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Cloning and expression of mink (Neovison vison) interferon-γ gene and development of an antiviral assay
Research in Veterinary Science 101 (2015) 93–98
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Cloning and expression of mink (Neovison vison) interferon-γ gene and development of an antiviral assay Hailing Zhang a,b, Jianjun Zhao a, Xue Bai a, Lei Zhang a, Sining Fan d, Bo Hu a, Hao Liu a, Dongliang Zhang c, Shujuan Xu a, Xijun Yan a,b,⁎ a
Division of Infectious Diseases of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, 4899 Juye Street, Changchun 130112, China State Key Laboratory for Molecular Biology, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China Jilin Teyan Biotechnological Co. Ltd, 388 Liuying West Road, Changchun 130122, China d Jilin Agricultural University, China b c
a r t i c l e
i n f o
Article history: Received 18 March 2015 Received in revised form 18 March 2015 Accepted 21 June 2015 Keywords: Mink Interferon-γ Antiviral activity
a b s t r a c t Minks (Neovison vison) farming is under a threat of a variety of viral infections with increasingly growing number of breeding in Northeastern and Western China. While interferon is effective in controlling viral infection, IFN among different species rarely share high homology enough to provide cross protective effect on inhibition of virus replication. We cloned, sequenced, phlogenetically analyzed and expressed the miIFN-γ gene in prokaryotic and eukaryotic cells. The anti-vesicular stomatitis virus (VSV) activity of miIFN-γ protein was tested in MDCK cells using in vitro cytopathic inhibition assay. The recombinant miIFN-γ could inhibit VSV replication in MDCK cells, which was confirmed by that pre-incubation of rabbit anti-miIFN-γ antibodies with miIFN-γ abrogated the miIFN-γ protective effect. Our findings implicated that the miIFN-γ gene may be a potential counter measure against viral infection in the mink farming. © 2015 Published by Elsevier Ltd.
1. Introduction Cytokines play a fundamental role in regulating immune responses. Interferons (IFNs) belong to cytokine family and has a wide range of physiological functions, such as inhibiting virus infection, regulating cell proliferation and differentiation, and modulating immune response (Schneider et al., 2014; Huang et al., 2012), thus being the first defensive barrier of viral infection (Janardhana et al., 2012; Gerlier and Lyles, 2011). IFNs are generally classified into types I, II and III (Sorgeloos et al., 2013) according to their cell surface receptors, acid stability, primary sequences and chromosomal localization (Lasfar et al., 2011). Type II IFN, also known as IFN-γ plays a key role in the development of innate and adaptive immunity and the regulation of Th1-type immune responses (Xi et al., 2012; Huang et al., 2012; Hoegen et al., 2004). IFN-γ plays a central role in orchestrating the immune response especially antiviral defense of the host cell by controlling viral infection and replication (Voigt et al., 2013; Schroder et al., 2004) therefore, IFN-γ
⁎ Corresponding author at: Division of Infectious Diseases of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, 4899 Juye Street, Changchun 130112, China. E-mail addresses: [email protected] (H. Zhang), [email protected] (J. Zhao), [email protected] (X. Bai), [email protected] (L. Zhang), [email protected] (S. Fan), [email protected] (B. Hu), [email protected] (H. Liu), [email protected] (D. Zhang), [email protected] (S. Xu), [email protected] (X. Yan).
http://dx.doi.org/10.1016/j.rvsc.2015.06.012 0034-5288/© 2015 Published by Elsevier Ltd.
has been used for evaluation of cellular immune response level and become an attractive candidate for immunotherapeutic agent for the treatment of cancer and infectious diseases (Biron, 1998; Chopra et al., 2015). Till now, recombinant human, equine, white rhinoceros, cattle, and fruit bat IFN-γ have been shown to inhibit a variety of virus replication in many cell lines (Morar et al., 2007; Huang et al., 2012), and the commercialized recombinant IFN-γ of some livestock animals such as pigs, dogs, and birds have been used in veterinary clinical treatment (Li and Sherry, 2010; Ma et al., 2009; Qu et al., 2013; Tian et al., 2014). Recombinant human, equine, white rhinoceros, cattle, and fruit bat IFN-γ have been shown to inhibit the replication of various viruses in many cell lines (Morar et al., 2007). In mammals, IFN-γ unlike IFN-α and IFN-β proteins which share a high degree of homology to allow functional cross-reactivity among different species; and different IFNγ proteins of different mammalian species share relatively low homology thereby seldom have cross protective activity. Mink (Neovison vison) is one of the major fur animals and its health problems have become an important issue for fur industry. The infection by canine distemper virus, parvovirus and Aleutian mink disease virus among minks has caused serious economic losses to the fur animal industry (Wang et al., 2012, 2014; Zhao et al., 2014). Currently, there is no effective treatment for mink viral disease due to the rare cross protective effect of IFN-γ among different mammalian species (Waldvogel et al., 2004). Therefore, cloning, sequencing, phylogenic analysis and expression of mink interferon-γ (miIFN-γ) gene are necessary for protection of mink from viral infection.
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2. Materials and methods
2.3. Sequence analysis
2.1. Reagents and animals
The miIFN-γ open reading frame (ORF) of the nucleotide sequence and the deduced IFN-γ amino acid sequence were blast aligned with those of IFN-γ genes of various species from GenBank, and a multispecies phylogenetic tree based on the nucleotide sequence sequences of arious IFNs was constructed using DNAStar 5.0 software. The signal peptide sequence and the glycosylation sites of the miIFN-γ protein were predicted with online SignalP 4.1 Server and NetNGlyc Servers (http://www.cbs.dtu.dk/services) respectively.
