Canine rabies in Rio Grande do Sul caused by an insectivorous bat rabies virus variant

Rabies is caused by rabies virus (RV), a RNA virus member of the Lyssavirus genus, family Rhabdoviridae. The aim of this study was to determine the some antigenic and characteristics of a rabies virus isolate (RV183-07) recovered from a stray bitch that died of rabies and to infer the most likely source of contamination since no urban rabies has been reported in the area in more than 20 years. The virus was identified by direct immunofluorescence and multiplied by one passage in mice. The antigenic profile of the isolate was determined with a panel of monoclonal antibodies to lyssavirus antigens on infected brain tissues. A fragment of the viral genome corresponding to the nucleoprotein (N) gene was submitted to reverse transcription/ polymerase chain reaction and the amplicon obtained was subjected to restriction enzyme analysis. A 303 base pair fragment of the N gene was cloned, sequenced and compared to other RV sequences available at Genbank. The isolate RV183-07 displayed antigenic and genomic characteristics of rabies virus variants whose natural reservoir is the non-hematophagous bat Tadarida brasiliensis. Therefore, the most likely source of contamination of the bitch was an incidental contact with an infected bat of that species, common inhabitants of the area. In view of that, the status of “urban rabies-free” of the area should not be compromised.


INTRODUCTION
Rabies is a widespread zoonotic disease caused by rabies virus (RV), a member of the Lyssavirus genus, family Rhabdoviridae [1].Epidemiologically, RV is maintained in distinct biological cycles, with distinct natural reservoir species.In the urban cycle, the domestic dog is the main RV reservoir [1].In the sylvatic cycle, in Latin America, the main natural reservoir is the vampire bat Desmodus rotundus.However, RV variants adapted to different bat species, including insectivorous and frugivorous bats, have been frequently reported.Such variants are antigenically and genetically distinct to variants whose natural reservoirs are haematophagous (vampire) bats [4].Antigenic differentiation can be achieved by monoclonal antibody analyses [4].Genomic comparisons can be accomplished by a number of molecular methods, including restriction enzyme analysis (REA) of genome fragments amplified by reverse transcription/polymerase chain reaction (RT-PCR) have been employed, often complemented by nucleotide sequencing [4].
In the state of Rio Grande do Sul (RS), southern Brazil, urban rabies has not been detected since 1988 [3].Nevertheless, rabies in vampire bats remains endemic [1].However, on a growing number of occasions, non-haematophagous bats have been found infected with RV in urban areas [1].In this study, a case is reported where rabies virus was isolated from a stray bitch which, as deduced from the antigenic and genomic characteristics of the isolate, was probably infected by a RV variant whose natural reservoirs are insectivorous bats of the species Tadarida Brasiliensis.

Virus isolation
In January 2007, a stray bitch in the city of Tapes (eastern RS, Brazil) was found dead by local residents.The bitch apparently did not develop any signs of disease.However, as it was occasionally fed by locals, citizens notified public health authorities.The public health service collected the dead animal, which was submitted to the laboratory for rabies diagnosis (sample number 183-07).
Routine identification of rabies virus antigens was performed by the direct fluorescent antibody test (DFAT) following standard diagnostic procedures [1].A polyclonal anti-rabies nucleocapsid fluorescein conjugate (Pasteur Institute, São Paulo, Brazil) was used.Virus identification was confirmed by mouse intracerebral inoculation followed by DFAT on brain tissues of infected mice [1].

Antigenic characterization
The antigenic profile of isolate RV183-07 was determined by indirect immunofluorescence (IIF) with a panel of eight monoclonal antibodies (Mabs) raised to lyssavirus antigens [4].Three other RV isolates, named 1024 (from an insectivorous bat), 576/RS (from a haematophagous bat) and 585/BA (from a case of urban dog rabies) were included in the tests for comparisons.

