Detection of Novel Poxvirus from Gray Seal (Halichoerus grypus), Germany.

We detected a novel poxvirus from a gray seal (Halichoerus grypus) from the North Sea, Germany. The juvenile animal showed pox-like lesions and deteriorating overall health condition and was finally euthanized. Histology, electron microscopy, sequencing, and PCR confirmed a previously undescribed poxvirus of the Chordopoxvirinae subfamily, tentatively named Wadden Sea poxvirus.

Emergence of Salmon Gill Poxvirus.

The Salmon gill poxvirus (SGPV) has emerged in recent years as the cause of an acute respiratory disease that can lead to high mortality in farmed Atlantic salmon presmolts, known as Salmon gill poxvirus disease. SGPV was first identified in Norway in the 1990s, and its large DNA genome, consisting of over 206 predicted protein-coding genes, was characterized in 2015. This review summarizes current knowledge relating to disease manifestation and its effects on the host immune system and describes dissemination of the virus. It also demonstrates how newly established molecular tools can help us to understand SGPV and its pathogenesis. Finally, we conclude and ask some burning questions that should be addressed in future research.

[Determination of the optimal immunizing dose of heterologous goat pox virus vaccine (Poxviridae: Chordopoxvirinae: Capripoxvirus) against lumpy skin disease].

INTRODUCTION: Lumpy skin disease (LSD), sheep pox and goat pox are dangerous diseases of domestic ruminants. Representatives of the genus of capripoxviruses are antigenically similar and can be used as a vaccine for three infections, as in the case of representatives of the genus of orthopoxviruses, which includes viruses of smallpox, monkeypox, and cowpox, that all belong to a single family Poxviridae. MATERIALS AND METHODS: In this study, the vaccine strain G20-LKV of the goat pox virus and the virulent strain RIBSP2019/K of the LSD virus were used. The experiments were carried out on clinically healthy cattle of the Kazakh White-headed breed, aged six to eight months. Virological and serological research methods were used in the work. RESULTS: All immunized animals that received different doses of the vaccine showed resistance to the infection challenge, without showing any clinical signs of the disease. In animals that received the lowest doses of the vaccine 15,000, 30,000 and 40,000 TCID50, no adverse events, skin and temperature reactions were observed at the injection site. Those vaccinated with high doses of the vaccine had a local reaction in the form of swelling at the site of vaccine administration. Control animals infected with a virulent virus showed clinical signs of the cattle lumpy skin disease . CONCLUSION: The vaccine, prepared based on the "G20-LKV" strain of the goat virus, is protective for cattle against infection with a virulent LSD virus at immunizing doses from 15,000 to 80,000 TCID50, which are dependent on the LSD epizootic situation in particular region.

Detection of Cetacean Poxvirus in Peruvian Common Bottlenose Dolphins (Tursiops truncatus) Using a Pan-Poxvirus PCR.

Cetacean poxviruses (CePVs) cause 'tattoo' skin lesions in small and large cetaceans worldwide. Although the disease has been known for decades, genomic data for these poxviruses are very limited, with the exception of CePV-Tursiops aduncus, which was completely sequenced in 2020. Using a newly developed pan-pox real-time PCR system targeting a conserved nucleotide sequence located within the Monkeypox virus D6R gene, we rapidly detected the CePV genome in typical skin lesions collected from two Peruvian common bottlenose dolphins (Tursiops truncatus) by-caught off Peru in 1993. Phylogenetic analyses based on the sequencing of the DNA polymerase and DNA topoisomerase genes showed that the two viruses are very closely related to each other, although the dolphins they infected pertained to different ecotypes. The poxviruses described in this study belong to CePV-1, a heterogeneous clade that infects many species of dolphins (Delphinidae) and porpoises (Phocoenidae). Among this clade, the T. truncatus CePVs from Peru were more related to the viruses infecting Delphinidae than to those detected in Phocoenidae. This is the first time that CePVs were identified in free-ranging odontocetes from the Eastern Pacific, surprisingly in 30-year-old samples. These data further suggest a close and long-standing pathogen-host co-evolution, resulting in different lineages of CePVs.

Engineered Promoter-Switched Viruses Reveal the Role of Poxvirus Maturation Protein A26 as a Negative Regulator of Viral Spread.

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Molecular and Biological Characterization of a Cervidpoxvirus Isolated From Moose with Necrotizing Dermatitis.

