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Due to our negligence, the original version of this article, published online on 12 January 2022, contained a mistake in Fig. 4A. The lane of β-actin in Western blotting was misused. The correct Fig. 4 is given below. We apologize for our oversight when preparing the figure and state that this does not change the scientific conclusions of the article in any way.[Formula presented] Fig. 4. Identification of interactions between RHDV nonstructural proteins and host factors of RCs. A NCL siRNA inhibited the formation of the RHDV RC. After HA tag affinity purification, the eluted proteins were resolved by SDS-PAGE. The protein bands were visualized with silver staining. PBS acted as a negative control; β-actin acted as an internal control and was detected by IB with mAb against β-actin. B Identification of these interactions by M2H assays. Bait and prey plasmids were co-transfected with pG5luc plasmids into subconfluent HEK-293T cells at a molar ratio of 1:1:1 for the pACT:pBIND:pG5luc vector. At 48 h post-transfection (hpt), the HEK-293T cells were lysed, and Rluc and Fluc activities were evaluated using the Promega Dual-Luciferase Reporter Assay System. All experimental groups were compared with the negative control group (ACT-Bind). Statistical analysis was performed by Student t-tests. ∗P < 0.05 and ∗∗P < 0.01. Data are shown as mean with SD. Replicate 1, 2, 3 means three independent experiments, and each experiment contains three technical replicate values. The number of cells used in all replicate experiments was similar. C These interactions were verified using Co-IP assays. RK-13 cells were co-transfected with bait and prey plasmids. Cell lysates were prepared 48 hpt and the proteins were subjected to IP followed by IB analysis. Myc fusion proteins acted as bait proteins and Flag fusion proteins acted as prey proteins. RHDV, rabbit hemorrhagic disease virus; RC, replication complex; IP, immunoprecipitation; IB, immunoblotting; mAb, monoclonal antibody; SD, standard deviation.

Interferons (IFNs) including type I/III IFNs are the major components of the host innate immune response against porcine epidemic diarrhea virus (PEDV) infection, and several viral proteins have been identified to antagonize type I/III IFNs productions through diverse strategies. However, the modulation of PEDV infection upon the activation of the host’s innate immune response has not been fully characterized. In this study, we observed that various IFN-stimulated genes (ISGs) were upregulated significantly in a time- and dose-dependent manner in LLC-PK1 cells infected with the PEDV G2 strain FJzz1. The transcriptions of IRF9 and STAT1 were increased markedly in the late stage of FJzz1 infection and the promotion of the phosphorylation and nuclear translocation of STAT1, implicating the activation of the JAK-STAT signaling pathway during FJzz1 infection. In addition, abundant type I/III IFNs were produced after FJzz1 infection. However, type I/III IFNs and ISGs decreased greatly in FJzz1-infected LLC-PK1 cells following the silencing of the RIG-I-like receptors (RLRs), including RIG-I and MDA5, and the Toll-like receptors (TLRs) adaptors, MyD88 and TRIF. Altogether, FJzz1 infection induces the production of type-I/III IFNs in LLC-PK1 cells, in which RLRs and TLRs signaling pathways are involved, followed by the activation of the JAK-STAT signaling cascade, triggering the production of numerous ISGs to exert antiviral effects of innate immunity.

Additional file 12: Supplementary Table 1. Information of bird species and library included in the present study.

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5'-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZalphaA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.