Isolation and phylogenetic analysis of orf virus from the sheep herd outbreak in northeast China

Background Orf is a zoonotic and epitheliotrophic contagious disease that mainly affects sheep, goats, wild ruminants, and humans with a worldwide distribution. To date, there is little information on the characterization of ORFV strains that are endemic in Mainland China. In addition, the relationship between the severity of disease and the molecular profile of ORFV strains has not been fully elucidated. Results From the recent outbreak of a sheep herd in Nongan, northeast of China, the novel orf virus (ORFV) strain NA1/11 was successfully isolated. Western blot analysis indicated that the NA1/11 strain cross reacts with monoclonal antibody A3 and infected sheep ORFV antiserum. The purified virions revealed the typical ovoid shape when observed by atomic force microscopy. To determine the genetic characteristics of the NA1/11 strain, the sequences of ORFV011 (B2L), ORFV059 (F1L), ORFV109, ORFV110 and ORFv132 (VEGF) genes were amplified and compared with reference parapoxvirus strains. Non-metric multidimensional scaling (nMDS) was performed to analyze the nucleotide similarities between different ORFV strains. Conclusions Phylogenetic analysis based on ORFV 011 nucleotide sequences showed that the NA1/11strain was closely related to Xinjiang and Gansu strains. ORFV110 and ORFV132 genes are highly variable. The results revealed that precise phylogenetic analysis might provide evidence for genetic variation and movement of circulating ORFV strains in Northeast China. In addition, nMDS analysis showed that geographic isolation and animal host are likely major factors resulting in genetic differences between ORFV strains.


Background
Orf, also known as contagious pustular dermatitis or contagious ecthyma, is an epitheliotrophic disease that mainly affects sheep, goats, wild ruminants, and humans with a worldwide distribution [1][2][3]. The lesions are characterized by maculopapular, vesicular pustules that mainly affect the skin around the lips, mouth muzzle, nostrils, teats, and oral mucosa and rarely extend into the esophagus, stomach, intestine, or the respiratory tract of sheep, goats and wild animals [3][4][5][6]. In humans, the most common lesions are self-limiting, painful pustules on the hands and fingers [3,4]. The infective lesions are usually confined to the areas surrounding the virus entry sites. The development stages include erythema, vesicles, pustules, and scabs [6,7]. Primary lesions are usually resolved within 1-2 months, however repeated and persistent infections can occur [6,8,9]. The mortality rate related to this disease is usually low but it can reach 93% in lambs with secondary bacterial or fungal infections [4,5]. To diagnose this disease, virus isolation is thought to be the gold standard, although gross clinical signs can be used as a good reference [10].
ORFV, the causative agent of orf, is the prototype member of the genus Parapoxvirus belong to the subfamily Chordopoxvirinae of the Poxviridae. This genus also includes pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) in cattle, squirrel parapoxvirus (SPPV), and parapoxvirus of red deer in New Zealand (PVNZ) [11,12]. Parapoxviruses are ovoid in shape, with a crisscross patterned tubule-like structure on the particle surface [13]. The ORFV genome consists of linear double-stranded DNA about 138kb long with~64% G+C content, which contains 132 putative genes that included 89 highly conserved genes and some variable genes [13,14]. Currently, there are four ORFV strains that have been isolated and completely sequenced, OVIA82 and OV-SA00 [14] in America, NZ2 in New Zealand [13], and D1701 in Germany [15,16].
The entire genomic sequence of the orf virus from mainland China is not available. Only partial sequences are listed in the GenBank database and phylogenetic analysis was based on the highly conserved genes of ORFV011 (B2L) and/or ORFV059 (F1L) [5,12,17]. Similarly, almost all the phylogenetic analysis data published worldwide are based on the highly conserved genes except for where the whole genomic sequence is available (Table 1). There is little information on the characterization of ORFV strains that are endemic to mainland China. In addition, the relationship between disease severity and the molecular profile of ORFV strains has not been fully elucidated.
Considering heterogeneity of different ORFV field strains, we focused on isolation and characterization of ORFV from lesions during an epidemic of multifocal, persistent, severe, proliferative dermatitis in lambs in China. The novel NA1/11 strain was successfully isolated and characterized by Western blotting, electronic morphological observation, and polymerase chain reaction analysis. Genetic diversity was also determined by comparing the full lengths of ORFV 011, 059, 109, 110 and 132 genes with reference strains in the literature. This study on the genetic diversity of ORFV in China may contribute to an improved understanding of ORFV pathogenesis, infection biology and epidemiology, and facilitate ORFV vaccine development.
Ordination is a method helpful to data clustering in multivariate analysis. Non-metric multidimensional scaling (nMDS) is an ordination technique similar to principal component analysis (PCA). Ordination can avoid constructing linear relationships. It was used in this study to visualize and analyze the relationship between genetic distances and distances in species or geographic space.

