Diagnosis and phylogenetic analysis of orf virus in Aleppo andSaanen goats from an outbreak in Turkey Dear Editor,
The Poxviridae family is divided into Chordopoxvirinaeand Entomopoxvirinae genera. Parapoxvirus is one of 11genera in the Chordopoxvirinae genus, including orf vir-us (ORFV), bovine papular stomatitis virus (BPSV),pseudocowpox virus (PCPV) and parapoxvirus of reddeer in New Zealand (PVNZ) (Skinner et al., 2012). Thisvirus, also known as contagious ecthyma or contagiouspustular dermatitis, is a viral skin disease causing ed-ematous proliferative lesions, most often in hairless skinregions around the muzzle and buccal cavity. Severalmolecular diagnostic methods including polymerasechain reaction (PCR) and quantitative PCR (qPCR) havebeen developed to detect orf virus (Gallina et al., 2006).In this study, multiplex PCR was used to amplify boththe B2L gene partial coding region and a housekeepinggene. A substantial amount of research has been conduc-ted on the B2L gene, and this presents opportunities forcomparisons between different strains around the world.Field strains have been compared with human, sheep,and goat orf viruses and with other parapoxviruses suchas PCPV and BPSV. Although the presence of the virusin Turkey was previously reported, its relationship withother orf viruses around the world has not yet beendemonstrated, in contrast to parapoxvirus infections ofcattle and humans in Turkey (Karakas et al., 2013;Oğuzoğlu et al., 2014). This study provides diagnosticand molecular characterization data for the economic-ally damaging parapoxvirus infection that circulatedamong goat flocks in Turkey in the 2013–2014 lambingseason.Scab samples were taken from five affected lambs
(aged 5–60 days) from a private goat herd reared fromAleppo and Saanen goats in Korkuteli, Antalya (SouthAnatolia), Turkey (Figure 1A). The herd was one-thirdAleppo and two-thirds Saanen goats. The herd with preg-nant goats comprised approximately 1,700 goats thatwere not pasturing outside the farm. The lambs, whichhad been vaccinated against orf virus before the out-break, had facial and oral lesions, with papillomatousproliferative lesions especially around the upper andlower labia (Figure 1B-1, 1B-2). There were also diffuse
nodular lesions in the lungs (Figure 1B-3). The farm had750 lactating female goats and 475 newborns, of which38 died during the outbreak.Scab materials were homogenized in phosphate-buf-
fered saline (PBS), frozen and thawed for three cycles,and centrifuged at 4000 rpm/min for 15 minutes. Theresulting supernatant was used for total DNA extractionby the phenol:chloroform:isoamylalcohol extractionmethod described by Sambrook et al. (2001). PCR wasperformed using a modification of the method of Inoshi-ma et al. (2000), using primers PPP-1 (5′-GTCGTCCAC-GATGAGCAGCT-3′) and PPP-4 (5′-TACGTGG-GAAGCGCCTCGCT-3′) specific for the parapoxvirusvirus B2L gene (Inoshima et al., 2000). Internal controlprimers were used in the same reaction, as described byMarkoulatos et al. (2000), for verification of the reac-tion, which was carried out in a total volume of 30 μL ofmix containing (5 U/μL) Taq DNA polymerase (MBI,Fermentas, Waltham, MA, USA), 10 × Taq Buffer (1.25mL including (NH4)2SO4, 25 mmol/L MgCl2, and water(18 Mohm/cm, Applichem, Darmstadt, Germany)),primers, and 3 μL of extracted DNA. An initial denatura-tion step at 96 °C for 5 min was followed by 35 cycles ofDNA denaturation at 96 °C for 30 s, primer annealing at48 °C for 30 s, and an amplification step at 72 °C for 45s. PCR products (595 bp) were purified with a Gene JETPCR purification Kit (Thermo Scientific, Waltham, MA,USA), and PCR fragments were sent to RefGen (Ankara,Turkey) for Sanger sequencing. BLAST searches in Gen-Bank revealed these sequences to be 98%–99% identicalto various orf virus strains, and they were submitted tothe NCBI GenBank database and assigned the accessionnumbers KP869116-KP869120. Homology analysesshowed that all five strains cluster together, with similarnucleotide variation. Among the five sequences, TR-ORF-2013-Caprine 2, 3, 4, and 5 shared 100% nucle-otide identities, and TR-ORF-2013-Caprine-1 showed a0.8% nucleotide difference from the other four fieldstrains. All five strains showed only a 0.8% differencefrom the KC491193.1 goat orf virus strain detected in2007 in Turkey (Figure 1C).BPSV Arkansas USA (AY386265.1), BPSV USA
