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. 2010 Nov 8;3:288. doi: 10.1186/1756-0500-3-288

Epidemiology of vampire bat-transmitted rabies virus in Goiás, central Brazil: re-evaluation based on G-L intergenic region

Shinji Hirano 1, Takuya Itou 1,✉, Adolorata AB Carvalho 2, Fumio H Ito 3, Takeo Sakai 1
PMCID: PMC2993726  PMID: 21059233

Abstract

Background

Vampire bat related rabies harms both livestock industry and public health sector in central Brazil. The geographical distributions of vampire bat-transmitted rabies virus variants are delimited by mountain chains. These findings were elucidated by analyzing a high conserved nucleoprotein gene. This study aims to elucidate the detailed epidemiological characters of vampire bat-transmitted rabies virus by phylogenetic methods based on 619-nt sequence including unconserved G-L intergenic region.

Findings

The vampire bat-transmitted rabies virus isolates divided into 8 phylogenetic lineages in the previous nucleoprotein gene analysis were divided into 10 phylogenetic lineages with significant bootstrap values. The distributions of most variants were reconfirmed to be delimited by mountain chains. Furthermore, variants in undulating areas have narrow distributions and are apparently separated by mountain ridges.

Conclusions

This study demonstrates that the 619-nt sequence including G-L intergenic region is more useful for a state-level phylogenetic analysis of rabies virus than the partial nucleoprotein gene, and simultaneously that the distribution of vampire bat-transmitted RABV variants tends to be separated not only by mountain chains but also by mountain ridges, thus suggesting that the diversity of vampire bat-transmitted RABV variants was delimited by geographical undulations.

Background

Rabies is a zoonosis that kills infected mammals, including humans, and is mainly transmitted by carnivores. In the Americas, chiropterans (insectivorous, frugivorous and hematophagous bat) are another reservoir of this disease. Although dog-transmitted rabies in central Brazil has been reduced by aggressive vaccination programs [1], chiroptera (particularly the common vampire bat, Desmodus rotundas)-transmitted rabies remains endemic in this region, and harms both the livestock industry and the public health sector [2,3].

To date, vampire bat-transmitted rabies in livestock has been controlled by reducing the population of vampire bats and by vaccinating livestock [3,4]. However, the depopulation of vampire bats has limitations and the effects are temporary, while vaccination of livestock is only carried out for some animals and is ineffective in decreasing rabies levels in vampire bats.

For the sustainable and effective control of vampire bat rabies, further knowledge of epidemiological features, such as vampire bat ecology and the dynamics of vampire bat-transmitted rabies, is necessary. Molecular epidemiological analysis of vampire bat-transmitted cattle rabies cases using the partial nucleoprotein gene, which is the most conserved gene in the rabies virus (RABV) genome, has suggested that the distribution of variants in Brazil is delimited by mountain chains and clustered in tens of thousands of square kilometers [5]. However, the vampire bats migrate several kilometers from their nests [6]. To elucidate a more detailed genetic clustering and geo-distribution of genetic clades of vampire bat-transmitted RABV, 204 isolates from Goiás, which includes the 185 isolates analyzed previously, were employed and analyzed by a phylogenetic method based on a nucleotide sequence encompassing the G-L intergenic region locating between glycoprotein (G) and polymerase (L) gene loci, which is the most divergent region in the RABV genome and is used for monitoring epidemiological changes in the evolution of RABV [7,8].

Results

Phylogenetic analysis

The 204 RABV isolates divided into 8 phylogenetic lineages in the previous nucleoprotein gene analysis were divided into 10 phylogenetic lineages with significant bootstrap values (Figure 1; details shown in Table 1). Isolates of the C-5 and C-6 lineages designated by Kobayashi et al. belonged to the A-lineage, while the B-lineage consisted of some isolates of C-5. The C-, D-, F- and J-lineages included isolates belonging to C-12, C-1, C-22 and C-3, respectively. Isolates of C-20 belonged to the I-lineage, and C-21 was divided into three lineages; G-, H- and I-lineages. Two isolates not belonging to any lineages in previous studies were assigned to the E-lineage.

