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Zoonotic orthopoxvirus infections continue to represent a threat to human health. The disease caused by distinct orthopoxviruses differs in terms of symptoms and severity, which may be explained by the unique repertoire of virus factors that modulate the host's immune response and cellular machinery. We report here on the construction of recombinant cowpox viruses (CPXV) which either lack the host range factor p28 completely or express truncated variants of p28. We show that p28 is essential for CPXV replication in macrophages of human or mouse origin and that the C-terminal RING finger domain of p28 is necessary to allow CPXV replication in macrophages., (© 2021. The Author(s).)

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 10 5 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD 50 . The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis., Competing Interests: Declaration of interests A U.S. provisional patent application (no. 62/848,255) has been filed for this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family and is endemic in western Eurasia. Based on seroprevalence studies in different voles from continental Europe and UK, voles are suspected to be the major reservoir host. Recently, a CPXV was isolated from a bank vole ( Myodes glareolus ) in Germany that showed a high genetic similarity to another isolate originating from a Cotton-top tamarin ( Saguinus oedipus ). Here we characterize this first bank vole-derived CPXV isolate in comparison to the related tamarin-derived isolate. Both isolates grouped genetically within the provisionally called CPXV-like 3 clade. Previous phylogenetic analysis indicated that CPXV is polyphyletic and CPXV-like 3 clade represents probably a different species if categorized by the rules used for other orthopoxviruses. Experimental infection studies with bank voles, common voles ( Microtus arvalis ) and Wistar rats showed very clear differences. The bank vole isolate was avirulent in both common voles and Wistar rats with seroconversion seen only in the rats. In contrast, inoculated bank voles exhibited viral shedding and seroconversion for both tested CPXV isolates. In addition, bank voles infected with the tamarin-derived isolate experienced a marked weight loss. Our findings allow for the conclusion that CPXV isolates might differ in their replication capacity in different vole species and rats depending on their original host. Moreover, the results indicate host-specific differences concerning CPXV-specific virulence. Further experiments are needed to identify individual virulence and host factors involved in the susceptibility and outcome of CPXV-infections in the different reservoir hosts., Competing Interests: The authors declare no conflict of interest.

Four cowpox virus (CPXV) outbreaks occurred in unrelated alpaca herds in Eastern Germany during 2012-2017. All incidents were initially noticed due to severe, generalized, and finally lethal CPXV infections, which were confirmed by testing of tissue and serum samples. As CPXV-infection has been described in South American camelids (SACs) only three times, all four herds were investigated to gain a deeper understanding of CPXV epidemiology in alpacas. The different herds were investigated twice, and various samples (serum, swab samples, and crusts of suspicious pox lesions, feces) were taken to identify additionally infected animals. Serum was used to detect CPXV-specific antibodies by performing an indirect immunofluorescence assay (iIFA); swab samples, crusts, and feces were used for detection of CPXV-specific DNA in a real-time PCR. In total, 28 out of 107 animals could be identified as affected by CPXV, by iIFA and/or PCR. Herd seroprevalence ranged from 16.1% to 81.2%. To investigate the potential source of infection, wild small mammals were trapped around all alpaca herds. In two herds, CPXV-specific antibodies were found in the local rodent population. In the third herd, CPXV could be isolated from a common vole ( Microtus arvalis ) found drowned in a water bucket used to water the alpacas. Full genome sequencing and comparison with the genome of a CPXV from an alpaca from the same herd reveal 99.997% identity, providing further evidence that the common vole is a reservoir host and infection source of CPXV. Only in the remaining fourth herd, none of the trapped rodents were found to be CPXV-infected. Rodents, as ubiquitous reservoir hosts, in combination with increasingly popular alpacas, as susceptible species, suggest an enhanced risk of future zoonotic infections., Competing Interests: The authors declare no conflict of interest.

Cowpox virus infections in captive cheetahs (Acinonyx jubatus) with high morbidity and mortality have already been reported in the UK and Russia in the 1970s. However, most of the reported cases have been singular events. Here, we report a total of five cowpox virus outbreaks in cheetahs in the same safari park in Denmark between 2010 and 2014. Nine cheetahs showed varying severity of clinical disease; two of them died (22%). All episodes occurred between August and October of the respective year. No other carnivores kept at the same institution nor the keepers taking care of the animals were clinically affected. The clinical picture of cowpox was confirmed by extensive laboratory investigations including histopathological and molecular analyses as well as cell culture isolation of a cowpox virus. High anti-orthopoxvirus antibody titers were detected in all 9 diseased cheetahs compared to seven contact cheetahs without clinical signs and 13 cheetahs not in direct contact. Additionally, whole genome sequencing from one sample of each cluster with subsequent phylogenetic analysis showed that the viruses from different outbreaks have individual sequences but clearly form a clade distinct from other cowpox viruses. However, the intra-clade distances are still larger than those usually observed within clades of one event. These findings indicate multiple and separate introductions of cowpox virus, probably from wild rodent populations, where the virus keeps circulating naturally and is only sporadically introduced into the cheetahs. Sero-positivity of voles (Arvicola amphibious) caught in zoo grounds strengthens this hypothesis. As a consequence, recommendations are given for medical and physical management of diseased cheetahs, for hygienic measures as well as for pre-shipment isolation before cheetah export from zoo grounds.

