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.

The orf virus (ORFV) protein OV20.0 interacts with the microprocessor complex subunit DGCR8 to regulate miRNA biogenesis and ORFV infection.

Cellular double-stranded RNA-binding proteins (DRBPs) play important roles in the regulation of innate immune responses and microRNA (miRNA) biogenesis. The current study aimed to understand whether OV20.0, a DRBP of orf virus (ORFV), is involved in cellular RNA biogenesis via association with host DRBPs. We found that OV20.0 interacts with DiGeorge syndrome critical region 8 (DGCR8), a subunit of the miRNA processor complex, and binds to primary- and precursor-miRNA. Additionally, OV20.0 regulates DGCR8 expression in multiple ways, including through interaction with the DGCR8 protein and binding to DGCR8 mRNA. Lastly, our data show that DGCR8 plays an antiviral role against ORFV infection, whereas it is beneficial for influenza virus propagation, indicating that the underlying mechanisms could be diverse among different viruses.

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.

Orf virus ORFV112, ORFV117 and ORFV127 contribute to ORFV IA82 virulence in sheep.

The parapoxvirus orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host innate and pro-inflammatory responses to infection. Using the ORFV IA82 strain as the parental virus, recombinant viruses with individual deletions in the genes encoding the IMPs chemokine binding protein (CBP; ORFV112), inhibitor of granulocyte-monocyte colony-stimulating factor and IL-2 (GIF, ORFV117) and interleukin 10 homologue (vIL-10; ORFV127) were generated and characterized in vitro and in vivo. The replication properties of the individual gene deletion viruses in cell culture was not affected comparing with the parental virus. To investigate the effect of the individual gene deletions in ORFV infection and pathogenesis, groups of four lambs were inoculated with each virus and were monitored thereafter. Lambs inoculated with either recombinant or with the parental ORFV developed characteristic lesions of contagious ecthyma. The onset, nature and severity of the lesions in the oral commissure were similar in all inoculated groups from the onset (3 days post-inoculation [pi]) to the peak of clinical lesions (days 11-13 pi). Nonetheless, from days 11-13 pi onwards, the oral lesions in lambs inoculated with the recombinant viruses regressed faster than the lesions produced by the parental virus. Similarly, the amount of virus shed in the lesions were equivalent among lambs of all groups up to day 15 pi, yet they were significantly higher in the parental virus group from day 16-21 pi. In conclusion, individual deletion of these IMP genes from the ORFV genome resulted in slight reduction in virulence in vivo, as evidenced by a reduction in the duration of the clinical disease and virus shedding.

K160 in the RNA-binding domain of the orf virus virulence factor OV20.0 is critical for its functions in counteracting host antiviral defense.

The OV20.0 virulence factor of orf virus antagonizes host antiviral responses. One mechanism through which it functions is by inhibiting activation of the dsRNA-activated protein kinase R (PKR) by sequestering dsRNA and by physically interacting with PKR. Sequence alignment indicated that several key residues critical for dsRNA binding were conserved in OV20.0, and their contribution to OV20.O function was investigated in this study. We found that residues F141, K160, and R164 were responsible for the dsRNA-binding ability of OV20.0. Interestingly, mutation at K160 (K160A) diminished the OV20.0-PKR interaction and further reduced the inhibitory effect of OV20.0 on PKR activation. Nevertheless, OV20.0 homodimerization was not influenced by K160A. The contribution of the dsRNA-binding domain and K160 to the suppression of RNA interference by OV20.0 was further demonstrated in plants. In summary, K160 is essential for the function of OV20.0, particularly its interaction with dsRNA and PKR that ultimately contributes to the suppression of PKR activation.

Dysbiosis of the gut microbiota maybe exacerbate orf pathology by promoting inflammatory immune responses.

