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Cowpox virus (CPXV; genus Orthopoxvirus ; family Poxviridae ) is the causative agent of cowpox, a self-limiting zoonotic infection. CPXV is endemic in Eurasia, and human CPXV infections are associated with exposure to infected animals. In the Fennoscandian region, five CPXVs isolated from cats and humans were collected and used in this study. We report the complete sequence of their genomes, which ranged in size from 220-222 kbp, containing between 215 and 219 open reading frames. The phylogenetic analysis of 87 orthopoxvirus strains, including the Fennoscandian CPXV isolates, confirmed the division of CPXV strains into at least five distinct major clusters (CPXV-like 1, CPXV-like 2, VACV-like, VARV-like and ECTV-Abatino-like) and can be further divided into eighteen sub-species based on the genetic and patristic distances. Bayesian time-scaled evolutionary history of CPXV was reconstructed employing concatenated 62 non-recombinant conserved genes of 55 CPXV. The CPXV evolution rate was calculated to be 1.65 × 10 -5 substitution/site/year. Our findings confirmed that CPXV is not a single species but a polyphyletic assemblage of several species and thus, a reclassification is warranted.

Cowpox virus, which has been used to protect humans against smallpox but may cause severe disease in immunocompromised persons, has reemerged in humans, domestic cats, and other animal species in Europe. Orthopoxvirus (OPV) DNA was detected in tissues (lung, kidney, spleen) in 24 (9%) of 263 free-ranging Eurasian lynx (Lynx lynx) from Sweden. Thymidine kinase gene amplicon sequences (339 bp) from 21 lynx were all identical to those from cowpox virus isolated from a person in Norway and phylogenetically closer to monkeypox virus than to vaccinia virus and isolates from 2 persons with cowpox virus in Sweden. Prevalence was higher among animals from regions with dense, rather than rural, human populations. Lynx are probably exposed to OPV through predation on small mammal reservoir species. We conclude that OPV is widely distributed in Sweden and may represent a threat to humans. Further studies are needed to verify whether this lynx OPV is cowpox virus.

Some orthopoxviruses produce large proteinaceous intracellular bodies, known as A-type inclusions (ATIs) during infection of host cells. Virions associate with ATIs resulting in distinct phenotypes referred to as V+, V+/ and V⁻. The phenotype V+ has the virions embedded in the ATI matrix; V⁻ has no virions embedded within or on the surface of the ATI matrix, whereas an aberrant phenotype, the V+/ has virions only on the surface of ATIs. Viruses that do not produce ATI are designated as V⁰. Recombinant viruses generated from a V+ cowpox virus (CPXV) and a V⁰ transgenic vaccinia virus (VACV) produced aberrant V+/ ATIs. ATI phenotype is dependent on the A-type inclusion protein (Atip) and the P4c protein. We sequenced the atip and p4c genes of parental and progeny recombinant viruses as well as their flanking sequences. The atip and p4c open reading frames were identical in parental V+ CPXV and hybrid V+/ progenies. Our results suggest that additional viral gene(s) are required for the formation of wild type V+ ATI.

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 forMVA to recombinewith naturally occurring orthopoxviruses in cells and hosts in which itmultiplies 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 semipermissive to MVA infection) and showed that recombination occurred in both coinfected 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 someMVA-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. [ABSTRACT FROM AUTHOR]

Abstract: Human orthopoxvirus (OPV) infections in Europe are usually caused by cowpox virus (CPXV). The genetic heterogeneity of CPXVs may in part be due to recombination with other OPV species. We describe the characterization of an atypical CPXV (CPXV-No-H2) isolated from a human patient in Norway. CPXV-No-H2 was characterized on the basis of A-type inclusion (ATI) phenotype as well as the DNA region containing the p4c and atip open reading frames. CPXV-No-H2 produced atypical V+/ ATI, in which virions are on the surface of ATI but not within the ATI matrix. Phylogenetic analysis showed that the atip gene of CPXV-No-H2 clustered closely with that of ectromelia virus (ECTV) with a bootstrap support of 100% whereas its p4c gene is diverged compared to homologues in other OPV species. By recombination analysis we identified a putative crossover event at nucleotide 147, downstream the start of the atip gene. Our results suggest that CPXV-No-H2 originated from a recombination between CPXV and ECTV. Our findings are relevant to the evolution of OPVs. [Copyright &y& Elsevier]