Logo of viroljBioMed CentralBiomed Central Web Sitesearchsubmit a manuscriptregisterthis articleVirology JournalJournal Front Page
Virol J. 2009; 6: 167.
Published online 2009 Oct 16. doi:  10.1186/1743-422X-6-167
PMCID: PMC2770494

Phylogenetic analysis and characterization of Korean orf virus from dairy goats: case report


An outbreak of orf virus infection in dairy goats in Korea was investigated. Suspected samples of the skin and lip of affected goats were sent to the laboratory for more exact diagnosis. Orf virus was detected by electron microscopy and viral DNA was identified by PCR. To reveal the genetic characteristics of the Korean strain (ORF/09/Korea), the sequences of the major envelope protein (B2L) and orf virus interferon resistance (VIR) genes were determined and then compared with published reference sequences. Phylogenetic analysis revealed that the ORF/09/Korea strain was closest to the isolates (Taiping) from Taiwan. This is believed to be the first report on the molecular characterization of orf virus in Korea.


Contagious ecthyma (contagious pustular dermatitis; orf) is a common epitheliotrophic viral disease of sheep, goats, and wild ruminants and is characterized by the formation of papules, nodules, or vesicles that progress into thick crusts or heavy scabs on the lips, gingiva, and tongue. Orf virus is an oval, enveloped virus containing dsDNA genome within the genus Parapoxvirus, family Poxviridae [1]. The genus also includes pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) in cattle and parapoxvirus of red deer in New Zealand. Zoonotic infection with orf virus is characterized by nodular and papillomatous lesions mainly on the hands, face, and mouth [2,3].

To reveal the genetic variation and characterization of parapoxvirus, the major virus envelope protein B2L and virus interferon resistance (VIR) genes have been used recently [4-8]. Orf virus infection has been diagnosed occasionally in Korea since the outbreak of orf was reported clinically in the 1990s. Although a few studies have been conducted, molecular epidemiology based on gene sequences of orf virus has not been performed because the population of sheep and goats is low in Korea. In the present study, orf virus infection in dairy goats was identified by clinical diagnosis and PCR. The complete B2L and VIR genes were sequenced, and their phylogenetic trees were constructed.

Case presentation

In April 2009, an exanthematic outbreak occurred in a farm with 400 dairy goats in the Chungbuk province. Sixty dairy goats presented with wart-like lesions on the lips, tongue, and around the anus (Fig. (Fig.1A1A and and1B).1B). The clinical diagnosis was contagious ecthyma. Morbidity was 15% (60/400), and goats of all the ages ranging from 2 weeks to 1 month were affected. Dried scabs collected from affected goats were stored in a -70°C freezer until the samples were used for further study.

Figure 1
Typical clinical cases of orf virus infection in diary goats (A, B), histopathological findings (C) showing intracytoplasmic eosinophilic inclusion bodies (arrows) in the keratinocytes indicated virus-induced lesions (HE, bar = 35 μm) and electron ...

The tissue samples were either fixed in 10% buffered formalin for histological examination, or were homogenized mechanically in PBS in a tube using a pellet pestle device. The homogenates were centrifuged at 3000 g for 5 min, and the supernatant was collected and negatively stained with 2% phosphotungstic acid for electron-microscopic examination of orf virus.

A PCR with OVS and OVA primers was performed as described previously [9]. In brief, the sequences of OVS and OVA primers are 5'-AGGCGGTGGAATGGAAAGA-3' and 5'-CCAGCAGGTATGCCAGGATG-3', respectively. The total viral DNA was extracted from scab samples using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The amplication were performed in a GeneAmp PCR System 2700 (Applied Biosystems) using the following conditions: a preliminary heating for 5 min at 94°C; 10 cycles of 30 s at 94°C, 45 s at 55°C, and 1 min at 72°C; 20cycles of 30 s at 94°C, 45 s at 57°C, and 1 min at 72°C; and the final elongation of 5 min at 72°C. For phylogenetic analysis, a PCR was also performed using previously described primers [4,5]. The amplified gene was purified using an agarose gel DNA extraction kit (INtRON, Seongnam, Korea) and further cloned into pGEM-T vector (Promega, Madison, WI, USA) according to the manufacturer's instructions. Automated nucleotide sequencing of the VP60 gene insert was performed using an ABI 3130XL genetic analyzer with the BigDye® Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The nucleotides at all positions were confirmed by three or more independent sequencing reactions in both directions. The published sequences of parapoxviruses were retrieved from GenBank for phylogenetic analyses (Table (Table11).

