Isolation and Selection of Duck Primary Cells as Pathogenic and Innate Immunologic Cell Models for Duck Plague Virus

Abstract

Duck plague virus (DPV) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. The mechanism of organ tropism and innate immune evasion of DPV has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. In the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (DEFs), neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages] to identify appropriate cell models for DPV, using tropism infection and innate immunologic assays. Cells responded differently to stimulation with DNA viruses or RNA virus analogs. DPV infection exhibited broad tropism, as the recombinant virulent strain (CHv-GFP) infected DEFs, neurons, astrocytes, and monocytes/macrophages, but not the PBMCs, as the expression of EGFP was negligible. The basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in DEFs and astrocytes. Conversely, the titer and genomic copy number of the attenuated virus strain was higher in DEFs and astrocytes than in neurons and monocytes/macrophages. The titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in DEFs, neurons, and astrocytes. The innate immune response was not significantly induced by either DPV strain in DEFs, neurons, or astrocytes. The virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. Blockage of IFNAR signaling promoted replication of the attenuated strain. Pre-activation of IFNAR signaling inhibited infection by the virulent strain. The selection assay results indicated that induction of innate immunity plays an essential role in controlling DPV infection, and monocytes/macrophages are an Tian et al.Duck Primary Cells Infection Models important cell model for further investigations. Our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating DPV and potentially other aerial bird pathogens.

SC75741 antagonizes vesicular stomatitis virus, duck Tembusu virus, and duck plague virus infection in duck cells through promoting innate immune responses

“…However, no obvious changes in these genes were detected (data not shown). Our previous study has shown that the basal expression level of STING, RIG-I, MDA5, MAVS, IRF7, IFN-β, and MX in monocytes or macrophages was higher than that in DEF cells, neurons, and astrocytes ( Tian et al., 2019 ). Hence, we hypothesized that SC75741 may promote innate immunity responses relying on the basal level of these genes.…”

“…We further determined whether SC75741 could inhibit virus infection in different duck cell types. For this reason, we isolated duck primary neurons, astrocytes, and monocytes or macrophages as previously described ( Tian et al., 2019 ). We observed that the viral titers of VSV-GFP ( Figure 4 A) and DTMUV ( Figure 4 B) were reduced in duck neurons and astrocytes under the addition of SC75741 as in the DEF cells.…”

“…DEFs were cultured to 100% confluence for 24 h and then passaged and subcultured for use in the subsequent assays. Duck primary neurons, astrocytes, and monocytes or macrophages were isolated, cultured, and identified as explained earlier ( Tian et al., 2019 ). All cells were cultured in an incubator at 37°C, 5% CO 2 .…”

“…RNA was extracted from the cells using TRIzol reagent (Takara, Kyoto, Japan), according to the manufacturer's instructions; the genomic DNA was digested and excluded with DNase I; total RNAs were reverse transcribed into cDNA with the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan); Q-PCR procedure was performed as described previously ( Tian et al., 2019 ). Expression levels of IL-1β, IL-6, C-C motif chemokine ligand 21 ( CCL21 ), interferon-beta ( IFN-β ), 2′-5′-oligoadenylate synthetase-like protein ( OASL ), and myxovirus resistance ( MX ) mRNAs was detected and presented as relative fold-change according to the 2 −ΔΔCT method.…”

Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-β signalling by interacting with STAT1

“…The data was analyzed by one-way ANOVA. ***p < 0.001 and persistently infects monocytes/macrophages [68], but the mechanism of immune evasion by DEV is unknown. In our study, we first found that DEV pUL47 significantly inhibits the IFN-β signalling induced by poly(I:C) and downregulates the STAT1-induced ISG expression.…”