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.

Duvenhage virus (DUVV) belongs to genotype (gt) 4 of the lyssavirus genus, in the family Rhabdoviridae, order Mononegavirales. This virus causes fatal rabies encephalitis and has only been reported from the African continent. To date there have been only five isolations of DUVV, three of which were from human fatalities and all of which were associated with insectivorous bat species. Genotype 4 lyssaviruses have not been well studied and as such little is known about them. The aim of this study was to determine the full genome sequence and investigate the epidemiology of this uniquely African lyssavirus. Standard methods of PCR and sequencing were used to determine the coding and non coding regions of various DUVV isolates. In order to determine the full genome sequence, an RNA circularization technique was used to obtain the genomic terminal sequences. Using various molecular techniques we then analyzed the sequence data, at both phylogenetic and evolutionary levels. Our analysis showed the evolutionary forces acting against DUVV, to be similar to that which has been found for its closest relative, European bat lyssavirus type 1 (EBLV1) (gt 5). Both these viruses have strong constraints against amino acid change, with no evidence of positive selection. Phylogenetic studies showed that not all Lyssavirus genes are equal for phylogenetic or lyssavirus classification analysis. High intergenotypic values at the nucleoprotein amino acid level emphasize that there is a need to reinvestigate the criteria for lyssavirus genotype classification. The strong support observed in our full genome studies suggests that full genomes may in fact be best for Lyssavirus analysis, so as to avoid the potential bias of individual gene analyses. Analysis of DUVV indicates that it is an older virus within the lyssavirus genus and as shown by the discovery of the most recent isolate, the genetic diversity and incidence of this virus is greatly underestimated. Poor surveillance of rabies-related lyssaviruses as well as the poor diagnostic capabilities through most of Africa are large contributors to our lack of information. Improved surveillance of the African rabies-related lyssaviruses will extend our knowledge on the geographic distribution, host species associations and epidemiology of these viruses. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

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.