Orf Virus in Eastern Turkish Goats Genetically Characterized

Orf virus, the cause of a highly contagious skin disease in sheep and goats, has been genetically mapped for the first time in goat herds across eastern Türkiye, revealing an unexpected picture of viral diversity in a geographically compact region. A new study published in Veterinary Medicine and Science analysed samples collected during outbreaks between 2022 and 2024 in the provinces of Elazığ, Diyarbakır and Tunceli, and found that while most Turkish isolates are nearly identical to strains circulating across Asia, one isolate from Tunceli stands apart with substantial nucleotide substitutions and a distinct evolutionary lineage.

The research team, working with the Elazığ Veterinary Control Institute of the Turkish Ministry of Agriculture and Forestry, set out to detect and molecularly characterise the strains of Orf virus (ORFV) currently circulating in eastern Türkiye. ORFV is the etiologic agent of contagious ecthyma, also known as orf, sore mouth or scabby mouth, a disease endemic wherever small ruminants are raised. It produces proliferative, often painful lesions on the lips, the skin around the nose and the oral mucosa, progressing through erythema, papules, pustules and scabbing, and usually healing within one to two months. Although the disease is generally regarded as mild in adults, mortality rates of up to 93 percent have been reported in goat kids, in which lesions on the lips prevent suckling and grazing, leading to rapid weight loss and death.

The outbreaks investigated in the study were severe. The affected animals were primarily hair goat kids aged three to four months, in which clinical signs were most pronounced and included facial oedema and anorexia. Thirteen kids died in a single herd. In adult goats, lesions were concentrated on the udders, a pattern consistent with transmission from nursing kids to their dams. The animals had failed to respond to prior treatment with oxytetracycline and enrofloxacin, underscoring that antibiotics are ineffective against the virus and that laboratory confirmation is essential.

Because the lesions of orf, while characteristic, are not sufficient on their own for definitive diagnosis, the researchers turned to molecular methods. Skin papule samples were homogenised in phosphate-buffered saline and centrifuged, and viral DNA was extracted using an automated QIAGEN QIAcube system. Screening was performed by real-time PCR targeting the viral DNA polymerase gene, with samples classified as positive when they produced a characteristic amplification curve with a cycle threshold value below 35. Seven samples were positive, and four representative isolates, one from each sampling context and province, were selected for deeper genetic analysis.

Rather than relying on a single gene, as many surveillance studies do, the team employed a multi-locus approach, amplifying and sequencing three genomic regions that together capture different dimensions of the virus’s biology. The F1L gene, located in the 59th open reading frame, encodes an envelope-associated immunogenic protein that mediates viral attachment and entry by binding heparan sulphate receptors on host cells. The B2L gene encodes the major envelope protein, a molecule with lipase activity and strong immunogenic properties that induces a robust antibody response and serves as the most widely used PCR target for ORFV detection. The third target, the VIR gene, encodes a double-stranded RNA–binding virulence protein that confers resistance to the host’s interferon-mediated antiviral defences; notably, the VIR protein has been shown to interact with the host tumour suppressor p53 and promote its degradation, blunting antiviral immune signalling.

Conventional PCR produced amplicons of 1023 base pairs for F1L, 1137 base pairs for B2L and 552 base pairs for VIR. These products were sequenced by the Sanger method, and the resulting chromatograms were quality-trimmed, compared against GenBank using BLASTn, and aligned with reference sequences using the ClustalW algorithm in MEGA X. Phylogenetic trees were constructed with the Maximum Likelihood method under the Tamura 3-parameter model with Gamma distribution, supported by 1000 bootstrap replicates, and rooted with outgroup sequences from related parapoxviruses, pseudocowpox virus and bovine papular stomatitis virus.

