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.

Generation and application of immortalized sheep fetal fibroblast cell line.

BACKGROUND: Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure. RESULTS: In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility. CONCLUSIONS: Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.

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 virus as an adjuvant enhances the immune response to a PCV2 subunit vaccine.

Orf virus (ORFV), a member of the genus Parapoxvirus, possesses an excellent immune activation capability, which makes it a promising immunomodulation agent. In this study, we evaluated ORFV as a novel adjuvant to enhance the immune response of mice to a subunit vaccine using porcine circovirus type 2 (PCV2) capsid (Cap) protein as a model. Our results showed that both inactivated and live attenuated ORFV activated mouse bone marrow-derived dendritic cells and increased expression of immune-related cytokines interleukin (IL)-1ß, IL-6, and TNF-α. Enhanced humoral and cellular immune responses were induced in mice immunized with PCV2 Cap protein combined with inactivated or live attenuated ORFV adjuvant compared with the aluminum adjuvant. Increased secretion of Th1 and Th2 cytokines by splenic lymphocytes in immunized mice further indicated that the ORFV adjuvant promoted a mixed Th1/Th2 immune response. Moreover, addition of the ORFV adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the spleen and lungs of PCV2-challenged mice and prevented pathological changes in lungs. This study demonstrates that ORFV enhances the immunogenicity of a PCV2 subunit vaccine by improving the adaptive immune response, suggesting the potential application of ORFV as a novel adjuvant.

Immunogenicity of chimeric hemagglutinins delivered by an orf virus vector platform against swine influenza virus.

Orf virus (ORFV) is a large DNA virus that can harbor and efficiently deliver viral antigens in swine. Here we used ORFV as a vector platform to deliver chimeric hemagglutinins (HA) of Influenza A virus of swine (IAV-S). Vaccine development against IAV-S faces limitations posed by strain-specific immunity and the antigenic diversity of the IAV-S strains circulating in the field. A promising alternative aiming at re-directing immune responses on conserved epitopes of the stalk segment of the hemagglutinin (HA2) has recently emerged. Sequential immunization with chimeric HAs comprising the same stalk but distinct exotic head domains can potentially induce cross-reactive immune responses against conserved epitopes of the HA2 while breaking the immunodominance of the head domain (HA1). Here, we generated two recombinant ORFVs expressing chimeric HAs encoding the stalk region of a contemporary H1N1 IAV-S strain and exotic heads derived from either H6 or H8 subtypes, ORFVΔ121cH6/1 and ORFVΔ121cH8/1, respectively. The resulting recombinant viruses were able to express the heterologous protein in vitro. Further, the immunogenicity and cross-protection of these vaccine candidates were assessed in swine after sequential intramuscular immunization with OV-cH6/1 and OV-cH8/1, and subsequent challenge with divergent IAV-S strains. Humoral responses showed that vaccinated piglets presented increasing IgG responses in sera. Additionally, cross-reactive IgG and IgA antibody responses elicited by immunization were detected in sera and bronchoalveolar lavage (BAL), respectively, by ELISA against different viral clades and a diverse range of contemporary H1N1 IAV-S strains, indicating induction of humoral and mucosal immunity in vaccinated animals. Importantly, viral shedding was reduced in nasal swabs from vaccinated piglets after intranasal challenge with either Oh07 (gamma clade) or Ca09 (npdm clade) IAV-S strains. These results demonstrated the efficiency of ORFV-based vectors in delivering chimeric IAV-S HA-based vaccine candidates and underline the potential use of chimeric-HAs for prevention and control of influenza in swine.

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.

Use of a local anaesthetic and antiseptic wound formulation for the treatment of lambs naturally infected with Orf virus.