Figure 1.

Figure 1

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NJ tree based on 619-nt sequence of the partial G and the G-L intergenic region. Full circles on internal branches indicate significant bootstrap values (>70%). The number of isolates retaining 100% nucleotide similarity with the representative isolate is shown in parentheses. Lineages of the isolates defined in a previous study [5] are shown in italics after a diagonal (Ud: undefined).

Table 1.

Isolates from Goiás

Grouping Accession No.
Sample Species Location Year This study Previous studyc N203-nt Partial G & GL
BRbv371 Cattle Caldas Novas 2002 A C-5 AB307182 AB544082
BRbv372 Cattle Água Limpa 2002 A C-5 AB307183 AB544083
BRbv373 Cattle Caldas Novas 2002 A C-5 AB307184 AB544084
BRbv375 Cattle Santa Cruz de Goias 2002 A C-5 AB307186 AB544085
BRbv379 Cattle Corumbaíba 2002 A C-5 AB307190 AB544089
BRbv380 Cattle Corumbaíba 2002 A C-5 AB307191 AB544090
BRbv381 Cattle Cristalina 2002 A C-5 AB307192 AB544091
BRbv386 Cattle Ipameri 2002 A C-5 AB307197 AB544095
BRbv391 Cattle Caldas Novas 2002 A C-5 AB307201 AB544099
BRbv392 Cattle Água Limpa 2002 A C-5 AB307202 AB544100
BRbv393 Cattle Itumbiara 2002 A C-5 AB307203 AB544101
BRbv395 Cattle Itumbiara 2002 A C-5 AB307205 AB544102
BRbv396 Cattle Itumbiara 2002 A C-5 AB307206 AB544103
BRbv397 Cattle Buriti Alegre 2002 A C-5 AB307207 AB544104
BRbv398 Cattle Itumbiara 2002 A C-5 AB307208 AB544105
BRbv400 Cattle Buriti Alegre 2002 A C-5 AB307210 AB544107
BRbv404 Cattle Buriti Alegre 2002 A C-5 AB307214 AB544111
BRbv405 Cattle Buriti Alegre 2002 A C-5 AB307215 AB544112
BRbv407 Cattle Goiandira 2002 A C-5 AB307217 AB544114
BRbv412 Cattle Itapaci 2002 A C-5 AB307222 AB544118
BRbv413 Cattle Caldas Novas 2002 A C-5 AB307223 AB544119
BRbv416 Cattle Morrinhos 2002 A C-5 AB307226 AB544122
BRbv421 Cattle Nova Aurora 2002 A C-5 AB307231 AB544124
BRbv429 Cattle Corumbaíba 2002 A Uda AB307238 AB544129
BRbv432 Cattle Ipameri 2002 A C-5 AB307241 AB544132
BRbv438 Cattle Buriti Alegre 2002 A C-5 AB307247 AB544137
BRhr441 Horse Buriti Alegre 2002 A Ud AB307251 AB544140
BRbv442 Cattle Buriti Alegre 2002 A C-5 AB307252 AB544141
BRbv447 Cattle Urutaí 2002 A C-6 AB307255 AB544143
BRbv449 Cattle Caldas Novas 2002 A C-5 AB307256 AB544145
BRbv451 Cattle Ipameri 2002 A C-5 AB307258 AB544147
BRbv453 Cattle São Luis de Montes Belos 2002 A C-5 AB307260 AB544149
BRbv456 Cattle Orizona 2002 A Ud AB307263 AB544150
BRbv457 Cattle Água Limpa 2002 A C-5 AB307264 AB544151
BRbv458 Cattle Buriti Alegre 2002 A C-5 AB307265 AB544152
BRbv466 Cattle Itumbiara 2002 A C-5 AB307273 AB544157
BRbv469 Cattle Ipameri 2002 A C-6 AB307275 AB544159
BRbv471 Cattle Santa Cruz de Goias 2002 A C-6 AB307277 AB544161
BRbv472 Cattle Ipameri 2002 A C-6 AB307278 AB544162
BRbv473 Cattle Itumbiara 2002 A C-5 AB307279 AB544163
BRbv475 Cattle Caldas Novas 2002 A C-5 AB307281 AB544165
BRbv477 Cattle Itumbiara 2002 A C-5 AB307283 AB544166
BRbv478 Cattle Itumbiara 2002 A C-5 AB307284 AB544167
