Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.
Rabies is a worldwide neglected fatal encephalitis, (1,2) which is listed amongst the ten major infectious causes of human deaths worldwide, estimated at 55,000 per year. (3) This infection is efficiently preventable by vaccination, (4) but treatment costs of rabid or exposed patients, diagnostic procedures, and vaccines make it a significant challenge for public health systems in endemic countries. (5,6)
The disease is caused by rabies virus (RABV) (family Rhabdoviridae, genus Lyssavirus), (7) an enveloped virus with a length of 110-250 nm and a diameter of 75 nm, usually transmitted by the saliva of an infected mammal. The RABV genome has a negative-stranded non-segmented ssRNA with 11,932-nucleotides that encodes the five structural proteins N (nucleoprotein), P (phosphoprotein), M (matrix), G (envelope glycoprotein), and L (large, RNA-dependent RNA-polymerase). (8,9) Between 2004 and 2005, 62 people died in the Brazilian Amazon of rabies transmitted by Desmodus rotundus vampire bat, a primary reservoir of rabies in Latin America. (10,11) Regarding frugivorous bats of the genus Artibeus, rabies has been reported in the species A. fimbriatus, A. jamaicensis, A. lituratus, and A. planirostris. (12) Artibeus spp. bats have been assigned an increasing importance in public health, as they are considered a rabies reservoir for humans in urban areas in Brazil, which is aggravated by the increasing population of these bats and the fact that they share roosts with D. rotundus. (12-14)
From 2003 to 2008, the Instituto Pasteur in Brazil tested 18,007 non-hematophagous bats for rabies, 252 of which were found to be positive. Also, from 2005 and 2007, 56 out of 160 non-hematophagous bats that tested positive for rabies were classified as A. lituratus.
Phylogenetic studies based on the N gene have suggested that RABV lineages from Artibeus sp. are not divergent from those from D. rotundus, (15) all belonging to the same genic (16) and antigenic variant 3 (AgV3). (17) Nonetheless, these studies have provided only inconclusive results, as they were based on a very restricted sampling regarding geographic area and sample number, mainly in the case of Artibeus spp. In addition, these studies were based on a single gene sequences for phylogenetic reconstructions.
The ability to determine the source of infection and the epidemiology of rabies cycles are paramount for accurate decision-making in public health, mainly regarding vaccination strategies and animal population control. This study aimed to evaluate the possibility of distinction between RABV genetic lineages related to D. rotundus and Artibeus spp. bats based on N and G genes sequences.
Materials and methods
Rabies virus strains
Twenty Artibeus spp. RABV strains were obtained from first-passage isolates in mice inoculated with 20% suspensions of A. lituratus and Artibeus spp. central nervous systems (CNS); 15 D. rotundus-related strains were obtained directly from naturally infected cattle brain tissues (Table 1). These 35 strains were collected in nine municipalities from Sao Paulo State, Southeastern Brazil (Fig. 1), between 2004 and 2005. All samples were diagnosed positive for rabies by direct immunofluorescence test (DIFT) targeted to the viral nucleoprotein. (18)
[FIGURE 1 OMITTED]
The nucleoprotein and the glycoprotein sequences generated in this study have been assigned the GenBank accession numbers JF682392-JF682426 and JF682427-JF682461, respectively.
Amplification and sequencing of the nucleoprotein and glycoprotein genes
Total RNA from the 35 RABV isolates CNS samples tested and the from positive and negative controls were extracted with TRIzol[TM] (Invitrogen--Carlsbad, CA, USA) method, following the manufacturer's instructions. The challenge virus standard (CVS) fixed strain of RABV isolated in mice brain and nuclease free-water were used as positive and negative controls, respectively.
Reverse transcription polymerase chain reaction (RT-PCR) to partially amplify the N and G genes was performed according to the protocol described by Carnieli et al., (19) using the primers described by Orciari et al. (20) for the N gene and those described by Sato et al. (21) for the G gene (Table 2). The PCR products were purified from the PCR reactions using the QIAquick[TM] Gel Extraction Kit[TM] (Qiagen--Valencia, CA, USA), according to the manufacturer's instructions. The products with nonspecific bands were purified using 1% agarose gel and the QIAquick[R] kit. After the purification step, the DNA samples were visually quantified in 2% agarose gel with Low Mass DNA Ladder (Invitrogen--Carlsbad, CA, USA), following the manufacturer's instructions.
