Human orf virus (family Poxviridae) infection following a lamb bite in Hungary.

Human orf disease (called ecthyma contagiosum or contagious/infectious pustular dermatitis in animals) was confirmed on the fingers of both hands of a 24-year-old female, after feeding diseased lambs with a nursing bottle in April 2023. In addition to skin symptoms, she had low-grade fever (37.6°C) and swollen lymph nodes in both axilla. The presence of orf virus (genus Parapoxvirus, family Poxviridae) was confirmed, and this strain, Baja/2023/HUN (OR372161-OR372163), was found to have > 98% nucleotide sequence identity to sheep-origin orf viruses in four tested genome regions (ORF011/B2L, ORF019, ORF020/VIR, and ORF056). This is the first report of a human case of infection with the neglected zoonotic orf virus in Hungary.

Finger nodules with a papulovesicular hands and feet eruption: a complicated human Orf virus infection.

BACKGROUND: Orf virus (ORFV) is the pathogen responsible for Orf, a zoonotic viral infection that can be spread to humans from sheep and goats. Here, we present a case of human Orf complicated by an immune-related reaction, to raise awareness of this under-recognized disease avoiding unnecessary investigations and overtreatment. CASE REPORT: A 51-year-old woman with no previous medical history presented with a one-week history of three asymptomatic swelling nodules with a grey necrotic center and red outer halo on her index finger. At physical examination there was also a pruritic papulovesicular eruption on her hands and feet. She reported a recent contact with a goat which had a similar nodular lesion in its mouth. A biopsy of the lesions was performed and a diagnosis of Orf complicated by widespread erythema multiforme was made based on the clinical and histopathological features. The lesions spontaneously resolved within the next 2 weeks. CONCLUSIONS: Orf is not very prevalent in our region, so we performed a biopsy of the lesion to guide us toward a diagnosis. However, we should remember that the diagnosis of ecthyma relies on clinical evaluation and epidemiological criteria.

Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

Orf, a Human Parapoxvirus Infection.

Despite being common worldwide, parapoxvirus infections are regarded as neglected zoonoses because their incidence is either unknown or grossly overestimated. In ruminants all throughout the world, parapoxvirus produces oral lesions and infectious pustular dermatitis. The pathogen is typically spread directly via items contaminated with parapoxvirus and indirectly via a near contact with dermatological lesions that contain the virus on affected animals. Animals infected with the parapoxvirus typically exhibit no clinical symptoms, and the mode of parapoxvirus transmission is occasionally unclear. For accurate etiological diagnosis and appropriate therapy of patients affected by zoonotic infections, the significance of adopting a "One Health" approach and cross-sector collaboration between human and veterinary medicine should be emphasized. The causative pathogen of ecthyma contagiosum in general people is the orf virus, which mostly infects various animals, either pets or wildlife species. The illness primarily affects minute wild ruminants, sheep, cattle, deer, and goats, and it can spread to people through contact with infected animals or contaminated meats anywhere in the world. Taxonomically speaking, the virus belongs to the parapoxvirus genus. Thus pathogen can be detected from crusts for a very long period (several months to several years), and the virus is found to be resistant to inactivation with a hot or dry atmosphere. In immunocompetent individuals, the lesions often go away on their own with a period as long 2 months. Nevertheless, it necessitates the applying of diverse strategies, such as antiviral, immunological modulator, or modest surgical excisions in immunosuppressed patients. The interaction of the virus with various host populations aids in the development of a defense mechanism against the immune system. The parapoxvirus illness in humans is covered in this chapter. The orf illness, a significant known human parapoxvirus infection, is given specific attention.

Detection and genetic characterization of orf virus from sheep and goats in Nigeria.

Orf is a contagious, viral epitheliotropic disease of small ruminants. We investigated the molecular epidemiology of orf virus (ORFV) in breeds of small ruminants to determine the evolutionary diversity in Nigeria. Out of 54 small ruminants screened, the number of animals that were positive for ORFV in the three locations were 25. The distribution of positive animals by location were FCT 45.0% (n = 9/20), Oyo State 42.9% (6/14), and Plateau State 50.0% (n = 10/20). ORFV sequences from this study clustered with viruses detected in Taiwan, Iran, USA, and France. Our findings highlight the risk of transmission across geographic boundaries in Nigeria and West Africa, and reinforces the need for increased surveillance to prevent and control spread. Comprehensive characterization of ORFV in small ruminants as well as in humans in Nigeria is required to better elucidate the epidemiological dynamics and the virus evolution.

Orf Nodule with Erythema Multiforme during a Monkeypox Outbreak, France, 2022.