Vesicular stomatitis virus (VSV) was provided by Dr. Haidong Zhi from the Institute of Harbin Veterinary Research, Chinese Academy of Agricultural Sciences, Harbin, China. The MDCK-line cells were purchased from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). RPM-I 1640 media were purchased from GIBCO (USA). Lymphocyte isolation kit was from Chinese Academy of Medical Sciences (Beijing, China). TRIzol, Ni-NTA protein purification system and anti-6 × His monoclonal antibodies were purchased from Invitrogen (USA). ExTaq polymerase, AMV reverse transcriptase, plant haemagglutinin (PHA) and pMD18-T vector were purchased from TaKaRa (Dalian, China), and PCR primers were synthesized at Shanghai Ying-Jun Biotech (Shanghai, China). TMB Chromogenic Reagent was purchased from Sigma (USA). Six-month old minks (American mink, N. vison) were provided by the Chinese Academy of Agricultural Sciences (Beijing, China). Two SPF rabbits (6-week old) were purchased from Changchun Institute of Biological Products Co., Ltd., Changchun, China. The animal experiments were performed according to the regulations for the administration of affairs concerning experimental animals, and was approved by the state council on October 21st, 1988 and promulgated by decree no. 2 of the State Science and Technology Commission on November 14th, 1988. 2.2. Total RNA extraction and cDNA cloning of mink IFN-γ Mink whole blood was diluted 1:1 with RPMI 1640 cell culture media (GIBCO, USA), added to an equal volume of lymphocyte separation media and centrifuged at 200 ×g at room temperature (RT) for 30 min. PBMCs were collected and washed with RPMI-1640 (GIBCO, USA) at RT. After centrifugation at 180 ×g for 10 min, resuspended in 10 ml of fresh RPMI supplemented with 2 mM L-glutamine and 10% fetal calf serum (Haclone, USA), plated at 1 × 106 cells/ml, stimulated with 100 μg/ml of phytohemagglutinin-L (PHA-L); (Sigma, USA) and incubated at 37 °C in 5% CO2 for 24 h for further RNA purification. PBMCs stimulated with PHA-L were centrifuged and total RNA was isolated according to RNeasy Mini Kit Operation Manual (Qiagen, USA). cDNA was synthesized from total RNA at 42 °C for 1 h after 9.5 μl RNA and 1 μl Oligod (T)15 primer was mixed and heated at 70 °C for 5 min, with 1 μl reverse transcription enzyme (TaKaRa, Japan), 0.5 μl RNA inhibitor and 4 μl 10 mM dNTP. The MiIFN-γ cDNA was amplified by PCR using the specific primers (Table 1) that were designed based on the canine IFN-γ cDNA sequence (GenBank no. AF126247.1). PCR reactions were subjected to 34 cycles of initial denaturation at 95 °C for 5 min, denaturation at 94 °C for 45 s, primer annealing at 53 °C for 45 s and DNA extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. Resultant PCR products were purified using a TaKaRa PCR Purification Kit (TaKaRa, China) and subcloned into pMD18-T vector for sequencing at Shanghai Ying-Jun BioTech (Invitrogen Shanghai, China). Table 1 Primers sequences. Cytokine
Predicted size (bp)
Primers
ORF
501
Mature peptide clone
444
P1 P2 P3
Eukaryotic expression
501
P4 P5 P6
5′-ATGGCCCTGCCCTGCTCCT-3′ 5′-TCACTTCCTGCTCCGCAATC-3′ 5′-GCGGATCCGGCTATTACTGTCAGGCC ATG-3′ 5′-GCAAGCTTTTATTTCGATGCTCTGCGG-3 5′-GCTGAATTCATGGCCCTGCCCTGCTCCT-3 5′-CACTCGAGTTACTTCCTGCTCCGCAATC-3
2.4. Expression of MiIFN-γ in Escherichia coli cells PCR product was separated on a 1.5% agarose gel. The purified DNA was digested with restriction enzymes BamH I and Hind III, ligated into pET32a vector, and transformed into competent E. coli JM109 cells. Positive clones were identified with PCR and restriction enzymes digestion and sequence analysis at Shanghai Ying-Jun BioTech. (Invitrogen, shanghai, China). The recombinant plasmid pET/miIFN-γ was transformed into E. coli BL21 competent cells and selected with. Positive clones were inoculated (1:200) in 5 ml of liquid LB media and cultured at 30 °C, and 1 mmol/L IPTG was added to induce the protein expression overnight when the OD600 reached to 0.6–0.9. For purification, the bacteria were centrifuged at 2000 ×g for 5 min, frozen and thawed 3 times, and the bacterial pellet was mixed with binding buffer (20 m M Na3PO4, 0.5 M NaCl, 20 mM imidazole, pH 7.4) and lysed by ultrasound for 5 s and left on ice for 5 s. Next, the mixture was centrifuged at 800 ×g and 4 °C for 10 min, and the supernatant was collected, incubated on ice and mixed with magnetic stirrer blender after the addition of 50% NI–NTA (Novagen) (4:1). Finally, the mixture was loaded onto a chromatography column (Novagen) and eluted by serial washing using elution buffer (20 mM Na3PO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4). The samples washed were separated on a 12% SDS-PAGE and transferred onto a PVDF membrane which was blocked with 5% milk in TBST (0.1 phosphate buffer containing, 0.5% Tween-20) and incubated with mouse anti-6×His monoclonal antibody at 4 °C overnight, followed by incubation with goat anti-mouse IgG-HRP at room temperature for 1 h. Finally, the membrane was developed. 2.5. Production of rabbit antiserum against recombinant MiIFN-γ Two SPF rabbits were immunized subcutaneously with 0.5 ml (0.3 mg) of purified miIFN-γ protein emulsed with 50% Freund's complete adjuvant (Sigma, USA). In 14 and 28 days after the first vaccination, the rabbits were boosted with 0.5 ml (0.3 mg) purified miIFN-γ protein in 50% Freund's incomplete adjuvant (Sigma, USA). 10 days after the last injection, the blood was collected from the rabbits ear vessels, antiserum against miIFN-γ (pAb) was separated at 1000 ×g and 4 °C for 10 min and frozen at −20 °C. 2.6. In vitro antiviral activity of MiIFN-γ The antiviral activity of the recombinant IFN-γ was determined by cell cytopathic inhibition assay. Briefly, MDCK cells were plated in 96well plates and incubated at 37 °C with 5% CO2 for 12 h; diluted recombinant IFN-γ was added to the monolayer cells and incubated at 37 °C with 5% CO2 overnight; and 100 TCID50 of VSV was defined as the virus amount that caused 50% of the cells to be cytopathic, which was assessed by a microscopic examination. Next, the cells were challenged with 100 TCID50 of VSV and incubated at 37 °C for 1–2 days, and the IFNγ was the concentration at which 50% of the cells were protected from VSV infection. To rest the miIFN-γ antiviral protective activity, neutralization assay was performed. The MDCK cells were treated with miIFN-γ first, neutralized with rabbit anti-miIFN-γ antibody (diluted 1:30) at 37 °C, and