The DNA sequencing reaction mixture consisted of 4 [micro]L of BigDye 3.1[TM] (Applied Biosystems--Foster City, CA, USA), 3.2pmol of both sense and antisense primer for each gene in separate reactions, 30-60 ng of target DNA and DNase-free water to a final reaction of 10 [micro]L. The reaction was performed in a Mastercycler Gradient thermal cycler[TM] (Eppendorf, NY, USA) with 35 cycles at 96[degrees]C for 10s, 50[degrees]C for 5s, and 60[degrees]C for 4min, with a ramp of 1[degrees]C/s between each temperature. Sequencing reaction products were purified with SephadexTMG-50 fine beads (GE Healthcare Biosciences) in 96-well multiscreen HV plates. After purification, the sequences were resolved in an ABI-3130[TM] Automatic Sequencer (Applied Biosystems--Foster City, CA, USA).
To construct the genealogic trees, nucleotide and putative amino acids sequences for the 35 RABV strains of N and G genes were aligned by the CLUSTAL/W multiple alignment algorithm method using the BioEdit program, (22) and then by manually checking the alignments for each set of aligned sequences. A score was assigned to each of the nucleotides shown on the electropherograms for each of the sequencing reactions using the online Phred application. Only positions with a Phred score >20 were used. (23) The final sequence for each strain was obtained using the Contig Assembly Program (CAP) in BioEdit v.5.0.9 (22) and submitted to BLASTn for homology confirmation.
Phylogenetic trees of the RABV isolates were built using the Neighbor-joining algorithm and the maximum composite likelihood (MCL) evolutionary model implemented in Mega 4.1 ([c] 1993-2008) (24) with 1,000 bootstrap replicates.
Additionally, 38 homologous sequences recovered from GenBank were included in the phylogenetic trees of the N and G genes (29 and none, respectively) and European bat Lyssavirus 1 (another specie in the Lyssavirus) was used as an outgroup.
The minimum, maximum, and mean nucleotide (with the MCL model) and amino acids (with the Poisson correction) identities for the clusters for the N and G gene sequences were calculated using Excel ([c] 1985-2003 Microsoft Corporation) based on the identity matrices calculated with the BioEdit program. The changes in the amino acids observed in the samples analyzed were studied using Mega 4.1 ([c] 1993-2008) (24) and BioEdit v.7.0.0 (22) programs.
After editing, the N gene was 1,281-nucleotides long, located between nucleotides 68 and 1,350 in relation to the N gene CVS reference strain (GenBank number AF406696) and had a putative amino acid sequence with 427 amino acids. Regarding the G gene, sequences were 1,571-nucleotides long, located between nucleotides 8 and 1,579 in relation to the G gene CVS reference strain (GenBank number FJ979833) and had a putative amino acid sequence with 520 amino acids.
[FIGURE 2 OMITTED]
The N gene nucleotide phylogenetic tree showed three clusters for the 35 RABV strains included in the study; one cluster for D. rotundus strains and two for the Artibeus spp. strains. Regarding N protein amino acids tree, the 35 strains segregated in only two clusters, one for D. rotundus and one formed mainly by Artibeus spp. strains (Fig. 2). Nucleotides and amino acids identities for the N region under analysis between D. rotundus and Artibeus spp. sequences ranged from 97.4% to 98.7% and 98.1% to 99.7%, respectively.
The G gene nucleotide phylogenetic tree showed two clusters for the 35 RABV strains included in the study; one cluster for D. rotundus strains and one for the Artibeus spp. strains. Regarding G amino acids tree, the 35 strains segregated in two clusters, one for D. rotundus and one formed by Artibeus spp. strains (Fig. 3). Nucleotides and amino acids identities for the G region under analysis between D. rotundus and Artibeus spp. sequences ranged from 97.0% to 99.1% and 96.1% to 99.0%, respectively.
Frugivorous bats of Artibeus spp. have an emerging importance for rabies epidemiology in Brazil, mainly in urban centers, and have been reported as carriers of RABV lineages close to those found in the vampire bat D. rotundus. (12,16) In the present investigation, RABV N and G phylogenies of strains recovered from these bats showed the existence of viral lineages that can be accurately attributed to D. rotundus or Artibeus spp. bats, a previously unknown fact.
