruses. Therefore, to reduce the risk of emergence of novel pandemic viruses, feeding mink with raw poultry by-products should not be permitted, and epidemiological surveillance of influenza viruses in mink farms should be urgently implemented.

H9N2 subtype avian influenza virus (AIV) is an ongoing threat causing substantial loss to the poultry industry and thus necessitating the development of safe and effective vaccines against AIV. Given that inactivated vaccines are less effective in activating the mucosal immune system, we aimed to generate a vaccine that can actively engage the mucosal immunity which is the front line of the immune system. We generated a group of flagellin-based hemagglutinin globular head (HA1) fusion proteins and characterized their immunogenicity and efficacy. We found that Salmonella typhimurium flagellin (fliC) lacking the hypervariable domain (called herein as HA1-ΔfliC) was recognized by TLR5 and induced a moderate innate immune response compared to N-terminus of fliC (HA1-fliC) and C-terminus of fliC (fliC-HA1). The HA1-ΔfliC protein had increased adherence to the nasal cavity and trachea than HA1-fliC and fliC-HA1 and significantly increased the HA-specific sIgA titers. Our in vivo results revealed that chickens treated with HA1-ΔfliC had a significantly reduced level of viral loads in the cloaca and throat compared with chickens treated with inactivated vaccine. Overall, these results revealed that HA1-ΔfliC can protect chickens against H9N2 AIV by eliciting the efficient mucosal immune responses., (Copyright © 2021. Published by Elsevier B.V.)

Influenza viruses cause severe respiratory infections in humans and birds, triggering global health concerns and economic burden. Influenza infection is a dynamic process involving complex biological host responses. The objective of this study was to illustrate global biological processes in ileum and cecal tonsils at early time points after chickens were infected with low pathogenic avian influenza virus (LPAIV) H9N2 through transcriptome analysis. Total RNA isolated from ileum and cecal tonsils of non-infected and infected layers at 12-, 24- and 72-h post-infection (hpi) was used for mRNA sequencing analyses to characterize differentially expressed genes and overrepresented pathways. Statistical analysis highlighted transcriptomic signatures significantly occurring 24 and 72 hpi, but not earlier at 12 hpi. Interferon (IFN)-inducible and IFN-stimulated gene (ISG) expression was increased, followed by continued expression of various heat-shock proteins (HSP), including HSP60, HSP70, HSP90 and HSP110. Some upregulated genes involved in innate antiviral responses included DDX60, MX1, RSAD2 and CMPK2. The ISG15 antiviral mechanism pathway was highly enriched in ileum and cecal tonsils at 24 hpi. Overall, most affected pathways were related to interferon production and the heat-shock response. Research on these candidate genes and pathways is warranted to decipher underlying mechanisms of immunity against LPAIV in chickens., (© 2021. The Author(s).)

Immunological memory is a cardinal feature of the immune system. The intestinal mucosa is the primary exposure and entry site of infectious organisms. For an effective and long-lasting safeguard, a robust immune memory system is required, especially by the mucosal immunity. It is well known that tissue-resident memory T cells (Trms) provide a first response against infections reencountered at mucosal tissues surfaces, where they accelerate pathogen clearance. However, their function in intestinal immunization remains to be investigated. Here, we report enhanced local mucosal and systemic immune responses through oral administration of H9N2 influenza whole inactivated virus (H9N2 WIV) plus Bacillus subtilis spores. Subsequently, H9N2 WIV plus spores led to the generation of CD103 + CD69 + Trms, which were independent of circulating T cells during the immune period. Meanwhile, we also found that Bacillus subtilis spores could stimulate Acrp30 expression in 3T3-L1 adipocytes. Moreover, spore-stimulated adipocyte supernatant also upregulated the expression of intercellular adhesion molecule-1 (ICAM1) in dendritic cells (DCs). Furthermore, the proportion of HA-tetramer + cells was severely curtailed upon suppressed ICAM1 expression, which also depended on HA-loaded DCs. Taken together, our data demonstrated that spore-promoted H9N2 WIV induced an immune response by enhancing Trms populations, which were associated with the activation of ICAM1 in DCs.

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

H9N2 avian influenza virus (AIV) has become endemic in many countries, causing great economic losses when co-infected with other pathogens. So far, several live vaccines based on Newcastle disease virus (NDV) vectors expressing influenza hemagglutinin (HA) have been developed. However, the thermostable recombinant NDV is rarely reported. In this study, using a thermostable NDV rAHR09 strain as the vector, three recombinant NDVs expressing native HA, chimeric HA ectodomain with transmembrane domain/C-terminal cytoplasmic tail domain from fusion protein of NDV, and HA ectodomain were generated, designated rAHR09-HA, rAHR09-HAF, and rAHR09-HAE. The MDT value of three recombinant NDVs was above 120 h, their ICPI value was about 0.03, and the recombinant NDVs were still infectious when treated for 100 min under 56 °C, which demonstrated that the recombinant NDVs kept the lentogenic and thermostable nature of rAHR09. The immunization data showed that rAHR09-HA and rAHR09-HAF induced a higher HI antibody titer against H9N2 AIV and NDV. After being challenged with H9N2 AIV, the rAHR09-HA and rAHR09-HAF could significantly reduce the virus shedding in cloacal and tracheal swab samples. Our results suggest that rAHR09-HA and rAHR09-HAF might be vaccine candidates against H9N2 AIV.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHA H5N6 + H9N2 BLP or a combination of tHA H5N6 BLP and tHA H9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

The protection of current influenza vaccines is limited due to the viral antigenic shifts and antigenic drifts. The universal influenza vaccine is a new hotspot in vaccine research that aims to overcome these problems. Polydopamine (PDA), a versatile biomaterial, has the advantages of an excellent biocompatibility, controllable particle size, and distinctive drug loading approach in drug delivery systems. To enhance the immunogenicities and delivery efficiencies of H9N2 avian influenza virus (AIV) epitope peptide vaccines, PDA nanoparticles conjugated with the BPP-V and BP-IV epitope peptides were used to prepare the nano BPP-V and BP-IV epitope peptide vaccines, respectively. The characteristics of the newly developed epitope peptide vaccines were then evaluated, revealing particle sizes ranging from approximately 240 to 290 nm (PDI<0.3), indicating that the synthesized nanoparticles were stable. Simultaneously, the immunoprotective effects of nano BPP-V and BP-IV epitope peptide vaccines were assessed. The nano BPP-V and BP-IV epitope vaccines, especially nano BP-IV epitope vaccine, quickly induced anti-hemagglutinin (HA) antibody production and a sustained immune response, significantly promoted humoral and cellular immune responses, reduced viral lung damage and provided effective protection against AIV viral infection. Together, these results reveal that PDA, as a delivery carrier, can improve the immunogenicities and delivery efficiencies of H9N2 AIV nano epitope vaccines, thereby providing a theoretical basis for the design and development of PDA as a carrier of new universal influenza vaccines., 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 © 2021 Liu, Wang, Zhou, Shi, Shen, Chen, Zhang, Liao and Wang.)