Genomic characterization
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) and submitted to reverse transcriptionpolymerase chain reaction (RT-PCR) with primers targeting the N gene as described by Tordo [5].
The amplicon obtained was subjected to restriction enzyme analysis (REA) with BglII and PvuII endonucleases.Cleavage was performed at 37°C for 1 hour.The products were submitted to electrophoresis in 0,7% agarose, stained with ethidium bromide by and visualized on a UV transilluminator [2].

Nucleotide sequence analysis
A fragment of the N gene was targeted for sequence amplification and comparative analyses.Primers corresponding to positions 66-82 (sense primer: 5'-CTACAATGGATGCCGAC-3') and 385-365 (anti-sense primer 5'-TGGGGTGATCTTRTCTCCTTT -3') were used to amplify the isolate RV183-07 by RT-PCR as described above.The amplicon obtained was cloned into plasmid pCR2.1 (Invitrogen) and sequenced three times in both senses, from three different clones.Sequencing was carried out with the DYEnamic ET terminators sequencing kit (GE Healthcare, Giles, United Kingdom) following the manufacturer's protocol, in a MegaBACE 500 automatic sequencer (GE).Assembled sequences with high quality were aligned using Clustal V method (weighted), gap penalty 10 and gap length penalty 10.Homology analyses were performed with basis on RV sequences available at the GenBank database.

Antigenic characterization
Mab analysis revealed that isolate RV183-07 displayed an antigenic pattern usually found in RV variants whose natural reservoirs are bats, indicating the likely origin of the recovered virus.Acta Scientiae Veterinariae.37(4): 391-394.

Genomic characterization
REA of the DNA fragment amplified from RV183-07 gave rise to two BglII fragments of 1000 and 530 bp, and two PvuII fragments of 1100 and 430 bp.The REA cleavage pattern obtained was similar to that observed with other bat RV variants, and unlike the pattern usually displayed by RV variants found in dogs [4].

Nucleotide sequence analysis
By aligning the sequenced fragment of RV183-07 with equivalent RV sequences available at Genbank, high homology was found between RV183-07 and RV variants whose natural reservoirs are the insectivorous bats of the Brazilian free-tailed bat species Tadarida brasiliensis (Figure 1).

DISCUSSION
The present study reports an investigation on the origin of a rabies virus variant isolated from a bitch in a canine rabies-free area.Antigenic and REA suggested that the virus was a RV variant whose natural reservoirs are bats [1,3,4].Phylogenetic analysis defined that the recovered virus had characteristics of RV variants recovered from insectivorous bats of the Brazilian freetailed bat species Tadarida brasiliensis [4], fairly abundant in the area of occurrence of the case.Thus, isolate RV183-07 is a variant adapted to Tadarida brasiliensis and its detection in a canine was more likely an occasional spillover from a distinct natural reservoir.
The determination of the species of origin of the case was important to allow appropriate management of sanitary vigilance resources; as the isolate RV183-07 was originated from an episode of incidental contamination of a bitch, the status of "canine rabies free" of the area was not compromised.Strict surveillance on rabies infections in bats must be maintained.In addition, these findings highlight the need of a better understanding of the biology of rabies in non-haematophagous bat species and its role in rabies epidemiology.

CONCLUSIONS
The RV isolate examined in this study (RV183-07), recovered from a bitch that died of rabies in the city of Tapes, RS, revealed antigenic and genomic characteristics of RV variants which have the "free tailed" bat Tadarida brasiliensis as natural host.Therefore, the more likely source of infection for the bitch was an incidental contact with an infected bat of that species.In view of these findings, the status of "urban rabiesfree" of the area should not be compromised.
Acknowledgements.All procedures involving animal care, handling and experimentations were performed under veterinary supervision and according to the recommendations of the Brazilian Committee on Animal Experimentation (COBEA; law no.6.638 of May 8 th , 1979).Work funded by FEPAGRO, CNPq and FAPERGS.H.B.C.R.B. is a doctorate fellow at the Programa de Pós-graduação em Ciências Veterinárias (PPGCV), UFRGS.P.M.Roehe is a CNPq researcher 1C.