Genetic studies of Hendra virus and other novel paramyxoviruses

Abstract

This thesis details genetic studies of Hendra virus (HeV), Mossman virus and J virus. It has as its main hypothesis that pteropid bats are the natural reservoir of HeV. Since it was first described in Australia in 1994, HeV has caused two outbreaks of fatal disease in horses and humans, in Brisbane and Mackay, and a third isolated fatal equine case in Cairns. The name equine morbillivirus was originally proposed for the virus which was isolated from four of the Brisbane horses and the first fatal human case. The primary aim of this thesis was to identify the natural reservoir host of He V and to subsequently isolate HeV from this host. The emergence of HeV prompted renewed interest in two other unclassified paramyxoviruses, Mossman virus and J virus which were also studied.When trying to identify the natural reservoir of HeV, we developed the hypothesis that pteropid bats were the most likely candidates, based on epidemiological and virological observation. Preliminary studies revealed a high prevalence of neutralising antibodies to HeV in bats of the genus Pteropus, but it was unclear whether this was due to infection with HeV or a related virus. We developed the hypothesis that HeV excretion from bats might be related to the birthing process and therefore targeted the reproductive tract for virus isolation. Three viral isolates were obtained from the uterine fluid and a pool of foetal lung and liver from one Grey-headed Flying-fox (Pteropus poliocephalus), and from the foetal lung of one Black Flying-fox (P alecto). Antigenically, these isolates appeared to be closely related to HeV, and sequencing of200 nucleotides in the M gene identified that these three isolates were identical to HeV, for the region sequenced.Isolations were confirmed after RNA extracted from original material was positive for HeV RNA when screened on a HeV TaqMan assay, which was developed for the purpose of confirming the presence of HeV RNA in diagnostic samples. Additional isolates of HeV were obtained from pteropid bats during sampling in 1996.Having isolated HeV from the suspected reservoir host, the next objective was to further characterise the pteropid bat HeV RNA. Viral genomic RNA of Hendra virus isolated from pteropid bats, was reverse transcribed, amplified and sequenced. In total, there were eight HeV isolates which were from seven pteropid bats representing three pteropid bat species from four distinct geographical locations. Sequencing revealed that all eight isolates were identical and that their sequence was almost identical to the Hendra virus isolate from the 1994 outbreak suggesting that this RNA virus has a highly conserved genome. Compared to the HeV isolated from horses in 1994, the bat isolate has nine amino acid changes, five of which are in the L gene. The most significant difference between the horse isolate and bat isolate was that the bat isolate had an extra potential phosphorylation site in the L protein. Extensive protein sequence comparisons showed that the bat isolate maintained the conservation of amino acids characterisitic of a wide range of paramyxoviruses studied.Investigations into the characterisation of the unclassified paramyxoviruses, Mossman virus and J virus, indicated that in a one-way neutralisation test, there was no serological cross-reactions between these viruses and two new unclassified paramyxoviruses, Hendra virus and Menangle virus. On the basis of RT-PCRs using universal paramyxovirus primers, it is unlikely that either Mossman virus or J virus are closely related to the prototype morbilliviruses and pneumoviruses. Polyclonal antisera to Mossman virus and J virus was produced in sheep. A cDNA library for J virus was constructed.Immunoglobulins G and M were prepared from pteropid bat sera by ammonium sulphate precipitation and affinity chromatography. Heavy and light chains were identified by polyacrylamide gel electrophoresis and Western blotting. Monoclonal antibodies to pteropid bat IgG (heavy and light chains) were also produced. The use of these pteropid bat specific immunological reagents was illustrated in an ELISA and Western blot. The unsuitability of other species-specific immunological reagents was demonstrated.In summary, the hypotheses that pteropid bats are the natural reservoir host of HeV, and that HeV excretion from bats is related to the birthing process, were confirmed. It was also shown that the pteropid bat isolate of HeV has a highly conserved genome, supporting the findings from previous outbreaks of HeV.