Table 1
Korean orf virus sequence and published parapoxvirus sequences used in the phylogenetic analysis

The B2L and VIR gene sequences of the orf virus, Korean strain were aligned with those of parapoxvirus sequences obtained from GenBank using Bioedit software (Ibis Biosciences, Carlsbad, CA, USA). A phylogenetic analysis was conducted using the Bioedit software and Molecular Evolutionary Genetics Analysis (MEGA) 3.1 software, with bootstrap values calculated from 1000 replicates [10]. The phylogenetic algorithm used for the building of the tree was the neighbor-joining method.

All clinical samples collected from the orf outbreak were positive using the previously described PCR method. The amplified PCR products were separated by electrophoresis and visualized by staining with ethidium bromide (EtBr). The size of amplified PCR product was 708 bp (data not shown). Keratinocytes in the stratum spinosum showed vacuolation and ballooning degeneration. Intracytoplasmic eosinophilic inclusion bodies were found characteristically in keratinocytes (Fig (Fig1C).1C). Negatively stained orf virus particles were examined by electron microscopy, which revealed the oval-shaped morphology of Parapoxvirus. The size of the observed virus particles was approximately 150-200 nm (Fig. (Fig.1D1D).

Additional PCR reactions were performed to obtain the full-length B2L and partial VIR genes. The amplified PCR products were 1206 bp for the full-length B2L gene and 552 bp for the partial VIR gene. The purified DNA was sequenced for comparative analysis. The sequence comparison of the ORF/09/Korea strain demonstrated a high degree of identity among our Korean strain and other orf virus strains. In phylogenetic analysis based on the complete B2L gene, the ORF/09/Korea strain was closer to the Taiping isolate (EU327506) from Taiwan (Fig. (Fig.2A).2A). The ORF/09/Korea strain and Taiping isolate showed 99.82% and 99.48% similarity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequences of the complete B2L gene of the ORF/09/Korea strain and the other strains were aligned using Bioedit software. Unique amino acid substitutions were found at two positions (A/R250→ D, I275→ M).

Figure 2
Phylogenetic analysis of different parapoxviruses based on nucleotide sequences of B2L gene (A) and VIR gene (B). The nucleotide sequences of diverse orf viruses were aligned using the Bioedit program and Mega 3 software. One thousand bootstrap replicates ...

In phylogenetic analysis based on the partial VIR gene, the ORF/09/Korea strain also was closer to the Taiping isolate (EU327508) from Taiwan (Fig. (Fig.2B).2B). The ORF/09/Korea strain and Taiping isolate showed 97.8% and 97.6% similarity at the nucleotide and amino acid level, respectively. The deduced amino acid sequences of the partial VIR gene of the ORF/09/Korea strain and the other strains were aligned, and unique amino acid substitutions were found at two positions (N/D250→ E, F275→ S).


Orf infection is endemic in Korea, and no vaccination has been implemented to control this disease. An increase in the dairy goat population in Korea has been observed over the past several years. Clinical diagnosis and electron microscopy have been used routinely to identify orf viruses in Korea. Recently, PCR methods have been used to detect the viruses [11,12]. The dairy goat is not a native species in Korea and has been imported from abroad. Several causes of orf outbreaks exist, and the phylogenetic analysis may indicate a hypothetical origin of the viral strains involved.

Although only one case from dairy goats that showed clinical signs was used in the present study, the ORF/09/Korea strain was closest to the isolates (Taiping) from Taiwan, according to phylogenetic analysis based on the B2L and VIR genes. However, it is difficult to conclude the precise route by which the Korean orf virus strain was introduced into Korea based on molecular analysis alone. Also, at present, all ruminants from Taiwan have been prohibited from importation into Korea because of the outbreak of foot-and-mouth disease in Taiwan. Therefore, more epidemiological data concerning the distribution of orf viruses in Korea and neighboring countries are needed. Our continuing work to characterize more isolates in Korea and neighboring countries should prove valuable in that regard.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JKO*, YHJ, and OSL participated in the planning of the project. KKL and HRK performed the PCR and phylogenetic analysis. ISR and KHL collected the samples and performed electron microscopy and histopathological examination. All authors read and approved the final manuscript.


This study was supported by the National Veterinary Research and Quarantine Service, Ministry of Agriculture, Anyang, Republic of Korea.


