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Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential for MVA to recombine with naturally occurring orthopoxviruses in cells and hosts in which it multiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semi-permissive to MVA infection) and showed that recombination occurred in both co-infected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that some MVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Diaz-Cánova, Moens, Brinkmann, Nitsche and Okeke.)

The ongoing worldwide monkeypox outbreak is caused by viral lineages (globally referred to as hMPXV1) that are related to but distinct from clade IIb MPXV viruses transmitted within Nigeria. Analysis of the genetic differences has indicated that APOBEC-mediated editing might be responsible for the unexpectedly high number of mutations observed in hMPXV1 genomes. Here, using 1,624 publicly available hMPXV1 sequences, we analyzed the mutations that accrued between 2017 and the emergence of the current predominant variant (B.1), as well as those that that have been accumulating during the 2022 outbreak. We confirmed an overwhelming prevalence of C-to-T and G-to-A mutations, with a sequence context (5'-TC-3') consistent with the preferences of several human APOBEC3 enzymes. We also found that mutations preferentially occur in highly expressed viral genes, although no transcriptional asymmetry was observed. A comparison of the mutation spectrum and context was also performed against the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV), as well as fowlpox virus (FWPV). The results indicated that in VARV genomes, C-to-T and G-to-A changes were more common than the opposite substitutions, although the effect was less marked than for hMPXV1. Conversely, no preference toward C-to-T and G-to-A changes was observed in CPXV and FWPV. Consistently, the sequence context of C-to-T changes confirmed a preference for a T in the -1 position for VARV, but not for CPXV or FWPV. Overall, our results strongly support the view that, irrespective of the transmission route, orthopoxviruses infecting humans are edited by the host APOBEC3 enzymes. IMPORTANCE Analysis of the viral lineages responsible for the 2022 monkeypox outbreak suggested that APOBEC enzymes are driving hMPXV1 evolution. Using 1,624 public sequences, we analyzed the mutations that accumulated between 2017 and the emergence of the predominant variant and those that characterize the last outbreak. We found that the mutation spectrum of hMPXV1 has been dominated by TC-to-TT and GA-to-AA changes, consistent with the editing activity of human APOBEC3 proteins. We also found that mutations preferentially affect highly expressed viral genes, possibly because transcription exposes single-stranded DNA (ssDNA), a target of APOBEC3 editing. Notably, analysis of the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV) indicated that in VARV genomes, TC-to-TT and GA-to-AA changes are likewise extremely frequent. Conversely, no preference toward TC-to-TT and GA-to-AA changes is observed in CPXV. These results suggest that APOBEC3 proteins have an impact on the evolution of different human-infecting orthopoxviruses.

Cowpox viruses (CPXVs) exhibit the broadest known host range among the Poxviridae family and have caused lethal outbreaks in various zoo animals and pets across 12 Eurasian countries, as well as an increasing number of human cases. Herein, we review the history of how the cowpox name has evolved since the 1700s up to modern times. Despite early documentation of the different properties of CPXV isolates, only modern genetic analyses and phylogenies have revealed the existence of multiple Orthopoxvirus species that are currently constrained under the CPXV designation. We further chronicle modern outbreaks in zoos, domesticated animals, and humans, and describe animal models of experimental CPXV infections and how these can help shaping CPXV species distinctions. We also describe the pathogenesis of modern CPXV infections in animals and humans, the geographic range of CPXVs, and discuss CPXV-host interactions at the molecular level and their effects on pathogenicity and host range. Finally, we discuss the potential threat of these viruses and the future of CPXV research to provide a comprehensive review of CPXVs.

