All research was approved by the relevant committees at the Zhejiang Academy of Agricultural Sciences.
Virus strain and cells
The bacterial pDEV-EF1/GS1783 which contains the full-length infectious BAC clone of DEV vaccine strain and corresponding virus rDEV-EF1 were constructed and stored in our laboratory [9, 10]. Chicken embryo fibroblasts (CEFs) were prepared from 10-day-old specific-pathogen-free (SPF) embryonated eggs (Zhejiang JianLiang Bioengineering Co., Ltd., Hangzhou, China) according to standard procedures and cultured in DMEM (Gibco-BRL) supplemented with 8% FBS, 100 U of penicillin/mL and 100 µg streptomycin/mL.
Construction of transfer vector pT-linker-mRFP-P1 and pT-linker-CFP-P2
The transfer vector pT-linker-mRFP-P1 was constructed as flows: primer pair mRFP-F1(gly) and mRFP-R1 (Table 1) was designed to amplify a red fluorescent protein (mRFP) gene prefixing with flexibility linker GGGGSGGG from plasmid pLVX-mRFP-C1 (Clontech). The PCR fragment linker-mRFP was cloned into a pMD19T-simple vector (Takara), and the forward inserted clone pT-linker-mRFP was selected by PCR amplification with primer pair M13(+) and mRFP-R1. Another primer pair kan_mRFP-for(PstI) and kan_mRFP-rev(PstI) (Table 1) was designed to amplify P1 fragment (containing Pst I-homologous arm a -kan-Pst I) from plasmid pEP-kan-S (a gift form Dr. Nikolaus Osterrieder, Freie Universität Berlin, Berlin, Germany).
And P1 fragment was cloned into the Pst I site present in pT-linker-mRFP to construct the transfer vector pT-linker-mRFP-P1 which containing linker-mRFP1 (the first part of mRFP gene)- Pst I -homologous arm a-kan-Pst I-mRFP2(the second part of mRFP gene) in sequence, that is, mRFP gene is divided into two parts by fragment “homologous arm a-kan-Pst I”, and homologous arm a (Hom A) matches exactly the front end sequence of mRFP2.
The construct strategy of transfer vector pT-linker-CFP-P2 was similar to pT-linker-mRFP-P1. Primer pair CFP-F1(gly-SacI) and CFP-R1(Table 1) was designed to amplify cyan fluorescent protein (CFP) gene prefixing with flexibility linker GGGGSGGG (linker-CFP) from plasmid pCMV-C-CFP(Beyotime Biotechnology). Primer CFP-F1(gly-SacI) was introduced flexibility linker GGGGSGGG and restriction enzyme Sac I site. The PCR fragment linker-CFP was cloned into a pMD19T-simple vector (Takara), and the forward inserted clone pT-linker-CFP was selected by PCR amplification with primer pair M13(+) and CFP-R1. Another primer pair kan_CFP-for(Sac I) and kan_CFP-rev(Sac I) (Table 1) was designed to amplify P2 fragment (containing Sac I -homologous arm b-kan- Sac I) from plasmid pEP-kan-S. And P2 fragment was cloned into the Sac I site present in pT-linker-CFP to construct the transfer vector pT-linker-CFP-P2 which containing linker-CFP1 (the first part of CFP gene)-Sac I-homologous arm b-kan-Sac I -CFP2 (the second part of CFP gene) in sequence. Homologous arm b (Hom B) matches exactly front end sequence of CFP2.
