The orf virus strain used was NA1/11, which was isolated from a sheep outbreak. The virus was grown in primary Ovine Fetal turbinate (OFTu) cell culture monolayers as described by Wei Li [16]. Cells were harvested when approximately 80–90% of the cells showed cytopathogenic effects (CPE). Cell debris was removed by centrifugation at 1000×g for 10 minutes, and orf virus in the supernatant was purified by sucrose gradient ultracentrifugation [16]. The purified viral particles were heat-inactivated at 96°C for 90 minutes before being disrupted by sonication. The purified native orf viral lysate was then frozen at −80°C until needed for subsequent ELISA and Western blot analyses.

All sera were obtained from the Animal Science College, Fujian Agriculture and Forest University, Fujian Province, China, and stored at −80°C. Non-immune sera were obtained from goat flocks that had never been infected with ORFV and were never inoculated with orf vaccine. Test sera were collected from 15 different goat farms in four districts of the Fujian Province of China (Fig.1; Table 1). Among of these farms, four of them were experiencing an active orf outbreak when the sera were collected; in these cases, samples of scabs from orf-infected lesions were also collected in sterile tubes and stored at −80°C for subsequent virus isolation and PCR analysis. A total of five orf isolates were obtained from these scabs (data not shown). Three farms had never experienced an ORFV outbreak. Eight farms had experienced at least one ORFV outbreak within two years of sera collection, and an ORFV outbreak occurred at one farm just one month after sera collection. All sera were stored in PBS with 50% glycerol at −80°C until needed for analysis.

The test procedure and calculation of results of indirect ELISA were as described by Yirrell, Reid, Norval and Howie (1989), with some modifications. Briefly, the plates were coated with the purified native orf viral lysate (50 ng per well) by incubation at 4°C overnight. Plates were then washed before incubation with diluted samples. Test sera were diluted in a two-step process to a final, effective dilution of 1∶2000 to 1∶16000. The plates were then incubated for 1 h at 37°C, and then washed. The appropriate dilution of an HRP-conjugated rabbit anti-ovine Ig (Sigma) was then added. The plates were incubated 1 h at 37°C, and then washed. A 3′,5,5′-Tetramethylbenzidine (TMB) (Sigma) substrate was then added and reacted 10 minutes at 37°C. After using 2% H₂SO₄ to end the reaction, the plates were read at 450 nm in an iMark Microplate Reader (Bio-Rad, U.S.A.). On each plate, two duplicate wells of diluted positive serum (1∶2000) was used as a positive control. Additionally, one well of diluted non-immune serum (1∶2000) and one well of ELISA sample diluent (without serum) was included in each plate as a sero-negative control and blank, respectively. In each experiment, the ELISA optical density (O.D.) reading of the reference positive serum was at least twice that of the negative sera at the same dilution. Any O.D. reading of a diluted serum sample measured as at least twice that of the sero-negative control at the same dilution was regarded as a sero-positive detection.

A total of 20 µg of purified native orf viral lysate was resolved by SDS-PAGE gel (10% acrylamide) and transferred by electroblotting to a nitrocellulose membrane, which was subsequently cut into multiple strips of approximately equal width. Strips were probed with goat sera (test sera). The strips were then incubated with HRP-conjugated rabbit anti-ovine IgG antibodies (Sigma) and washed before incubation with an ECL chemiluminescent substrate as a reporter (Pierce-Thermo Scientific, U.S.A.).

From December 31, 2011 to October 1, 2012, ten scabs from the outbreaks in ten different farms were collected, and eight virus strains have been isolated from them. The viral genomic DNAs were isolated from tissue suspensions or cell-cultured virions (200 µl each) by using QIAamp DNA blood kit (QIAGEN, Germany) following the manufacture's instructions. All animal protocols used in this study have been approved by the Department of Science and Technology of Fujian Province, and were in compliance with the guidelines of the Animal Care Committee, Fujian Agriculture and Forestry University (Certification Number: CNFJAC0027).

Sites in China at which orf isolate sequences of ORFV011 and ORFV059 genes used for phylogenetic analysis are depicted in Figures 1 and 2 [16].