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the controls were untreated for 24 h, Then, all cells were challenged with 100 TCID50 of VSV. 2.7. Immunofluorescence assay PCR was used to generate a construct for the eukaryotic expression of complete ORF of MiIFN-γ using the forward primer 5′-GTGAATTC CAGGCCATGTTTTTTAAAGA-3′ containing the EcoRIsite and reverse primer 5′-GACTCGAGGCAGGATGACCGTTATTTCG-3′ containing the XhoIsite. After digestion with EcoR I and Xho I, the PCR products were subcloned into the corresponding sites of pcDNA3.1 (+) (Novagen, USA) with T4 DNA Ligase (Thermo Scientific, USA). The recombinant transfer vector pcDNA3.1 (+)-ORF or the negative control vector pcDNA3.1 was transfected into MDCK cell using Lipofectamine (Roche, USA). The cells were then incubated with rabbit anti-MiIFN-γ serum (1:10) at 37 °C for 3 h washed with PBS three times, and incubated with fluorescein isothiocyanate conjugated (FITC) goat anti-rabbit IgG (1:500 ) for 1 h at 37 °C with fluorescein isothiocyanate conjugated (FITC) goat anti-rabbit IgG diluted 1:500. The immunofluorescence was observed under a LEICA DMI3000 B fluorescence microscope. 3. Result 3.1. Analysis of MiIFN-γ gene The mink IFN-γ cDNAs were successfully amplified by RT-PCR, predicted to be 501 bp, and subcloned into plasmid pET32a vector to derive recombinant plasmid pET/miIFN-γ, which was confirmed by restriction enzyme digestion analysis. DNAStar software analysis demonstrated that the mink IFN-γ cDNA was 501 bp in length. The ORF of the MiIFN-γ sequence was deposited into GenBank (GenBank accession no. KJ888148). The deduced miIFN-γ protein consists of 167 amino acids, including 3 cysteine amino acid residues at positions 13, 17 and 23. The analysis by NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/ NetNGlyc/) indicated that miIFN-γ had three potential N-glycosylation sites. SignalP 4.1 server software http://www.cbs.dtu.dk/services/ SignalP/ analysis predicted that the N terminal 19 amino acids were signal peptide. The ORF of miIFN-γ was compared with that of several mammalian IFN-γs (Fig. 2). Both mink and ferret belong to Mustelidae (Ochi et al., 2008). The homology between the miIFN-γ and the domestic ferret nucleotide and amino acid sequences (GenBank registration number: EF492064) were 99.4% and 98.2%, respectively. The nucleotide sequence alignment analysis (Table 2) showed that miIFN-γ shared 98.6% identity with Eurasian badger, 93.4% identity with giant panda, 93.0% identity with dog and fox, 87.2% identity with cat, 80.0–82.6% identity with
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cattle, horse, bottlenosed dolphin and pig, and less than 80% identity with the IFN-γs of other mammalian species (Table 2). Phylogenetic analysis (Fig. 1) further indicated that IFN-γ was subdivided into two monophyletic lineages: chicken (avian) and mammalian. Mammalian branch was divided into carnivores and herbivores. The mink, domestic ferret, Eurasian badger, giant panda, dog, fox and cat IFN-γs nucleotide sequences shared higher homology because they formed a carnivore monophyletic group that was distinct from other herbivores IFN-γs (Fig. 2). 3.2. Expression and purification of MiIFN-γ in E. coli cells To evaluate the biological activity of miIFN-γ, we subcloned miIFN-γ cDNAs into prokaryotic expression vector pET32a and induced its expression by IPTG in E. coli BL21. Western blot analysis miIFN-γ with an anti-6 × His antibody revealed an about 17 kDa miIFN-γ protein (Fig. 3). The protein solubility analysis showed that the recombinant miIFN-γ protein accounted for 80% of the total soluble proteins. 3.3. In vitro bioactivity of recombinant miIFN-γ To investigate whether pre-treatment with mink IFN-γ could protect cells from virus infection, we analyzed antiviral activity using cytopathic inhibition assays in vitro (Wallach, 1983). Different extents of cytopathic effects caused by VSV were observed in MDCK cells pretreated with different concentrations of IFN-γ, viral replication was completely inhibited when cell hole was inoculated with 52.5 μg/ml 100 μl. The protein even at 1:1024 dilution conferred a protective effect on MDCK at different levels (Fig. 4); and cellular changes were observed through a microscope. The negative controls were normal MDCK, and the positive control showed completely (100%) cytopathic effect, which was significantly inhibited by soluble MiIFN-γ. 3.4. Immunofluorescence assay MiIFN-γ cDNA encoding the signal sequence and mature protein, including the stop codon, was cloned into the expression vector pcDNA3.1. The recombinant pcDNA3.1/MiIFN-γ plasmid DNAs were transfected into MDCK cells which were incubated with rabbit antimiIFN-γ serum and FITC-goat-anti-rabbit IgG. Some cells showed obvious green fluorescence (Fig. 5A), indicating that the miIFN-γ was mainly expressed in the cytoplasm of MDCK cells. The cells transfected with plasmid pcDNA3.1 without MiIFN-γ gene showed no fluorescence (Fig. 5 B). The experimental results showed that rabbit anti-miIFN-γ
Table 2 Homology of mink IFN-γ (KJ888148) gene with that of different species. Species
Nucleotide homology (%)
Amino acid homology (%)
Genbank accession no.
Domestic ferret Eurasian badger Giant panda Dog Fox Cat Chimpanzee Cattle Horse Bottlenosed dolphin Pig Human Rabbit Chicken
99.4 98.6 93.4 93.0 93.0 87.2 74.3 80.0 82.6 82.6 80.6 74.5 75.4 41.0
98.2 98.2 91.0 87.4 87.4 82.0 63.5 72.5 79.0 74.9 74.3 63.5 61.1 29.7
EF492064 Y11647 GQ386864 FJ194478 EF428257 NM001009873 NM001193665 NM174086 NM001081949 AB022044 NM213948 NM000619 AB010386 NM205149
Fig. 1. Phylogenetic tree of IFN-γ gene nucleotide sequences of several mammalian and Chichen. (Genbank accession No.: mink/KJ888148; domestic ferret/EF492064; Eurasian badger/Y11647; giant pandger/GQ386864; dog/FJ194478; fox/EF428257; cat/NM001009873; bottlenosed dolphin/AB022044; cattle/NM174086; chimpanzee/NM001193665; horse/ NM001081949; human/NM000619; pig/NM213948; rabbit/AB010386).
