Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir

F. Bernardi1, S. A. Nadin-Davis2, A. I. Wandeler2, J. Armstrong2, A. A. B. Gomes3, F. S. Lima3, F. R. B. Nogueira3 and F. H. Ito1

1 Department of Preventive Veterinary Medicine and Animal Health, FMVZ-USP, Av. Professor Dr Orlando Marques de Paiva 87, 05508-000 Cidade Universitária, São Paulo-SP, Brazil
2 Rabies Centre of Expertise, Canadian Food Inspection Agency, Ottawa Laboratory-Fallowfield, 3851 Fallowfield Road, Ottawa, Canada K2H 8P9
3 Department of Veterinary Medicine - DMV, ‘Centro de Saúde e Tecnologia Rural - CSTR’, Federal University of Campina Grande, Caixa Postal 64, 58700-000 Patos-PB, Brazil

Correspondence
S. A. Nadin-Davis
nadindaviss{at}inspection.gc.ca


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fifty Brazilian rabies viruses, collected from many different animal species and several regions of the country, were characterized by partial sequencing of the central, variable region of the P gene, a locus useful for sensitive molecular epidemiological studies. Phylogenetic analysis of the sequences, which included comparison with other rabies strains recovered from throughout the Americas, identified three main groups of Brazilian viruses, arbitrarily designated BRL-1 to BRL-3. BRL-1 was found in terrestrial carnivores and clusters with other American strains of the cosmopolitan lineage. BRL-2 comprised two distinct isolates, recovered from two species of non-haematophagous bats, that had evolutionary links to insectivorous-bat-derived strains of North America. BRL-3 consisted of isolates from vampire bats and from livestock species probably infected via contact with vampire bats. The terrestrial group was further subdivided into three subtypes: BRL-1a was associated exclusively with dogs and cats, while BRL-1b and BRL-1c were found exclusively in hoary foxes. These observations strongly support the role of the Brazilian hoary fox as a rabies reservoir. Screening of representative Brazilian rabies viruses against a collection of anti-rabies monoclonal antibodies (mAbs) identified a small panel of mAbs that could be used to discriminate between all Brazilian subgroups as defined by genetic classification in this study.

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences determined in this work are AY962047–AY962096.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Lyssavirus, family Rhabdoviridae, comprises a group of negative-sense RNA viruses having genomes of approximately 12 kb that encode five genes (N, P, M, G and L) and which are all capable of eliciting clinical rabies in mammalian species (Tordo et al., 1998). All members of this genus so far recovered in the Americas belong exclusively to the sero-genotype group 1 that comprises all classical rabies viruses (Bourhy et al., 1993; Kissi et al., 1995; Badrane & Tordo, 2001; Nadin-Davis et al., 2001, 2002); however, the epidemiology of rabies on the American continent is complex. In Canada and the United States, where dog rabies was controlled in the 1940s–1950s, the disease remains a significant public health concern due to its persistence in a variety of terrestrial- and aerial-wildlife hosts (Krebs et al., 2003). Similarly in Mexico, where intensive urban rabies-control efforts have substantially reduced both dog and subsequent human cases of rabies in recent years, the existence of distinct rabies virus variants associated with specific terrestrial hosts (skunks and foxes) and chiropteran species (the Brazilian free-tailed bat and the vampire bat) has been recognized (Velasco-Villa et al., 2002). As additional Latin American countries strive to reduce human rabies through the control of the disease in dogs, it is probable that sylvatic rabies, maintained in a variety of mammalian hosts, will emerge as a significant problem. Public health authorities will thus need to adjust their control and surveillance efforts in response to these changes in disease demographics.