MxA protein is expressed in response to type I and type III Interferon and constitute an important antiviral factor with broad antiviral activity to diverse RNA viruses. In addition, some studies expand the range of MxA antiviral activity to include particular DNA viruses like Monkeypox virus (MPXV) and African Swine Fever virus (ASFV). However, a broad profile of activity of MxA to large DNA viruses has not been established to date. Here, we investigated if some well characterized DNA viruses belonging to the Poxviridae family are sensitive to human MxA. A cell line inducibly expressing MxA to inhibitory levels showed no anti-Vaccinia virus (VACV) virus activity, indicating either lack of susceptibility of the virus, or the existence of viral factors capable of counteracting MxA inhibition. To determine if VACV resistance to MxA was due to a virus-encoded anti-MxA activity, we performed coinfections of VACV and the MxA-sensitive Vesicular Stomatitis virus (VSV), and show that VACV does not protect VSV from MxA inhibition in trans. Those results were extended to several VACV strains and two CPXV strains, thus confirming that those Orthopoxviruses do not block MxA action. Overall, these results point to a lack of susceptibility of the Poxviridae to MxA antiviral activity.

Human cowpox virus (CPXV) infections are rare, but can result in severe and sometimes fatal outcomes. The majority of recent cases were traced back to contacts with infected domestic cats or pet rats. The aim of the present study was to evaluate a three-dimensional (3D) skin model as a possible replacement for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with two CPXV strains of different pathogenic potential: a recent pet rat isolate (RatPox09) and the reference Brighton red strain. Infected 3D skin cultures exhibited histological alterations that were similar to those of mammal skin infections, but there were no differences in gene expression patterns and tissue damage between the two CPXV strains in the model system. In conclusion, 3D skin cultures reflect the development of pox lesions in the skin very well, but seem not to allow differentiation between more or less virulent virus strains, a distinction that is made possible by experimental infection in suitable animal models., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus' zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing.

Unlabelled: The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species., Importance: We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Most poxvirus proteins are either highly conserved and essential for basic steps in replication or less conserved and involved in host interactions. Homologs of the CPXV219 protein, encoded by cowpox virus, are present in nearly all chordopoxvirus genera and some species have multiple copies. The CPXV219 homologs have estimated masses of greater than 200 kDa, making them the largest known poxvirus proteins. We showed that CPXV219 was expressed early in infection and cleaved into N- and C-terminal fragments that remained associated. The protein has a signal peptide and transited the secretory pathway where extensive glycosylation and proteolytic cleavage occurred. CPXV219 was located by immunofluorescence microscopy in association with the endoplasmic reticulum, Golgi apparatus and plasma membrane. In non-permeabilized cells, CPXV219 was accessible to external antibody and biotinylation. Mutants that did not express CPXV219 replicated normally in cell culture and retained virulence in a mouse respiratory infection model., (Published by Elsevier Inc.)

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases., (Published by Elsevier Inc.)

Objectives: ST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target., Methods: Vaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity., Results: Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences., Conclusions: Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown., Importance: It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virus CrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Some orthopoxviruses including cowpox virus embed virus particles in dense bodies, comprised of the A-type inclusion (ATI) protein, which may provide long-term environmental protection. This strategy could be beneficial if the host population is sparse or spread is inefficient or indirect. However, the formation of ATI may be neutral or disadvantageous for orthopoxviruses that rely on direct respiratory spread. Disrupted ATI open reading frames in orthopoxviruses such as variola virus, the agent of smallpox, and monkeypox virus suggests that loss of this feature provided positive selection. To test this hypothesis, we constructed cowpox virus mutants with deletion of the ATI gene or another gene required for embedding virions. The ATI deletion mutant caused greater weight loss and higher replication in the respiratory tract than control viruses, supporting our hypothesis. Deletion of the gene for embedding virions had a lesser effect, possibly due to known additional functions of the encoded protein., (Published by Elsevier Inc.)

Classical inbred mice are extensively used for virus research. However, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. Experiments described here indicated that the 50% lethal dose of vaccinia virus (VACV) and cowpox virus (CPXV) were two logs lower in wild-derived inbred CAST/Ei mice than classical inbred BALB/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex virus. Live bioluminescence imaging was used to follow spread of pathogenic and attenuated VACV strains and CPXV virus from nasal passages to organs in the chest and abdomen of CAST/Ei mice. Luminescence increased first in the head and then simultaneously in the chest and abdomen in a dose-dependent manner. The spreading kinetics was more rapid with VACV than CPXV although the peak photon flux was similar. These data suggest advantages of CAST/Ei mice for orthopoxvirus studies., (© 2013 Published by Elsevier Inc.)

