ICTV Virus Taxonomy Profile: Poxviridae 2023.

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae, whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae, which is available at ictv.global/report/poxviridae.

Inhibition of Poxvirus Gene Expression and Genome Replication by Bisbenzimide Derivatives.

Virus infection of humans and livestock can be devastating for individuals and populations, sometimes resulting in large economic and societal impact. Prevention of virus disease by vaccination or antiviral agents is difficult to achieve. A notable exception was the eradication of human smallpox by vaccination over 30 years ago. Today, humans and animals remain susceptible to poxvirus infections, including zoonotic poxvirus transmission. Here we identified a small molecule, bisbenzimide (bisbenzimidazole), and its derivatives as potent agents against prototypic poxvirus infection in cell culture. We show that bisbenzimide derivatives, which preferentially bind the minor groove of double-stranded DNA, inhibit vaccinia virus infection by blocking viral DNA replication and abrogating postreplicative intermediate and late gene transcription. The bisbenzimide derivatives are potent against vaccinia virus and other poxviruses but ineffective against a range of other DNA and RNA viruses. The bisbenzimide derivatives are the first inhibitors of their class, which appear to directly target the viral genome without affecting cell viability.IMPORTANCE Smallpox was one of the most devastating diseases in human history until it was eradicated by a worldwide vaccination campaign. Due to discontinuation of routine vaccination more than 30 years ago, the majority of today's human population remains susceptible to infection with poxviruses. Here we present a family of bisbenzimide (bisbenzimidazole) derivatives, known as Hoechst nuclear stains, with high potency against poxvirus infection. Results from a variety of assays used to dissect the poxvirus life cycle demonstrate that bisbenzimides inhibit viral gene expression and genome replication. These findings can lead to the development of novel antiviral drugs that target viral genomes and block viral replication.

Enhancing the toolbox to study IL-17A in cattle and sheep.

The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

Infection with Possible Novel Parapoxvirus in Horse, Finland, 2013.

A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus.

Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity.

Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) cells, and a more diffuse population of CD14(+)CD16(+)CD163(+) cells. In contrast, ovine monocytes consisted of only a major CD14(+)CD16(+) subset and a very low percentage of CD14(-)CD16(++)cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14(-)CD16(++) monocytes were CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14(+) monocytes. The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1ß, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14(-)CD16(++) cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

The genome of pseudocowpoxvirus: comparison of a reindeer isolate and a reference strain.

Parapoxviruses (PPV), of the family Poxviridae, cause a pustular cutaneous disease in sheep and goats (orf virus, ORFV) and cattle (pseudocowpoxvirus, PCPV and bovine papular stomatitis virus, BPSV). Here, we present the first genomic sequence of a reference strain of PCPV (VR634) along with the genomic sequence of a PPV (F00.120R) isolated in Finland from reindeer (Rangifer tarandus tarandus). The F00.120R and VR634 genomes are 135 and 145 kb in length and contain 131 and 134 putative genes, respectively, with their genome organization being similar to that of other PPVs. The predicted proteins of F00.120R and VR634 have an average amino acid sequence identity of over 95%, whereas they share only 88 and 73% amino acid identity with the ORFV and BPSV proteomes, respectively. The most notable differences were found near the genome termini. F00.120R lacks six and VR634 lacks three genes seen near the right terminus of other PPVs. Four genes at the left end of F00.120R and one in the middle of both genomes appear to be fragmented paralogues of other genes within the genome. VR634 has larger than expected inverted terminal repeats possibly as a result of genomic rearrangements. The high G+C content (64%) of these two viruses along with amino acid sequence comparisons and whole genome phylogenetic analyses confirm the classification of PCPV as a separate species within the genus Parapoxvirus and verify that the virus responsible for an outbreak of contagious stomatitis in reindeer over the winter of 1999-2000 can be classified as PCPV.

