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Monkeypox virus (MPXV) has caused a large pandemic outbreak in 2022 with more than 90.000 confirmed cases and 181 deaths. Notably, signs of microevolution and host adaption have been observed. Here, we demonstrate that viral genomes from Franconia, Bavaria acquired different mutations. Three isolates obtained from diagnostic samples, submitted from suspected Mpox cases, show differences in their replication capacities. One MPXV isolate which shows the fastest replication kinetics and higher viral loads, possesses a unique non-synonymous mutation (D616L) in the A11L protein (gene OPG136), which encodes for a protein that is part of a major viral core structure. In regard to pandemic preparedness and future outbreaks, we analyzed the antiviral activity of dihydroorotate dehydrogenase (DHODH) inhibitors, and show that they are active against MPXV, vaccinia virus (VACV), and cowpox virus (CPXV) and therefore likely against orthopoxviruses in general. In agreement with that, we also demonstrated that chemical optimization leads to compounds with EC 50 values in the sub-nanomolar range, associated with low cytotoxicity, which forms a good basis for future drug development from this chemical series., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: C.G. and H.K. have a patent application WO2023/118576 filed covering the new DHODH inhibitors., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.