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family. It infects a broad range of vertebrates and can cause zoonotic infections. CPXV has the largest genome among the orthopoxviruses and is therefore considered to have the most complete set of genes of all members of the genus. Since CPXV has also become a model for studying poxvirus genetics and pathogenesis, we created and characterized a complete set of single gene knockout bacterial artificial chromosome (BAC) clones of the CPXV strain Brighton Red. These mutants allow a systematic assessment of the contribution of single CPXV genes to the outcome of virus infection and replication, as well as to the virus host range. A full-length BAC clone of CPXV strain Brighton Red (pBRF) harboring the gene expressing the enhanced green fluorescent protein under the control of a viral late promoter was modified by introducing the mrfp1 gene encoding the monomeric red fluorescent protein driven by a synthetic early vaccinia virus promoter. Based on the modified BAC (pBRFseR), a library of targeted knockout mutants for each single viral open reading frame (ORF) was generated. Reconstitution of infectious virus was successful for 109 of the 183 mutant BAC clones, indicating that the deleted genes are not essential for virus replication. In contrast, 74 ORFs were identified as essential because no virus progeny was obtained upon transfection of the mutant BAC clones and in the presence of a helper virus. More than 70% of all late CPXV genes belonged to this latter group of essential genes.

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Orthopoxvirus vector has a broad prospect in recombinant vaccine research, but the rarely severe side-effect impedes its development. Vaccinia virus and Cowpox virus of Orthopoxvirus have broad host range, and they have typical host range genes as K1L, CP77 and C7L. These three genes affect host range of Vaccinia virus, disturb the cell signaling pathways, suppress immune response and are related to virulence.

The last years, cowpox infections are being increasingly reported through Eurasia. Cowpox viruses (CPXVs) have been reported to have different genotypes and may be subdivided in at least five genetically distinct monophyletic clusters. However, little is known about their in vitro and in vivo features. In this report, five genetically diverse CPXVs, including one reference strain (CPXV strain Brighton) and four clinical isolates from human and animal cases, were compared with regard to growth in cells, pathogenicity in mice and inhibition by antivirals. While all CPXVs replicated similarly in vitro and showed comparable antiviral susceptibility, marked discrepancies were seen in vivo, including differences in virulence with recorded mortality rates of 0%, 20% and 100%. The four CPXV clinical isolates appeared less pathogenic than two reference strains, CPXV Brighton and vaccinia virus Western-Reserve. Disease severity seemed to correlate with high viral DNA loads in several organs, virus titers in lung tissues and levels of IL-6 cytokine in the sera. Our study highlighted that the species CPXV consists of viruses that not only differ considerably in their genotypes but also in their in vivo phenotypes, indicating that CPXVs should not be longer classified as a single species. Lung virus titers and IL-6 cytokine level in mice may be used as biomarkers for predicting disease severity. We further demonstrated the potential benefit of cidofovir, CMX001 and ST-246 use as antiviral therapy.

Vaccinia virus (VACV) enters cells by a low pH endosomal route or by direct fusion with the plasma membrane. We previously found differences in entry properties of several VACV strains: entry of WR was enhanced by low pH, reduced by bafilomycin A1 and relatively unaffected by heparin, whereas entry of IHD-J, Copenhagen and Elstree were oppositely affected. Since binding and entry modes may have been selected by specific conditions of in vitro propagation, we now examined the properties of three distinct, recently isolated cowpox viruses and a monkeypox virus as well as additional VACV and cowpox virus strains. The recent isolates were more similar to WR than to other VACV strains, underscoring the biological importance of endosomal entry by orthopoxviruses. Sequence comparisons, gene deletions and gene swapping experiments indicated that viral determinants, other than or in addition to the A26 and A25 "fusion-suppressor" proteins, impact entry properties., (Published by Elsevier Inc.)

Recently, several cases of human cowpox virus (CPXV) infections were reported in France and Germany, which had been acquired through close contact with infected pet rats. The animals exhibited respiratory signs or skin lesions and died shortly after purchase. After natural infection of white rats with CPXV in the USSR in 1978, a peracute pulmonary form, a milder dermal form, and a mixed form exhibiting features of both have been described. To the best of the authors' knowledge, 3 experimental cowpox virus infection studies using rats have been performed to date; however, neither results of histomorphological examinations nor immunohistochemical analyses have yet been reported in rats after experimental infections. To investigate the impact of the infection route on the clinical course, the development of lesions, and tropism, rats were infected intradermally, intranasally, or by a combination of both routes. The authors found a correlation between clinical manifestation, pathology, and infection routes. Intradermal and contact exposure yielded a mild dermal form, characterized by the development of vesiculopustular dermatitis. In contrast, intranasally infected animals died peracutely, showing severe dyspnea. Occasionally, a combination of the dermal and the respiratory form occurred after intranasal infection. Immunohistochemically, CPXV antigen was detected in the epithelial and mesenchymal cells of the upper respiratory tract and affected skin lesions and rarely in mesenchymal cells of lymph nodes. This is the first histomorphological and immunohistochemical analysis of CPXV in rats after experimental infection.