[FIGURE 3 OMITTED]
RABV lineages heterogeneity expressed phylogenetically as host-specific lineages is a widely documented epidemiological phenomenon, (16,19,25,26) and is influenced by geographic barriers rather than by species barriers only. (27) Accordingly, the two Artibeus spp. clusters found for N gene sequences might also represent regional sub lineages of RABV, a fact already described for D. rotundus RABV lineages in Brazil. (28,29) These observations agree with the proposition that, regarding rabies in bats, host species are as important as geographic variations. (30) Regarding viral lineages, genetic variations that occur within a host species are different from those that occur in another, and this variation, coupled with the host's geographical isolation, may explain the RABV genetic differences reported in this work. (31)
Seven strains (05/Art7270, 05/Art8456, 05/Art7436, 05/Art8921, 05/Art8688, 05/Art3738, and 08IacriSP3577B) were classified in the Artibeus spp. group for the N nucleotides tree; however they segregated into the D. rotundus cluster of the N amino acids tree. This fact suggests the possibility that Artibeus spp. strains are still under an adaptation process after the spill-over event from D. rotundus, as already suggested by Kissi, (31) who have already experimentally reported this type of stepped adaptation of RABV. This phenomenon can occur as a consequence of the predominance of synonymous mutations over non-synonymous nucleotides mutations, which leads to greater differences among the nucleotide sequences than among the amino acids sequences. (32,33)
Regarding the Bov7525 and Bov6314 strains, in cattle, both segregated in the Artibeus spp. RABV cluster; this was unexpected, as cattle rabies is related to that of D. rotundus and not to that of frugivorous bats. (34,35) The most plausible explanation is that these two strains have been transmitted from an Artibeus spp. to a D. rotundus, and then from this bat to the cattle in a rare class of spill-over transmission.
Kobayashi et al., (16) analyzing the N gene of the RABV isolated from frugivorous bats, insectivorous bats, and D. rotundus, reported lineages associated with Artibeus spp. bats (frugivorous), D. rotundus, and insectivorous bats, suggesting that there are species-specific lineages. (26) However, they have not provided significant results to distinguish between RABV isolated from D. rotundus and Artibeus spp., possibly due to a restricted number of samples and sampling area. In the present study, this problem was compensated by the inclusion of 35 RABV isolates of bats (20 from Artibeus spp. and 15 from D. rotundus) from a broad geographical area of Sao Paulo. In addition, this study also analyzed the complete G gene, which provides a more specific distinction between the genetic sequences.
Different hosts pose different challenges for rabies control. This is more complex in bats due to the large number of species, the different ecologic niches that they occupy, and the impossibility to vaccinate these animals. For instance, the population of D. rotundus in Latin America can be legally controlled with vampiricide anticoagulants applied on cattle or on the bats themselves. (34) Conversely, non-hematophagous bats are under legal protection, and only now the knowledge of rabies epidemiology in these bats species is increasing. (35,36)
In this context, the results obtained in this study are valuable, because based on the partial amino acid sequences for the N gene it is possible to differentiate RABV strains from Artibeus spp. and D. rotundus for the purpose of defining the infection sources in molecular epidemiology. These results show the close host relationship of RABV transitions, and have an invaluable application for determining the sources of rabies infections transmitted mainly to dogs and cats in urban centers.
In conclusion, for rabies virus isolates related to frugivorous bats of the Artibeus spp. and to the vampire bat D. rotundus, the phylogeny based on sequences of the N and G genes shows segregation patterns in genus-specific agreement in each of these bats. Data from this study suggest that a lineage of RABV is possibly being established in the Artibeus spp. genus.
Received 18 May 2012
Accepted 26 July 2012
Available online 10 November 2012
Conflict of interest
All authors declare to have no conflict of interest.
The authors are grateful to CAPES (W.O. Fahl's PhD fellowship) and CNPq (P.E. Brandao's PQ-2 fellowship).
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Willian Oliveira Fahl (a), *, Pedro Carnieli Jr. (a), Juliana Galera Castilho (a), Maria Luiza Carrieri (a), Ivanete Kotait (a), Keila lamamoto (a), Rafael Novaes Oliveira (a), Paulo Eduardo Brandao (b)
(a) Instituto Pasteur, Sao Paulo, SP, Brazil
(b) Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
* Corresponding autho at: Av. Paulista 393, 01311-001, Sao Paulo, SP, Brazil.
E-mail address: email@example.com (W.O. Fahl).