Contagious ecthyma (CE) is a worldwide highly contagious zoonotic viral skin disease of sheep and goats. Treatment for Orf virus (ORFV) infection usually involves topical and oral antibiotics. An anaesthetic and antiseptic topical gel (Multisolfen® or Tri-Solfen®; MS®, Medical Ethics, Australia) has been documented as an efficacious therapy for lesions from mucosal and epithelial viral infections in ruminants. The present study tested a new treatment protocol of MS® for CE therapy on-farm in 150 lambs naturally infected with ORFV. Lambs were divided into three cohorts of 50 lambs each (C, D and E). Cohort C was treated with MS® 3 times with an interval of 3 days between treatments, cohort D was treated daily with hypochlorous acid, whilst cohort E served as untreated controls. The lambs were examined clinically every two days, weight measured weekly, with whole blood and sterile swabs from ORFV lesions collected for haematological analysis and specific ORFV PCR. Cohort C presented fewer lambs displaying ORFV-associated lesions than other cohorts at different times of the experiment. Further, lesions treated with MS® were milder compared with other cohorts. However, following cessation of therapy, most of the lambs again developed ORFV-associated lesions. No differences between cohorts were observed in weight, haematological and PCR results. These findings suggest that topical treatment with MS® is effective for CE in field conditions, especially in the first stages of the clinical course, although treatment with MS® may need to be extended a minimum of 4 weeks.

Development of an indirect ELISA against Orf virus using two recombinant antigens, partial B2L and F1L.

Orf is a highly contagious viral disease affecting goats and sheep. It is caused by Orf virus (ORFV) and has caused severe economic losses to the global goat industry, including in China. In this study, an indirect ELISA method for recombinant proteins based on truncated dominant antigenic epitopes of B2L and F1L genes of ORFV was established. A series of conditions and its performance were comprehensively evaluated. The optimized ELISA reaction conditions were: the optimal coating amount of antigen was 0.25 µg/mL, 5% skim milk powder was closed for 1 h, the optimal dilution of serum was 1:200, the optimal incubation time of the rabbit anti-goat IgG was 1:8000, the optimal color development time of TMB was 15 mins, and the threshold value of negative-positive was 0.358. The method specifically detects anti-ORFV antibodies and does not cross-react with positive sera for other common goat pathogenic bacteria antiserum. ORFV-positive sera were still positive after 1:512 dilution, with intra-batch coefficient of variation (CV) between 7.1% and 9.5% and inter-batch CV between 5.0% and 7.6%; 51% (92/180) of immunized goat serum samples were tested positive and 14.44% (14/63) of non-immunized goat serum samples were positive. The results show that the indirect ELISA antibody assay established in this study has good specificity, sensitivity and reproducibility, and provides a technical tool for clinical ORFV serum antibody detection and epidemiological investigation.

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.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.

An evaluation of the ecological niche of Orf virus (Poxviridae): Challenges of distinguishing broad niches from no niches.

Contagious ecthyma is a skin disease, caused by Orf virus, creating great economic threats to livestock farming worldwide. Zoonotic potential of this disease has gained recent attention owing to the re-emergence of disease in several parts of the world. Increased public health concern emphasizes the need for a predictive understanding of the geographic distributional potential of Orf virus. Here, we mapped the current distribution using occurrence records, and estimated the ecological niche in both geographical and environmental spaces. Twenty modeling experiments, resulting from two- and three-partition models, were performed to choose the candidate models that best represent the geographic distributional potential of Orf virus. For all of our models, it was possible to reject the null hypothesis of predictive performance no better than random expectations. However, statistical significance must be accompanied by sufficiently good predictive performance if a model is to be useful. In our case, omission of known distribution of the virus was noticed in all Maxent models, indicating inferior quality of our models. This conclusion was further confirmed by the independent final evaluation, using occurrence records sourced from the Centre for Agriculture and Bioscience International. Minimum volume ellipsoid (MVE) models indicated the broad range of environmental conditions under which Orf virus infections are found. The excluded climatic conditions from MVEs could not be considered as unsuitable owing to the broad distribution of Orf virus. These results suggest two possibilities: that the niche models fail to identify niche limits that constrain the virus, or that the virus has no detectable niche, as it can be found throughout the geographic distributions of its hosts. This potential limitation of component-based pathogen-only ENMs is discussed in detail.

The Global Evolutionary History of Orf Virus in Sheep and Goats Revealed by Whole Genomes Data.