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N -linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46 + NKG2A + memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-μ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46 + NKG2A + NK cells were recruited to infected lung and modulated viral clearance of virus and CD8 + T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response. IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46 + NKG2A + NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection., (Copyright © 2021 American Society for Microbiology.)

H9N2 avian influenza virus has been continuously circulating among poultry and can infect mammals, indicating that this virus is a potential pandemic strain. During influenza pandemics, secondary bacterial (particularly pneumococcal) pneumonia usually contributes to excessive mortality. In the present study, we observed the dynamic effect of H9N2 virus infection on host defense against secondary pneumococcal infection in mice. BALB/c mice were intranasally inoculated with 1.2 × 10 5 PFU of H9N2 virus followed by 1 × 10 6 CFU of Streptococcus pneumoniae at 7, 14, or 28 days post-H9N2 infection (dpi). The bacterial load, histopathology, body weight, and survival were assessed after pneumococcal infection. Our results showed that H9N2 virus infection had no significant impact on host resistance to secondary pneumococcal infection at 7 dpi. However, H9N2 virus infection increased pulmonary pneumococcal clearance and reduced pneumococcal pneumonia-induced morbidity after secondary pneumococcal infection at 14 or 28 dpi, as reflected by significantly decreased bacterial loads, markedly alleviated pulmonary histopathological changes, and significantly reduced weight loss in mice infected with H9N2 virus followed by S. pneumoniae compared with mice infected only with S. pneumoniae Further, the significantly decreased bacterial loads were observed when mice were previously infected with a high dose (1.2 × 10 6 PFU) of H9N2 virus. Also, similar to the results obtained in BALB/c mice, improvement in pulmonary pneumococcal clearance was observed in C57BL/6 mice. Overall, our results showed that pulmonary pneumococcal clearance is improved after resolution of H9N2 virus infection in mice., (Copyright © 2021 Li et al.)

Influenza A viruses (IAV) of subtype H9N2, endemic in world-wide poultry holdings, are reported to cause spill-over infections to pigs and humans and have also contributed substantially to recent reassortment-derived pre-pandemic zoonotic viruses of concern, such as the Asian H7N9 viruses. Recently, a H9N2 bat influenza A virus was found in Egyptian fruit bats ( Rousettus aegyptiacus ), raising the question of whether this bat species is a suitable host for IAV. Here, we studied the susceptibility, pathogenesis and transmission of avian and bat-related H9N2 viruses in this new host. In a first experiment, we oronasally inoculated six Egyptian fruit bats with an avian-related H9N2 virus (A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2)). In a second experiment, six Egyptian fruit bats were inoculated with the newly discovered bat-related H9N2 virus (A/bat/Egypt/381OP/2017 (H9N2)). While R. aegyptiacus turned out to be refractory to an infection with H9N2 avian-type, inoculation with the bat H9N2 subtype established a productive infection in all inoculated animals with a detectable seroconversion at day 21 post-infection. In conclusion, Egyptian fruit bats are most likely not susceptible to the avian H9N2 subtype, but can be infected with fruit bat-derived H9N2. H9-specific sero-reactivities in fruit bats in the field are therefore more likely the result of contact with a bat-adapted H9N2 strain.

A semiannual immunization with a commercial inactivated H9 subtype avian influenza virus (AIV) vaccine developed for poultry has been used to prevent and control the avian influenza (AI) infections among captured wild birds in Shanghai Zoo. However, the overall safety and effectiveness of the poultry vaccine for housed birds in the zoo remain unclear. To verify the safety and efficacy of the commercial inactivated H9 AI vaccine on zoo birds and to explore a more reasonable and effective immunization procedure, 48 zoo birds, including 11 Oriental white storks, 25 peafowl, and 12 silver pheasants, were administered the AI vaccine developed for poultry use. Then, the clinical signs of the immunized birds were observed for 2 weeks, and the antibodies against H9 AI were determined via the hemagglutination inhibition test. Results showed that no harmful effects related to the vaccination were observed, and the antibody titers of the Oriental white stork, peafowl, and silver pheasants were all higher than 7 log 2 at 21 days, 30 days, 60 days, 120 days, and 180 days postimmunization. For further study, the H9 AIV titers of 11 peafowls and 6 Oriental storks, which were raised in the nursing ground, were continuously monitored for 15 months. All of their antibody titers were above the national standards of China (5 log 2 ; GB/T18936-2003), even at 12 months and 15 months postimmunization. We concluded that the commercial inactivated H9 AI vaccine used at the present time in Shanghai Zoo can induce high and prolonged immune responses in vaccinated birds.

Avian influenza H9N2 viruses circulate in all types of poultry species, including turkeys, and cause significant losses for the poultry industry in many parts of the word. The aim of this study was to assess the pathogenesis of the Moroccan avian influenza virus (AIV) H9N2 under experimental conditions in turkeys and the protection efficacy of an inactivated commercial vaccine against AIV H9N2. Unvaccinated turkeys showed marked depression sinusitis, respiratory distress characterized by bronchiolar and tracheal rales of moderate severity, and a mortality rate of 50%. Postmortem examinations of dead and euthanatized birds revealed the presence of fibrinous tracheitis and airsacculitis lesions. Vaccination reduced the mortality rate to 20%. Vaccinated birds recovered at day 10 postchallenge, and only 12.5% (1/8) and 37.5% of birds still displayed fibrinous and nonfibrinous airsacculitis lesions, respectively, at day 15 postinoculation. Viral shedding in cloacal and tracheal swabs was lower in vaccinated than in control birds. Although viral RNA was detected in the cloacal swabs of all unvaccinated turkeys at day 3 postinoculation, only 50% of the vaccinated turkeys were positive for virus detection. At day 11 postinoculation, no viral RNA was detected in oropharyngeal swabs of vaccinated turkeys, whereas 40% of the unvaccinated turkeys were still shedding virus.

Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections., Methods: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction., Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism., Conclusions: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

H9N2 avian influenza viruses (AIV) continue to circulate in vaccinated chicken flocks in China, which prompted us to investigate the differential immune protection factors induced by H9N2 AIV infection and immunization for analyzing the reason of protection deficiency of H9N2 AIV inactivated vaccine. In this study, we firstly explored virus-induced optimal immune responses in chicken after H9N2 AIV infection. And, we found that H9N2 hemagglutination inhibition (HI) antibody level, antiviral interferon-stimulated genes including 2',5'-oligoadenylate synthetase-like and myxovirus resistance 1, CD8 + T cell response in peripheral blood lymphocytes (PBL) accompanied by the cytotoxicity-associated genes, including poly (ADP-ribose) polymerase and IFN-r play important roles in defending against H9N2 infection. Besides, we observed that vaccine immunization triggered the similar H9N2 HI antibody level as viral infection, the increase of CD4 + T cell percentage instead of CD8 + T cell percentage in PBL. Moreover, we further made a comparative analysis of immune-related gene expression profile in PBL and lung after H9N2 AIV infection and immunization, respectively. The results showed that vaccine immunization contributed to the up-regulation of Th2 cytokine. But the deficiency of cytotoxicity-associated genes induced by H9N2 AIV inactivated vaccine may be the potential key reason of protection deficiency. These findings provide evidence and direction for developing effective H9N2 AIV vaccines., (Copyright © 2020. Published by Elsevier Inc.)

Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is "fit" and able to overcome vaccine pressure.

Inactivated H9N2 influenza vaccines required adjuvants to induce strong immune responses to protect poultry from the infections of H9N2 influenza viruses. Recently, positively charged nanoparticles-based adjuvant delivery systems have been extensively investigated as the novel vaccine adjuvant due to the protection antigens and drugs from degradation, promoting antigens and drugs uptake by antigen presenting cells (APCs), and inducing strong humoral and cellular immune responses. In this study, the immunostimulant Angelica sinensis polysaccharide (ASP) was encapsulated into Poly (lactic-co-glycolic acid) PLGA nanoparticles, and the Polyethylenimine (PEI) was coated on the nanoparticles to develop a novel adjuvant (ASP-PLGA-PEI). To further investigate the adjuvant activities of ASP-PLGA-PEI nanoparticles for H9N2 vaccines in chickens and compare the adjuvant activities of nanoparticles adjuvant and conventional adjuvants (Alum and oil-based adjuvant), the H9N2 antigen was incubated with three different adjuvants and then immunized with chickens to evaluate the ability of inducing humoral and cellular immune responses. The results revealed that compared to Alum adjuvant, ASP-PLGA-PEI nanoparticles adjuvant stimulated higher antibody responses, promoted the activation of CD4 + T cells and CD8 + T cells, increased the expression of Th1 cytokines IFN-γ. Compared to oil-based adjuvant (ISA-206), ASP-PLGA-PEI nanoparticles adjuvant induced comparable antibody immune responses at later period after immunization, improved the activation of CD4 + T cells and CD8 + T cells. Therefore, compared to Alum and oil-based adjuvant, the ASP-PLGA-PEI nanoparticles serve as an efficient adjuvant for H9N2 vaccine and have the potential to induce vigorous humoral and cellular immune responses in chickens., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Avian Influenza (AI), caused by Alphainfluenzaviruses (AIVs), is a contagious respiratory disease in birds and mammals. AIVs have been reported in poultry worldwide and the impact of AIVs on human health is immense. In this study, a serological survey of AIV subtype H5 and H9 was conducted in a live bird market (LBM) in Yangon, Myanmar during February 2016 to September 2016. A total of 621 serum samples were collected from chickens (n = 489) and ducks (n = 132) from 48 vendors in the LBM. The samples were examined for antibodies against influenza viruses by using NP-ELISA and specific antibodies against AIV-H5N1 (Clade 2.3.4) and AIV-H9N2 (Clade 9.4.2) by using Hemagglutination Inhibition (HI) assay. The result of NP-ELISA assay showed that 12.88 % (80/621) of poultry in LBM was positive for AIV antibodies. In detail, 38.06 % (51/134) of layers, 7.08 % (8/113) of backyard chicken, 2.07 % (5/242) of broilers and 12.12 % (16/132) of ducks were AIV positive. The HI test for specific antibodies against AIV-H5N1 and AIV-H9N2 were 1.77 % (11/621) and 4.51 % (28/621), respectively. Our findings revealed the evidence of AIV-H5N1 and AIV-H9N2 exposure in both chicken and ducks in the LBM in Yangon, Myanmar. Risks of influenza infections and transmission among poultry and humans in the LBMs could not be ignored., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: H9N2 Low pathogenic avian influenza virus (LPAIV) raises public health concerns and its eradication in poultry becomes even more important in preventing influenza. AJSAF is a purified active saponin fraction from the stem bark of Albizzia julibrissin. In this study, AJSAF was evaluated for the adjuvant potentials on immune responses to inactivated H9N2 avian influenza virus vaccine (IH9V) in mice and chicken in comparison with commercially oil-adjuvant., Results: AJSAF significantly induced faster and higher H9 subtype avian influenza virus antigen (H9-Ag)-specific IgG, IgG1, IgG2a and IgG2b antibody titers in mice and haemagglutination inhibition (HI) and IgY antibody levels in chicken immunized with IH9V. AJSAF also markedly promoted Con A-, LPS- and H9-Ag-stimulated splenocyte proliferation and natural killer cell activity. Furthermore, AJSAF significantly induced the production of both Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 and Th2 cytokines and transcription factors in splenocytes from the IH9V-immunized mice. Although oil-formulated inactivated H9N2 avian influenza vaccine (CH9V) also elicited higher H9-Ag-specific IgG and IgG1 in mice and HI antibody titer in chicken, this robust humoral response was later produced. Moreover, serum IgG2a and IgG2b antibody titers in CH9V-immunized mice were significantly lower than those of IH9V alone group., Conclusions: AJSAF could improve antigen-specific humoral and cellular immune responses, and simultaneously trigger a Th1/Th2 response to IH9V. AJSAF might be a safe and efficacious adjuvant candidate for H9N2 avian influenza vaccine.

