Evolutionary genomics of cowpox virus and recombination in vitro between a naturally occurring cowpox virus and a vaccinia virus vectored influenza vaccine

Abstract

Modified vaccinia virus Ankara (MVA) is a promising orthopoxvirus (OPXV) vector vaccine candidate due to its host range restriction and good safety profile as a smallpox vaccine. It has been widely tested in clinical trials as a recombinant vector for vaccination against infectious diseases and cancers in humans and animals. Furthermore, it is being used as a smallpox and Mpox vaccine. However, the extensive use of MVA and MVA vectored vaccines have the potential for MVA or MVA vectored vaccine to recombine with naturally circulating OPXV. Cowpox virus (CPXV) as a close relative of MVA is a potential candidate for recombination. Hence, the genetic diversity and evolution of CPXV was assessed in this work, as well as recombination in vitro between a naturally occurring CPXV and MVA vectored vaccine in cells in which MVA multiplies poorly. CPXV is classified as a single species; however, we demonstrated that CPXV might be an assemblage of several species based on its high genetic diversity, lack of monophyly, and close phylogenetic relationship with other OPXV. CPXV strains were separated into five major clusters rather than one monophyletic cluster. Furthermore, we described a new, distinct cluster closely related to Ectromelia virus (ECTV) and Abatino macacapox virus (Abatino) named “ECTV-Abatino-like CPXV”. Additionally, we showed evidence that a Norwegian CPXV isolate was a natural occurring recombinant CPXV that might have emerged following multiple recombination events between different OPXV species from the Old World and North America. Under in vitro conditions, the progeny viruses obtained from co-infection and superinfection of Vero cells with MVA-HANP and CPXV-No-F1 had mosaic genomes and displayed parental and non-parental plaque phenotypes. Furthermore, some progeny viruses contained the transgene from MVA-HANP and regained genes that were deleted or fragmented in MVA-HANP. Overall, these findings will contribute to the environmental risk assessment of MVA and MVA vectored vaccines and to the improvement of the biosafety of MVA vectored vaccines.