lunes, 2 de abril de 2012

Typing of the rabies virus in Chile, 2002–2008 V. YUNG, M. FAVI AND J. FERNANDEZ 2012


Typing of the rabies virus in Chile, 2002–2008
V. YUNG1*, M. FAVI1 AND J. FERNANDEZ2
1 Sección Rabia, Subdepartamento Virologı´a, Instituto de Salud Pu´blica de Chile, Santiago, Chile
2 Subdepartamento Gene´tica Molecular, Instituto de Salud Pu´blica de Chile, Santiago, Chile
Received 20 July 2011; Final revision 23 February 2012; Accepted 4 March 2012
 Epidemiol. Infect., Page 1 of 6. f Cambridge University Press 2012
doi:10.1017/S0950268812000520

SUMMARY
In Chile, dog rabies has been controlled and insectivorous bats have been identified as the main
rabies reservoir. This study aimed to determine the rabies virus (RABV) variants circulating in
the country between 2002 and 2008. A total of 612 RABV isolates were tested using a panel with
eight monoclonal antibodies against the viral nucleoprotein (N-mAbs) for antigenic typing, and
a product of 320-bp of the nucleoprotein gene was sequenced from 99 isolates. Typing of the
isolates revealed six different antigenic variants but phylogenetic analysis identified four clusters
associated with four different bat species. Tadarida brasiliensis bats were confirmed as the main
reservoir. This methodology identified several independent rabies enzootics maintained by
different species of insectivorous bats in Chile.
Key words: Antigenic variant, bats, phylogenetic analysis, rabies.
Rabies is a fatal viral zoonosis caused by viruses of
the genus Lyssavirus in the family Rhabdoviridae. It
is transmitted between mammals, including bats, primarily
through bite inoculation of the rabies virus
(RABV) present in the saliva of infected individuals
[1]. Members of the Lyssavirus genus constitute a
single monophyletic clade, distinct from other rhabdoviruses.
The genus consists of 11 genotypes (seven
established genotypes and four newly described lyssaviruses
from Eurasia). Genotype 1 (RABV, classical
RABV) has worldwide distribution and at present is
the only genotype to be isolated in the Americas
(South, Central and North) that forms endemic cycles
within terrestrial mammals and bats [2].
Rabies occurs in two different epidemiological
forms : urban rabies, with dogs and domestic animals
as the principal reservoir and transmitter, and sylvatic
rabies, with various wild species in the Carnivora
and Chiroptera orders acting as reservoirs and transmitters.
In Chile, dog rabies has been controlled, and
since 1985 insectivorous bats have been identified
as the country’s main rabies reservoirs and infection
source for sporadic cases of rabies in domestic animals
[3, 4].
At least four genera of insectivorous bats (Tadarida,
Myotis, Histiotus, Lasiurus) are widely distributed in
Chile. The role of these species as reservoirs hosts and
transmitters supports the theory that diverse viral
variants of rabies are circulating. Recent evidence
suggests that all RABV variants affecting terrestrial
carnivores may have originated from cross-species
transmission events from long-term enzootic batassociated
variants. A molecular-clock model based
on genetic divergence of RABV variants in bats
of different species suggests that in North America,
the divergence of extant bat-associated RABVs from
a common ancestor took place between 1651 and
* Author for correspondence: Dr V. Yung, Marathon 1000, co´ digo
postal 7780050, N˜ un˜ oa, Santiago, Chile.
(Email: vyung@ispch.cl)
Epidemio1660 C.E. The bat RABV variants found in Latin
America in common vampire bats (Desmodus
rotundus) and free-tailed bats (genus Tadarida, family
Mollosidae) are the closest ones to the earliest common
ancestor [5].

The purpose of this study was to investigate
which RABV variants were circulating in Chile in
2002–2008. During this period, 11 342 animals from
areas around the country were submitted for rabies
testing. Of this number, 653 insectivorous bats, one
dog and one cat tested positive using the fluorescent
antibody test (FAT). Applying the mouse inoculation
test (MIT) [6], 612 samples were successfully isolated
and then typed using a panel of eight monoclonal
antibodies against the viral nucleoprotein (N-mAbs)
provided by the Centers for Disease Control and
Prevention (USA). The reaction patterns obtained
with different mAbs for determining the antigenic
variant have been described in a previous report [4].