Cowpox is caused by a DNA virus known as the cowpox virus (CPXV) belonging to the Orthopoxvirus genus in the family Poxviridae. Cowpox is a zoonotic disease with the broadest host range among the known poxviruses. The natural reservoir hosts of CPXV are wild rodents. Recently, the cases of orthopoxviral infections have been increasing worldwide, and cowpox is considered the most common orthopoxviral infection in Europe. Cowpox is often a self-limiting disease, although cidofovir or anti-vaccinia gammaglobulin can be used in severe and disseminated cases of human cowpox. In this computational study, a molecular docking analysis of thymine- and arabinofuranosyl-thymine-related structures ( 1-21 ) on two cowpox-encoded proteins was performed with respect to the cidofovir standard and a 3D ligand-based pharmacophore model was generated. Three chemical structures (PubChem IDs: 123370001, 154137224, and 90413364) were identified as potential candidates for anti-cowpox agents. Further studies combining in vitro and in silico molecular dynamics simulations to test the stability of these promising compounds could effectively improve the future design of cowpox virus inhibitors, as molecular docking studies are not sufficient to consider a ligand a potential drug.

The genome of cowpoxvirus (CPXV) could be considered prototypical for orthopoxviridae (OXPV) since it contains many open reading frames (ORFs) absent or lost in other OPXV, including vaccinia virus (VACV). These additional ORFs are non-essential for growth in vitro but are expected to contribute to the broad host range, virulence and immune evasion characteristics of CPXV. For instance, unlike VACV, CPXV encodes proteins that interfere with T cell stimulation, either directly or by preventing antigen presentation or co-stimulation. When studying the priming of naïve T cells, we discovered that CPXV, but not VACV, encodes a secreted factor that interferes with activation and proliferation of naïve CD8+ and CD4+ T cells, respectively, in response to anti-CD3 antibodies, but not to other stimuli. Deletion mapping revealed that the inhibitory protein is encoded by CPXV14, a small secreted glycoprotein belonging to the poxvirus immune evasion (PIE) family and containing a smallpoxvirus encoded chemokine receptor (SECRET) domain that mediates binding to chemokines. We demonstrate that CPXV14 inhibition of antibody-mediated T cell activation depends on the presence of Fc-gamma receptors (FcγRs) on bystander cells. In vitro, CPXV14 inhibits FcγR-activation by antigen/antibody complexes by binding to FcγRs with high affinity and immobilized CPXV14 can trigger signaling through FcγRs, particularly the inhibitory FcγRIIB. In vivo, CPXV14-deleted virus showed reduced viremia and virulence resulting in reduced weight loss and death compared to wildtype virus whereas both antibody and CD8+ T cell responses were increased in the absence of CPXV14. Furthermore, no impact of CPXV14-deletion on virulence was observed in mice lacking the inhibitory FcγRIIB. Taken together our results suggest that CPXV14 contributes to virulence and immune evasion by binding to host FcγRs., Competing Interests: The authors have declared that no competing interests exist.

Cowpox virus (CPXV) has an animal reservoir and is typically transmitted to humans by contact with infected animals. In 2017, CPXV infection of a pregnant woman in France led to the death of her fetus. Fetal death after maternal orthopoxvirus (smallpox) vaccination has been reported; however, this patient had not been vaccinated. Investigation of the patient's domestic animals failed to demonstrate prevalence of CPXV infection among them. The patient's diagnosis was confirmed by identifying CPXV DNA in all fetal and maternal biopsy samples and infectious CPXV in biopsy but not plasma samples. This case of fetal death highlights the risk for complications of orthopoxvirus infection during pregnancy. Among orthopoxviruses, fetal infection has been reported for variola virus and vaccinia virus; our findings suggest that CPXV poses the same threats for infection complications as vaccinia virus.

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.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 ( g7tGP ) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284 , encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model. IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts., (Copyright © 2020 American Society for Microbiology.)

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that infects a wide range of mammals. CPXV-specific DNA and antibodies were detected in different vole species, such as common voles ( Microtus arvalis ) and bank voles ( Myodes glareolus ). Therefore, voles are the putative main reservoir host of CPXV. However, CPXV was up to now only isolated from common voles. Here we report the detection and isolation of a bank vole-derived CPXV strain (GerMygEK 938/17) resulting from a large-scale screening of bank voles collected in Thuringia, Germany, during 2017 and 2018. Phylogenetic analysis using the complete viral genome sequence indicated a high similarity of the novel strain to CPXV clade 3 and to OPV "Abatino" but also to Ectromelia virus (ECTV) strains. Phenotypic characterization of CPXV GerMygEK 938/17 using inoculation of embryonated chicken eggs displayed hemorrhagic pock lesions on the chorioallantoic membrane that are typical for CPXV but not for ECTV. CPXV GerMygEK 938/17 replicated in vole-derived kidney cell lines but at lower level than on Vero76 cell line. In conclusion, the first bank vole-derived CPXV isolate provides new insights into the genetic variability of CPXV in the putative reservoir host and is a valuable tool for further studies about CPXV-host interaction and molecular evolution of OPV.