Clinical gross pathological changes
In the sheep herd in Nongan clinical lesions with characteristic symptoms of orf such as papules, pustules, and scabs were recorded in 11 lambs from the flock. Two lambs showing proliferative lesions on the lips, nostrils, and eyelids were examined ( Figure 1). Both lambs presented with weight loss, anorexia, and proliferative papillomatous nodules ranging from 3-8 cm in diameter. Of the 11 infected lambs, 10 recovered about 30 days after clinical signs first appeared. One two-month old lamb died of secondary infection. The morbidity and mortality of the outbreak were 9.6% (11 out of 115) and 0.9% (1 out of 115) respectively. No cases of disease were recorded in breed ewes or the farm staff.

Orf virus isolation and purification
All the homogenates from tissues collected from the outbreak were inoculated into cultures of OFTu cells. The cytopathic effect (CPE), indicated by cell rounding, pyknosis, and cell detachment, was observed in cell cultures but not in mock-infected cells. CPE was evident 1 to 2 days after the third blind passage (Figure 2). To isolate a single clone of the viral strain, a plaque assay was performed to isolate a monoclonal plaque, caused by a single virus. The isolated virus from the single plaque was amplified, titrated, and stored at −80°C. The virus isolate was designated ORFV/Nongan-C1/2011 (NA1/11). The mature virions were purified by sucrose gradient ultra-centrifugation. A major virus band was obtained after centrifugation of virus infected OFTu cells in the 32%-36% sucrose gradient.

Western blot analysis
The cross reaction of the NA1/11 strain with sheep serum and monoclonal antibodies against other ORFV strains was determined using Western blot analysis. As shown in Figure 3A, the purified viral protein interacted with ORFV-positive antiserum, which was collected from a separate orf outbreak [5]. The strongest reactive protein bands were 39, 25, 20 and 15 kDa but 28, 35, 49, 55, 60 and 90 kDa bands were also detectable ( Figure 3A, Lane 2). In addition, a 39 kDa protein was detected when the NA1/11 viral protein blot was incubated with Mab A3 against ORFV-Jilin ORFV059 protein ( Figure 3B, Lane 2) [23], suggesting a cross reaction between NA1/11 and ORFV-Jilin strain.
Morphological determination of the NA1/11 strain Two types of orf virus particles were visualized by transmission electron microscopy of ultra thin sections: the immature form ( Figure 4A, arrows) and mature form ( Figure 4A arrowheads and B). The NA1/11 viral particles consisted of complete, ovoid-shaped virions characteristic of parapoxvirus with a crisscross patterned tubule-like structure measuring about 260 nm×150 nm on the particle surface when observed under the MFP-3D atomic force microscope ( Figure 4C). No other viruses were detected.

PCR amplification
DNA template for PCR was prepared from tissue suspensions, infected cell culture supernatant, or purified viral particles. DNA from non-infected OFTu cells was used as a negative control. The full length of the coding regions of ORFV011, 059, 109, 110 and 132 -134 gene

Phylogenetic analysis
To compare and determine the phylogenetic relationship of NA1/11 strain with other ORFV strains, PCPV and BPSV, the corresponding sequences obtained from GenBank were used in this study and are presented in Table 1.