Orf is a contagious disease caused by the epitheliotropic orf virus (ORFV) that mainly affects goats and sheep. Orf occurs worldwide and can cause great losses to livestock production. Mounting evidence has shown that gut microbiota plays a pivotal role in shaping the immune responses of the host and thus affecting the infection process of a wide range of pathogens. However, it is unclear whether gut microbiota plays a role during orf development. In this study, we exploited asymptomatic ORFV-carrier goats to explore the potential effects of gut microbiota on orf pathogenesis. The results showed that antibiotics-induced gut microbiota disruption significantly aggravated orf, as indicated by the greater disease severity and higher percentage of animals manifesting clinical orf symptoms. Further analysis suggested IL-17-induced excessive neutrophil accumulation in the diseased lips was potentially responsible for the tissue pathology. In addition, skin γδT cells may be an important source of IL-17. In conclusion, our study showed that the gut microbiota of ORFV-carrier goats plays a central role in controlling inflammatory pathology during ORFV infection, partly through suppressing IL-17-mediated local proinflammatory immune responses. This finding can provide help for elucidating the pathogenesis of orf and also suggests an efficient strategy to minimize the inflammatory pathology by maintaining a healthy gut microbiota during orf development.

Comparison of the sensitivity of three cell cultures to ORFV.

BACKGROUND: Contagious ecthyma (CE) appears in the countries and regions containing goat and sheep farms, and it is considered a global epidemic. CE not only severely endangers the healthy development of the sheep and goat industries but also threatens human health. For viral infectious diseases, fast and effective isolation and culture of the pathogen is critical for CE diagnosis, and for disease prevention and control. Therefore, the sensitivity of bovine Sertoli cells to ORFV was estimate in this study. RESULTS: The sensitivities of bovine Sertoli cells, primary neonatal bovine testicular cells, and Madin-Darby bovine kidney (MDBK) cell line to ORFV were compared. Our results showed that the isolated bovine Sertoli cells were sensitive to inoculated ORFV, and viral titers were approximately 1 log higher than those in primary neonatal bovine testicular cells and in MDBK cell lines. CONCLUSION: Appropriately sensitive cells for the highly efficient isolation and culture of the ORFV were obtained. Culture of ORFV using the Sertoli cells showed good consistency and stability and also avoided the risk of other pathogens presenting during viral culture using a primary cell line. In addition, using these passaged bovine Sertoli cells to proliferate ORFV may simplify the CE diagnosis process, thereby reducing detection time and cost. Hence, this test has important practical significance for the diagnosis of CE and the research on the pathogenic mechanism of ORFV.

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.

The re-emerging of orf virus infection: A call for surveillance, vaccination and effective control measures.

Orf disease is known to be enzootic among small ruminants in Asia, Africa, and some other parts of the world. The disease caused by orf virus is highly contagious among small ruminant species. Unfortunately, it has been neglected for decades because of the general belief that it only causes a self-limiting disease. On the other hand, in the past it has been reported to cause huge cumulative financial losses in livestock farming. Orf disease is characterized by localized proliferative and persistent skin nodule lesions that can be classified into three forms: generalized, labial and mammary or genitals. It can manifest as benign or malignant types. The later type of orf can remain persistent, often fatal and usually causes a serious outbreak among small ruminant population. Morbidity and mortality rates of orf are higher especially in newly infected kids and lambs. Application of antibiotics together with antipyretic and/or analgesic is highly recommended as a supportive disease management strategy for prevention of subsequent secondary microbial invasion. The presence of various exotic orf virus strains of different origin has been reported in many countries mostly due to poorly controlled cross-border virus transmission. There have been several efforts to develop orf virus vaccines and it was with variable success. The use of conventional vaccines to control orf is a debatable topic due to the concern of short term immunity development. Following re-infection in previously vaccinated animals, it is uncommon to observe the farms involved to experience rapid virus spread and disease outbreak. Meanwhile, cases of zoonosis from infected animals to animal handler are not uncommon. Despite failures to contain the spread of orf virus by the use of conventional vaccines, vaccination of animals with live orf virus is still considered as one of the best choice. The review herein described pertinent issues with regard to the development and use of potential effective vaccines as a control measure against orf virus infection.