(AY424973.1), sea lion parapoxvirus USA (DQ163058.1),
Phylogenetic analysis of orf virus in goats in Turkey
2 VIROLOGICA SINICA
pseudocowpox virus Turkey (KF554010.1), and pseudo-cowpox virus USA (AY424972.1) were used as outlierstrains. These parapoxviruses were found to be distantlyrelated to goat and sheep orf viruses, except for thepseudocowpox virus USA strain, which was closely re-lated to our field strains, with 99.5% sequence homo-logy. The Turkish pseudocowpox virus showed a largedistance from our field strains, with a 100 bootstrapvalue and 84.9% sequence identity. Strain Hubei/2010/goat/China/KF913722, FJSL/2012/goat/China/KC568398.1,and B047/1987/capra_hircus/Germany/KF478799.1 se-quences were strongly similar to our five field strains,with 99.84% identity.TR-ORF-1987-Sheep-87/B2L gene/Turkey/KF714241.1,
and TR-ORF-2009-Sheep-13/B2L gene/Turkey/KF714238.1shared 99.84% identity at the nucleotide level with fourof the strains characterized in this study, with TR-ORF-2013-Caprine 1 being the only exception. They also dis-played 99.76% similarity with TR-ORF-2013-Caprine 1.TR-ORF-2008-Sheep-12/B2L gene/Turkey/KF714237.1showed 99.92% and 99.84% identity with TR-ORF-2013-Caprine 2, 3, 4, and 5, and TR-ORF-2013-Caprine1, respectively. TR-ORF-2010-Goat-2/B2L gene/Turkey/KF714240.1, TR-ORF-2010-Goat-2/B2L gene/Turkey/KF714240.1, and TR-ORF-2007-Sheep-10/B2Lgene/Turkey/KF714236.1 strains exhibited 99.76% and99.68% identity with TR-ORF-2013-Caprine 2, 3, 4, and5 and TR-ORF-2013-Caprine 1, respectively.CTF_GAntep_n1/2012/B2L gene/human/Turkey/
KC776922.1 showed 99.84 and 99.76% identity withTR-ORF-2013-Caprine 2, 3, 4, and 5, and TR-ORF-2013-Caprine 1, respectively. CTF_GAntep_2/2012/B2Lgene/human/Turkey/KC776923.1, which displayed99.76% and 99.67% identity with TR-ORF-2013-Caprine 2, 3, 4, and 5, and TR-ORF-2013-Caprine 1, re-spectively, showed a greater similarity to these strainsthan TR-ORF-S-Human/2011/B2L gene/Turkey/JQ936990.1, which had 99.51% and 99.60% identitywith TR-ORF-2013-Caprine 2, 3, 4, and 5, and TR-ORF-2013-Caprine 1, respectively.The other strains from different geographical regions
(China's Taiwan, South Korea, China, India, and USA)also demonstrated 99.67%–99.84% identity globally. Fi-
nally, EEV envelope phospholipase gene/reindeer/Fin-land/JF773696.1 had 99.68% similarity with all fivestrains characterized in this study.Amino acid (aa) sequence comparisons between the
Turkish goat orf strains and the selected reference orfstrains showed that they were nearly identical but con-tained a few changes (Figure 1D). Like other referenceorf strains, the Lysine (K) at position 18 of the aa se-quence in all the Turkish strains was changed to Glutam-ic acid (E). The Alanine (A) at position 51 was changedto Threonine (T), and the Leucine (L) at position 131was changed to Threonine (T) concurrently in all Turk-ish goat orf strains. Some other individual aa changeswere observed in TR-ORF-1 and TR-ORF-4. In TR-ORF-1; the Glutamic acid, (E) at position 35 of the aasequence was changed to Glutamine (Q). In TR-ORF-4,the Histidine (H) at position 120 was changed to Tyr-osine (Y), and Arginine (R) at position 144 was changedto Cysteine (C).Animal husbandry in Turkey includes 10 million