To better understand rabies epidemiology, antigenic and genetic methods of virus characterization are being increasingly applied to collections of Latin American rabies viruses. Most antigenic methods of strain discrimination target the viral nucleoprotein, the product of the N gene, which is expressed in substantial quantities in infected tissues and exhibits sufficient antigenic variation that most strains can be distinguished. In particular, a panel of eight anti-N protein monoclonal antibodies (mAbs), which can differentiate between 11 distinct rabies virus variants harboured by a variety of terrestrial and chiropteran hosts, was reported (Diaz et al., 1994; Delpietro et al., 1997). Application of this panel to rabies virus collections from many Latin American countries, including Brazil (Diaz et al., 1994; Roehe et al., 1997; Morais et al., 2000; Favoretto et al., 2002), has identified two major variants, associated with the dog and the vampire bat (Desmodus rotundus), as well as other variants associated with several insectivorous bats, including the free-tailed bat (Tadarida brasiliensis) and the hoary bat (Lasiurus cinereus). In the Ceará state of Brazil, yet another distinct rabies variant associated with a small non-human primate, Callithrix jacchus, commonly referred to as the white-tufted-ear marmoset, has been reported (Favoretto et al., 2001). In some cases the classification of certain rabies virus isolates by this panel can be confounded by non-typical reactivity patterns not assigned to any known variant, as found for certain Argentinian rabies viruses (Cisterna et al., 2005). The application of molecular genetic techniques for characterization of viral collections can assist in resolving such typing difficulties; moreover, nucleotide sequencing provides data amenable for prediction of the evolutionary relationships between strains. Several studies, targeting either partial or complete N gene sequences, have been reported for isolates from Brazil (Ito et al., 2001a, b, 2003; Romijn et al., 2003; Schaefer et al., 2005), Chile (de Mattos et al., 2000; Yung et al., 2002), Colombia (Páez et al., 2003) and Venezuela (de Mattos et al., 1996). Another study on Brazilian rabies viruses targeted the more variable G gene, encoding the viral glycoprotein, and the highly variable G–L intergenic region (Sato et al., 2004). All these studies again identified two principal viral types, associated with dog and vampire-bat hosts, and also established the existence of other reservoirs in various species of insectivorous bats. Several species of rabid frugivorous bats of the genus Artibeus were found to harbour the viral strain normally associated with vampire bats, presumably via spillover from this reservoir (Shoji et al., 2004).

In Brazil, rabies is still endemic in many parts of the country and 29 human rabies cases were reported in 2004 (Ministério Da Saúde, 2004). Up until a few years ago, transmission from dogs was the most frequent means of human exposure, but reports of bat-transmitted rabies are becoming increasingly common (Araújo, 2002). Indeed, 22 of the human rabies deaths reported in 2004 occurred in the Amazonian state of Para, where the population reports frequent vampire-bat bites (www.promedmail.org, archive number 20040527.1428). In Brazil, diagnosis of animal rabies is conducted using the World Health Organization approved fluorescent antibody test (FAT) and the mouse inoculation test (MIT), by approved state laboratories under the supervision of public or animal health authorities. However, some Northern and North-Eastern states lack their own rabies diagnostic service, so specimens must be sent to laboratories located in other states (Gomes, 2004), a situation that probably places significant limitations on rabies surveillance and diagnosis in these areas. Laboratory testing is performed primarily on domestic animals; wild animals or captive wild animals are tested only sporadically (OPAS, 2001), and so knowledge of the role of wildlife in maintaining rabies reservoirs is very limited.

