Contagious Pustular Dermatitis Research Articles

Immunomodulatory effect of tibetan medicine compound extracts against ORFV in vitro by metabolomics

Ovine contagious pustular dermatitis (ORF) is one of the main diseases of sheep and is a zoonotic disease caused by Ovine contagious pustular dermatitis virus (ORFV) infection, posing a significant constraint on sheep breeding industry and human health. The Tibetan medical formulation composed of Polygonum leucoides, Polygonum xanthoxylum and Acanthophora rotunda significantly regulated lymphocyte immune function following ORFV stimulation, although the mechanism remains unclear. In order to study the immunomodulatory effects and mechanism of three Tibetan medicinal extracts (Polygonum leucoides, Polygonum xanthoxylum, and Acanthophora rotunda) against ORFV in vitro, sheep peripheral blood lymphocytes were isolated in vitro and treated with different concentrations of Tibetan medicine compound extract solution after ORFV infection. The cytokine expression levels in lymphocytes were measured at 4 h, 8 h and 12 h. Additionally endogenous metabolites in lymphocytes at 0 h, 4 h, 8 h and 12 h were quantified by untargeted metabolomics method. The results showed that, the extracts could regulate the lymphocyte immune factors altered by ORFV, and regulate the lymphocyte immune function through cysteine and methionine metabolic pathways as well as the pyrimidine metabolic pathways, potentially alleviating the immune evasion induced by ORFV.

Genistein is effective in inhibiting Orf virus infection in vitro by targeting viral RNA polymerase subunit RPO30 protein.

Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.

A novel triplex real-time PCR assay for the differentiation of lumpy skin disease virus, goatpox virus, and sheeppox virus.

Three members of Capripoxvirus (CaPV) genus, including lumpy skin disease virus (LSDV), goatpox virus (GTPV), and sheeppox virus (SPPV), are mentioned as notifiable forms by World Organization for Animal Health. These viruses have negatively impacted ruminant farming industry worldwide, causing great economic losses. Although SPPV and GTPV cause more severe clinical disease in only one animal species, they can transfer between sheep and goats. Both homologous and heterologous immunization strategies are used to protect animals against CaPVs. However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of CaPV infection. Therefore, we developed a novel triplex real-time PCR (qPCR) for the differentiation of LSDV, GTPV, and SPPV. Universal primers were designed to detect pan-CaPV sequences. Species-specific minor groove binder (MGB)-based probes were designed, which were labeled with FAM for LSDV, HEX for GTPV, and ROX for SPPV. The sensitivity, specificity, reproducibility, and ability of detecting mixed infections were evaluated for the triplex qPCR. Further, 226 clinical samples of the infection and negative controls were subjected to the triplex qPCR, and the results were verified using PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods for PRO30 gene. The triplex qPCR could successfully distinguish LSDV, GTPV, and SPPV in one reaction, and the assay sensitivity was 5.41, 27.70, and 17.28 copies/μL, respectively. No cross-reactivity was observed with other viruses causing common ruminant diseases, including des petits ruminants virus, foot-and-mouth disease virus, bluetongue virus, ovine contagious pustular dermatitis virus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea-mucosal disease virus. Inter-and intra-assay variabilities were < 2.5%. The results indicated that the triplex qPCR was highly specific, sensitive, and reproducible. Simulation experiments revealed that this assay could successfully distinguish two or three viruses in case of mixed infections without any cross-reaction. For clinical samples, the results were completely consistent with the results of PCR-RFLP and sequencing. This demonstrated that the assay was reliable for clinical application. The triplex qPCR is a robust, rapid, and simple tool for identifying various types of CaPV as it can successfully distinguish LSDV, GTPV, and SPPV in one reaction. Furthermore, the assay can facilitate more accurate disease diagnosis and surveillance for better control of CaPV infection.

Molecular Characterization of Contagious Pustular Dermatitis Virus in Goats of Sri Lanka

Molecular Characterization of Contagious Pustular Dermatitis Virus in Goats of Sri Lanka

Rapid Onsite Visual Detection of Orf Virus Using a Recombinase-Aided Amplification Assay.