A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling.

Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb.

Virus orf en humanos, confirmación molecular de un caso clínico en Chile / Orf virus in human, confirmation in case report from Chile

Resumen La infección por el virus orf, también conocida como ectima contagioso, es reconocida una zoonosis ocupacional. Se diagnostica por lesiones cutáneas que evolu cionan rápidamente desde máculas a pápulas, vesículas y pústulas. Se presenta el caso clínico de una estudiante de medicina veterinaria que había tenido contacto con caprinos, clínicamente sanos y sin lesiones aparentes, hacía 19 días. Presentó dos lesiones vesiculares que coalescieron hasta formar una lesión de mayor tamaño rodeada por un halo eritematoso. Las lesiones fueron compatibles con la presentación clásica de las producidas por el virus orf en humanos. Se confirmó la presencia del virus orf mediante una RPC anidada del tejido de biopsia. Es uno de los primeros casos confirmados mediante técnicas moleculares en seres humanos en Chile.

Infection with the orf virus, also known as contagious ecthyma, is recognized as an occupational zoonosis worldwide. It is diagnosed by cutaneous lesions that progress rapidly from macules to papules, vesicles and pustules. The clinical case of a student of veterinary medicine who had had contact with goats, clinically healthy and without apparent lesions, which occured 19 days ago, is reported. She presented two vesicular lesions that coalesced to form a larger lesion surrounded by an erythematous halo. The lesions were compatible with the classical presentation of those produced by the orf virus in humans. The presence of the orf virus was confirmed by a nested PCR from biopsy tissue. It is one of the first cases confirmed by molecular techniques in humans in Chile.

A parapoxviral virion protein inhibits NF-κB signaling early in infection.

Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKß, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection.

An Overwiev of ORF Virus Infection in Humans and Animals.

BACKGROUND: Orf virus is a DNA virus that belongs to the Parapoxvirus genus. The virus is a causative agent of orf in humans or contagious ecthyma in animals which is mostly seen in sheep, goat and cattle. DISCUSSION: Orf is an emerging zoonosis with an increasing number of worldwide outbreaks that have been reported. It is a contagious disease that tends to spread very fast among livestock. The morbidity rate is very high, particularly among young unvaccinated animals. The fatality rate is low but can be seen due to secondary infections. The disease has a significant effect on livestock health and may lead to economical losses. Humans may become infected if they have a direct contact with animal lesions. The disease is seen as a cutaneous lesion with a mild clinical outcome. Human to human transmission exists but is very rare. Nosocomial transmission was reported with one outbreak in a burn unit. The diagnosis is mostly based on the history of animal contact and clinical findings. Molecular tests are used to confirm clinical diagnose. There is no specific treatment but a live vaccine is available for animals. Surveillance implementations and infection control measurements are very important for the prevention of infection. Currently, there are limited studies on orf or contagious ecthyma. It has been observed that there are few studies that have resulted in patents. CONCLUSION: The aim of this paper was to review the current relevant patents, epidemiological features, clinical presentations, the diagnosis and treatment of orf.

Genetic characterization of orf virus associated with an outbreak of severe orf in goats at a farm in Lusaka, Zambia (2015).

Orf or contagious ecthyma is a neglected and economically important zoonotic disease caused by a dermatotropic parapoxvirus that commonly affects domestic small ruminants. Although orf is globally distributed, there is a paucity of information on the disease in many African countries. Here, a suspected severe outbreak of orf in goats at a farm in Lusaka was investigated. Orf virus (ORFV) infection was confirmed by PCR amplification of viral DNA (RNA polymerase, B2L and virus interferon-resistance genes) in clinical samples. Some detected genes were sequenced and phylogenetically analyzed. This is the first report on molecular characterization of ORFV in goats in Zambia.

