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.

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