journa l homepage: www.e lsev ier .com/ locate / jv i romet
eview
ne-step protocol for amplification of near full-length cDNA of the rabies virusenome
ngélica Cristine de Almeida Camposa,∗, Fernando Lucas Melob, Camila Malta Romanob,c,anielle Bastos Araujoa, Elenice Maria Sequetin Cunhaf, Débora Regina Veiga Sacramentoa,e,aolo Marinho de Andrade Zanottob, Edison Luiz Durigona, Silvana Regina Favorettoa,d
Núcleo de Pesquisas em Raiva do Laboratório de Virologia Clínica e Molecular da Universidade de São Paulo, Av Prof. Lineu Prestes, 1374, sala 225 CEP: 05508-900, São Paulo, BrazilLaboratório de Evolucão Molecular e Bioinformática, Instituto de Ciências Biomédicas da Universidade de São Paulo, Av Prof. Lineu Prestes, 1374, sala 215 CEP: 05508-900,ão Paulo, BrazilLaboratório de Virologia, Instituto de Medicina Tropical da Faculdade de Medicina, Universidade de São Paulo, Av. Professor Enéas de Carvalho Aguiar, 470, CEP: 05403-000,ão Paulo, BrazilInstituto Pasteur de São Paulo, Av Paulista, 393, CEP: 01311-000, São Paulo, BrazilGenomic Engenharia Molecular, Rua Itapeva, 500, Cj 5AB, CEP: 01332-903, São Paulo, BrazilLaboratório de Encefalites do Centro de P&D de Sanidade Animal do Instituto Biológico da Agência Paulista de Tecnologia dos Agronegócios da Secretaria da Agricultura do Estadoe São Paulo, Brazil
rticle history:eceived 17 June 2010eceived in revised form 24 March 2011ccepted 28 March 2011vailable online 5 April 2011
eywords:
a b s t r a c t
Full-length genome sequencing of the rabies virus is not a routine laboratory procedure. To understandfully the epidemiology, genetic variation and evolution of the rabies virus, full-length viral genomes needto be obtained. For rabies virus studies, cDNA synthesis is usually performed using nonspecific oligonu-cleotides followed by cloning. When specific primers are used, the cDNA obtained is only partial and islimited to the coding regions. Therefore, the development of methods for synthesizing long cDNA usingrabies virus-specific primers is of fundamental importance. A new protocol for the synthesis of long cDNA
abies virusong cDNAequencingethod development
everse-transcriptase polymerase chaineaction
and the development of 19 new primers are described in this study. This procedure allowed the efficientamplification of the full-length genome of the rabies virus variant maintained by hematophagous bat(Desmodus rotundus) populations following the synthesis of a complete long cDNA. Partial sequencing ofthe rabies virus genome was performed to confirm rabies-specific PCR amplification. Because degenerateprimers were employed, this technique can be adapted easily to other variants. Importantly, this newmethod is faster and less expensive than cloning methods.
The Rabies virus, which belongs to the Lyssavirus genus ofhe Rhabdoviridae family, has a single-stranded, nonsegmentedegative-sense RNA genome of about 12 kb comprising fiverotein-coding genes, N, P, M, G and L (Tordo, 1996). These genesncode for the nucleoprotein (N), phosphoprotein (P), matrix pro-ein (M), glycoprotein (G) and large, virion-associated transcriptaseL) protein (Tordo et al., 1986a,b; Wunner et al., 1988) and are sep-rated by short intergenic regions and a long pseudogene betweenhe G and L genes. This sequence is flanked by a 3′ leader regionLe) and a 5′ trailer region (Tr) (Wunner, 2002).
Rabies, a zoonotic virus disease characterized by acute evolu-ion, is caused by a group of neurotropic viruses that infect domesticnd wild animals. Rabies is distributed widely and defies all thevailable technological resources for control of this disease, result-ng in thousands of human deaths worldwide annually. Accordingo the World Health Organization (WHO), rabies is a public healthroblem that can cause serious environmental and economic dam-ge despite the existence of effective vaccines for human andeterinary use (WHO, 2005).