PCR was performed on DNA extracted from skin scabs or infected cell cultures. Two sets of primers were designed based on the genomic sequences of OV-IA82 to amplify the entire open reading frame of two highly conserved genes, ORFV011 (B2L) and ORFV059 (F1L). The primer sequences were as follows: ORFV011Fw1: 5′- AAGCGGAGACCTAATGCC-3′; ORFV011Rv1: 5′-ACCTTTCCCCAGAACCC-3′; ORFV059Fw1: 5′-CTCGGTCAAGGACTGGATA-3′; ORFV059Rv1: 5′-GATGGCTGGATGGTGCA-3′. PCR was carried out in a 50 µl reaction volume containing 10 µl of 5X PCR buffer (10 mM Tris-HCL and 50 mM KCL), 2 µl of DNA template, 200 µM dATP, dTTP, dCTP, dGTP, 1 µM of each primer, 25 µM MgCl2 and 0.5 µl of Taq polymerase (Takara Co., Japan). PCR was performed in a thermocycler (Bio-Rad, U.S.A.) for 35 cycles, consisting of denaturation at 98°C for 1 min, annealing at 58°C for 45 s and extension at 72°C for 90 s, and a final elongation of 10 min at 72°C. The amplified DNA products were resolved by 1% agarose gel electrophoresis and analyzed with an IS-1000 Digital Imaging System (Bio-Rad, U.S.A.). The amplified gene was purified using an agarose gel DNA extraction kit (OMEGA, U.S.A.) according to the manufacturer's instructions. Automated nucleotide sequencing of ORFV011 and ORFV059 was performed using 3730XL DNA Analyzer with the BigDye terminator v3.1 by Shanghai ShengGong Biological Engineering Technology Service Co., LTD, China.

Sera from a total of 349 goats from 15 farms were tested for antibodies reactive to native orf viral lysate proteins in this survey (Table 1). Sero-positive reactivity to the native orf lysate would be consistent with prior or active infection with ORFV. Based on statements of the farmers and our own investigations, the 15 farms were divided into three categories. In the first category, three herds that reportedly had never been infected by ORFV were classified as “healthy” herds. In the second category, the eight farms that suffered at least one ORFV outbreak within two years of sera collection were classified as recovering herds. In the third category, the four farms experiencing an orf disease outbreak contemporaneously with sera collection were classified as infected herds.

Table 1 illustrates the results of reactivity of test sera from goats at these 15 farms to purified orf viral lysates, as tested by indirect ELISA at dilution ratios of 1∶2000, 1∶4000, 1∶8000 and 1∶16000. From the three “healthy” herds, among the 106 goat sera tested, 16 percent were sero-positive for ORFV at one or more dilution ratios tested (see Table 2). In the recovering herds, among 182 goat sera tested, 83% were sero-positive for ORFV at one or more dilution ratios tested (see Table 3). In the infected herds, among 61 goat sera tested, 93.4% were sero-positive for ORFV at one or more dilution ratios tested (see Table 4). Figure 3 depicts differences in serum titers to orf virus in goats from the three categories of herd infection status. In the “healthy” herds, more than 80% of serum titers were less than 2000 (i.e., sero-negative at a dilution ratio of 1∶2000), and no test serum was sero-positive at a dilution ratio of 1∶16000. In the recovering herds, about 20% of serum titers were below 2000, about 30% of serum titers equaled 2000 (i.e., were sero-positive for ORFV only at the 1∶2000 dilution ratio), more than 30% of serum titers equaled 4000 (i.e., were sero-positive for ORFV at dilution ratios of 1∶4000 and 1∶2000), about 20% of serum titers were 8000 (i.e., were sero-positive for ORFV at dilutions ratios of ≤1∶8000), and only a few sera had titers of 16000 (i.e., were sero-positive for ORFV at all dilutions, including 1∶16000). By contrast, in the infected herds, nearly 80% of sera had titers of ≥8000, about 15% had titers of 4000, and less than 10% of serum titers were ≤2000.

To analyze the relationships of these serum titers, we calculated the distance between serum titers measured at each of 15 farms (Table 5) to construct a serum titer clustering tree (Figure 4). Interestingly, the serum titer clustering tree demonstrated that the 15 farm serum titers clustered into three branches: The three “healthy” herds all clustered together, all eight recovering herds share great homology and clustered together, and the four infected herds also clustered together. This further confirms specificity of the ELISA in our survey.

Autoradiographs prepared using sera from nine farms are presented in Figure 5. Lane 9 contains serum from a “healthy” herd, and lane 11 represents serum from an infected herd. Both of them had serum titers of <2000 as determined by indirect ELISA and reacted with none of the purified native orf viral protein bands in the Western blot. However, a band with an approximate molecular weight (MW) of 40 kDa is evident in the other lanes, all of which correspond to sera that also reacted positively to the orf viral lysate by indirect ELISA. Most of positive sera in this study also reacted with a band of an approximate MW of 18 kDa. In some samples, 35 kDa, 38 kDa and 130 kDa bands were also detectable. However, the autoradiographic images of these bands were less intense than the 40 kDa and 18 kDa bands. Sera from nine farm animals that demonstrated negative reactivity by indirect ELISA also generated no detectable bands in the Western blot. Moreover, all sera demonstrating positive detection via indirect ELISA also identified a 40 kDa protein in the Western blot, validating the reliability of ELISA test results.