Construction of recombinant BAC clones
Mutated clone pDEV-UL35(c)CFP-gC(c)mRFP carrying a red fluorescent protein (mRFP) gene attached to the viral envelope protein gC combined with a cyan fluorescent protein (CFP) gene fused to the viral capsid UL35 gene was constructed by three times of two-step Red (en passant) recombination [11, 12]. Firstly, pDEV-dEF1GFP deleting open reading frame (ORF) of green fluorescent protein gene (GFP) (pEF1-GFP) was constructed, then mRFP gene was inserted to C terminus of DEV gC gene to construct recombinant clone pDEV-gC(c)mRFP, at last, pDEV-UL35(c)CFP-gC(c)mRFP was obtained by inserting CFP gene to C terminus of DEV UL35 gene. In details, to construct pDEV-dEF1GFP, ORF of pEF1-GFP was deleted from pDEV-EF1 by two-step Red (en passant) recombination. Brief, PCR was done with primer pair DEV-dEF1-gfp-for and DEV- dEF1-gfp -rev (Table 1) using pEP-kan-S as template. Then, 100 ng of purified PCR product (1200 bp in length) was electroporated into competent GS1783 bacteria containing pDEV-EF1 and bacteria of transformation were plated on LB plates containing chloramphenicol (34 µg/mL) and kanamycin (50 µg/mL) and incubated at 32℃ for 48 h. Double-resistant colonies were selected for the 2nd Red recombination to remove the kanamycin resistance gene. Colonies resistant to chloramphenicol but sensitive to kanamycin were selected. Following, positive clonies were determined by RFLP (restriction fragment length polymorphisms) with Xba I and sequencing the PCR products amplifying with primer pairs dgfp-JD-F and dgfp-JD-R flanking the region of pEF1-GFP deletion. Following, to construct pDEV-gC(c)mRFP, PCR was done with primer pair mRFP-gC(c)-F1 and mRFP-gC(c)-R1 (Table 1) using pT-linker-mRFP-P1 as template. And 100 ng of purified PCR product (1856 bp in length) was electroporated into competent GS1783 bacteria containing pDEV-dEF1GFP to carry out two-step Red (en passant) recombination as described earlier. Positive clones were determined by RFLP with Bgl II and sequencing the PCR products amplifying with primer pair gC(c).mRFP-JD-F and gC(c).mRFP-JD-R (Table 1) flanking the region of linker-mRFP insertion. At last, to construct pDEV-UL35(c)CFP-gC(c)mRFP, PCR was done with primer pair CFP-UL35 (c)-F1 and CFP-UL35 (c)-R1 (Table 1) using pT-linker-CFP-P2 as template. And 100 ng of purified PCR product (1898 bp in length) was electroporated into competent GS1783 bacteria containing pDEV-gC(c)mRFP to carry out two-step Red (en passant) recombination in the same way. Positive clones were determined by RFLP with Bgl II and sequencing the PCR products amplifying with primer pair CFP-UL35-JD-F and CFP-UL35-JD-R flanking the region of linker-CFP insertion.
Rescue of recombinant viruses
BAC DNAs of pDEV-dEF1GFP, pDEV-gC(c)mRFP and pDEV-UL35(c)CFP-gC(c)mRFP were extract from bacterial by alkaline lysis method. Next, 4 µg of BAC DNA was transfected into CEFs by calcium phosphate precipitation according to Chen L . The cells were then cultured with DMEM supplemented with 8% FBS for 3 ~ 6 days until 70 ~ 80% cytopathogenic effect (CPE) occurred. The virus collected were separately named rDEV-dEF1GFP, rDEV-gC(c)mRFP and rDEV-UL35(c)CFP-gC(c)mRFP.
Measure sizes of virus plaque
The plaque sizes of rDEV-UL35(c)CFP-gC(c)mRFP, rDEV-gC(c)mRFP, rDEV-dEF1GFP and rDEV-EF1 were measured as follow. The viruses were serially diluted and plated onto CEFs seeded in a 12-well plate, and 2 h later, the culture medium was replaced with DMEM containing 1.5% methylcellulose (Sigma). After a 2-day-incubation at 37 °C, for every virus, 100 fluorescent plaques were randomly selected and taken photo under fluorescence microscope (Nikon, Japan), then their sizes were measured using Image J software (http://rsb.info.nih.gov/ij/). Statistical analyses to compare the differences in the plaque sizes between the four strains were conducted using one-way ANOVA with SPSS 11.5 software.