Species of the Carnivora and Chiroptera orders are recognizeds wild reservoirs. Lyssavirus strains that are adapted to bats areenetically different from carnivore-related rabies viruses (WHO,005). This fact confirms the need to develop methods that allowore precise complementary studies concerning the biology and
pidemiology of rabies. Corroborating this idea, Faber et al. (2004)apped the complete genome of rabies virus isolated from Lasy-
nicteris noctivagans, an important reservoir of the rabies virus inorth America. Given the importance of increased understandingf this virus, Delmas et al. (2008), Szanto et al. (2008) and Mochizukit al. (2009) mapped recently other variants of the rabies virus: oneirus isolated from an insectivore bat, one virus from Procyon lotornd one virus from Dusicyon sp. respectively.
The rabies virus strain maintained among populations ofematophagous bats (Desmodus rotundus), which are a naturaleservoir for the virus in the region spanning from Mexico to theorth of Argentina, causes economic loss to herds.
Furthermore, this strain is transmitted to humans, posing a seri-us public health problem in several countries, particularly in themazon region.
In the early 1980s, the development of molecular biology tech-iques allowed improved diagnostic tests and epidemiologicaltudies regarding the rabies virus. The detection of viral RNA byeverse transcription followed by polymerase chain reaction ampli-cation (RT-PCR) permits diagnosis in tissue samples (Sacramentot al., 1991; Kamolvarin et al., 1993; Whitby et al., 1997; Nadin-avis, 1998; Heaton et al., 1999; Black et al., 2000; Smith et al.,000; Echevarría et al., 2001), even in highly decomposed orormalin-fixed samples, from which viral isolation is impossibleDavid et al., 2002; Favoretto et al., 2005, Rojas-Anaya et al., 2006;raujo et al., 2008; Lopes et al., 2010). Several research groupsave improved these techniques to optimize molecular studies. It
s currently possible to distinguish the 11 viral species of the genusyssavirus using molecular analysis methods (ICTV, 2009).
For studies involving the rabies virus, cDNA synthesis is usuallyerformed using nonspecific oligonucleotides and cloning. Whenpecific primers are used, only partial cDNA is obtained (Marstont al., 2007; Inoue et al., 2003; Ito et al., 2001a,b).
To create a simple, cost-effective and specific method for study-ing the Rabies virus, we developed a new protocol for long cDNAsynthesis and subsequent amplification. Specific primers comple-mentary to the different coding regions of the viral genome andintergenic regions were developed.
Several methods for viral RNA transcription exist; however,these methods, which are intended for the study of short genefragments, are not efficient enough for the analysis of long genefragments or complete genomes. The methods used for geneticstudies of the rabies virus are usually limited to the transcription ofcoding genes for different viral proteins. The development of meth-ods for synthesizing long cDNA using specific primers for the rabiesvirus is therefore of great importance.
2. Time required for this experiment: 6 h 40 min
(i) Total RNA extraction: 2 h 30 min.(ii) Reverse transcriptation reaction (RT-PCR) – (cDNA synthesis):
4 h 10 min.
3. Materials and methods
3.1. Viral specimens
Five different rabies virus samples from 4 different variantswere selected from the Sample Bank of the Center for RabiesResearch/Biosafety Level 3 Laboratory of the Clinical and Molec-ular Virology Laboratory of the Institute of Biomedical SciencesII at the University of Sao Paulo (ICBII-USP), São Paulo, Braziland maintained at −70 ◦C. The sample brdrusp01/09 was isolatedfrom hematophagous bats, and antigenic and genetic identifica-tion showed that it was related to the variant maintained amongD. rotundus bat populations (Accession number GU592648). A first-passage virus was made in Swiss albino mouse brains.
The sample brsgusp32/07 was isolated from Brazilian mar-mosets, and antigenic and genetic identification showed acharacteristic pattern of variant maintained by marmosets. Thesample brmnusp91/05 was isolated from insectivorous bat (Myotisnigricans) and segregated with samples isolated from insectivorousbats (accession number HM173087). The sample brcvsusp47/05was a Challenge Virus Standard maintained in cell culture, andthe sample brbvusp01/06 of bovine origin was previously charac-terized as a hematophagous bat variant and utilized as a positivecontrol (accession number GU592649).
3.2. Description of the oligonucleotide primers
To determine the complete genome of any rabies virus variant,19 oligonucleotide primers (Table 1) were designed based on theGenBank published complete genome nucleotide sequence fromwild strains (accession numbers AY705373, AY956319, EF437215)and vaccine strains (AB009663, EF206707, M31046, NC 001542)
using OLIGO 6.83 software (Molecular Biology Insights, Inc., Cas-cade, CO). The same program was used to analyze the quality of theprimers by calculating annealing temperatures and assessing thepresence of false base-pairing regions and ‘hairpins’.
