Candidate Genes Associated with Survival Following Highly Pathogenic Avian Influenza Infection in Chickens.

Highly pathogenic strains of avian influenza (HPAI) devastate poultry flocks and result in significant economic losses for farmers due to high mortality, reduced egg production, and mandated euthanization of infected flocks. Within recent years, HPAI outbreaks have affected egg production flocks across the world. The H5N2 outbreak in the US in 2015 resulted in over 99% mortality. Here, we analyze sequence data from chickens that survived (42 cases) along with uninfected controls (28 samples) to find genomic regions that differ between these two groups and that, therefore, may encompass prime candidates that are resistant to HPAI. Blood samples were obtained from survivors of the 2015 HPAI outbreak plus age and genetics-matched non-affected controls. A whole-genome sequence was obtained, and genetic variants were characterized and used in a genome-wide association study to identify regions showing significant association with survival. Regions associated with HPAI resistance were observed on chromosomes 1, 2, 5, 8, 10, 11, 15, 20, and 28, with a number of candidate genes identified. We did not detect a specific locus which could fully explain the difference between survivors and controls. Influenza virus replication depends on multiple components of the host cellular machinery, with many genes involved in the host response.

Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination.

Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.

Genetic insertion of mouse Myxovirus-resistance gene 1 increases innate resistance against both high and low pathogenic avian influenza virus by significantly decreasing replication in chicken DF1 cell line.

Avian influenza virus (AIV) is a constant threat to animal health with recent global outbreaks resulting in the death of hundreds of millions of birds with spillover into mammals. Myxovirus-resistance (Mx) proteins are key mediators of the antiviral response that block virus replication. Mouse (Mu) Mx (Mx1) is a strong antiviral protein that interacts with the viral nucleoprotein to inhibit polymerase function. The ability of avian Mx1 to inhibit AIV is unclear. In these studies, Mu Mx1 was stably introduced into chicken DF1 cells to enhance the immune response against AIV. Following infection, titers of AIV were significantly decreased in cells expressing Mu Mx1. In addition, considerably less cytopathic effect (CPE) and matrix protein staining was observed in gene-edited cells expressing Mu Mx1, suggesting Mu Mx1 is broadly effective against multiple AIV subtypes. This work provides foundational studies for use of gene-editing to enhance innate disease resistance against AIV.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

Comprehensive genome­wide analysis of the chicken heat shock protein family: identification, genomic organization, and expression profiles in indigenous chicken with highly pathogenic avian influenza infection.

BACKGROUND: Heat shock proteins (HSPs) function as molecular chaperones with critical roles in chicken embryogenesis, immune response to infectious diseases, and response to various environmental stresses. However, little is known on HSP genes in chicken. In this study, to understand the roles of chicken HSPs, we performed genome-wide identification, expression, and functional analyses of the HSP family genes in chicken. RESULTS: A total of 76 HSP genes were identified in the chicken genome, which were further classified into eight distinct groups (I-VIII) based on phylogenetic tree analysis. The gene-structure analysis revealed that the members of each clade had the same or similar exon-intron structures. Chromosome mapping suggested that HSP genes were widely dispersed across the chicken genome, except in chromosomes 16, 18, 22, 25, 26, and 28-32, which lacked chicken HSP genes. On the other hand, the interactions among chicken HSPs were limited, indicating that the remaining functions of HSPs could be investigated in chicken. Moreover, KEGG pathway analysis showed that the HSP gene family was involved in the regulation of heat stress, apoptotic, intracellular signaling, and immune response pathways. Finally, RNA sequencing data revealed that, of the 76 chicken HSP genes, 46 were differentially expressed at 21 different growth stages in chicken embryos, and 72 were differentially expressed on post-infection day 3 in two indigenous Ri chicken lines infected with highly pathogenic avian influenza. CONCLUSIONS: This study provides significant insights into the potential functions of HSPs in chicken, including the regulation of apoptosis, heat stress, chaperone activity, intracellular signaling, and immune response to infectious diseases.