Determination of the growth curve
The growth curve of those reconstructed virus were determined and compared with the parent virus rDEV-EF1. Briefly, CEFs were infected with approximately 0.02 MOI((multiplicity of infection) of cell-free viruses of rDEV-EF1, rDEV-dEF1GFP, rDEV-gC(c)mRFP and rDEV-UL35(c)CFP-gC(c)mRFP. The cells and culture supernatant were harvested at different time (6, 12, 24, 36, 48, 60, 72, 84, 96, 108 h) after virus infection. And cells were collected by trypsin digestion following washing twice with phosphate-buffered saline (PBS, pH7.0) and then suspended with 1 mL DMEM-2% FBS as the equal volume of supernatant. Then the collected cells and supernatants were stored at -70℃until all samples have been collected. Before titrated on CEFs, cells were treated by Tissuelyser-24 at 65 Hz for 90 s and centrifuged at 6000 rpm for 5 min, then 100 µL of lysis supernatant and culture supernatant were taken to titer by TCID50 test according to standard virological method. The growth curves were computed from three independent experiments.
Western blot analysis of UL35-CFP and gC-mRFP protein expressed in recombinant virus-infected CEFs
Expression of UL35-CFP and gC-mRFP protein was verified by Western blot analysis using rabbit anti-GFP polyclonal antibody (Beyotime Biotechnology), rabbit anti-mRFP polyclonal antibody (Abcam) and mouse anti-gC polyclonal antibody (Preparation in our laboratory) and rabbit anti-UL35 polyclonal antibody (Preparation by Genscript Co.). In details, the cells infected with 1.0 MOI rDEV-EF1, rDEV-dEF1GFP and rDEV-gC(c)mRFP, rDEV-UL35(c)CFP-gC(c)mRFP which occurred 80% CPE were collected and treated with sample buffer, then subjected to 10% SDS-PAGE and electrophoretically transferred to a PVDF membrane (Millipore). To eliminate possible non-specific binding, the membranes were block with 10% skimmed milk in PBST (0.5% Tween20 in PBS) overnight at 4℃ and incubated with rabbit anti-GFP polyclonal antibody (1: 500) or rabbit anti-mRFP polyclonal antibody (1:500) or mouse anti-gC polyclonal antibody (1: 500) or rabbit anti-UL35 polyclonal antibody (1: 250) for 1 h at 37℃. The horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP- conjugated rabbit anti-mouse IgG (Santacruz) was used as the secondary antibody. And the protein bands were visualized by SuperSignal® West Pico Chemiluminescent Substrate (Pierce) and chemiluminescence was observed using the CCD camera-equipped Chemi Lux detection system by Intas.
Protein Localization observed by confocal microscopy
The viruses were plated onto the slides of CEFs seeded in a 6-well plate, and 24 h, 36h,48 h, 60h༌72 h, 96 h after infection, the cells were staining with SYTO® Green-Fluorescent Nucleic Acid Stains (Life technologies)and visualized by laser scanning confocal microscopy (Leica TCS-SP5) with a 100 × oil immersion objective.
Transmission Electron Microscopy
CEFs were infected at a MOI of 0.01 and processed for sectioning at 84 hpi. Briefly, cell pellets were collected and washed three times with PBS,then were fixed in a solution containing 2.5% glutaraldehyde plus 1% paraformaldehyde in 0.1 M cacodylic buffer for 2–4 h at 4 ºC, further fixed with 1% buffered OSO4 (Sigma) for 2 h at RT, washed three times in 0.1 M PBS. Samples were then dehydrated in an ethanol-water series(25–100% ethanol) and embedded in resin using an Embed 812 kit (SPI, USA), and polymerized at 60℃ for 48 h. Then ultrathin sections were obtained by sectioning the polymerized samples at 60 ~ 80 nm with a diamond knife Ultra 45 ° (Diatome) using a Leica UC7 (Leica Microsystems,Wetzlar, Germany). Sections were stained with uranium acetate and lead salts. Then cells were examined with a transmission electron microscope HT7700 (hitachi) at 120 kV.