Influenza virus neuraminidase (NA) contains a universally conserved epitope (NAe, NA 222-230 ). However, no studies have reported vaccines targeting this NA conserved epitope and inducing antibodies recognizing NAe. The extracellular domain of M2 (M2e) is considered as an attractive target for a universal influenza vaccine. We generated recombinant influenza H1N1 viruses expressing conserved epitopes in hemagglutinin (HA) molecules: NAe (NAe-HA) or M2e (M2e-HA) within the HA head domain. Inactivated recombinant NAe-HA and M2e-HA viruses were more effective in inducing IgG antibodies specific for an inserted conserved epitope than live recombinant virus. Recombinant inactivated M2e-HA virus vaccination induced cross protection against H3N2 virus with less weight loss compared to NAe-HA and was more effective in inducing humoral and cellular M2e immune responses. This study provides insight into developing recombinant influenza virus vaccines compatible with current platforms to induce antibody responses to conserved poorly immunogenic epitopes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (10 6 EID 50 /bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7-8 log 2 ) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The H9N2 avian influenza virus has been widely spread in poultry around the world. It is proved to the world that the avian influenza virus can directly infect human beings without any intermediate host adaptation in "1997 Hong Kong avian influenza case," which shows that the avian influenza virus not only causes significant losses to the poultry industry but also affects human health. In this study, we aimed to address the problem of low protection of avian H9N2 subtype influenza virus vaccine against H9N2 wild-type virus. We have rescued the H9.4.2.5 branched avian influenza virus isolated in South China by reverse genetics technology. We have recombined these virus (rHA/NA-GD37 and rHA/NA-GD38) which contain hemagglutinin and neuraminidase genes from the H9N2 avian influenza virus (MN064850 or MN064851) and 6 internal genes from the avian influenza virus (KY785906). We compared the biological properties of the virus for example virus proliferation, virus elution, thermostability, and pH stability. Then, we evaluated the immune effects between rHA/NA-GD37 and GD37, which show that the recombinant avian influenza virus-inactivated vaccine can stimulate chickens to produce higher antibody titers and produce little inflammatory response after the challenge. It is noticeable that the recombinant virus-inactivated vaccine had better immune impact than the wild-type inactivated vaccine. Generally speaking, this study provides a new virus strain for the development of a H9N2 vaccine., (Copyright © 2020. Published by Elsevier Inc.)

Selenium is a trace mineral that has antioxidant activities and can influence the immune system. However, antiviral effects of selenium have not been well studies in chickens. Chickens were therefore fed diets supplemented with two levels of two different sources of selenium (organic: selenium enriched yeast; SEY or inorganic: sodium selenite; SS). Chickens in the control groups did not receive supplemental dietary selenium. At 14 and 21 days of age, chickens were vaccinated with an inactivated low pathogenicity avian influenza virus (AIV, subtype H9N2) vaccine and blood samples were collected to determine the level of antibodies using hemagglutination inhibition (HI) and ELISA. At 30 days of age, chickens were also challenged with the same virus and swab samples were collected to assess the amount of virus shedding. Antibody levels, as measured by HI, increased significantly in the chickens that received higher levels of SEY at 16 days post vaccination. ELISA titers for IgM and IgY were higher in selenium supplemented chickens. Comparing to challenged control, virus shedding was lower in organic as well as inorganic selenium treated groups. Therefore, it may be concluded that supplemental dietary selenium could enhance vaccine conferred immunity thereby impacting protection against viral challenge in chickens., (Copyright © 2020. Published by Elsevier B.V.)

Background: The global prevalence of economic primary infection of poultry by H9N2 virus, including the Lineage A, panzootic group ME1, and associated with secondary infection by Mycoplasma gallisepticum (MG), is alarming to the sustainability of the poultry sector. This research evaluated in broilers the immunity and protection induced by aerosolization of liposomal nanoparticles vaccine, encapsulating antigens of H9N2 virus and MG, with or without the incorporation of Echinacea extract (EE) immuno-stimulant. Six different treatments (TRTs) of broilers were included in the experimental design, with three replicate pens/TRT and stocking of 20 day-old birds/replicate., Results: The tracheobronchial washings of birds subjected to aerosolization of liposomal nanoparticles, encapsulating antigens of H9N2 and MG and EE had the highest significant mean levels of each of IgA and IgG specific to H9N2 and MG, associated with lowest tracheal MG colonization, tracheal H9N2 recovery, tracheal histopathologic lesions, mortality, and best performance in body weight and feed conversion compared to all other challenged birds allocated to different treatments (P < 0.05). However, the control broilers, free from challenge with MG and H9N2, had the lowest mortality and tracheal lesions, and the highest production performance., Conclusion: The aerosolization of liposomal nanoparticles, encapsulating antigens of H9N2 and MG and EE resulted in enough local immunity for protection of broilers against infection, and in attaining the highest production performance in challenged birds. The potential implication of vaccinating with safe killed nanoparticle vaccines is of utmost importance to the global poultry sector.

Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses. IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam., (Copyright © 2020 American Society for Microbiology.)

Newcastle disease virus (NDV) and H9N2 subtype Avian influenza virus (AIV) are two notorious avian respiratory pathogens that cause great losses in the poultry industry. Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses, while an attenuated live vaccine bears the risk of mutation. Dendritic cell (DC) targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens. In this study, DC-binding peptide (DCpep)-decorated chimeric virus-like particles (cVLPs), containing NDV haemagglutinin-neuraminidase (HN) and AIV haemagglutinin (HA), were developed as a DC-targeting mucosal vaccine candidate. DCpep-decorated cVLPs activated DCs in vitro, and induced potent immune stimulation in chickens, with enhanced secretory immunoglobulin A (sIgA) secretion and splenic T cell differentiation. 40 μg cVLPs can provide full protection against the challenge with homologous, heterologous NDV strains, and AIV H9N2. In addition, DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly, as indicated by robust sIgA secretion and a reduced virus shedding period. Taken together, this chimeric VLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.