Orf is an important zoonotic disease caused by the Orf virus (ORFV) which can cause contagious pustular dermatitis in goats and sheep. Orf is widespread in most sheep-raising countries in the world, causing huge economic losses. Although diagnostic methods for ORFV infection already exist, it is still necessary to develop a time-saving, labor-saving, specific, low-cost and visual diagnostic method for rapid detection of ORFV in the field and application in grassroots laboratories. This study establishes a DNA extraction-free, real-time, visual recombinase-aided amplification (RAA) method for the rapid detection of ORFV. This method is specific to ORFV and does not cross-react with other common DNA viruses. The detection limits of the real-time RAA and visual judgment of the RAA assay at 95% probability were 13 and 21 copies per reaction for ORFV, respectively. Compared with qPCR, the sensitivity and specificity of the real-time RAA assay were 100%, and those of the visual RAA assay were 92.31% and 100.0%, respectively. The DNA extraction-free visual detection method of RAA established in this study can meet the needs of rapid onsite detection and grassroots laboratories and has important reference value and significance for the early diagnosis of diseased animals.

Immune Response, Gene sequence to Contagious Ecthyma Virus (Orfv) Infected Lambs in Diyala Governorate, Iraq

Contagious ecyhyma Known (Orf, contagious pustular dermatitis, sore mouth, scabby mouth) a disease spreads easily and brought by the epitheliotropic. It’s endemic around the world and has a serious economic impact. This study aimed to understand the immune response mechanisms of the lambs to defense against Contagious ecthyma virus and molecular detection of the virus with the determining the sequence and mutant of the viral genome to 91 lambs (41 has clinical signs of Orfv and 50 control do not have clinical sign of Orfv) for the period extended from September 2022 to April 2023. Blood samples were collected from all above groups to apply for detection the level of IgM, IgG, IL-10, and IFN-γ of Orfv by ELISA test. Positive Orfv IgM was 75.6% infected lamb and 72% none infected, and positive Orfv IgG was 90.2% infected lamb and 88% none infected. The result was showed the level of IL-10 a significant increasing, in all Lambs positive IgM and IgG compared with control group. IFN-γ level showed non significant differences between all Lambs positive IgM and IgG compared with control group. Molecular detection of Orfv result was confirmed the positivity of all samples positive Orfv IgM. And the sequencing of the Orfv DNA showed a mutant in the some Allel compared with References strain registered at NCBI. Concluded: high prevalence of Orfv among lambs and happened mutants to the viral genome increased the virus’s virulence by affecting the immune response.

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.

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.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-022-05443-5.

Possibility of mechanical transmission of parapoxvirus by houseflies (Musca domestica) on cattle and sheep farms.

Parapoxvirus (PPV) causes papular stomatitis and contagious pustular dermatitis in ruminants worldwide. The virus is generally transmitted through close contact with skin lesions containing PPV in infected animals and indirectly through PPV-contaminated materials. PPV-infected animals frequently do not show clinical signs and the route of PPV transmission is sometimes unclear. In this study, the possibility of mechanical transmission of PPV by houseflies (Musca domestica) was investigated using polymerase chain reaction (PCR) gene surveillance. Samples were collected from cattle, sheep, barn environments, direct wash solution of the body surface of houseflies, and indirect wash solution of the body surface and feces of the flies. Bovine papular stomatitis virus, pseudocowpox virus, and orf virus were detected in the oral cavity and body surface of cattle and sheep without clinical signs of PPV infection or barn environments; PPV was considered to have been retained on the farm. PPVs were also detected in the direct wash solution of the body surface of houseflies, and the indirect wash solution of the body surface and feces of the flies. The viral sequence determined from the indirect wash solution of the body surface and feces of the flies was identical to that determined from the body surface of cattle and barns. These results suggested that houseflies may mechanically transmit PPV to both cattle and sheep.