The objectives of this study were twofold. First, we wished to examine the molecular epidemiology of vampire-bat rabies in Brazil to explore whether regional variation in isolates could be identified and thereby used to monitor disease spread. Second, we wished to compare isolates taken from domestic animals with those obtained from hoary foxes (Dusicyon vetulus). Up until recently, few positive cases of rabies were diagnosed in Brazilian hoary foxes (Barros et al., 1989), and it was thought that these cases were due to infection by dog bites. However, numerous individuals of this species from the state of Paraiba have recently been diagnosed as rabid, and the possible reservoir role played by this wild species was felt to be worthy of further investigation. To achieve these goals, a molecular epidemiological study of a collection of viruses from Brazil was undertaken by characterization of isolates at their P gene locus, a highly variable region of the genome that previously has been proven useful for sensitive viral typing and phylogenetic studies (Nadin-Davis et al., 2002, 2003). Finally, we sought to develop antigenic-typing tools that would allow for the rapid discrimination of rabies virus variants identified by genetic analysis.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Rabies viruses.
Fifty brain samples, which were diagnosed as rabies-positive by both the FAT (Dean et al., 1996) and the MIT (Koprowski, 1996), were used in these studies. As shown in Table 1 and Fig. 1, these isolates came from several distinct geographical regions of Brazil and from several animal species. For the purpose of importing the bovine isolates into Canada, these viruses were passaged once in mice and the infected mice brains were used as the source of the virus. For other species, virus was recovered from the original brains.


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Table 1. Rabies viruses employed in these studies

GO, Goias; MG, Minas Gerais; MS, Mato Grosso do Sul; MT, Mato Grosso; PB, Paraiba; SP, São Paulo; TO, Tocantins. Dashes indicate that the year of isolation is unknown.

 


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Fig. 1. Map of Brazil showing the locations of the states from which samples were collected for this study. States are identified according to the abbreviations defined in Table 1.

 
RNA extraction and RT-PCR.
Total RNA was recovered from rabies-positive brain tissue using TRIzol reagent, as recommended by the manufacturer (Invitrogen). RNA (2 µg) was used to synthesize cDNA in a 20µl reaction, as detailed elsewhere (Nadin-Davis, 1998). Reverse transcription was primed with the oligonucleotide RabP-for, sequence 5'-CTACTTCTCCGGGGAAACCAGAAG-3', corresponding to bases 1249–1272 of the positive-sense N gene sequence of the PV strain (Tordo et al., 1986). For amplification of the complete P gene, 5 µl cDNA was used together with the Expand high fidelity system (Roche Diagnostics), according to the manufacturer's specifications, and reverse primer RabP-rev 5'-GGRAGCCAYAGGTCRTCGTCAT-3', corresponding to bases 2575–2596 of the negative-sense M gene sequence of the PV strain. Thermal cycling was performed in an Applied Biosystems 9700 thermal cycler using the following profile: 93 °C hold, 2 min, followed by 35 cycles of 93 °C, 10 s; 48 °C, 1 min; 68 °C, 2 min, with a final 5 min hold at 68 °C. For those samples that did not yield a detectable product, a second round of amplification was performed using internal primers Pseqfor 5'-GAGATGGCAGAGGARACTGTAGATCT-3' (corresponding to bases 1568–1593 of the PV strain) and Pseqrev 5'-CCTTAACTATGTCRTCAAGRTTCA-3' (corresponding to bases 2208–2231 of the PV strain) and the Expand high fidelity system. The cycling profile was similar to that used for the first round, except that an annealing temperature of 50 °C was employed. The amplified products were purified using the Wizard PCR purification system (Promega).

Nucleotide sequencing and phylogenetic analysis.
Purified PCR products were sequenced using a Li-Cor 4200L automated sequencing system, a Thermosequenase cycle sequencing kit (Amersham Biosciences) and custom infared (IR)-dye labelled primers (Li-Cor) corresponding in sequence to the nested PCR primers. A 528 bp region in the central part of the P gene was targeted. Eseq v2 software was used for base calling and, after manual review and editing, sequences were saved in FASTA format for subsequent alignment using CLUSTALX v1.8 (Thompson et al., 1997) and phylogenetic analysis using PHYLIP v3.63 (Felsenstein, 1993). Trees were generated by a neighbour-joining algorithm as detailed previously (Nadin-Davis et al., 2002), and presented graphically using TREEVIEW software (Page, 1996).

