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.

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.

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.

The whole genomic analysis of the Orf virus strains ORFV-SC and ORFV-SC1 from the Sichuan province and their weak pathological response in rabbits.

The Orf virus (ORFV) is a member of the Parapoxvirus genus of the Poxviridae family and can cause contagious diseases in sheep, goats, and wild ungulates. In the present study, two ORFV isolates (ORFV-SC isolated from Sichuan province and ORFV-SC1 produced by 60 passages of ORFV-SC in cells) were sequenced and compared to multiple ORFVs. The two ORFV sequences had entire genome sizes of 14,0707 bp and 141,154 bp, respectively, containing 130 and 131 genes, with a G + C content of 63% for the ORFV-SC sequence and 63.9% for the ORFV-SC1 sequence. Alignment of ORFV-SC and ORFV-SC1 with five other ORFV isolates revealed that ORFV-SC, ORFV-SC1, and NA1/11 shared > 95% nucleotide identity with 109 genes. Five genes (ORF007, ORF20, ORF080, ORF112, ORF116) have low amino acids identity between ORFV-SC and ORFV-SC1. Mutations in amino acids result in changes in the secondary and tertiary structure of ORF007, ORF020, and ORF112 proteins. The phylogenetic tree based on the complete genome sequence and 37 single genes revealed that the two ORFV isolates originated from sheep. Finally, animal experiments demonstrated that ORFV-SC1 is less harmful to rabbits than ORFV-SC. The exploration of two full-length viral genome sequences provides valuable information in ORFV biology and epidemiology research. Furthermore, ORFV-SC1 demonstrated an acceptable safety profile following animal vaccination, indicating its potential as a live ORFV vaccine.

Orf virus induces complete autophagy to promote viral replication via inhibition of AKT/mTOR and activation of the ERK1/2/mTOR signalling pathway in OFTu cells.

Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.

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.

Are Parapoxvirus zoonotic diseases doomed to remain neglected?

Parapoxvirus (PPV) infections are considered neglected zoonoses because their incidence is often unknown or greatly underestimated despite being endemic globally. Here, we report the comprehensive diagnostic workflow that led to the identification of two cases of persistent PPV infections. The results obtained underline the importance of adopting a "One Health" approach and cross-sectoral collaboration between human and veterinary medicine for precise aetiological diagnosis and correct management of patients affected by zoonotic diseases.

Orf Virus-Based Therapeutic Vaccine for Treatment of Papillomavirus-Induced Tumors.

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.

Identification and screening of host proteins interacting with ORFV-ORF047 protein.

BACKGROUND: Orf virus (ORFV) is a member of the genus Parapoxvirus and family Poxviridae. The virus has a worldwide distribution and infects sheep, goats, humans, and wild animals. However, due to the complex structure of the poxvirus, the underlying mechanism of the entry and infection by ORFV remains largely unknown. ORFV ORF047 encodes a protein named L1R. Poxviral L1R serves as the receptor-binding protein and blocks virus binding and entry independently of glycosaminoglycans (GAGs). The study aimed to identify the host interaction partners of ORFV ORF047. METHODS: Yeast two-hybrid cDNA library of sheep testicular cells was applied to screen the host targets with ORF047 as the bait. ORF047 was cloned into a pBT3-N vector and expressed in the NMY51 yeast strain. Then, the expression of bait proteins was validated by Western blot analysis. RESULTS: Sheep SERP1and PABPC4 were identified as host target proteins of ORFV ORF047, and a Co-IP assay further verified their interaction. CONCLUSIONS: New host cell proteins SERP1and PABPC4 were found to interact with ORFV ORF047 and might involve viral mRNA translation and replication.

Lentivirus expressing shRNAs inhibit the replication of contagious ecthyma virus by targeting DNA polymerase gene.

BACKGROUND: Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. RNAi particularly shRNA provides us with the chance to tackle this obstacle by an encouraging new approach. The current study indicates the design and experiment of third-generation lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. Given the importance of DNA-pol gene in virus replication, in this study, three shRNAs against this gene were designed and cloned into lentiviral vectors to stabilize the expression of shRNAs. After producing lentivectors expressing ORFV-DNA- pol in HEK293T cells, the synthesized shRNAs were applied to downregulate viral replication and gene expression. The reduction in viral titer and RNA was evaluated by TCID50 test as well as real-time RT-PCR. The results were then analyzed in comparison with the control group. RESULTS: Designed shRNAs significantly reduced virus yield approximately 90 to 97% and 96.8 to 99.4%, respectively compared to the control groups (cells infected with ORFV and infected with ORFV and scrambled vector) by TCID50 test. Real-time RT-PCR revealed a dramatic reduction in the expression of viral RNA approximately 99% compared to cells infected with ORFV and from 92.6 to 99%, respectively compared to cells infected with ORFV and scrambled vector. CONCLUSIONS: Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.