Cervidpoxvirus is one of the more recently designated genera within the subfamily Chordopoxvirinae, with Deerpox virus (DPV) as the only recognized species to date. In this study, the authors describe spontaneous disease and infection in the North American moose (Alces americanus) by a novel Cervidpoxvirus, here named Moosepox virus (MPV). Three 4-month-old moose calves developed a multifocal subacute-to-chronic, necrotizing, suppurative-to-granulomatous dermatitis that affected the face and the extremities. Ultrastructurally, all stages of MPV morphogenesis-that is, crescents, spherical immature particles, mature particles, and enveloped mature virus-were observed in skin tissue. In vitro infection with MPV confirmed that its morphogenesis was similar to that of the prototype vaccinia virus. The entire coding region, including 170 putative genes of this MPV, was sequenced and annotated. The sequence length was 164,258 bp with 98.5% nucleotide identity with DPV (strain W-1170-84) based on the whole genome. The genome of the study virus was distinct from that of the reference strain (W-1170-84) in certain genes, including the CD30-like protein (83.9% nucleotide, 81.6% amino acid), the endothelin precursor (73.2% nucleotide including some indels, 51.4% amino acid), and major histocompatibility class (MHC) class I-like protein (81.0% nucleotide, 68.2% amino acid). This study provides biological characterization of a new Cervidpoxvirus attained through in vivo and in vitro ultrastructural analyses. It also demonstrates the importance of whole-genome sequencing in the molecular characterization of poxviruses identified in taxonomically related hosts.

Crocodilepox Virus Evolutionary Genomics Supports Observed Poxvirus Infection Dynamics on Saltwater Crocodile (Crocodylus porosus).

Saltwater crocodilepox virus (SwCRV), belonging to the genus Crocodylidpoxvirus, are large DNA viruses posing an economic risk to Australian saltwater crocodile (Crocodylus porosus) farms by extending production times. Although poxvirus-like particles and sequences have been confirmed, their infection dynamics, inter-farm genetic variability and evolutionary relationships remain largely unknown. In this study, a poxvirus infection dynamics study was conducted on two C. porosus farms. One farm (Farm 2) showed twice the infection rate, and more concerningly, an increase in the number of early- to late-stage poxvirus lesions as crocodiles approached harvest size, reflecting the extended production periods observed on this farm. To determine if there was a genetic basis for this difference, 14 complete SwCRV genomes were isolated from lesions sourced from five Australian farms. They encompassed all the conserved genes when compared to the two previously reported SwCRV genomes and fell within three major clades. Farm 2's SwCRV sequences were distributed across all three clades, highlighting the likely mode of inter-farm transmission. Twenty-four recombination events were detected, with one recombination event resulting in consistent fragmentation of the P4c gene in the majority of the Farm 2 SwCRV isolates. Further investigation into the evolution of poxvirus infection in farmed crocodiles may offer valuable insights in evolution of this viral family and afford the opportunity to obtain crucial information into natural viral selection processes in an in vivo setting.

Hypsugopoxvirus: A Novel Poxvirus Isolated from Hypsugo savii in Italy.

Interest in bat-related viruses has increased considerably during the last decade, leading to the discovery of a rising number of new viruses in several bat species. Poxviridae are a large, diverse family of DNA viruses that can infect a wide range of vertebrates and invertebrates. To date, only a few documented detections of poxviruses have been described in bat populations on three different continents (America, Africa, and Australia). These viruses are phylogenetically dissimilar and have diverse clinical impacts on their hosts. Herein, we report the isolation, nearly complete genome sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (Hypsugo savii) in Northern Italy. The virus is tentatively named Hypsugopoxvirus (HYPV) after the bat species from which it was isolated. The nearly complete genome size is 166,600 nt and it encodes 161 genes. Genome analyses suggest that HYPV belongs to the Chordopoxvirinae subfamily, with the highest nucleotide identity (85%) to Eptesipoxvirus (EPTV) detected from a microbat Eptesicus fuscus in WA, USA, in 2011. To date, HYPV represents the first poxvirus detected in bats in Europe; thus, its viral ecology and disease associations should be investigated further.

Spread and impact of goat pox ("sagolay bohonta") in a village smallholder community around Kaziranga National Park, Assam, India.

During September and October 2017, a highly fatal outbreak of a disease clinically indistinguishable from goat pox occurred in the villages around the Kaziranga National Park, Assam, India. This was investigated through clinical examination of affected animals, individual interviews with goat keepers and participatory village meetings. Laboratory confirmation was impractical due to the isolation and poverty of the affected community and unnecessary due to the specific nature of the clinical signs. Respondents reported not having encountered the disease previously, and it would appear that a naïve local population developed within an endemically affected region because of a trend to avoid purchasing animals from outside the village. Local grazing practices appear to have had a role in both the spread and control of the outbreak. Goats are an important form of savings and cash income to people in the locality, and the outbreak may result in considerable financial hardship for affected goat keepers. We provide a detailed description of the clinical disease and the spread of the outbreak in the locality. Awareness of the disease with reference to farming practices will provide opportunities for future disease control to enhance animal welfare and rural prosperity.