Cowpox virus (CPXV) host range factor CP77 was identified to be required for virus replication in Chinese hamster ovary (CHO) cells, but the underlying molecular mechanism by which CP77 modulates host range has remained unclear. Therefore, a CPXVΔCP77 deletion mutant was constructed by applying bacterial artificial chromosome (BAC) technology. Integrity of BAC-derived viral DNA was confirmed by whole genome sequencing. In vitro growth characteristics of CPXV wild type (WT), BAC-derived vCPXV WT and vCPXVΔCP77 were virtually indistinguishable in HEK293T cells, whereas in CHO-K1 cells replication of virus lacking CP77 was unambiguously attenuated. This block of viral replication was confirmed by lack of late viral protein expression. The replication defect of various Orthopoxviruses lacking CP77 in CHO cells could be restored by recombinant expression of CP77. Thus, for the first time, the described CP77-dependent host range effect in CHO cells was shown in the background of CPXV as well as Camelpox virus. To further characterize the mutant virus, cells of several different species were comparably infected with vCPXV WT and vCPXVΔCP77, respectively. Interestingly, except for CHO-K1 cells, vCPXV WT and vCPXVΔCP77 showed no significant difference in terms of morphology of cytopathic effects, expression of a late transcribed virus-encoded green fluorescent protein and virus reproduction, even in other hamster-derived cells. Additionally, in ovo inoculation with either virus revealed the same red-pock phenotype on chicken egg chorioallantoic membranes. Since the data presented indicate a CP77-dependent host range effect only for CHO cells, we conclude that the protein might mediate additional functions not identified yet. The vCPXVΔCP77 deletion mutant generated can now be applied as a useful tool to investigate the function of the putative host range protein CP77., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Features of spread of cowpox in the contemporary conditions are examined. A decrease of population immunity to pox in the population of Russia caused by cancellation of pox immunization, hidden circulation of cowpox virus in various species of rodents, as well as lack of vigilance to pathogenic orthopoxviurses in healthcare workers were noted to create the real preconditions for the emergence of infection of humans caused by cowpox virus. Thereby presence of means of express laboratory diagnostics of cowpox and means of effective medical protection for the prevention of development of this disease in the population of Russia becomes an actual necessity.

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC(50) of 0.25 μM against cowpox and 0.8 μM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5 mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5 mg/kg mitoxantrone and 100 mg/kg cidofovir than with 100 mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model., (Published by Elsevier B.V.)

The public health threat of orthopoxviruses from bioterrorist attacks has prompted researchers to develop suitable animal models for increasing our understanding of viral pathogenesis and evaluation of medical countermeasures (MCMs) in compliance with the FDA Animal Efficacy Rule. We present an accessible intrabronchial cowpox virus (CPXV) model that can be evaluated under biosafety level-2 laboratory conditions. In this dose-ranging study, utilizing cynomolgus macaques, signs of typical orthopoxvirus disease were observed with the lymphoid organs, liver, skin (generally mild) and respiratory tract as target tissues. Clinical and histopathological evaluation suggests that intrabronchial CPXV recapitulated many of the features of monkeypox and variola virus, the causative agent of smallpox, infections in cynomolgus macaque models. These similarities suggest that CPXV infection in non-human primates should be pursued further as an alternative model of smallpox. Further development of the CPXV primate model, unimpeded by select agent and biocontainment restrictions, should facilitate the development of MCMs for smallpox.

A pet rat derived cowpox virus strain, which was also the source of human infections, was used to infect young Wistar and fancy rats. After an incubation period of 6 days the animals developed a severe, often fatal disease with high amounts of virus detected in oropharyngeal secretions., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hemorrhagic smallpox was a rare but severe manifestation of variola virus infection that resulted in nearly 100% mortality. Here we describe intravenous (IV) inoculation of cowpox virus Brighton Red strain in cynomolgus macaques (Macaca fascicularis) which resulted in disease similar in presentation to hemorrhagic smallpox in humans. IV inoculation of macaques resulted in a uniformly lethal disease within 12 days post-inoculation in two independent experiments. Clinical observations and hematological and histopathological findings support hemorrhagic disease. Cowpox virus replicated to high levels in blood (8.0-9.0 log(10) gene copies/mL) and tissues including lymph nodes, thymus, spleen, bone marrow, and lungs. This unique model of hemorrhagic orthopoxvirus infection provides an accessible means to further study orthopoxvirus pathogenesis and to identify virus-specific and nonspecific therapies. Such studies will serve to complement the existing nonhuman primate models of more classical poxviral disease., (Published by Elsevier Inc.)