СОСТОЯНИЕ КОЖИ ПРИ ЭКСПЕРИМЕНТАЛЬНОМ ЗАРАЖЕНИИ ОВЕЦ КОНТАГИОЗНЫМ ДЕРМАТИТОМ

As a result of the study, it was found that with contagious pustular dermatitis, the damage affects the epidermis and dermis of the skin. The most severe damage is recorded in the epidermis on day 9, fragments of organelles and viroplast clusters are observed in the cytoplasm. On the 22nd day, regenerative and regenerative processes prevail in the epidermis and dermis, in the form of proliferation, cell differentiation and organization of intercellular substance.

Evolution of humoral immunity effectors in four flocks of sheep with symptoms of contagious ecthyma, from Santău village, Romania

Contagious ecthyma or contagious pustular dermatitis is a debilitating condition located on the skin of sheep and goats, with a zoonotic character. Contagious ecthyma is a disease with economic and animal welfare implications, rarely being fatal if the host organism suffers from immunosuppression or if secondary infections occur. Similar to other members of the Poxviridae family, immunity to contagious ecthyma is mediated by both innate and adaptive immune responses. Following the invasion of the contagious ecthyma virus, immunoglobulins are mobilized that specifically bind to the contagious ecthyma virus to form immune complexes, which are then eliminated by defense system to protect tissues from damage. The level of circulating immune complexes and immunoglobulins can be quantified and can be an indicator of the stage of infection. In the present study we aimed to investigate whether there is a link between the individual characteristics (sex, age, severity of clinical signs) of sheep with orf symptoms and the evolution of circulating immune complexes and total immunoglobulins. In addition to these investigations, we aimed to follow the evolution of these immunological parameters compared to the evolution in clinically healthy animals. Investigations of the serum revealed that levels of these two parameters (total Ig and CIC), can be influenced by the individual characteristics of sheep affected by the contagious ectima and shows differences compared to clinically healthy animals.

Orf Virus VIR Antagonizes p53-Mediated Antiviral Effects to Facilitate Viral Replication.

As a zoonotic disease, ovine contagious pustular dermatitis (Orf) is a serious threat to sheep as well as humans. Orf virus (ORFV) interferon resistance protein (VIR) is the principal virulence protein that encodes a dsRNA-binding protein to inhibit host antiviral response. p53 is one of the key proteins of the host antiviral innate immunity. It not only enhances type I interferon secretion but also induces apoptosis in infected cells, and plays a crucial role in the immune response against various viral infections. However, it remains to be elucidated what role p53 plays in ORFV replication and whether ORFV's own protein VIR regulates p53 expression to promote self-replication. In this study, we showed that p53 has an antiviral effect on ORFV and can inhibit ORFV replication. In addition, ORFV nonstructural protein VIR interacts with p53 and degrades p53, which inhibits p53-mediated positive regulation of downstream antiviral genes. This study provides new insight into the immune evasion mediated by ORFV and identifies VIR as an antagonistic factor for ORFV to evade the antiviral response.

A Study on Therapeutic Management, Prevention and Control of Contagious Pustular Dermatitis in Teddy Goat Herd in Pakistan

A Study on Therapeutic Management, Prevention and Control of Contagious Pustular Dermatitis in Teddy Goat Herd in Pakistan

Identification and phylogenetic analysis of an orf virus strain isolated in Anhui Province, East-central China, in 2018.

Orf, also called contagious ecthyma or contagious pustular dermatitis, is a significant zoonotic disease that primarily affects goat and sheep globally. Currently, the infection by orf virus (ORFV) has been observed in different host species worldwide, including China. Here, a suspected outbreak of orf infection in a goat farm in Anhui Province in 2018 was investigated. Through PCR, electron microscopy, and cell culture techniques, we confirmed that the outbreak was caused by ORFV. Consequently, the orf virus strain was named the AH/LA/2018 strain. The amplified and sequenced ORFV011 (B2L) and ORFV059 (F1L) genes were used to construct phylogenetic trees to elucidate the genetic characteristics of the ORFV and the molecular epidemiology of orf. The present study is the first systematic evolution analysis of the ORFV strain isolated in Anhui Province. The results of this study will be helpful to better understand the characteristics of ORFV, to help prevent and control the transmission of ORFV at an early stage in China. Keywords: Anhui Province; goat; orf virus; phylogenetic analysis.

