Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants.

The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate.

Recent emergence of the Arctic rabies virus lineage.

The rabies viruses that circulate in Arctic countries and in much of northern and central Asia are phylogenetically closely related and collectively referred to as the Arctic/Arctic-like (AL) lineage. The emergence and spread of this lineage is of significant interest given that rabies remains a serious zoonotic disease in many parts of Asia, especially in India where the prevalence of dog rabies leads to frequent human exposures and deaths. Previous molecular epidemiological studies of rabies viruses in India identified the AL lineage as the type circulating across much of the country.

Generation and characterization of a panel of anti-phosphoprotein monoclonal antibodies directed against Mokola virus.

The generation of a new panel of 21 monoclonal antibodies (MAbs) reactive with the P protein of Mokola virus (MOKV) is described. Through competitive ELISA and immunoblotting analyses, these MAbs were classified into several groups. Consistent with prior studies on lyssavirus P protein antigenic structure, many of the sites recognized by these Mabs appear to correspond to sites identified previously.

Characterization of a panel of anti-phosphoprotein monoclonal antibodies generated against the raccoon strain of rabies virus.

The generation of a new panel of 32 monoclonal antibodies (MAbs) reactive with the P protein of the raccoon strain of rabies virus is described. Through a series of analyses employing competitive ELISA and immunoblotting, these MAbs were classified into eight groups, each defining an antigenic site, thereby increasing the number of sites now recognized along the length of the P protein. Studies on MAb reactivity with a collection of diverse lyssaviruses identified sites that were highly conserved, moderately conserved and highly variable.

Spatial and temporal dynamics of rabies virus variants in big brown bat populations across Canada: footprints of an emerging zoonosis.

Phylogenetic analysis of a collection of rabies viruses that currently circulate in Canadian big brown bats (Eptesicus fuscus) identified five distinct lineages which have emerged from a common ancestor that existed over 400 years ago. Four of these lineages are regionally restricted in their range while the fifth lineage, comprising two-thirds of all specimens, has emerged in recent times and exhibits a recent demographic expansion with rapid spread across the Canadian range of its host. Four of these viral lineages are shown to circulate in the US.

Development of real-time reverse transcriptase polymerase chain reaction methods for human rabies diagnosis.

To improve timely ante-mortem human rabies diagnosis, methods to detect viral RNA by TaqMan-based quantitative reverse transcriptase polymerase chain reactions (qRT-PCRs) have been developed. Three sets of two primers and one internal dual-labeled probe for each primer set that target distinct conserved regions of the rabies virus N gene were designed and evaluated. Using a collection of 203 isolates representative of the world-wide diversity of rabies virus, all three primers/probe sets were shown to detect a wide range of rabies virus strains with very few detection failures; the RABVD1 set in particular was the most broadly reactive.

In vitro and in vivo genetic stability studies of a human adenovirus type 5 recombinant rabies glycoprotein vaccine (ONRAB).

Investigation into the genetic stability of a replication-competent human adenovirus rabies glycoprotein recombinant (ONRAB) developed for use as an oral vaccine for wildlife rabies prevention is of major importance due to the vaccine's intended placement in the environment. Using a collection of murine monoclonal antibodies directed to six distinct antigenic sites on the rabies glycoprotein, preservation of all main immunogenic epitopes of the protein after virus growth in vitro was established. A competition experiment which involved the in vitro passaging of a mixture of ONRAB and wild-type human adenovirus type 5 demonstrated that the two viruses do not exhibit noticeably different fitness levels in this environment.

Origins of the rabies viruses associated with an outbreak in Newfoundland during 2002-2003.

After being free of rabies of terrestrial mammals since 1988, an outbreak of rabies occurred on the Island of Newfoundland in December 2002 and continued into the middle of 2003. Twenty-one cases, all due to the arctic fox strain of rabies virus, were reported. To explore the immediate origins of this outbreak, viruses from the Newfoundland epizootic were genetically compared to two other rabies viruses recovered in mid-2002 from Cartwright, a mainland coastal community near the Island.

Mokola virus in domestic mammals, South Africa.

We recently identified 2 Mokola viruses from domestic mammals (a dog and a cat) in South Africa. These cases occurred 8 years after the last reported case of infection with this virus. Our findings emphasize the endemicity of rabies-related lyssaviruses in South Africa and the need to better understand the epidemiology of Mokola viruses.

Diagnosis and analysis of a recent case of human rabies in Canada.

Background: On September 30, 2000, staff at the Canadian Food Inspection Agency's Centre of Expertise for Rabies, located at the Animal Diseases Research Institute in Ottawa, Ontario, diagnosed rabies in a child from Quebec. This was the first case of rabies in a human in Canada in 15 years and in 36 years in the province of Quebec. After spending a week in intensive care in a Montreal hospital, the nine-year-old boy succumbed to this nearly always fatal disease.

A molecular epidemiological study of rabies in Puerto Rico.

The mongoose is the principal reservoir for rabies on the island of Puerto Rico. This report describes a molecular epidemiological study of representative rabies viruses recovered from the island in 1997. Two closely related but distinct variants circulating in regionally localised parts of the island were identified.

Emergence of Arctic-like rabies lineage in India.

A collection of 37 rabies-infected samples, 10 human saliva and 27 animal brain, were recovered during 2001-2004 from the cities of Bangalore and Hyderabad in southern India and from Kasauli, a mountainous region in Himachal Pradesh, northern India. Phylogenetic analysis of partial N gene nucleotide sequences of these 37 specimens and 1 archival specimen identified 2 groups, divided according to their geographic (north or south) origins. Comparison of selected Indian viruses with representative rabies viruses recovered worldwide showed a close association of all Indian isolates with the circumpolar Arctic rabies lineage distributed throughout northern latitudes of North America and Europe and other viruses recovered from several Asian countries.

Isolation of Lagos bat virus from water mongoose.

A genotype 2 lyssavirus, Lagos bat virus (LBV), was isolated from a terrestrial wildlife species (water mongoose) in August 2004 in the Durban area of the KwaZulu-Natal Province of South Africa. The virus isolate was confirmed as LBV by antigenic and genetic characterization, and the mongoose was identified as Atilax paludinosus by mitochondrial cytochrome b sequence analysis. Phylogenetic analysis demonstrated sequence homology with previous LBV isolates from South African bats.

Persistence of genetic variants of the arctic fox strain of Rabies virus in southern Ontario.

Genetic-variant analysis of rabies viruses provides the most sensitive epidemiologic tool for following the spread and persistence of these viruses in their wildlife hosts. Since its introduction by a southern epizootic movement that began in the far north, the arctic fox (AFX) strain of Rabies virus has been enzootic in Ontario for almost 50 y. Prior genetic studies identified 4 principal genetic variants (ONT.

Use of discriminatory probes for strain typing of formalin-fixed, rabies virus-infected tissues by in situ hybridization.

An in situ hybridization (ISH) method has been developed to overcome difficulties encountered in the viral typing of formalin-fixed rabies virus-infected brain tissue. Rabies viruses representative of all strains normally encountered in diagnostic submissions throughout Canada, including 3 strains of terrestrial hosts (arctic fox, western skunk, mid-Atlantic raccoon), 10 strains circulating in several bat reservoirs (BBCAN1 to BBCAN7, LACAN, SHCAN, and MYCAN), and the Evelyn-Rokitniki-Abelseth (ERA) strain, used as an oral vaccine for fox rabies control in Ontario, were targeted. Partial phosphoprotein gene fragments generated from reverse transcription (RT)-PCR products of specimens of each viral type were molecularly cloned and used to produce negative-sense digoxigenin-labeled RNA transcripts.