P1 virus genomic DNA
The full-length genome of the P1 virus JSNJ was isolated from the serum of a piglet with PMWS. All sample collection was conducted between December 2013 and January 2014 in Jiangsu Province, China. Permission to collect the study samples was granted by the pig farms. All procedures involving animals throughout the study were approved by the Committee on the Ethics of Animal Experiments at the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, China. The viral sequences were determined after the genome was amplified with PCR. The genome was digested singly BamHI, tandem dimers were ligated with T4 DNA ligase, and the dimeric genomes inserted into the BamHI site of the pBluescript SK (pSK) vector (Stratagene). The cloned constructs were used for in vitro transfection assays.
Cell culture and transfection
A PK15 cell line [2] free of PCV2, PCV1, and mycoplasma contamination was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 0.01% penicillin–streptomycin in 5% CO2 at 37°C. The cells were transfected with LipofectamineTM 2000 (Invitrogen), according to theprotocol recommended by the manufacturer. DNA (4 μg) was used to transfect 106 cells in six-well plates. The transfected cells and the culture media were harvested at different times (0, 12, 24, 48, 72, 96, and 120 h) and frozen at −80°C before RNA extraction. The pSK vector control was used to check for any nonspecific responses, and no specific bands were detected with either northern blotting or reverse transcription polymerase chain reaction (RT-PCR).
RNA isolation and preparation
Total RNA was extracted from the transfected cells with TRIzol Reagent (invitrogen), according to the manufacture’s instructions. The quality of the RNA template was assessed by the ratio of 28S:18S RNAs on a denaturing formaldehyde agarose gel after it was stained with ethidium bromide. The RNA concentrations were determined spectrophotometrically at 260 nm.
The total RNAs isolated at selected times for northern blotting analysis were not treated with DNaseI, and the vector DNA was used as an internal control. Any residual DNA was removed from the RNA samples for both RT-PCR and the random amplification of cDNA ends (RACE) with the TURBO DNA-freeTM Kit (Ambion), according to the manufacture’s instructions. A control PCR reaction without the RT step was also conducted to ensure that the input P1 plasmid DNA was completely digested by DNaseI.
For RT-PCR, 1 μg of RNA was reverse transcribed at 42°C for 1 h using avian myeloblastosis virus (AMV) reverse transcriptase and an oligo(dT)18 primer, according to the protocol of the manufacturer (TaKaRa, China).
5′- and 3′- RACE products were generated from first-strand cDNA synthesized from 1 μg of total RNA in a 10 μL reaction mixture using the SMARTerTM RACE cDNA amplification kit (Clontech catalog no. 634923), according to the manufacturer’s protocol. In this reaction, RNA was reverse transcribed with SMARTScribe Reverse Transcriptase at 42°C for 90 min. The 3′-CDS primer A or 5′-CDS primer A and SMARTer IIA oligonucleotide were used to synthesize 3′-RACE-Ready cDNA and 5′-RACE-Ready cDNA, respectively.
Northern blotting analysis
Single-stranded RNAs were prepared by in vitro transcription using T7 RNA polymerase. The P1 molecular DNA clone was used as the template for PCR to generate the (+) and (−) P1 transcripts. The primer sets were probe R (F1: 5′-GCGCTAATACGACTCACTATAGGGATCTTCAACACCCGCCTCT-3′, R1: 5′-GGATATTGTAGTCCTGGTCGTAT-3′), and probeF (F2: 5′-ATCTTCAACACCCGCCTCT-3′, R2: 5′-GCGCTAATACGACTCACTATAGGGGGATATTGTAGTCCTGGTCGTAT-3′). Two digoxigenin (DIG)-labelled viroid-specific riboprobes were synthesized using the DIG RNA Labelling Kit (SP6/T7) (Roche Applied Science), as recommended by the manufacturer.
The RNAs were separated with 1% formaldehyde agarose gel electrophoresis (25 V overnight) and electroblotted (400 mA for 1 h) onto positively charged nylon membranes (HyBond N+, Amersham Pharmacia Biotech) by capillary transfer in 20 × saline sodium citrate (SSC) and immobilized for 2 h at 80°C. The membranes were prehybridized for 2 h at 68°C with Roche DIG Easy Hyb and then hybridized overnight at 68°C in DIG Easy Hyb containing the denatured probe. After hybridization, the membranes were washed twice (15 min each) in 0.1 × SSC/0.1% sodium dodecyl sulfate (SDS) solution at 68°C, equilibrated for 2–5 min in washing buffer, blocked in blocking solution with gentle agitation for 1 h, incubated for 30 min in alkaline-phosphatase-conjugated anti-DIG antibody, washed twice (15 min each) in washing buffer, and incubated for 5 min in CSPD® Substrate (Roche Applied Science). Following the chemiluminescent detection procedure, the membranes were exposed to X-ray films for 10–30 min.
