IFN-γ establishes interferon-stimulated gene-mediated antiviral state against Newcastle disease virus in chicken fibroblasts

Abstract

Newcastle disease virus (NDV) causes severe economic losses through severe morbidity and mortality and poses a significant threat to the global poultry industry. Significant efforts have been made to develop novel vaccines and therapeutics; however, the interaction of NDV with the host is not yet fully understood. Interferons (IFNs), an integral component of innate immune signaling, act as the first line of defense against invading viruses. Compared with the mammalian repertoire of IFNs, limited information is available on the antiviral potential of IFNs in chickens. Here, we expressed chicken IFN-γ (chIFN-γ) using a baculovirus expression vector system, characterized its antiviral potential against NDV, and determined its antiviral potential. Priming of chicken embryo fibroblasts with chIFN-γ elicited an antiviral environment in primary cells, which was mainly due to interferon-stimulated genes (ISGs). A genome-wide transcriptomics approach was used to elucidate the possible signaling pathways associated with IFN-γ-induced immune responses. RNA-sequencing (RNA-seq) data revealed significant induction of ISG-associated pathways, activated temporal expression of ISGs, antiviral mediators, and transcriptional regulators in a cascade of antiviral responses. Collectively, we found that IFN-γ significantly elicited an antiviral response against NDV infection. These data provide a foundation for chIFN-γ-mediated antiviral responses and underpin functional annotation of these important chIFN-γ-induced antiviral influencers.

Keywords: NDV; RNA-seq; chicken embryo fibroblast; interferon; interferon-stimulated gene.

Figure 1

Schematic representation of the silkworm BEVS It demonstrated the construction of recombinant baculovirus and expression of recombinant protein.

Figure 2

chIFN-γ inhibited NDV (strain F48E9) infection in CEFs CEFs were either left untreated or treated with chIFN-γ (4.19 × 10³ IU/ml) for 24 h before infection with NDV (strain F48E9). The supernatant containing NDV RNA was measured by RT-qPCR at 12, 24, and 36 h post-infection. Surprisingly, we observed that pre-treatment with chIFN-γ at a concentration of 4.19 × 10³ IU/ml significantly reduced the viral replication in CEFs at a regular interval (12, 24, and 36 hpi). The results are presented as the mean ± SEM (n = 3). ^*P-value <0.05 by unpaired t-test.

Figure 3

Gene expression illustration volcano plots and a Venn diagram (A) DEGs in NDV-treated CEFs. (B) DEGs in chIFN-γ-treated CEFs. (C) DEGs in chIFN-γ + NDV-treated CEFs. (D) DEGs in chIFN-γ + NDV-treated CEFS with NDV treated CEFS (chIFN-γ + NDV vs NDV). Red and green dots demonstrate upregulated and downregulated DEGs, respectively. Differential expression patterns represent the temporal expression of genes expressed in the four groups.

Figure 4

A hierarchical heat map representing the expressional values of DEGs in the different groups (A) The color bar represents the level of differential expression compared with the control group. GAMMA (chIFN-γ-treated CEF), Control (only CEF with blank silkworm blood), NDV (NDV-treated CEF), and G_NDV (chIFN-γ + NDV-treated CEF). Red and blue colors represent up and down-regulation, respectively. (B) Venn diagram representing the common gene distribution among the three distinct groups.

Figure 5

Verification of the relative expression levels by quantitative real-time PCR Expression patterns of selected DEGs associated with the immune response as determined by qPCR. The x-axis shows the annotations of the selected genes. The y-axis shows expression levels that are normalized to β-actin expression. Gene expression was quantified relative to β-actin expression using the 2^-ΔΔCT method. The results are presented as the mean ± SEM (three biological replicates and three technical replicates). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by unpaired t-test.

Figure 6

Gene network describing the association of genes observed in the present study (kmeans = 4) String protein network demonstrating the interlinked genes associated with immune response, immune system process, MHC protein complex, MHC class II protein complex, cytokine activity, and cytokine receptor binding (NDV + IFN-γ group) (https://string-db.org/).

Figure 7

GO enrichment analysis for DEGs (A) GO analyses of DEGs in NDV-treated CEF compared with control. (B) GO analyses of DEGs in chIFN-γ-treated CEFs compared with control. (C) GO analyses of DEGs in NDV + chIFN-γ-treated CEF compared with control. (D) GO analyses of DEGs in NDV + chIFN-γ-treated CEFs compared with NDV-treated CEF. Among various enriched GO terms, cytokine-mediated immune processes were the most enriched processes based on the criteria (P < 0.05).

Figure 8

KEGG pathway enrichment analysis for all upregulated DEGs (A) KEGG pathway enrichment in NDV-treated CEFs compared with the control group. (B) KEGG pathway enrichment in chIFN-γ-treated CEFs compared with control group. (C) KEGG pathway enrichment in NDV + chIFN-γ-treated CEFs compared with control group. (D) KEGG pathway enrichment in NDV + chIFN-γ-treated CEFs compared with NDV-treated CEFs group. Circles represent the number of genes, while the colors illustrate the magnitude of richness factor.