Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus (EHDV)

Abstract

The core proteins of epizootic haemorrhagic disease virus (EHDV) have important roles to perform in maintaining the structure and function of the virus. A complete genetic and phylogenetic analysis was therefore performed on these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G + C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The inner core proteins (VP1, VP3, VP4 and VP6) were characterised by high levels of sequence conservation, and the ability to topotype isolates very strongly into eastern or western groups. This is particularly evident in genome segment 9 (VP6) which exists as two different sized homologues. VP7 did not topotype, but rather exhibited a more random, radial phylogeny suggestive of genetic drift. With the exception of VP6, all of the core proteins also showed high numbers of synonymous mutations in the third base position, suggesting they have been evolving for a long period of time. Interestingly, VP6 did not show this, and possible reasons for this are discussed.

Introduction

Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious insect transmitted disease of ruminants caused by viruses belonging to the species Epizootic haemorrhagic disease virus (EHDV). Along with Bluetongue virus (BTV), EHDV is classified within the genus Orbivirus of the family Reoviridae (Mertens et al., 2005). It was first formally identified in 1955 by Richard Shope and colleagues in New Jersey (USA) following reports of a fatal epizootic in white-tailed deer (Odocoileus virginianus) (Shope et al., 1955).

EHDV and BTV are both icosahedral viruses, ∼80 nm in diameter, with 10-segmented, double-stranded RNA genomes (Huismans and Van Dijk, 1990, Mertens, 1999, Verwoerd et al., 1972, Verwoerd et al., 1970). Each of these 10 segments codes for 1 of 10 distinct viral proteins, 7 of which are structural components of the virus particle, and three of which are non-structural (Huismans et al., 1979, Mecham and Dean, 1988, Mertens et al., 1984). As demonstrated for BTV, the seven structural proteins form the viral capsid and are arranged as three concentric capsid shells surrounding the viral dsRNA (Grimes et al., 1998, Hewat et al., 1992a, Hewat et al., 1992b, Huismans and Van Dijk, 1990, Roy, 1992, Verwoerd et al., 1972).

The viral core is made up of VP1, VP3, VP4, VP6 and VP7, encoded by genome segments 1, 3, 4, 9 and 7, respectively (Huismans et al., 1979, Mecham and Dean, 1988). VP3 forms the innermost sub-core capsid shell, and its self-assembly controls the overall size and organization of the capsid structure (Grimes et al., 1998, Mertens and Diprose, 2004). VP3 is also an RNA binding protein and interacts with both the viral RNA genome and with the minor proteins VP1, VP4 and VP6 (Mertens et al., 1992, Ramadevi et al., 1998, Ramadevi and Roy, 1998). VP7 is the main immuno-dominant viral protein (Gumm and Newman, 1982, Huismans and Erasmus, 1981, Thevasagayam et al., 1996), can bind dsRNA and can be involved in cell entry, particularly insect cells for which cores have a high specific infectivity—as demonstrated for BTV (Diprose et al., 2002, Mertens et al., 1996, Roy et al., 1990). VP7 is antigenically specific to each of the Orbivirus species, and therefore represents a suitable target for the design of group-specific diagnostic assays (Gumm and Newman, 1982, Luo and Sabara, 2005, Mecham and Jochim, 2000, White et al., 1991).

Within the orbiviral core, the 10 dsRNA segments of the viral genome form a highly ordered, liquid crystal structure. This gives each genome segment considerable freedom of movement within the central space, allowing them to act as templates for ssRNA synthesis by the viral transcriptase complexes (TC—composed of VP1, VP4 and VP6). The TCs are situated on the inner surface of the sub-core shell at each of the 12 icosahedral vertices, as demonstrated for cypovirus and rotavirus (Miura et al., 1969, Prasad et al., 1988) and studies with other reoviruses, including cytoplasmic polyhedrosis viruses (genus Cypovirus) have suggested that each TC may be associated with a separate genome segment (Yazakia and Miura, 1980); perhaps explaining why no member of the Reoviridae contains more than 12 RNA segments (Mertens, 2004). Within this TC, VP1 functions as the viral RNA-dependant RNA polymerase (RdRP), and is highly conserved (Boyce et al., 2004, Urakawa et al., 1989, Wehrfritz et al., 2007). VP4 is the capping enzyme (type 1), which has guanylyltransferase and two transmethylase activities (Mertens et al., 1992, Mertens and Diprose, 2004, Ramadevi et al., 1998, Ramadevi and Roy, 1998) and VP6 is believed to be the viral helicase, as demonstrated in both BTV and African horse sickness virus (de Waal and Huismans, 2005, Kar and Roy, 2003).

It is significant that our knowledge of the structure and function of the EHD viral core is largely based on assumption for what we know to be true for BTV, or other related reoviruses. For example the high conservation in VP1, or the ability of VP3 to topotype virus isolates are well documented for BTV—but there is only very limited data currently for EHDV. A full-genome database was recently completed for 11 different strains of EHDV (Anthony et al., submitted for publication-a, Anthony et al., submitted for publication-b). The genetic and phylogenetic analyses of the core proteins of EHDV are described in this report.