An RNA molecular weight marker I, DIG-labeled (0.3–6.9 kb) (Roche Applied Science) was used as the size standard.
RT-PCR
PCR was used to amplify the cDNA generated from about 20 ng of total RNA in triplicate, in a final volume of 25 μL, using specific primers. An aliquot (10 μL) of the reaction mixture was analyzed with gel electrophoresis after 35 cycles of amplification. The primers used for RT-PCR were 170F: 5′-TTTGTTATTTGGTTGGAAGTAATCAATAGT-3′; 462R: 5′-CCAGGAGGGGGGACCAACAAA-3′; 485F: 5′-AATCTCATCATGTCCACCGCCCAGGAG-3′; 579R: 5′-GGCATCTTCAACACCCGCCTC-3′; 288F: 5′-GGTCATAGGTTTGGGCCGTGG-3′; 577F: 5′-GCCATTTTTCCTTCTCCAACG-3′; and 36R: 5′-TTAATCTTAAGGGCCCCCCAC-3′.
RACE
5′- and 3′-RACE PCR amplification were performed using the 5′-RACE-Ready and 3′-RACE-Ready cDNAs, respectively. RACE-PCR was performed with the Universal Primer A mix (UPM) from the SMARTerRACE cDNA Amplification Kit, and the primers appropriate to 5′-RACE or 3′-RACE. The conditions for amplification were those recommended by the manufacturer: five cycles of denaturation at 94°C for 30 s, annealing-extension at 72°C for 3 min; followed by five cycles at 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min; and finally, 25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min. All the products were analyzed with gel electrophoresis on 2% agarose gels, purified after gel elution, cloned into the pMD-18 T vector (TaKaRa, China), and sequenced. Two controls were also used to exclude the possibility of nonspecific amplification. One control reaction was performed with only one viral-specific primer, and the other was performed with UPM alone. No bands were amplified in either control.
The primers used for RT-PCR were 267F: 5′-CGGGAGGAGTAGTTACATATGGG-3′; 540F: 5′-TATATCCGAAGGTACGGGAGAGG-3′; 536R: 5′-TCAAGGCTACCACAGTCAGAACG-3′; 640F: 5′-AGTGGATCCTCATTTAGGGTTTAAGTGG-3′; 299R: 5′-AAACCTATGACCCATATGTAACT-3′; 343R: 5′-CCCTGTTATTCTAGATGATAACTTTG-3′; 425R: 5′-CCCACTACAGAATAAGAAAGGTTAAG-3′;248R: 5′-CTTCTCCTACCACTCCCGTTACT-3′; 562R: 5′-CCTCTCCCGTACCTTCGGATATA-3′;541F: 5′-ATATCCGAAGGTACGGGAGAGGC-3′; 30R: 5′-CTAAAGACCCCCCACTTAAACCC-3′; and 285F: 5′-ATGGGTCATAGGTTTGGGCCGTG-3′.
Virus and expression of recombinant ORF1 protein
The purified P1 virus was produced as previously described [15]. Briefly, PK15 cells were transfected with the P1 DNA clone and harvested at 96 h. The cells were frozen and thawed three times, and centrifuged in two rounds of CsCl density gradient ultracentrifugation. The P1 virus was collected by aspiration and dialysis.
To construct an expression plasmid encoding the ORF1 protein, ORF1 of P1 was amplified by PCR using primers 5′-GGCGGATCCATGAGATTTAATATTGACGAC-3′ and 5′-ATACTCGAGGCCAAAGCTGATTCCTTTTG-3′ (the italics sequences identify the BamHI and XhoI sites). The PCR products were cloned into the pMD-18 T vector, doubly digested with BamHI and XhoI, and cloned into the prokaryotic expression vector pET-32a (+) (Novagen, Germany). The resulting recombinant expression plasmid was used to transform Escherichia coli DH5α cells and was identified by restriction enzyme digestion and DNA sequencing.
To express the cloned ORF1, E. coli BL21 (DE3) pLys cells were transformed with the pET32a–ORF1 plasmid. Single colonies of transformants were grown in Luria–Bertani medium at 37°C to an optical density at 600 nm (OD600) of about 0.4–0.6, and isopropylthio-β-d-galactopyranoside was added to a final concentration of 1 mM. After induction at 20°C for 12 h, the bacteria were collected by centrifugation at 5000 × g for 10 min and lysed by boiling for 10 min. The fusion proteins from the whole-cell lysates and the virus purified by CsCl gradient centrifugation were analyzed with SDS polyacrylamide gel electrophoresis (PAGE) and immunoblotting, respectively. The rabbit anti-P1 hyperimmune serum used for immunoblotting was prepared as described previously [2].