BRbv482 Cattle Panamá 2002 A C-5 AB307288 AB544170
BRhr483 Horse Panamá 2002 A Ud AB307291 AB544171
BRbv486 Cattle Itumbiara 2002 A C-5 AB307293 AB544172
BRbv489 Cattle Itumbiara 2002 A Ud AB307295 AB544174
BRbv493 Cattle Ipameri 2002 A C-6 AB307298 AB544177
BRbv495 Cattle Panamá 2002 A C-5 AB307300 AB544179
BRbv496 Cattle Panamá 2002 A C-5 AB307301 AB544180
BRbv497 Cattle Itumbiara 2002 A C-5 AB307302 AB544181
BRbv498 Cattle Bela Vista de Goiás 2002 A C-5 AB307303 AB544182
BRbv502 Cattle Itumbiara 2002 A C-5 AB307306 AB544186
BRbv503 Cattle Orizona 2002 A Ud AB307307 AB544187
BRbv509 Cattle Buriti Alegre 2002 A C-5 AB307311 AB544192
BRbv514 Cattle Panamá 2002 A C-5 AB307316 AB544196
BRbv516 Cattle Orizona 2002 A Ud AB307318 AB544198
BRbv521 Cattle Urutaí 2002 A Ndb Nd AB544201
BRbv524 Cattle Urutaí 2002 A C-6 AB307323 AB544204
BRbv526 Cattle Panamá 2002 A C-5 AB307325 AB544206
BRbv527 Cattle Campos Belos 2002 A C-5 AB307326 AB544207
BRbv684 Cattle Ipameri 2001 A C-5 AB307412 AB544211
BRbv685 Cattle Corumbaíba 2001 A Nd Nd AB544212
BRbv686 Cattle Ipameri 2001 A C-5 AB307413 AB544213
BRbv687 Cattle Corumbaíba 2001 A C-5 AB307414 AB544214
BRbv690 Cattle Ipameri 2001 A C-5 AB307417 AB544215
BRbv691 Cattle Ipameri 2001 A C-5 AB307418 AB544216
BRbv694 Cattle Iporá 2001 A Nd Nd AB544219
BRbv697 Cattle Campo Alegre de Goiás 2001 A C-5 AB307423 AB544221
BRbv698 Cattle Campo Alegre de Goiás 2001 A C-5 AB307424 AB544222
BRbv699 Cattle Campo Alegre de Goiás 2001 A C-5 AB307425 AB544223
BRbv700 Cattle Caldas Novas 2001 A Nd Nd AB544224
BRbv701 Cattle Caldas Novas 2001 A C-5 AB307426 AB544225
BRhr704 Horse Corumbaíba 2001 A Nd Nd AB544227
BRbv705 Cattle Ipameri 2001 A C-5 AB307428 AB544228
BRbv707 Cattle Corumbaíba 2001 A Nd Nd AB544229
BRsp708 Sheep Ouvidor 2001 A Nd Nd AB544230
BRbv710 Cattle Morrinhos 2001 A C-5 AB307431 AB544232
BRbv711 Cattle Morrinhos 2001 A C-5 AB307432 AB544233
BRbv717 Cattle Ipameri 2001 A C-5 AB307438 AB544235
BRbv718 Cattle Ipameri 2001 A C-5 AB307439 AB544236
BRbv741 Cattle Marzagão 2001 A C-5 AB307450 AB544244
BRbv742 Cattle Ipameri 2001 A C-5 AB307451 AB544245
BRbv745 Cattle Buriti Alegre 2001 A C-5 AB307454 AB544248
BRbv747 Cattle Corumbaíba 2001 A Ud AB307456 AB544250
BRbv748 Cattle Campo Alegre de Goiás 2001 A C-5 AB307457 AB544251
BRbv752 Cattle Catalão 2001 A Ud AB307461 AB544254
BRhr755 Horse Cumari 2001 A Nd Nd AB544257
BRbv770 Cattle Ipameri 2002 A C-5 AB307476 AB544269
BRbv771 Cattle Caldas Novas 2002 A C-5 AB307477 AB544270
BRbv780 Cattle Nova Crixás 2002 A C-5 AB307484 AB544274
BRbv785 Cattle Corumbaíba 2002 A C-5 AB307486 AB544277
BRbv786 Cattle Buriti Alegre 2002 A Ud AB307487 AB544278
BRbv796 Cattle Pires do Rio 2002 A C-6 AB307495 AB544283
BRbv797 Cattle Cristalina 2002 A C-6 AB307496 AB544284
BRbv402 Cattle Divinópolis de Goiás 2002 B C-5 AB307212 AB544109
BRbv406 Cattle Posse 2002 B C-5 AB307216 AB544113
BRbv459 Cattle Monte Alegre de Goiás 2002 B C-5 AB307266 AB544153
BRbv461 Cattle Monte Alegre de Goiás 2002 B C-5 AB307268 AB544155
