Orthopoxvirus Evolution: Automated Genome Annotation, Advanced Phylogeny and Analysis of Genome Plasticity

Poxviruses within the genus Orthopoxvirus (OPXV) exhibit significant zoonotic potential,
leading frequently to human spill-over infections. The natural reservoir for
the major Cowpox virus (CPXV) strains within OPXV is believed to be voles or
small rodents, which can be further supported by the findings of this thesis.
The OPXV-specific genome assembly pipeline OPVassembly and the genome
annotation pipeline OPVanno were implemented to conduct a comprehensive gene
content analysis and phylogenetic analysis. Both command-line tools were made
available at https://doi.org/10.5281/zenodo.10784666.
Out of 172 OPXV genomes that were (re-)annotated in this study, 41 CPXV
genomes provided by the National Reference Laboratory for Monkeypox (NRL) at
the Friedrich-Loeffler-Institute (FLI) were newly assembled and annotated. The
samples were collected from veterinary sources in Germany, England, Italy, Slovenia
and Lithuania as part of frequent rodent screenings or case studies from 2006 to
2020. 15 newly assembled genomes fell within the stable CPXV-Myodes clade containing
CPXV genomes that were predominantly sampled from Myodes glaerolus in
Germany between 2017 and 2020. Remarkably, the clade showed similarities with
New World poxviruses based on their gene conservation states. The large gene content
of both clades was distinct from CPXV-like 1 and 2. Overall, the gene content
analysis indicated that the genomic composition and macro-evolution of OPXV was
mainly driven by gene loss.
A set of 210 OPXV-specific orthologous gene groups was identified, which divided
further into 114 accessory genes and 96 core genes that were present in all 172
poxvirus genomes. For each core gene, a Maximum-Likelihood (ML) tree was computed
separately on the basis of DNA and AA. Three different topological distance
measures were compared against each other in a comprehensive tree space analysis.
The pairwise distances were computed on a set of 197 phylogenetic trees, including
two ML-trees, which were computed via concatenated DNA/AA alignment of the
core genes using a partitioning model. With respect to their topology, the DNA core
gene trees were found to form a denser cluster around the concatenated-alignment
tree than the AA gene trees. The concatenated alignment of 96 core genes, while
20,000 bp shorter than the estimated core genome length of 120,000 bp, presented a
methodically sound underlying approach. Among the set of core genes, the gene tree
of the large DNA polymerase protein OPG071 was found to approximate OPXV’s
phylogeny. The additional computation of a supernetwork based on five core genes
revealed further evidence of recombination between the major CPXV clades.