Background: The PD-1/PD-L1 pathway is an inhibitory signaling pathway that maintains the balance between the immune response and immunotolerance, and its overactivation in cancer and viral infections inhibits T cell function. The target cells of various viruses, microvascular endothelial cells (MECs) have been shown to be key regulatory points in immune regulation and virion diffusion in vivo during infection with multiple influenza virus subtypes. Furthermore, avian influenza virus (AIV) infection can induce immunosuppression by causing imbalances in immune responses and immune organ damage. Thus, the aim of this study was to investigate whether the H9N2 virus inhibited the immune function of T cells that migrated across MECs by upregulating PD-L1 expression on MECs., Methods: The susceptibility of rat pulmonary microvascular endothelial cells (RPMECs) to the H9N2 virus was evaluated by a plaque-forming assay and immunofluorescence staining. Then, we quantified the mRNA and protein levels of PD-L1 in RPMECs induced by H9N2 virus infection using quantitative real-time PCR and flow cytometry. The interaction between the activated T cells and RPMECs infected with the H9N2 virus was revealed using a coculture system. The effect of endothelial-derived PD-L1 on T cell function was investigated by using ELISA and flow cytometry with or without a PD-L1-specific antibody., Results: Surface staining and the plaque-forming assay showed that the H9N2 virus infected and replicated in RPMECs. Both the PD-L1 mRNA level and PD-L1 protein level were upregulated in RPMECs infected with the H9N2 virus. H9N2 virus-induced PD-L1 expression significantly reduced the secretions of IL-2, IFN-γ and granzyme B and perforin expression in T cells. The above data were significantly increased after treatment with an anti-PD-L1 antibody, confirming the above mentioned findings. In addition, the induction of PD-L1 expression decreased the proliferative capacity of the cocultured T cells but did not affect the apoptosis rate of T cells., Conclusions: Taken together, the results suggest that the H9N2 virus is able to inhibit the T cell immune response by upregulating PD-L1 expression in pulmonary microvascular endothelial cells.

In recent decades, multiple subtypes (i.e. H9N2, H5N1 and H7N9) of avian influenza virus (AIV) have become widespread in China, which has caused enormous economic losses and posed considerable threats to public health. In this review, with the aim to provide insights into and guidelines for the control of AIV spread in China and globally in the future, we analysed the reasons why AIV has persisted in China based on socio-economic features, including poultry biosecurity, live bird markets, live bird transportation, wild birds, poultry waterfowl, poultry density, poultry population and infected birds. We also described the present status of the AIV subtypes H9, H5 and H7 in China to elucidate the effectiveness of the strategies currently employed in China (i.e. culling, mass vaccination and biosecurity improvement) to control the disease based on a literature review and our unpublished surveillance data collected over a 12-year period from 2007 to 2018. We then summarized the lessons to be learned from the control experience in China, including whether culling of infected birds is of limited value for disease control and whether improved biosecurity is a better option than culling and vaccination for the long-term control of AIV, and when the vaccine strain should be updated., (© 2020 Blackwell Verlag GmbH.)

Biodegradable nanomaterials can protect antigens from degradation, promote cellular absorption, and enhance immune responses. We constructed a eukaryotic plasmid [pCAGGS-opti441-hemagglutinin (HA)] by inserting the optimized HA gene fragment of H9N2 AIV into the pCAGGS vector. The pCAGGS-opti441-HA/DGL was developed through packaging the pCAGGS-opti441-HA with dendrigraft poly-l-lysines (DGLs). DGL not only protected the pCAGGS-opti441-HA from degradation, but also exhibited high transfection efficiency. Strong cellular immune responses were induced in chickens immunized with the pCAGGS-opti441-HA/DGL. The levels of IFN-γ and IL-2, and lymphocyte transformation rate of the vaccinated chickens increased at the third week post the immunization. For the vaccinated chickens, T lymphocytes were activated and proliferated, the numbers of CD3+CD4+ and CD4+/CD8+ increased, and the chickens were protected completely against H9N2 AIV challenge. This study provides a method for the development of novel AIV vaccines, and a theoretical basis for the development of safe and efficient gene delivery carriers., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1β and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4 + and CD8 + T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4 + and CD8 + T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.

Chitosan nanoparticles (CNPs) represent an efficient vaccination tool to deliver immunogenic antigens to the antigen-presenting cells (APCs), which subsequently stimulate protective immune responses against infectious diseases. Herein, we prepared CNPs encapsulating mRNA molecules followed by surface coating with conserved H9N2 HA2 and M2e influenza proteins. We demonstrated that CNPs efficiently delivered mRNA molecules into APCs and had effectively penetrated the mucosal barrier to reach to the immune initiation sites. To investigate the potential of CNPs delivering influenza antigens to stimulate protective immunity, we intranasally vaccinated chickens with empty CNPs, CNPs delivering HA2 and M2e in both mRNA and protein formats (CNPs + RNA + Pr) or CNPs delivering antigens in protein format only (CNPs + Pr). Our results demonstrated that chickens vaccinated with CNPs + RNA + Pr elicited significantly (p < 0.05) higher systemic IgG, mucosal IgA antibody responses and cellular immune responses compared to the CNPs + Pr vaccinated group. Consequently, upon challenge with either H7N9 or H9N2 avian influenza viruses (AIVs), efficient protection, in the context of viral load and lung pathology, was observed in chickens vaccinated with CNPs + RNA + Pr than CNPs + Pr vaccinated group. In conclusion, we show that HA2 and M2e antigens elicited a broad spectrum of protection against AIVs and incorporation of mRNAs in vaccine formulation is an effective strategy to induce superior immune responses.