Ninety-nine of the RABV isolates were selected for
performing nucleotide sequence analyses. Of these, 66
were from T. brasiliensis bats (the most common species
submitted for testing), 31 were from the remaining
insectivorous bat species (L. cinereus, L. borealis,
H. macrotus, M. chiloensis) and two were taken from
domestic animals (dog and cat). All 99 were collected
in Chile’s central region (Fig. 1).
Total RNA extraction was conducted using
TRIzol (Invitrogen, USA) in accordance with the
manufacturer’s instructions. Complementary DNA
(cDNA) was produced by reverse transcription–
polymerase chain reaction using primers 10 g and 304
as described previously and a product of 320-bp of
the nucleoprotein gene (1157–1476) was sequenced
using the BigDye Terminator Cycle Sequence kit v3.1
(Applied Biosystems, USA) [7]. Nucleotide sequences
were analysed with an ABI PRISM 3130 genetic analyser
(Applied Biosystems).
A phylogenetic tree was reconstructed for aligned
nucleotide sequences by means of a neighbour-joining
(NJ) analysis with 1000 bootstrap replicates using the
MEGA 3>1 software tool [8]. Bootstrap resampling
analysis of 1000 replicates was employed to estimate
the reliability of the prediction tree. For the phylogenetic
analysis, sequences from other countries
in the Americas were included (GenBank accession
numbers are given in Fig. 2). Two non-rabies
lyssaviruses, European bat 1 (EBLV Genbank accession
no. U22845) and Duvenhage virus (DUVV
Genbank accession no. U22848) were used as outgroups
[9].
Reaction patterns using a panel of eight mAbs of
613 rabies isolates revealed six different antigenic
variants in the Chilean bat species (Table 1). Of
this total, 572 isolates were antigenic variant 4 (568
from T. brasiliensis bats, two from M. chiloensis bats
and one each from a dog and a cat) and 18 were
antigenic variant 6 (14 from L. cinereus bats, four
Map of Chile Region Species
Year
2002 2003 2004 2005 2006 2007 2008 Total
III Tb 1 1
IV Tb 1 2 3
My 1 1 2
V Tb 1 2 1 1 1 1 2 9
My 1 1
H 1 1 1 1 1 5
Lc 1 1
VI Tb 2 4 6
My 1 1
H 1 1
Lc 1 1
VII Tb 1 1 2 1 4 9
My 1 1
Dog 1 1
Cat 1 1
VII Tb 2 4 1 1 1 1 2 12
H 1 1
Lb 1 1 2
XI Tb 1 1 1 1 4
My 1 1 2
X Tb 2 2 1 1 1 7
Lc 1 1
RM Tb 1 2 2 5 5 15
H 1 1 2
Lc 1 1 1 1 2 1 1 8
Lb 1 1 2
Rabies-positive bats
Rabies-positive dog
Rabies-positive cat
Fig. 1 [colour online]. Geographical distribution of sequenced rabies cases (Chile, 2002–2008).l.
1660 C.E. The bat RABV variants found in Latin
America in common vampire bats (Desmodus
rotundus) and free-tailed bats (genus Tadarida, family
Mollosidae) are the closest ones to the earliest common
ancestor [5].
The purpose of this study was to investigate
which RABV variants were circulating in Chile in
2002–2008. During this period, 11 342 animals from
areas around the country were submitted for rabies
testing. Of this number, 653 insectivorous bats, one
dog and one cat tested positive using the fluorescent
antibody test (FAT). Applying the mouse inoculation
test (MIT) [6], 612 samples were successfully isolated
and then typed using a panel of eight monoclonal
antibodies against the viral nucleoprotein (N-mAbs)
provided by the Centers for Disease Control and
Prevention (USA). The reaction patterns obtained
with different mAbs for determining the antigenic
variant have been described in a previous report [4].
