-
Journal of Innate Immunity Jun 2024Inactivated parapoxvirus ovis (iPPVO) exerts strong immunomodulatory effects on innate immune cells, making it an attractive therapeutic candidate. However, little is...
TLR9 dependent activation by inactivated parapoxvirus ovis (iPPVO) in murine bone marrow-derived dendritic cells is associated with specific strain-dependent dendritic cell subsets.
INTRODUCTION
Inactivated parapoxvirus ovis (iPPVO) exerts strong immunomodulatory effects on innate immune cells, making it an attractive therapeutic candidate. However, little is known about the signaling pathways involvIing in iPPVO-induced immune responses.
METHODS
In this study, we systematically analyzed how different types of dendritic cells (DCs) react to iPPVO (Zylexis, strain D1710) in both BALB/c and C57BL/c mice by Flow cytometry and ELISAs, and investigated which signaling pathway is related to DC activation by Western blotting and Protein profiling.
RESULTS
We demonstrated that bone marrow-derived conventional DCs (BM-cDCs) and bone marrow-derived plasmacytoid DCs (BM-pDCs) matured and secreted IFN-α/β in response to Zylexis stimulation in both mouse strains. Similarly, Zylexis promoted the secretion of IL-12/23p40 and TNF by pDCs. However, IL-12/23p40 and TNF secretion by cDCs was induced in BALB/c mice but not in C57BL/6 mice. Analyzing the underlying signaling pathways revealed that iPPVO-induced maturation of cDCs was TLR9 independent, while the maturation of pDCs partially depended on the TLR9 pathway. Moreover, the production of proinflammatory cytokines by cDCs and the secretion IFN-α/β by pDCs partially depended on the TLR9 pathway in both mouse strains. Therefore, other signaling pathways seem to participate in the response of DCs to iPPVO, supported by protein profiling.
CONCLUSION
Our data provide useful insights into the diversity of iPPVO sensors and their varying effects across different strains and species.
PubMed: 38852581
DOI: 10.1159/000538625 -
One Health (Amsterdam, Netherlands) Jun 2024Peru was one of the most affected countries during the COVID-19 pandemic. Moreover, multiple other viral diseases (enteric, respiratory, bloodborne, and vector-borne)...
Peru was one of the most affected countries during the COVID-19 pandemic. Moreover, multiple other viral diseases (enteric, respiratory, bloodborne, and vector-borne) are endemic and rising. According to Peru's Ministry of Health, various health facilities in the country were reallocated for the COVID-19 pandemic, thereby leading to reduced action to curb other diseases. Many viral diseases in the area are under-reported and not recognized. The One Health approach, in addition to clinical testing, incorporates environmental surveillance for detection of infectious disease outbreaks. The purpose of this work is to use a screening tool that is based on molecular methods, high throughput sequencing and bioinformatics analysis of wastewater samples to identify virus-related diseases circulating in Trujillo-Peru. To demonstrate the effectiveness of the tool, we collected nine untreated wastewater samples from the Covicorti wastewater utility in Trujillo-Peru on October 22, 2022. High throughput metagenomic sequencing followed by bioinformatic analysis was used to assess the viral diversity of the samples. Our results revealed the presence of sequences associated with multiple human and zoonotic viruses including Orthopoxvirus, Hepatovirus, Rhadinovirus, Parechovirus, Mamastrovirus, Enterovirus, Varicellovirus, Norovirus, Kobuvirus, Bocaparvovirus, Simplexvirus, Spumavirus, Orthohepevirus, Cardiovirus, Molliscipoxvirus, Salivirus, Parapoxvirus, Gammaretrovirus, Alphavirus, Lymphocryptovirus, Erythroparvovirus, Sapovirus, Cosavirus, Deltaretrovirus, Roseolovirus, Flavivirus, Betacoronavirus, Rubivirus, Lentivirus, Betapolyomavirus, Rotavirus, Hepacivirus, Alphacoronavirus, Mastadenovirus, Cytomegalovirus and Alphapapillomavirus. For confirmation purposes, we tested the samples for the presence of selective viruses belonging to the genera detected above. PCR based molecular methods confirmed the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), monkeypox virus (MPXV), noroviruses GI and GII (NoVGI and NoVGII), and rotavirus A (RoA) in our samples. Furthermore, publicly available clinical data for selected viruses confirm our findings. Wastewater or other environmental media surveillance, combined with bioinformatics methods, has the potential to serve as a systematic screening tool for the identification of human or zoonotic viruses that may cause disease. The results of this method can guide further clinical surveillance efforts and allocation of resources. Incorporation of this bioinformatic-based screening tool by public health officials in Peru and other Latin American countries will help manage endemic and emerging diseases that could save human lives and resources.