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl. IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine., (Copyright © 2019 American Society for Microbiology.)

Poxviruses encode many proteins that enable them to evade host anti-viral defense mechanisms. Spi-2 proteins, including Cowpox virus CrmA, suppress anti-viral immune responses and contribute to poxviral pathogenesis and lethality. These proteins are 'serpin' protease inhibitors, which function via a pseudosubstrate mechanism involving initial interactions between the protease and a cleavage site within the serpin. A conformational change within the serpin interrupts the cleavage reaction, deforming the protease active site and preventing dissociation. Spi-2 proteins like CrmA potently inhibit caspases-1, -4 and -5, which produce proinflammatory cytokines, and caspase-8, which facilitates cytotoxic lymphocyte-mediated target cell death. It is not clear whether both of these functions are equally perilous for the virus, or whether only one must be suppressed for poxviral infectivity and spread but the other is coincidently inhibited merely because these caspases are biochemically similar. We compared the caspase specificity of CrmA to three orthologs from orthopoxviruses and four from more distant chordopoxviruses. All potently blocked caspases-1, -4, -5 and -8 activity but exhibited negligible inhibition of caspases-2, -3 and -6. The orthologs differed markedly in their propensity to inhibit non-mammalian caspases. We determined the specificity of CrmA mutants bearing various residues in positions P4, P3 and P2 of the cleavage site. Almost all variants retained the ability to inhibit caspase-1, but many lacked caspase-8 inhibitory activity. The retention of Spi-2 proteins' caspase-8 specificity during chordopoxvirus evolution, despite this function being readily lost through cleavage site mutagenesis, suggests that caspase-8 inhibition is crucial for poxviral pathogenesis and spread., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

We report a case of atypical cowpox virus infection in France in 2016. The patient sought care for thoracic lesions after injury from the sharp end of a metallic guardrail previously stored in the ground. We isolated a cowpox virus from the lesions and sequenced its whole genome. The patient reported that he had been previously vaccinated against smallpox. We describe an alternative route of cowpox virus infection and raise questions about the immunological status of smallpox-vaccinated patients for circulating orthopoxviruses.

Taterapox virus (TATV) is phylogenetically the closest related virus to variola-the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice; however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.

Competing Interests: The authors have declared that no competing interests exist.

Annotated whole genome sequences of three isolates of the Akhmeta virus (AKMV), a novel species of orthopoxvirus (OPXV), isolated from the Akhmeta and Vani regions of the country Georgia, are presented and discussed. The AKMV genome is similar in genomic content and structure to that of the cowpox virus (CPXV), but a lower sequence identity was found between AKMV and Old World OPXVs than between other known species of Old World OPXVs. Phylogenetic analysis showed that AKMV diverged prior to other Old World OPXV. AKMV isolates formed a monophyletic clade in the OPXV phylogeny, yet the sequence variability between AKMV isolates was higher than between the monkeypox virus strains in the Congo basin and West Africa. An AKMV isolate from Vani contained approximately six kb sequence in the left terminal region that shared a higher similarity with CPXV than with other AKMV isolates, whereas the rest of the genome was most similar to AKMV, suggesting recombination between AKMV and CPXV in a region containing several host range and virulence genes.