BRbv462 Cattle Monte Alegre de Goiás 2002 B C-5 AB307269 AB544156
BRbv479 Cattle Colinas do Sul 2002 B C-5 AB307285 AB544168
BRbv481 Cattle Serranópolis 2002 B C-5 AB307287 AB544169
BRbv490 Cattle Colinas do Sul 2002 B C-5 AB307296 AB544175
BRbv494 Cattle Divinópolis de Goiás 2002 B C-5 AB307299 AB544178
BRbv500 Cattle Colinas do Sul 2002 B C-5 AB307304 AB544184
BRbv505 Cattle Colinas do Sul 2002 B C-5 AB307309 AB544188
BRhr506 Horse Colinas do Sul 2002 B Nd Nd AB544189
BRhr507 Horse Colinas do Sul 2002 B Nd Nd AB544190
BRbv510 Cattle Monte Alegre de Goiás 2002 B C-5 AB307312 AB544193
BRbv511 Cattle Divinópolis de Goiás 2002 B C-5 AB307313 AB544194
BRbv518 Cattle Campos Belos 2002 B C-5 AB307319 AB544199
BRbv525 Cattle Panamá 2002 B C-5 AB307324 AB544205
BRbv753 Cattle Monte Alegre de Goiás 2001 B C-5 AB307462 AB544255
BRbv762 Cattle Divinópolis de Goiás 2001 B C-5 AB307470 AB544263
BRbv387 Cattle Doverlândia 2002 C C-12 AB307198 AB544096
BRbv401 Cattle Mineiros 2002 C C-12 AB307211 AB544108
BRbv424 Cattle Doverlândia 2002 C C-12 AB307234 AB544125
BRbv692 Cattle Mineiros 2001 C C-12 AB307419 AB544217
BRbv712 Cattle Doverlândia 2001 C C-12 AB307433 AB544234
BRbv751 Cattle Mineiros 2001 C C-12 AB307460 AB544253
BRbv383 Cattle Nova América 2002 D C-1 AB307194 AB544092
BRbv384 Cattle Nova América 2002 D C-1 AB307195 AB544093
BRbv409 Cattle Rubiataba 2002 D C-1 AB307219 AB544115
BRbv411 Cattle Morrinhos 2002 D C-1 AB307221 AB544117
BRbv452 Cattle Rubiataba 2002 D C-1 AB307259 AB544148
BRbv767 Cattle Rubiataba 2002 D C-1 AB307474 AB544267
BRbv793 Cattle Urutaí 2002 D C-1 AB307493 AB544282
BRbv440 Cattle Nova Crixás 2002 E Ud AB307249 AB544139
BRbv519 Cattle Mundo Novo 2002 E Ud AB307320 AB544200
BRhr389 Horse Nova Crixás 2002 F Nd Nd AB544098
BRbv434 Cattle Itapuranga 2002 F Ud AB307243 AB544133
BRbv439 Cattle Mundo Novo 2002 F C-22 AB307248 AB544138
BRbv488 Cattle Carmo do Rio Verde 2002 F Ud AB307294 AB544173
BRbv749 Cattle Nova Crixás 2001 F C-22 AB307458 AB544252
BRbv757 Cattle Monte Alegre de Goiás 2001 F C-22 AB307465 AB544259
BRbv763 Cattle Aruanã 2002 F C-22 AB307471 AB544264
BRbv773 Cattle Aruanã 2002 F C-22 AB307479 AB544272
BRbv774 Cattle Mozarlândia 2002 F C-22 AB307480 AB544273
BRsp787 Sheep Ipameri 2002 F Nd Nd AB544279
BRbv378 Cattle Novo Brasil 2002 G C-21 AB307189 AB544088
BRbv425 Cattle Novo Brasil 2002 G C-21 AB307235 AB544126
BRbv445 Cattle Morrinhos 2002 G Ud AB307254 AB544142
BRbv448 Cattle Buriti de Goiás 2002 G C-21 AB307256 AB544144
BRhr467 Horse Buriti de Goiás 2002 G Nd Nd AB544158
BRbv474 Cattle Moiporá 2002 G C-21 AB307280 AB544164
BRbv501 Cattle Anicuns 2002 G C-21 AB307305 AB544185
BRbv515 Cattle Córrego do Ouro 2002 G C-21 AB307317 AB544197
BRbv522 Cattle Buriti de Goiás 2002 G Nd Nd AB544202
BRbv523 Cattle Córrego do Ouro 2002 G C-21 AB307322 AB544203
BRbv764 Cattle Córrego do Ouro 2002 G C-21 AB307472 AB544265
BRbv385 Cattle Ivolândia 2002 H C-21 AB307196 AB544094
BRbv414 Cattle Ivolândia 2002 H C-21 AB307224 AB544120
