Identification and phylogenetic analysis of contagious ecthyma virus from camels (Camelus dromedarius) in Iran

Contagious ecthyma is a highly contagious disease affecting domestic and wild ruminants such as sheep, goats and camels. The identification and characterisation of a parapoxvirus (PPV) infecting camels is described here. The virus was detected in dromedary camels (Camelus dromedarius) from Kerman and Shiraz in Iran. PPV-specific amplification by polymerase chain reaction (PCR) further confirmed that the disease was associated with PPV infection. Phylogenetic analysis of ORF011 (B2L) gene sequences showed 99.79% and 82.13% similarity of the PPV identified in this study with the Jodhpur isolate and the bovine papular stomatitis virus (BPSV) isolates (CE41), respectively. Moreover, phylogenetic analysis of the ORF045 gene indicated that the Shiraz sample was in all probability closely related to VR634 and to F00.120R and PCPV776. In conclusion, the results suggest that camel PPV (CPPV) is a likely cause of contagious ecthyma in dromedary camels in Iran.


Introduction
Camel contagious ecthyma (CCE), also known as orf, Auzdik or contagious pustular dermatitis, is a worldwide skin disease in camelids characterised by self-limiting proliferative lesions (Nagarajan et al. 2010). These nodular lesions followed by papules and vesicles mostly appear around the mouth, nares, lips and eyes or in some instances, other sites (Abubakr et al. 2007;Venkatesan et al. 2010). They then develop into scabs and healing may take up to 20 days -30 days or even more (Nagarajan et al. 2010). This disease is caused by pseudocowpox virus (PCPV) in cattle, whereas its main causative agent in sheep, goats, camels and humans is Orf virus (ORFV) (Davari et al. 2013;Nagarajan et al. 2010). However, PCPV has been reported as the etiologic agent of the disease in camels (Abubakr et al. 2007;Nagarajan et al. 2010). The ORFV or CCE virus belongs to the genus parapoxvirus (PPV), the subfamily Chordopoxvirinae of the Poxviridae family Harvey et al. 2015;Klein & Tryland 2005;Venkatesan et al. 2010). Morbidity is high, whereas its mortality and fatality rates are low to moderate. Nevertheless, this debilitating disease can lead to high mortality in younger animals due to secondary infections and trouble in suckling and eating (Hosamani et al. 2006). The genome of ORFV contains a linear double-stranded DNA molecule of approximately 134 kbp -139 kbp in length (Hautaniemi et al. 2010;Li et al. 2013;Rovozze & Burke 1973). The genes at the terminal ends are mostly responsible for viral replication, whereas those located in the central sites are essential for virus virulence and pathogenesis (Li et al. 2013). For instance, the well-conserved ORF045 gene encodes the late transcription factor VLTF-1, whereas the envelope or ORF011 (B2L) gene encodes a highly immunogenic envelope protein stimulating a strong antibody response (Kottaridi et al. 2006;Zhang et al. 2014). ORFV is sometimes transmitted to humans via direct contact with the infected animal (Torfason & Gunadottir 2002). Clinical signs of pox, contagious ecthyma and papillomatosis are similar and difficult to distinguish (Khalafalla, Al-Busada & El-Sabagh 2015a;Khalafalla et al. 2015b;Mosadeghhesari et al. 2014;Nagarajan et al. 2010). Polymerase chain reaction (PCR) and sequencing methods may be useful for genetic characterisation and classification of PPVs (Inoshima et al. 2001;Khalafalla et al. 2015a). The PPVs in Iranian dromedaries (Camelus dromedarius) have not yet been characterised at the genetic level, whereas their relationship to PPVs in domestic animals also remains unclear. Also, the ORF011 and ORF045 genes of camel PPV (CPPV) were used for viral detection, examining the outbreak of CPPV infection in camels by PCR and for phylogenetic analysis.

Sampling
Sixty skin samples were collected from two outbreaks of ORFV which occurred in 2013 in dromedary camels of Shiraz and Kerman cities, southern and eastern Iran, respectively. Biopsies Contagious ecthyma is a highly contagious disease affecting domestic and wild ruminants such as sheep, goats and camels. The identification and characterisation of a parapoxvirus (PPV) infecting camels is described here. The virus was detected in dromedary camels (Camelus dromedarius) from Kerman and Shiraz in Iran. PPV-specific amplification by polymerase chain reaction (PCR) further confirmed that the disease was associated with PPV infection. Phylogenetic analysis of ORF011 (B2L) gene sequences showed 99.79% and 82.13% similarity of the PPV identified in this study with the Jodhpur isolate and the bovine papular stomatitis virus (BPSV) isolates (CE41), respectively. Moreover, phylogenetic analysis of the ORF045 gene indicated that the Shiraz sample was in all probability closely related to VR634 and to F00.120R and PCPV776. In conclusion, the results suggest that camel PPV (CPPV) is a likely cause of contagious ecthyma in dromedary camels in Iran.

Identification and phylogenetic analysis of contagious ecthyma virus from camels (Camelus dromedarius) in Iran
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of the skin and the lip of the affected camels were sent to Razi Research Vaccine and Serum Institute. The samples collected from the diseased camels were analysed for PCR assay.