Molecular characterization of the first saltwater crocodilepox virus genome sequences from the world's largest living member of the Crocodylia.

Crocodilepox virus is a large dsDNA virus belonging to the genus Crocodylidpoxvirus, which infects a wide range of host species in the order Crocodylia worldwide. Here, we present genome sequences for a novel saltwater crocodilepox virus, with two subtypes (SwCRV-1 and -2), isolated from the Australian saltwater crocodile. Affected belly skins of juvenile saltwater crocodiles were used to sequence complete viral genomes, and perform electron microscopic analysis that visualized immature and mature virions. Analysis of the SwCRV genomes showed a high degree of sequence similarity to CRV (84.53% and 83.70%, respectively), with the novel SwCRV-1 and -2 complete genome sequences missing 5 and 6 genes respectively when compared to CRV, but containing 45 and 44 predicted unique genes. Similar to CRV, SwCRV also lacks the genes involved in virulence and host range, however, considering the presence of numerous hypothetical and or unique genes in the SwCRV genomes, it is completely reasonable that the genes encoding these functions are present but not recognized. Phylogenetic analysis suggested a monophyletic relationship between SwCRV and CRV, however, SwCRV is quite distinct from other chordopoxvirus genomes. These are the first SwCRV complete genome sequences isolated from saltwater crocodile skin lesions.

Molecular and microscopic characterization of a novel Eastern grey kangaroopox virus genome directly from a clinical sample.

Poxviruses are large DNA viruses with varying zoonotic potential, and are recognised in a broad range of wildlife. Although poxviruses have been detected in kangaroos, their genetic relationships to poxviruses in other animals and humans is not well understood. Here, we present a novel genome sequence of a marsupial poxvirus, the Eastern grey kangaroopox virus (EKPV-NSW), isolated from a wild eastern grey kangaroo. In the present study, histopathologically confirmed epidermal pox lesions were used to recover the full-length viral genome and perform electron microscopic analysis, with both immature virions and intracellular mature virions detected. Subsequent analysis of the EKPV-NSW genome demonstrated the highest degree of sequence similarity with EKPV-SC strain (91.51%), followed by WKPV-WA (87.93%), and MOCV1 (44.05%). The novel EKPV-NSW complete genome encompasses most of the chordopoxviruses protein coding genes (138) that are required for genome replication and expression, with only three essential protein coding genes being absent. The novel EKPV-NSW is missing 28 predicted genes compared to the recently isolated EKPV-SC, and carries 21 additional unique genes, encoding unknown proteins. Phylogenetic and recombination analyses showed EKPV-NSW to be the distinct available candidate genome of chordopoxviruses.

Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae.

UNLABELLED: Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species, including reptiles, birds, and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu, and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon. In the present study, we used quantitative PCR and immunohistochemistry to determine aspects of salmon gill poxvirus disease, which are described here. The gill was the main target organ where immature and mature poxvirus particles were detected. The particles were detected in detaching, apoptotic respiratory epithelial cells preceding clinical disease in the form of lethargy, respiratory distress, and mortality. In moribund salmon, blocking of gas exchange would likely be caused by the adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in control fish with gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR of archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus genome encompasses most key chordopoxvirus genes that are required for genome replication and expression, although the gene order is substantially different from that in other chordopoxviruses. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membrane biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins, many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures. IMPORTANCE: Aquaculture is an increasingly important global source of high-quality food. To sustain the growth in aquaculture, disease control in fish farming is essential. Moreover, the spread of disease from farmed fish to wildlife is a concern. Serious poxviral diseases are emerging in aquaculture, but very little is known about the viruses and the diseases that they cause. There is a possibility that viruses with enhanced virulence may spread to new species, as has occurred with the myxoma poxvirus in rabbits. Provision of the first fish poxvirus genome sequence and specific diagnostics for the salmon gill poxvirus in Atlantic salmon may help curb this disease and provide comparative knowledge. Furthermore, because salmon gill poxvirus represents the deepest branch of chordopoxvirus so far discovered, the genome analysis provided substantial insight into the evolution of different functional modules in this important group of viruses.

Genome variability and gene content in chordopoxviruses: dependence on microsatellites.

To investigate gene loss in poxviruses belonging to the Chordopoxvirinae subfamily, we assessed the gene content of representative members of the subfamily, and determined whether individual genes present in each genome were intact, truncated, or fragmented. When nonintact genes were identified, the early stop mutations (ESMs) leading to gene truncation or fragmentation were analyzed. Of all the ESMs present in these poxvirus genomes, over 65% co-localized with microsatellites-simple sequence nucleotide repeats. On average, microsatellites comprise 24% of the nucleotide sequence of these poxvirus genomes. These simple repeats have been shown to exhibit high rates of variation, and represent a target for poxvirus protein variation, gene truncation, and reductive evolution.