A 26-year-old patient in France who worked as a butcher sought care initially for erythema multiforme. Clinical examination revealed a nodule with a crusty center, which upon investigation appeared to be an orf nodule. Diagnosis was confirmed by PCR. The patient was not isolated and had a favorable outcome after basic wound care.

Deletion of gene OV132 attenuates Orf virus more effectively than gene OV112.

Orf virus (ORFV), a Parapoxvirus in Poxviridae, infects sheep and goats resulting in contagious pustular dermatitis. ORFV is regarded as a promising viral vector candidate for vaccine development and oncolytic virotherapy. Owing to their potential clinical application, safety concerns have become increasingly important. Deletion of either the OV132 (encoding vascular endothelial growth factor, VEGF) or OV112 (encoding the chemokine binding protein, CBP) genes reduced ORFV infectivity, which has been independently demonstrated in the NZ2 and NZ7 strains, respectively. This study revealed that the VEGF and CBP gene sequences of the local strain (TW/Hoping) shared a similarity of 47.01% with NZ2 and 90.56% with NZ7. Due to the high sequence divergence of these two immunoregulatory genes among orf viral strains, their contribution to the pathogenicity of Taiwanese ORFV isolates was comparatively characterized. Initially, two ORFV recombinants were generated, in which either the VEGF or CBP gene was deleted and replaced with the reporter gene EGFP. In vitro assays indicated that both the VEGF-deletion mutant ORFV-VEGFΔ-EGFP and the CBP deletion mutant ORFV-CBPΔ-EGFP were attenuated in cells. In particular, ORFV-VEGFΔ-EGFP significantly reduced plaque size and virus yield compared to ORFV-CBPΔ-EGFP and the wild-type control. Similarly, in vivo analysis revealed no virus yield in the goat skin biopsy infected by ORFV-VEGFΔ-EGFP, and significantly reduced the virus yield of ORFV-CBPΔ-EGFP relative to the wild-type control. These results confirmed the loss of virulence of both deletion mutants in the Hoping strain, whereas the VEGF-deletion mutant was more attenuated than the CBP deletion strain in both cell and goat models. KEY POINTS: • VEGF and CBP genes are crucial in ORFV pathogenesis in the TW/Hoping strain • The VEGF-deletion mutant virus was severely attenuated in both cell culture and animal models • Deletion mutant viruses are advantageous vectors for the development of vaccines and therapeutic regimens.

Recent advances in diagnostic approaches for orf virus.

Orf virus (ORFV), the prototype species of the Parapoxvirus genus, is an important zoonotic virus, causing great economic losses in livestock production. At present, there are no effective drugs for orf treatment. Therefore, it is crucial to develop accurate and rapid diagnostic approaches for ORFV. Over decades, various diagnostic methods have been established, including conventional methods such as virus isolation and electron microscopy; serological methods such as virus neutralization test (VNT), immunohistochemistry (IHC) assay, immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA); and molecular methods such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and recombinase-aided amplification (RAA) assay. This review provides an overview of currently available diagnostic approaches for ORFV and discusses their advantages and limitations and future perspectives, which would be significantly helpful for ORFV early diagnosis and surveillance to prevent outbreak of orf. KEY POINTS: • Orf virus emerged and reemerged in past years • Rapid and efficient diagnostic approaches are needed and critical for ORFV detection • Novel and sensitive diagnostic methods are required for ORFV detection.

Identification and molecular characterization of Orf virus infection in occupationally exposed women in South America.

Contagious Ecthyma (CE) is a severe exanthematous dermatitis caused by the Orf virus (ORFV) that mainly affects domestic small ruminants such as sheep and goats. It is a worldwide-distributed occupational zoonosis, particularly infecting those in close contact with animals or animal products such as shepherds, farmers and veterinarians, among others. In the present work, we report the first human CE case confirmed in Argentina. A phylogenetic analysis based on four gene sequences of the isolated strain responsible for the disease showed that this isolate grouped with other ORFV sequences that caused reported CE cases in sheep from the same Argentine province. We also sequenced a sample from a Chilean human case reported in 2017, whose phylogenetic analysis showed that it groups together with other Argentine isolates from locations close to the border with Chile.

Orf Virus ORF120 Protein Positively Regulates the NF-κB Pathway by Interacting with G3BP1.

