Journal of Clinical Virology 26 (2003) 317–330
Discrimination between dog-related and vampire bat-related
rabies viruses in Brazil by strain-specific reverse
transcriptase-polymerase chain reaction and restriction
fragment length polymorphism analysis
Mikako Ito a, Takuya Itou a, Youko Shoji a, Takeo Sakai a,*, Fumio H. Ito b,
Yohko T. Arai c, Tomohiko Takasaki c, Ichiro Kurane c
a Department of Pre
enti
e Veterinary Medicine and Animal Health, Nihon Uni
ersity School of Veterinary Medicine,
1866 Kameino, Fujisawa 252-8510, Japan
b Departamento de Medicina Veterina´ria Pre
enti
a e Sau´de Animal,
Faculdade de Medicina Veterina´ria e Zootecnia da Uni
ersidade de Sa˜o Paulo, A
. Prof. Dr. Orlando Marques de Pai
a, 87,
Cidade Uni
ersita´ria, Sa˜o Paulo 05508-000, Brazil
c Department of Virology 1, National Institute of Infectious Disease, 1-23-1 Toyama, Shinjyuku-ku, Tokyo 162-8640, Japan
Accepted 17 May 2002
Abstract
Background: There is a geographical overlap between the two main rabies epidemiological cycles maintained by
dogs and vampire bats in Latin America. The geographical and temporal coincidence of rabies outbreaks of respective
origins is not unusual in rural areas of Latin America. These circumstances make it difficult to discriminate the
intraspecies and interspecies transmission pathways of rabies. Objecti
e: This study was conducted to develop
techniques to discriminate dog-related and vampire bat-related rabies virus isolates (DRRV and VRRV, respectively)
in Brazil. Study design: The 1396 nucleotides of the nucleoprotein gene of a total of 27 DRRV and VRRV were
sequenced. Strain-specific (SS) primers were developed based on these sequences. Forty-nine rabies virus strains
isolated from animals and humans in several parts of Brazil were examined by reverse transcriptase-polymerase chain
reaction (RT-PCR) with SS primers. These rabies viruses were also amplified by RT-PCR with general rabies primers
and the PCR products were cut by three restriction enzymes, Blp I, Bsu36 I and BspE I. Results: All the DRRV and
VRRV were distinguished by RT-PCR with SS primers. The PCR products obtained from DRRV were cut at one
site by Blp I, but not by Bsu36 I. The PCR products obtained from VRRV were cut at one or two sites by Bsu36
I, but not by Blp I. Blp I and Bsu36 I clearly discriminated DRRV and VRRV in restriction fragment length
polymorphysim (RFLP) assays. The results of SS RT-PCR and RFLP were consistent. Conclusion: SS RT-PCR and
RFLP assays have been developed for determining the origins of rabies virus isolates in Brazil. These assays are
simple and rapid, and will be useful for identifying the rabies virus reservoirs of field isolates in Brazil, especially when
used together. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Rabies virus; Vampire bat; RT-PCR; RFLP; Brazil
www.elsevier.com/locate/jcv
* Corresponding author. Tel./fax: +81-466-84-3650.
E-mail address: sakai@brs.nihon-u.ac.jp (T. Sakai).
1386-6532/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S1386 -6532 (02 )00048 -3

M. Ito et al. / Journal of Clinical Virology 26 (2003) 317–330318
1. Introduction
There is a geographical overlap between the
two primarily epidemiological cycles, dogs and
vampire bats, in Latin America. Dogs are the
principal terrestrial vector, while vampire bats
(Desmodus rotundus) are the major sylvatic rabies
vector. The vampire bats are responsible for
heavy losses in livestocks and are increasingly
involved in human rabies transmission (Diaz et
al., 1994; World Health Organization, 1992). We
reported that 50 Brazilian rabies viruses isolated
from various species of animals and humans were
divided into two groups and co-circulated in dis-
crete reservoirs, dogs and vampire bats, according
to the sequence analysis of 203 nucleotides of the
nucleoprotein (N) gene (Ito et al., 2001b). We also
reported that each phylogenetic lineage showed a
complex overlap of host species. The geographical
and temporal coincidences of rabies outbreaks of
respective origins are not unusual in rural areas.
These circumstances make it difficult to discrimi-
nate the intraspecies and interspecies transmission
pathways.
Genetic sequencing enables an improved geno-
typing analysis to differentiate rabies cycles, al-
though it takes several days to perform
(Crawford-Miksza et al., 1999; Nadin-Davis et al.,
1999). However, it is often difficult to perform
systematic sequencing, particularly in non-special-
ized laboratories in developing countries. Mono-
clonal antibodies (Mabs) against the N protein of
rabies virus were used to differentiate between
dog-related and vampire bat-related rabies viruses
(DRRV and VRRV, respectively) (Delpietro et
al., 1997; Diaz et al., 1994). However, there were
reports that Mabs did not differentiate virus vari-
ants in which sequences of the N gene differed by
5.5% (Rohde et al., 1997; Smith et al., 1992). It
has been reported that reverse transcriptase-poly-
merase chain reaction (RT-PCR) with variants-
specific primers and restriction fragment length
polymorphism (RFLP) can be used to distinguish
rabies virus variants (Black et al., 2000; Heaton et
al., 1997; Loza-Rubio et al., 1999; Nadin-Davis,
1998; Nadin-Davis et al., 1993, 1994, 1996;
Sabouraud et al., 1999; Smith et al., 2000; Warner
et al., 1996). These tests are rapid, sensitive and
specific, when coupled to genetic sequencing, and
provide both genotyping and epidemiological in-
formation (Heaton et al., 1999).
The purpose of the present study is to develop
strain-specific (SS) RT-PCR and RFLP assays for
differentiation between DRRV and VRRV circu-
lating in Brazil. We first sequenced the 1396 nu-
cleotides of the N gene. Based on these sequences
data, SS primers were prepared and viral SS
RT-PCR was performed. The SS RT-PCR am-
plified only specific viral strains as expected. The
PCR product of the N gene was obtained by
RT-PCR and digested by three restriction en-
donucleases. Unique numbers of fragments with
respective size were produced from DRRV and
VRRV. The results suggest that SS RT-PCR and
RFLP assays for Brazilian isolates were developed
and that these two new methods are useful for
diagnosis and epidemiological studies of rabies in
Brazil.
2. Materials and methods
2.1. Viruses
We used 49 Brazilian street rabies viruses iso-
lated from 12 dogs, 11 cats, five vampire bats, 14
cattle, two horses, one pig, one sheep and three
humans at different regions of Brazil from 1987 to
1999. The summary of the viruses used in the
study is shown in Table 1, and the details of these
isolates were previously reported (Ito et al.,
2001a,b).
2.2. RNA extraction
RNAs were extracted directly from rabid brain
or mouse brain emulsions with a commercial
reagent kit (QIAamp Viral RNA Mini Kit., QIA-
GEN K.K. Japan) in Brazil, according to the
manufacture’s instructions and transported to our
laboratory in Japan (Ito et al., 2001a,b).
2.3. RT-PCR
Representative 27 rabies viruses (Table 1) were
chosen from 49 viruses, based on the 203 nucle-