In 1796, Edward Jenner developed the smallpox vaccine consisting of pustular material obtained from lesions on cows affected by so-called cow-pox. The disease, caused by cowpox virus, confers crossprotection against smallpox. However, historical evidence suggests that Jenner might have used vaccinia virus or even horsepox virus instead of cowpox virus. Mysteries surrounding the origin and nature of the smallpox vaccine persisted during the 19th century, a period of intense exchange of vaccine strains, including the Beaugency lymph. This lymph was obtained from spontaneous cases of cow-pox in France in 1866 and then distributed worldwide. A detailed Historical Review of the distribution of the Beaugency lymph supports recent genetic analyses of extant vaccine strains, suggesting the lymph was probably a vaccinia strain or a horsepox-like virus. This Review is a historical investigation that revisits the mysteries of the smallpox vaccine and reveals an intricate evolutionary relationship of extant vaccinia strains., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The emergence of Variola virus-like viruses by natural evolution of zoonotic Orthopoxviruses, like Cowpox virus (CPXV), is a global health threat. The proteasome is essential for poxvirus replication, making the viral components interacting with the ubiquitin-proteasome system attractive antiviral targets. We show that proteasome inhibition impairs CPXV replication by prevention of uncoating, suggesting that uncoating is mediated by proteasomal degradation of viral core proteins. Although Orthopoxvirus particles contain considerable amounts of ubiquitin, distinct modification sites are largely unknown. Therefore, for the first time, we analyzed globally ubiquitination sites in CPXV mature virion proteins using LC-MS/MS. Identification of 137 conserved sites in 54 viral proteins among five CPXV strains revealed extensive ubiquitination of structural core proteins. Moreover, since virions contained primarily K48-linked polyubiquitin, we hypothesized that core proteins are modified accordingly. However, quantitative analysis of ubiquitinated CPXV proteins early in infection showed no proteasomal degradation of core proteins. Instead, our data indicate that the recently suggested proteasomal regulation of the uncoating factor E5 is a prerequisite for uncoating. Expanding our understanding of poxvirus uncoating and elucidating a multitude of novel ubiquitination sites in poxvirus proteins, the present study verifies the major biological significance of ubiquitin in poxvirus infection.

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.

DNA viruses, like poxviruses, possess a highly stable genome, suggesting that adaptation of virus particles to specific cell types is not restricted to genomic changes. Cowpox viruses are zoonotic poxviruses with an extraordinarily broad host range, demonstrating their adaptive potential in vivo. To elucidate adaptation mechanisms of poxviruses, we isolated cowpox virus particles from a rat and passaged them five times in a human and a rat cell line. Subsequently, we analyzed the proteome and genome of the non-passaged virions and each passage. While the overall viral genome sequence was stable during passaging, proteomics revealed multiple changes in the virion composition. Interestingly, an increased viral fitness in human cells was observed in the presence of increased immunomodulatory protein amounts. As the only minor variant with increasing frequency during passaging was located in a viral RNA polymerase subunit and, moreover, most minor variants were found in transcription-associated genes, protein amounts were presumably regulated at transcription level. This study is the first comparative proteome analysis of virus particles before and after cell culture propagation, revealing proteomic changes as a novel poxvirus adaptation mechanism., Competing Interests: The authors declare no conflict of interest.

Transcripts are known to be incorporated in particles of DNA viruses belonging to the families of Herpesviridae and Mimiviridae , but the presence of transcripts in other DNA viruses, such as poxviruses, has not been analyzed yet. Therefore, we first established a next-generation-sequencing (NGS)-based protocol, enabling the unbiased identification of transcripts in virus particles. Subsequently, we applied our protocol to analyze RNA in an emerging zoonotic member of the Poxviridae family, namely Cowpox virus. Our results revealed the incorporation of 19 viral transcripts, while host identifications were restricted to ribosomal and mitochondrial RNA. Most viral transcripts had an unknown and immunomodulatory function, suggesting that transcript incorporation may be beneficial for poxvirus immune evasion. Notably, the most abundant transcript originated from the D5L/I1R gene that encodes a viral inhibitor of the host cytoplasmic DNA sensing machinery., Competing Interests: The authors declare no conflict of interest.