Cowpox virus infection induces interleukin-10 (IL-10) production from mouse bone marrow-derived dendritic cells (BMDCs) or cells of the mouse macrophage line (RAW264.7) at about 1800 pg/ml, whereas infections with vaccinia virus (strains WR or MVA) induced much less IL-10. Similarly, in vivo, IL-10 levels in bronchoalveolar lavage fluids of mice infected with cowpox virus were significantly higher than those after vaccinia virus infection. However, after intranasal cowpox virus infection, although dendritic and T-cell accumulations in the lungs of IL-10 deficient mice were greater than those in wild-type mice, weight-loss and viral burdens were not significantly different. IL-10 deficient mice were more susceptible than wild-type mice to re-infection with cowpox virus even though titers of neutralizing antibodies and virus-specific CD8 T cells were similar between IL-10 deficient and wild-type mice. Greater bronchopneumonia in IL-10 deficient mice than wild-type mice suggests that IL-10 contributes to the suppression of immunopathology in the lungs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

Terameprocol (TMP) is a methylated derivative of nordihydroguaiaretic acid, a phenolic antioxidant originally derived from creosote bush extracts. TMP has previously been shown to have antiviral and anti-inflammatory activities, and has been proven safe in phase I clinical trials conducted to evaluate TMP as both a topical and parenteral therapeutic. In the current study, we examined the ability of TMP to inhibit poxvirus growth in vitro, and found that TMP potently inhibited the growth of both cowpox virus and vaccinia virus in a variety of cell lines. TMP treatment was highly effective at reducing infectious virus yield in multi-step virus growth assays, but it did not substantially inhibit the synthesis of infectious progeny viruses in individual infected cells. These contrasting results showed that TMP inhibits poxvirus growth in vitro by preventing the efficient spread of virus particles from cell to cell. The canonical mechanism of poxvirus cell-to-cell spread requires morphogenesis of cell-associated, enveloped virions. The virions then trigger the formation of actin tails to project them from the cell surface. The number of actin tails present at the surface of poxvirus-infected cells was reduced dramatically by treatment with TMP. Whether TMP inhibits poxvirus morphogenesis, or subsequent events required for actin tail formation, remains to be determined. The results of this study, together with the clinical safety record of TMP, support further evaluation of TMP as a poxvirus therapeutic., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Downregulation of MHC class I on the cell surface is an immune evasion mechanism shared by many DNA viruses, including cowpox virus. Previously, a cowpox virus protein, CPXV203, was shown to downregulate MHC class I. Here we report that CPXV12 is the only other MHC class I-regulating protein of cowpox virus and that it uses a mechanism distinct from that of CPXV203. Whereas CPXV203 retains fully assembled MHC class I by exploiting the KDEL-mediated endoplasmic reticulum retention pathway, CPXV12 binds to the peptide-loading complex and inhibits peptide loading on MHC class I molecules. Viruses deleted of both CPXV12 and CPXV203 demonstrated attenuated virulence in a CD8 T cell-dependent manner. These data demonstrate that CPXV12 and CPXV203 proteins combine to ablate MHC class I expression and abrogate antiviral CD8 T cell responses.

Cowpox virus encodes an extensive array of putative immunomodulatory proteins, likely contributing to its wide host range, which includes zoonotic infections in humans. Unlike Vaccinia virus, cowpox virus prevents stimulation of CD8(+) T cells, a block that correlated with retention of MHC class I in the endoplasmic reticulum by the cowpox virus protein CPXV203. However, deletion of CPXV203 did not restore MHC class I transport or T cell stimulation. Here, we demonstrate the contribution of an additional viral protein, CPXV12, which interferes with MHC class I/peptide complex formation by inhibiting peptide translocation by the transporter associated with antigen processing (TAP). Importantly, human and mouse MHC class I transport and T cell stimulation was restored upon deletion of both CPXV12 and CPXV203, suggesting that these unrelated proteins independently mediate T cell evasion in multiple hosts. CPXV12 is a truncated version of a putative NK cell ligand, indicating that poxviral gene fragments can encode new, unexpected functions.

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.

Background: Poxvirus-vectored vaccines against infectious diseases and cancer are currently under development. We hypothesized that the extensive use of poxvirus-vectored vaccine in future might result in co-infection and recombination between the vaccine virus and naturally occurring poxviruses, resulting in hybrid viruses with unpredictable characteristics. Previously, we confirmed that co-infecting in vitro a Modified vaccinia virus Ankara (MVA) strain engineered to express influenza virus haemagglutinin (HA) and nucleoprotein (NP) genes with a naturally occurring cowpox virus (CPXV-NOH1) resulted in recombinant progeny viruses (H Hansen, MI Okeke, Ø Nilssen, T Traavik, Vaccine 23: 499-506, 2004). In this study we analyzed the biological properties of parental and progeny hybrid viruses., Results: Five CPXV/MVA progeny viruses were isolated based on plaque phenotype and the expression of influenza virus HA protein. Progeny hybrid viruses displayed in vitro cell line tropism of CPXV-NOH1, but not that of MVA. The HA transgene or its expression was lost on serial passage of transgenic viruses and the speed at which HA expression was lost varied with cell lines. The HA transgene in the progeny viruses or its expression was stable in African Green Monkey derived Vero cells but became unstable in rat derived IEC-6 cells. Hybrid viruses lacking the HA transgene have higher levels of virus multiplication in mammalian cell lines and produced more enveloped virions than the transgene positive progenitor virus strain. Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein. The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles., Conclusion: Our results suggest that homologous recombination between poxvirus-vectored vaccine and naturally circulating poxviruses, genetic instability of the transgene, accumulation of non-transgene expressing vectors or hybrid virus progenies, as well as cell line/type specific selection against the transgene are potential complications that may result if poxvirus vectored vaccines are extensively used in animals and man.

