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. 2016 Dec;52(6):877-882.
doi: 10.1007/s11262-016-1374-2. Epub 2016 Jul 28.

Short communication: antiviral activity of porcine IFN-λ3 against porcine epidemic diarrhea virus in vitro

Haiyan Shen  1 , Chunhong Zhang  1 , Pengju Guo  2 , Zhicheng Liu  1 , Minhua Sun  1 , Junying Sun  1 , Linlin Li  1 , Jiawen Dong  1 , Jianfeng Zhang  3
Affiliations

Short communication: antiviral activity of porcine IFN-λ3 against porcine epidemic diarrhea virus in vitro

Haiyan Shen et al. Virus Genes. 2016 Dec.

Abstract

A new family of IFNs called type III IFN or IFN-λ has been described, and shown to induce antiviral activity against several viruses in the cell culture. In this study, the molecular cloning, expression, and antiporcine epidemic diarrhea virus (PEDV) activity of porcine IFN-λ3 (poIFN-λ3) were reported. The full-length poIFN-λ3 cDNA sequence encoded 196 amino acids with a 23 amino acid signal peptide. Sequence alignments showed that poIFN-λ3 had an amino acid sequence similarity to Ovis aries (78.1 %), Bos taurus (76.0 %), Tupaia belangeri (71.3 %), Equus caballus (69.9 %), and Homo sapiens (69.9 %). The phylogenetic analysis based on the genomic sequences indicated that poIFN-λ3 is located in the same branch as B. taurus and O. aries IFN-λ3. The poIFN-λ3 without a signal anchor sequence was efficiently expressed in Escherichia coli, and the purified recombinant poIFN-λ3 exhibited significant antiviral effects against PEDV in a dose- and time-dependent manner. This inhibitory effect of poIFN-λ3 on PEDV was observed under three different treatment conditions. The highest inhibition of PEDV was observed in Vero E6 cell cultures pretreated with poIFN-λ3 (prior to PEDV infection). In addition, poIFN-λ3 was able to induce the expression of IFN-stimulated genes, including ISG15, OAS1, and Mx1 in Vero E6 cells. These data demonstrate that poIFN-λ3 has antiviral activity against PEDV and may serve as a useful biotherapeutic candidate to inhibit PEDV or other viruses in swine.

Keywords: Antiviral activity; PEDV; Porcine IFN-λ3.

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Conflict of interest statement

All the authors have no conflict of interest to declare.

Figures

Fig. 1
Fig. 1
Cloning, expression, and purification of recombinant poIFN-λ3 in R. gami B. a Molecular cloning of poIFN-λ3. b SDS-PAGE analysis of samples. M, molecular weight marker; lane 1 the supernatant of bacterial cells harboring pET-poIFN-λ3 without IPTG induced; lane 2 the precipitation of bacterial cells harboring pET-poIFN-λ3 without IPTG induced; lane 3 the supernatant of bacterial cells harboring pET-poIFN-λ3 with 0.4 mM IPTG induced for 16 h at 20 °C; lane 4 the precipitation of bacterial cells harboring pET-poIFN-λ3 with 0.4 mM IPTG induced for 16 h at 20 °C. c Western blot using an anti-His antibody. M, molecular weight marker; lane 1 R. gami B extract transformed by pET-32a (+) (negative control); lane 2 R. gami B extract transformed by pET-poIFN-λ3
Fig. 2
Fig. 2
Amino acid sequences analysis, phylogenetic tree, and prediction of a signal peptide for poIFN-λ3. a Alignment of amino acid sequences for porcine, B. taurus, O. aries, T. belangeri, Pan troglodytes, Equus caballus, Mus musculus, Gallus silurana, and Homo sapiens IFN-λ3. The numbers indicate the amino acid position. Predicted signal peptide: amino acids 1–23 (boxed in blue). Putative N-glycosylation site: amino acids 71–74 (boxed in red). b A phylogenetic tree of the nine identified or predicted IFN-λ3 from different species. The uprooted tree was built using the neighbor-joining method based on the alignment of IFN-λ3 amino acid sequences. The scale bar is 0.1. c The signal peptide of poIFN-λ3 was predicted by SignalP3.0 Server. C, S, and Y scores indicate cleavage sites, ‘signal peptide-ness,’ and combined cleavage site predictions, respectively (Color figure online)
Fig. 3
Fig. 3
Effect of poIFN-λ3 on PEDV infection of Vero E6 cells. a Effect of poIFN-λ3 on PEDV infection and replication under three different conditions. Vero E6 cells were pretreated with poIFN-λ3 (100 ng/mL) for 24 h, and then infected with PEDV CV777 strain (Before) or coincubated with poIFN-λ3 (100 ng/mL) and PEDV CV777 strain at the same time (during), or 12 h after PEDV infection, Vero E6 cells were treated with 100 ng/mL poIFN-λ3 (After). b Time-dependent effect of poIFN-λ3 on PEDV infection. Vero E6 cells were infected with PEDV CV777 strain for 12 h and then treated with poIFN-λ3 at a concentration of 100 ng/mL. The PEDV titer in the supernatant at the indicated time point was titrated by TCID50. c Dose-dependent effect of poIFN-λ3 on PEDV infection. Vero E6 cells were infected with PEDV CV777 strain for 12 h and then treated with poIFN-λ3 at the indicated concentrations. The PEDV titer in the supernatant was analyzed at 48 h postinfection. d, e Real-time quantitative RT-PCR detection of PEDV-N mRNA transcripts relative to β-actin transcripts in the same sample. The mean of three repeat experiments performed in triplicate is shown and error bars represent the SD. d is under three different conditions and e is at different doses of poIFN-λ3
Fig. 4
Fig. 4
Expressions of OAS1, Mx1, and ISG15 induced by poIFN-λ3. The levels of OAS1, Mx1, and ISG15mRNA were measured by quantitative PCR, and the results were normalized by the GAPDH levels of each sample. Values represent the mean ± SD of three independent tests. P < 0.01 compared with the untreated Vero E6 cells
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