The vertebrate immune system uses multiple, sometimes redundant, mechanisms to contain pathogenic microorganisms that are always evolving to evade host defenses. Thus, the cowpox virus (CPXV) uses genes encoding CPXV12 and CPXV203 to prevent direct MHC class I presentation of viral peptides by infected cells. However, CD8 T cells are effectively primed against CPXV by cross-presentation of viral Ags in young mice. Old mice accumulate defects in both CD8 T cell activation and cross-presentation. Using a double-deletion mutant (∆12∆203) of CPXV, we show that direct priming of CD8 T cells in old mice yields superior recall responses, establishing a key contribution of this mechanism to host antipoxvirus responses and enhancing our fundamental understanding of how viral manipulation of direct presentation impacts pathogenesis. This also provides a proof of principle that suboptimal CD8 T cell in old organisms can be optimized by manipulating Ag presentation, with implications for vaccine design., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Cowpox virus (CPXV) was considered as uniform species within the genus Orthopoxvirus (OPV). Previous phylogenetic analysis indicated that CPXV is polyphyletic and isolates may cluster into different clades with two of these clades showing genetic similarities to either variola (VARV) or vaccinia viruses (VACV). Further analyses were initiated to assess both the genetic diversity and the evolutionary background of circulating CPXVs. Here we report the full-length sequences of 20 CPXV strains isolated from different animal species and humans in Germany. A phylogenetic analysis of altogether 83 full-length OPV genomes confirmed the polyphyletic character of the species CPXV and suggested at least four different clades. The German isolates from this study mainly clustered into two CPXV-like clades, and VARV- and VACV-like strains were not observed. A single strain, isolated from a cotton-top tamarin, clustered distantly from all other CPXVs and might represent a novel and unique evolutionary lineage. The classification of CPXV strains into clades roughly followed their geographic origin, with the highest clade diversity so far observed for Germany. Furthermore, we found evidence for recombination between OPV clades without significant disruption of the observed clustering. In conclusion, this analysis markedly expands the number of available CPXV full-length sequences and confirms the co-circulation of several CPXV clades in Germany, and provides the first data about a new evolutionary CPXV lineage., Competing Interests: The authors declare no conflict of interest.

Traditionally, virus taxonomy relied on phenotypic properties; however, a sequence-based virus taxonomy has become essential since the recent requirement of a species to exhibit monophyly. The species Cowpox virus has failed to meet this requirement, necessitating a reexamination of this species. Here, we report the genomic sequences of nine Cowpox viruses and, by combining them with the available data of 37 additional genomes, confirm polyphyly of Cowpox viruses and find statistical support based on genetic data for more than a dozen species. These results are discussed in light of the current International Committee on Taxonomy of Viruses species definition, as well as immediate and future implications for poxvirus taxonomic classification schemes. Data support the recognition of five monophyletic clades of Cowpox viruses as valid species.

Background: A 17-year-old boy on long-term immunosuppression following renal transplantation for chronic kidney disease (CKD), the result of dysplastic kidneys, initially presented with a swelling in his neck while attending hospital for an unrelated problem. A clinical diagnosis of tonsillitis was made, and he was treated with broad-spectrum antibiotics. Over a few days, his condition deteriorated, and he developed multiple vesicopustular skin lesions and required an emergency tonsillectomy due to respiratory distress., Case Diagnosis/treatment: Histological investigation of the skin and tonsillar tissue suggested a viral aetiology, and subsequent electron microscopy and polymerase chain reaction (PCR) tissue examination proved disseminated cowpox infection. The family cat, which was reported as having self-resolving sores on its skin, was likely the source of the infection. The child failed to respond to antiviral treatment and succumbed to multiorgan failure within a month of admission., Conclusions: We report this case of fatal disseminated cowpox infection to highlight an increasing risk of this illness in the post-transplant population and to detail some unusual features not previously described, such as tonsillar involvement, disseminated skin lesions and multiorgan failure.

We investigated the first laboratory-confirmed human case of cowpox virus infection in Russia since 1991. Phylogenetic studies of haemagglutinin, TNF-α receptor-like protein and thymidine kinase regions showed significant differences with known orthopoxviruses, including unique amino-acid substitutions and deletions. The described cowpox virus strain, taking into account differences, is genetically closely related to strains isolated years ago in the same geographical region (European part of Russia and Finland), which suggests circulation of viral strains with common origin in wild rodents without spread over long distances and appearance in other parts of the world.