Poxviral E3L ortholog (Viral Interferon resistance gene) of orf viruses of sheep and goats indicates species-specific clustering with heterogeneity among parapoxviruses.

Orf is a contagious disease posing a serious threat to animal and human health. E3L is one of the evolutionarily acquired immunomodulatory proteins present in orf virus (ORFV) and is responsible for conferring resistance to interferons among poxviruses. Genetic analysis of ORFV isolates of different geographical regions including Indian subcontinent targeting viral interferon resistance (VIR) gene (a homolog of vaccinia virus E3L gene) revealed a high percentage of identity among themselves and other ORFV isolates at both nt and aa levels as compared to low identity among parapoxviruses (PPVs). Phylogenetic analysis showed species-specific clustering among PPVs along with sub-clusters based on host species of origin among ORFVs infecting sheep and goats. Conserved amino acids in N-terminal Z-DNA binding domain and C-terminal ds RNA binding domain of VIR proteins of PPVs corresponding to ORFV VIR positions namely N37, Y41, P57, and W59 (necessary for Z-DNA binding) and E116, F127, F141, and K160 (necessary for dsRNA binding) were found. Further, the predicted protein characteristics and homology model of VIR protein of ORFV showed high structural conservation among poxviruses. This study on E3L genetic analysis of ORFV isolates may provide a better understanding of the molecular epidemiology of circulating strains in India and neighboring countries. Also, E3L deleted or mutated ORFV may be an as vaccine candidate and/or compounds blocking E3L may prove as an effective method for treating broad spectrum poxviral infections, suggesting a wider application in control of poxvirus infections.

Genomic characterization of two Orf virus isolates from Jilin province in China.

Orf virus (ORFV), a typical member of the Parapoxvirus genus within the family Poxviridae, which is the causative agent of Orf, a common epitheliotropic viral disease of sheep, goats, wild ruminants, and humans. In the present study, we sequenced the complete genomic sequences of two ORFV strains (ORFV-SY17, isolated from sheep, and ORFV-NA17, isolated from goat) and conducted the comparative analysis of multiple ORFVs. The complete genomic sequence of ORFV-SY17 was at length of 140,413 bp, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 4267 bp at both ends. The ORFV-NA17 strain displayed the similar genome structure with ORFV-SY17. The whole genomic sequence of ORFV-NA17 strain was 139,287 bp in length and contained 132 ORFs flanked by ITRs of 3974 bp. The overall G+C contents of ORFV-SY17 and ORFV-NA17 genome sequences were about 63.8% and 63.7%, respectively. The ITR sequences analysis showed that ORFV-SY17 and ORFV-NA17 contained the terminal BamHI sites and conserved telomere resolution sequences at both ends of their genome. In addition, comparative analysis of ORFs among ORFV-SY17, ORFV-NA17, and other ORFV strains revealed several sequence variations caused by insertions or deletions, especially in ORFs 005 and 116, which were very likely associated with host species. Phylogenetic analysis based on the complete genome sequences revealed that ORFV-SY17 was genetically closely related to NA1/11 and HN3/12 strains derived from sheep, while ORFV-NA17 was closely related to YX strain derived from goat. The multiple alignment of deduced amino acid sequences further revealed the genetic relationship between host species and genetic variations of ORFV strains. Taken together, the availability of genomic sequences of ORFV-SY17 and ORFV-NA17 strains from Jilin Province will aid in our understanding of the genetic diversity and evolution of ORFV strains in this region and can assist in distinguishing between ORFV strains that originate in sheep and goats.

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.

Cyclophilin B facilitates the replication of Orf virus.

BACKGROUND: Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. METHODS: Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID50) assay and qRT-PCR detection. RESULTS: In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. CONCLUSIONS: Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

Identification and characterization of Orf virus 050 protein proteolysis.