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Fig. 2. Analysis of amino acid sequences of several mammals IFN-γ (mink, domestic ferret, Eurasian badger, giant panda, dog, fox, cat, bottlenosed dolphin, cattle, chimpanzee, horse, human, pig and rabbit). Black triangle represents the predicted cysteine residues; and red and boxed areas indicate potential N-glycosylation sites. Underlined is the predicted signal sequence of mink IFN-γ.
antibody produced with miIFN-γ proteins expressed in the prokaryotic expression system specifically bound to MiIFN-γ in MDCK cells. 4. Discussion Mink fur is an economically important fur raw material in the garment industry worldwide; therefore mink breeding provides enormous
Fig. 3. Soluble expression and purification of rMiIFN-γ was observed on SDS-PAGE. Lane A: non-induced crude; Lane B: induced crude; Lane C: supernatant of lysate; Lane D: precipitation of lysate; Lane E: recombinant rMiIFN-γ purified by NTA-Ni chromatography at elution buffer pH 7.4; and MK: molecular weight marker.
economic value for fur animal industry. In recent years, mink distemper, mink parvovirus enteritis, aeutian disease and other infectious diseases have been endangering the survival of mink (Wang et al., 2014; Zhang et al., 2015). Thus, the prevention and treatment of these diseases cannot be ignored. IFNs, were identified long ago but have recently been intensively re-investigated as the interdependence of the innate and adaptive immune responses have become more evident (Guo et al., 2014). To determine whether miIFN-γ in exhibits antiviral activity, we cloned, sequenced and phlogenetically analyzed and expressed MiIFNγ gene and tested the antiviral activity against virus replication.
Fig. 4. Cytopathic inhibition rate in MDCK pretreated with rMiIFN-α and infected with VSV.
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Fig. 5. Immunofluorescence micrographs of transfected MDCK. A: MDCK (pcDNA3.1-MiIFN-γ ORF group); and B: MDCK (pcDNA3.1 group).
The deduced miIFN-γ protein consists of 167 amino acids, with 3 cysteine amino acid residues at positions 13, 17 and 23, two of these three cysteines (at positions 13 and 23) are conserved among most of the mammalian IFN-γ proteins (Altmann et al., 2003; Schultz et al., 1995). Domestic ferret, also belonging to Mustelidae, is very genetically close to kinship with the mink. Phylogenetic analysis (Fig. 2) further indicated that IFN-γ was subdivided into two monophyletic lineages: chicken (avian) and mammalian, and the mammalian branch was divided into carnivores and herbivores. The mink, domestic ferret, Eurasian badger, giant panda, dog, fox and cat IFN-γs shared higher homology and formed a carnivore monophyletic group that was distinct from other herbivore IFN-γs. MiIFN-γ ORF size is 501 bp and completely consistent with domestic ferret, Eurasian badger, giant panda, dog, pig and horse. As expected from the close phylogenetic relationship, the highest amino acids homology up to 98% was observed with the domestic ferret and Eurasian badger IFN-γ sequences. The MiIFN-γ gene was predicted to encode a signal sequence of 19 aa and a mature protein of 147 aa residues. Sequence analysis revealed there are three codon differences between mink and domestic ferret. These three codons encode the 66th, 111th and 122nd amino acids, respectively. Among them, the 66th aa site AAC encodes Thr in mink and AAA Lys in domestic ferret; the 111th aa site CGG encodes Arg in mink and ctg encodes Leu in domestic ferret; and the 122nd aa site was CCG in mink and CCT in domestic ferret which both encodes the same aa (Pro). These results indicate that although mink and domestic ferret all belong to Mustelidae species, certain gene mutations occurred in the long process of genetic evolution. Currently, E. coli is still the easiest, cheapest, and most widely used protein expression system. In our study, soluble MiIFN-γ was easy to be produced, this may be because only one cysteine and one glycosylation site existed in the mature protein aa sequence. To see whether MiIFN-γ possesses biological function, we measured the bioactivity of MiIFN-γ protein using cytopathic effect (CPE) inhibition assays in vitro. These results demonstrated that MiIFN-γ protected MDCK cells from challenge with VSV, indicating that MiIFN-γ protein showed certain degrees of cross-species activity. This finding was surprising considering the close relationship in evolutionary terms between mink and canine. The antiviral activity of the MiIFN-γ protein was almost completely neutralized with polyclonal antibody against MiIFNγ at 1:20 but was almost not neutralized at 1:100. In fact, these antibodies were inhibitory only under certain experimental conditions (Zhang et al., 2008). Many previous studies reported that anti-HBV effect was mediated by recombinant eukaryotic expression vector for IFN-α (Yu et al., 2013). In our study, immunofluorescence experiments showed that the mink IFN-γ was highly expressed in MDCK cells by pcDNA3.1 Vector, we will carry out anti-viral effect of recombinant eukaryotic expression in the future. In conclusion, the present study reports the successful cloning, sequencing and expression of IFN-γ from mink, and the monoclonal
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/yrvsc
Cloning and expression of mink (Neovison vison) interferon-γ gene and development of an antiviral assay Hailing Zhang a,b, Jianjun Zhao a, Xue Bai a, Lei Zhang a, Sining Fan d, Bo Hu a, Hao Liu a, Dongliang Zhang c, Shujuan Xu a, Xijun Yan a,b,⁎ a
Division of Infectious Diseases of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, 4899 Juye Street, Changchun 130112, China State Key Laboratory for Molecular Biology, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China Jilin Teyan Biotechnological Co. Ltd, 388 Liuying West Road, Changchun 130122, China d Jilin Agricultural University, China b c
a r t i c l e
i n f o
Article history: Received 18 March 2015 Received in revised form 18 March 2015 Accepted 21 June 2015 Keywords: Mink Interferon-γ Antiviral activity
a b s t r a c t Minks (Neovison vison) farming is under a threat of a variety of viral infections with increasingly growing number of breeding in Northeastern and Western China. While interferon is effective in controlling viral infection, IFN among different species rarely share high homology enough to provide cross protective effect on inhibition of virus replication. We cloned, sequenced, phlogenetically analyzed and expressed the miIFN-γ gene in prokaryotic and eukaryotic cells. The anti-vesicular stomatitis virus (VSV) activity of miIFN-γ protein was tested in MDCK cells using in vitro cytopathic inhibition assay. The recombinant miIFN-γ could inhibit VSV replication in MDCK cells, which was confirmed by that pre-incubation of rabbit anti-miIFN-γ antibodies with miIFN-γ abrogated the miIFN-γ protective effect. Our findings implicated that the miIFN-γ gene may be a potential counter measure against viral infection in the mink farming. © 2015 Published by Elsevier Ltd.