Ninety-nine of the RABV isolates were selected for
performing nucleotide sequence analyses. Of these, 66
were from T. brasiliensis bats (the most common species
submitted for testing), 31 were from the remaining
insectivorous bat species (L. cinereus, L. borealis,
H. macrotus, M. chiloensis) and two were taken from
domestic animals (dog and cat). All 99 were collected
in Chile’s central region (Fig. 1).
Total RNA extraction was conducted using
TRIzol (Invitrogen, USA) in accordance with the
manufacturer’s instructions. Complementary DNA
(cDNA) was produced by reverse transcription–
polymerase chain reaction using primers 10 g and 304
as described previously and a product of 320-bp of
the nucleoprotein gene (1157–1476) was sequenced
using the BigDye Terminator Cycle Sequence kit v3.1
(Applied Biosystems, USA) [7]. Nucleotide sequences
were analysed with an ABI PRISM 3130 genetic analyser
(Applied Biosystems).
A phylogenetic tree was reconstructed for aligned
nucleotide sequences by means of a neighbour-joining
(NJ) analysis with 1000 bootstrap replicates using the
MEGA 3>1 software tool [8]. Bootstrap resampling
analysis of 1000 replicates was employed to estimate
the reliability of the prediction tree. For the phylogenetic
analysis, sequences from other countries
in the Americas were included (GenBank accession
numbers are given in Fig. 2). Two non-rabies
lyssaviruses, European bat 1 (EBLV Genbank accession
no. U22845) and Duvenhage virus (DUVV
Genbank accession no. U22848) were used as outgroups
[9].
Reaction patterns using a panel of eight mAbs of
613 rabies isolates revealed six different antigenic
variants in the Chilean bat species (Table 1). Of
this total, 572 isolates were antigenic variant 4 (568
from T. brasiliensis bats, two from M. chiloensis bats
and one each from a dog and a cat) and 18 were
antigenic variant 6 (14 from L. cinereus bats, four
Map of Chile Region Species
Year
2002 2003 2004 2005 2006 2007 2008 Total
III Tb 1 1
IV Tb 1 2 3
My 1 1 2
V Tb 1 2 1 1 1 1 2 9
My 1 1
H 1 1 1 1 1 5
Lc 1 1
VI Tb 2 4 6
My 1 1
H 1 1
Lc 1 1
VII Tb 1 1 2 1 4 9
My 1 1
Dog 1 1
Cat 1 1
VII Tb 2 4 1 1 1 1 2 12
H 1 1
Lb 1 1 2
XI Tb 1 1 1 1 4
My 1 1 2
X Tb 2 2 1 1 1 7
Lc 1 1
RM Tb 1 2 2 5 5 15
H 1 1 2
Lc 1 1 1 1 2 1 1 8
Lb 1 1 2
Rabies-positive bats
Rabies-positive dog
Rabies-positive cat
Fig. 1 [colour online]. Geographical distribution of sequenced rabies cases (Chile, 2002–2008).
2 V. Yung and others
from L. borealis bats). Eleven isolates from H. macrotus
bats and one from T. brasiliensis bats were associated
with an atypical antigenic variant described
previously in Chile that is unrelated to any previously
described reaction panel using a panel with eight
N-mAbs [7]. Five isolates from M. chiloensis
bats were characterized as antigenic variant 3, two
from M. chiloensis bats as variant 8, and four from
T. brasiliensis bats as variant 9 (associated with
T. brasiliensis mexicana).
Tb-2519_AV4
AY233427 Tb Arg.
Tb-732_AV4
Tb-1123_AV4
Tb-373_AV4
Tb-398_AV4
Ct-2427_AV4
Tb-789_AV4
Dg-2426_AV4
Tb-534_AV4
Tb-2701_AV4
Tb-610_AV4
Tb-3049_AV4
Tb-2899_AV9
Tb-815_AV4
Tb-188_AV4
AY233428 Tb Arg.