The Orf virus 050 (ORFV050) gene is located in the core region of the ORFV genome. It is similar to Vaccinia virus (VV) Copenhagen L4R, and encodes the DNA-binding virion core protein VP8, which has structures similar to the VV P25K core protein and may undergo similar proteolytic processing during virus assembly. Three conserved Ala-Gly-X motifs at putative cleavage sites were identified in ORFV050. To investigate the proteolysis of ORFV050 and its participation in viral assembly, full-length and site-directed mutant ORFV050 recombinant proteins were constructed and expressed. Two distinct protein bands of 28.5 and 25 kDa were detected in the infected cells using anti-ORFV050 polyclonal antiserum. A potential cleavage site was identified at amino acids 30-32 of ORFV050. Mutation of AG/A to (R) in ORFV050 abolished the process of proteolysis. ORFV050 is a late gene synthesized during viral replication in the host cytoplasm. According to these results, we conclude that ORFV050 undergoes proteolysis and plays an important role in viral assembly.

The whole genomic analysis of orf virus strain HN3/12 isolated from Henan province, central China.

BACKGROUND: The Orf virus (ORFV) is the causative agent of orf, a globally-occurring, acute, pustular, contagious disease affecting sheep, goats and humans with a worldwide distribution. Currently, the genomic analysis of four ORFV strains from the Fujian province in southern China and a NA1/11 strain isolated from the Jilin province in northeast China have been reported. However, little is known about the genomic information of ORFV strains from central China. RESULTS: From a recent outbreak in a sheep herd in the Henan province of central China, a novel ORFV strain (HN3/12) was isolated and cultured in ovine fetal turbinate (OFTu) cells. The strain was identified as HN3/12 and verified by PCR based on the DNA sequences of 011 and 059 genes. The whole genomic sequence of this isolate was determined by Next Generation Sequencing technology. To determine the genetic characteristics of the HN3/12 strain, phylogenetic analysis of the 011 and 059 genes and amino acid sequence alignment of the HN3/12 strain were performed and compared with reference parapoxvirus strains. CONCLUSIONS: The HN3/12 genome is 136,643 bp in length, contains 63.67% G + C and encodes 132 putative genes. Phylogenetic analysis of the 011 and 059 nucleotide sequences showed that this viral strain was similar to the NA1/11 isolate. The homology analysis indicates that HN3/12 has 93% to 98% identity with published ORFV strains at amino acid level. When open reading frames (ORFs) were aligned among the HN3/12 and four Fujian ORFV strains, most of them have identities greater than 90% and only a few less than 60%. The availability of the whole genomic sequence of HN3/12 aids in our understanding of, and provides new insights into, the genetic diversity of ORFV.

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.

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.

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.

Construction of a Triple-Gene Deletion Mutant of Orf Virus and Evaluation of Its Safety, Immunogenicity and Protective Efficacy.

Contagious ecthyma is a zoonotic disease caused by the orf virus (ORFV). Since there is no specific therapeutic drug available, vaccine immunization is the main tool to prevent and control the disease. Previously, we have reported the construction of a double-gene deletion mutant of ORFV (rGS14ΔCBPΔGIF) and evaluated it as a vaccine candidate. Building on this previous work, the current study reports the construction of a new vaccine candidate, generated by deleting a third gene (gene 121) to generate ORFV rGS14ΔCBPΔGIFΔ121. The in vitro growth characteristics, as well as the in vivo safety, immunogenicity, and protective efficacy, were evaluated. RESULTS: There was a minor difference in viral replication and proliferation between ORFV rGS14ΔCBPΔGIFΔ121 and the other two strains. ORFV rGS14ΔCBPΔGIFΔ121 induced continuous differentiation of PBMC to CD4+T cells, CD8+T cells and CD80+CD86+ cells and caused mainly Th1-like cell-mediated immunity. By comparing the triple-gene deletion mutant with the parental strain and the double-gene deletion mutant, we found that the safety of both the triple-gene deletion mutant and the double-gene deletion mutant could reach 100% in goats, while the safety of parental virus was only 50% after continually observing immunized animals for 14 days. A virulent field strain of ORFV from an ORF scab was used in the challenge experiment by inoculating the virus to the hairless area of the inner thigh of immunized animals. The result showed that the immune protection rate of triple-gene deletion mutant, double-gene mutant, and the parental virus was 100%, 66.7%, and 28.6%, respectively. In conclusion, the safety, immunogenicity, and immune-protectivity of the triple-gene deletion mutant were greatly improved to 100%, making it an excellent vaccine candidate.