Sheep herds and tissue collection
The case described presently originated on a farm keeping about 400 sheep located in a village in Wuyang, Henan province, in the north-central region of China(113.6 E, 33.43 N), in January, 2012. Eleven lambs, aged one to six months, and six ewes presented with typical skin lesions on the muzzle, lips, and teats, possibly due to an Orf virus infection. The afflicted animals were cured within 5 weeks and suffered no deaths. Skin tissue samples with gross pathologic changes and scabs were collected, using a pair of sterilized tweezers, from four lambs and two ewes and stored at −80 °C for virus isolation and further analyses. Sera from the corresponding animals were collected from the jugular vein and preserved at −80 °C for further testing. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at South China Agricultural University (the certification number: CNAS BL0011).
Isolation and culture of Orf virus
The isolation of the virus was performed according to previous methods with some modifications [33, 34]. Briefly, tissue samples were mechanically homogenized with a disposable pestle (Sangon Biotech, Shanghai, China) in 1 × MEM medium (Hyclone, Logan, UT, USA) supplemented with gentamicin (50 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml), followed by centrifugation at 3000×g for 10 min. Clarified supernatant was passed through 0.45 μm filters and inoculated onto OFTu cells. The cells were incubated at 37 °C in 5% CO2. Cells were observed daily with an inverted microscope for monitoring the progression of virus-induced CPE. Infected cells were passaged thrice after CPE appeared. When 60%–80% CPE was observed, cells and medium were harvested and stored at −80 °C for further experiments. The virus was isolated from the OFTu cell culture medium and a single clone of the viral strain was isolated by a plaque assay, performed according to previous reports [6, 9]. Briefly, OFTu cells were infected with serial dilutions of virus from 10−1 to 10−6 for 1 h at 37 °C in a 5% CO2 incubator. 3 ml MEM containing 5% FBS and 0.5% low melting point agarose (Sea KemW GTGW, Lonza, Rockland, ME, USA) was added after removing the old medium. Plaques caused by a single virus were visualized and picked at 4 or 5 dpi. Each isolate was acquired with a minimum of 2 or 3 plaques.
Polymerase chain reaction (PCR) and gene sequencing
Total DNA was extracted directly from tissue suspensions (200ul) of the lip lesions or purified virion materials using a QIAamp DNA blood kit (QIAGEN, Duesseldorf, Germany), according to the manufacturer’s instructions. PCR of the ORFV011 and ORFV059 genes was performed. Two sets of primers were designed based on the OV-NZ2 genomic sequence to amplify these two highly conserved genes [20]. Primer sets were as follows: NZ2-ORFV011Fw: 5′- ACACCTTTCCCCAGAACCCCA-3′; NZ2-ORFV011Rv:5′- GTCCGAGCTCCAGTTGCTGACTT-3′; NZ2-ORFV059Fw: 5′- ACGTCATCACATGCGGGTCAGAG-3′; NZ2ORFV059Rv:5′- CTTCCTGTTCCTGGCGGGCAT-3′. PCR reactions were carried out in 50 μl reaction volumes, which contained 10 μl of 5 × PCR buffer (10 mMTris–HCl and 50 mMKCl), 2 μl of DNA template, 200 μMof each dNTP, 0.4 μM of each primer, 25 μM MgCl2 and 0.5 μl of Taq polymerase (Takara, Dalian, China). PCR reactions were performed in a thermocycler (GeneAmp PCR 2400, Perkin Elmer, Shelton, CT, USA) for 32 cycles of denaturation at 95 °C for 1 min, annealing at 56 °C for 30s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The amplified products were resolved by 1% agarose gel electrophoresis and analyzed with an IS-1000 Digital Imaging System (Alpha Innotech Corp. San Leandro, CA, USA).
The PCR products were individually purified using a MiniElute gel extraction kit (QIAGEN, Duesseldorf, Germany), according to manufacturer’s procedures. Purified gene fragments were cloned into pMD-19 T vector (Takara, Dalian, China) and transformed into E.coli Top10 competent cells. The existence of the insert was determined by restriction endonuclease (EcoRI and BamHI) digestion and agarose gel electrophoresis. Two or three positive clones for each gene were sent to BGI-Shenzhen for sequencing.
Genomic DNA extraction and sequencing
Infected cells were harvested when 80% CPE was observed by scraping the bottom of the tissue culture flasks. The mature virions were purified from the infected cells by sucrose gradient ultra-centrifugation as previously described [9, 16]. Viral genomic DNA was extracted from tissue purified virion material using a Virus DNA purification Kit (Roche, Basel, Switzerland), following the manufacturer’s instructions. This genomic DNA preparation was sequenced using the Paired-End method on a high-throughput Illumina system [35, 36]. Briefly, viral genomic DNA was interrupted randomly by sonication to produce a series DNA fragments with sizes less than or equal to 800 bp. The DNA fragments were then repaired and adapters ligated to both ends. Sequence data were assembled with the Phrap and CAP3 software programs. Gaps were closed by primer walking and verified by sequencing of PCR products.
Genomic DNA composition, structure, repeats, and restriction enzyme patterns were analyzed using the Genetics Computer Group (GCG) version 10 software package. Open reading frames (ORFs) longer than 30 codons were evaluated for coding potential and ORFs greater than 60 codons were subjected to homology searches with BLAST in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). In addition, the Hexamer (ftp.sanger.ac.uk/pub/rd) and Glimmer programs were used to evaluate coding potential. Based on these criteria, 132 ORFV putative genes were annotated and orthologous ORFs were similarly numbered. The genome sequence was submitted to GenBank with the Accession Number: HN3/12: KY053526.
Sequences alignment and phylogenetic analysis
The nucleotide and deduced amino acid sequences of the HN3/12 isolate were aligned with Sequencer version 3.0 (Gene Codes Corp., Ann Arbor, MI, USA) and compared to sequences of the corresponding genes of those of PPVs available in the GenBank database using the online BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Two phylogenetic trees ofORFV011 and 059 gene sequences were constructed by the neighbor-joining method, with 1000 bootstrap replicates, using MEGA version 5.2. Phylogenetic comparisons were done based on the whole genomic nucleotide sequences, including terminal repetition, of PPV strains with MEGA 5.2 software using the maximum-likelihood method. The 13 PPVs genomic sequences were deposited into GenBank with the following Accession Numbers: HN3/12: KY053526, OV-YX: KP010353; OV-GO: KP010354; OV-NP: KP010355; OV-SJ1: KP010356; IA82: AY386263; SA00: AY386264; NZ2: DQ184476and F00.120R: GQ329669; VR634: GQ329670; BV-AR02: AY386265.
Amino acid sequence alignment of ORFVs
Pairwise sequence alignments of 132 ORFs from the HN3/12 strain with eight ORFV strains (OV-GO, OV-YX, OV-NP, OV-SJ1, NA1/11, IA82, NZ2 and SA00) were performed using EMBOSS Needle in the EMBL-EBI database (available at http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Multiple amino acid alignment for ORF001, ORF005, ORF116 and ORF120 from the Fujian ORFV strains and the Henan strain were performed by Clustal Omega (available at http://www.ebi.ac.uk/Tools/msa/clustalo/) and DNAMAN software.