A.C.A. Campos et al. / Journal of Virological Methods 174 (2011) 1–6 3
Table 1Oligos for the amplification and sequencing of rabies virus genome.
Primer Gene position Sequence 5′–3′ Use Annealing temperature
Início Posicão1 F 1–22 Leader ACgCTTAACAACAARATCARAg PCR and Sequencing 55 ◦CSEQ NucleoPr F.750 749–766 Nucleoprotein ggCACAgTWgTCACTgCT PCR and Sequencing 55 ◦CNPM 1 F.1342 1342–1361 Nucleoprotein TgTCTCAgTCAgYTCCAATC PCR and Sequencing 55 ◦CNPM 1 R.2479 2501–2480 Phosphoprotein gTTCATYTTATCAgTggTgTTg PCR and Sequencing 55 ◦CNPM 2 F.2207 2207–2226 Phosphoprotein ATgAACCTTgATgAYATAgT PCR and Sequencing 50 ◦CNPM 2 R.3428 3447–3428 Glycoprotein ggRCAgCTgAgRTgATgTAT PCR and Sequencing 50 ◦CGlyco F pcr – A 3284–3303 Intergenic M-G CTATCAACATCCCTCAAARg PCR and Sequencing 55 ◦CGlyco F Sato pcr – C 3219–3237 Intergenic M-G CgCTgCATTTTRTCARAgT PCR and Sequencing 55 ◦CGlyco R pcr – D 5443–5426 Pseudogene � CgggTCATCATARACCTC PCR and Sequencing 55 ◦CGlyco seqF 1 3987–4007 Glycoprotein gACTTgSggMTTTgTRgATgA PCR and Sequencing 55 ◦CGlyco seqR 1 4138–4120 Glycoprotein gATCMggRgggCACCATTT PCR and Sequencing 55 ◦CGlyco seqF 2 4120–4138 Glycoprotein AAATggTgCCCYCCKgATC PCR and Sequencing 55 ◦CGlyco seqR 2 4538–4520 Glycoprotein TCCAACARYTCCATATgTT PCR and Sequencing 55 ◦CGlyco seqF 3 4370–4390 Glycoprotein ggACTTggAACgARRTCATCC PCR and Sequencing 55 ◦CGlyco seqR 3 4656–4637 Glycoprotein gAgAYCTgTTTgTGMACATC PCR and Sequencing 55 ◦CGliPol 1 R.6270 6287–6270 Glycoprotein AgTTTCCRCACATRgACA PCR and Sequencing 55 ◦CPol 3 F.10226 10225–10242 L Protein gATCARgARgTKCgCCAT PCR and Sequencing 56 ◦C
gATgACAAA
3
weth(pc
aa11wpswc
TF
3
(
1piailC3Ort2baia
Pol 4 R.11616 11636–11616 L Protein TCCAgTgAPosicão FINAL R.11904 11924–11904 Trailer ACgCTTAA
.3. Total RNA extraction
To each fragment of macerated brain, 500 �L of DEPC® H2Oas added, and samples were vortex homogenized. Total RNA was
xtracted using 500 �L of Trizol LS Reagent® (Invitrogen Corpora-ion, Carlsbad, CA) in an ice bath and refrigerated centrifuge. Twoundred microliters of 25:24:1 phenol:chloroform:isoamyl alcoholInvitrogen Corporation, Carlsbad, CA) was then added, and sam-les were vortex homogenized for 15 s, chilled on ice for 5 min andentrifuged at 12,000 × g at 4 ◦C for 5 min.
