Genetic tracking of the raccoon variant of rabies virus in eastern North America.

To gain insight into the incursion of the raccoon variant of rabies into the raccoon population in three Canadian provinces, a collection of 192 isolates of the raccoon rabies virus (RRV) strain was acquired from across its North American range and was genetically characterized. A 516-nucleotide segment of the non-coding region between the G and L protein open reading frames, corresponding to the most variable region of the rabies virus genome, was sequenced. This analysis identified 119 different sequences, and phylogenetic analysis of the dataset supports the documented history of RRV spread. Three distinct geographically restricted RRV lineages were identified. Lineage 1 was found in Florida, Alabama and Georgia and appears to form the ancestral lineage of the raccoon variant of rabies. Lineage 2, represented by just two isolates, was found only in Florida, while the third lineage appears broadly distributed throughout the rest of the eastern United States and eastern Canada. In New York State, two distinct spatially segregated variants were identified; the one occupying the western and northern portions of the state was responsible for an incursion of raccoon rabies into the Canadian province of Ontario. Isolates from New Brunswick and Quebec form distinct, separate clusters, consistent with their independent origins from neighboring areas of the United States. The data are consistent with localized northward incursion into these three separate areas with no evidence of east-west viral movement between the three Canadian provinces.

Re-assessment of direct fluorescent antibody negative brain tissues with a real-time PCR assay to detect the presence of raccoon rabies virus RNA.

The first report of the raccoon variant of rabies virus was in Ontario, Canada in 1999. As part of the control of this outbreak a Point Infection Control (PIC) strategy of trapping and euthanizing vector species was implemented. To evaluate whether this strategy was indeed removing diseased animals, rabies diagnosis was performed on these specimens. During a PIC program conducted in 2003, 721 animals (raccoons, striped skunks and red foxes) were collected and euthanized and brain material from each specimen was divided into two halves; one half was submitted for rabies diagnosis by a direct fluorescent antibody (DFA) test while the other was tested using a sensitive real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), to detect raccoon rabies virus (RRV) RNA. This latter assay can detect less than ten viral copies in 200ng of total cellular RNA. All 721 PIC brain samples were negative by the DFA test but ten of them (5 raccoons, 5 skunks) tested positive for raccoon rabies virus by the RT-qPCR assay albeit at low levels. Three of these samples were confirmed by sequencing of the PCR products. Little correlation was observed between clinical rabies DFA positive scoring categories and viral copy number as determined by RT-qPCR.

Complete genome sequence of a raccoon rabies virus isolate.

The entire genome of a mid-Atlantic raccoon strain rabies virus (RRV) isolated in Canada was sequenced; this is the second North American wildlife rabies virus isolate to be fully characterized. The overall organization and length of the genome was similar to that of other lyssaviruses. The nucleotide sequence identity of the raccoon strain ranged between 32.7% and 85.0% when compared to other lyssaviruses, while the deduced amino acid sequence identity ranged between 22.9% and 94.2% with the nucleoprotein and polymerase being the most conserved. Notable features of RRV include the phosphoprotein's four amino acid extension compared to most other rabies viruses, and a nucleotide substitution immediately prior to the normal start codon that results in an additional methionine at the beginning of the L protein. This is the first report of the RRV L gene sequence and its 2128 amino acid product. Rates of non-synonymous and synonymous nucleotide changes within the lyssavirus L gene identified the conserved blocks II, III and IV as being most constrained. Analysis of L gene codon substitution patterns favoured models that supported positive selection, but only one site, corresponding to Leu62 of the RRV L protein, was identified as being under weak positive selection.