1. Introduction Cytokines play a fundamental role in regulating immune responses. Interferons (IFNs) belong to cytokine family and has a wide range of physiological functions, such as inhibiting virus infection, regulating cell proliferation and differentiation, and modulating immune response (Schneider et al., 2014; Huang et al., 2012), thus being the first defensive barrier of viral infection (Janardhana et al., 2012; Gerlier and Lyles, 2011). IFNs are generally classified into types I, II and III (Sorgeloos et al., 2013) according to their cell surface receptors, acid stability, primary sequences and chromosomal localization (Lasfar et al., 2011). Type II IFN, also known as IFN-γ plays a key role in the development of innate and adaptive immunity and the regulation of Th1-type immune responses (Xi et al., 2012; Huang et al., 2012; Hoegen et al., 2004). IFN-γ plays a central role in orchestrating the immune response especially antiviral defense of the host cell by controlling viral infection and replication (Voigt et al., 2013; Schroder et al., 2004) therefore, IFN-γ
⁎ Corresponding author at: Division of Infectious Diseases of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, 4899 Juye Street, Changchun 130112, China. E-mail addresses: [email protected] (H. Zhang), [email protected] (J. Zhao), [email protected] (X. Bai), [email protected] (L. Zhang), [email protected] (S. Fan), [email protected] (B. Hu), [email protected] (H. Liu), [email protected] (D. Zhang), [email protected] (S. Xu), [email protected] (X. Yan).
http://dx.doi.org/10.1016/j.rvsc.2015.06.012 0034-5288/© 2015 Published by Elsevier Ltd.
has been used for evaluation of cellular immune response level and become an attractive candidate for immunotherapeutic agent for the treatment of cancer and infectious diseases (Biron, 1998; Chopra et al., 2015). Till now, recombinant human, equine, white rhinoceros, cattle, and fruit bat IFN-γ have been shown to inhibit a variety of virus replication in many cell lines (Morar et al., 2007; Huang et al., 2012), and the commercialized recombinant IFN-γ of some livestock animals such as pigs, dogs, and birds have been used in veterinary clinical treatment (Li and Sherry, 2010; Ma et al., 2009; Qu et al., 2013; Tian et al., 2014). Recombinant human, equine, white rhinoceros, cattle, and fruit bat IFN-γ have been shown to inhibit the replication of various viruses in many cell lines (Morar et al., 2007). In mammals, IFN-γ unlike IFN-α and IFN-β proteins which share a high degree of homology to allow functional cross-reactivity among different species; and different IFNγ proteins of different mammalian species share relatively low homology thereby seldom have cross protective activity. Mink (Neovison vison) is one of the major fur animals and its health problems have become an important issue for fur industry. The infection by canine distemper virus, parvovirus and Aleutian mink disease virus among minks has caused serious economic losses to the fur animal industry (Wang et al., 2012, 2014; Zhao et al., 2014). Currently, there is no effective treatment for mink viral disease due to the rare cross protective effect of IFN-γ among different mammalian species (Waldvogel et al., 2004). Therefore, cloning, sequencing, phylogenic analysis and expression of mink interferon-γ (miIFN-γ) gene are necessary for protection of mink from viral infection.
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2. Materials and methods
2.3. Sequence analysis
2.1. Reagents and animals
The miIFN-γ open reading frame (ORF) of the nucleotide sequence and the deduced IFN-γ amino acid sequence were blast aligned with those of IFN-γ genes of various species from GenBank, and a multispecies phylogenetic tree based on the nucleotide sequence sequences of arious IFNs was constructed using DNAStar 5.0 software. The signal peptide sequence and the glycosylation sites of the miIFN-γ protein were predicted with online SignalP 4.1 Server and NetNGlyc Servers (http://www.cbs.dtu.dk/services) respectively.
Vesicular stomatitis virus (VSV) was provided by Dr. Haidong Zhi from the Institute of Harbin Veterinary Research, Chinese Academy of Agricultural Sciences, Harbin, China. The MDCK-line cells were purchased from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). RPM-I 1640 media were purchased from GIBCO (USA). Lymphocyte isolation kit was from Chinese Academy of Medical Sciences (Beijing, China). TRIzol, Ni-NTA protein purification system and anti-6 × His monoclonal antibodies were purchased from Invitrogen (USA). ExTaq polymerase, AMV reverse transcriptase, plant haemagglutinin (PHA) and pMD18-T vector were purchased from TaKaRa (Dalian, China), and PCR primers were synthesized at Shanghai Ying-Jun Biotech (Shanghai, China). TMB Chromogenic Reagent was purchased from Sigma (USA). Six-month old minks (American mink, N. vison) were provided by the Chinese Academy of Agricultural Sciences (Beijing, China). Two SPF rabbits (6-week old) were purchased from Changchun Institute of Biological Products Co., Ltd., Changchun, China. The animal experiments were performed according to the regulations for the administration of affairs concerning experimental animals, and was approved by the state council on October 21st, 1988 and promulgated by decree no. 2 of the State Science and Technology Commission on November 14th, 1988. 2.2. Total RNA extraction and cDNA cloning of mink IFN-γ Mink whole blood was diluted 1:1 with RPMI 1640 cell culture media (GIBCO, USA), added to an equal volume of lymphocyte separation media and centrifuged at 200 ×g at room temperature (RT) for 30 min. PBMCs were collected and washed with RPMI-1640 (GIBCO, USA) at RT. After centrifugation at 180 ×g for 10 min, resuspended in 10 ml of fresh RPMI supplemented with 2 mM L-glutamine and 10% fetal calf serum (Haclone, USA), plated at 1 × 106 cells/ml, stimulated with 100 μg/ml of phytohemagglutinin-L (PHA-L); (Sigma, USA) and incubated at 37 °C in 5% CO2 for 24 h for further RNA purification. PBMCs stimulated with PHA-L were centrifuged and total RNA was isolated according to RNeasy Mini Kit Operation Manual (Qiagen, USA). cDNA was synthesized from total RNA at 42 °C for 1 h after 9.5 μl RNA and 1 μl Oligod (T)15 primer was mixed and heated at 70 °C for 5 min, with 1 μl reverse transcription enzyme (TaKaRa, Japan), 0.5 μl RNA inhibitor and 4 μl 10 mM dNTP. The MiIFN-γ cDNA was amplified by PCR using the specific primers (Table 1) that were designed based on the canine IFN-γ cDNA sequence (GenBank no. AF126247.1). PCR reactions were subjected to 34 cycles of initial denaturation at 95 °C for 5 min, denaturation at 94 °C for 45 s, primer annealing at 53 °C for 45 s and DNA extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. Resultant PCR products were purified using a TaKaRa PCR Purification Kit (TaKaRa, China) and subcloned into pMD18-T vector for sequencing at Shanghai Ying-Jun BioTech (Invitrogen Shanghai, China). Table 1 Primers sequences. Cytokine