Analysis of miRNA expression in the trachea of Ri chicken infected with the highly pathogenic avian influenza H5N1 virus.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) is considered a global threat to both human health and the poultry industry. MicroRNAs (miRNA) can modulate the immune system by affecting gene expression patterns in HPAIV-infected chickens. OBJECTIVES: To gain further insights into the role of miRNAs in immune responses against H5N1 infection, as well as the development of strategies for breeding disease-resistant chickens, we characterized miRNA expression patterns in tracheal tissues from H5N1-infected Ri chickens. METHODS: miRNAs expression was analyzed from two H5N1-infected Ri chicken lines using small RNA sequencing. The target genes of differentially expressed (DE) miRNAs were predicted using miRDB. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were then conducted. Furthermore, using quantitative real-time polymerase chain reaction, we validated the expression levels of DE miRNAs (miR-22-3p, miR-146b-3p, miR-27b-3p, miR-128-3p, miR-2188-5p, miR-451, miR-205a, miR-203a, miR-21-3p, and miR-200a-3p) from all comparisons and their immune-related target genes. RESULTS: A total of 53 miRNAs were significantly expressed in the infection samples of the resistant compared to the susceptible line. Network analyses between the DE miRNAs and target genes revealed that DE miRNAs may regulate the expression of target genes involved in the transforming growth factor-beta, mitogen-activated protein kinase, and Toll-like receptor signaling pathways, all of which are related to influenza A virus progression. CONCLUSIONS: Collectively, our results provided novel insights into the miRNA expression patterns of tracheal tissues from H5N1-infected Ri chickens. More importantly, our findings offer insights into the relationship between miRNA and immune-related target genes and the role of miRNA in HPAIV infections in chickens.

In 'proof of concept,' CRISPR-edited chickens shrug off flu.

Edits to one gene can't completely stop infections, however.

Creating resistance to avian influenza infection through genome editing of the ANP32 gene family.

Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.

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

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

The SUMO-interacting motif in NS2 promotes adaptation of avian influenza virus to mammals.

Species differences in the host factor ANP32A/B result in the restriction of avian influenza virus polymerase (vPol) in mammalian cells. Efficient replication of avian influenza viruses in mammalian cells often requires adaptive mutations, such as PB2-E627K, to enable the virus to use mammalian ANP32A/B. However, the molecular basis for the productive replication of avian influenza viruses without prior adaptation in mammals remains poorly understood. We show that avian influenza virus NS2 protein help to overcome mammalian ANP32A/B-mediated restriction to avian vPol activity by promoting avian vRNP assembly and enhancing mammalian ANP32A/B-vRNP interactions. A conserved SUMO-interacting motif (SIM) in NS2 is required for its avian polymerase-enhancing properties. We also demonstrate that disrupting SIM integrity in NS2 impairs avian influenza virus replication and pathogenicity in mammalian hosts, but not in avian hosts. Our results identify NS2 as a cofactor in the adaptation process of avian influenza virus to mammals.

Surveillance of avian influenza viruses from 2014 to 2018 in South Korea.

Surveillance of influenza A viruses (IAVs) among migratory waterfowl is a first step in understanding the ecology, biology, and pathogenicity of IAVs. As part of the nationwide surveillance effort for IAVs in fowl in South Korea, we collected environmental fecal samples in different migratory bird stopover sites in South Korea during the winter seasons within November 2014 through January 2018. We collected a total of 6758 fecal samples, 75 of which were positive for IAV (1.11% positivity). Prevalence of IAVs varied per site and per year. Based on sequencing, the most prevalent hemagglutinin (HA) subtypes were H1, H6, and H5, and the most prevalent neuraminidase (NA) subtypes were N1, N3, and N2. Phylogenetic analyses showed that the genes we isolated clustered with reported isolates collected from other locations along the East Asian-Australasian Flyway. All the H5 and H7 isolates collected in this study were of low pathogenicity. None of the N1 and N2 genes carried amino acid markers of resistance against NA inhibitors. The winter 2016-2017 subset were primarily borne by migratory geese (Anser spp.). These results suggest that majority of the IAVs circulating among migratory wild fowl in South Korea in 2014-2018 were of low pathogenicity.

Mammalian ANP32A and ANP32B Proteins Drive Differential Polymerase Adaptations in Avian Influenza Virus.

ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.

Effect of avian influenza virus subtype H9N2 on the expression of complement-associated genes in chicken erythrocytes.

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.

H7N9 bearing a mutation in the nucleoprotein leads to increased pathology in chickens.

The zoonotic H7N9 avian influenza (AI) virus first emerged in 2013 as a low pathogenic (LPAI) strain, and has repeatedly caused human infection resulting in severe respiratory illness and a mortality of ~39% (>600 deaths) across five epidemic waves. This virus has circulated in poultry with little to no discernible clinical signs, making detection and control difficult. Contrary to published data, our group has observed a subset of specific pathogen free chickens infected with the H7N9 virus succumb to disease, showing clinical signs consistent with highly pathogenic AI (HPAI). Viral genome sequencing revealed two key mutations had occurred following infection in the haemagglutinin (HA 226 L>Q) and nucleoprotein (NP 373 A>T) proteins. We further investigated the impact of the NP mutation and demonstrated that only chickens bearing a single nucleotide polymorphism (SNP) in their IFITM1 gene were susceptible to the H7N9 virus. Susceptible chickens demonstrated a distinct loss of CD8+ T cells from the periphery as well as a dysregulation of IFNγ that was not observed for resistant chickens, suggesting a role for the NP mutation in altered T cell activation. Alternatively, it is possible that this mutation led to altered polymerase activity, as the mutation occurs in the NP 360-373 loop which has been previously show to be important in RNA binding. These data have broad ramifications for our understanding of the pathobiology of AI in chickens and humans and provide an excellent model for investigating the role of antiviral genes in a natural host species.

ProbeTools: designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa.

BACKGROUND: Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral genome sequencing. Hybridization probes are widely used for enrichment in many fields, but their application to viral sequencing faces a major obstacle: it is difficult to design panels of probe oligo sequences that broadly target many viral taxa due to their rapid evolution, extensive diversity, and genetic hypervariability. To address this challenge, we created ProbeTools, a package of bioinformatic tools for generating effective viral capture panels, and for assessing coverage of target sequences by probe panel designs in silico. In this study, we validated ProbeTools by designing a panel of 3600 probes for subtyping the hypervariable haemagglutinin (HA) and neuraminidase (NA) genome segments of avian-origin influenza A viruses (AIVs). Using in silico assessment of AIV reference sequences and in vitro capture on egg-cultured viral isolates, we demonstrated effective performance by our custom AIV panel and ProbeTools' suitability for challenging viral probe design applications. RESULTS: Based on ProbeTool's in silico analysis, our panel provided broadly inclusive coverage of 14,772 HA and 11,967 NA reference sequences. For each reference sequence, we calculated the percentage of nucleotide positions covered by our panel in silico; 90% of HA and NA references sequences had at least 90.8 and 95.1% of their nucleotide positions covered respectively. We also observed effective in vitro capture on a representative collection of 23 egg-cultured AIVs that included isolates from wild birds, poultry, and humans and representatives from all HA and NA subtypes. Forty-two of forty-six HA and NA segments had over 98.3% of their nucleotide positions significantly enriched by our custom panel. These in vitro results were further used to validate ProbeTools' in silico coverage assessment algorithm; 89.2% of in silico predictions were concordant with in vitro results. CONCLUSIONS: ProbeTools generated an effective panel for subtyping AIVs that can be deployed for genomic surveillance, outbreak prevention, and pandemic preparedness. Effective probe design against hypervariable AIV targets also validated ProbeTools' design and coverage assessment algorithms, demonstrating their suitability for other challenging viral capture applications.

Enhanced stability of M1 protein mediated by a phospho-resistant mutation promotes the replication of prevailing avian influenza virus in mammals.

