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Concept: Poxviruses

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Poxviruses are widespread pathogens, which display extremely different host ranges. Whereas some poxviruses, including variola virus, display narrow host ranges, others such as cowpox viruses naturally infect a wide range of mammals. The molecular bases for differences in host range are poorly understood but apparently depend on the successful manipulation of the host antiviral response. Some poxvirus genes have been shown to confer host tropism in experimental settings and are thus called host range factors. Identified host range genes include vaccinia virus K1L, K3L, E3L, B5R, C7L and SPI-1, cowpox virus CP77/CHOhr, ectromelia virus p28 and 022, and myxoma virus T2, T4, T5, 11L, 13L, 062R and 063R. These genes encode for ankyrin repeat-containing proteins, tumor necrosis factor receptor II homologs, apoptosis inhibitor T4-related proteins, Bcl-2-related proteins, pyrin domain-containing proteins, cellular serine protease inhibitors (serpins), short complement-like repeats containing proteins, KilA-N/RING domain-containing proteins, as well as inhibitors of the double-stranded RNA-activated protein kinase PKR. We conducted a systematic survey for the presence of known host range genes and closely related family members in poxvirus genomes, classified them into subgroups based on their phylogenetic relationship and correlated their presence with the poxvirus phylogeny. Common themes in the evolution of poxvirus host range genes are lineage-specific duplications and multiple independent inactivation events. Our analyses yield new insights into the evolution of poxvirus host range genes. Implications of our findings for poxvirus host range and virulence are discussed.

Concepts: Vaccination, Gene, Poxviruses, Cowpox, Vaccinia, Poxviridae, Smallpox, Virus

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Ecthyma contagiosum, also called contagious pustular dermatosis, is a zoonotic disease caused by the orf virus (OrfV). As a member of the poxviridae family and parapoxvirus genus, this dermatotropic virus has developed an array of mechanisms by which to evade the host immune system in both humans and animals. The ubiquitousness of this pathogen in sheep, goats, and deer has led to the development of orf in diverse areas around the world. Human disease occurs via direct contact with infected animals or fomites. Rarely, human-to-human transmission has been reported. The disease progresses through six clinically distinct dermatologic stages and lesions usually heal in three to six weeks without scarring. Farmers, veterinarians, and hunters represent high-risk groups, as their repeated contact with livestock and wild animals predisposes them to infection. With an increasing number of cattle, livestock, and wild animals being kept as pets, human orf may become more prevalent in the future. Taken with the lack of a widely accepted and successful antiviral treatment regimen, this demonstrates the importance of conducting additional research to further elucidate the pathogenic effects of the OrfV in humans. J Drugs Dermatol. 2017;16(7):684-689.

Concepts: Bacteria, Poxviruses, Orf, Disease, Immune system, Infection, Infectious disease

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The orf virus, a member of poxvirus family, is a zoonotic parapoxvirus endemic in many countries, mostly seen among sheep, goats, oxen, and may be transmitted to humans. Orf virus infections may induce ulceration, papulonodular, pustular, or ecthyma lesions in the skin. Rarely, orf virus provokes extensive vasculoendothelial proliferation by encoding an apparent homolog of the mammalian vascular endothelial growth factor family of molecules. The vascular endothelial growth factor-like viral gene product is expressed early during infection and could be responsible for the induction of endothelial proliferation. Here, a 6-year-old male patient with poxvirus-induced widespread vascular angiogenesis is presented, which developed ten days after a thermal burn.

Concepts: Angiogenesis, Pus, Vascular endothelial growth factor, Poxviruses, Poxviridae, Inflammation, Immune system, Virus

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Poxviruses such as Vaccinia virus (VACV) undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms, and includes a crucial “wrapping” step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways were required for this wrapping step we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor complex GARP (Golgi-associated retrograde pathway complex), a central component of retrograde transport. VACV multi-step replication was significantly impaired in cells transfected with siRNA targeting the GARP complex or in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small molecule inhibitors of retrograde transport strongly suppressed VACV multi-step growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.

Concepts: Poxviruses, Smallpox, Poxviridae, DNA, Vaccinia, Virus, Cell membrane, Protein

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Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real-time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory-based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory-based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory-based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings.

Concepts: Animal virology, Milk, Infectious disease, Cattle, Nucleic acid, DNA, Poxviruses, Polymerase chain reaction

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Sheeppox (SPP) and goatpox (GTP) caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively of the genus Capripoxvirus in the family Poxviridae, are severely afflicting small ruminants' production systems in Africa and Asia. In endemic areas, SPP and GTP are controlled using vaccination with live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). Sometimes outbreaks occur following vaccination. In order to successfully control the spread of the virus, it is essential to identify whether the animals were infected by the field strain and the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or in rare occasions, re-gain virulence. Thus, diagnostic tools for differentiation of virulent strains from attenuated vaccine strains of the virus are needed. The aim of this study was to identify an appropriate diagnostic target region in the capripoxvirus genome by comparing the genomic sequences of SPPV field isolates with those of the most widely used SPP vaccine strains.