  • Robinson AJ, Balassu TC. Contagious pustular dermatitis (orf) Vet Bull. 1981;51:771–82.
  • Haig DM, Thomson J, Mclnnes C, McCaughan C, Imlach W, Mercer A, Fleming S. Orf virus immuno-modulation and the host immune response. Vet Immunol Immunopathol. 2002;87:395–99. doi: 10.1016/S0165-2427(02)00087-9. [PubMed] [Cross Ref]
  • Haig DM, Mercer AA. Ovine diseases. Orf. Vet Res. 1998;29:311–26. [PubMed]
  • Hosamani M, Bhanuprakash V, Scagliarini A, Singh RK. Comparative sequence analysis of major envelope protein gene (B2L) of Indian orf viruses isolated from sheep and goats. Vet Microbiol. 2006;116:317–24. doi: 10.1016/j.vetmic.2006.04.028. [PubMed] [Cross Ref]
  • Guo J, Zhang Z, Edwards JF, Ermel RW, Taylor C, Jr, de la Concha-Bermejillo A. Characterization of a North American orf virus isolated from a goat with persistent, proliferative dermatitis. Virus Res. 2003;93:169–79. doi: 10.1016/S0168-1702(03)00095-9. [PubMed] [Cross Ref]
  • Chan KW, Lin JW, Lee SH, Liao CJ, Tsai MC, Hsu WL, Wong ML, Shih HC. Identification and phylogenetic analysis of orf virus from goats in Taiwan. Virus Genes. 2007;35:705–12. doi: 10.1007/s11262-007-0144-6. [PubMed] [Cross Ref]
  • Kottaridi C, Nomikou K, Teodori L, Savini G, Lelli R, Markoulatos P, Mangana O. Phylogenetic correlation of Greek and Italian orf virus isolates based on VIR gene. Vet Microbiol. 2006;116:310–16. doi: 10.1016/j.vetmic.2006.04.020. [PubMed] [Cross Ref]
  • Mondal B, Bera AK, Hosamani M, Tembhurne PA, Bandyopadhyay SK. Detection of Orf virus from an outbreak in goats and its genetic relation with other parapoxviruses. Vet Res Commun. 2006;30:531–39. doi: 10.1007/s11259-006-3270-z. [PubMed] [Cross Ref]
  • Zheng M, Liu Q, Jin N, Guo J, Huang X, Li H, Zhu W, Xiong Y. A duplex PCR assay for simultaneous detection and differentiation of capripoxvirus and orf virus. Mol Cell Probes. 2007;21:276–81. doi: 10.1016/j.mcp.2007.01.005. [PubMed] [Cross Ref]
  • Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 2004;5:150–63. doi: 10.1093/bib/5.2.150. [PubMed] [Cross Ref]
  • Inoshima Y, Morooka A, Sentsui H. Detection and diagnosis of parapoxvirus by the polymerase chain reaction. J Virol Methods. 2000;84:201–208. doi: 10.1016/S0166-0934(99)00144-5. [PubMed] [Cross Ref]
  • Chan KW, Hsu WL, Wang CY, Yang CH, Lin FY, Chulakasian S, Wong ML. Differential diagnosis of orf viruses by a single-step PCR. J Virol Methods. 2009;160:85–9. doi: 10.1016/j.jviromet.2009.04.025. [PubMed] [Cross Ref]
  • Sullivan JT, Mercer AA, Fleming SB, Robinson AJ. Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K. Virol. 1994;202:968–73. doi: 10.1006/viro.1994.1420. [PubMed] [Cross Ref]
  • Delhon G, Tulman ER, Afonso CL, Lu Z, de la Concha-Bermejillo A, Lehmkuhl HD, Piccone ME, Kutish GF, Rock DL. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus. J Virol. 2004;78:168–77. doi: 10.1128/JVI.78.1.168-177.2004. [PMC free article] [PubMed] [Cross Ref]
  • Mercer AA, Ueda N, Friederichs SM, Hofmann K, Fraser KM, Bateman T, Fleming SB. Comparative analysis of genome sequences of three isolates of Orf virus reveals unexpected sequence variation. Virus Res. 2006;116:146–58. [PubMed]
  • McInnes CJ, Wood AR, Mercer AA. Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L. Virus Genes. 1998;17:107–15. doi: 10.1023/A:1026431704679. [PubMed] [Cross Ref]
  • Chan KW, Yang CH, Lin JW, Wang HC, Lin FY, Kuo ST, Wong ML, Hsu WL. Phylogenetic analysis of parapoxviruses and the C-terminal heterogeneity of viral ATPase proteins. Gene. 2009;432:44–53. doi: 10.1016/j.gene.2008.10.029. [PubMed] [Cross Ref]

Articles from Virology Journal are provided here courtesy of BioMed Central
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence and PMC links.
  • MedGen
    Related information in MedGen
  • Nucleotide
    Primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • Protein
    Protein translation features of primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...