key: cord-0029109-ianpm4k9
authors: Pirbaluty, Azadeh Moradi; Mehrban, Hossein; Kadkhodaei, Saeid; Ravash, Rudabeh; Oryan, Ahmad; Ghaderi-Zefrehei, Mostafa; Smith, Jacqueline
title: Network Meta-Analysis of Chicken Microarray Data following Avian Influenza Challenge—A Comparison of Highly and Lowly Pathogenic Strains
date: 2022-02-26
journal: Genes (Basel)
DOI: 10.3390/genes13030435
sha: f0cbbe80a295e016e449ca07b86cc21b4ea409c0
doc_id: 29109
cord_uid: ianpm4k9

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.

Global monitoring of influenza is crucial for improvements in disease management, rapid intervention and decreasing the potential impact of an influenza pandemic. Avian Influenza (AI) is caused by three types of viruses: types A, B and C. Influenza A viruses (IAVs) are potentially zoonotic viruses that can cause infection in birds and a small number of mammals [1] . Influenza A virus is the only species of the alpha influenza virus genus in the Orthomyxoviridae family. Most human influenza pandemics of the 20th century were caused by IAVs that originated, either wholly or in part, from avian influenza A viruses [2] . The virus can be transmitted from wild birds to native poultry, which provides (multigraph). Figure 1 delineates both directed and undirected graphs with their adjacency matrices.

Genes 2022, 13, 435 3 of 16 (multigraph). Figure 1 delineates both directed and undirected graphs with their adjacency matrices. Potential applications within the field of biology include the identification of drug targets and the design of effective control strategies for infectious diseases [27] , to name but two. A basic premise of designing a gene network is that knowledge regarding the structure of genetic relationships enriches existing knowledge of the function of each individual gene. Supplementary Table S1 summarizes some network-based studies that have been applied in poultry transcriptomic research. However, these network-based analyses have not been utilized for studying the host response to influenza infection in chicken. Gene network analysis goes beyond knowledge of single gene effects and shows relational interaction of numerous genes. This is therefore an effective way to construct gene modules to gain a deeper understating of the biological pathways and networks underpinning response to disease. In this study, for the first time, we use indirect graph analyses to look at the biological effects of influenza infection in chickens and identify the core genes involved in transcriptional change. We believe it is vital to understand the path and pace of the virus which culminates in the identified host-virus interactions, with a view to formulating decisions regarding mitigation strategies, virus containment, antiviral therapy and vaccination.

Microarray expression profiles from chicken after influenza challenge were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo (accessed on 22 October 2018). Data relating to LPAI studies included challenge with H2N3, H5N3, H9N2, H5N2 and H1N1 strains and data relating to HPAI involved H5N1 challenge. Each dataset consisted of infected and control groups. For HPAI, 24 samples were examined (consisting of 16 infected and 8 control samples) and for LPAI, 76 samples (consisting of 56 infected and 20 controls) were studied ( Figure 2 ). Potential applications within the field of biology include the identification of drug targets and the design of effective control strategies for infectious diseases [27] , to name but two. A basic premise of designing a gene network is that knowledge regarding the structure of genetic relationships enriches existing knowledge of the function of each individual gene. Supplementary Table S1 summarizes some network-based studies that have been applied in poultry transcriptomic research. However, these network-based analyses have not been utilized for studying the host response to influenza infection in chicken. Gene network analysis goes beyond knowledge of single gene effects and shows relational interaction of numerous genes. This is therefore an effective way to construct gene modules to gain a deeper understating of the biological pathways and networks underpinning response to disease. In this study, for the first time, we use indirect graph analyses to look at the biological effects of influenza infection in chickens and identify the core genes involved in transcriptional change. We believe it is vital to understand the path and pace of the virus which culminates in the identified host-virus interactions, with a view to formulating decisions regarding mitigation strategies, virus containment, antiviral therapy and vaccination.