The seasonality of recurrent epidemics has been largely neglected, especially where patterns are not driven by forces external to the population. Here, we use data on cowpox virus in field voles to explore the seasonal patterns in wildlife (variable abundance) populations and compare these with patterns previously found in humans. Timing in our system was associated with both the number and the rate of recruitment of susceptible hosts. A plentiful and sustained supply of susceptible hosts throughout the summer gave rise to a steady rise in infected hosts and a late peak. A meagre supply more limited in time was often insufficient to sustain an increase in infected hosts, leading to an early peak followed by a decline. These seasonal patterns differed from those found in humans, but the underlying association found between the timing and the supply of susceptible hosts was similar to that in humans. We also combine our data with a model to explore these differences between humans and wildlife. Model results emphasize the importance of the interplay between seasonal infection and recruitment and suggest that our empirical patterns have a relevance extending beyond our own system.

Diagnoses of ongoing viral infections commonly rely on PCR methodology. Sample material that may contain hazardous virus should be efficiently inactivated in biological containment or bed-side before diagnostic PCR analysis. Surprisingly little documentation is available for inactivation of human viral pathogens by inactivation reagents that allow for subsequent PCR diagnostics. It is now shown that pathogenic DNA viruses (orthopoxvirus) are completely inactivated by a commercially available Roche MagNA Pure lysis/binding buffer as evaluated by subsequent cell culture. However, inactivation reagents are typically toxic and therefore problematic in cell culture. Using the relatively large orthopoxvirus, a method was developed in which virus is precipitated by high-speed centrifugation after inactivation but prior to application onto the target cells, thereby eliminating the cytotoxic effect of the lysis buffer. The results from quantitative PCR analysis indicate that the viral DNA from the completely inactivated virus particles, remain associated to macromolecules and aggregates. The use of inactivation buffers for bed-side inactivation of special patient samples taken for PCR diagnostics should be considered in cases where high containment would otherwise be required.

Major histocompatibility complex (MHC) class I molecules assemble with peptides in the ER lumen and are transported via Golgi to the plasma membrane for recognition by T cells. Inhibiting MHC assembly, transport, and surface expression are common viral strategies of evading immune recognition. Cowpox virus, a clinically relevant orthopoxvirus, downregulates MHC class I expression on infected cells. However, the viral protein(s) and mechanisms responsible are unknown. We identify CPXV203 as a cowpox virus protein that associates with fully assembled MHC class I molecules and blocks their transport through the Golgi. A C-terminal KTEL motif in CPXV203 closely resembles the canonical ER retention motif KDEL and is required for CPXV203 function, indicating that a physiologic pathway is exploited to retain MHC class I in the ER. This viral mechanism for MHC class I downregulation may explain virulence differences between clinical isolates of orthopoxviruses.

Variola virus and other members of the genus Orthopoxviruses constitute a prominent bioterrorism and public health threat. Treatment with the anti-viral drug cidofovir inhibits replication of orthopoxviruses in vitro and in vivo. In this study, we visualized the effect of cidofovir on viral kinetics in orthopoxvirus infected mice by using whole-body fluorescence imaging (FI). We engineered a cowpox virus (CPV) expressing the enhanced green fluorescent protein (GFP). Single-step growth curves and calculated 50% lethal doses (LD(50)) of wild-type CPX (Wt-CPV) and GFP-expressing CPX (GFP-CPV) were comparable. Whole-body FI first detected GFP fluorescence in the mesenteric tissue of untreated animals on post-infection day (PID) 1. On PID 3 GFP signal was detected throughout the mesentery, in all abdominal organs by PID 5 and in most major organs, except for the heart and brain by PID 6. Infected animals treated with 25mg/kg of cidofovir also began showing signs of viral replication on PID 1, however, the fluorescent signal was limited only to discrete foci throughout the course of the infection. This work describes the first use of an established Orthopox model of infection to evaluate drug efficacy and track virus progression on a macroscopic level.

Appropriation of signalling pathways facilitates poxvirus replication. Poxviruses, as do most viruses, try to modify the host cell environment to achieve favourable replication conditions. In the present study, we show that the early growth response 1 gene (egr-1) is one of the host cell factors intensely modulated by the orthopoxviruses VV (vaccinia virus) and CPV (cowpox virus). These viruses stimulated the generation of both egr-1 mRNA and its gene product, throughout their entire replication cycles, via the requirement of MEK [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway. We showed that, upon VV infection, EGR-1 translocates into the nucleus where it binds to the EBS (egr-1-binding site) positioned at the 5' region of EGR-1-regulated genes. In spite of both viruses belonging to the same genus, several lines of evidence, however, revealed a remarkable contrast between them as far as the roles played by the MEK/ERK/EGR-1 pathway in their biological cycles are concerned. Hence (i) the knocking-down of egr-1 by siRNA (small interfering RNA) proved that this transcription factor is of critical relevance for VV biology, since a decrease of about one log cycle in virus yield was verified, along with a small virus plaque phenotype, whereas the gene silencing did not have a detrimental effect on either CPV multiplication or viral plaque size; (ii) while both pharmacological and genetic inhibition of MEK/ERK resulted in a significant decrease in VV yield, both approaches had no impact on CPV multiplication; and (iii) CPV DNA replication was unaffected by pharmacological inhibition of MEK/ERK, but phosphorylation of MEK/ERK was dependent on CPV DNA replication, contrasting with a significant VV DNA inhibition and VV DNA replication-independence to maintain ERK1/2 phosphorylation, observed under the same conditions.

