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.

Detection of genetic variations in Orf virus isolates epidemic in Xinjiang China.

Orf is an exanthemous viral disease seriously threatening the goat and sheep industry and widely epidemic in the goat and sheep populations in Xinjiang, China. In order to investigate the genetic variability of the orf virus (ORFV), three virus isolates (SHZ1, SHZ2, and SHZ3) were isolated by PCR and Vero cell culture using the clinical samples from the lips of the lambs suspected of ORFV infection. The isolates were further verified by electron microscopy and animal infection experiments. The protective antigen genes B2L, F1L, and virulence genes VIR, GIF, and VEGF in the isolates were cloned, sequenced and analyzed for genetic evolution. The results showed that B2L and F1L were relatively conservative with homology 86.7-97.9%, while VIR, GIF, particularly VEGF were considerably variable with homology 71.5-97.9% at amino acid sequence level, respectively. Phylogenetic tree analysis based on B2L and VIR showed that the isolates SHZ1 and SHZ2 were closely related with the Taiwan isolates. This is the first report to confirm that there have been genetic variations in the Xinjiang ORFV isolates. The findings provide molecular evidence about the genetic variability of the major antigenic and virulence genes in the virus isolates epidemic in Xinjiang.

Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [corrected] (HPMPC, cidofovir, CDV, Vistide) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of codofovir [corrected] and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved in milder lesions that resolved [corrected] more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

A real time PCR assay for the detection and quantification of orf virus.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.

Heparin binding activity of orf virus F1L protein.

The orf virus is the type species of the Parapoxvirus genus and is the causative agent of contagious echtyma, a debilitating skin disease of sheep and goats, which can also affect man. The virus exhibits a restricted host range, even if it has been shown to bind to a wide range of tissues of non-permissive species. This ability is an argument for its potential use as an expression vector. Since most mammalian cell types express heparan sulfate (HS) surface receptors, we assumed that HS could serve as receptors to mediate orf virus binding. In this study, we showed that orf virus is inhibited by the addition of soluble heparin in cell cultures. Affinity chomatography using heparin agarose demonstrated that orf virus F1L is the major heparin binding protein. Furthermore, the recombinant F1L protein was visualised on the cell surface by confocal microscopy, and rabbits immunised with recombinant F1L protein produced virus neutralising antibodies. These results confirm that the F1L immunodominant protein is also involved in virus binding to cells as for the vaccinia homologue H3L protein. Heparin also inhibited the binding of the F1L protein to cells showing that this protein has a role in the early stages of infection.

In vivo and in vitro characteristics of contagious ecthyma virus isolates: host response mechanism.

Three Vero cell culture-adapted contagious ecthyma virus (CEV) isolates were compared by plaque morphology, ability to induce vesicles in skin and in vivo growth curve characteristics by sampling sequentially experimental skin lesions produced in four sheep and one goat. Two of the isolates (CEV-29A and CEV-378) were from outbreaks of ecthyma in sheep and one (CEV-102) from a human case of orf. When replicating in Vero cells, the viruses exhibited similar growth parameters, but were distinguishable from each other on the basis of plaque morphology. In vivo latent periods for these isolates were 48 h (CEV-29A), 96 h (CEV-102), and 120 h (CEV-378). When isolates CEV-102 and CEV-29A were passaged into another sheep, they produced similar patterns of growth. Isolate CEV-102 produced the highest infectivity titer [1.4 X 10(9) plaque forming units (PFU) g-1], followed by CEV-29A (6.8 X 10(7) PFU g-1) and CEV-378 (2.5 X 10(7) PFU g-1). In addition, these viruses varied in their ability to induce vesicle formation. Virus was no longer detectable at the inoculation sites at 288 h post-infection (PI). We conclude that plaque morphology, ability to induce vesicle formation in the skin and growth curves in the skin can be considered as important criteria to differentiate CEV isolates. A comparison of the growth curves of CEV-378 in the skin of sheep and goats suggested differences in virus-host interaction between the two animal species. Since intravenous injection of 1 X 10(9) PFU of CEV failed to produce lesions in the sham-scarified skin of sheep, virus spread via the hematogenous route from one site to another appears unlikely. No virus-neutralizing antibody or interferons were found in serum samples or in skin homogenates collected between 0 and 24 days PI. Virus-neutralizing antibody was present in the circulation as late as 24 days PI. Lymphocytes collected from CEV-exposed sheep as early as 12 days PI responded specifically to stimulation with CEV antigen. As this was about the time when infectious virus disappeared from the sites, we assume that cell-associated immune mechanisms may play a larger role in virus clearance from skin lesions than virus-neutralizing antibody.

Genetic characterization of orf viruses isolated from various ruminant species of a zoo.