goats, constituting 2.5% of total milk production. In re-cent years, Aleppo and Saanen goats have been increas-ingly exported to Turkey for milk production. However,importing animals increases the risk of exposure associ-ated with infectious agents such as orf virus, which canlead to outbreaks in goats and associated economic dam-age. Orf virus infection has a worldwide distribution(Fleming et al., 2007, Kumar et al., 2014) and causes theformation of lesions around the mouth and nostrils sothat animals are unable to graze and become weak(Mazur et al., 1989). The disease mainly infects lambsyounger than 1 year. Viral circulation happens very rap-idly through direct contact or scab shedding (Fleming etal., 2007). Orf virus can also cause mortality in younganimals, although the rate is lower than the incidence ofthe disease, which can reach 90% (Mazur et al., 1989;Fleming et al., 2007). Mortality is either due to second-ary bacterial and fungal infections or because the lambsare unable to suckle (Haig et al., 2002). In the presentoutbreak, the kid fatality rate was 8% (38/475). Few in-vestigations have focused on Turkish goat orf viruses(Oguzoglu et al., 2009). The present study illustrates therelationship of Turkish goat orf viruses with other orf
Figure 1. (A) Samples collected from Antalya city, Korkuteli county, South Anatolia, Turkey in 2013–2014. (B-1, B-2)Kids showing multiple proliferative lesions on the upper and lower labia and the junction of lips. (B-3) Lung of a kid withnodules. (C) Phylogenic analysis of local goat orf viruses based on partial B2L protein coding sequences. The se-quences obtained were aligned using CLC Main Workbench v5.5 (CLCBio, Aarhus, Denmark), and analyzed with Mega6.06 (Tamura et al., 2013). The Maximum-Likelihood trees were generated based on the Hasegawa-Kishino-Yano mod-el with 1,000 bootstrap replicates. Virus strains represented are indicated by name following GenBank accession num-ber. Sea lion parapoxvirus, pseudocowpox viruses and bovine papular stomatitis viruses are included as an outlier. Allsequences were compared against nucleotide numbers 11,053–11,557 of their complete genome. (D) Multiple align-ment of the deduced amino acid sequences of B2L genes of Turkish goat orf strains with those of orf strains from vari-ous countries. Dots denote amino acid identity with a goat orf virus from China (KC568399).
Zeynep Akkutay-Yoldar et al
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and parapoxviruses around the world. All five strainsshared 99.92% identity at the nucleotide level withKC491193.1. Few amino acid changes were seen in theB2L gene between Turkish goat orf strains from the sameherd and orf strains from different countries (Figure 1-D). However, this strain was localized in a separatebranch on the phylogenetic tree. The phylogenetic ana-lysis results, which also included sequence data fromseveral years in Turkey (1975, 1987, 2006, 2007, 2008,2009, and 2010), showed that these orf virus outbreaksare closely related antigenically. Moreover, homology-based phylogenetic analysis showed that the goat orf vir-us B2L gene partial coding region encoding the majorenvelope protein p42K is not only similar to that of goatorf viruses but also to that of sheep, deer, human, andcow parapoxviruses (Sullivan et al., 1994). This indic-ates that orf virus infection is endemic in small rumin-ants in Turkey, with continuous risk of human transmis-sion. The phylogenetic characterization among para-poxvirus strains indicates that the B2L region is a highlyconserved region in orf viruses. As has already been car-ried out by some researchers, future studies can employdifferent open reading frames (ORFs) to detect variableregions in the virus genome (Peralta et al., 2015). Appro-priate vaccination programs and sanitation measureswould both decrease economic losses and reduce the riskof human infection.
FOOTNOTES
We thank veterinary surgeon Osman Talayman for his helpsampling scab materials from lambs. This study did not receiveany funding. All institutional and national guidelines for the careand use of animals were followed. The authors declare that thereare no conflicts of interests.
Department of Virology, Faculty of Veterinary Medicine, AnkaraUniversity, Ankara, Diskapi 06110, Turkey* Correspondence:Phone: +90-312-3170315-4294, Fax: +90-312-3164472,Email: [email protected]: 0000-0002-1178-5347
Published Online: 20 January 2016
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