It has been reported previously that some thiosemicarbazone compounds have prophylactic activity against smallpox disease and therapeutic activity against vaccinia virus (VV) infections. In these studies, isatin-beta-thiosemicarbazone (IBT) and marboran were administered once daily by intraperitoneal (ip) injection to mice using 30, 10 or 3 mg/kg for 5 days beginning 24, 48 or 72 h after inoculation with VV or cowpox virus (CV). Both compounds were highly effective (p < 0.01) at preventing mortality due to VV even when treatment was delayed up to 72 h postinfection. In CV-infected mice, neither IBT nor Marboran were effective in preventing mortality at any dosage tested when administered at 24 h postinoculation. Viral replication in liver, spleen and kidney was delayed or reduced by 100-to 10,000-fold by 10 mg/kg of marboran, but not IBT, in VV infections. Neither compound was effective against CV infection. Neither IBT nor marboran treatment of mice cutaneously infected with VV or CV reduced viral replication or clinical disease. These results suggest that this class of compound has little therapeutic potential for orthopoxvirus infections since the in vivo activity against CV, a surrogate virus for variola, is lacking.

The serpin SPI-3 and the hemagglutinin (HA) encoded by cowpox virus (CPV) block cell-cell fusion, and colocalize at the cell surface. wtCPV does not fuse cells, but inactivation of either gene leads to fusion. SPI-3 mAb added to wtCPV-infected cells caused fusion, confirming that SPI-3 protein at the cell surface prevents fusion. The SPI-3 mAb epitope mapped to an 85-amino acid region at the C-terminus. Removal of either 44 residues from the SPI-3 C-terminus or 48 residues following the N-terminal signal sequence resulted in fusion. Interaction between SPI-3 and HA proteins in infected cells was shown by coimmunoprecipitation. SPI-3/HA was not associated with the A27L "fusion" protein. SPI-3 and HA were able to associate in uninfected cells in the absence of other viral proteins. The HA-binding domain in SPI-3 resided in the C-terminal 229 residues, and did not include helix D, which mediates cofactor interaction in many other serpins.

Electron microscopy was used to study the reproduction of cowpox virus strain EP-2 in the cells of a primary fibroblast cultures (PFC) and chorion-allantoic membrane (CAM) of chick embryos (CE). The sequential stages of viral morphogenesis and the structure of A-type inclusions were described. The parameters of viral reproduction in PFC and CE CAM were compared. The formation of crystalloid tubular structures in PFC, unusual electron dense inclusions in the cells of CE CAN, and different variants of A-type inclusions in the cells of a pock was found. The histological and ultrastructural characteristics of pocks in CE CAM are described.

Cowpox virus (CPXV) strain GRI-90 contains six genes encoding kelch-like proteins. All six proteins contain both, the N-terminal BTB domain and the C-terminal kelch domain. We constructed mutant variants of a CPXV strain with targeted deletions of one to four genes of the kelch family, namely D11L, C18L, G3L, and A57R. As kelch genes are located in terminal variable regions of the CPXV genome, we studied the relationship of these genes with integral biological characteristics such as virulence, host range, reproduction in vitro and in ovo (in chicken embryos). It was demonstrated that the following effects occurred in a gene dose dependent manner with an increase of the number of genes deleted: (1) range of sensitive cells altered--deletion mutants lacking three genes displayed a considerably decreased ability to reproduce in MDCK cells; mutants lacking four genes lost this ability completely; (2) analysis of pocks formed by mutants with deletion of three and four kelch-like genes on chorioallantoic membranes of chicken embryos demonstrated that pock size and virus yield were significantly decreased; (3) light microscopic analysis of the pocks revealed impaired proliferation and reduced vascularisation in the pock region. More alterations were detected by electron microscopic analysis: the reproduction of mutants results in a reduction of the number of mature virions formed, and in many cells this process was arrested at the stage of assembly of immature virions; and (4) the evaluation of LD(50) and body weight loss in BALB/c mice infected intranasally with CPXVs revealed a reduction of the virulence of the deletion mutants, which became statistically significant when four kelch-like genes were excised.

The specific features of reproduction of EP-2 strain of cowpox virus (CPV) were studied in intranasally infected BALC/C mice by light and electron microscopy. Virus replication was found in the ciliated, intercalary, basal, and goblet cells (the nasal respiratory area), basal and supporting cells (the nasal olfactory area), ciliated, intercalary, goblet cells (the tracheal and bronchial epithelium), and collagen-producing, Schwann's, endothelial, smooth muscle, and adventitial cells. It has been shown that the CPV strain EP-2 locally replicates in the nasal cavity, trachea, and large bronchi and that there is no generalized infection.

