This thesis describes the molecular characterisation of Broome virus (BroV), a new fusogenic orthoreovirus species that was isolated from a little red flying-fox (Pteropus scapulatus) in Broome, Western Australia in 2002. The BroV genome consists of ten segments of dsRNA, each containing a plus-strand with a 3’ terminal pentanucleotide sequence that is conserved amongst all viruses in the genus Orthoreovirus, family Reoviridae, and a 5’ terminal pentanucleotide sequence that is unique to BroV. With the exception of S4, all genome segments are predicted to encode a single translation product producing a total of seven structural and four nonstructural proteins. All BroV proteins were identified as homologues of known orthoreovirus proteins and shown to have similar secondary structure and possess key conserved amino acid sequence motifs and structural features implicated in biological function. Notably, no cell-attachment protein gene homologue was identified in the BroV genome suggesting the use of an alternate cell entry mechanism to that employed by most orthoreoviruses. The amino acid sequence identity between cognate BroV proteins and those of other orthoreoviruses ranges from 13-50%, which is too low for BroV to be considered a new isolate of any established orthoreovirus species group. Phylogenetic analyses based on both structural and nonstructural proteins provide additional evidence to support this claim. It is proposed that BroV is the prototype member of a new sixth species group Broome virus, in the genus Orthoreovirus. The complete genome characterisation of BroV provided an opportunity to produce recombinant proteins in Escherichia coli and to generate polyclonal antibodies in rabbits for use in research and surveillance. Such reagents proved valuable in the experimental identification of the fusion-associated small transmembrane (FAST) protein p13 that is responsible for the syncytia observed in BroV-infected cells. Despite the low amino acid sequence identity between the FAST proteins of different orthoreovirus species they possess conserved structural features that have been implicated in biological function. Of these conserved features, the BroV p13 protein is predicted to possess one transmembrane domain, a C-terminal polybasic region, a C-terminal hydrophobic patch and an N-terminal myristoylation consensus sequence. The unique repertoire and arrangement of sequence-predicted structural features identified in p13 indicate that it is a novel fifth member of the FAST protein family. The BroV-specific immunological reagents were also used to develop an enzyme-linked immunosorbent assay (ELISA) suitable for serological screening. A survey of flying-foxes from Papua New Guinea (PNG) revealed that BroV or BroV-like viruses are currently circulating in these animals. This demonstrates that BroV is not limited to the Australian continent.
| Identifer | oai:union.ndltd.org:ADTP/285518 |
| Creators | Claudia Thalmann |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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