Tb-3190_AV4
Tb-1107_AV9
Tb-780_AV4
Tb-101_AV4
Tb-992_AV4
Tb-1275_AV4
Tb-131_AV4
Tb-26_AV4
EF377701 Colombia
AB201803
AB297635
AF045166
AF394876
AY959947
AY170236
DQ416111
AB297658 I.bat Brazil
AY233449 Argentina
Mch-2544_AV3
Mch-1110_AV8
Mch-2821_AV3
Mch-421_AV8
Mch-2672_AV3
Mch-462_AV3
Mch-3171_AV4
EF377703 I bat Colombia
AY233448 Argentina
DQ416123 Mexico.
Hm-860_NT
Hm-2106_NT
Tb-1980_NT
Hm-908_NT
Hm-419_NT
Hm-462_NT
Hm-1465_NT
Hm-880_NT
Hm-592_NT
Hm-2386_NT
EF377705 Colombia
AY039224 Texas
DQ416120 Mexico
Tb1952_AV9
AF351845 USA
AF394883 Texas
AB297653 Brazil
AF351844 USA
Lc-03_AV6
Lb-1193_AV6
Lc-2125_AV6
Lc-2665_AV6
Lc-1252_AV6
Lc-3043_AV6
Lc-299_AV6
Lc-2593_AV6
Lc-543_AV6
Lc-804_AV6
Lc-1320_AV6
Lb-679_AV6
Lb-198_AV6
Lb-3215_AV6
Lc-1236_AV6
AY233401
AY340785
AY233423
EBLV
DUVV
99
94
99
88
98
94
73
64
81
85
50
87
94
0.05
I
II
III
IV
AgV1-AgV2
Haematophagous bats
T.b . North
America
Fig. 2. Phylogenetic relationships among 99 RABV isolates from Chile (GenBank accession nos. HQ385325–HQ385423) and
rabies strains of bats from the Americas based on nucleotide homology of a 320-bp region of the nucleoprotein gene.
Neighbour-joining tree with bootstrap values >50% obtained from 1000 resamplings are shown in the nodes. Tb, Tadarida
brasiliensis ; My, Myotis chiloensis ; Hm, Histiotus macrotus; Lc, Lasiurus cinereus; Lb, Lasiurus borealis.
Rabies virus in Chile 3
Although it offers a rapid, simple and inexpensive
means of typing for epidemiological studies, antigenic
analysis with mAbs is lacking in precision. To obtain
a more accurate determination of the diversity of the
RABV in bat populations, partial sequencing and
phylogenetic analyses of 99 Chilean RABV isolates
were conducted. Four monophyletic clusters associated
with four different bat species were thus identified,
each one defined as a group of related sequences
that share specific patterns of nucleotide variation and
are associated with rabies maintained and transmitted
by the same or some other bat species according to
taxonomic identification of specimens (Fig. 2).
Cluster I contained 66 isolates obtained from 64
T. brasiliensis bats and two domestic animals (a dog
and a cat), but due to the large number of isolates
with 100% nucleotide similarity we took only representative
sequences for the phylogenetics analyses.
The overall average identity in these isolates
was 95.9%. This variant is distantly related to the
genetic variant circulating in the North American
T. brasiliensis bat population but is very closely related
to the genetic variants in Argentinean and
Colombian bats. The RABV found in T. brasiliensis
in Chile does not seem to be closely related to rabies in
the same species in North America, where the RABV
lineage found in T. brasiliensis is related primarily to
the vampire viruses [10]. Since RABV circulates
in Chile in insectivorous bats only, it is not found in
haematophagous bat species.
Cluster II was represented by isolates from six
M. chiloensis bats (colonial and non-migratory) with
an overall average identity of 95.5%. They were antigenically
identified as variants 3 and 8 (Table 1), but
in the genetic analysis they segregated into a different
cluster associated with Argentinean Myotis bats.