BRbv415 Cattle Ivolândia 2002 H C-21 AB307225 AB544121
BRbv426 Cattle Amorinópolis 2002 H C-21 AB307236 AB544127
BRbv427 Cattle Ivolândia 2002 H C-21 AB307237 AB544128
BRbv431 Cattle Palestina de Goiás 2002 H Ud AB307240 AB544131
BRbv437 Cattle Goiandira 2002 H Ud AB307246 AB544136
BRbv693 Cattle Amorinópolis 2001 H C-21 AB307420 AB544218
BRhr703 Horse Ivolândia 2001 H Nd Nd AB544226
BRbv736 Cattle Amorinópolis 2001 H C-21 AB307446 AB544242
BRbv744 Cattle Amorinópolis 2001 H C-21 AB307453 AB544247
BRbv370 Cattle Caiapônia 2002 I C-21 AB307181 AB544081
BRbv376 Cattle Caiapônia 2002 I C-21 AB307187 AB544086
BRbv377 Cattle Bom Jardim de Goiás 2002 I C-21 AB307188 AB544087
BRbv388 Cattle Piranhas 2002 I Ud AB307199 AB544097
BRbv399 Cattle Rio Verde 2002 I C-21 AB307209 AB544106
BRbv403 Cattle Piranhas 2002 I C-20 AB307213 AB544110
BRbv410 Cattle Piranhas 2002 I C-21 AB307220 AB544116
BRbv420 Cattle Piranhas 2002 I C-21 AB307230 AB544123
BRbv430 Cattle Buriti Alegre 2002 I Ud AB307239 AB544130
BRbv435 Cattle Bom Jardim de Goiás 2002 I C-20 AB307244 AB544134
BRbv436 Cattle Piranhas 2002 I Ud AB307245 AB544135
BRbv450 Cattle Palestina de Goiás 2002 I C-21 AB307257 AB544146
BRbv460 Cattle Caiapônia 2002 I C-20 AB307267 AB544154
BRbv470 Cattle Piranhas 2002 I C-20 AB307276 AB544160
BRbv491 Cattle Caiapônia 2002 I C-20 AB307297 AB544176
BRbv499 Cattle Bom Jardim de Goiás 2002 I Nd Nd AB544183
BRbv513 Cattle Caiapônia 2002 I C-20 AB307315 AB544195
BRbv681 Cattle Piranhas 2001 I Nd Nd AB544208
BRbv682 Cattle Piranhas 2001 I Nd Nd AB544209
BRbv683 Cattle Piranhas 2001 I C-21 AB307411 AB544210
BRbv696 Cattle Piranhas 2001 I C-21 AB307422 AB544220
BRbv709 Cattle Piranhas 2001 I C-21 AB307430 AB544231
BRbv720 Cattle Arenópolis 2001 I C-21 AB307440 AB544237
BRbv721 Cattle Arenópolis 2001 I C-21 AB307441 AB544238
BRbv722 Cattle Arenópolis 2001 I C-21 AB307442 AB544239
BRbv734 Cattle Arenópolis 2001 I C-21 AB307444 AB544240
BRbv735 Cattle Arenópolis 2001 I Ud AB307445 AB544241
BRbv738 Cattle Piranhas 2001 I C-20 AB307448 AB544243
BRbv743 Cattle Marzagão 2001 I C-20 AB307452 AB544246
BRbv746 Cattle Caiapônia 2001 I C-20 AB307455 AB544249
BRbv756 Cattle Arenópolis 2001 I C-21 AB307464 AB544258
BRbv758 Cattle Iporá 2001 I C-21 AB307466 AB544260
BRbv759 Cattle Diorama 2001 I C-21 AB307467 AB544261
BRbv760 Cattle Palestina de Goiás 2001 I C-21 AB307468 AB544262
BRbv765 Cattle Caiapônia 2002 I C-20 AB307473 AB544266
BRbv769 Cattle Palestina de Goiás 2002 I C-21 AB307475 AB544268
BRbv772 Cattle Arenópolis 2002 I C-21 AB307478 AB544271
BRhr782 Horse Palestina de Goiás 2002 I Nd Nd AB544275
BRbv784 Cattle Piranhas 2002 I C-20 AB307485 AB544276
BRbv790 Cattle Pires do Rio 2002 I C-21 AB307490 AB544280
BRbv508 Cattle Porangatatu 2002 J C-3 AB307310 AB544191
BRbv754 Cattle Alvorada do Norte 2001 J C-3 AB307463 AB544256
BRbv792 Cattle Morrinhos 2002 J C-3 AB307492 AB544281
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aUndefined; bNo data; cKobayashi et al. (2008)