Microarray expression profiles from chicken after influenza challenge were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih. gov/geo (accessed on 22 October 2018). Data relating to LPAI studies included challenge with H2N3, H5N3, H9N2, H5N2 and H1N1 strains and data relating to HPAI involved H5N1 challenge. Each dataset consisted of infected and control groups. For HPAI, 24 samples were examined (consisting of 16 infected and 8 control samples) and for LPAI, 76 samples (consisting of 56 infected and 20 controls) were studied ( Figure 2 ). Figure 3 shows the bioinformatics pipeline used in this study. The .CEL files each dataset were normalized using the RMA normalization method in the Affym Expression console software package (version: 1.1). RMA is a normalization algorith microarray data, correcting background, normalizing and summarizing the probe information without the use of the information from the Mis-Match probe. Correct malization is an absolute prerequisite for accurate evaluation of gene expression [28 Figure 3 shows the bioinformatics pipeline used in this study. The CEL files from each dataset were normalized using the RMA normalization method in the Affymetrix Expression console software package (version: 1.1). RMA is a normalization algorithm for microarray data, correcting background, normalizing and summarizing the probe level information without the use of the information from the Mis-Match probe. Correct normalization is an absolute prerequisite for accurate evaluation of gene expression [28] . Figure 3 shows the bioinformatics pipeline used in this study. The .CEL files from each dataset were normalized using the RMA normalization method in the Affymetrix Expression console software package (version: 1.1). RMA is a normalization algorithm for microarray data, correcting background, normalizing and summarizing the probe level information without the use of the information from the Mis-Match probe. Correct normalization is an absolute prerequisite for accurate evaluation of gene expression [28] . 

We used the rank product meta-analysis algorithm on normalized microarray data from the various avian influenza challenge experiments shown in Figure 2 . The rank product is a non-parametric statistic that was originally aimed at identifying differentially expressed genes (DEGs) in a dataset [29] . Using the RankProd (RP) package (Version 1.1.383) [30] , an add-on R package, a set of DEGs with FDR < 0.05 were obtained. The RP is a 

We used the rank product meta-analysis algorithm on normalized microarray data from the various avian influenza challenge experiments shown in Figure 2 . The rank product is a non-parametric statistic that was originally aimed at identifying differentially expressed genes (DEGs) in a dataset [29] . Using the RankProd (RP) package (Version 1.1.383) [30] , an add-on R package, a set of DEGs with FDR < 0.05 were obtained. The RP is a statistical approach that is widely used to detect differentially expressed features in -omics data e.g., transcriptomics, metabolomics and proteomics studies. Standardized expression values of meta-genes from all samples were used to rank and select the most important genes.

Up-regulated and down-regulated DEGs were used to train the network. Differentially expressed genes were obtained using meta-analysis by comparing fold change between disease and control groups from each data series. The gene expression data were then standardized. The Pearson correlation between the gene expression data was then obtained and genes with correlation values higher than 95% (ad-hoc) were used in Networkx (version 2.4 for python3.7 via anaconda) (https://networkx.github.io/ (accessed on 16 November 2018) to draw the undirected gene network and its metric measures cited in Table 1 . NeworkX is a Python-based software package, used to extract the network metric measures, manipulate data, and study the structure, dynamics, and functions of complex networks (Supplementary Table S2 ). It has many standard graph algorithms for data structures for graphs, digraphs, and multigraphs. We developed individual networks based on both up-regulated and down-regulated genes. Investigation of complete sets of DEGs did not produce meaningful networks. LPAI-lowly pathogenic avian influenza; HPAI-highly pathogenic avian influenza; nodes = genes; edges = interactions.