NMDS
The results of nMDS analyses are displayed as scatter plots ( Figure 9). Analysis of similarities between different strains grouped by geographic isolation indicated that different strains from the same region are in close proximity, e.g. strains from China, Taiwan, India, USA, Brazil, or Finland. Strains from the same continent are closer to each other than strains from other continents except D1701 and NZ2 ( Figure 9A). The similarities among different strains grouped by host species showed that strains with the same host species did not scatter or group together because of their different geographic locations. However, they did gather into clusters ( Figure 9B). Interestingly, the similarities between different strains grouped by host species plus A B C  geographic isolation ( Figure 9C) are similar to the pattern of in Figure 9B. Strains in the same region isolated from different hosts have a big genetic variation. In nMDS analysis NA1/11 strain clusters with the other eight strains from China ( Figure 9A) and is very close in distance to strain No.7 (Xinjiang, sheep), which is in accordance with the results of phylogenetic analysis.

Discussion
In recent years there have been increased reports of ORFV infections in sheep, goats, wild animals, and humans worldwide [5,10,12,19,20,[24][25][26][27]. The clinical features of the infected animals varied from subclinical to multiple lesions around the lips, mouth, muzzle, nostrils, teats, and oral mucosa and occasionally within the buccal cavity esophagus, stomach, intestine, or the respiratory tract [3][4][5]24,27]. Recently genital lesions [24] and multifocal cutaneous infections [25] were also reported. However, virus isolation and characterization is seldom achieved in China. In this outbreak, the lesions were located around the mouth, muzzle, and the upper and lower eyelids (Figure 1). These are classical clinical signs and strong evidence of an orf virus infection.  The identity of NA1/11 strain was confirmed by Western blot, electron microscopy, amplification of specific genes from viral DNA by PCR, and DNA sequencing. Western blot analysis revealed four strong reactive protein bands and six weak bands detected by sheep ORFV antiserum ( Figure 3). Furthermore, a 39 kDa protein was detected in the NA1/11 viral protein blot using a Mab A3 against ORFV-Jilin ORFV059 protein [23], suggesting a cross reaction between the two different isolates and providing a tool for ORFV epidemiological surveillance and diagnosis.
The observed shape of NA1/11 virus was similar to the typical ovoid-shape of orf virus and the specific PCR products from the genomic DNA of the isolated virus confirmed our diagnosis of orf infection from this sheep herd. In China, from 2005 to 2006, ORFV outbreaks occurred in the provinces of Inner-Mongolia, Guangxi, Shanxi, Fujian, Jilin, Jiangsu and Beijing, and the government strengthened the vaccination program to control this disease [12]. However, in 2010, Jilin and Hubei provinces reported outbreaks of ORFV infected sheep herds [5,12]. In 2011, we found an outbreak in sheep herds in Nongan, northeast China. Because of the persistence or repetition of ORFV infections, it is very difficult to eliminate this virus. ORFV infection is considered a re-emerging disease in China and other countries [24,25].
In the ORFV genome, ORFV011, 059, 109, 110 and 132 genes have distinct functions for orf virus infections and pathogenesis. The ORFV011 gene encodes the major envelope immunogenic protein [28], while the ORFV059 gene encodes an immunodominant protein and plays a role in virus maturation and adsorption [23]. Both of these proteins are conserved and are used for orf detection, molecular characterization, and phylogenetic analysis [5,28]. ORFV109 and 110 encode envelope type II glycoproteins, which are expressed in inter-and extracellular enveloped virions [13]. The ORFV132 protein is an apparent homolog of the mammalian vascular endothelial growth factor (VEGF) family and plays an important role in the development of lesions induced by the orf virus [29]. These three genes are highly variable. According to the phylogenetic relationships based on the ORFV011, NA1/11 clustered with two Chinese strains, Xinjiang and Gansu, isolated from Northwest China, and was close to the Brazilian strain MT-05, American strain OV-IA82 and New Zealand strain NZ2. Usually phylogenetic analysis indicates a hypothetical origin of virus strains; the results presented here suggest that the OV-IA82 and NZ2 strains were introduced into China, but it is difficult to determine the route through which the new strains entered into China. Theoretically, the diversity of viral sequences is linked to viral strain differences, or other factors such as flock immunity, genetic susceptibility, or method of exposure. This implies that we may use the NZ2 strain, which is licensed and widely used in Europe [19] to vaccinate sheep herds and reduce ORFV outbreaks in China. We propose that the phylogenetic analysis of parapoxvirus should use entire genomic sequences to obtain more precise results.
Our results indicated that the nucleotide sequences similarities (or distances) among different ORFV strains are divergent (Figure 8 and 9). We hypothesize that the genetic differences among ORFV virus strains mainly relate to geographic location and animal host. However, as the variables are non-metric, the nMDS results need further complete genome comparisons to understand fully the genetic diversity and epidemiology of this complex group of viruses.