Chordopoxvirus protein F12 implicated in enveloped virion morphogenesis is an inactivated DNA polymerase.

UNLABELLED: Through the course of their evolution, viruses with large genomes have acquired numerous host genes, most of which perform function in virus reproduction in a manner that is related to their original activities in the cells, but some are exapted for new roles. Here we report the unexpected finding that protein F12, which is conserved among the chordopoxviruses and is implicated in the morphogenesis of enveloped intracellular virions, is a derived DNA polymerase, possibly of bacteriophage origin, in which the polymerase domain and probably the exonuclease domain have been inactivated. Thus, F12 appears to present a rare example of a drastic, exaptive functional change in virus evolution. REVIEWERS: This article was reviewed by Frank Eisenhaber and Juergen Brosius.

Novel host-related virulence factors are encoded by squirrelpox virus, the main causative agent of epidemic disease in red squirrels in the UK.

Squirrelpox virus (SQPV) shows little evidence for morbidity or mortality in North American grey squirrels (Sciurus carolinensis), in which the virus is endemic. However, more recently the virus has emerged to cause epidemics with high mortality in Eurasian red squirrels (S. vulgaris) in Great Britain, which are now threatened. Here we report the genome sequence of SQPV. Comparison with other Poxviridae revealed a core set of poxvirus genes, the phylogeny of which showed SQPV to be in a new Chordopoxvirus subfamily between the Molluscipoxviruses and Parapoxviruses. A number of SQPV genes were related to virulence, including three major histocomaptibility class I homologs, and one CD47 homolog. In addition, a novel potential virulence factor showing homology to mammalian oligoadenylate synthetase (OAS) was identified. This family of proteins normally causes activation of an endoribonuclease (RNaseL) within infected cells. The putative function of this novel SQPV protein was predicted in silico.

Squirrelpox virus: assessing prevalence, transmission and environmental degradation.

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25 °C) and dry conditions than in cooler (5 and 15 °C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species.

Phylogenetic analysis of the large family of poxvirus ankyrin-repeat proteins reveals orthologue groups within and across chordopoxvirus genera.

Ankyrin-repeat (ANK) protein-interaction domains are common in cellular proteins but are relatively rare in viruses. Chordopoxviruses, however, encode a large number of ANK domain-containing ORFs of largely unknown function. Recently, a second protein-interaction domain, an F-box-like motif, was identified in several poxvirus ANK proteins. Cellular F-box proteins recruit substrates to the ubiquitination machinery of the cell, a putative function for ANK/poxviral F-box proteins. Using publicly available genome sequence data we examined all 328 predicted ANK proteins encoded by 27 chordopoxviruses that represented the eight vertebrate poxvirus genera whose members encode ANK proteins. Within these we identified 15 putative ANK protein orthologue groups within orthopoxviruses, five within parapoxviruses, 23 within avipoxviruses and seven across members of the genera Leporipoxvirus, Capripoxvirus, Yatapoxvirus, Suipoxvirus and Cervidpoxvirus. Sequence comparisons showed that members of each of these four clusters of orthologues were not closely related to members of any of the other clusters. Of these ORFs, 67% encoded a C-terminal poxviral F-box-like motif, whose absence could largely be attributed to fragmentation of ORFs. Our findings suggest that the large family of poxvirus ANK proteins arose by extensive gene duplication and divergence that occurred independently in four major genus-based groups after the groups diverged from each other. It seems likely that the ancestor ANK proteins of poxviruses contained both the N-terminal ANK repeats and a C-terminal F-box-like domain, with the latter domain subsequently being lost in a small subset of these proteins.

GC content-based pan-pox universal PCR assays for poxvirus detection.

Chordopoxviruses of the subfamily Chordopoxvirinae, family Poxviridae, infect vertebrates and consist of at least eight genera with broad host ranges. For most chordopoxviruses, the number of viral genes and their relative order are highly conserved in the central region. The GC content of chordopoxvirus genomes, however, evolved into two distinct types: those with genome GC content of more than 60% and those with a content of less than 40% GC. Two standard PCR assays were developed to identify chordopoxviruses based on whether the target virus has a low or high GC content. In design of the assays, the genus Avipoxvirus, which encodes major rearrangements of gene clusters, was excluded. These pan-pox assays amplify DNA from more than 150 different isolates and strains, including from primary clinical materials, from all seven targeted genera of chordopoxviruses and four unclassified new poxvirus species. The pan-pox assays represent an important advance for the screening and diagnosis of human and animal poxvirus infections, and the technology used is accessible to many laboratories worldwide.

Characterization of a newly identified 35-amino-acid component of the vaccinia virus entry/fusion complex conserved in all chordopoxviruses.

The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function.