After centrifugation, the supernatants were precipitated bydding isopropanol 100% (v/v) (Sigma–Aldrich Co. St. Louis, MO),nd samples were vortex homogenized and incubated on ice for5 min. The mixtures were then centrifuged at 12,000 × g for5 min, supernatants were discarded and the pellets were washedith 1 mL of 75% ethanol (Merck, Darmstadt, Germany). The sus-ensions were then centrifuged at 7500 × g at 4 ◦C for 8 min. Theupernatants were again discarded, and after drying, the sedimentsere resuspended in 30 �L of DEPC H2O containing 40 U of ribonu-
lease inhibitor (RNAse OUT, Invitrogen Corporation, Carlsbad, CA).Quantitation of the extracted RNA was performed using a
To synthesize rabies virus long cDNA, the reverse transcriptionRT) reaction method described below was developed:
Twenty-five microliters of extracted RNA was added to 2 �L of0 mM dNTP (Invitrogen Corporation, Carlsbad, CA) and 5 �L ofrimer “Posicao Final” (10 pmol/�L), which anneals at the end pos-
tive sense RNA (3′ extremity). These samples were then incubatedt 65 ◦C for 5 min. Nineteen microliters of a second mixture contain-ng 200 U of reverse transcriptase (RT) (MMLV – Moloney-murineeukemia virus/Super ScriptTM – Invitrogen Corporation, Carlsbad,A), 8 �L of enzyme buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl,mM MgCl2), 4 mM DTT, 40 U of ribonuclease inhibitor (RNAseUT, Invitrogen Corporation, Carlsbad, CA) and H2O DEPC (Invit-
ogen Corporation, Carlsbad, CA) were added. The first stage ofranscription was performed at 45 ◦C for 105 min. An additional00 U of RT (MMLV/Super ScriptTM – Invitrogen Corporation, Carls-
ad, CA) was then added, and the reaction was incubated fornother 105 min. After this final reverse transcription step, RT wasnactivated at 70 ◦C for 15 min followed by an incubation of 20 mint 37 ◦C.
VAgACTCA PCR and Sequencing 56 ◦CTAAACAACA RT-PCR and Sequencing 56 ◦C
To amplify each viral gene individually, PCRs were performedusing cDNA diluted in 10× reaction buffer, 10 mM Tris–HCl (pH9.0), 50 mM KCl, 2.5 mM MgCl2, 200 �M of each dNTP, 50 pmolof each primer, 2.5 U of Taq DNA polymerase (Invitrogen Cor-poration, Carlsbad, CA) and H2O DEPC in a final volume of50 �L.
In addition to the primers presented in Table 1, a pair of primersdescribed previously (Smith et al., 1995) was also used to amplifyand sequence part of the nucleoprotein coding region.
Amplifications were carried out using the following cyclingconditions: samples were preheated for 5 min at 95 ◦C, subjectedto 35 cycles of denaturation for 45 s at 94 ◦C, primer annealingfor 45 s at temperatures between 45 ◦C and 58 ◦C depending onthe pair of primers employed (Table 1) and primer extensionfor 90 s at 72 ◦C, and terminated with a final extension step for5 min at 72 ◦C before being held at 4 ◦C in a MasterCycler Gradi-ent Eppendorf thermocycler (Eppendorf AG, Hamburg, Germany).Each PCR experiment included samples lacking RNA template asnegative controls and H2O as contamination controls. The ampli-fied products were identified by 1.5% agarose gel electrophoresis(Invitrogen Corporation, Carlsbad, CA) and visualized by ethid-ium bromide [0.5 �g/mL] (Edt Br, Sigma–Aldrich Co. St. Louis,MO) staining under UV light. One kilobase DNA markers (Fermen-tas International Inc., Burlington, Ontario, Canada) were used todetermine the size of each product. The results obtained weredocumented.
Double-stranded PCR-amplified products were purified usingthe ExoSAP-IT system (GE Healthcare Bio-Sciences Ltd – USB Corpo-ration, Cleveland, OH) according to the manufacturer’s instructions.
To amplify the full-length rabies genome, 300 ng of long cDNAwere diluted in 10× reaction buffer, 25 mM dNTP Mix, 40 pmol ofeach primer (Inicio + Pol 4R), and 2.5 U of Expand Long DNA poly-merase (Roche Diagnostics, Mannheim, Germany) in a final volumeof 50 �L.
The thermal cycling conditions were as follows: 1 cycle of 2 minat 94 ◦C, 20 cycles of 10 s at 94 ◦C, primer annealing for 30 s at tem-peratures between 62 ◦C and 54 ◦C with a decrease of −0.5 ◦C percycle and 10 min at 68 ◦C, 25 cycles of 10 s at 94 ◦C, 30 s at 53 ◦C and10 min at 68 ◦C and a final extension step of 10 min at 68 ◦C. PCR
products were analyzed by agarose gel electrophoresis and the sizesof products were estimated based on their electrophoretic mobili-ties relative to those of the 1 kb DNA ladder (Invitrogen, Carlsbad,CA).