The obtained results were used to construct a Venn diagram to identify genes overlapping between different strains. This was done using the Jvenn package (http://jvenn. toulouse.inra.fr/app/example.html (accessed on 20 May 2019). Gene IDs of DEGs were also uploaded to the PANTHER [31] website (http://www.pantherdb.org/ (accessed on 1 July 2019) to investigate the differences in gene expression between the samples after the viral challenge compared to the control samples. Network ranking algorithms created using Cytoscape software package ClueGo + Clue Pedia (version: 3.8.0.) (https://cytoscape.org/ (accessed on 28 September 2019) were then used to identify genes overlapping with the Python-based network results. Biological pathways, functions and networks relating to identified DEGs were investigated by means of Ingenuity Pathway Analysis (IPA) software (Qiagen: https://digitalinsights.qiagen.com/products-overview/discovery-insightsportfolio/analysis-and-visualization/qiagen-ipa/ (accessed on 12 April 2021). Differentially expressed genes with adjusted p < 0.05 and FC > 1.5 were used in the IPA analysis.

In the LPAI datasets, 6004 probes (3535 genes) were differentially expressed (p < 0.05) with 31% of DEGs being up-regulated and 69% of DEGs down-regulated. For the H1N1 strain there were 9 common probes between 2 h and 10 h after viral infection ( Figure 4A ). For H5N2 there were 24 probes expressed at all times and there were 64 common probes expressed between 2 h and 10 h after viral infection ( Figure 4B ). For H9N2, there were 15 probes expressed across all times and there were 57 common probes between 2 h and 10 h after viral infection ( Figure 4C ). In terms of probes commonly expressed between different viral sub-types, we see 1520 common probes between H5N2 and H9N2, 271 probes between H5N2 and H1N1, 102 probes between H1N1 and H9N2, 294 probes between H5N2 and H5N3, 171 probes between H5N2, H9N2 and H1N1 and 210 probes between H5N2, H9N2 and H5N3. All LPAI DEGs (FC > +/−1.5) are shown in Supplementary Table S3. es 2022, 13, 435 6 of 16

In the LPAI datasets, 6004 probes (3535 genes) were differentially expressed (p < 0.05) with 31% of DEGs being up-regulated and 69% of DEGs down-regulated. For the H1N1 strain there were 9 common probes between 2 h and 10 h after viral infection ( Figure 4A ). For H5N2 there were 24 probes expressed at all times and there were 64 common probes expressed between 2 h and 10 h after viral infection ( Figure 4B ). For H9N2, there were 15 probes expressed across all times and there were 57 common probes between 2 h and 10 h after viral infection ( Figure 4C ). In terms of probes commonly expressed between different viral sub-types, we see 1520 common probes between H5N2 and H9N2, 271 probes between H5N2 and H1N1, 102 probes between H1N1 and H9N2, 294 probes between H5N2 and H5N3, 171 probes between H5N2, H9N2 and H1N1 and 210 probes between H5N2, H9N2 and H5N3. All LPAI DEGs (FC > +/−1.5) are shown in Supplementary Table  S3 . 

The Venn diagram in Figure 4D shows that for H5N1 infections, there were 31 commonly expressed probes between Ck10 12 h and Ck10 24 h datasets; 23 between Ck-pax 12 h and Ck-pax 24 h and 2778 between H5N1 ty-Ty and H5N1 50-92. The largest set of 

The Venn diagram in Figure 4D shows that for H5N1 infections, there were 31 commonly expressed probes between Ck10 12 h and Ck10 24 h datasets; 23 between Ck-pax 12 h and Ck-pax 24 h and 2778 between H5N1 ty-Ty and H5N1 50-92. The largest set of common probes was found between Ck-pax 24 h, 50-92 and ty-Ty datasets, with 550 probes shared ( Figure 4D ). The result of the meta-analysis for chicken influenza microarray data showed that in the HPAI datasets examined, 390 probes (265 genes) were differentially expressed (p < 0.05). For DEGs, 29% were found to be up-regulated while 71% were down-regulated. A list of all significant differentially expressed genes is shown in Supplementary Table S4.