Avian influenza virus (AIV) can evolve multiple strategies to combat host antiviral defenses and establish efficient infectivity in mammals, including humans. H9N2 AIV and its reassortants (such as H5N6 and H7N9 viruses) pose an increasing threat to human health; however, the mechanisms involved in their increased virulence remain poorly understood. We previously reported that the M1 mutation T37A has become predominant among chicken H9N2 isolates in China. Here, we report that, since 2010, this mutation has also been found in the majority of human isolates of H9N2 AIV and its emerging reassortants. The T37A mutation of M1 protein enhances the replication of H9N2 AIVs in mice and in human cells. Interestingly, having A37 instead of T37 increases the M1 protein stability and resistance to proteasomal degradation. Moreover, T37 of the H9N2 M1 protein is phosphorylated by protein kinase G (PKG), and this phosphorylation induces the rapid degradation of M1 and reduces viral replication. Similar effects are also observed in the novel H5N6 virus. Additionally, ubiquitination at K187 contributes to M1-37T degradation and decreased replication of the virus harboring T37 in the M1 protein. The prevailing AIVs thereby evolve a phospho-resistant mutation in the M1 protein to avoid viral protein degradation by host factors, which is advantageous in terms of replication in mammalian hosts.

Comparative trachea transcriptome analysis in SPF broiler chickens infected with avian infectious bronchitis and avian influenza viruses.

Infectious bronchitis virus (IBV) and avian influenza virus (AIV) are two major respiratory infections in chickens. The coinfection of these viruses can cause significant financial losses and severe complications in the poultry industry across the world. To examine transcriptome profile changes during the early stages of infection, differential transcriptional profiles in tracheal tissue of three infected groups (i.e., IBV, AIV, and coinfected) were compared with the control group. Specific-pathogen-free chickens were challenged with Iranian variant-2-like IBV (IS/1494), UT-Barin isolates of H9N2 (A/chicken/Mashhad/UT-Barin/2017), and IBV-AIV coinfection; then, RNA was extracted from tracheal tissue. The Illumina RNA-sequencing (RNA-seq) technique was employed to investigate changes in the Transcriptome. Up- and downregulated differentially expressed genes (DEGs) were detected in the trachea transcriptome of all groups. The Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology databases were examined to identify possible relationships between DEGs. In the experimental groups, upregulated genes were higher compared to downregulated genes. A more severe immune response was observed in the coinfected group; further, cytokine-cytokine receptor interaction, RIG-I-like receptor signaling, Toll-like receptor signaling, NOD-like receptor signaling, Janus kinase/signal transducer, and activator of transcription, and apoptotic pathways were important upregulated genes in this group. The findings of this paper may give a better understanding of transcriptome changes in the trachea during the early stages of infection with these viruses.

Network Meta-Analysis of Chicken Microarray Data following Avian Influenza Challenge-A Comparison of Highly and Lowly Pathogenic Strains.

The current bioinformatics study was undertaken to analyze the transcriptome of chicken (Gallus gallus) after influenza A virus challenge. A meta-analysis was carried out to explore the host expression response after challenge with lowly pathogenic avian influenza (LPAI) (H1N1, H2N3, H5N2, H5N3 and H9N2) and with highly pathogenic avian influenza (HPAI) H5N1 strains. To do so, ten microarray datasets obtained from the Gene Expression Omnibus (GEO) database were normalized and meta-analyzed for the LPAI and HPAI host response individually. Different undirected networks were constructed and their metrics determined e.g., degree centrality, closeness centrality, harmonic centrality, subgraph centrality and eigenvector centrality. The results showed that, based on criteria of centrality, the CMTR1, EPSTI1, RNF213, HERC4L, IFIT5 and LY96 genes were the most significant during HPAI challenge, with PARD6G, HMG20A, PEX14, RNF151 and TLK1L having the lowest values. However, for LPAI challenge, ZDHHC9, IMMP2L, COX7C, RBM18, DCTN3, and NDUFB1 genes had the largest values for aforementioned criteria, with GTF3C5, DROSHA, ATRX, RFWD2, MED23 and SEC23B genes having the lowest values. The results of this study can be used as a basis for future development of treatments/preventions of the effects of avian influenza in chicken.

Special Issue-Immunity to Influenza Viruses.

Influenza viruses remain a constant global threat with significant health and socioeconomic impact every year and have the potential to cause devastating pandemics [...].

Proteomic analysis of differential expression of lung proteins in response to highly pathogenic avian influenza virus infection in chickens.

Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.