Antigenic analysis.
Antigenic analysis was undertaken essentially as described previously using an indirect FAT applied to virus propagated in murine neuroblastoma cell culture (Nadin-Davis et al., 2001). Representative rabies viruses (see Table 2) were grown in murine neuroblastoma cell culture and tested individually with 473 mAbs; most had anti-N specificity but a few were anti-P specific. Those mAbs exhibiting differential reactivities were further examined on all 50 Brazilian rabies samples, and a panel of 10 mAbs capable of differentiating the viruses represented by all phylogenetic clades was assembled.


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Table 2. Reactivity profiles for selected mAbs tested against several Brazilian rabies viruses

 

   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phylogenetic studies
The P gene sequences produced during these studies include the region encoding amino acids 42–218 and, as noted previously (Nadin-Davis et al., 2002), the portions of the gene encoding residues 61–80 and 134–180 were particularly variable in a manner reflecting the phylogenetic relationships of the isolates. Phylogenetic analysis revealed three main clades of Brazilian rabies viruses: these may be classified as terrestrial (BRL-1), which consists of cases in dogs, cats and foxes; insectivorous bat (BRL-2), comprising just two specimens in two species of non-haematophagous bat; and vampire bat (BRL-3), which includes all cases in herbivores, a single case in a cat (V977), as well as all vampire-bat-derived specimens (see Fig. 2). Within the terrestrial clade, several further subdivisions are very strongly supported. For example, a group of ten isolates (BRL-1a) all originated from either dogs or cats, while the six specimens of subgroup BRL-1b, as well as a group of three isolates forming subgroup BRL-1c, all represent isolates from foxes of the Paraiba region. The segregation of the two fox clades, labelled ‘fox 1’ (1b) and ‘fox 2’ (1c), from the domestic clade is very highly supported by bootstrap values (1000 and 962, respectively).



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Fig. 2. Phylogeny of Brazilian rabies virus strains. Fifty Brazilian isolates were characterized by partial nucleotide sequencing (528 bp) of the P gene coding region. An alignment of these data, together with the CVS sequence used as an outgroup, was generated using CLUSTALX. Phylogenies were predicted from the aligned sequences using a neighbour-joining algorithm in the PHYLIP (version 3.63) software package. Bootstrap values, determined by using 1000 replicates of the data, indicate the number of times that the clade to the right of the branch is predicted in the consensus tree. The main Brazilian groupings referred to in the text are indicated to the right of the tree. Tree branches reflect the genetic distance between isolates according to the scale shown at the bottom of the figure. GenBank accession numbers are given in parenthesis.

 
Although the viruses from the two non-haematophagous bats clustered together with strong bootstrap support (952), the genetic distance between these two isolates (0·138) would, based on guidelines proposed previously (Nadin-Davis et al., 2002), support the placement of these two viruses into distinct lineages. However, the investigation of the role of these two chiropteran species, Histiotus velatus and Molossus molossus, as rabies reservoirs will require further viral isolations and type determinations from these species.

The BRL-3 viruses represent the variant that circulates in Brazilian vampire bats with frequent spillover into domestic species, particularly livestock such as bovines and equines. Although this group could be further subdivided into several smaller clusters that segregated with moderate to strong bootstrap support, these subdivisions did not exhibit any obvious trends with respect to either temporal or regional localization of subtypes.