In order to shed some light on the differential gene expression between HPAI and LPAI challenge, Venn diagrams were used to identify overlapping DEGs between both the up-regulated and down-regulated gene sets ( Figure 5 ). Collectively, this analysis, based on direct comparison of gene expression across the two influenza groups, demonstrated that there are 91 genes in common between down-regulated HPAI and LPAI datasets-the largest number of common genes. When up-regulated data are compared, there are seen to be 69 genes differentially expressed after both LPAI and HPAI challenge.

Genes 2022, 13, 435 7 of 16 common probes was found between Ck-pax 24 h, 50-92 and ty-Ty datasets, with 550 probes shared ( Figure 4D ). The result of the meta-analysis for chicken influenza microarray data showed that in the HPAI datasets examined, 390 probes (265 genes) were differentially expressed (p < 0.05). For DEGs, 29% were found to be up-regulated while 71% were down-regulated. A list of all significant differentially expressed genes is shown in Supplementary Table S4 .

In order to shed some light on the differential gene expression between HPAI and LPAI challenge, Venn diagrams were used to identify overlapping DEGs between both the up-regulated and down-regulated gene sets ( Figure 5 ). Collectively, this analysis, based on direct comparison of gene expression across the two influenza groups, demonstrated that there are 91 genes in common between down-regulated HPAI and LPAI datasets-the largest number of common genes. When up-regulated data are compared, there are seen to be 69 genes differentially expressed after both LPAI and HPAI challenge. The purpose of this study was to identify the most important genes involved in the avian flu response, based on the location of these genes in a gene network. Meta-analysis was performed individually on LPAI and HPAI datasets for genes up-regulated and for those down-regulated. Table 1 shows the information relating to the Python networks generated for each challenge group. The nodes represent genes and edges represent interactions in the gene network. For LPAI data, the up-regulated network includes 1842 nodes and 60,606 edges, while the down-regulated network contains 4162 nodes and 243,604 edges. With the HPAI data, we see an up-regulated network including 305 nodes and 114 edges and a down-regulated network that includes 1813 nodes and 276 edges. Results from our network analyses are summarized in Table 2 , which describes the hub genes identified in each of the up-or down-regulated groups. The purpose of this study was to identify the most important genes involved in the avian flu response, based on the location of these genes in a gene network. Meta-analysis was performed individually on LPAI and HPAI datasets for genes up-regulated and for those down-regulated. Table 1 shows the information relating to the Python networks generated for each challenge group. The nodes represent genes and edges represent interactions in the gene network. For LPAI data, the up-regulated network includes 1842 nodes and 60,606 edges, while the down-regulated network contains 4162 nodes and 243,604 edges. With the HPAI data, we see an up-regulated network including 305 nodes and 114 edges and a down-regulated network that includes 1813 nodes and 276 edges. Results from our network analyses are summarized in Table 2 , which describes the hub genes identified in each of the up-or down-regulated groups. As the centrality measure of the genes in the networks reduces, the proportion of the genes remaining in the network also reduces. Genes with high 'centrality', 'harmonic', 'degree', 'closeness', 'Eigenvector' and 'Subgraph' metrics achieve similar performance, whereas 'betweenness centrality' and 'load centrality' have a lower performance. Results from the 'core number' and 'cluster' criteria were not appropriate for finding modules. The 'degree centrality' metric in gene networks defines the importance of a gene in a graph as being measured based on its degree; the higher the degree of a gene, the more important it is in a graph. According to the results of the present study, the 'degree centrality' criterion obtained more accurate results than other criteria, as the results from this criterion are consistent with the results of the gene network based on the correlation between the genes. Centrality criteria for different DEGs are as shown in Table 2 .