PubMed: 38798735
DOI: 10.1016/j.onehlt.2024.100756 -
Vaccines May 2024Prime-2-CoV_Beta is a novel Orf virus (ORFV)-based COVID-19 vaccine candidate expressing both the nucleocapsid and spike proteins of SARS-CoV-2 with the receptor-binding...
Prime-2-CoV_Beta is a novel Orf virus (ORFV)-based COVID-19 vaccine candidate expressing both the nucleocapsid and spike proteins of SARS-CoV-2 with the receptor-binding domain (RBD) of the Beta strain. This candidate was shown to be safe and immunogenic in a first-in-human Phase I clinical trial. With the shift in the immune landscape toward the Omicron variant and the widespread vaccine- and/or infection-derived immunity, further pre-clinical research was needed to characterize Prime-2-CoV. Here, we quantified the humoral and cellular response to Prime-2-CoV_Beta in pre-immunized mice and compared the protective efficacy of mono- and bivalent variant-based Prime-2-CoV vaccine candidates in hamsters. Prime-2-CoV_Beta induced robust humoral and cellular immune responses in naïve animals but did not further boost antibody titers in the tested setting when given as repeat booster at short interval. We furthermore showed that Prime-2-CoV_Beta-based mono- and bivalent immunization strategies produced comparable immunogenicity and protection from infection. Our results highlight the potential of the Orf virus as a vaccine platform against SARS-CoV-2 and potentially other infectious viruses.
PubMed: 38793740
DOI: 10.3390/vaccines12050490 -
Virus Research Aug 2024Parapoxviruses (PPV) of animals are spread worldwide. While the Orf virus (ORFV) species is a molecularly well-characterized prototype pathogen of small ruminants, the...
Parapoxviruses (PPV) of animals are spread worldwide. While the Orf virus (ORFV) species is a molecularly well-characterized prototype pathogen of small ruminants, the genomes of virus species affecting large ruminants, namely Bovine papular stomatitis virus (BPSV) and Pseudocowpox virus (PCPV), are less well known. Using Nanopore sequencing we retrospectively show the whole genome sequences (WGS) of six BPSV, three PCPV isolates and an attenuated ORFV strain, originating from different geographic locations. A phylogenetic tree shows that the de novo assembled genomes belong to PPV species including WGS of reference PPV. Remarkably, Nanopore sequencing allowed the molecular resolution of inverted terminal repeats (ITR) and the hairpin loop within the de novo assembled WGS. Additionally, peculiarities regarding map location of two genes and the heterogeneity of a genomic region were noted. Details for the molecular variability of an interferon response modulatory gene (ORF116) and the PCPV specificity of gene 073.5 are reported. In summary, WGS gained by Nanopore sequencing allowed analysis of complete PPV genomes and confident virus species attribution within a phylogenetic tree avoiding uncertainty of limited gene-based diagnostics. Nanopore-based WGS provides robust comparison of PPV genomes and reliable identity determination of new Poxviruses.
Topics: Animals; Phylogeny; Genome, Viral; Cattle; Parapoxvirus; Poxviridae Infections; Whole Genome Sequencing; Retrospective Studies; Cattle Diseases; Nanopore Sequencing; DNA, Viral
PubMed: 38782262
DOI: 10.1016/j.virusres.2024.199404 -
BMC Veterinary Research May 2024Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However,...
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.
Topics: Animals; Fibroblasts; Sheep; Mice; Orf virus; Mice, Nude; Cell Proliferation; Simian virus 40; Cell Line; Apoptosis; Antigens, Viral, Tumor
PubMed: 38745180
DOI: 10.1186/s12917-024-04054-3 -
Applied Microbiology and Biotechnology May 2024Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of...
Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.
Topics: Animals; Goats; T-Lymphocytes, Regulatory; Antibodies, Monoclonal; Flow Cytometry; Viral Load; Ecthyma, Contagious; Interleukin-2 Receptor alpha Subunit; Orf virus; Fluorescent Antibody Technique; CD4 Antigens; Goat Diseases
PubMed: 38717623
DOI: 10.1007/s00253-024-13115-4 -
Veterinary Pathology Apr 2024ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like...
ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the and genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.
PubMed: 38613413
DOI: 10.1177/03009858241241794 -
Frontiers in Immunology 2024The immune system of Asian elephants (Elephas maximus) is poorly studied, compared to that of livestock, rodents or humans. The innate immune response has become a focus...
The immune system of Asian elephants (Elephas maximus) is poorly studied, compared to that of livestock, rodents or humans. The innate immune response has become a focus of interest in relation to Elephant endotheliotropic herpesviruses (EEHVs). EEHVs cause a fatal hemorrhagic disease (EEHV-HD) and are a significant threat to captive Asian elephant populations worldwide. Similar to other herpesvirus infections, nearly all animals become infected, but only some develop disease. As progression to EEHV-HD is often acute, a robust innate immune response is crucial to control EEHV infections. This is invariably true of the host in the first instance, but it can also potentially be modulated by intervention strategies. Here, two immunostimulant veterinary medicinal products, authorized for use in domestic species, were tested for their ability to induce innate anti-viral immune responses in Asian elephant blood cells. Sequence data were obtained for a range of previously unidentified Asian elephant immune genes, including C-X-C motif chemokine ligand 10 (CXCL10), interferon stimulated gene 15 (ISG15) and myxovirus GTPase 1 (Mx1), and were employed in the design of species-specific qPCR assays. These assays were subsequently used in analyses to determine fold changes in gene expression over a period of 24 hours. This study demonstrates that both immunostimulant medications are capable of inducing significant innate anti-viral immune responses which suggests that both could be beneficial in controlling EEHV infections in Asian elephants.
Topics: Humans; Animals; Sheep; Herpesviridae Infections; Elephants; DNA, Bacterial; Herpesviridae; Blood Cells; Immunity, Innate; Plasmids; Immunization; Adjuvants, Immunologic; Gene Expression
PubMed: 38590526
DOI: 10.3389/fimmu.2024.1329820 -
Animals : An Open Access Journal From... Mar 2024Parapoxviruses are worldwide epitheliotropic viruses that affect ruminants. Viruses of this genus have a narrow host range; however, the pseudocowpox virus (PCPV) also...
Parapoxviruses are worldwide epitheliotropic viruses that affect ruminants. Viruses of this genus have a narrow host range; however, the pseudocowpox virus (PCPV) also infects humans. Unfortunately, these cases are not well documented, and the epidemiology and the properties of the causative agents are not properly described. Here, we report the first case of PCPV in northern Russia (the Irkutsk region). The infection occurred in non-immune herds where no new arrivals of animals had been reported. Moreover, clinical signs of infection (skin lesions) were observed in humans. Based on the nucleotide identity and phylogenetic analysis of the partial-length B2L gene, the Irkutsk 2019 isolate was classified as PCPV. Phylogenetic analysis based on the nucleotide sequence of the B2L gene fragment of PCPV revealed a close phylogenetic relationship between the Irkutsk 2019 isolate and the PCPV strains isolated in Europe and the USA. The high degree of conservatism of the B2L gene does not allow for finding a correlation between their geographical origin and the results of phylogenetic analysis.
PubMed: 38540067
DOI: 10.3390/ani14060969 -
Vaccine: X Jun 2024The Orf virus (ORFV) is a promising vector platform for the generation of vaccines against infectious diseases and cancer, highlighted by its progression to clinical...
The Orf virus (ORFV) is a promising vector platform for the generation of vaccines against infectious diseases and cancer, highlighted by its progression to clinical testing phases. One of the critical steps during GMP manufacturing is the clarification of crude harvest because of the enveloped nature and large size of ORFV. This study presents the first description of ORFV clarification process from a HEK suspension batch process. We examined various filter materials, membrane pore sizes, harvest timings, and nuclease treatments. Employing the Ambr® crossflow system for high-throughput, small-volume experiments, we identified polypropylene-based Sartopure® PP3 filters as ideal. These filters, used in two consecutive stages with reducing pore sizes, significantly enhanced ORFV recovery and addressed scalability challenges. Moreover, we demonstrated that the time of harvest and the use of a nuclease play a decisive role to increase ORFV yields. With these findings, we were able to establish an efficient and scalable clarification process of ORFV derived from a suspension production process, essential for advancing ORFV vaccine manufacturing.
PubMed: 38523620
DOI: 10.1016/j.jvacx.2024.100474