Conclusions
In summary, Orf infection is endemic in China. Although vaccination has been implemented in some regions to control the disease, an increase of orf infection in the dairy sheep and goat population in China has been observed in recent years, causing significant veterinary and economic losses. Based on the clinical signs, morphology, PCR amplification, and ORFV011, 059, 109, 110 and 132 gene sequences, we conclude that the isolated NA1/11 virus is a novel ORFV strain. The phylogenetic analysis revealed that NA1/11 clusters together with Xinjiang and Gansu strains. Therefore, more epidemiological surveillance is needed in China. Our continuing work to isolate and characterize ORFV isolates from Orf virus Pseudocowpox BPSV Figure 6 Phylogenetic analysis based on nucleotide sequences of ORFV011. The phylogenetic relationship was constructed by the neighborjoining algorithm using MEGA 5.0 software; one thousand bootstrap replicates were subjected to nucleotide sequence distance (cut-off value of 50% from 1000 bootstrap replicates

Sheep herds and tissue collection
The recent outbreak of orf in sheep occurred in 2011 in northeast China. It was recorded in a farm with 115 Figure 7 Phylogenetic analysis based on nucleotide sequences of ORFV059. The phylogenetic relationship was constructed by using the same method as described in Figure 6. All bootstrap values are displayed above branches. •: NA1/11 isolated in this study.  small-tailed Han sheep, including 95 breed ewes and 20 lambs aged 1 to 5 months, located in northeast Nongan county, China (124°31' E, 44°55' N). Skin biopsies with gross pathologic changes were collected with a surgical punch 5 mm in diameter (Miltex Stainless, Germany) and stored at −80°C for virus isolation and PCR analysis. Serum was also collected from corresponding animals and also stored at −80°C for future use. 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).

Virus isolation
The virus was isolated from tissues collected as described with some modifications [8,30]. Briefly, 40% w/v tissue suspension in 1× MEM medium (Invitrogen) supplemented with gentamicin (50 μg/ml), penicillin (100 U/ml), streptomycin (100 μg/ml), and 5 mg/ml of fungizone were homogenized in tissue mortars. The suspensions were centrifuged at 3000 ×g for 10 min, after which the supernatants were transferred and inoculated into OFTu cells. Inoculated cells were observed daily for the presence of cytopathic effect (CPE), and passaged three times. When 60% CPE was observed, cells and media were harvested and frozen at −80°C. Polymerase chain reactions together with sequencing reactions were performed to confirm Orf virus nucleic acid specificity. In order to isolate the virus strain, a plaque assay was carried out for further viral purification [31]. Briefly, 7×10 5 OFTu cells per well were transferred into 6-well plates one day before infection. On the next day, the cells were infected with serial dilutions of viruses from 10 -1 to 10 -6 . The viruses were allowed to absorb to the cells for 1 h at 37°C in a 5% CO 2 incubator. The medium was removed and the cells were overlaid with 3 ml of MEM containing 5% FBS and 0.5% low melting point agarose (Sea Kem W GTG W , Lonza, Rockland, ME, USA). Plaques were visualized and picked at 4 or 5 dpi. A minimum of 2 or 3 plaques were picked for each isolate.