Table 1--GenBank accession numbers for the reference sequences of N gene and G gene used in phylogenetic analysis in this study showing strain, the specific-host from which the AgV3 RABV lineages were isolated, city, and year in which the samples were obtained. GenBanknumber N gene G gene Strain City JF682392 JF682427 05/Art3250 Catanduva JF682393 JF682428 05/Art4578 Monte Mor JF682394 JF682429 05/Art3598 Ribeirao Preto JF682395 JF682430 05/Art3738 Ribeirao Preto JF682396 JF682431 05/Art4850 Ribeirao Preto JF682397 JF682432 05/Art4932 Ribeirao Preto JF682398 JF682433 05/Art5459 Ribeirao Preto JF682399 JF682434 05/Art6734 Ribeirao Preto JF682400 JF682435 05/Art6956 Ribeirao Preto JF682401 JF682436 05/Art7045 Ribeirao Preto JF682402 JF682437 05/Art7436 Ribeirao Preto JF682403 JF682438 05/Art8688 Ribeirao Preto JF682404 JF682439 05/Art8921 Ribeirao Preto JF682405 JF682440 05/Art10509 Ribeirao Preto JF682406 JF682441 05/Art7270 Paraguacu Paulista JF682407 JF682442 05/Art7547 Paraguacu Paulista JF682408 JF682443 05/Art8456 Paraguacu Paulista JF682409 JF682444 05/Art11206 Paraguacu Paulista JF682410 JF682446 05/Art8639 Mar'lia JF682413 JF682445 05/Art7848 Mar'lia JF682411 JF682460 05/Bov6314 Garca JF682412 JF682461 05/Bov7535 Platina JF682414 JF682447 05/Bov451 Altinopolis JF682415 JF682449 05/Bov3924 Altinopolis JF682416 JF682451 05/Bov10339 Altinopolis JF682417 JF682450 04/Bov8967 Altinopolis JF682418 JF682448 04/Bov2196 Altinopolis JF682419 JF682452 05/Bov2579 Santo Antonio da Alegria JF682420 JF682453 04/Bov3441 Santo Antonio da Alegria JF682421 JF682454 04/Bov3833 Santo Antonio da Alegria JF682422 JF682455 04/Bov4698 Santo Antonio da Alegria JF682423 JF682456 04/Bov6930 Santo Antonio da Alegria JF682424 JF682457 04/Bov11044 Santo Antonio da Alegria JF682425 JF682458 04/Bov11817 Santo Antonio da Alegria JF682426 JF682459 04/Bov11818 Santo Antonio da Alegria GenBanknumber N gene G gene Specific-host Year JF682392 JF682427 Artibeus spp. 2005 JF682393 JF682428 Artibeus lituratus 2005 JF682394 JF682429 Artibeus lituratus 2005 JF682395 JF682430 Artibeus lituratus 2005 JF682396 JF682431 Artibeus spp. 2005 JF682397 JF682432 Artibeus spp. 2005 JF682398 JF682433 Artibeus lituratus 2005 JF682399 JF682434 Artibeus lituratus 2005 JF682400 JF682435 Artibeus spp. 2005 JF682401 JF682436 Artibeus spp. 2005 JF682402 JF682437 Artibeus lituratus 2005 JF682403 JF682438 Artibeus lituratus 2005 JF682404 JF682439 Artibeus lituratus 2005 JF682405 JF682440 Artibeus spp. 2005 JF682406 JF682441 Artibeus lituratus 2005 JF682407 JF682442 Artibeus lituratus 2005 JF682408 JF682443 Artibeus lituratus 2005 JF682409 JF682444 Artibeus lituratus 2005 JF682410 JF682446 Artibeus lituratus 2005 JF682413 JF682445 Artibeus spp. 2005 JF682411 JF682460 Bovine 2005 JF682412 JF682461 Bovine 2005 JF682414 JF682447 Bovine 2005 JF682415 JF682449 Bovine 2005 JF682416 JF682451 Bovine 2005 JF682417 JF682450 Bovine 2004 JF682418 JF682448 Bovine 2004 JF682419 JF682452 Bovine 2005 JF682420 JF682453 Bovine 2004 JF682421 JF682454 Bovine 2004 JF682422 JF682455 Bovine 2004 JF682423 JF682456 Bovine 2004 JF682424 JF682457 Bovine 2004 JF682425 JF682458 Bovine 2004 JF682426 JF682459 Bovine 2004 Table 2--Primers for RT-PCR and genetic sequencing of RABV N and G genes. Primers Orientation Sequence Gene 21G Sense 5' ATGTAACACCTCTACAATG 3' N 304 Antisense 5' TTDACGAAGATCTTGCTCAT 3' N GA 3222-40 Sense 5'CGCTGCATTTTRTCARAGT 3' G GB 4119-39 Antisense 5' GGAGGGCACCATTTGGTMTC 3' G GS 3994 Sense 5'GGGMTTTGTGGATGAAAGRGGC 3' G GantiBR2072 Antisense 5' TGCTGATTGCRCCTACATT 3' G
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|Title Annotation:||Original article|
|Author:||Fahl, Willian Oliveira; Carnieli, Pedro, Jr.; Castilho, Juliana Galera; Carrieri, Maria Luiza; Kotai|
|Publication:||The Brazilian Journal of Infectious Diseases|
|Date:||Nov 1, 2012|
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