The networks created are too large to present pictorially, but as an example, we show a section of the HPAI networks as depicted using Cytoscape software. Figure 6A shows a section of the up-regulated network and Figure 6B part of the down-regulated gene network. Examining genes differentially expressed between the HPAI and LPAI data sets identified the genes summarized in Table 3 . CMTR1, HERC4L, IFIT5, LY96, RNF213 and EP-STI1 were all more highly expressed after HPAI challenge compared to LPAI challenge. It is interesting to note that several of these genes (IFIT5, LY96, RNF213 and EPST1) were previously identified in a study examining LPAI and HPAI infection in ducks and chickens [32] . However, RBM18, NDUFB1, DCTN3, COX7C, IMMP2 and LZDHHC9 all showed higher expression during the host response to LPAI. Examining genes differentially expressed between the HPAI and LPAI data sets identified the genes summarized in Table 3 . CMTR1, HERC4L, IFIT5, LY96, RNF213 and EPSTI1 were all more highly expressed after HPAI challenge compared to LPAI challenge. It is interesting to note that several of these genes (IFIT5, LY96, RNF213 and EPST1) were previously identified in a study examining LPAI and HPAI infection in ducks and chickens [32] . However, RBM18, NDUFB1, DCTN3, COX7C, IMMP2 and LZDHHC9 all showed higher expression during the host response to LPAI. 

We also performed a general analysis across all up- (Table 4 ) and all down-regulated (Table 5 ) genes from both HPAI and LPAI datasets and identified significant genes. In general, we see a fairly unique response to LPAI and HPAI infections. However, from our network analysis, some genes were identified as important hub genes across both types of challenge. Amongst genes up-regulated, SELENOK, NDUFA1, PPP1R7, SMDT1, COX7C, PRELID3B, CIB1, OST4 and NDUFB2 were highlighted. Some of these (SELENOK, SMDT1, CIB1) are involved in calcium signaling, while others (NDUFA1, COX7C, NDUFB2) play a role in the mitochondrial respiratory chain. Ca 2+ -dependent signaling plays a crucial role in influenza viral internalization and infection, as well as being implicated in apoptosis of viral-infected cells [33] . An increase in activity of the mitochondrial respiratory chain is also known to occur after influenza infection [34] . Significant down-regulated genes included PUS10, ERBIN, SYDE2, PCGF6, FZD6, ROR1, LRIG2, SUPT7L, EXOC8, KIF1C and PCM1. These genes have a variety of functions including miRNA processing (PUS10), RNA polymerase II-specific transcription repressor activity (PCGF6), and negative regulation of biological processes including cell proliferation and apoptosis (FZD6) and chromatin modification (SUPT7L). Many of these genes are thus seen to have a role in how other genes are regulated. 

Analysis of functional ontologies associated with the genes being differentially expressed in response to influenza infection was carried out. Supplementary Tables S5-S8 show the biological processes, molecular functions and cellular components associated with DEGs identified as being up-regulated in response to HPAI (Supplementary Table S5) , down-regulated in response to HPAI (Supplementary Table S6 ), up-regulated in response to LPAI (Supplementary Table S7 ) and down-regulated in response to LPAI (Supplementary  Table S8 ). Based on KEGG analysis, important pathways affected during response to HPAI include those of Influenza A, Cytokine-cytokine receptor interaction, NOD-like receptor signaling and Toll-like receptor signaling, whereas after LPAI challenge cell adhesion molecules, MAPK signaling, ErbB signaling and phagosome activity are modified.

In order to explore the biological pathways and functional processes associated with differentially expressed genes, Ingenuity Pathway Analysis software was used. Figure 7 shows a comparison of some of the most significant biological activities related to the DEGs with respect to LPAI challenge compared to HPAI disease. Viral infection and replication is lower after LPAI challenge and apoptosis higher. This higher level of cell death is mirrored by the lower levels of cell survival indicated. This reflects the host ability to kill AI-infected cells and ultimately overcome LPAI infection-a situation not enabled when chickens succumb to HPAI virus. Upon investigation of the gene networks being modified upon HPAI challenge, we see that IFNG and IFNB are two of the main activators of gene expression. Conversely, genes such as IL4, IL10 and STAT6 are inhibited, restricting a Th2type immune response (Supplementary Table S9 ), directing the host toward an antiviral Th1 response. Supplementary Figure S1 shows the interferon-stimulated inflammatory response that is initiated after the H5N1 HPAI infection investigated in this study.