Population genomics of louping ill virus provide new insights into the evolution of tick-borne flaviviruses.

The emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the United Kingdom (UK). However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland, but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. We sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. The 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors.

Immunological Homeostasis at the Ovine Placenta May Reflect the Degree of Maternal Fetal Interaction.

Successful mammalian pregnancies are a result of complex physiological, endocrinological, and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents.

Immunity and counter-immunity during infection with the parapoxvirus orf virus.

Orf virus is a DNA parapoxvirus that causes orf, an acute debilitating skin disease of sheep, goats and humans. In sheep, a vigorous immune response involving neutrophils, dermal dendritic cells, T cells, B cells and antibody is generated after infection. CD4(+) T cells, IFN-gamma and to a lesser extent CD8(+) T cells are involved in partial protection against infection. In spite of this, orf virus can repeatedly infect sheep albeit with reduced lesion size and time to resolution compared to primary infection. This is due at least in part to the action of virus immuno-modulator proteins that interfere with host immune and inflammatory responses. These include: an interferon resistance protein; a viral orthologue of mammalian IL-10 (vIL-10) that is an anti-inflammatory cytokine; and a novel inhibitor of the cytokines GM-CSF and IL-2 (GIF). The virus also encodes a virulence protein that is an orthologue of mammalian vascular endothelial growth factor. The study of the immuno-modulator proteins provides an insight into disease pathogenesis and important elements of a host protective response. This information will be used to devise a rational disease control strategy.

A comparison of the anti-inflammatory and immuno-stimulatory activities of orf virus and ovine interleukin-10.

Orf virus causes pustular skin lesions (orf) in sheep, goats and humans. The virus encodes an interleukin-10 (orfvIL-10) that is identical in amino acid composition to ovine IL-10 (ovIL-10) over the C terminal two-thirds of the polypeptide, but not in the N terminal third. The immuno-suppressive and immuno-stimulatory activities of orfvIL-10 and ovIL-10 were compared. Both orfvIL-10 and ovIL-10 inhibited TNF-alpha and IL-8 cytokine production from stimulated ovine macrophages and keratinocytes and IFN-gamma and GM-CSF production from peripheral blood lymphocytes. OrfvIL-10 and ovIL-10 co-stimulated both ovine and murine mast cell proliferation in conjunction with IL-3 (ovine) or IL-4 (murine). Isoleucine at position 87 (Ile(87)) of the mature human IL-10 (huIL-10) has been reported as essential for the immuno-stimulatory activity of huIL-10. In spite of the differences in amino acids within the N-terminal third of orfvIL-10 compared with ovIL-10 and substitution of Ile(87) with Ala(87) in ovIL-10, these variants of ovIL-10 and orfvIL-10 all co-stimulated mast cell proliferation and inhibited macrophage IL-8 production. As ovIL-10 and orfvIL-10 have a similar structure to huIL-10 and conserved receptor-binding residues, it was concluded that Ile(87) is not essential for IL-10 immuno-stimulatory activity. Finally, ovine keratinocytes do not express ovIL-10. This might explain why orf virus has evolved a viral IL-10.

Preliminary characterisation of Pentlands paramyxovirus-1, -2 and -3, three new paramyxoviruses of rodents.

A paramyxovirus was discovered by chance during the primary culture of grey squirrel (Sciurus carolinensis) kidney cells from the UK. Amplification, sequencing and phylogenetic analysis of part of the genome encoding a region of the RNA polymerase (L gene) confirmed that the virus was a member of the Paramyxovirinae subfamily, but that it did not partition with any of the currently recognised paramyxovirus genera and instead segregated with the unclassified rodent viruses, J-virus, Beilong virus and Tailam virus as well as paramyxoviruses recently detected in rodents in Africa. A subsequent examination of kidney samples from red squirrels (Sciurus vulgaris) revealed that they too harboured a paramyxovirus, but sequence analysis of the corresponding region of the L gene revealed that it was approximately 67% identical to the grey squirrel virus, suggesting the presence of a second species of virus. In addition, one of the red squirrels examined harboured a second virus with approximately 69% identity to the grey squirrel virus, but only approximately 63% identity to the other red squirrel viruses, signifying the presence of a third species of paramyxovirus. In a sample of 22 red and grey squirrels 68% of those examined were found to harbour virus suggesting that paramyxovirus infection in squirrels may be common within the UK.