Orf virus (ORFV) belongs to the genus Parapoxvirus (Poxviridae family). It is the causative agent of contagious ecthyma (CE) that is an economically detrimental disease affecting small ruminants globally. Contagious ecthyma outbreaks are usually reported in intensive breeding of sheep and goats but they have also been reported in wildlife species. Notably, ORFV can infect humans, leading to a zoonotic disease. This study aims to elucidate the global evolutionary history of ORFV genomes in sheep and goats, including the first genomes from Central America in the analyses. In comparison to the last study on ORFV whole genomes, the database now includes 11 more sheep and goat genomes, representing an increase of 42%. The analysis of such a broader database made it possible to obtain a fine molecular dating of the coalescent time for ORFV S and G genomes, further highlighting the genetic structuring between sheep and goat genomes and corroborating their emergence in the latter half of 20th century.

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.

Contagious ecthyma in Egypt: Clinical, virological and molecular explorations.

Contagious Ecthyma (CE) is a highly contagious viral disease of sheep and goats with worldwide distribution. The present study aimed to provide a clinical description of contagious ecthyma in four sheep flocks and screen the possible genetic variation in the B2L gene of the detected isolates. Oral lesions were collected and inoculated into chorioallantoic membrane (CAM) of 11 days embryonated chicken eggs. Polymerase chain reaction and direct sequencing of the B2L gene was conducted. Infected sheep exhibited anorexia with a development of nodular lesions evolving in proliferative thick scabs around oral commissures. The inoculated CAM showed small-sized white pock lesions accompanied with thickening of CAM. The partial length of B2L gene (592 bp) was successfully amplified in samples collected from four flocks. The isolated strains belong to genotype I/I and I/II. Sequence and evolutionary analysis illustrate that B2L gene (ORF011) are highly conserved among Orf viruses isolated from different countries.

Dual analysis of wild-type and attenuated Orf virus and host cell transcriptomes revealed novel virus-host cell interactions.

IMPORTANCE: Currently, the only available commercial vaccines for Orf virus (ORFV) are live attenuated vaccines, which present a potential risk of reversion to virulence. Therefore, understanding the pathogenic mechanisms of different virulent strains of ORFV and host immune responses triggered by these viruses is crucial for developing new vaccines and interventions. In this study, we found that the attenuated strain downregulates the host innate immune response and antiviral activity. In addition, we noted that the wild-type strain can induce the immune response pattern centered on interferon-stimulated genes and interferon regulatory factor gene family. We predicted that STAT1 and STAT2 are the main transcription factors upstream of target gene promoters through gene regulatory networks and exert significant regulatory effects on co-expressed genes. Our study elucidated the complex interaction between ORFV strains and host cell immune responses, providing new insights into vaccine research for ORFV.

The parapoxvirus Orf virus inhibits dsDNA-mediated type I IFN expression via STING-dependent and STING-independent signalling pathways.

Type I interferons (IFNs) are critical in the host defence against viruses. They induce hundreds of interferon-stimulated genes (ISGs) many of which have an antiviral role. Poxviruses induce IFNs via their pathogen-associated molecular patterns, in particular, their genomic DNA. In a majority of cell types, dsDNA is detected by a range of cytoplasmic DNA sensors that mediate type I IFN expression via stimulator of interferon genes (STING). Orf virus (ORFV) induces cutaneous pustular skin lesions and is the type species of the Parapoxvirus genus within the Poxviridae family. The aim of this study was to investigate whether ORFV modulates dsDNA-induced type I IFN expression via STING-dependent signalling pathways in human dermal fibroblasts (hNDF) and THP-1 cells. We showed that ORFV infection of these cell types treated with poly(dA:dT) resulted in strong inhibition of expression of IFN-ß. In hNDFs, we showed using siRNA knock-down that STING was essential for type I IFN induction. IFN-ß expression was further reduced when both STING and RIG-I were knocked down. In addition, HEK293 cells that do not express STING or Toll-like receptors also produce IFN-ß following stimulation with poly(dA:dT). The 5' triphosphate dsRNA produced by RNA polymerase III specifically results in the induction of type I IFNs through the RIG-I receptor. We showed that ORFV infection resulted in strong inhibition of IFN-ß expression in HEK293 cells stimulated with poly(dA:dT). Overall, this study shows that ORFV potently counteracts the STING-dependent and STING-independent IFN response by antagonizing dsDNA-activated IFN signalling pathways.

Poxvirus sensitivity of a novel diploid sheep embryonic heart cell line.

