No description available.

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2 data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p&lt 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan® PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in v mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer &gt 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres &gt 2 IU/mL had cross-neutralising ABLV titres &gt 1:154. A specifically developed ELISA detected a strong but transient response to KLH.

No description available.

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.

No description available.

No description available.

No description available.

No description available.

Chapter one introduces the concept of disease emergence and factors associated with emergence. The role of wildlife as reservoirs of emerging diseases and specifically the history of bats as reservoirs of zoonotic diseases is previewed. Finally, the aims and structure of the thesis are outlined. In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating wildlife reservoirs of disease, and the modelling of disease in wildlife populations is reviewed. Chapter three describes the search for the origin of Hendra virus and investigations of the ecology of the virus. In a preliminary survey of wildlife, feral and pest species, 6/21 Pteropus alecto and 5/6 P. conspicillatus had neutralizing antibodies to Hendra virus. A subsequent survey found 548/1172 convenience-sampled flying foxes were seropositive. Analysis using logistic regression identified species, age, sample method, sample location and sample year, and the interaction terms age*species and age* sample method as significantly associated with HeV serostatus. Analysis of a subset of the data also identified a significant or near-significant association between time of year of sampling and HeV serostatus. In a retrospective survey, 16/68 flying fox sera collected between 1982 and 1984 were seropositive. Targeted surveillance of non-flying fox wildlife species found no evidence of Hendra virus. The findings indicate that flying foxes are a likely reservoir host of Hendra virus, and that the relationship between host and virus is mature. The transmission and maintenance of Hendra virus in a captive flying fox population is investigated in Chapter four. In study 1, neutralizing antibodies to HeV were found in 9/55 P. poliocephalus and 4/13 P. alecto. Titres ranged from 1:5 to 1:160, with a median of 1:10. In study 2, blood and throat and urogenital swabs from 17 flying foxes from study 1 were collected weekly for 14 weeks. Virus was isolated from the blood of a single aged non-pregnant female on one occasion. In study 3, a convenience sample of 19 seropositive and 35 seronegative flying foxes was serologically monitored monthly for all or part of a two-year period. Three individuals (all pups born during the study) seroconverted, and three individuals that were seropositive on entry became seronegative. Two of the latter were pups born during the study period. Dam serostatus and pup serostatus at second bleed were strongly associated when data from both years were combined (p<0.001; RR=9, 95%CI 1.42 to 57.12). The serial titres of 19 flying foxes monitored for 12 months or longer showed a rising and falling pattern (10), a static pattern (1) or a falling pattern (8). The findings suggest latency and vertical transmission are features of HeV infection in flying foxes. Chapter five describes Australian bat lyssavirus surveillance in flying foxes, insectivorous bats and archived museum bat specimens. In a survey of 1477 flying foxes, 69/1477 were antigen-positive (all opportunistic specimens) and 12/280 were antibody-positive. Species (p<0.001), age (p=0.02), sample method (p<0.001) and sample location (p<0.001) were significantly associated with fluorescent antibody status. There was also a significant association between rapid focus fluorescent inhibition test status and species (p=0.01), sample method (p=0.002) and sample location (p=0.002). There was a near-significant association (p=0.067) between time of year of sampling and fluorescent antibody status. When the analysis was repeated on P. scapulatus alone, the association stronger (p=0.054). A total of 1234 insectivorous bats were surveyed, with 5/1162 antigen–positive (all opportunistic specimens) and 10/390 antibody-positive. A total of 137 archived bats from 10 species were tested for evidence of Australian bat lyssavirus infection by immunohistochemistry (66) or rapid focus fluorescent inhibition test (71). None was positive by either test but 2 (both S. flaviventris) showed round basophilic structures consistent with Negri bodies on histological examination. The findings indicate that Australian bat lyssavirus infection is endemic in Australian bats, that submitted sick and injured bats (opportunistic specimens) pose an increased public health risk, and that Australian bat lyssavirus infection may have been present in Australian bats 15 years prior to its first description. In Chapter six, deterministic state-transition models are developed to examine the dynamics of HeV infection in a hypothetical flying fox population. Model 1 outputs demonstrated that the rate of transmission and the rate of recovery are the key parameters determining the rate of spread of infection, and that population size is positively associated with outbreak size and duration. The Model 2 outputs indicated that that long-term maintenance of infection is inconsistent with lifelong immunity following infection and recovery. Chapter seven discusses alternative hypotheses on the emergence and maintenance of Hendra virus and Australian bat lyssavirus in Australia. The preferred hypothesis is that both Hendra virus and Australian bat lyssavirus are primarily maintained in P. scapulatus populations, and that change in the population dynamics of this species due to ecological changes has precipitated emergence. Future research recommendations include further observational, experimental and/or modeling studies to establish or clarify the route of HeV excretion and the mode of transmission in flying foxes, the roles of vertical transmission and latency in the transmission and maintenance of Hendra virus in flying foxes, and the dynamics of Hendra virus infection in flying foxes.