Cluster III was composed of 10 isolates, nine from
H. macrotus bats and one from a T. brasiliensis bat,
with an overall average identity of 98.6%. These isolates
clustered with viruses associated with H. macrotus
in Argentina and a Histiotus-like bat found in
Mexico [11]. Very little is known about the biology
and distribution of this bat species, which may be
found in other parts of the Americas in addition to
Chile, Argentina and Mexico [10].
Finally, Cluster IV was made up of 16 isolates,
of which four were from L. borealis bats, 11 from
L. cinereus bats and one from T. brasiliensis. The
overall average identity was 99.5%. The Lasiurus
genus is solitary and often described as a tree-dweller
due to its roosting preference. It is also migratory
and hence has a more southerly range during the
winter. All three of these species share the same
phylogenetic lineage as Lasiurus bats in North
America. Some bat species seem able to maintain the
same virus variant in geographically distant territories.
The two T. brasiliensis cases observed in this
cluster are probably spillovers of an endemic cycle
maintained by Lasiurus sp. This spillover transmission
mechanism may be due to the fact that solitary
bat species such as Lasiurus spp. can develop
furious rabies, in which case they may actively attack
bats or other animals [12].
One isolate (Mch-3171), obtained from a
M. chiloensis bat and antigenically identified as variant
4, segregated into a different cluster, with an
insectivorous bat from Colombia. It was more narrowly
related to cluster II. However, given the lack of
Table 1. Antigenic typing with monoclonal antibodies (mAbs) of rabies isolates from Chile
Antigenic
variant 2002 2003 2004 2005 2006 2007 2008 Total
4 98Tb 66Tb 74 Tb 92 Tb 99 Tb 71 Tb 68 Tb 572
1 Mch 1 Mch 1 Dog
1 Cat
6 2 Lc 2 Lc 2 Lb 3 Lc 3 Lc 1 Lc 2 Lc 18
1 Lc 2 Lb
NT 2 Hm 1 Tb 1 Hm 1 Hm 1 Hm 3 Hm 2 Hm 11
3 1 Mch 1 Mch 1 Mch 1 Mch 1 Mch 5
8 1 Mch 1 Mch 2
9 2 Tb 2 Tb 4
NT, Not typed; Tb, Tadarida brasiliensis ; Lc, Lasiurus cinereus; Lb, Lasiurus borealis; Hm, Histiotus macrotus; Mch,
Myotis chiloensis.
Rabies isolates are grouped according to patterns of reaction with eight N-mAbs.
4 V. Yung and others
statistical support for its potential association with
other RABVs so far reported, complete nucleoprotein
sequences and a more comprehensive sampling encompassing
RABV diversity in the region are needed
to help identify whether it is a new variant or the reservoir
host associated with it.
Although antigenic typing of RABV using mAbs
may distinguish diverse variants of the virus, distinguishing
different types within a variant may become
difficult using this method, which could be more easily
and accurately done with molecular characterization
via nucleotide and amino-acid sequence determinations.
These molecular analyses may help to unravel
the precise genetic diversity of a RABV and
the sequence characteristic of RABVs specifically
associated with each host species. The first phylogenetic
investigation into bat RABV using partial N
gene sequencing established that there were distinct
lineages of bat RABV associated with different bat
species [13].
RABV is widespread in the Americas and genetic
differentiation in RABVs is believed to have occurred
in response to their association with particular host
species [14]. However, topography may play a less
significant role in shaping the phylogeny of bat RABV
than it potentially does for terrestrial mammal RABV
[15]. When a physical barrier is considerable (e.g. the
Andes mountain range) genetic isolation may occur,
as demonstrated by the separation of the Chilean
strains from isolate samples obtained in other Latin
American locations [12].
In Chile, where long-term enzootic canine RABVs
have not been detected since 1990, the disease is confined
to the wild cycle mainly due to T. brasiliensis
bats. Although no human rabies cases have been reported
since 1996, rabies remains a public health risk
in Chile and other parts of Latin America because of
the frequency of contact between humans and bats.
The coexistence of an abundant bat population with
humans and their domestic animals in the urban centres
of these countries poses a new challenge to the
understanding of rabies epidemiology in metropolitan
areas [16, 17].