Orf virus (ORFV) is a highly epitheliotropic parapoxvirus with zoonotic significance that induces proliferative lesions in the skin of sheep, goats, and humans. Several viral proteins carried by ORFV, including nuclear factor-κB (NF-κB) inhibitors, play important roles in hijacking host-associated proteins for viral evasion of the host innate immune response. However, the roles of proteins with unknown functions in viral replication and latent infection remain to be explored. Here, we present data demonstrating that the ORF120, an early-late ORFV-encoded protein, activates the NF-κB pathway in the early phase of infection, which implies that ORFV may regulate NF-κB through a biphasic mechanism. A DUAL membrane yeast two-hybrid system and coimmunoprecipitation experiments revealed that the ORF120 protein interacts with Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1). The overexpression of the ORF120 protein can efficiently increase the expression of G3BP1 and nuclear translocation of NF-κB-p65 in primary ovine fetal turbinate (OFTu) and HeLa cells. The knockdown of G3BP1 significantly decreased ORF120-induced NF-κB activation, indicating that G3BP1 is involved in ORF120-induced NF-κB pathway activation. A dual-luciferase reporter assay revealed that ORF120 could positively regulate the NF-κB pathway through the full-length G3BP1 or the domain of G3BP1RRM+RGG. In conclusion, we demonstrate, for the first time, that the ORF120 protein is capable of positively regulating NF-κB signaling by interacting with G3BP1, providing new insights into ORFV pathogenesis and a theoretical basis for antiviral drug design. IMPORTANCE As part of the host innate response, the nuclear factor-κB (NF-κB) pathway plays a partial antiviral role in nature by regulating the innate immune response. Thus, the NF-κB pathway is probably the most frequently targeted intracellular pathway for subversion by anti-immune modulators that are carried by a wide range of pathogens. Various viruses, including poxviruses, carry several proteins that prepare the host cell for viral replication by inhibiting cytoplasmic events, leading to the initiation of NF-κB transcriptional activity. However, NF-κB activity is hypothesized to facilitate viral replication to a great extent. The significance of our research is in the exploration of the activation mechanism of NF-κB induced by the Orf virus (ORFV) ORF120 protein interacting with G3BP1, which helps not only to explain the ability of ORFV to modulate the immune response through the positive regulation of NF-κB but also to show the mechanism by which the virus evades the host innate immune response.

Genetic analysis of two viroceptor genes of orf virus.

In the present study, we analyzed the chemokine-binding protein (CBP) and the GM-CSF/IL-2 inhibition factor (GIF) of orf virus (ORFV) isolates of sheep and goat origin from different geographical regions of India. Both are immunomodulatory proteins known for their unique strategy of establishing short-term immunity and re-infection in their host. The GIF gene is highly conserved, whereas the CBP gene is highly variable. Both the proteins have conserved potential N-glycosylation sites. The GIF protein contains the "WDPWV" motif responsible for receptor activation. In addition, the SUSHI/short consensus repeats (SCR) domain is reported for the first time in ORFV. Both proteins could potentially be used as immunotherapeutic agents in inflammatory diseases related to the overexpression of specific cytokines.

Recombination drives the emergence of orf virus diversity: evidence from the first complete genome sequence of an Indian orf virus isolate and comparative genomic analysis.

Contagious pustular dermatitis is a disease that primarily infects small ruminants and possesses zoonotic potential. It is caused by orf virus (ORFV), a member of the genus Parapoxvirus. In this study, we evaluated an ORFV outbreak in goats in Madhya Pradesh, a state in central India, during 2017. The transboundary potential of this virus was evaluated by constructing phylogenetic trees. The complete genome sequence of an ORFV isolate named Ind/MP/17 was found to be 139,807 bp in length with 63.7% GC content and 132 open reading frames (ORFs) flanked by 3,910-bp inverted terminal repeats (ITRs). An investigation into evolutionary parameters such as selection pressure (θ = dN/dS) and nucleotide diversity (π) demonstrated that ORFV has undergone purifying selection. A total of 40 recombination events were identified, 21 of which were evident in the Ind/MP/17 genome, indicating its ability to generate new variants.

Complete genomic sequences and comparative analysis of two Orf virus isolates from Guizhou Province and Jilin Province, China.

Orf virus (ORFV, species Orf virus) belongs to the typical species of the Parapoxvirus genus of the family Poxviridae, which infects sheep, goats, and humans with worldwide distribution. Although outbreaks of Orf have been reported sequentially in several Chinese provinces, the epidemiology of Orf and genetic diversity of ORFV strains still needs to be further characterized. To further reveal the genomic organization of the ORFV-GZ18 and ORFV-CL18 isolates, the complete genome sequences of two recently obtained ORFV isolates were sequenced using the next-generation sequencing technology and analyzed, which had been deposited in the GenBank database under accession number MN648218 and MN648219, respectively. The complete genomic sequence of ORFV-CL18 was 138,495 bp in length, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 3481 bp at both ends, which has genomic structure typical Parapoxviruses. The overall genomic organization of the fully sequenced genome of ORFV-GZ18 was consistent with ORFV-CL18 genome, with a complete genome size of 138,446 nucleotides, containing 131 ORFs flanked by ITRs of 3469 bp. Additionally, the overall G + C contents of ORFV-GZ18 and ORFV-CL18 genome sequences were about 63.9% and 63.8%, respectively. The phylogenetic analysis showed that both ORFV-GZ18 and ORFV-CL18 were genetically closely related to ORFV-SY17 derived from sheep. In summary, the complete genomic sequences of ORFV-GZ18 and ORFV-CL18 are reported, with the hope it will be useful to investigate the host range, geographic distribution, and genetic evolution of the virus in Southern West and Northern East China.