Effects of parasitism and morphology on squirrelpox virus seroprevalence in grey squirrels (Sciurus carolinensis).

Invasive species have been cited as major causes of population extinctions in several animal and plant classes worldwide. The North American grey squirrel (Sciurus carolinensis) has a major detrimental effect on native red squirrel (Sciurus vulgaris) populations across Britain and Ireland, in part because it can be a reservoir host for the deadly squirrelpox virus (SQPV). Whilst various researchers have investigated the epizootiology of SQPV disease in grey squirrels and have modelled the consequent effects on red squirrel populations, less work has examined morphological and physiological characteristics that might make individual grey squirrels more susceptible to contracting SQPV. The current study investigated the putative relationships between morphology, parasitism, and SQPV exposure in grey squirrels. We found geographical, sex, and morphological differences in SQPV seroprevalence. In particular, larger animals, those with wide zygomatic arch widths (ZAW), males with large testes, and individuals with concurrent nematode and/or coccidial infections had an increased seroprevalence of SQPV. In addition, males with larger spleens, particularly those with narrow ZAW, were more likely to be exposed to SQPV. Overall these results show that there is variation in SQPV seroprevalence in grey squirrels and that, consequently, certain individual, or populations of, grey squirrels might be more responsible for transmitting SQPV to native red squirrel populations.

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.

European red squirrel population dynamics driven by squirrelpox at a gray squirrel invasion interface.

Infectious disease introduced by non-native species is increasingly cited as a facilitator of native population declines, but direct evidence may be lacking due to inadequate population and disease prevalence data surrounding an outbreak. Previous indirect evidence and theoretical models support squirrelpox virus (SQPV) as being potentially involved in the decline of red squirrels (Sciurus vulgaris) following the introduction of the non-native gray squirrel (Sciurus carolinensis) to the United Kingdom. The red squirrel is a major UK conservation concern and understanding its continuing decline is important for any attempt to mitigate the decline. The red squirrel-gray squirrel system is also exemplary of the interplay between infectious disease (apparent competition) and direct competition in driving the replacement of a native by an invasive species. Time series data from Merseyside are presented on squirrel abundance and squirrelpox disease (SQPx) incidence, to determine the effect of the pathogen and the non-native species on the native red squirrel populations. Analysis indicates that SQPx in red squirrels has a significant negative impact on squirrel densities and their population growth rate (PGR). There is little evidence for a direct gray squirrel impact; only gray squirrel presence (but not density) proved to influence red squirrel density, but not red squirrel PGR. The dynamics of red SQPx cases are largely determined by previous red SQPx cases, although previous infection of local gray squirrels also feature, and thus, SQPV-infected gray squirrels are identified as potentially initiating outbreaks of SQPx in red squirrels. Retrospective serology indicates that approximately 8% of red squirrels exposed to SQPV may survive infection during an epidemic. This study further highlights the UK red squirrel - gray squirrel system as a classic example of a native species population decline strongly facilitated by infectious disease introduced by a non-native species. It is therefore paramount that disease prevention and control measures are integral in attempts to conserve red squirrels in the United Kingdom.

Characterization of a novel poxvirus in a North American red squirrel (Tamiasciurus hudsonicus).