The article describes the isolation of a cowpox virus (CPXV) isolate originating from a horse. The skin of a foal, aborted in the third trimester, displayed numerous cutaneous papules. The histological examination showed A-type inclusion bodies within the lesion, typical for CPXV infections. This suspicion was confirmed by real-time PCR where various organs were analyzed. From skin samples, virus isolation was successfully performed. Afterwards, the whole genome of this new isolate "CPXV Amadeus" was sequenced by next-generation technology. Phylogenetic analysis clearly showed that "CPXV Amadeus" belongs to the "CPXV-like 1" clade. To our opinion, the study provides important additional information on rare accidental CPXV infections. From the natural hosts, the voles, species such as rats, cats, or different zoo animals are occasionally infected, but until now only two horse cases are described. In addition, there are new insights toward congenital CPXV infections.

Buffalopox is a contagious viral disease affecting milch buffaloes (Bubalus Bubalis) and, rarely, cows. The disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. Buffalopox is associated with high morbidity (80%). The clinical symptoms of the disease are characterized by wartline lesions on the udder, teats, inguinal region, base of the ears, and over the parotid. In the severe form, generalized rash is observed. Although the disease does not lead to high mortality, it has an adverse effect on the productivity and working capacity of the animals resulting in large economic losses. The outbreaks of buffalopox occurred frequently in India, Pakistan, Bangladesh, Nepal, Iran, Egypt, and Indonesia, where buffaloes are reared as milch animals. The buffalopox is closely related with other Orthopoxviruses. In particular, it is close to the vaccinia virus. There is a view that the buffalopox virus might be derived from the vaccinia virus. It is possible that it became pathogenic to humans and animals through adaptive evolution of the genome by obtaining the virulence genes. PCR is performed for the C18L gene for the purpose of specific detection and differentiation of the buffalopox virus from other orthopoxviruses. The C18L gene encodes the ankyrin repeat protein, which determines the virus host range. The open reading frame of this gene is only 150-nucleotide long as against 453 nucleotide in the vaccinia virus, 756 - in the camelpox virus, and 759 - in the cowpox virus. It can be concluded that a systematic study based on the epidemiology of the virus, existence of reservoirs, biological transmission, and the molecular organization of the buffalopox virus from buffalo, cow, and humans may pave the way to a better understanding of the circulating virus and contribute to the control of the disease using the suitable diagnostic and prophylactic measures.

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.)

Cowpox viruses are orthopoxviruses that may survive in the environment for years. Rodents are regarded as the primary hosts, but transmission to other species has been reported. This report describes a cowpox virus infection in a cat with subsequent transmission to its owner leading to protracted, atypical and severe clinical signs. A young cat presented with multiple crusts and plaques on the neck, muzzle and tail base. The owner developed an erythematous lesion with elevated margins, central necrosis and crust formation below the left breast, a neurogenic inflammation, enlarged regional lymph nodes, a colliquative lymphadenitis and concomitant flu-like symptoms. Cultures were taken at the first visit from the cat's lesional skin and the patient's skin, and polymerase chain reaction with sequencing of the haemagglutinin region of both were positive for cowpox virus. The patient was treated with various antibiotics and methylprednisolone and was in clinical remission after 7 months., (© 2015 British Association of Dermatologists.)

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs., (© 2015 by The American Society for Biochemistry and Molecular Biology, 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.)

The species cowpox virus (CPXV), genus Orthopoxvirus (OPV), consists of isolates highly variable in their biological properties and their genotypes. A TaqMan PCR assay for the specific detection of CPXV DNA based on sequences of the ORF D11L has been developed recently. (Gavrilova et al., 2010; Shchelkunov et al., 2011); however, a rather limited panel of CPXV stains has been used. When a much larger panel of 47 CPXV DNAs has been tested, three strains could not be amplified at all because of large deletions in their respective ORF D11L. In addition, a deletion of 23bp led to low-efficiency detection of five other CPXV strains. To solve this problem a new primer/probe combinations was selected based on sequences of ORF D8L, and a new real-time PCR method for (i) a genus-specific detection of OPVs and (ii) a simultaneous CPXV-specific differentiation is described in this study. The specificity and sensitivity were assessed by analyzing DNA of 67 strains belonging to human-pathogenic OPV species, including variola virus, as well as specimens of CPXV-infected mice., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.

CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

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.)

Background: Cowpox virus (CPXV), a rodent-borne Orthopoxvirus (OPV) that is indigenous to Eurasia can infect humans, cattle, felidae and other animals. Molecular characterization of CPXVs isolated from different geographic locations is important for the understanding of their biology, geographic distribution, classification and evolution. Our aim was to characterize CPXVs isolated from Fennoscandia on the basis of A-type inclusion (ATI) phenotype, restriction fragment length polymorphism (RFLP) profiles of atip gene fragment amplicon, and phylogenetic tree topology in conjunction with the patristic and genetic distances based on full length DNA sequence of the atip and p4c genes., Methods: ATI phenotypes were determined by transmission electron microcopy and RFLP profiles were obtained by restriction enzyme digestion of the atip gene fragment PCR product. A 6.2 kbp region spanning the entire atip and p4c genes of Fennoscandian CPXV isolates was amplified and sequenced. The phylogenetic affinity of Fennoscandian CPXV isolates to OPVs isolated from other geographic regions was determined on the basis of the atip and p4c genes., Results: Fennoscandian CPXV isolates encoded full length atip and p4c genes. They produce wild type V+ ATI except for CPXV-No-H2. CPXVs were resolved into six and seven species clusters based on the phylogeny of the atip and p4c genes respectively. The CPXVs isolated from Fennoscandia were grouped into three distinct clusters that corresponded to isolates from Norway, Sweden and Finland., Conclusion: CPXV is a polyphyletic assemblage of six or seven distinct clusters and the current classification in which CPXVs are united as one single species should be re-considered. Our results are of significance to the classification and evolution of OPVs.

Cowpox virus (CPXV) was isolated from a fatal outbreak among cotton-top tamarins. Samples from healthy common marmosets in contact were also CPXV genome positive. The CPXV isolated from the cotton-top tamarins exhibited a unique hemagglutinin sequence. Pathogenicity investigations using a Wistar rat model characterized the isolate as low pathogenic.

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.)

The availability of adequate treatments for poxvirus infections would be valuable not only for human use but also for veterinary use. In the search for novel antiviral agents, a 1'-methyl-substituted 4'-thiothymidine nucleoside, designated KAY-2-41, emerged as an efficient inhibitor of poxviruses. In vitro, KAY-2-41 was active in the micromolar range against orthopoxviruses (OPVs) and against the parapoxvirus orf. The compound preserved its antiviral potency against OPVs resistant to the reference molecule cidofovir. KAY-2-41 had no noticeable toxicity on confluent monolayers, but a cytostatic effect was seen on growing cells. Genotyping of vaccinia virus (VACV), cowpox virus, and camelpox virus selected for resistance to KAY-2-41 revealed a nucleotide deletion(s) close to the ATP binding site or a nucleotide substitution close to the substrate binding site in the viral thymidine kinase (TK; J2R) gene. These mutations resulted in low levels of resistance to KAY-2-41 ranging from 2.7- to 6.0-fold and cross-resistance to 5-bromo-2'-deoxyuridine (5-BrdU) but not to cidofovir. The antiviral effect of KAY-2-41 relied, at least in part, on activation (phosphorylation) by the viral TK, as shown through enzymatic assays. The compound protected animals from disease and mortality after a lethal challenge with VACV, reduced viral loads in the serum, and abolished virus replication in tissues. In conclusion, KAY-2-41 is a promising nucleoside analogue for the treatment of poxvirus-induced diseases. Our findings warrant the evaluation of additional 1'-carbon-substituted 4'-thiothymidine derivatives as broad-spectrum antiviral agents, since this molecule also showed antiviral potency against herpes simplex virus 1 in earlier studies.

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.