H7N9 viruses pose a threat to human health and they are no less harmful to the poultry industry than the H5N1 avian influenza viruses. However, the pathogenesis, transmissibility, and the host immune response of the H7N9 virus in chickens and mice remain unclear. In this study, we found that H7N9 viruses replicated in multiple organs of the chicken and viral shedding persisted up to 30 days postinoculation (DPI). The viruses were efficiently transmitted between chickens through direct contact. Notably, chickens infected with H7N9 had high antibody levels throughout the entire observation period and their antibody response lasted for 30 DPI. The expression levels of the pattern-recognition receptors and pro-inflammatory cytokines were found to be significantly upregulated in the brain using quantitative real-time PCR. The expression of TLR3, TLR7, MDA5, Mx, IL-1β, IL-6, IFN-α, and IFN-γ were also significantly different in the lungs of infected chickens. We found that the viruses isolated from these birds had low pathogenicity in mice, produced little weight loss and could only replicate in the lungs. Our findings suggested that the H7N9 viruses could replicate in chickens and mice and be efficiently transmitted between chickens, which presented a significant threat to human and poultry health.

Duck Tembusu virus (DTMUV) infection has caused great economic losses to the poultry industry in China, since its first discovery in 2010. Understanding of host anti-DTMUV responses, especially the innate immunity against DTMUV infection, would be essential for the prevention and control of this viral disease. In this study, transcriptomic analysis of duck embryonic fibroblasts (DEFs) infected with DTMUV reveals that several innate immunity-related pathways, including Toll-like, NOD-like, and retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathways, are activated. Further verification by RT-qPCR confirmed that RIG-I, MAD5, TLR3, TLR7, IFN-α, IFN-β, MX, PKR, MHCI, MHCII, IL-1β, IL-6, (IFN-regulatory factor 1) IRF1, VIPERIN, IFIT5, and CMPK2 were upregulated in cells infected with DTMUV. Through overexpression and knockdown/out of gene expression, we demonstrated that both VIPERIN and IRF1 played an important role in the regulation of DTMUV replication. Overexpression of either one significantly reduced viral replication as characterized by reduced viral RNA copy numbers and the envelope protein expression. Consistently, down-regulation of either one resulted in the enhanced replication of DTMUV in the knockdown/out cells. We further proved that IRF1 can up-regulate VIPERIN gene expression during DTMUV infection, through induction of type 1 IFNs as well as directly binding to and activation of the VIPERIN promoter. This study provides a genome-wide differential gene expression profile in cells infected with DTMUV and reveals an important function for IRF1 as well as several other interferon-stimulated genes in restricting DTMUV replication.

Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host’s innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-β mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host’s immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.

For industrial vaccine production, overwhelming the existing antiviral innate immune response dominated by type I interferons (IFN-I) in cells would be a key factor improving the effectiveness and production cost of vaccines. In this study, we report the construction of an IFN-I receptor 1 (IFNAR1)-knockout DF-1 cell line (KO-IFNAR1), which supports much more efficient replication of the duck Tembusu virus (DTMUV), Newcastle disease virus (NDV) and gammacoronavirus infectious bronchitis virus (IBV). Transcriptomic analysis of DTMUV-infected KO-IFNAR1 cells demonstrated that DTMUV mainly activated genes and signaling pathways related to cell growth and apoptosis. Among them, JUN, MYC and NFKBIA were significantly up-regulated. Furthermore, knockdown of zinc-fingered helicase 2 (HELZ2) and interferon-α-inducible protein 6 (IFI6), the two genes up-regulated in both wild type and KO-IFNAR1 cells, significantly increased the replication of DTMUV RNA. This study paves the way for further studying the mechanism underlying the DTMUV-mediated IFN-I-independent regulation of virus replication, and meanwhile provides a potential cell resource for efficient production of cell-based avian virus vaccines.