In 2009, a novel poxvirus was identified in a North American red squirrel (Tamiasciurus hudsonicus) from Yukon, Canada. Initial molecular analyses indicated that this virus was likely to be distinct from all other known mammalian poxviruses, including those previously associated with disease in tree squirrels--squirrel fibroma virus in North America and squirrelpox virus in the UK (UK SQPV). We characterize the Canadian squirrelpox virus (Canadian SQPV) using DNA sequence analysis and negative-contrast transmission electron microscopy (TEM). Sequence analysis revealed that the Canadian SQPV is distinct from all known mammalian poxviruses but most closely related to the parapoxviruses, followed by UK SQPV. In contrast, TEM showed that the ultrastructure of Canadian SQPV is distinct from that of the parapoxviruses and UK SQPV but indistinguishable from that of other chordopoxviruses (a morphological group that includes the orthopoxviruses and leporipoxviruses). Overall, our analyses suggest a potential evolutionary relationship between UK SQPV and Canadian SQPV and supports our assertion that the Canadian virus represents a newly identified poxvirus in North American tree squirrels.

Analysis of deletion within the reindeer pseudocowpoxvirus genome.

Cases of contagious pustular stomatitis have been reported in Finnish reindeer for many years. Two species of the genus Parapoxvirus of the family Poxviridae have been identified as the causative agent of the disease; orf virus (ORFV) was found during the 1992-1993 epidemic and pseudocowpoxvirus (PCPV) was connected to the 1999-2000 epidemic. The genome of reindeer parapoxvirus from the latter outbreak, isolate F00.120R, was recently sequenced and confirmed as PCPV. The six gene deletion of the right terminus of the F00.120R genome, in comparison to ORFV, was investigated in an attempt to use it in differentiating viruses causing pustular stomatitis in reindeer. The present study describes discovery and analysis of genes 116-121 in reindeer PCPV and in an Italian field isolate of bovine PCPV. The results show that a 5431 bp sequence containing genes 116-121 was likely to have been deleted from the F00.120R genome between the 6th and 7th passage in cell culture, and that these genes are present in other isolates of reindeer and bovine PCPV isolated in Finland during the years 2005-2010. The data presented here extends our knowledge of the PCPV genome, confirming that it contains homologues of all known ORFV genes and further reinforces their close genetic relationship. The similarity between the EEV envelope and GM-CSF inhibitory factor genes from reindeer PCPV and ORFV isolates, Finnish sheep ORFV and cattle PCPV isolates indicate that these viruses have been circulating among Finnish reindeer, cattle and sheep over a long period of time.

Specific antibodies induced by inactivated parapoxvirus ovis potently enhance oxidative burst in canine blood polymorphonuclear leukocytes and monocytes.

We have recently shown that inactivated parapoxvirus ovis (iPPVO) effectively stimulates canine blood phagocytes. However, a potential link between innate and adaptive immunity induced by iPPVO remained open. The objective of this study was to define the effects of repeated iPPVO treatment of dogs to evaluate (i) iPPVO-specific antibody production, and (ii) modulation of iPPVO-induced oxidative burst by anti-iPPVO antibodies. Serum analysis of dogs treated repeatedly with iPPVO (Zylexis) showed transient production of non-neutralising iPPVO-specific IgG. There was a correlation between iPPVO-specific IgG levels and enhanced oxidative burst rates in vitro upon transfer of immune sera. Even four years after Zylexis treatment considerably stronger oxidative burst rates in response to iPPVO were observed in monocytes and PMN, whereas only moderate burst rates were detected in monocytes, but not in PMN, from dogs treated with a placebo. Depletion of serum IgG by protein A-sepharose or by parapoxvirus ovis coupled to sepharose abolished the increase of oxidative burst responses and resulted in burst rates similar to blood leukocytes from control dogs. However, uptake of viral particles was found to be independent of iPPVO-specific IgG and restricted to cells with dendritic and monocytic morphology. These data demonstrate that non-neutralising iPPVO-specific IgG is produced during treatment with Zylexis. Moreover, for the first time the interaction of iPPVO with antibodies is shown to enhance oxidative burst.