The approach adopted in this study enabled the
identification of several rabies enzootics maintained
independently by different species of insectivorous
bat through transmission events involving bat-to-bat
or bat-to-terrestrial species. The investigation also
confirmed T. brasiliensis as the main RABV reservoir
and the existence of compartmentalization in Chile in
other bat species.
Finally, we note that studies of RABV characterization
are a valuable asset in supporting epidemiological
surveillance systems for the disease and
selecting control strategies and monitoring programmes,
which can have major impacts on both
human health and ecosystems.
ACKNOWLEDGEMENTS
We thank the laboratory staff who assisted in this
study for their excellent technical assistance and also
thank Kenneth Rivkin for his valuable contribution
in the translation of this paper. The authors are also
grateful for funding provided by the Public Health
Institute of Chile.

REFERENCES
1. Johnson N, et al. Phylogenetics comparison of the genus
Lyssavirus using distal coding sequences of the glycoprotein
and nucleoprotein genes. Archives of Virology
2002; 147: 2111–2123.
2. Kuzmin IV, et al. The rhabdoviruses: biodiversity,
phylogenetics, and evolution. Infection, Genetics and
Evolution 2009; 9: 541–53.
3. Nun˜ ez S, et al. Wild rabies in insectivorous bats in
Chile. Bulletin of Pan American Health Organization
1987; 103: 140–145.
4. Favi M, et al. Role of insectivorous bats in the
transmission of rabies in Chile. Archivos de Medicina
Veterinaria 1999; 31: 157–165.
5. Calisher C, et al. Bats: important reservoir hosts of
emerging viruses. Clinical Microbiology Reviews 2006;
19: 531–545.
6. Koprowsky H. The mouse inoculation test. In:
Kaplan MN, Koprowsky H, eds. Rabies. Laboratory
Techniques. Ginebra: OMS, 1976, pp 88–97.
7. Yung V, Ferna´ ndez J, Favi M. Genetic and antigenic
typing of rabies virus in Chile. Archives of Virology
2002; 147: 197–205.
8. Kumar S, Tamura K, Nei M. MEGA 3: Integrated
software for molecular evolutionary genetics analysis
and sequence alignment. Briefings in Bioinformatics
2004; 5: 150–63.
9. Kissi B, Tordo N, Bourhy H. Genetics polymorphism in
the rabies virus nucleoprotein gene. Virology 1995; 209:
526–537.
10. Velasco-Villa A, et al. Molecular diversity of rabies
viruses associated with bats in Mexico and other
countries of the Americas. Journal of Clinical Microbiology
2006; 44: 1697–710.
Rabies virus in Chile 5
11. Cisterna D, et al. Antigenic and molecular characterization
of rabies virus in Argentina. Virus Research
2005; 109: 139–147.
12. Kuzmin I, Rupprech C. Bat rabies. In: Jackson A,
Wunner B, eds. Rabies, 2nd edn, 2009, pp. 259–381.
13. Smith J S, et al. Epidemiologic and historical relationships
among 87 rabies virus isolates as determine by
limited sequence analysis. Journal of Infectious Disease
1992; 166: 296–307.
14. Hughes GJ, Orciari LA, Rupprecht CE. Evolutionary
timescale of rabies virus adaptation to North American
bats inferred from the substitution rate of the nucleoprotein
gene. Journal of General Virology 2005; 86:
1467–1474.
15. Davis PL, Bourhy H, Holmes EC. The evolutionary
history and dynamics of bat rabies virus. Infection,
Genetics and Evolution 2006; 6: 464–473.
16. Favi M, et al. First case of human rabies in Chile due to
an insectivorous bats virus variant. Emerging Infectious
Disease 2002; 8: 79–81.
17. De Mattos C, et al. Bats rabies in urban centers in Chile.
Journal of Wildlife Disease 2000; 36: 231–240.
6 V. Yung and others

No hay comentarios:

Publicar un comentario en la entrada