In order to explore the biological pathways and functional processes associa differentially expressed genes, Ingenuity Pathway Analysis software was used. shows a comparison of some of the most significant biological activities relate DEGs with respect to LPAI challenge compared to HPAI disease. Viral infection lication is lower after LPAI challenge and apoptosis higher. This higher level of c is mirrored by the lower levels of cell survival indicated. This reflects the host a kill AI-infected cells and ultimately overcome LPAI infection-a situation not when chickens succumb to HPAI virus. Upon investigation of the gene networ modified upon HPAI challenge, we see that IFNG and IFNB are two of the main a of gene expression. Conversely, genes such as IL4, IL10 and STAT6 are inhibited ing a Th2-type immune response (Supplementary Table S9 ), directing the host to antiviral Th1 response. Supplementary Figure S1 shows the interferon-stimulated matory response that is initiated after the H5N1 HPAI infection investigated in th 

Here we present a network analysis of a variety of microarray datasets repr avian influenza challenge in chicken. These datasets represent both lowly pathog highly pathogenic infections. Our network modelling identifies core genes centr response to both kinds of challenge, whether that be through up-or down-regu gene expression.

We observed that degree, harmonic and closeness centrality methods genera significant results. These methods are reliable applicants for use in practice to ide hub genes related to a particular disease [35] . Load and betweenness centrality do not have a higher statistical significance than the baseline method [34] in high known avian influenza genes. However, degree, harmonic and closeness centr rameters can identify the previously unknown genes which are involved in the response of interest. These methods can be used to generate new hypotheses on h interaction, and highlight candidates for experimental validation. Gene netwo 

Here we present a network analysis of a variety of microarray datasets representing avian influenza challenge in chicken. These datasets represent both lowly pathogenic and highly pathogenic infections. Our network modelling identifies core genes central to the response to both kinds of challenge, whether that be through up-or down-regulation of gene expression.

We observed that degree, harmonic and closeness centrality methods generate highly significant results. These methods are reliable applicants for use in practice to identify the hub genes related to a particular disease [35] . Load and betweenness centrality methods do not have a higher statistical significance than the baseline method [34] in highlighting known avian influenza genes. However, degree, harmonic and closeness centrality parameters can identify the previously unknown genes which are involved in the disease response of interest. These methods can be used to generate new hypotheses on host-virus interaction, and highlight candidates for experimental validation. Gene networks were used to identify the functional relevance of a gene interacting with communicating nodes in a biological network. The higher the value, the higher the relevance of the gene in connecting the regulatory molecules [36] . One can easily read basic features of the graph (degree, hierarchical structure, etc.) as well as more nuanced features, e.g., the relationship between a vertex and the hierarchical position of its neighbors. The present visualization strategy is a useful tool in discriminating between networks with different topological properties and structural arrangement, and may be also used for comparison of models with real data, providing an additional tool for model validation [37] .

For each type of challenge (LPAI and HPAI), unique and common host response genes were identified. During LPAI infection, genes involved in the process of apoptosis are activated, compared with the HPAI response. This may be one of the reasons why birds are able to overcome lowly pathogenic strain infections but not that of the highly pathogenic variety. On the other hand, immune stimulation and an interferon response is seen after HPAI challenge. However, as chickens are unable to survive the HPAI challenge, this response is either (1) inadequate in overcoming the viral infection or (2) too extreme and causes the so-called 'cytokine storm' often responsible for mortality in the face of HPAI infection. The levels of cytokine expression that are seen here do not appear to be excessive however, so it is likely that the response that is being initiated, in this instance, is insufficient. Indeed, only 96 genes are seen to be significantly up-regulated in response to HPAI (with 4.9-fold maximum expression change). A strong down-regulation of several genes is however seen. These include immune genes such as BLB1, CXCR4, IRF2BP2, TLR5 and TNFRSF1B. So it may be that HPAI infection causes an overall down-regulation of the immune system, thus rendering the host incapable of mounting a sufficient response to the virus.