Solitary Subungual Orf.

Orf-ecthyma contagiosum-is an endemic cutaneous disease caused by parapoxvirus that is transmitted via direct contact with contagious animals. The lesions are located frequently on the hands and fingers. Subungual presentation of orf is very rare. We report a case of solitary subungual orf. Suspicious subungual nodular lesions may be cases of orf, especially in endemic areas. Orf disease should be considered in the differential diagnosis of subungual nodular lesions to avoid unnecessary surgical interventions.

Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan.

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408 bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

Orf progressiva: giant progressive and destructive infections in the immunocompromised.

Orf virus causes a self-limited infection in humans that resolves without scarring within 6-12 weeks. However, lesions in the immunocompromised can be progressive and disfiguring. The lesions frequently recur after treatment. To our knowledge, there are eleven published cases of these infections. We propose the name orf progressiva to call attention to this progressive, treatment-resistant entity. We present a 43-year-old male ranch owner with a history of renal transplantation who contracted an orf infection from his lamb. The infection recurred despite attempts at debridement, but achieved near complete resolution after treatment with imiquimod and valacyclovir. The histologic findings of orf progressiva are identical to the early stages of classic orf infection and are characterized by epithelial hyperplasia, intracytoplasmic eosinophilic inclusions, and an edematous, vascular dermis. There is no standard treatment for orf progressiva. Surgical excision has frequently resulted in rapid reoccurrence. Topical therapies such as imiquimod and cidofovir cream in combination with excision have been successful in some cases. Acyclovir or valacyclovir with imiquimod has been reported to be effective. Two patients achieved cure with imiquimod alone. We summarize these cases to prompt recognition of orf progressiva as a distinct clinical entity that requires treatment.

The genetic identification of camel contagious ecthyma virus as the causative agent of contagious ecthyma in dromedary camels (Camelus dromedarius) in Qatar.

Camel contagious ecthyma is a contagious viral disease of camels caused by either Orf virus (ORFV) or camel contagious ecthyma virus (CCEV). It has been previously reported and shown to cause economic losses in some camel-rearing countries in Asia and Africa, but has not been detected in Qatar. The purpose of this study was to identify and genetically characterize the contagious ecthyma causative agent in Qatari dromedary camels between 2017 and 2018. Accordingly, we made diagnoses of camel contagious ecthyma based on the clinical signs and genetic analysis of the entire major envelop protein (B2L) gene. The sequence analysis showed that CCEV was the infecting virus, and the B2L gene sequences were highly conserved between the locally infected camels with 100% similarity with isolates from Bahrain. This is the first study reporting the detection of CCEV in Qatar. We suggest that sequencing of the CCEV genome is necessary to determine the origin and relationship of this virus with other members of the parapoxvirus genus.

Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication.

Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine-to-inosine (A-to-I)-editing activity. Finally, the association with OV20.0 suppressed the A-to-I-editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.IMPORTANCE Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection.

Feast of Sacrifice and Orf, Milan, Italy, 2015-2018.

Orf (ecthyma contagiosum) is an infection of the skin caused by a DNA virus belonging to the genus Parapoxvirus. We recently observed 7 cases of orf in Muslim men living in the metropolitan area of Milan, Italy, who acquired the infection after the Feast of Sacrifice.

Human Infection with Orf Virus and Description of Its Whole Genome, France, 2017.

Zoonotic transmission of parapoxvirus from animals to humans has been reported; clinical manifestations are skin lesions on the fingers and hands after contact with infected animals. We report a human infection clinically suspected as being ecthyma contagiosum. The patient, a 65-year-old woman, had 3 nodules on her hands. She reported contact with a sheep during the Aïd-el-Fitr festival in France during 2017. We isolated the parapoxvirus orf virus from these nodules by using a nonconventional cell and sequenced the orf genome. We identified a novel orf virus genome and compared it with genomes of other orf viruses. More research is needed on the genus Parapoxvirus to understand worldwide distribution of and infection by orf virus, especially transmission between goats and sheep.