Abstract
In order to develop novel strategies to protect against increasingly virulent bird-linked pathogens, a better understanding of the avian antiviral response mechanism is essential. Type I interferons (IFNs) are recognized as the first line of defense in a host’s antiviral response; and it has been suggested that IRF7, a member of the IFN regulatory factor (IRF) family, plays an important role in modulating the immune response to avian influenza virus infection in chickens. The objective of this study was to identify candidate genes and pathways associated with IRF7 regulation at the transcriptome level as a first step towards elucidating the underlying cellular mechanisms of IRF7 modulation in the chicken antiviral response. IRF7 overexpression and knockdown DF-1 cell lines were established and stimulated by various pathogen-associated molecular patterns. Significant IRF7 and type I IFN expression changes were observed in both the IRF7 overexpression cell line and the IRF7 knockdown cell line upon exposure to the double stranded RNA (dsRNA) analog poly(I:C). Using RNA-seq based transcriptome analysis, we identified potential novel genes that IRF7 may help regulate as part of the host immune response to dsRNA; potential biomarkers and therapeutic targets revealed as a result of this study warrant further investigation. Based on our results, we suggest that IRF7 may have conserved functional activity in the avian antiviral response, and plays a crucial role in type I IFN regulation.
Highlights
Zoonotic viral pathogens are a serious threat to both poultry production and to human health
IRF7 expression is driven by the CMV immediate-early enhancer/ promoter, with GFP and Puromycin resistance serving as dual selection markers; empty vector, encoding only the selection markers, was used to generate control cell lines
A single IRF7 overexpression DF-1 cell line was selected based on its stable expression of both the selection markers and IRF7; the cell line selected demonstrated approximately 200-fold higher expression of IRF7 relative to the control cell line as determined by Quantitative reverse transcriptase PCR (qRT-PCR) (Fig 1C)
Summary
Zoonotic viral pathogens are a serious threat to both poultry production and to human health. Upon activation by pathogen recognition receptors (PRRs), type I interferons (IFNs) serve as the primary trigger of a host’s innate immune response against viral infection by initiating a signaling pathway that includes more than 300 IFN-stimulated genes (ISGs) in mammals [4]. Interferon regulatory factors (IRFs), transcription factors of type I IFNs, further modulate the immune response and are especially important for broad aspects of host defense such as adaptive immunity, oncogenesis regulation, and cell lineage differentiation [5]. IRF3 and IRF7, two key transcription factors that modulate type I IFN expression, are phosphorylated, undergo homo/heterodimerization, and translocate into the nucleus upon viral infection [6]. It has been demonstrated in mice that upon ssRNA viral infection, along with IRF3, IRF7 plays a key role in the induction and modulation of type I IFN expression [7]. Robust expression of type I IFNs is ensured via positive feedback regulation, as downstream components of the IFN signaling cascade drive the expression of IRF7 [6]
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