DNA extraction and primer design
DNA of the samples from the skin and lip lesions was extracted by High Pure ® Extraction Kit (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, 50 mg of the tissue samples was homogenised in 200 µL of phosphate buffer saline (PBS) and mixed with 200 µL of binding buffer and 50 µL of proteinase K then incubated at 72 °C for 10 min. The process was continued according to the manufacturer's instructions.

Polymerase chain reaction
For amplification of the ORF011 gene, the PCR assay was carried out as follows: 1 µg of DNA isolated from the infected or uninfected cells was added to 50 µL of the PCR mixture containing 0.2 mM primers (PPP-1 and PPP-4), 0.2 mM dATP, dCTP, dGTP and dTTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 and 1 U of AmpliTaq Gold DNA polymerase. DNA was amplified with a DNA thermal cycler by a two-step reaction. The mixture was denatured at 95 °C for 9 min, five cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, 25 repeated cycles at 94 °C for 1 min, 55 °C for 1 min and finally 72 °C for 1 min (Inoshima et al. 2000).
For amplification of the ORF045 gene, the PCR assay was carried out in a reaction mixture of 50 µL containing 1 µg (10 µL) of extracted DNA, 5 µL of dNTP (10 mM), 5 µL of 10x PCR buffer, 2 µL of MgCl 2 (50 mM), 2 U/tube of Taq polymerase, 50 pmol of each of the primers and 33 µL of distilled water. Forty cycles of denaturation (at 95 °C, for 10 s), annealing (at 47 °C for 10 s) and extension (at 74 °C for 10 s) were followed by a 15-min incubation at 78 °C to final extension of the primers. The amplification products (10 µL) were analysed by 2% agarose gel in Tris-boric acid-EDTA (TBE) buffer and stained with SYBR Green (1 µg/mL) (Kottaridi et al. 2006). The amplicons were visualised using a UV transilluminator 'gel doc' system.

Sequencing of the polymerase chain reaction products containing ORF011 and ORF045 genes
The PCR products with the expected sizes (594 bp and 393 bp) were purified from the gel using the Bioneer (Daejeon, South Korea) gel extraction kit, according to the manufacturer's procedures, and then were sequenced by the use of neighbour-joining method (Bioneer).

Phylogenetic analysis
Comparison among the isolates of this study and those of the ORFV available in GenBank, NCBI database, was made using the online BLAST program. Sequence identities of the nucleotides were analysed by CLC program version 5.5. The sequences were assembled into multiple sequence alignment. A phylogenetic tree derived from the nucleotide sequences was constructed for the ORFV, using neighbour-joining method of CLC program version 5.5. The sequences of the ORF011 and ORF045 genes of ORFV used in phylogenetic analysis are presented in Tables 1 and 2.

Polymerase chain reaction and DNA sequencing
The PCR method was performed to amplify the ORF011 gene. This method successfully detected the DNA from members of the PPV genus and was able to diagnose the ORFV infection.
To confirm the causative agent, a partial region of the ORF011 gene was amplified. The primers PPP-1 and PPP-4 were described as the universal primers to amplify most species of ORFVs that have been used in the present study. A 594-bp product representing the region (157 nt -750 nt) of the ORF011 gene was amplified from Shiraz samples. The 594-bp DNA fragment was sequenced and the nucleotide sequence data confirmed ORFV. Another PCR test was performed on DNA purified from Kerman samples as template and the pair of 045F/045R primers to amplify the ORF045 gene. PCR resulted in the amplification of the gene ORF045 with an expected DNA fragment (392 bp).

Phylogenetic analysis
The samples used in this study included Kerman (MSOKE) and Shiraz (MSOSH) isolates. The sequences aligned with MSOKE and MSOSH were based on the ORF011 gene (Table 1) and the gene ORF045 (Table 2), respectively. As stated previously, the samples of this study and the previous isolates were aligned and the phylogenetic tree was drawn by CLC program version 5.5. Results of the phylogenetic tree related to the ORF011 gene have been shown in Figure 1a  (VR634), another PCPV from Finland (F00.120R) and then also to PCPV from Austria (PCPV776).

Discussion
Several procedures have been developed to detect ORFV, while most of them are time-consuming, costly and sometimes associated with low specificity, reduced sensitivity and with cross reactions (Inoshima et al. 2001;Khalafalla et al. 2015a;Kottaridi et al. 2006). Assays based on PCR are considered as rapid, sensitive and specific tests for identifying the causative agents of diseases (Andrade et al. 1983;Inoshima et al. 2001Inoshima et al. , 2002. Several PCR protocols have been described for detecting the DNA of PPV (Inoshima et al. 2000(Inoshima et al. , 2001(Inoshima et al. , 2002Torfason & Gunadottir 2002     http://www.ojvr.org Open Access attenuation of the virus in Iran would therefore be necessary for protection of camels. A recent study performed by Mombeni et al. (2013) in Iran reported severe cases of CCE that caused a morbidity and mortality rate of 70.6% and 6.0%, respectively. This points to the potential importance of this disease in Iran. This study has indicated that ORFV with an apparent close similarity to PCPV can be a major cause of contagious ecthyma in Iranian camels.