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Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.

Introduction

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).

Phylogenetic analyses

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.

Results

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.

Discussion

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.

ARTICLE INFO

Article history:

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.

Acknowledgements

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: wfahl@yahoo.com.br (W.O. Fahl).

http://dx.doi.org/10.1016/j.bjid.2012.07.002
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
Words:3734
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