Characterisation of Australian bat lyssavirus and an Evaluation of the Rabies Vaccines.

Australian bat lyssavirus (ABLV) is an endemic Lyssavirus first identified in Australia in 1996 and is most closely related to Rabies virus (RABV). There are two genetically distinct strains of ABLV, the first is transmitted by the four flying foxes (Pteropus sp.) found on the Australian mainland and the second is transmitted by the insectivorous bat Saccolaimus flaviventris. Australian bat lyssavirus has been responsible for the deaths of two Queensland women, in 1996 and 1998 and as such is of public health significance. The main aim of this thesis was to evaluate the efficacy of the rabies vaccines currently used in Australia to vaccinate humans and animals against ABLV infection. The neuroinvasiveness, phenotypic and genetic characteristics of a number of ABLV isolates was also evaluated and compared with specific RABV strains. The full genome of ABLV isolated from a Yellow bellied sheath tailed bat (Saccolaimus flaventris) was sequenced for the first time (chapter 2). Additionally the full genomes of ABLV isolates from each of the four mainland hosts (Pteropus alecto, P. scapulatus, P. conspicillatus and P. poliocephalus) were sequenced for the first time. Phylogenetic trees of each of the five genes were constructed along with the pseudo gene (thought to be a remnant gene located between the G and L genes on the ABLV genome) and a whole genome comparison undertaken. Previous studies of Lyssaviruses had grouped viruses based on host species or geographic location after phylogenetic analysis of whole genome sequences and pseudo gene sequences. However, there was not enough variation between ABLV isolates of the same strain to identify isolates from different bat species or location by sequence data, which correlates with previous studies of individual genes. Chapter 3 identified a pteropid ABLV (Pt-ABLV) isolate with a high neuroinvasiveness (ability to cause disease when the virus is inoculated peripherally) for the vaccine trial. This Pt-ABLV isolate along with a Yellow bellied ABLV (Yb-ABLV) isolate and three Rabies isolates, challenge vaccine standard 24 (CVS-24), a bat Rabies isolate (BRV) and a coyote Rabies isolate (COSRV) were titrated in mice by intra-cerebral (i.c.) and peripheral (footpad f.p.) inoculation. These titrations allowed the neuroinvasiveness of each virus to be calculated for comparison with previous studies. A Pt-ABLV isolate from the salivary gland of a P. alecto was most neuroinvasive followed by the brain isolate of the same Pt-ABLV virus, BRV and CVS-24. The neuroinvasiveness of COSRV and Yb-ABLV could not be calculated as these viruses did not cause sufficient disease by f.p. inoculation. This chapter also examined the difference between incubation periods of Pt-ABLV isolates isolated from the brain and salivary gland of the same flying fox. Additionally, despite Yb-ABLV displaying a low titre by f.p inoculation in mice it had a shorter incubation period on average when compared with Pt-ABLV. Chapter 4 is based on studies of a RABV isolated from a Silver haired bat (Lasionycteris noctivagans) that was responsible for a number of human deaths in the United States of America that had no history of exposure to rabies vectors. It was found that this virus had a higher affinity for epithelial and fibroblast cells when compared with a terrestrial RABV strain and it was concluded that this virus could cause disease from less severe bite wounds. These cell infection assays were repeated with both ABLV strains along with the three RABV strains CVS-24, COSRV and BRV. It was concluded that both ABLV strains possessed similar phenotypic characteristics to SHBRV with Yb-ABLV displaying a high affinity for epithelial cells. In chapter 5 the efficacy of the Rabipur® (human) and Nobivac® (veterinary) rabies vaccines was evaluated, to ascertain protection against Pt-ABLV, the stain of ABLV to which humans are most likely to be exposed. Three vaccination methods, pre exposure, post exposure and post exposure with anti-rabies immune globulin (RIG) were also compared. The study showed that the Rabipur® vaccine protected 100% of mice against Pt-ABLV challenge by both pre exposure and post exposure vaccination with RIG. One hundred percent of mice challenged with CVS-24 were protected by pre exposure vaccination but only 75% were protected by post exposure vaccination with RIG. Additionally when post exposure vaccination without RIG was used only 5% of mice were protected against CVS-24 and 95% against Pt-ABLV. The Nobivac® vaccine protected 100% and 95% of mice vaccinated by pre and post exposure vaccination and challenged with Pt-ABLV, with 90% and 0% protected against challenge with CVS-24. It was also observed that although all mice were challenged with 25MFPLD50 of each virus, the incubation period of Pt-ABLV was four to five times longer than that of CVS-24, aiding successful vaccination