4 A.C.A. Campos et al. / Journal of Virological Methods 174 (2011) 1–6
Fig. 1. Ethidium bromide-stained 1.5% agarose gels containing amplicons generated from different host samples: brdrusp01/09, brsgusp32/07, brmnusp91/05, CVS strain(brcvsusp47/05), brbvusp01/06 and negative control. Lanes 1 and 20: 1 kb DNA size marker (Invitrogen). Lanes 7, 13, 19, 26 and 32: negative controls. Lanes 2–6: partialP es 8–1N P2F anp otein
3
NttBDvv
ed9
t(u
p(
D
3
mta
4
1M
ocwf
tc
gg1wa
CR nucleoprotein products (765 bp) amplified with primer pair SeqN and 304; lanPM1R; lanes 14–18: PCR matrix products (1224 bp) amplified with primer pair NMair GlycoA and GlycoD and lanes 27–31: PCR products of 1699 bp from partial L pr
.6. Sequence reaction and purification
Products were quantitated using a Thermo ScientificanoDropTM 1000 Spectrophotometer (Thermo Fisher Scien-
ific, Waltham, MA), and 30–100 ng of each product were addedo microtubes containing 2 �L of 5× Sequencing buffer (Appliediosystems, Foster City, CA), 3.2 pmol of primer, 2 �L of ABI PRISMyeTM Terminator Cycle Sequencing Ready Reaction kit (Big Dye3.1, Applied Biosystems, Foster City, CA) and DEPC water in a finalolume of 10 �L.
The homogenized mixture was placed in a MasterCycler Gradi-nt Eppendorf thermocycler (Eppendorf AG, Hamburg, Germany),enatured for 5 min at 96 ◦C, and subjected to 25 cycles of 10 s at6 ◦C, 15 s at 50 ◦C and 4 min at 60 ◦C.
The excess dideoxynucleotide terminators were removed withhe Applied Biosystems Big Dye XTerminatorTM Purification KitApplied Biosystems, Foster City, CA) in accordance with the man-facturer’s recommendations.
Purified samples were subjected to electrophoresis in POP6olymer using an automatic sequencer ABI-PRISM model 3100Applied Biosystems, Foster City, CA).
The samples were tracked automatically using the AutomaticNA Analyzer software package of the ABI-PRISM model 3100.
.6.1. Processing and alignment of the rabies virus sequencesThe nucleotide sequences obtained from individual gene frag-
ents generated after long cDNA amplification was analyzed usinghe BLAST program (www.ncbi.nlm.nih.gov/blast.html) to confirmmplification of the specific product.
. Results
RNAs were quantitated using a Thermo Scientific NanoDropTM
000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham,A). The RNA concentrations obtained were above 145.42 ng/�L.The quantity of each cDNA used for PCR was based on the size
f the fragment to be amplified. One hundred fifty nanograms ofDNA were used for nucleoprotein, glycoprotein and polymerase,hile quantities between 80 and 150 ng of cDNA were sufficient
or amplification of phosphoprotein and matrix genes.Positive PCR results were obtained for each rabies genes and for
he entire viral genome of the five samples studied and positiveontrol, confirming the viability of long cDNA synthesis (Fig. 1).
Fig. 1 shows the amplification of the N, P, M, G and L genesenerated from the long cDNA. Amplification of the nucleoprotein
ene using the Inicio + 304 primers produced a single amplicon of550 bp. The primer pairs NPM1F + NPM1R and NPM2F + NPM2Rere used to amplify the phosphoprotein and matrix genes, gener-
ting fragments of 1147 bp and 1224 bp, respectively. As expected,
2: PCR phosphoprotein products (1147 bp) amplified with primer pair NPM1F andd NPM2R; lanes 21–25: PCR glycoprotein products (2164 bp) amplified with primerthat were amplified with primer pair Pol3F and Final.
the glycoprotein amplicon was detected at 2164 bp. A partial ampli-fication of L polymerase using the Pol3F + Final primers was alsodetected, resulting in a product of 1699 bp (Fig. 1).
The complete amplification of the rabies virus genome using theprimer pair Inicio + Pol 4R produced a single amplicon of 11,622 bp(Fig. 2).
