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Taxonomy of the Lyssavirus genus
LBV is part of the Lyssavirus genus in the family Rhabdoviridae (Rhabdos in Greek:
rod). The Rhabdoviridae family belongs to the Mononegavirales order (Tordo et al., 2005), a group of single stranded, negative sense, non-segmented RNA genome viruses, also comprising of other important virus families associated with human and animal diseases such as Paramyxoviridae, Filoviridae and Bornaviridae. Lyssavirus, Vesiculovirus, Ephemerovirus, Novirhabdovirus, Cytorhabdovirus and Nucleorhabdovirus are all genera in the Rhabdoviridae family and have very similar bullet shaped morphology, structure and replication mechanisms but infect a wide range of hosts varying from mammals, birds, fish, arthropods, plants and other invertebrates (Tordo et al., 2005). In earlier taxonomy the bullet shaped morphology and serological cross reactivity with other rhabdoviruses determined placement of a virus in the Rhabdoviridae family, leading to more than a hundred unassigned rhabdoviruses (Tordo et al., 2005). Further analysis is needed to justify their inclusion into an existing genus or create new groups. Recently DNA sequence similarity has been used to differentiate between genera and define genotypes (species) in a genus. Based on the phylogenetic analysis of the polymerase gene of assigned and unassigned rhabdoviruses it has been suggested that phylogenetic analyses combined with data on genome organization may be a more useful guide for taxonomic classification than serological cross-reactivity (Bourhy et al., 2005).
Sequence similarity in the nucleoprotein has been used for the identification of lyssaviruses (Arai et al., 2003; Bourhy et al., 1993b; Kuzmin et al., 2003), vesiculoviruses (Masters and Banerjee, 1987) and ephemeroviruses (Walker et al., 1994; Wang et al., 1995).
RABV, the prototype Lyssavirus, was believed to be antigenically unique until the identification of the first rabies-related lyssavirus from Africa (Boulger and Porterfield, 1958; Shope et al., 1970). After the recognition of the rabies-related lyssaviruses, the lyssavirus genus was first divided into 4 serotypes (RABV, LBV, Mokola virus (MOKV) and Duvenhage virus (DUVV)) on the basis of sero-neutralization (Schneider et al., 1973) and monoclonal antibody reactions (Dietzschold et al., 1988).
European bat lyssaviruses (EBLV) were first believed to belong to serotype 4 (DUVV) (Schneider et al., 1985) but were subsequently shown to represent a separate serotype, viz serotype 5 (Dietzschold et al., 1988). Within this serotype two biotypes (lineages) have been identified and designated, EBLV-1 and EBLV-2 (Montano- Hirose et al., 1990). With the development of molecular biology techniques, the lyssavirus genus was divided into six genotypes (gts) based on the amino acid identity of the nucleoprotein (Bourhy et al., 1993b). Gt 1 (RABV) occurs worldwide whereas LBV (gt 2), MOKV (gt 3) and DUVV (gt 4) have only been isolated from the African continent and EBLV-1 (gt 5) and EBLV-2 (gt 6) are present in Europe. In 1996 a new lyssavirus was isolated in Australia, constituting gt 7 (Gould et al., 1998).
All these gts have been reported to be pathogenic for animals and with the exception of gt 2, were also reported to cause rabies encephalitis in humans. Recently a number of new lyssavirus isolates were identified from Eurasia; Irkut (Botvinkin et al., 2003), Aravan (Arai et al., 2003; Kuzmin et al., 2003), Khujand (Kuzmin et al., 2003) and West Caucasian bat virus (WCBV) (Botvinkin et al., 2003). These viruses are currently listed as tentative species (gts) in the Lyssavirus genus (Tordo et al., 2005). It has been proposed that a new lyssavirus gt is defined if there are less than 80% nucleoprotein (nt) identity and less than 92% amino acid (aa) identity (Kissi et al., 1995) or less than 93.3-97.1% aa identity (Bourhy et al., 1993b) in the nucleoprotein gene. Tordo and Kouknetzoff (1993) also suggested that a threshold of 80% aa identity could be used for the first 133 aa of the glycoprotein to define a gt.