Developing an effective universal influenza vaccine against influenza virus with highly conserved antigenic epitopes could induce a broad-spectrum immune response to prevent infection. The soluble protein M1 that can induce the M1 specific immune response was first confirmed in our previous study. In this study, we characterized the immune response induced by DNA prime-subunit protein boost strategy based on the relatively conserved matrix protein 1 (M1) in the BALB/c mouse model, and evaluated its protection ability against a lethal challenge of homologous H9N2 avian influenza virus (A/Chicken/Jiangsu/11/2002). The results showed that 100 μg DNA prime + 100 μg M1 subunit protein boost-strategy significantly increased antibody levels more than vaccination with M1 DNA or M1 subunit protein alone, and induced a more balanced Th1 / Th2 immune response, which not only can provide protection against the homologous virus but also can provide part of the cross-protection against the heterosubtypic PR8 H1N1 strain. In addition, we used an Elispot assay to preliminary screen the T cell epitope in M1 protein, and identified that p22 (M1 11-25 VLSIIPSGPLKAEIA) epitope was the only immunodominant M1-specific CD4 + T cell epitopes, which could be helpful in understanding the function of influenza virus T cell epitopes.

Background: Avian influenza H9 is endemic in commercial and backyard poultry in Pakistan and is a serious occupational health hazard to industry workers. This study aimed to determine the seroprevalence of avian influenza H9 infection in people working with poultry in Rawalpindi, Pakistan and assess the measures they took to protect themselves from infection., Methods: A cross-sectional study was conducted from December 2016 to May 2017 of 419 people working with poultry in Rawalpindi Division, including farm workers, vaccinators, field veterinarians, butchers and staff working in diagnostic laboratories. Potential participants were randomly approached and gave written consent to participate. Data were collected using a standardized questionnaire and serum samples were processed to detect H9 antibodies using the haemagglutination inhibition test., Results: Of the 419 participants, 406 (96.9%) were male. The mean age of the participants was 36.4 (SD 10.86) years. A total of 332 participants agreed to a blood test, 167 of whom were positive for A(H9) antibodies, giving an overall seroprevalence of 50.3%. Laboratory staff had the highest seroprevalence (100%) and veterinarians the lowest (38.5%). Vaccinators, butchers and farm workers had a seroprevalence of 83.3%, 52.4% and 45.5% respectively. Personals who used facemasks had significantly lower (P<0.002) seroprevalence (29.6%) than those who never used them (90.6%). Similarly, those who always used gloves and washed their hands with soap had a seroprevalence of 32.8% compared with 89.0% in those who never took these precautions. Of the participants who handled antigens, 92.3% were seropositive., Conclusion: Laboratory staff and vaccinators are exposed to viral cultures and influenza vaccines respectively which may explain their high seroprevalence., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Avian influenza virus (AIV) H9N2 is endemic in Iran and its large-scale circulation in the poultry industry of the country is devastating. This virus was first reported in the industrial poultry populations of Iran in July 1998. Some of the published studies showed that inactivated avian influenza (AI) vaccines are capable of inducing an immune response and providing protection against morbidity and mortality in different countries (Vasfi et al., 2002; Tavakkoli et al., 2011). Low pathogenicity avian influenza subtype H9N2 virus has been reported to have a zoonotic potential and widespread distribution in Iran. Therefore, water-in-oil emulsion vaccines are employed to control the disease in chickens (Nili and Asasi, 2003). This cohort study was conducted during July 2016-November 2017 in broiler chicken farms of Qazvin province, Iran to investigate the serological change trends in broiler chickens in this region. Level of immunity against the H9N2 virus was evaluated by hemagglutination inhibition assay. Fifteen farms out of thirty enrolled units used AI H9N2 killed vaccines. The minimum of mean antibody titers (MATs) was 4.54-2.42 and the maximum of MATs was 4.54+2.42 on day 3. In addition, the minimum and maximum MATs on day 50 were 0.4-0.64 and 0.4+0.064, respectively. The transfer rate of H9N2 AIV antibodies from the serum of breeders to the serum of chickens was calculated as 60.35% in our study. A significant difference was revealed between the maternal mean antibody titers (MMATs) and the MATs on day 3 (P&lt;0.001). In addition, the difference between the MATs on day 3 and the MATs on day 10 was found to be significant (P&lt;0.01). Moreover, MATs were significantly different between the vaccinated and unvaccinated herds on day 40 (P&lt;0.05), while no significant difference was observed on days 3, 10, 20, and 30 (P&gt;0.05). According to the results of this study, antibody titers in the vaccinated farms did not reach the protective level until the end of the rearing period. Most of the unvaccinated herds experienced a spurt in antibody titers due to exposure to the virus. Consequently, biosecurity measures must be implemented more seriously and strictly in broiler farms., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

The H9N2 avian influenza viruses cause significant economic losses in poultry worldwide and could potentially cause human pandemic. Currently, the available vaccines have limited efficacy due to antigenic drift of H9N2. To improve vaccine efficacy, we developed monovalent vaccine strain via the modification of neutralizing epitopes on hemagglutinin (HA) to broaden the protection against H9N2 viruses. In this study, single and multiple mutation were introduced to amino acid at position 148, 150 (site I) and 183, 186, 188 (site II) on the full-length HA gene of H9N2 strain (A/Hong Kong/33982/2009). These mutant HA constructs were displayed on the baculovirus surface (BacH9), and evaluated for their cross-protective efficacy against H9N2 viruses in a mouse model. Our findings indicate that mice immunized with multiple BacH9 mutant constructs (148-150 183 and 186) induced cross-protective immunity against circulating H9N2 in the viral challenge study and prove to be a promising vaccine candidate for H9N2., 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 © 2019 Elsevier Ltd. All rights reserved.)