To place the Brazilian strains identified in this report into a more global context, selected viral sequences were compared with representative viruses from throughout the Americas in another phylogenetic analysis. As shown in Fig. 3, all of the BRL-3 viruses clustered within a clade that included vampire-bat rabies viruses from other Latin American countries (Paraguay and Mexico), and Trinidad in the Caribbean. The strongly supported monophyletic nature of this clade indicates that all these vampire-bat-derived viruses have a common origin and a progenitor shared with the free-tailed-bat-derived strain represented in this tree by the V235.FTB isolate from Texas. Notably there was no strong association of any Brazilian vampire-bat-derived isolates to a specific lineage within this clade, further indicating the lack of any clear regional variation within this strain. The two Brazilian insectivorous-bat-derived isolates (group BRL-2) again grouped together and within a region of the tree representing many insectivorous-bat-derived strains recovered primarily in Canada. While the Brazilian variants were not strongly associated with any particular variant from Canada, it is apparent that these viruses are evolutionarily more closely related to the North-American-bat-derived strains than to the vampire-bat-derived strain. The Brazilian BRL-1 viruses all clustered within a clade representing many terrestrial strains of the Americas, including dog-derived isolates from Peru, Paraguay and Mexico, the grey-fox- and coyote-derived strains from Texas in the USA, the western-Canadian-skunk-derived strain, and an isolate representative of mongoose rabies on the island of Puerto Rico. Samples of this clade were previously assigned to a grouping known as the cosmopolitan lineage (Nadin-Davis et al., 2002).



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Fig. 3. Phylogeny showing the relationships of representative Brazilian rabies viruses to other rabies virus strains of the Americas. A 528 bp sequence from the central portion of the P gene coding region was used to compare 11 representative Brazilian isolates and 29 other isolates, all of which were described previously (Nadin-Davis et al., 2002), except for sample 3306.99RAC, which is an isolate of the mid-Atlantic-raccoon-derived strain recovered from Ontario, Canada, in 1999. The tree depicts a neighbour-joining analysis employing two members of the ARCTIC lineage from Canada as an outgroup. Genetic distances between isolates are reflected in branch lengths according to the scale at the bottom of the figure. GenBank accession numbers are given in parenthesis.

 
Antigenic discrimination
Of the 473 mAbs tested, 10 were selected based on their ability to differentiate between the Brazilian rabies virus variants as identified by genetic analysis. Their reactivities with representative viruses of the genetic groupings are indicated in Table 2, and the following observations are of particular note. mAbs 11DD1 and M1745 differentiate the single M. molossus specimen from all others, while both BRL-2 specimens were exceptional by not reacting to mAb 24FF11, 32FF1 reacted to all BRL-3 viruses but not to those of the other groups, M1386 reacted weakly if at all to BRL-1a viruses but was positive to all other groups, while M1748 reacted with all types except group BRL-1b. Thus, when used in combination with the other mAbs of the panel, mAbs M1386 and M1748 could be used to discriminate the three subgroups of clade BRL-1 viruses. Some of these mAbs (M1495, M1590) reacted differentially to certain vampire-bat-derived isolates but these specificities did not correlate with the genetic relationship of the specimens.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The continued characterization of rabies viruses in countries of the American continent is necessary to more fully define the extent of virus variation, and to assist in the identification of all reservoir species involved in maintaining the disease at the regional level. The situation must be regarded as a continually evolving process in light of evidence that rabies virus spillover into new hosts can, under specific circumstances, lead to viral adaptation to the new host, thereby resulting in the emergence of new viral–host relationships (Badrane & Tordo, 2001) and even new viral biotypes (Fooks, 2004).

In this study, phylogenetic analysis of a collection of Brazilian viruses employed the variable, central region of the P gene. Although the choice of genomic target employed for phylogenetic studies of lyssaviruses does not, in general, greatly impact on the general conclusions of the studies, greater variability within the database provides for a more robust and sensitive analysis, and hence, due to higher bootstrap values, more strongly supported conclusions. The central portion of the P gene is amongst the more variable coding regions of the lyssavirus genome (Le Mercier et al., 1997), second only to the region encompassing the 3' terminus (coding sense) of the G gene which, together with the contiguous, non-coding G–L intergenic region, has been used for molecular epidemiological studies (Nel et al., 1997; Páez et al., 2003, 2005). Similarly, use of the P gene for sensitive and robust phylogenetic studies has been previously reported (Nadin-Davis et al., 2003). An extensive database of lyssavirus P gene sequences is now publicly available to provide comparative data (Nadin-Davis et al., 2002). Moreover, due to the multifunctional nature of the lyssavirus P protein, including its ability to interact with host-cell proteins (Poisson et al., 2001), exploration of structural variations that may confer some measure of host adaptation should be continued (Nadin-Davis et al., 2002).