To confirm rabies-specific PCR amplification, each product wassubmitted to direct sequencing of both strands. Following purifica-tion, the amplified material was quantitated. For the N, P, M and Ggenes, between 10 and 30 ng of each purified PCR product was used,while for the polymerase, between 10 and 100 ng of the PCR productwas required. Sequence comparisons revealed extensive nucleotidesequence identity between amplified products and representativeLyssaviruses belonging to genotype 1.
5. Discussion
Since the first complete sequence of the fixed rabies virusstrain was published in 1986 (Tordo et al., 1986a,b), there havebeen numerous reports documenting partial rabies genome char-acterization. The reason for this is that generating a single, nearlyfull-length PCR amplicon using RNA as the template has been rela-tively difficult. A good strategy for amplification and sequencing ofnearly full-length cDNA is therefore needed.
The aims of this study were to optimize a method for syn-thesizing long cDNA of the rabies virus using a specific primerat the 3′ position to and to develop new primers that canbe employed either for amplification or sequencing based onsequences deposited in GenBank. Previous studies have reportedprimers that were designed based for a specific group of sam-ples. The primers presented in this paper were designed to be ableto detect most rabies virus strains. Based on current knowledgeof the rabies virus, these primers bind conserved regions of thegenome (Tordo et al., 1986a,b), but contain degenerate oligonu-cleotides. This feature allows the primers to be used for manyvariants isolated from different animals. Additionally, melting tem-peratures near 55 ◦C were evaluated to abolish some secondarystructural elements and prevent interference with the activity ofRT.
Optimal conditions for reverse transcription and PCR amplifica-tion were defined, resulting in a simple, sensitive, fast and effectiveassay for sequencing of nearly full-length rabies virus cDNA fromall fresh samples and samples maintained at −70 ◦C. Because longcDNA synthesis is performed only once, while synthesizing cDNAfor each viral gene would entail repeating the process at least five
times, the methods reported here proved to be less expensive andfaster than the current protocols (Ito et al., 2001a,b; Mochizuki et al.,2009). The fact that samples are manipulated only once also min-imizes the risk of contamination or mixed samples, resulting in
Fig. 2. Ethidium bromide-stained 1% agarose gel containing two amplicons fromthe rabies virus complete genome using long cDNA. Lane 1: 1 kb DNA size marker(ca
ici
baaingfit
rot
Invitrogen); lane 2: negative control and lanes 3 and 4: bruspdr01/09 and positiveontrol PCR products (brbvusp01/06), of 11,622 bp amplified with primer pair Iniciond Pol 4R.
mproved quality control. Long cDNA synthesis is extremely effi-ient, allowing for amplification of the complete viral genome,ncluding the 3′ leader region.
Methods reported previously are more complex, can introduceias and require PCR products to be cloned with a commerciallyvailable cloning kit. These methods are more time-consumingnd require special equipment, reagents and lab space, resultingn increased cost. Furthermore, in the method described above, theumber of reverse transcription reactions is reduced: instead of 5ene-specific reactions, this method uses a single reaction. There-ore, this new method provides a viable alternative that can bentroduced and implemented in any molecular biology laboratoryo map the complete genome of the rabies virus.
Studies using the long cDNA are being conducted by severalesearch groups (Marston et al., 2007; Rousseau et al., 2006). Theptimization of the time dedicated to laboratorial practice andhese techniques will make larger scale analyses possible. It is
gical Methods 174 (2011) 1–6 5
important to note that PCR amplifications performed using a longcDNA as a template ensure that all fragments amplified come fromthe same virion instead of from a whole population. Further, thismethod consists in synthesize a long cDNA molecule and it is idealfor studying the epidemiology of the rabies virus as our resultsalready reflect the currently circulating virus. Finally, this methodresults in synthesis of a long cDNA molecule, it can be adapted forother viruses, including flavivirus and retroviruses.
The development of new primers and new methods that leadto the standardization of laboratory procedures allows a broadervariety of questions to be addressed. New data must be obtained toelucidate the role of circulating viral variants and their interactionwith their host. Thus, increased numbers of phylogenetic studieswill provide a better knowledge of viral evolution and allow forimproved decision making regarding the epidemiological controlof the rabies virus.
Acknowledgements
This work was supported financially by Foundation for ResearchSupport of Sao Paulo State (FAPESP Process: 07/01843-0).
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