[Orf virus in human, confirmation in case report from Chile]. / Virus orf en humanos, confirmación molecular de un caso clínico en Chile.

Infection with the orf virus, also known as contagious ecthyma, is recognized as an occupational zoonosis worldwide. It is diagnosed by cutaneous lesions that progress rapidly from macules to papules, vesicles and pustules. The clinical case of a student of veterinary medicine who had had contact with goats, clinically healthy and without apparent lesions, which occured 19 days ago, is reported. She presented two vesicular lesions that coalesced to form a larger lesion surrounded by an erythematous halo. The lesions were compatible with the classical presentation of those produced by the orf virus in humans. The presence of the orf virus was confirmed by a nested PCR from biopsy tissue. It is one of the first cases confirmed by molecular techniques in humans in Chile.

Effect of ORF119 gene deletion on the replication and virulence of orf virus.

Orf is a severe infectious disease of sheep and goats caused by orf virus (ORFV). To investigate the role of ORF119 gene of ORFV, we constructed ORFV with deleted ORF119 gene and LacZ as reporter gene (ORFV-Δ119-LacZ) via homologous recombination. The results showed that wild-type ORF-SHZ1 and ORFV-Δ119-LacZ deletion viruses replicated in Vero cells to similar titers. Relative transcriptional levels of virulence genes OVIFNR, GIF, VEGF and VIL-10 of ORFV-Δ119-LacZ deletion virus were slightly but not significantly lower after 24 hr compared with the wtORF-SHZ1 virus. In vivo experiments showed that 2-month-old lambs inoculated with ORFV-Δ119-LacZ deletion virus exhibited a similar total clinical score compared with those inoculated with wtORF-SHZ1 virus. Based on these results, we conclude that deletion of the ORF119 gene has no significant effect on ORFV replication and virulence.

Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.IMPORTANCE Our previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol 89:4966-4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds directly to the RNA binding domains (RBDs) of PKR, and this interaction does not require dsRNA. Moreover, OV20.0 interacts with or occupies the RBD2 and the kinase domain of PKR, which then prevents PACT binding to PKR. Finally, OV20.0 associates with PACT via the RBDs, which may reduce the ability of PACT to induce PKR activation. The findings in this study provide new concepts in relation to how ORFV modulates PKR activation.

Pathogenesis in lambs and sequence analysis of putative virulence genes of Brazilian orf virus isolates.

The parapoxvirus orf virus (ORFV) is the agent of contagious ecthyma, an ubiquitous mucocutaneous disease of sheep and goats that may present variable clinical presentations. We herein studied the pathogenesis of ORFV infection in lambs and analyzed three putative virulence genes of four Brazilian ORFV isolates. Lambs inoculated in the labial commissures with each ORFV isolate (n=4, viral titer 10(5.6) TCID50/ml) developed classical orf lesions, characterized by a progressive course of erythema/macules, vesicles, pustules and proliferative scabs. Lesions lasted an average of 22.9 days (18-26) and virus shedding was detected for approximately 24.6 days (18-30). Two isolates (SV269/11 and SV820/10) produced more severe, long-lasting lesions resulting in highest clinical scores. Lambs inoculated with isolate SV581/11 developed lesions markedly milder (lower clinical scores [p<0.05]) and more limited than the other groups. Virus shedding by SV581/11 group, however, lasted similarly or even longer than the other groups. Sequence analysis of three virulence genes (VEGF, VIR and IL-10v) revealed amino acid deletions and mutations in VEGF and IL-10v genes of SV581/11 and SV252/11, the isolate(s) producing milder lesions. Additionally, the VEGF gene of isolate SV581/11 presented the lowest amino acid identity with the other isolates and with ORFV standard strain OV-IA82. Thus, these results demonstrate that ORFV isolates may display differential virulence in lambs and these differences might be associated with genetic changes in putative virulence genes.