Viral protein purification
OFTu cells were grown in T150 tissue flasks up to 90% confluency and infected with 1 MOI of NA1/11 isolate. Cells were cultured in Eagle's minimal essential medium with Earle's salts, supplemented with 1% penicillinstreptomycin (10,000 U of penicillin and 10 mg of streptomycin/ml in physiological saline), 1% l-glutamine (200 mM), 1% nonessential amino acids, and 10% (v/v) heat-inactivated fetal calf serum. All reagents were purchased from Life Sciences (Invitrogen). Cells were harvested when approximately 80-90% of the cells showed CPE. Debris was removed by centrifugation at a speed of 1,000 ×g for 10 min at 4°C. The supernatant containing mature viral particles was used for further sucrose gradient ultracentrifugation [32]. The purified viral particles were heat-inactivated at 96°C for 90 min and sonicated. The protein concentration was measured with Bio-Rad protein assay reagent (Bio-Rad Laboratories). The purified viral protein was frozen at −80°C for future use.

Western blots
20 μg of total OFTu cell lysates or 2 μg of purified viral protein as described above were resolved by SDS-PAGE gel (10%) and blotted to nitrocellulose membranes. Blots were probed with orf virus infected sheep serum or Mab A3 against ORFV059 [23], followed by incubation with donkey anti-sheep or goat anti-mouse HRP-conjugated IgG antibodies (Santa Cruz) and developed using a chemiluminescent substrate ECL (Pierce-Thermo Scientific).

Electron microscopy
The infected cell cultures of 24 hpi and the purified virions were fixed for 60 min at 4°C in a solution containing 0.1% glutaraldehyde, 4% freshly prepared formaldehyde, 1% picric acid and 3.5% sucrose in 0.1 M cacodylate buffer pH 7.2. The morphology of the virions was observed under an MFP-3D atomic force microscope from Asylum Research, Inc. [33]. For transmission electron microscopy, ultrathin sections of fixed infected cultured cells were cut on the LKB ultratome and mounted on copper grids. The sections were stained by a combination of uranyl acetate-lead citrate [34].

DNA extraction from scab suspensions and purified virions
Tissue suspensions (200ul) or purified virion materials were prepared for viral genomic DNA isolation by using QIAamp DNA blood kit (QIAGEN, Germany) following the manufacturer's instructions.
The amplicons were ligated into the TA cloning Vector (Invitrogen) following the manufacturer's instructions. Nucleotide sequencing was performed in both orientations by automated sequencing using sequencing primers T7-promotor and M13-reverse −24. Sequences were read on an automated sequencer (Applied Biosystems DNA Sequencer 373A, Norfolk, CT) and then edited using Sequencer version 3.0 (Gene Codes Corp., Ann Arbor, MI).

Phylogenetic analysis
Using the Neighbour-Joining method, a bootstrap consensus tree was used to represent the evolutionary history of the ORFV011 [35,36]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches [36]. Evolutionary analyses were carried out using the MEGA5 software (MEGA, version5) [37,38] and expressed based on the number of nucleotide substitutions per site. The numbers used in the phylogenetic trees represent the Geninfo Identifier (GI) sequence identification number in GenBank (http://www.ncbi.nlm.nih.gov/ Genbank/). Alignment and comparison of the nucleotide and amino acid sequences among NA1/11 and reference strains were performed using ClustalW [39].
The alignment results of ORFV011 nucleotide sequences of 43 strains (all are ORFV) were applied to pairwise distance calculations using the MEGA5 software (MEGA, version5). Distance matrixes were also used for nMDS analysis below. The variance estimation method is bootstrap method (no. of bootstrap replications is 1000) and the substitution model used is Tajima-Nei model.

NMDS
NMDS were performed based on pairwise distance matrix. The nMDS scatting plots were constructed to investigate the similarities between different ORFV strains using the free software PAST (Copyright Hammer and Harper, http://folk.uio.no/ohammer/past). The similarity measure method is Gower method. In nMDS analysis, strains were grouped by geographic isolation, animal host species, geographic isolation plus animal host species. Different groups were marked by different colors.