Predicted size (bp)
Primers
ORF
501
Mature peptide clone
444
P1 P2 P3
Eukaryotic expression
501
P4 P5 P6
5′-ATGGCCCTGCCCTGCTCCT-3′ 5′-TCACTTCCTGCTCCGCAATC-3′ 5′-GCGGATCCGGCTATTACTGTCAGGCC ATG-3′ 5′-GCAAGCTTTTATTTCGATGCTCTGCGG-3 5′-GCTGAATTCATGGCCCTGCCCTGCTCCT-3 5′-CACTCGAGTTACTTCCTGCTCCGCAATC-3
2.4. Expression of MiIFN-γ in Escherichia coli cells PCR product was separated on a 1.5% agarose gel. The purified DNA was digested with restriction enzymes BamH I and Hind III, ligated into pET32a vector, and transformed into competent E. coli JM109 cells. Positive clones were identified with PCR and restriction enzymes digestion and sequence analysis at Shanghai Ying-Jun BioTech. (Invitrogen, shanghai, China). The recombinant plasmid pET/miIFN-γ was transformed into E. coli BL21 competent cells and selected with. Positive clones were inoculated (1:200) in 5 ml of liquid LB media and cultured at 30 °C, and 1 mmol/L IPTG was added to induce the protein expression overnight when the OD600 reached to 0.6–0.9. For purification, the bacteria were centrifuged at 2000 ×g for 5 min, frozen and thawed 3 times, and the bacterial pellet was mixed with binding buffer (20 m M Na3PO4, 0.5 M NaCl, 20 mM imidazole, pH 7.4) and lysed by ultrasound for 5 s and left on ice for 5 s. Next, the mixture was centrifuged at 800 ×g and 4 °C for 10 min, and the supernatant was collected, incubated on ice and mixed with magnetic stirrer blender after the addition of 50% NI–NTA (Novagen) (4:1). Finally, the mixture was loaded onto a chromatography column (Novagen) and eluted by serial washing using elution buffer (20 mM Na3PO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4). The samples washed were separated on a 12% SDS-PAGE and transferred onto a PVDF membrane which was blocked with 5% milk in TBST (0.1 phosphate buffer containing, 0.5% Tween-20) and incubated with mouse anti-6×His monoclonal antibody at 4 °C overnight, followed by incubation with goat anti-mouse IgG-HRP at room temperature for 1 h. Finally, the membrane was developed. 2.5. Production of rabbit antiserum against recombinant MiIFN-γ Two SPF rabbits were immunized subcutaneously with 0.5 ml (0.3 mg) of purified miIFN-γ protein emulsed with 50% Freund's complete adjuvant (Sigma, USA). In 14 and 28 days after the first vaccination, the rabbits were boosted with 0.5 ml (0.3 mg) purified miIFN-γ protein in 50% Freund's incomplete adjuvant (Sigma, USA). 10 days after the last injection, the blood was collected from the rabbits ear vessels, antiserum against miIFN-γ (pAb) was separated at 1000 ×g and 4 °C for 10 min and frozen at −20 °C. 2.6. In vitro antiviral activity of MiIFN-γ The antiviral activity of the recombinant IFN-γ was determined by cell cytopathic inhibition assay. Briefly, MDCK cells were plated in 96well plates and incubated at 37 °C with 5% CO2 for 12 h; diluted recombinant IFN-γ was added to the monolayer cells and incubated at 37 °C with 5% CO2 overnight; and 100 TCID50 of VSV was defined as the virus amount that caused 50% of the cells to be cytopathic, which was assessed by a microscopic examination. Next, the cells were challenged with 100 TCID50 of VSV and incubated at 37 °C for 1–2 days, and the IFNγ was the concentration at which 50% of the cells were protected from VSV infection. To rest the miIFN-γ antiviral protective activity, neutralization assay was performed. The MDCK cells were treated with miIFN-γ first, neutralized with rabbit anti-miIFN-γ antibody (diluted 1:30) at 37 °C, and
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the controls were untreated for 24 h, Then, all cells were challenged with 100 TCID50 of VSV. 2.7. Immunofluorescence assay PCR was used to generate a construct for the eukaryotic expression of complete ORF of MiIFN-γ using the forward primer 5′-GTGAATTC CAGGCCATGTTTTTTAAAGA-3′ containing the EcoRIsite and reverse primer 5′-GACTCGAGGCAGGATGACCGTTATTTCG-3′ containing the XhoIsite. After digestion with EcoR I and Xho I, the PCR products were subcloned into the corresponding sites of pcDNA3.1 (+) (Novagen, USA) with T4 DNA Ligase (Thermo Scientific, USA). The recombinant transfer vector pcDNA3.1 (+)-ORF or the negative control vector pcDNA3.1 was transfected into MDCK cell using Lipofectamine (Roche, USA). The cells were then incubated with rabbit anti-MiIFN-γ serum (1:10) at 37 °C for 3 h washed with PBS three times, and incubated with fluorescein isothiocyanate conjugated (FITC) goat anti-rabbit IgG (1:500 ) for 1 h at 37 °C with fluorescein isothiocyanate conjugated (FITC) goat anti-rabbit IgG diluted 1:500. The immunofluorescence was observed under a LEICA DMI3000 B fluorescence microscope. 3. Result 3.1. Analysis of MiIFN-γ gene The mink IFN-γ cDNAs were successfully amplified by RT-PCR, predicted to be 501 bp, and subcloned into plasmid pET32a vector to derive recombinant plasmid pET/miIFN-γ, which was confirmed by restriction enzyme digestion analysis. DNAStar software analysis demonstrated that the mink IFN-γ cDNA was 501 bp in length. The ORF of the MiIFN-γ sequence was deposited into GenBank (GenBank accession no. KJ888148). The deduced miIFN-γ protein consists of 167 amino acids, including 3 cysteine amino acid residues at positions 13, 17 and 23. The analysis by NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/ NetNGlyc/) indicated that miIFN-γ had three potential N-glycosylation sites. SignalP 4.1 server software http://www.cbs.dtu.dk/services/ SignalP/ analysis predicted that the N terminal 19 amino acids were signal peptide. The ORF of miIFN-γ was compared with that of several mammalian IFN-γs (Fig. 2). Both mink and ferret belong to Mustelidae (Ochi et al., 2008). The homology between the miIFN-γ and the domestic ferret nucleotide and amino acid sequences (GenBank registration number: EF492064) were 99.4% and 98.2%, respectively. The nucleotide sequence alignment analysis (Table 2) showed that miIFN-γ shared 98.6% identity with Eurasian badger, 93.4% identity with giant panda, 93.0% identity with dog and fox, 87.2% identity with cat, 80.0–82.6% identity with