Most of the up-regulated LPAI DEGs are involved in functions associated with the mitochondria or Golgi apparatus. Flu infection is known to lead to alterations in mitochondria morphology, release of pro-apoptotic proteins, loss of mitochondrial membrane potential, and eventually cell death [38] . When we look at the up-regulated HPAI DEGs on the other hand, we see that several are involved in the innate immune response, with CMTR1 and IFIT5 being well-known interferon-stimulated genes, LY96 enhancing TLR4dependent activation of NF-kappa-B [39] and EPSTI1 mediating RELA/p65 and STAT1 phosphorylation and nuclear localization upon activation of macrophages [40] .

Looking at core genes identified across all viral challenges, genes up-regulated in both highly and lowly pathogenic infections include those involved in calcium signaling -a process central to biological activity after viral infection. Various viruses enter host cells via endocytosis, but the underlying molecular mechanisms are unknown. The influenza A viruses (IAVs) enter cells via redundant pathways of clathrin-mediated and clathrinindependent endocytosis, with intracellular calcium having a central role in regulation of both pathways, by activating a signaling axis comprising RhoA, Rho-kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and phospholipase C (PLC). IAV infection induces oscillations in the cytosolic Ca 2+ concentration of host cells, the prevention of which markedly attenuates virus internalization and infection. The small GTPase RhoA is found both to function downstream of the virus-induced Ca 2+ response and to induce Ca 2+ oscillations in a manner dependent on Rho-kinase and subsequent PIP5K-PLC signaling. This signaling circuit regulates both clathrin-mediated and clathrin-independent endocytosis during virus infection and seems to constitute a key mechanism for regulation of IAV internalization and infection [33] .

Other genes with a role in the mitochondrial respiratory chain were also identified. Genes commonly down-regulated encompass a variety of functions, but many were involved in gene regulation. The Cap Methyltransferase 1 (CMTR1) gene was identified as a core up-regulated gene in the HPAI gene expression network. This gene has been identified as a host dependency factor, vital for efficient viral cap-snatching and regulating cell autonomous immune response. It also provides synergistic protection with the influenza endonuclease inhibitor Xofluza [41] . CMTR1 also has a potential role in the pathogenesis of asthma exacerbations [42] . Also in the HPAI gene expression network, the PARD6G gene (Par-6 Family Cell Polarity Regulator Gamma) was seen to be down-regulated. It is thought to play a role in the formation of epithelial tight junctions [43] . The expression changes occurring in these genes could help explain the differences in mortality seen between LPAI and HPAI infection in chickens.

We have used network analysis methods to predict hub gene associations from a collection of microarray datasets known to be related to the avian response to influenza infection, and created gene-interaction networks by correlation amongst differentially regulated genes. Next, we used degree, eigenvector, closeness, betweenness, subgraph, harmonic centrality, clustering and core number metrics to rank the genes in the network according to their relevance in the system. Our method has enabled the identification of core genes involved in the general host response to influenza infection in chicken e.g., SELENOK, NDUFA1, PPP1R7, SMDT1, COX7C, PRELID3B, CIB1, OST4 and NDUFB2 amongst upregulated data and PUS10, ERBIN, SYDE2, PCGF6, FZD6, ROR1, LRIG2, SUPT7L, EXOC8, KIF1C and PCM1 from down-regulated data. Comparison between different pathogenic strains identifies up-regulation of CMTR1, HERC4L, IFIT5, LY96, RNF213 and EPSTI1 as being significant during HPAI challenge, with RBM18, NDUFB1, DCTN3, COX7C, IMMP2 and LZDHHC9 being central to the LPAI expression network.

Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13030435/s1, Table S1 : Previous network based studies in chicken [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] , Table S2 : Network metrics used in this study [61] [62] [63] [64] [65] [66] [67] , Table S3 : Differentially expressed genes from LPAI data analysis, Table S4 : Differentially expressed genes from HPAI data analysis, Table S5 : Gene Ontology of up-regulated HPAI DEGs, Table S6 : Gene Ontology of downregulated HPAI DEGs, Table S7 : Gene Ontology of up-regulated LPAI DEGs, Table S8 : Gene Ontology of down-regulated LPAI DEGs, Table S9 : HPAI upstream activators. 

The authors declare no conflict of interest, but would like to state that J.S. is a member of the Genes Editorial Board.

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Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance

Statistical mechanics of complex networks

Normal and pathological oscillatory communication in the brain

Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets

Rank products: A simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments

RankProd: A bioconductor package for detecting differentially expressed genes in meta-analysis

PANTHER version 11: Expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements

A comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferoninduced transmembrane proteins in viral resistance

A Ca 2+ -dependent signalling circuit regulates influenza A virus internalization and infection

Transcriptomic profiling of a chicken lung epithelial cell line (CLEC213) reveals a mitochondrial respiratory chain activity boost during influenza virus infection

Identifying gene-disease associations using centrality on a literature mined geneinteraction network

Identification of hub subnetwork based on topological features of genes in breast cancer

K-core decomposition of internet graphs: Hierarchies, self-similarity and measurement biases

Dikic, I. Mitochondrial Functions in Infection and Immunity

MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4

Regulation of inflammatory gene expression in macrophages by epithelial-stromal interaction 1 (Epsti1)

Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection

CMTR1 is associated with increased asthma exacerbations in patients taking inhaled corticosteroids

Differential day-night expression of tight junction components in murine retinal pigment epithelium

Transcriptional and pathway analysis in the hypothalamus of newly hatched chicks during fasting and delayed feeding

Transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection

Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease

Transcriptome analysis reveals an activation of major histocompatibility complex 1 and 2 pathways in chicken trachea immunized with infectious laryngotracheitis virus vaccine

RNA-seq analysis of broiler liver transcriptome reveals novel responses to high ambient temperature

Transcriptional profiles of host-pathogen responses to necrotic enteritis and differential regulation of immune genes in two inbreed chicken lines showing disparate disease susceptibility

Thymus transcriptome reveals novel pathways in response to avian pathogenic Escherichia coli infection

A Genome-Wide mRNA Screen and Functional Analysis Reveal FOXO3 as a Candidate Gene for Chicken Growth

De novo transcriptomic analysis of peripheral blood lymphocytes from the Chinese goose: Gene discovery and immune system pathway description

Immediate and long-term transcriptional response of hind muscle tissue to transient variation of incubation temperature in broilers

Liver transcriptome response to hyperthermic stress in three distinct chicken lines

Comparative transcriptome analysis reveals induction of apoptosis in chicken kidney cells associated with the virulence of nephropathogenic infectious bronchitis virus

Identification of the crucial genes in the elimination and survival process of Salmonella enterica ser. Pullorum in the chicken spleen

Transcriptome Analysis on Single Small Yellow Follicles Reveals That Wnt4 Is Involved in Chicken Follicle Selection

Thyroid transcriptome analysis reveals different adaptive responses to cold environmental conditions between two chicken breeds

Transcriptome profile analysis of leg muscle tissues between slow-and fast-growing chickens

Comprehensive Transcriptome Analysis Reveals Competing Endogenous RNA Networks During Avian Leukosis Virus, Subgroup J-Induced Tumorigenesis in Chickens

A set of measures of centrality based on betweenness

The structure and function of complex networks

Closeness Centrality Extended to Unconnected Graphs: The Harmonic Centrality Index

On the Distributed Computation of Load Centrality and its Application to DV Routing

Subgraph centrality in complex networks

Generalizations of the clustering coefficient to weighted complex networks