The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments.

The cowpox virus (CPV) glycoprotein serpin SPI-3, a functional protease inhibitor, and the viral hemagglutinin (HA) are required to prevent fusion of wt CPV infected cells. SPI-3 and HA from CPV infected cells co-localize to the plasma membrane and are found in extracellular enveloped virus (EEV). We also show that an N-terminal SPI-3 signal sequence, but not glycosylation, is required for membrane localization and fusion inhibition. In the absence of HA (CPVDeltaHA), no SPI-3 is found on the membrane and infected cells fuse. Conversely, HA from both wt CPV and CPVDeltaSPI-3 infections is on the membrane, indicating a requirement of HA for SPI-3 plasma membrane localization. In the absence of HA, secretion of SPI-3 or SPI-3 N-glyc(-) was markedly enhanced, suggesting HA serves to retain SPI-3 on the plasma membrane,thereby preventing cell fusion.

Cowpox virus (Brighton Red strain) possesses one of the largest genomes in the Orthopoxvirus genus. Sequence analysis of a region of the genome that is type-specific for cowpox virus identified a gene, vCD30, encoding a soluble, secreted protein that is the fifth member of the tumor necrosis factor receptor family known to be encoded by cowpox virus. The vCD30 protein contains 110 aa, including a 21-residue signal peptide, a potential O-linked glycosylation site, and a 58-aa sequence sharing 51-59% identity with highly conserved extracellular segments of both mouse and human CD30. A vCD30Fc fusion protein binds CD153 (CD30 ligand) specifically, and it completely inhibits CD153/CD30 interactions. Although the functions of CD30 are not well understood, the existence of vCD30 suggests that the cellular receptor plays a significant role in normal immune responses. Viral inhibition of CD30 also lends support to the potential therapeutic value of targeting CD30 in human inflammatory and autoimmune diseases.

Cidofovir ([(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] [HPMPC])-resistant forms of camelpox, cowpox, monkeypox, and vaccinia viruses were developed by prolonged passage in Vero 76 cells in the presence of drug. Eight- to 27-fold-higher concentrations of cidofovir were required to inhibit the resistant viruses than were needed to inhibit the wild-type (WT) viruses. Resistant viruses were characterized by determining their cross-resistance to other antiviral compounds, examining their different replication abilities in two cell lines, studying the biochemical basis of their drug resistance, and assessing the degrees of their virulence in mice. These viruses were cross resistant to cyclic HPMPC and, with the exception of vaccinia virus, to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. Three of the four resistant cowpox and monkeypox viruses exhibited reduced abilities to infect and replicate in 3T3 cells compared to their abilities in Vero 76 cells. Compared to the WT virus polymers the resistant cowpox virus DNA polymerase was 8.5-fold less sensitive to inhibition by cidofovir diphosphate, the active form of the drug. Intracellular phosphorylation of [3H]cidofovir was not stimulated or inhibited by infection with resistant cowpox virus. In intranasally infected BALB/c mice, WT cowpox virus was 80-fold more virulent than the resistant virus. Cidofovir treatment (100 mg/kg of body weight, given one time only as early as 5 min after virus challenge) of a resistant cowpox virus infection could not protect mice from mortality. However, the drug prevented mortality in 80 to 100% of the mice treated with a single 100-mg/kg dose at 1, 2, 3, or 4 days after WT virus challenge. By application of these results to human orthopoxvirus infections, it is anticipated that resistant viruses may be untreatable with cidofovir but their virulence may be attenuated. Studies will need to be conducted with cidofovir-resistant monkeypox virus in monkeys to further support these hypotheses.

NF-kappaB comprises a family of transcription factors that regulate key immune processes. In this study, the effects of orthopoxvirus infection upon the activation of NF-kappaB were examined. During the early phase of infection, cowpox virus can inhibit the induction of NF-kappaB-regulated gene expression by interfering with the process of IkappaBalpha degradation. Although either okadaic acid or tumor necrosis factor (TNF) treatment of infected cells can induce IkappaBalpha phosphorylation, further processing of IkappaBalpha is inhibited. These results suggest that cowpox virus is capable of inhibiting the activation of NF-kappaB at a point where multiple signal transduction pathways converge. Other orthopoxviruses affect NF-kappaB activity, but in a type-specific manner. Raccoonpox virus and vaccinia virus (Copenhagen strain) negatively affect NF-kappaB induction by TNF. In contrast, the modified vaccinia virus Ankara strain induces NF-kappaB activation, even in the absence of other stimuli. These findings suggest that orthopoxviruses may affect a broad range of virus-host interactions through their effects upon NF-kappaB activation. Moreover, because of the central role for NF-kappaB in immune processes and disease, these type-specific effects may contribute significantly to the immunogenic and pathogenic properties of poxviruses., (Copyright 2001 Academic Press.)