While two principal rabies cycles, maintained by dogs and vampire bats, have been well established in many parts of Latin America, including Brazil (Ito et al., 2001a), recent reports indicate that the situation is much more complex, particularly with respect to the role played by insectivorous bats, as reported in studies undertaken in Argentina and Chile (de Mattos et al., 2000; Cisterna et al., 2005), and also in Colombia (Páez et al., 2003). Although this study did not focus on the role of insectivorous bats as rabies reservoirs, and our sample set included only two rabies isolates from non-haematophagous bats (H. velatus and M. molossus), which comprised group BRL-2, these isolates were quite distinct from all others, and apparently represented two different viral variants. In a continental context, these isolates were evolutionarily more closely related to specimens from North American insectivorous bats than to Brazilian vampire bats. Further isolation of viruses from these two chiropteran species will be necessary to establish these bats as the reservoirs of these rabies variants. However, it should be noted that a group of rabies isolates recovered from species of the genus Histiotus from Chile and Argentina, and which exhibited a distinctive antigenic profile, formed a monophyletic group that may represent a previously unidentified reservoir (Yung et al., 2002; Cisterna et al., 2005). The histiotus-derived specimen in this study may represent this same variant, but unfortunately, since the N gene was targeted in those earlier studies, direct comparison between those histiotus isolates and the one reported in this study was not possible. Moreover, a case of rabies in a M. molossus specimen in Colombia yielded a rabies variant that clearly segregated together with other rabies variants associated with insectivorous bats of the Americas (Páez et al., 2003), thereby strengthening the possibility that this bat species may act as a rabies reservoir.

The Brazilian isolates representative of the vampire-bat-derived strain were relatively homogeneous, and clustered together with vampire-bat-derived isolates recovered from several other countries in Latin America and the Caribbean, an observation supporting the concept of a common origin of all isolates of this strain despite its extensive geographical range. Despite a fairly extensive sampling of this variant, no obvious temporal or spatial trends with regards to the emergence of Brazilian subvariants were identified. Additionally, no consistent differences in reactivity with a large mAb panel could be discerned for viruses of this group, supporting the conclusion that no clear regional variants of this strain circulate.

Spillover of a rabies virus strain from its reservoir host to other species is not uncommonly reported, as documented, for example, between wildlife in South Africa (Nel et al., 1997), for the raccoon rabies strain to skunks in the United States (Guerra et al., 2003), and even more recently in Northern Colombia, where cases of rabies in humans, dogs and grey foxes were due to a single genetic variant (Páez et al., 2005). In addition, there are multiple examples in the literature that clearly indicate the long-term emergence of independent cycles of disease in new reservoir hosts subsequent to such spillover events. For example, Bourhy et al. (1999) presented evidence that during the westward movement of rabies across Europe during the early 20th century, rabies crossed species from the dog to become established in the red fox population. Johnson et al. (2003) reported on the apparent recent transmission of rabies virus variants from dogs to foxes in Turkey, while the existence of two independent cycles of transmission involving foxes and domestic animals has been reported within the Federal Republic of Yugoslavia (Stankov, 2001). Indeed, the present situation in Northern Colombia, which involves dogs and grey foxes, may represent the very initial stages of such a species jump, which, without intervention, might eventually lead to the emergence of a new fox-adapted strain.

