Abstract
Raccoon rabies remains a serious public health problem throughout much of the eastern seaboard of North America due to the urban nature of the reservoir host and the many challenges inherent in multi-jurisdictional efforts to administer co-ordinated and comprehensive wildlife rabies control programmes. Better understanding of the mechanisms of spread of rabies virus can play a significant role in guiding such control efforts. To facilitate a detailed molecular epidemiological study of raccoon rabies virus movements across eastern North America, we developed a methodology to efficiently determine whole genome sequences of hundreds of viral samples. The workflow combines the generation of a limited number of overlapping amplicons covering the complete viral genome and use of high throughput sequencing technology. The value of this approach is demonstrated through a retrospective phylogenetic analysis of an outbreak of raccoon rabies which occurred in the province of Ontario between 1999 and 2005. As demonstrated by the number of single nucleotide polymorphisms detected, whole genome sequence data were far more effective than single gene sequences in discriminating between samples and this facilitated the generation of more robust and informative phylogenies that yielded insights into the spatio-temporal pattern of viral spread. With minor modification this approach could be applied to other rabies virus variants thereby facilitating greatly improved phylogenetic inference and thus better understanding of the spread of this serious zoonotic disease. Such information will inform the most appropriate strategies for rabies control in wildlife reservoirs.
Highlights
Over the last 25 years tools to characterise RNA virus genomes have evolved considerably and the information gained from such studies is improving our understanding of the evolution of these viruses and the factors that impact their emergence and spread
Amplicons A and B were amplified well using either sequence-specific primers (RT1 protocol) or random primers (RT2 protocol); a single cDNA generated by the RT2 protocol was employed for generation of these PCRs so as to minimise the number and cost of reverse transcription reactions
While single gene sequencing still has an important role to play in rabies virus (RABV) strain identification for routine viral typing purposes, whole genome sequencing (WGS) clearly has vastly superior capability to reveal the detailed phylogeny of a selected viral strain
Summary
Over the last 25 years tools to characterise RNA virus genomes have evolved considerably and the information gained from such studies is improving our understanding of the evolution of these viruses and the factors that impact their emergence and spread. Since the initial recognition of the RRV strain in Florida in the 1940s (Jenkins et al, 1988) it has spread by both natural and anthropogenic means throughout the eastern seaboard of the USA reaching as far north as the state of Maine and causing incursions into southern parts of eastern Canada on several separate occasions (Nadin-Davis et al, 2006; Rees et al, 2011; Wandeler et al, 2000; Wandeler and Salsberg, 1999) As a result, this epizootic has come under significant scrutiny and has been the target of various control efforts (Rosatte et al, 2009; Slate et al, 2005). Studies employing partial genome characterisation of RRV have explored how landscape features influence evolution and spread of this virus over time (Szanto et al, 2011) and demonstrated how viral genetic signatures could yield information on the viral population history even in the absence of surveillance data (Biek et al, 2007)
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