PCR amplified full length transcripts of the ORFV011 and ORFV059 genes in eight cell culture isolates (ORFV-FJ-GO, ORFV-FJ-GT, ORFV-FJ-DS, ORFV-FJ-SL, ORFV-FJ-SJ1, ORFV-FJ-YX, ORFV-FJ-NP, ORFV-FJ-FQ) and three scabs samples (ORFV-FJ-GS, ORFV-FJ-ZX, ORFV-FJ-SJ2). The sizes of products were approximately 1460 bp (ORFV011, an open reading frame of 1,137 bp) and 1268 bp (ORFV059, an open reading frame of 1023 bp) (data not shown). The identity of each PCR product was confirmed by automated DNA sequencing. The sequences were edited, aligned, and submitted to GenBank with the following Accession Numbers:

ORFV011-FJ-DS:KC568390; ORFV011-FJ-FQ:KC568391; ORFV011-FJ-GO:KC568392; ORFV011-FJ-GS:KC568393; ORFV011-FJ-GT:KC568394; ORFV011-FJ-NP:KC568395; ORFV011-FJ-SJ1:KC568396; ORFV011-FJ-SJ2:KC568397; ORFV011-FJ-SL:KC568398; ORFV011-FJ-YX:KC568399; ORFV011-FJ-ZX:KC568400; ORFV059-FJ-DS:KC568401; ORFV059-FJ-FQ:KC568402; ORFV059-FJ-GO:KC568403; ORFV059-FJ-GS:KC568404; ORFV059-FJ-GT:KC568405; ORFV059-FJ-NP:KC568406; ORFV059-FJ-SJ1:KC568407; ORFV059-FJ-SJ2:KC568408; ORFV059-FJ-SL:KC568409; ORFV059-FJ-YX:KC568410; ORFV059-FJ-ZX:KC568411.

DNA sequences comparisons of the full length ORFV011 and ORFV059 sequences obtained by PCR amplification were conducted to determine the phylogenetic relationships between isolates. Using GenBank nucleotide sequences of either ORFV011, ORFV059 or both, the phylogenetic relationships between 11 various Fujian isolates and with other ORFV isolates was constructed. The phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA) 5.0 software, with bootstrap values calculated from 1000 replicates. The phylogenetic algorithm used for the building of the tree was the Maximum Likelihood Methods.

The phylogenetic analysis of 11 Fujian Province (China) ORFV 011 sequences (Figure 6) shows that they do not cluster together. Among the 11 Fujian ORFV sequences, only FJ-GO (KC568392) and FJ-GS (KC568393) shared 100% nucleotide identities, suggesting that they are the same virus, and the other nine sequences shared 98.2%–99.3% nucleotide identity. Interestingly, FJ-SJ1 (KC568407) and FJ-SJ2 (568408) shared 99% identity at the nucleotide level, despite the fact that they came from scabs collected from the same farm eight months apart. This suggests that the evolution of ORFV is rapid.

The phylogenetic analysis of 11 Fujian Province (China) ORFV 059 sequences (Figure 7) shows that they nearly cluster together. Among 11 Fujian ORFV sequences, FJ-GO (KC568403) and FJ-GS (KC568404) shared 100% nucleotide identities, further suggesting they are the same virus, and the other 9 sequences shared 95.9%–99.3% nucleotide identity. FJ-SJ1 (KC568396) and FJ-SJ2 (KC568397) shared 98.9% similarities at the nucleotide level.

Phylogenetic analysis based on nucleotide sequences of ORFV011 (Figure 8A) shows Fujian Province 11 isolates do not cluster together. However, based on nucleotide sequences of ORFV0059 (Figure 8 B), the 11 Fujian Province isolates cluster together. If the phylogenetic analysis is based on the concatenated distances between the nucleotide sequences of ORFV011-059,(Figure 8C), the 11 Fujian Province isolates also cluster together. This reveals phylogenetic based on single gene maybe have some bias.

To compare and determine the phylogenetic relationship of 11 Fujian isolates, we drew a ground-distance comparison based on ORFV011?ORFV059 and ORFV011-059, where the Euclidian distance (D011-059)  =  ((D011)?2+(D059)?2)?0.5) (Figure 9). The data indicate that there is no correlation between geographical distance and phylogenetic distance.