Characteristics analysis of Orf virus from goats to human

Contagious pustular dermatitis (orf) is an exanthemous disease affecting sheep and goats primarily. As a zoonotic infectious disease caused by parapoxvirus, orf should be managed not only in animals but also in humans. In this study, the typical orf clinical symptoms in goat and humans were observed. Human and goat samples were drawn. The Orf virus (orfv) was identified using an electron microscope, and PCR was used to amplify the target for B2L gene sequence. Molecular analysis of other B2L gene sequences downloaded from GenBank was performed by Mega4 soft. Results indicated that a 21-year old girl who worked in a goat farmcontacted orfv infection from infected goats directly. Eleven amino acid (AA) mutations were detected in the goat orfv transmitted to human orfv. Phylogenetic analysis showed that human orfv in this study was closely related genetically to FJ-SJ2 (KC568397), which was isolated from Fujian province 2012. The results facilitate the development of programs to control Orf virus infections not only in goats but also in humans.

Pathogenicity of blood orf virus isolates in the development of dairy goat contagious pustular dermatitis

Contagious pustular dermatitis is an exanthematous zoonotic disease caused by the orf virus. Pandemic outbreaks of this disease cause great economic losses, while the pathogenesis of this disease still remains obscure. In this study, blood samples were collected from 628 asymptomatic goats across China for PCR-based virus detection. We detected the orf virus in the blood of asymptomatic goats. Moreover, the orf virus obtained from the blood of infected goats was infectious and induced typical symptoms of contagious pustular dermatitis after inoculation of uninfected dairy goats. In summary, our data provide evidence that asymptomatic animals may be carriers of orf virus. Our findings should contribute to elucidating the details underlying the pathogenesis of contagious pustular dermatitis.

Identification of host cellular proteins LAGE3 and IGFBP6 that interact with orf virus protein ORFV024

ObjectiveOrf virus (ORFV) is the pathogen causing contagious pustular dermatitis in goats, sheep and herdsmen. Evidence has confirmed that ORFV can be used as a preventive and therapeutic immunomodulatory agent in several animal models. Our previous data demonstrated that ORFV024 is able to inhibit activation of the NF-κB signaling pathway and act as an important modulator for early immune responses against viral infection. However, the molecular mechanism by which ORFV024 exerting biological function remains unclear. In the present study, we explored and analyzed the function of host cellular proteins that interact with ORFV024. MethodsThe yeast two-hybrid (Y2H) assay was performed to screen proteins interacting with ORFV024 using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). Two of the screened proteins were further confirmed by confocal microscopy, His-tag pull-down assay and CO-Immunoprecipitation (CO-IP) assay. In addition, the ORFV024 interaction network was constructed using the STRING database. ResultsIn this study, 11 ovine cellular proteins were found to interact with ORFV024. In view of the importance of LAGE3 and IGFBP6 in the ORFV024 functional analysis, we further constructed LAGE3 and IGFBP6 interaction networks. The interactions between ORFV024 and LAGE3 or IGFBP6 were confirmed by confocal microscopy, LAGE3 was further confirmed in the His-tag pull-down assay and CO-IP assay. ConclusionsOur findings indicate that ORFV024 can interact with ovine cellular proteins LAGE3 and IGFBP6.