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cattle, horse, bottlenosed dolphin and pig, and less than 80% identity with the IFN-γs of other mammalian species (Table 2). Phylogenetic analysis (Fig. 1) further indicated that IFN-γ was subdivided into two monophyletic lineages: chicken (avian) and mammalian. Mammalian branch was divided into carnivores and herbivores. The mink, domestic ferret, Eurasian badger, giant panda, dog, fox and cat IFN-γs nucleotide sequences shared higher homology because they formed a carnivore monophyletic group that was distinct from other herbivores IFN-γs (Fig. 2). 3.2. Expression and purification of MiIFN-γ in E. coli cells To evaluate the biological activity of miIFN-γ, we subcloned miIFN-γ cDNAs into prokaryotic expression vector pET32a and induced its expression by IPTG in E. coli BL21. Western blot analysis miIFN-γ with an anti-6 × His antibody revealed an about 17 kDa miIFN-γ protein (Fig. 3). The protein solubility analysis showed that the recombinant miIFN-γ protein accounted for 80% of the total soluble proteins. 3.3. In vitro bioactivity of recombinant miIFN-γ To investigate whether pre-treatment with mink IFN-γ could protect cells from virus infection, we analyzed antiviral activity using cytopathic inhibition assays in vitro (Wallach, 1983). Different extents of cytopathic effects caused by VSV were observed in MDCK cells pretreated with different concentrations of IFN-γ, viral replication was completely inhibited when cell hole was inoculated with 52.5 μg/ml 100 μl. The protein even at 1:1024 dilution conferred a protective effect on MDCK at different levels (Fig. 4); and cellular changes were observed through a microscope. The negative controls were normal MDCK, and the positive control showed completely (100%) cytopathic effect, which was significantly inhibited by soluble MiIFN-γ. 3.4. Immunofluorescence assay MiIFN-γ cDNA encoding the signal sequence and mature protein, including the stop codon, was cloned into the expression vector pcDNA3.1. The recombinant pcDNA3.1/MiIFN-γ plasmid DNAs were transfected into MDCK cells which were incubated with rabbit antimiIFN-γ serum and FITC-goat-anti-rabbit IgG. Some cells showed obvious green fluorescence (Fig. 5A), indicating that the miIFN-γ was mainly expressed in the cytoplasm of MDCK cells. The cells transfected with plasmid pcDNA3.1 without MiIFN-γ gene showed no fluorescence (Fig. 5 B). The experimental results showed that rabbit anti-miIFN-γ
Table 2 Homology of mink IFN-γ (KJ888148) gene with that of different species. Species
Nucleotide homology (%)
Amino acid homology (%)
Genbank accession no.
Domestic ferret Eurasian badger Giant panda Dog Fox Cat Chimpanzee Cattle Horse Bottlenosed dolphin Pig Human Rabbit Chicken
99.4 98.6 93.4 93.0 93.0 87.2 74.3 80.0 82.6 82.6 80.6 74.5 75.4 41.0
98.2 98.2 91.0 87.4 87.4 82.0 63.5 72.5 79.0 74.9 74.3 63.5 61.1 29.7
EF492064 Y11647 GQ386864 FJ194478 EF428257 NM001009873 NM001193665 NM174086 NM001081949 AB022044 NM213948 NM000619 AB010386 NM205149
Fig. 1. Phylogenetic tree of IFN-γ gene nucleotide sequences of several mammalian and Chichen. (Genbank accession No.: mink/KJ888148; domestic ferret/EF492064; Eurasian badger/Y11647; giant pandger/GQ386864; dog/FJ194478; fox/EF428257; cat/NM001009873; bottlenosed dolphin/AB022044; cattle/NM174086; chimpanzee/NM001193665; horse/ NM001081949; human/NM000619; pig/NM213948; rabbit/AB010386).
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Fig. 2. Analysis of amino acid sequences of several mammals IFN-γ (mink, domestic ferret, Eurasian badger, giant panda, dog, fox, cat, bottlenosed dolphin, cattle, chimpanzee, horse, human, pig and rabbit). Black triangle represents the predicted cysteine residues; and red and boxed areas indicate potential N-glycosylation sites. Underlined is the predicted signal sequence of mink IFN-γ.
antibody produced with miIFN-γ proteins expressed in the prokaryotic expression system specifically bound to MiIFN-γ in MDCK cells. 4. Discussion Mink fur is an economically important fur raw material in the garment industry worldwide; therefore mink breeding provides enormous
Fig. 3. Soluble expression and purification of rMiIFN-γ was observed on SDS-PAGE. Lane A: non-induced crude; Lane B: induced crude; Lane C: supernatant of lysate; Lane D: precipitation of lysate; Lane E: recombinant rMiIFN-γ purified by NTA-Ni chromatography at elution buffer pH 7.4; and MK: molecular weight marker.
economic value for fur animal industry. In recent years, mink distemper, mink parvovirus enteritis, aeutian disease and other infectious diseases have been endangering the survival of mink (Wang et al., 2014; Zhang et al., 2015). Thus, the prevention and treatment of these diseases cannot be ignored. IFNs, were identified long ago but have recently been intensively re-investigated as the interdependence of the innate and adaptive immune responses have become more evident (Guo et al., 2014). To determine whether miIFN-γ in exhibits antiviral activity, we cloned, sequenced and phlogenetically analyzed and expressed MiIFNγ gene and tested the antiviral activity against virus replication.
Fig. 4. Cytopathic inhibition rate in MDCK pretreated with rMiIFN-α and infected with VSV.
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Fig. 5. Immunofluorescence micrographs of transfected MDCK. A: MDCK (pcDNA3.1-MiIFN-γ ORF group); and B: MDCK (pcDNA3.1 group).