In the present study, an outbreak of proliferative dermatitis in musk ox (Ovibos moschatus), Sichuan takin (Budorcas taxicolor tibetana) and domestic Shetland sheep (Ovis aries) in a zoo is described. Skin lesions consisted of severe, persistent, multifocal, proliferative dermatitis in musk ox, and mild, transient, focal, dermatitis in the Sichuan takin and Shetland sheep. Parapoxviruses were isolated from skin lesions, and characterized by restriction enzyme analysis and partial gene sequencing. The results of this investigation indicate that the outbreak of proliferative dermatitis was due to infection by a single parapoxvirus, which is genetically closely related to other orf virus (ORFV) strains but distant to bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV).

Comparison of native and subunit antigens as ELISA reagents for the detection of antibodies against scabby mouth virus.

A simple ELISA test to detect antibodies against scabby mouth virus (SMV) has been developed. Native whole virions and subunits of SMV generated by boiling the virus in the presence of sodium dodecyl sulphate (SDS) detergent and beta-mercaptoethanol were compared as ELISA assay reagents using naive and hyperimmune sera from sheep and rabbits. Approximately 2 x 10(4) intact virus particles per microtiter well were required to generate a positive to negative signal of 0.8:0.3 ELISA O.D. units when the serum was used at a dilution of 1/100. In contrast, total subunit antigen generated by disrupting and coupling of 250-500 virions per well provided a signal ratio of 1.58:0.3 ELISA O.D. units at a serum dilution of 1/250. Total subunit antigens were therefore 400 times more economical to use than intact virions. In addition, subunit antigens could be readily bound to microtiter plates without the need for removal of the SDS. Secondly, it was not necessary to block non-specific binding sites on the plate with blockers such as gelatin and skim-milk, thereby shortening the time needed to complete the ELISA assay. The total subunit antigen ELISA test was used to detect seroconversion in new born lambs where there was an occurrence of SMV infection in housed sheep. Three bleeds were taken at fortnightly intervals and the ELISA results showed that 9 out of 15 lambs were seropositive for all bleed points. Four of the lambs showed a sequential rise in titer while only one lamb failed to seroconvert.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions.

In sheep infected with the parapoxvirus orf virus, primary infection orf skin lesions developed and resolved within 8 weeks. Reinfection lesions were smaller and resolved within 3 weeks. The host response in the skin was characterized by an accumulation of neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, B cells and T19+ gammadelta T cells. The magnitude of this accumulation paralleled orf virus replication in the skin. In situ hybridization was used to detect cells expressing interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) mRNAs in orf skin. Cells expressing IL-4 mRNA were not detected at any time after infection. Cells expressing IFN-gamma mRNA were detected after reinfection but not after primary infection. Cells expressing TNF-alpha mRNA included epidermal cells, vascular endothelium and uncharacterized cells that increased more rapidly in the skin after reinfection compared to primary infection. The results are consistent with a prominent role for IFN-gamma in the host immune response controlling the severity of the disease.

Vaccination of sheep with cell culture grown orf virus.

Orf virus, derived from contagious pustular dermatitis (scabby mouth) lesions in sheep, was adapted to cell culture and subsequently evaluated as a potential vaccine for sheep. The traditional vaccine virus, prepared from the infected scabs of orf virus lesions in sheep, was used to vaccinate sheep by scratching with an applicator (mounted pins) dipped in virus. Less than 10 TCID50 (50% tissue culture infectious doses) of virus was required to produce large lesions (greater than 5 mm diameter) which developed during a period of 10 to 14 d prior to onset of healing which was complete by 28 to 30 d. A serum neutralising antibody response was also detected and protection against challenge by application of virulent virus to abraded skin was demonstrated in that challenge lesions developed and healed more quickly (14 d against 30 d). However, cell culture-adapted virus required more than 10(5) TCID50 to induce even small lesions (less than 2 mm diameter). An antibody response could not be detected and no evidence of protection against challenge with virulent virus was demonstrated. In contrast, a recent field isolate has yielded a cell culture-adapted virus preparation that readily infects sheep, produces large lesions, detectable antibody and protects against challenge. This isolate is distinct from the traditional vaccine strain on the basis of restriction enzyme analysis but provides cross-protection in sheep inmmunisation and challenge studies. These results demonstrate that a cell culture produced scabby mouth vaccine is feasible.

Investigation of orf virus structure and morphogenesis using recombinants expressing FLAG-tagged envelope structural proteins: evidence for wrapped virus particles and egress from infected cells.

Orf virus (ORFV) is the type species of the genus Parapoxvirus, but little is known about the structure or morphogenesis of the virus. In contrast, the structure and morphogenesis of vaccinia virus (VACV) has been extensively studied. VACV has two main infectious forms, mature virion (MV) and extracellular virion (EV). The MV is wrapped by two additional membranes derived from the trans-Golgi to produce a wrapped virion (WV), the outermost of which is lost by cellular membrane fusion during viral egress to form the EV. Genome sequencing of ORFV has revealed that it has homologues of almost all of the VACV structural genes. Notable exceptions are A36R, K2L, A56R and B5R, which are associated with WV and EV envelopes. This study investigated the morphogenesis and structure of ORFV by fusing FLAG peptide to the structural proteins 10 kDa, F1L and ORF-110 to form recombinant viruses. 10 kDa and F1L are homologues of VACV A27L and H3L MV membrane proteins, whilst ORF-110 is homologous to VACV A34R, an EV membrane protein. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-F1L and FLAG-10 kDa were displayed on the surface of infectious particles, whereas ORF-110-FLAG could not be detected. Western blot analysis of solubilized recombinant ORF-110-FLAG particles revealed that ORF-110-FLAG was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking. Fluorescent microscopy confirmed the prediction that ORF-110-FLAG localized to the Golgi in virus-infected cells. Finally, immunogold labelling of EVs showed that ORF-110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell.