The results place the Turkish isolates squarely within the broader Asian ORFV phylogenetic landscape. For the F1L gene, three of the four isolates were 100 percent identical to each other and shared 98.64 percent nucleotide similarity with strains from China, Malaysia and India. Overall similarity to globally circulating goat ORFV strains ranged from 96.29 to 98.64 percent, while similarity to other parapoxviruses dropped to between 75.2 and 90.9 percent, confirming that the samples were unequivocally orf virus. The B2L analysis mirrored this pattern: the Elazığ and Diyarbakır isolates were identical to one another and shared 99.48 percent similarity with an Iranian strain, while the overall range of similarity to other goat ORFV isolates spanned 96.56 to 99.48 percent.

The VIR gene told a more nuanced story. Consistent with its role as a rapidly evolving virulence locus, the VIR sequences showed the greatest divergence from reference strains, and the VIR-based trees placed the Turkish isolates closest to strains from China and India, with 98.81 to 99.01 percent similarity, and, for the Tunceli isolate, closest to strains from the United States and Taiwan. Overall VIR similarity to goat ORFV strains in GenBank ranged from 95.63 to 99.01 percent. This variability is precisely what makes the VIR gene valuable, the authors argue: because it evolves faster than the more conserved F1L and B2L regions, it acts as a more sensitive molecular marker for tracking viral evolution, host immune evasion and adaptation.

The most striking finding, however, was the behaviour of the Tunceli isolate. Across all three genes, the Tunceli sample clustered separately from the Elazığ and Diyarbakır isolates, which were identical to each other, and showed nucleotide similarities of only 96.57 to 98.64 percent relative to its Turkish counterparts. In the F1L analysis, it formed its own cluster, and in the VIR analysis it aligned with a group containing American and Taiwanese strains rather than with its geographic neighbours. The authors interpret this as evidence of localised evolutionary dynamics and genetic heterogeneity, suggesting that even within geographically close provinces, ORFV populations can display unique clustering patterns that single-gene surveillance would miss.

From an epidemiological standpoint, the close relationship between the Turkish isolates and Asian strains raises questions about the routes by which the virus has moved through the region. The authors suggest that the molecular correlations could reflect recent transmission events, shared ancestral lineages, or historical livestock trade routes that have long connected these geographic areas. Livestock movement across Anatolia and into the Middle East and South Asia provides plausible pathways for such exchange, and the study’s finding that Turkish strains cluster with isolates from China, India, Iran and Malaysia is consistent with earlier reports from other Turkish research groups.

The broader significance of the work lies in its implications for disease control. ORFV is currently endemic in Türkiye, and outbreaks in goats are increasing, yet high-resolution genomic data on circulating strains have remained scarce. Detailed genomic characterisation of local strains can inform the design of vaccines tailored to the genetic landscape of the region, an important consideration because immunity to orf is largely strain-specific and commercial vaccines have had mixed success. The VIR gene, with its high mutation rate and central role in defeating host antiviral responses, is a particularly compelling target for such efforts, and the authors call for up-to-date molecular and phylogenetic analyses of VIR in currently circulating strains as a prerequisite for rational vaccine development.

The study also carries public health weight. Orf is zoonotic, and humans are at high risk of infection through direct contact with infected animals or indirectly via contaminated fomites, with lesions typically appearing on the hands and fingers of farmers, veterinarians and abattoir workers. Continuous monitoring of genetic variation in circulating strains is therefore essential not only for veterinary surveillance but for assessing cross-species transmission risk.

The authors acknowledge the limitations of a small sample size, which constrains how broadly the findings can be generalised, and they recommend wider geographic sampling across Türkiye together with whole-genome sequencing to capture the full extent of national viral diversity. Nevertheless, the work stands as a pilot reference for future epidemiological surveillance in the country and as a demonstration of methodological principle: combining conserved structural genes with the hyper-variable VIR locus yields a far finer resolution picture of viral evolution than any single gene can provide, revealing that the orf viruses circulating in eastern Türkiye are not a uniform population but a mosaic of lineages shaped by geography, trade and the ongoing arms race between virus and host immunity.