Geographical plotting

In this study, geographical areas in Goiás were divided by mountain chains into the Northwest, North central, Northeast and South regions (Figure 2). Most isolates of the A-lineage were distributed in the South region. The B-lineage was likely to exist in the Northeast and North central regions. The isolates belonging to the C-, E-, F-, G-, H- and I-lineages were distributed in the Northwest region. The G-, H- and I-lineages have narrow distributions and are apparently separated by mountain ridges (Figure 2; Area I), while the A-lineage was distributed in a wide range throughout a Southeastern basin (Figure 2; Area II). The isolates of the D-lineage were plotted on an eastern edge of the North central region. The J-lineage had a wide geographical distribution in Goiás.

Figure 2.

Figure 2

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Distribution of vampire bat-transmitted RABV isolates in Goiás. Area I: distribution of G-, H- and I-lineages in mountain ridges. Area II: distribution of A-lineage in the basin. Symbols are same as those used in Fig. 1. Numbers inside the symbols indicate the number of isolates obtained in the same region. Small and open symbols indicated that only one isolate was obtained.

Discussion

Previous studies had elucidated that the distributions of vampire bat and transmitted RABV are delimited by mountain chains [5]. In the present study, it was reconfirmed that mountain chains divide the distribution patterns of each viral lineage. Furthermore, the isolates belonging to C-5 having a wide range in Goiás were divided into the A- and B-lineages, and were found to be distributed in the South and North regions on either side of a mountain chain. This finding supports Kobayashi's hypothesis that distribution of vampire bat-transmitted RABV is affected by mountain chains.