[Adaptation of ecthyma contagiosum virus to cell culture]. / Opiti na adaptirane na virusa na zaraznata ektima vurkhu kletuchni kulturi.

Series of attempts were made to adapt the Phylaxia vaccinal strain (Hungary) to various cell cultures in vitro. The work with twenty-one successive passages of the virus in a culture of lamb testis resulted in the production of an adapted strain that could produce a characteristic cytopathic effect at the 24th--48th hour of the infection, reaching a titer of 10(4.5) ID/cm3. Cytologic and electron-microscope investigations confirmed the fact that this cell strain belonged to the Parapoxvirus genus. The experimental infection of 4-5-month-old lambs showed that the virus strain retained its capacity to cause typical clinical signs of infectious ectyma. It could be used in the production of a cell-cultural vaccine.

Ultrastructural study of cell cultures infected with swinepox and orf viruses.

Monolayer cells of a porcine kidney cell line were infected with the PP-1 strain of swinepox virus, while secondary or third subcultured monolayer cells of African green monkey kidney were inoculated with the Iwate BT-9 strain of orf virus. Those infected cells were fixed when CPE became remarkable in the monolayers and examined by electron microscope. In the cytoplasm of cells infected with both viruses, various immature forms of viral particles in different developmental stages were observed. Micells were detected very close to the opening of immature particles. Mature particles asssociated with a double membrane were frequently observed. From those observations it was suggested that the developmental sequence of both viruses is essentially the same as that of vaccinia (Dales, 1973). Besides various virus forms as well as factories, the following ultrastructural changes were noted in swinepox infected cells, i.e. 1) intranuclear inclusions which consist of very fine filaments, 2) fibrillar structures with cross striations located in the nuclear inclusions, and 3) similar striated fibrillar structures in or just adjacent to virus factories (B type inclusions) in the cytoplasm. Those observations made with in vitro cells are in good accordance with the descriptions by previous investigators on in vivo materials. Accordingly those ultrastructural changes characterize the swinepox infection.

Orf virus replication in bovine testis cells: kinetics of viral DNA, polypeptide, and infectious virus production and analysis of virion polypeptides.

The replication of orf virus in bovine testis cells was analysed in one-step growth experiments. Newly replicated viral DNA was detected 4 to 6 hours post-infection (p.i.), accumulated rapidly between 8 and 16 hours p.i. and reached a plateau between 25 and 30 hours p.i. Most virus-induced polypeptides were first detected in a two hour period beginning 10 hours p.i., reached a peak rate of synthesis between 14 and 16 hours p.i., and continued at that rate for at least 10 hours. Host polypeptide synthesis declined to very low levels by 20 hours p.i. From these results, the transition between early and late events appears to occur between 8 and 10 hours p.i. Infectious virus was first detected between 16 and 18 hours p.i. and continued to be produced at a steady rate till 40 hours p.i. Up to 35 polypeptides were detected in SDS-polyacrylamide gels of purified orf virions disrupted in SDS/2-ME. Virions treated with NP40/2-ME were separable into soluble and insoluble components by centrifugation. Some 13 polypeptides were found in the soluble fraction and a polypeptide of molecular weight 38,500 believed to be the basic subunit of the virion surface tubule structure. Little difference was found between polypeptide profiles of five independently isolated NZ orf virus strains.

[Recent information about the orf virus of small ruminants]. / Neue Erkenntnisse über das Orf-Virus der kleinen Wiederkäuer.

New scientific findings in the field of immunobiology and diagnosis of parapoxvirus ovis (Orf-virus) as the causal agent of a zoonosis are presented. The adaptation of Orf-virus to cell lines and its in vitro multiplication without difficulties offer the possibility for extensive studies into the biology of parapoxviruses. The development of monoclonal antibodies (MAB) against an attenuated Orf-virus strain (D-1701) led to the elaboration of a simplified, cheap and highly sensitive "antigen detection ELISA" as a diagnostic tool. Moreover the MAB's can be used to identify parapoxvirus in cells of infected tissues (indirect immuno-peroxidase technique) which is an important precondition for more detailed surveillance of the pathogenesis of parapoxvirus infections. The immunobiological behavior of the Orf-virus in its natural hosts (small ruminants) as well as in non-susceptible species (e.g. laboratory animals) is explained by the course of cellular and humoral immune reactions following infection or vaccination. The problems of combat measures against contagious ecthyma (scabby mouth) of sheep and the human Orf-virus infections are discussed.