Lumpy skin disease virus (LSDV), camelpox virus (CPV), and orf virus (ORFV) are members of the family Poxviridae. These viruses are usually isolated or produced in embryonated eggs or primary cells because continuous cell lines are less sensitive to infection. Disadvantages of the use of eggs or primary cells include limited availability, potential endogenous contaminants, and a limited ability to perform multiple passages. In this study, we developed a diploid cell culture from sheep embryonic hearts (EHs) and demonstrated its high proliferative and long-term storage capacities. In addition, we demonstrated its sensitivity to representatives of three genera of the family Poxviridae: Capripoxvirus (LSDV), Orthopoxvirus (CPV), and Parapoxvirus (ORFV). The cell culture had a doubling time of 24 h and reached 40 passages with satisfactory yield. This is comparable to that observed in primary lamb testis (LT) cells at passage 5 (P5). After infection, each poxvirus titer was 7.0-7.6 log TCID50/mL for up to five passages and approximately 6.8, 6.4, and 5.6 for the three viruses at P6-P25, P30, and P40, respectively. The sensitivity of sheep EH cells to poxvirus infection did not decrease after long-term storage in liquid nitrogen and was higher than that of primary LT cells, which are used for capripoxvirus and parapoxvirus detection and growth, and Vero cells, which are used for orthopoxvirus detection and growth. Thus, EH diploid cells are useful for poxvirus isolation and production without embryonated eggs or primary cells.

ORFV entry into host cells via clathrin-mediated endocytosis and macropinocytosis.

Orf virus (ORFV), also known as infectious pustular virus, leads to an acute contagious zoonotic infectious disease. ORFV can directly contact and infect epithelial cells of skin and mucosa, causing damage to tissue cells. So far, the pathway of ORFV entry into cells is unclear. Therefore, finding the internalization pathway of ORFV will help to elucidate the cellular and molecular mechanisms of ORFV infection and invasion, which in turn will provide a certain reference for the prevention and treatment of ORFV. In the present study, chemical inhibitors were used to analyze the mechanism of ORFV entry into target cells. The results showed that the inhibitor of clathrin-mediated endocytosis could inhibit ORFV entry into cells. However, the inhibitor of caveolae-mediated endocytosis cannot inhibit ORFV entry into cells. In addition, inhibition of macropinocytosis pathway also significantly reduced ORFV internalization. Furthermore, the inhibitors of acidification and dynamin also prevented ORFV entry. However, results demonstrated that inhibitors inhibited ORFV entry but did not inhibit ORFV binding. Notably, extracellular trypsin promoted ORFV entry into cells directly, even when the endocytic pathway was inhibited. In conclusion, ORFV enters into its target cells by clathrin-mediated endocytosis and macropinocytosis, while caveolae-dependent endocytosis has little effects on this process. In addition, the entry into target cells by ORFV required an acid environment and the effect of dynamin. Meanwhile, we emphasize that broad-spectrum antiviral inhibitors and extracellular enzyme inhibitors are likely to be effective strategies for the prevention and treatment of ORFV infection.

Recombinant Orf virus induced antibody production against capsid protein of porcine circovirus type 3 in mice.

The emerging worldwide distributed porcine circovirus type 3 (PCV3) infection poses a serious threat to swine herds. An important means of preventing and controlling PCV3 infection is the development of the vaccine, while, the inability to cultivate in vitro has become the biggest obstacle. Orf virus (ORFV), the prototypic member of the Parapoxviridae, has been proven to be a novel valid vaccine vector for preparing various candidate vaccines. Here, recombinant ORFV expressing capsid protein (Cap) of PCV3 was obtained and proved its favorable immunogenicity inducing antibody against Cap in BALB/c mice. Based on the enhanced green fluorescent protein (EGFP) as a selectable marker, the recombinant rORFVΔ132-PCV3Cap-EGFP was generated. Then, recombinant ORFV expressing Cap only, rORFVΔ132-PCV3Cap, was obtained based on rORFVΔ132-PCV3Cap-EGFP using a double homologous recombination method by screening single non-fluorescent virus plaque. Results of the western blot showed that the Cap can be detected in rORFVΔ132-PCV3Cap infected OFTu cells. The results of immune experiments in BALB/c mice indicated that a specific antibody against Cap of PCV3 in serum was induced by rORFVΔ132-PCV3Cap infection. The results presented here provide a candidate vaccine against PCV3 and a feasible technical platform for vaccine development based on ORFV.