Avian influenza subtype H9N2 infection is a mild but highly contagious disease that is associated with a decrease in the efficacy of vaccine interventions, and an increase in susceptibility to secondary infections in poultry. However, the immune evasion mechanism of H9N2 avian influenza viruses (AIVs) in chickens is poorly understood. Dendritic cells (DCs) are immune cells of major importance, involved in innate immune responses against viruses, but also in the setting of adaptive immune response due to their high ability to present viral antigen. Therefore, in the present study we used high-throughput RNA-sequencing technology at the transcriptome level to identify the differentially expressed genes (DEGs) between chicken DCs infected with H9N2 virus and mock-infected DCs. We identified 4151 upregulated DEGs and 2138 downregulated DEGs. Further enrichment analysis showed that the upregulated DEGs were enriched in the biological processes mainly involved in signal transduction, transmembrane transport, and innate immune/inflammatory responses. In contrast, the downregulated DEGs were associated with the biological processes mainly including metabolic process, and MHC class I antigen processing and presentation. In addition, 49 of these immune-related DEGs were validated by reverse transcription quantitative PCR (RT-qPCR). Collectively, these data suggest that H9N2 virus infection may enhance the signal transduction, and innate immune responses in chicken DCs, but impair their metabolic functions and antigen-presenting responses, which provide helpful insight into the pathogenesis of H9N2 AIVs in chickens and managing this infection in poultry farms., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Low pathogenicity avian influenza (LPAI) H9N2, highly pathogenic avian influenza (HPAI) H5N1, and H5N8 circulate in Egyptian poultry and cause veterinary and public health burdens. In response, AIV vaccines are commonly used. The main objective of this study was to develop a broad, cross-protective, trivalent vaccine based on circulating AIVs in Egypt. We generated highly replicating avirulent AIVs, H5N1, and H5N8, to be used in combination with H9N2 strain for the generation of an inactivated vaccine. Immunogenicity and protective efficacy of this vaccine were tested. Results showed that a single immunization dose enhanced humoral immune responses giving full protection against challenges with LPAI H9N2, HPAI H5N1, and H5N8 viruses. This efficacious vaccine will reduce the cost of vaccination for poultry growers and is expected to be effective in the field as it is based on contemporary viruses currently in circulation among Egyptian poultry., (© 2019 Poultry Science Association Inc.)

The outbreak of H9N2 avian influenza virus (H9N2 AIV) brings high mortality and huge economic losses every year. Sensitive and reliable detection methods are essential to timely diagnosis and treatment. Herein, a dual-modality immunoassay is proposed for H9N2 AIV detection by employing fluorescent-magnetic-catalytic nanospheres (FMCNs) as labels and alkaline phosphatase (ALP)-induced metallization as a signal amplification strategy. The excellent magnetic properties of FMCNs endow the assay a potential application in complex samples. Also, the excellent fluorescence properties of FMCNs enable fluorescence modality readout. The antibodies on the FMCN surface can achieve efficient capture and separation of targets. Amplified electrochemical modality readout can be obtained through ALP-catalyzed silver deposition. Dual-modality immunoassay combined the advantages of electrochemical assay with fluorescence assay and provides accurate detection results to meet different testing needs. With two quantitative analysis forms, H9N2 AIV can be detected by electrochemical signals with a quantitation range of 0.1 to 1000 ng/mL and a detection limit of 10 pg/mL. The linear range is 300 to 1000 ng/mL with a detection limit of 69.8 ng/mL by the fluorescence signal readout. Moreover, the specificity, anti-interference ability, accuracy, and diversity of the proposal have unlimited potential for early diagnosis of suspect infections.

In late 2017, increased mortality was detected in chicken farms in Algeria undergoing A(H9N2) influenza outbreaks. Analysis of viruses isolated from affected farms showed that they were monophyletic, were of the G1 hemagglutinin (HA) lineage, and were antigenically and genetically similar to viruses detected contemporaneously in other countries in Northern Africa and the Middle East. The virus was able to spread via contact transmission between ferrets but did not cause disease in intravenously inoculated chickens., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Variations in the composition of commensal gut microbiota have been reported to be major contributors to differences in responses to vaccination among individuals. In chickens, there is limited information on the role of gut microbiota in responses to vaccination. The current study studied the role of gut microbiota in cell- and antibody-mediated immune responses to vaccination with a whole inactivated avian influenza virus, subtype H9N2. A total of 166 one-day-old specific pathogen free layer chickens (SPF) were randomly assigned to treatments, where a combination of antibiotic depletion, and probiotics (a combination of five Lactobacillus species) or fecal microbial transplant (FMT) reconstitution were used to study the dynamics of cell- and antibody-mediated immune responses to primary and secondary vaccinations at days 15 and 29 of age, respectively. Overall, at days 7 and 14 post primary vaccination (p.p.v.), administration of probiotics to non-depleted chickens resulted in significantly higher mean hemagglutination (HI) titre compared to antibiotic treated chickens. Furthermore, at day 21 p.p.v., chickens treated with probiotics or FMT post-antibiotic treatment showed a significantly higher mean HI titre compared to non-depleted chickens treated with probiotics. At day 7 p.p.v., a significantly higher virus specific IgM and IgG titres were observed in non-depleted chickens administered with probiotics compared to antibiotic depleted chickens, and a significantly higher IgG titre was observed in chickens treated with FMT following antibiotic treatment compared to only antibiotic treatment. Analysis of interferon gamma expression in splenocytes to assess cell-mediated immune responses showed a significantly lower expression in antibiotic-treated chickens compared to non-depleted chickens and FMT reconstituted chickens. Taken together, the current study suggests that shifts in the composition of gut microbiota of chickens may result in changes in cell- and antibody-mediated immune responses to vaccination against influenza viruses. Further studies will be needed to highlight the mechanisms involved in this modulation., (Copyright © 2019. Published by Elsevier Ltd.)