The same variants of vampire bat-transmitted RABV were spread widely in flat low lands (< 800 m), but at higher elevations (800-1600 m), they had a narrower distribution [9]. However, the G-, H- and I-lineages were found to be separated by mountain ridges in low areas (400-800 m) located in a southern undulating area of the Northwestern region (Figure 2; Area I). Furthermore, the A-lineage was located in an eastern basin of the South region (> 800 m; Figure 2; Area II). Considering that the higher lands showed an undulating landscape, the results suggest that the distribution patterns of vampire bat-transmitted RABV variants depend on such undulations. On the other hand, the distribution of common vampire bats in a valley is limited by the ridges that form the valley [10], thus supporting the notion that the distribution of RABV variants is affected by smaller topography than mountain chains.

Conclusions

The present study analyzed the epidemiology of vampire bat-transmitted RABV using a 619-nt region containing the partial glycoprotein gene and the G-L intergenic region, and indicated that the isolates can be further divided into several phylogenetic lineages with significant bootstrap values when compared to characterization based on the 203-nt partial N gene. Furthermore, the phylogenetic lineages were divided by both mountain chains and mountain ridges. In future studies, it will be important to analyze samples from different time points and to elucidate the dynamics of vampire bat-transmitted rabies in order to establish effective and sustainable control measures for preventing rabies circulation among vampire bats.

Methods

Samples

A total of 204 samples obtained from 192 cattle, 10 horses and 2 sheep in Goiás from October 2001 to August 2002 were employed, which had been confirmed as rabies positive through fluorescent antibody test and mouse inoculation test (Table 1). Viral RNA was extracted from the brain as described previously [11]. Lineages of the 164 cattle isolates, C-1, C-3, C-5, C-6, C-12, C-20, C-21 and C-22, were previously characterized based on a 203-nt sequence of the nucleoprotein gene [5], and are shown in Table 1.

Determination of nucleotide sequences

RT-PCR and direct sequencing with the HmG5-1302 (4619TGTTGAAGTTCACCTYCCMGATGT4642, positions relative to PV strain genome (Accession No. M13215) and RVLa-1 (5435ATRGGGTCATCATAAACCTC5416) primer pair were performed as described previously [11]. The target sequence includes the partial glycoprotein gene and G-L intergenic region. Nucleotide sequences were determined using the ATGC program version 4.02 (GENETYX Co., Tokyo, Japan).

Phylogenetioc analysis

Multiple nucleotide sequence alignments of the partial glycoprotein gene and G-L intergenic region were generated by the ClustalW package in MEGA ver. 4.0 [12]. A phylogenetic tree was constructed by the neighbor joining (NJ) method with bootstrap analysis (1000 psuedoreplicates) under the p-distance model. Phylogenetic clustering supported by a bootstrap value exceeding 70% was regarded as a reliable lineage [13]. Results were validated by the maximum likelihood method using PhyML [14]. In order to reconfirm the shape of the NJ tree, the ML tree was constructed under HKY substitution model justified by MODELTEST packaged in Hyphy program [15].

Geographical plotting

The 204 RABV isolates were plotted onto a geographical map described using the DIVA-GIS program [16] with GIS data from Instituto Brasileiro de Geografia e Estatística [17] and DIVA-GIS gData [18].

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SH participated in the design of the study, performed the experimental procedures and the data analysis, and wrote the manuscript. TI, AABC, FHI and TS elaborated the study design, management, coordination, and drafting the manuscript. The authors have read and approved the final manuscript.

Contributor Information

Shinji Hirano, Email: s.hirano19821007@gmail.com.

Takuya Itou, Email: itou.takuya@nihon-u.ac.jp.

Adolorata AB Carvalho, Email: adbianco@fcav.unesp.br.

Fumio H Ito, Email: fumio@usp.br.

Takeo Sakai, Email: sakai.takeo@nihon-u.ac.jp.

Acknowledgements

This work was partly supported by the "Strategic Research Base Development" Program for Private Universities subsidized by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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