Comparative and Evolutionary Genomics

Motivation: Orthopoxviruses are a large group of closely related viruses displaying a high homology of their genomic nucleotide sequences. These viruses differ also in the severities of the diseases they cause and have different host ranges. Evolutionary relationships of various orthopoxvirus species are yet vague (Marennikova, Shchelkunov, 1998). Results: Analysis of the structures of important virulent factors, such as virus growth factor, gamma-interferon receptor, hemagglutinin, and fusion protein, demonstrated that the corresponding variola virus genes display most numerous distinctions from the other orthopoxviruses, including the functionally important gene regions. In phylogenetic relationships, the genes in question fall into natural groups according to their species attribution. Phylogenetic relationships between the studied orthopoxviruses found while analyzing different genes display certain distinctions. Analysis of the EGF-like region of growth factors demonstrated that orthopoxvirus growth factors clustered with epiregulin, whereas leporipoxvirus growth factors, with transforming growth factors and those of orf viruses, with epidermal growth factors. Presumably, this suggests that different cellular genes from the family of EGF-like growth factors were donated to different poxviruses during their evolutionary development. Introduction Poxviruses are the largest, intricately organized animal viruses. The viruses from the genus Orthopoxvirus are most well studied. In particular, this may be explained by the fact that such viruses as variola and vaccinia belong to orthopoxviruses. However, evolutionary relationships of orthopoxviruses are yet unclear. The viruses closely related to variola virus (VAR), such as monkeypox (MPV) and cowpox (CPV) viruses, persist in nature. MPV causes a smallpox-like human disease with a certain mortality rate (Marennikova, Shchelkunov, 1998). CPV is also capable of causing a generalized infection with a fatal outcome in immunocompromised persons. However, both MPV and CPV differ from VAR by their incapability of causing large-scale human epidemic outbreaks (low contagiousness). However, these viruses might acquire such properties in nature through evolutionary changes. Consequently, it is interesting to compare organizations of several orthopoxvirus genes to find out whether these genes evolve independently or according to a common pattern and to determine evolutionary relationships between poxviruses and the variation limits of their individual genes. This work is both experimental and computer-based. The goal of this work was to sequence orthopoxvirus structural genes and those determining their virulence and to carry out their computer analysis. Orthopoxvirus A27L, A56R, B8R, and С11R genes (according to the nomenclature of vaccinia virus (VAC) strain Copenhagen) were analyzed. A27L gene encodes the 14K fusion protein of surface membrane of intracellular mature virus (IMV), providing formation of the envelope and release from the cell of extracellular enveloped viruses (EEV). This protein forms a stable complex with protein A17 (VAC), activates cell-mediated immune response in infected organism, and is essential for formation of EEV. A56R gene encodes hemagglutinin (HA), an envelope glycoprotein of EEV and membrane of infected cell. This protein inhibits fusion of infected cells and activates proteolytically the infectivity of virions. It is known that HA interacts with protein F13 (VAC). B8R gene encodes a homologue of γ-interferon receptor, secreted from the cell. C11R encodes the protein that is a secreted growth factor (VGF). It is known that the viral growth factor is not essential for virus replication. However, the experiments on its genetic inactivation have demonstrated an essential role of this factor in increasing the virulence and stimulating the cell proliferation at the site of primary infection. Growth factor plays an important role in the mechanism of virus penetration causing local hyperplasia and increasing the number of metabolically active cells, accessible for viral infection (Fenner et al., 1989; Shchelkunov, 1996). Methods and Algorithms Polymerase chain reaction was used to amplify in vitro the selected loci of viral genome containing target genes A27L, A56R, B8R, and С11R of the orthopoxviruses in question for further sequencing. PCR primers were selected so that the DNA fragments synthesized would contain the full-sized genes. The program Oligo 3.3 (Breslauer et al., 1986) was used to calculate the primers. Various strains of variola, monkeypox, cowpox, vaccinia, ectromelia, and camelpox were used in