Detection of Parapoxvirus in goats during contagious ecthyma outbreak in Ceará State, Brazil by transmission electron microscopy techniques

Contagious ecthyma or contagious pustular dermatitis, is a viral skin disease that occurs in sheep, goats and wild ruminants and is characterized by the formation of papules, nodules or vesicles that progress into thick crusts or heavy scabs on the lips, gingiva and tongue, caused by a member of the Parapoxvirus genus. Humans are occasionally affected constituting important zoonosis. The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfare. In this study, a contagious ecthyma outbreak which occurred in one flock with 90 goats located in the Ceara State, Brazil, was described. Twenty-two goats older than 6 months were affected. The animals presented crusted lesions on the buccal region, tongue, udder and teats, which began with swelling in the mouth area. Dried crusts and serum collected were processed for transmission electron microscopy utilizing, negative staining (rapid preparation), Immunocytochemistry (immunolabelling with colloidal gold particles) and resin embedding techniques. At the Philips EM 208 transmission electron microscopy all the samples were analyzed by negative staining technique and a great number of parapoxvirus particles ovoid or cylindrical, showing two morphological forms, a mulberry (M) form with a distinctive crisscross filament pattern derived from the superimposition of upper and lower virion surfaces and a capsular (C) form caused by stain penetration and distention of the virion core, measuring 300 x 180 nm was observed. Antigen antibody reaction was increased by the colloidal gold particles. In the ultrathin sections of crusts, we verified the presence of three types of intracytoplasmic inclusion bodies, type A or Bollinger inclusion bodies, outlined by membrane, presented in it is interior, oval, mature or complete viral particles, measuring on the average, 225nm x 130 nm, showing an inner dumbell-shaped core, two lateral bodies and an external envelope, or cigar shaped core. In the type B electron dense inclusions bodies, were visualized parapoxvirus particles budding of dense and amorphous material. Fibrillar intracytoplasmic inclusions were also found located between the virions, consisting of groups of fibrils, arranged in groups or concentrically in the middle of the granular material. Intracytoplasmic vesicles outlined by membranes, measuring 560 x 420 nm, containing granular material in its interior were also observed. The nuclei showed an aspect deformed.

Association of two clusters of Orf virus isolates in outbreaks of infection in goat in the Central Anatolian region of Turkey.

Orf virus (ORFV) is the etiological agent of contagious pustular dermatitis and can cause skin disease in sheep and goats. In this study, two outbreaks of ORFV infection in goats in the Central Anatolian region of Turkey were investigated. Samples were collected from 1- to 4-month-old kids (n=9) in two different flocks in the Aksaray and Konya Provinces during the months of March and May 2016. The presence of ORFV in suspected samples was confirmed by PCR using primers specific to envelope gene (B2L). The analysis of the B2L gene sequences revealed that the nucleotide homology between the two isolates in the present study was 100%, whereas the similarity with Parapoxvirus isolates from different regions ranged from 83.6 to 99%. Phylogenetic analysis of the B2L gene revealed that there are two main clusters of ORFV isolates which were responsible for past outbreaks in Turkey. The information presented here will provide an insight into genetic diversity of field isolates of ORFV circulating in the Central Anatolian region of Turkey.

Phylogenetic Analysis of ORF Virus from Goats in Tanzania: Short Communication

ORF virus is a double-stranded DNA virus belonging to the genus Parapoxvirus (PPV) in the family poxviridae. ORF virus is the causative agent of contagious pustular dermatitis which is a zoonotic and neglected disease of humans and small ruminants. It causes severe exanthematous dermatitis that afflicts domestic and small wild ruminants. Cases of ORFV infections in goats in Tanzania have been reported for many years. The basis of reporting ORFV cases were mainly on clinical signs with no confirmatory tests (Ministry of Livestock Development reports-Tanzania). In this study, a case of proliferative dermatitis in goats was confirmed using PCR. A physical examination of the animal was carried out and tissue scrapings were collected for molecular analysis in the laboratory. The presence of ORFV in tissue scrapings from the lips was tested by GIF/IL-2gene polymerase chain reaction (PCR), DNA sequencing and phylogenetic analysis of the GIF/IL-2 gene. The results of this study indicated that the disease was caused by infection with a virus which is closely and genetically related to several ORF virus found in the data base. This is the first report to provide phylogenetic information about the ORF virus in Tanzania, which will be useful in prospective public health studies.