Our genetic analysis of all Brazilian isolates of terrestrial-host origin (see Fig. 2) defined one major group (BRL-1) for which further division into three subgroups was strongly supported. All isolates recovered from domestic animals, including V986, which came from a dog of Paraiba state, belonged to subgroup BRL-1a, while subgroups BRL-1b and BRL-1c were associated exclusively with hoary foxes. Despite the limited number of isolates studied, these results clearly support the existence of genetically distinct strains of rabies, derived from a common ancestor, that now circulate independently in dogs and hoary foxes. The phylogenetic analysis presented in Fig. 3 indicates that the Brazilian BRL-1 rabies viruses cluster within the cosmopolitan lineage believed to have been introduced into the Americas during colonial times, probably by transportation of infected dogs from Europe (Nadin-Davis & Bingham, 2004). Of the specimens included in this study, the isolate most closely related to the Brazilian viruses came from a dog in Paraguay, suggesting a common origin for the viruses circulating in these neighbouring countries. Notably the Brazilian fox-derived strain, represented by isolates V997 and V1001 in Fig. 3, does not associate closely with the Texas grey-fox-derived strain, represented in this tree by isolate V224.FX, thereby suggesting that these two fox reservoirs have emerged independently from the progenitor of the cosmopolitan lineage. Our identification of the hoary fox of the Paraiba region of Brazil as a rabies reservoir maintaining a viral strain evolutionarily related to the urban rabies variant also circulating in Brazil thus appears to mirror situations reported elsewhere, particularly in Europe, where dog to fox transmission, followed by persistence in the wildlife reservoir, has been documented. While it cannot be inferred from our data whether these dog- and fox-derived strains were originally urban strains that were subsequently transmitted to the sylvatic reservoir, historically transmission from dog to fox appears to be the more common occurrence. The fact that the hoary fox plays a much more extensive role in the maintenance and dissemination of rabies in Brazil than was previously supposed has significant public health implications since in this region, where public awareness of rabies is low and animal vaccination rarely undertaken, hoary foxes are not infrequently raised as pets.

To maximize the effectiveness of rabies-control programmes, strain-typing regimens that identify the reservoirs responsible for disease outbreaks are an essential tool, and several methods currently exist. Genetic methods employing either PCR and nucleotide sequencing (Bordignon et al., 2005), strain-specific RT-PCR and restriction fragment length polymorphism analysis (Ito et al., 2003), or multiplex PCR (Sato et al., 2005) have all been applied to collections of Brazilian isolates. However, to date, these methods discriminate only between dog-related and vampire-bat-related virus variants, and these methods require specialized technical facilities and expertise. Systematic typing by genetic methods is time consuming and costly and, even in laboratories of developed countries, is performed on selected cases only. Routine application of antigenic-typing methods, employing a limited panel of mAbs in an indirect FAT procedure, is more practical, especially in developing countries. Thus, there is a need for a rational approach to the development of strain-typing methods in which genetic characterization of representative isolates can be used to direct the identification of mAbs exhibiting reactivity patterns that will differentiate between distinct rabies variants. Such an approach has been described here in which a panel of mAbs, capable of discriminating between the distinct Brazilian viral variants identified by genetic methods, was developed. In particular, the ability to discriminate between the fox- and dog-associated variants may be of considerable importance to future control efforts, where the roles of both species in the continued maintenance of the disease should be clearly established, and the range of the variant associated with fox populations needs to be better defined. The mAbs described here may be a useful addition to the current CDC mAb panel (Diaz et al., 1994) being employed for strain discrimination. Indeed the utility of the mAbs described in this report to identify other rabies variants unavailable for these studies (e.g. isolates from the marmoset primate of the Ceará region that neighbours Paraiba state, and from other insectivorous bat species) should be the subject of future investigations.


   ACKNOWLEDGEMENTS
 
We thank the Inter-American Institute for Cooperation on Agriculture (IICA) for providing assistance to Dr Fernanda Bernardi to work as a collaborating visiting scientist at the Rabies Centre of Expertise, Ottawa Laboratory-Fallowfield, Canadian Food Inspection Agency.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 31 May 2005; accepted 4 July 2005.



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