Concepts: Animal virology, Smallpox, Orf, Virology, Poxviruses, Infectious disease, Vaccination, Vaccine

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Orf virus (ORFV), a member of the genus Parapoxvirus in the family Poxviridae, is the cause of orf, a highly contagious zoonotic viral disease that affects mainly sheep and goats. In the present study, the sequence and phylogenetic analysis of Indian ORFV isolates (n = 15) from natural outbreaks in sheep and goats belonging to different geographical regions were analysed on the basis of F1L gene along with homology modelling of F1L protein. Multiple sequence alignments revealed highly conserved C-terminus and heterogeneity of N-terminus region of F1L among all orf viruses studied. Further, a comparative sequence alignment indicated conservation of various motifs such as glycosaminogalycan (GAG), Asp/Glu-any residue-Asp (D/ExD) and a Cx3C chemokine like motif among all poxviruses and unique motifs (proline rich region [PRR] and Lys-Gly-Asp [KGD]), in parapoxviruses including ORFV isolates irrespective of geography and host species. Phylogenetically, two major clusters were noticed which included Indian orf isolates along with foreign isolates. Structurally, ORFV F1L resembled the topology as exhibited by its homologue vaccinia virus H3 protein with mixed β/α folds and ligand binding specificity in docking models. We noted that despite differences in host cell specificity and pathogencity, poxvirus proteins especially ORFV F1L protein and its homologues presumed to share similarities as they are highly conserved irrespective of species and countries of origin. Further, the study also indicated the possibilities of differentiation of ORFV strains based on N-terminal heterogeneity despite highly conserved C-terminal region with conserved motifs.

Concepts: Protein structure, DNA, C-terminus, N-terminus, Poxviruses, Virus, Poxviridae, Protein

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Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed whenA36Ris deleted.

Concepts: Vaccine, Vaccination, Monkeypox, Vaccinia, Poxviruses, Poxviridae, Orthopoxvirus, Smallpox

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Molluscum contagiosum virus (MCV) causes persistent, benign skin neoplasm in children and adults. MCV is refractive to growth in standard tissue culture and there is no relevant animal model of infection. Here we investigated whether another poxvirus (vaccinia virus; VACV) could be used to examine MCV immunoevasion protein properties in vivo. The MCV MC159L or MC160L genes, which encode NF-κB antagonists, were inserted into an attenuated VACV lacking an NF-κB antagonist (vΔA49), creating vMC159 and vMC160. vMC160 slightly increased vΔA49 virulence in the intranasal and intradermal routes of inoculation. vMC159 infection was less virulent than vΔA49 in both inoculation routes. vMC159-infected ear pinnae did not form lesions, but virus replication still occurred. Thus, the lack of lesions was not due to abortive virus replication. This system provides a new approach to examine MCV immunoevasion proteins within the context of a complete and complex immune system.

Concepts: Toxin, Vaccinia, Poxviruses, Smallpox, Poxviridae, Infectious disease, Virus, Molluscum contagiosum

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The Orthopoxvirus (OPV) genus of the Poxviridae family contains several human pathogens, including Vaccinia virus (VACV), which have been implicating in outbreaks of a zoonotic disease called Bovine Vaccinia in Brazil. So far, no approved treatment exists for OPV infections, but ST-246 and Cidofovir (CDV) are now in clinical development. Therefore, the objective of this work was to evaluate the susceptibility of five strains of Brazilian VACV (Br-VACV) to ST-246 and Cidofovir. The susceptibility of these strains to both drugs was evaluated by plaque reduction assay, extracellular virus’s quantification in the presence of ST-246 and one-step growth curve in cells treated with CDV. Besides that, the ORFs F13L and E9L were sequenced for searching of polymorphisms associated with drug resistance. The effective concentration of 50% (EC50) from both drugs varies significantly for different strains (from 0.0054 to 0.051 μM for ST-246 and from 27.14 to 61.23 μM for CDV). ST-246 strongly inhibits the production of extracellular virus for all isolates in concentrations as low as 0.1 μM and it was observed a relevant decrease of progeny production for all Br-VACV after CDV treatment. Sequencing of the F13L and E9L ORFs showed that Br-VACV do not present the polymorphism(s) associated with resistance to ST-246 and CDV. Taken together, our results showed that ST-246 and CDV are effective against diverse, wild VACV strains and that the susceptibility of Br-VACV to these drugs mirrored the phylogenetic split of these isolates into two groups. Thus, both ST-246 and CDV are of great interest as compounds to treat individuals during Bovine Vaccinia outbreaks in Brazil.

Concepts: Bird, Cowpox, Poxviruses, Vaccinia, Pharmacology, Virus, Poxviridae, Smallpox