Previously proposed criteria were acceptable for gt 1-7 where intragenotype identities were greater than intergenotype identities and strong bootstrap values supported the phylogenetic grouping of seven independent genera. With the discovery of new lyssaviruses it became apparent that the guidelines for the inclusion of new gts in the lyssavirus genus are lacking (Kuzmin et al., 2003) since not all genes could provide clear results on taxonomic status of the new lyssaviruses. It has been suggested that the nucleoprotein gene should be used for taxonomic purposes since it provided the clearest division of lyssavirus gts (Kuzmin et al., 2005). When analyzing the nucleoprotein gene of newly discovered lyssaviruses, Aravan virus had the highest sequence homology with gt 4, 5 and 6 (Kuzmin et al., 2003) and Khujand virus with gt 6 (Kuzmin et al., 2003). Irkut virus had the highest sequence homology with gt 4 and 5 (Kuzmin et al., 2005) and Aravan virus was the closest related to Khujand virus (78.8% nucleoprotein nucleotide identity). Analyses of the nucleoprotein gene sequences of WCBV indicated that it is the most divergent representative of the lyssavirus genus discussed up to date (Kuzmin et al., 2005). Based on the current proposed criteria for a new lysssavirus gt, these four viruses may all be considered new lyssavirus gts (Kuzmin et al., 2005) (Figure 2.1).
Morphology and structure of a lyssavirus virion
The morphology and structure of RABV were reviewed in Tordo and Poch, (1988).
The virion is bullet shaped with an average diameter of 75 nm and length of 180 nm (Davies et al., 1963). Variation in length (100-300 nm) may be due to strain differences or defective interfering (DI) particles consisting of truncated genomes and defectiveness in various viral functions (Holland, 1987). The RNA genome, together with the nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L), are condensed into a helical nucleocapsid. A lipoprotein envelope, derived from the host’s cell membrane during viral budding, surrounds the nucleocapsid. A 2:1 ratio of N:P molecules occur per virion and the L protein is present in the minority.
Surface projections, consisting of glycoprotein monomers, extend from the envelope and are anchored in the membrane by a 22 aa hydrophobic transmembrane domain. The M protein forms a layer between the envelope and the nucleocapsid (Figure 2.2).
Properties of the lyssavirus genome
The lyssavirus genome consists of a single stranded negative sense non-infectious molecule of RNA (Sokol et al., 1969). The first complete genome sequence available for lyssaviruses was the Pasteur virus (PV) (Tordo et al., 1986a; Tordo et al., 1986b; Tordo et al., 1988; Bourhy et al., 1989) and nucleotide sequence information of complete lyssavirus genomes are now available for certain vaccine strains of RABV (gt 1) (Conzelmann et al., 1990), MOKV (gt 3) (Le Mercier et al., 1997; Bourhy et al., 1989), ABLV (gt 7) (Warrilow et al., 2002; Gould et al., 1998; Gould et al., 2002), EBLV-1 (gt 5) (Marston et al., 2007) and EBLV-2 (gt 6) (Marston et al., 2007).
Sequence analyses indicated that lyssaviruses share the same genomic organization with slight differences in the length of genes and intergenic regions (Bourhy et al., 1989; Bourhy et al., 1993b; Conzelmann et al., 1990; Warrilow, 2005). The 3’ end of the genome encodes a short leader RNA, followed by the nucleo (N), phospho (P), matrix (M), glyco (G) and RNA polymerase (L) gene, each encoding a protein (Figure 2.3).
Chapter I: Introduction
1.1 Background and motivation
1.2 Layout of the thesis
Chapter II: Literature review
2.1 History of rabies
2.2 Taxonomy of the Lyssavirus genus
2.3 Morphology and structure of a lyssavirus virion
2.4 Properties of the lyssavirus genome
2.5 Analysis of lyssavirus infection
2.6 Diagnostics of lyssaviruses
2.7 Preventative measures against lyssavirus infection
2.8 Global epidemiology of lyssaviruses
2.9 Molecular epidemiology of lyssaviruses
2.10 Lagos bat virus
2.11 Aims of this study
Chapter III: Identification and characterization of new Lagos bat virus isolates from South Africa
3.1 Introduction
3.2 Materials and methods
3.3 Results
3.4 Discussion
Chapter IV: Non-neuronal viral tissue distribution and serology of naturally infected frugivorous bats with Lagos bat virus
4.1 Introduction
4.2 Materials and methods
4.3 Results
4.4 Discussion
Chapter V: Molecular epidemiology of Lagos bat virus
5.1 Introduction
5.2 Materials and methods
5.3 Results
5.4 Discussion
Chapter VI: Pathogenesis of Lagos bat virus
6.1 Introduction
6.2 Materials and methods
6.3 Results
6.4 Discussion
Chapter VII: Conclusions