Avian influenza viruses (AIV) are very active in several parts of the globe and are the cause of huge economic loss for the poultry industry and also human fatalities. Three dimensional modeling was carried out for neuraminidase (NA) and hemagglutinin (HA) proteins of AIV. The C-score, estimated TM-Score, and estimated root-mean-square deviation (RMSD) score for NA of H5N1 were -1.18, 0.57 ± 0.15, and 9.8 ± 7.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for NA of H9N2 were -1.43, 0.54 ± 0.15, and 10.5 ± 4.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H5N1 were -0.03, 0.71 ± 0.12, and 7.7 ± 4.3, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H9N2 were -0.57, 0.64 ± 0.13, and 8.9 ± 4.6, respectively. Intrinsically disordered regions were identified for the NA and HA proteins of H5N1 and H9N2 with the use of PONDR program. Linear B cell epitope was predicted using BepiPred 2 program for NA and HA of H5N1 and H9N2 avian influenza strains. Discontinuous epitopes were predicted by Discotope 2 program. The linear epitopes that were considered likely to be immunogenic and within the intrinsically disordered region for the NA of H5N1 was TKSTNSRSGFEMIWDPNGWTGTDSSFSVK, and for H9N2 it was VGDTPRNDDSSSSSNCRDPNNERGAP. In the case of HA of H5N1, it was QRLVPKIATRSKVNGQSG and ATGLRNSPQRERRRKK; for H9N2 it was INRTFKPLIGPRPLVNGLQG and SLKLAVGLRNVPARSSR. The discontinuous epitopes of NA of H5N1 and H9N2 were identified at various regions of the protein structure spanning from amino acid residue positions 90 to 449 and 107 to 469, respectively. Similarly, the discontinuous epitopes of HA of H5N1 and H9N2 were identified in the amino acid residue positions 27 to 517 and 136 to 521, respectively. This study has identified potential and highly immunogenic linear and conformational B-cell epitopes towards developing a vaccine against AIV both for human and poultry use., (© 2019 Wiley Periodicals, Inc.)

The stimulator of interferon genes (STING) protein has been shown to play a pivotal role in response to both cytosolic RNA and dsDNA to elicit interferon (IFN) production in mammals. However, the role of duck STING (DuSTING) in antiviral innate immunity, especially in anti-RNA virus infection, has yet to be elucidated. In this study, the function of DuSTING in IFN induction and its role in anti-RNA virus infections were studied. DuSTING was amplified via reverse transcription-polymerase chain reaction (RT-PCR) from Pekin duck, showing that its cDNA sequence contains an open reading frame (ORF) of 1,149 bp and encodes 382 amino acids (aa). Sequence alignment showed that DuSTING protein shares 71.1, 43.4, and 33.3% identity with chickens, humans, and zebra fish, respectively. Overexpression of DuSTING in duck embryo fibroblasts (DEFs) strongly activated IFN-β promotor activity. Deletion mutant analysis revealed that the first 42 aa containing the first transmembrane (TM) domains and the last 32 aa containing a part of the C-terminal tail (CTT) are essential for its IFN-β activation. In vitro experiments showed that the mRNA levels of DuSTING and IFNs were all upregulated when the DEFs were infected with H9N2 avian influenza virus (AIV) SH010, while overexpression of DuSTING inhibited the replication of this virus. In vivo studies showed that DuSTING mRNA was widely expressed in different tissues, and was up-regulated in the spleen and lung of ducks challenged with SH010. In conclusion, our results indicate that DuSTING is an essential IFN mediator and plays a role in anti-RNA virus innate immunity., (Copyright © 2019 Cheng, Liu, Shi, Niu, Zhu, Wang, Ma, Wang, Yan and Sun.)

In the present study, the potency and immunogenicity of the inactivated bivalent vaccine of Newcastle disease (ND) and avian influenza (AI) produced by Razi institute in Iran were compared with a similar imported vaccine administered by standard methods to specific-pathogen-free (SPF) chicken. A total of 150 twenty-one-day-old SPF chickens were used for evaluating Razi and imported inactivated H9N2/ND vaccines. The chickens were divided into eight groups. The subjects in groups 1 and 3 were vaccinated with 0.3 ml/bird by subcutaneous route in the back of the neck with Razi and imported vaccines, respectively. Chickens in group 2 received Razi vaccine based on the recommended dose of the manufacturer (0.2 ml/bird) by the same route. The birds in groups 4 and 5 received 0.01 ml/bird of Razi and imported vaccine, respectively. Groups 7 and 8 were considered for studying the safety of the two vaccines and received a double amount of the full dose of vaccines. Moreover, group6 was regarded as the negative control. Sera were collected weekly from each chicken for antibody analysis against Newcastle disease virus (NDV) and AI virus (AIV) and the study continued for 15 weeks after vaccination. An immunological evaluation was carried out using haemagglutination inhibition (HI) antibody test against NDV and AIV. The results showed that up to 15 weeks after vaccination, the Razi vaccine induced a higher level of protective antibody against AI and ND in comparison with the imported ones at the dose of 0.3 ml/bird. The mean HI titer was significantly different between Razi vaccine and imported vaccine at the dose of 0.3 ml/bird. There was no statically significant difference between Razi vaccine (0.2 ml/bird) and the imported vaccine (0.3 ml/bird) against NDV and AIV. According to the findings, 15 weeks after vaccination, HI titers were still detectable at a high level. The mean HI titer was found as 5.2 log2 against NDV and 5 log2 against H9N2 with Razi vaccine (0.3 ml/bird). In addition, the mean HI titer with Razi vaccine (0.2 ml/bird) was 4.1 log2 and 4.7 log2 against ND and AI, respectively. In summary, our results indicated that Razi inactivated vaccine (0.3 ml/bird) induced a strong and rapid antibody response in vaccinated chickens and is more effective in chicken against AIVs and NDVs, in comparison with the imported vaccine., (Copyright © 2019, Archives of Razi Institute. Published by Kowsar.)

Some immune escape mutants of H9N2 virus and the corresponding mutations in hemagglutinin (HA) have been documented, but little is known about the impact of a single mutation on the antigenicity and pathogenesis of H9N2. In this study, seven critical sites in HA associated with the antigenicity were identified and the effects of a HA mutation (N166D) derived from a H9N2 escape mutant (m3F2) were investigated. Although N166D did not significantly affect viral replication in Madin-Darby canine kidney (MDCK) cells and viral shedding in the larynx and cloaca of chicken, N166D attenuated the pathogenesis of the virus in mice. Compared to the rescued RgPR8-H9&#95;166D, RgPR8-H9&#95;166N caused greater body weight loss and higher viral titers in the lungs of the infected mice. Moreover, hemagglutination inhibition (HI) assay for the sera from the chickens infected with wild type H9N2 and mutant m3F2 showed that N166D mutation could result in weak antibody response in chickens. Considering the field strains of H9N2 with N166D mutation are frequently isolated in the countries with H9N2 vaccination, the findings that the single mutation in HA, N166D, affected both the antigenicity and pathogenesis of H9N2 highlight the significance of surveillance on such mutation that may contribute to the failure of H9N2 vaccination in the field.