Chapter one introduces the concept of disease emergence and factors associated with emergence. The role of wildlife as reservoirs of emerging diseases and specifically the history of bats as reservoirs of zoonotic diseases is previewed. Finally, the aims and structure of the thesis are outlined. In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating wildlife reservoirs of disease, and the modelling of disease in wildlife populations is reviewed. Chapter three describes the search for the origin of Hendra virus and investigations of the ecology of the virus. In a preliminary survey of wildlife, feral and pest species, 6/21 Pteropus alecto and 5/6 P. conspicillatus had neutralizing antibodies to Hendra virus. A subsequent survey found 548/1172 convenience-sampled flying foxes were seropositive. Analysis using logistic regression identified species, age, sample method, sample location and sample year, and the interaction terms age*species and age* sample method as significantly associated with HeV serostatus. Analysis of a subset of the data also identified a significant or near-significant association between time of year of sampling and HeV serostatus. In a retrospective survey, 16/68 flying fox sera collected between 1982 and 1984 were seropositive. Targeted surveillance of non-flying fox wildlife species found no evidence of Hendra virus. The findings indicate that flying foxes are a likely reservoir host of Hendra virus, and that the relationship between host and virus is mature. The transmission and maintenance of Hendra virus in a captive flying fox population is investigated in Chapter four. In study 1, neutralizing antibodies to HeV were found in 9/55 P. poliocephalus and 4/13 P. alecto. Titres ranged from 1:5 to 1:160, with a median of 1:10. In study 2, blood and throat and urogenital swabs from 17 flying foxes from study 1 were collected weekly for 14 weeks. Virus was isolated from the blood of a single aged non-pregnant female on one occasion. In study 3, a convenience sample of 19 seropositive and 35 seronegative flying foxes was serologically monitored monthly for all or part of a two-year period. Three individuals (all pups born during the study) seroconverted, and three individuals that were seropositive on entry became seronegative. Two of the latter were pups born during the study period. Dam serostatus and pup serostatus at second bleed were strongly associated when data from both years were combined (p<0.001; RR=9, 95%CI 1.42 to 57.12). The serial titres of 19 flying foxes monitored for 12 months or longer showed a rising and falling pattern (10), a static pattern (1) or a falling pattern (8). The findings suggest latency and vertical transmission are features of HeV infection in flying foxes. Chapter five describes Australian bat lyssavirus surveillance in flying foxes, insectivorous bats and archived museum bat specimens. In a survey of 1477 flying foxes, 69/1477 were antigen-positive (all opportunistic specimens) and 12/280 were antibody-positive. Species (p<0.001), age (p=0.02), sample method (p<0.001) and sample location (p<0.001) were significantly associated with fluorescent antibody status. There was also a significant association between rapid focus fluorescent inhibition test status and species (p=0.01), sample method (p=0.002) and sample location (p=0.002). There was a near-significant association (p=0.067) between time of year of sampling and fluorescent antibody status. When the analysis was repeated on P. scapulatus alone, the association stronger (p=0.054). A total of 1234 insectivorous bats were surveyed, with 5/1162 antigen–positive (all opportunistic specimens) and 10/390 antibody-positive. A total of 137 archived bats from 10 species were tested for evidence of Australian bat lyssavirus infection by immunohistochemistry (66) or rapid focus fluorescent inhibition test (71). None was positive by either test but 2 (both S. flaviventris) showed round basophilic structures consistent with Negri bodies on histological examination. The findings indicate that Australian bat lyssavirus infection is endemic in Australian bats, that submitted sick and injured bats (opportunistic specimens) pose an increased public health risk, and that Australian bat lyssavirus infection may have been present in Australian bats 15 years prior to its first description. In Chapter six, deterministic state-transition models are developed to examine the dynamics of HeV infection in a hypothetical flying fox population. Model 1 outputs demonstrated that the rate of transmission and the rate of recovery are the key parameters determining the rate of spread of infection, and that population size is positively associated with outbreak size and duration. The Model 2 outputs indicated that that long-term maintenance of infection is inconsistent with lifelong immunity following infection and recovery. Chapter seven discusses alternative hypotheses on the emergence and maintenance of Hendra virus and Australian bat lyssavirus in Australia. The preferred hypothesis is that both Hendra virus and Australian bat lyssavirus are primarily maintained in P. scapulatus populations, and that change in the population dynamics of this species due to ecological changes has precipitated emergence. Future research recommendations include further observational, experimental and/or modeling studies to establish or clarify the route of HeV excretion and the mode of transmission in flying foxes, the roles of vertical transmission and latency in the transmission and maintenance of Hendra virus in flying foxes, and the dynamics of Hendra virus infection in flying foxes.