The deduced miIFN-γ protein consists of 167 amino acids, with 3 cysteine amino acid residues at positions 13, 17 and 23, two of these three cysteines (at positions 13 and 23) are conserved among most of the mammalian IFN-γ proteins (Altmann et al., 2003; Schultz et al., 1995). Domestic ferret, also belonging to Mustelidae, is very genetically close to kinship with the mink. Phylogenetic analysis (Fig. 2) further indicated that IFN-γ was subdivided into two monophyletic lineages: chicken (avian) and mammalian, and the mammalian branch was divided into carnivores and herbivores. The mink, domestic ferret, Eurasian badger, giant panda, dog, fox and cat IFN-γs shared higher homology and formed a carnivore monophyletic group that was distinct from other herbivore IFN-γs. MiIFN-γ ORF size is 501 bp and completely consistent with domestic ferret, Eurasian badger, giant panda, dog, pig and horse. As expected from the close phylogenetic relationship, the highest amino acids homology up to 98% was observed with the domestic ferret and Eurasian badger IFN-γ sequences. The MiIFN-γ gene was predicted to encode a signal sequence of 19 aa and a mature protein of 147 aa residues. Sequence analysis revealed there are three codon differences between mink and domestic ferret. These three codons encode the 66th, 111th and 122nd amino acids, respectively. Among them, the 66th aa site AAC encodes Thr in mink and AAA Lys in domestic ferret; the 111th aa site CGG encodes Arg in mink and ctg encodes Leu in domestic ferret; and the 122nd aa site was CCG in mink and CCT in domestic ferret which both encodes the same aa (Pro). These results indicate that although mink and domestic ferret all belong to Mustelidae species, certain gene mutations occurred in the long process of genetic evolution. Currently, E. coli is still the easiest, cheapest, and most widely used protein expression system. In our study, soluble MiIFN-γ was easy to be produced, this may be because only one cysteine and one glycosylation site existed in the mature protein aa sequence. To see whether MiIFN-γ possesses biological function, we measured the bioactivity of MiIFN-γ protein using cytopathic effect (CPE) inhibition assays in vitro. These results demonstrated that MiIFN-γ protected MDCK cells from challenge with VSV, indicating that MiIFN-γ protein showed certain degrees of cross-species activity. This finding was surprising considering the close relationship in evolutionary terms between mink and canine. The antiviral activity of the MiIFN-γ protein was almost completely neutralized with polyclonal antibody against MiIFNγ at 1:20 but was almost not neutralized at 1:100. In fact, these antibodies were inhibitory only under certain experimental conditions (Zhang et al., 2008). Many previous studies reported that anti-HBV effect was mediated by recombinant eukaryotic expression vector for IFN-α (Yu et al., 2013). In our study, immunofluorescence experiments showed that the mink IFN-γ was highly expressed in MDCK cells by pcDNA3.1 Vector, we will carry out anti-viral effect of recombinant eukaryotic expression in the future. In conclusion, the present study reports the successful cloning, sequencing and expression of IFN-γ from mink, and the monoclonal
antibody specific for rMiIFN provides an essential tool for development of an assay to detect the IFN-γ response to infection and cellular immune status after virus vaccination in mink. Acknowledgments We thank Dr. Zhi Haidong (Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China) for kindly providing VSV. This study was supported by Jilin Province Science and Technology Development Program (20140520172JH, 20130206026NY), and Jilin City Science and Technology Development Program (No. 2013625018, 2013222014). References Altmann, S.M., Mellon, M.T., Distel, D.L., Kim, C.H., 2003. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. J. Virol. 77, 1992–2002. Biron, C.A., 1998. Role of early cytokines, including alpha and beta interferons (IFN-alpha/ beta), in innate and adaptive immune responses to viral infections. Semin. Immunol. 10, 383–390. Chopra, A., Marak, C., Alappan, N., Shim, C., 2015. Organizing pneumonia associated with pegylated interferon α and ribavirin therapy. Case Rep. Pulmonol. 2015, 794592. Gerlier, D., Lyles, D.S., 2011. Interplay between innate immunity and negative-strand RNA viruses: towards a rational model. Microbiol. Mol. Biol. Rev. 75, 468–490. Guo, F., Zhao, X., Gill, T., Zhou, Y., Campagna, M., Wang, L., Liu, F., Zhang, P., DiPaolo, L., Du, Y., Xu, X., Jiang, D., Wei, L., Cuconati, A., Block, T.M., Guo, J.T., Chang, J., 2014. An interferon-beta promoter reporter assay for high throughput identification of compounds against multiple RNA viruses. Antivir. Res. 107, 56–65. Hoegen, B., Saalmüller, A., Röttgen, M., Rziha, H.J., Geldermann, H., Reiner, G., Pfaff, E., Büttner, M., 2004. Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine. Vet. Immunol. Immunopathol. 102, 389–397. Huang, L., Cao, R.B., Wang, N., Liu, K., Wei, J.C., Isahg, H., Song, L.J., Zuo, W.Y., Zhou, B., Wang, W.W., Mao, X., Chen, P.Y., 2012. The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α. Cytokine 57, 37–45. Janardhana, V., Tachedjian, M., Crameri, G., Cowled, C., Wang, L.F., Baker, M.L., 2012. Cloning, expression and antiviral activity of IFNγ from the Australian fruit bat, Pteropus alecto. Dev. Comp. Immunol. 36, 610–618. Lasfar, A., Abushahba, W., Balan, M., Cohen-Solal, K.A., 2011. Interferon lambda: a new sword in cancer immunotherapy. Clin. Dev. Immunol. 2011, 1–11. Li, L., Sherry, B., 2010. IFN-alpha expression and antiviral effects are subtype and cell type specific in the cardiac response to viral infection. Virology 396, 59–68. Ma, D., Jiang, D., Qing, M., Weidner, J.M., Qu, X., Guo, H., Chang, J., Gu, B., Shi, P.Y., Block, T.M., Guo, J.T., 2009. Antiviral effect of interferon lambda against West Nile virus. Antivir. Res. 83, 53–60. Morar, D., Tijhaar, E., Negrea, A., Hendriks, J., van Haarlem, D., Godfroid, J., Michel, A.L., Rutten, V.P., 2007. Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies. Vet. Immunol. Immunopathol. 115, 146–154. Ochi, A., Danesh, A., Seneviratne, C., Banner, D., Devries, M.E., Rowe, T., Xu, L., Ran, L., Czub, M., Bosinger, S.E., Cameron, M.J., Cameron, C.M., Kelvin, D.J., 2008. Cloning, expression and immunoassay detection of ferret IFN-gamma. Dev. Comp. Immunol. 32, 890–897. Qu, H., Yang, L., Meng, S., Xu, L., Bi, Y., Jia, X., Li, J., Sun, L., Liu, W., 2013. The differential antiviral activities of chicken interferon α (ChIFN-α) and ChIFN-β are related to distinct interferon-stimulated gene expression. PLoS One 8, e59307. Schneider, W.M., Chevillotte, M.D., Rice, C.M., 2014. Interferon-stimulated genes: a complex web of host defenses. Annu. Rev. Immunol. 32, 513–545. Schroder, K., Hertzog, P., Ravasi, T., Hume, D.A., 2004. Interferon-gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75, 163–189.
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