Infection, Genetics and Evolution 10 (2010) 278–283

Low genetic diversities of rabies virus populations within different hosts in Brazil

Yuki Kobayashi a,b, Yoshiyuki Suzuki b, Takuya Itou a,*, Adolorata A.B. Carvalho c, Elenice M.S. Cunha d,Fumio H. Ito e, Takashi Gojobori b, Takeo Sakai a

a Nihon University Veterinary Research Center, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japanb Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japanc Faculty of Agriculture and Veterinary Science, Department of Preventive Veterinary Medicine, UNESP, Via de Acesso Prof. Paulo Donato Castellane, Jaboticabal,

Sao Paulo 14884-900, Brazild Research and Development Center for Animal Sanitation, Biological Institute-Sao Paulo State Agency of Agribusiness (APTA)-Sao Paulo State Secretary of Agriculture and Supply,

Av. Conselheiro Rodrigues Alves, 1252 CEP 04014-002 Sao Paulo, Brazile Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine and Zootechny, University of Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87,

Cidade Universtiatria, Sao Paulo 05508-000, Brazil

A R T I C L E I N F O

Article history:

Received 11 September 2009

Received in revised form 3 December 2009

Accepted 5 December 2009

Available online 14 December 2009

Keywords:

Evolution

Genetic heterogeneity

Low genetic diversity

Rabies virus

A B S T R A C T

The low rates of nonsynonymous evolution observed in natural rabies virus (RABV) isolates are

suggested to have arisen in association with the structural and functional constraints operating on the

virus protein and the infection strategies employed by RABV within infected hosts to avoid strong

selection by the immune response. In order to investigate the relationship between the genetic

characteristics of RABV populations within hosts and the virus evolution, the present study examined the

genetic heterogeneities of RABV populations within naturally infected dogs and foxes in Brazil, as well as

those of bat RABV populations that were passaged once in suckling mice. Sequence analyses of complete

RABV glycoprotein (G) genes showed that RABV populations within infected hosts were genetically

highly homogeneous whether they were infected naturally or experimentally (nucleotide diversities of

0–0.95 � 10�3). In addition, amino acid mutations were randomly distributed over the entire region of

the G protein, and the nonsynonymous/synonymous rate ratios (dN/dS) for the G protein gene were less

than 1. These findings suggest that the low genetic diversities of RABV populations within hosts reflect

the stabilizing selection operating on the virus, the infection strategies of the virus, and eventually, the

evolutionary patterns of the virus.

� 2009 Elsevier B.V. All rights reserved.

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1. Introduction

The rabies virus (RABV), which belongs to genotype 1 of thegenus Lyssavirus in the family Rhabdoviridae, has a single-strandedand negative-sense RNA genome containing the genes encodingthe nucleoprotein, phosphoprotein (P), matrix protein, glycopro-tein (G), and the RNA polymerase proteins (Dietzschold et al.,2008). While the principal reservoirs of RABV include the membersof the orders Carnivora and Chiroptera, the RABV in the saliva ofrabid animals can infect all warm-blooded animals, resulting indeath by a form of encephalitis. RABV has an almost globaldistribution and approximately 50,000 people die each year fromthe highly neurotropic disease (WHO, 2007).

In most cases of natural infection, the transmission of RABVoccurs through a bite of a rabid animal. After the transmission, thevirus invades the nervous system, which has no lymphoid

* Corresponding author. Tel.: +81 466 84 3375; fax: +81 466 84 3380.

E-mail address: itou.takuya@nihon-u.ac.jp (T. Itou).

1567-1348/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2009.12.003

structures and antigen presenting cells (Barker and Billingham,1977; Shankar et al., 1991). The virus is then transported to thebrain along spinal cord by retrograde transportation beforespreading to the salivary glands (Murphy, 1977; Dietzscholdet al., 2008). In the nervous system, the P protein of RABV inhibitsthe response of type 1 IFN, which is one of the primary defensemechanisms of the host response system against viral infection(Camelo et al., 2001; Brzozka et al., 2005; Wang et al., 2005). Inaddition, T cells are destroyed by apoptosis, resulting from an up-regulation of Fas ligand due to RABV infection (Baloul et al., 2004).It has also been proposed that the apoptosis/necrosis in thenervous system, which can induce host immune response, isinhibited by regulating the expression level of the RABV G gene(Dietzschold et al., 2008). Consequently, RABV may not besubjected to a strong immune response within hosts (Badraneand Tordo, 2001; Holmes et al., 2002; Dietzschold et al., 2008).

Generally, when RNA viruses are subjected to the actions of thehost immune response, positive selection is frequently observed inthe antigenic sites, where amino acid substitutions are rapidlyaccumulated to generate escape mutants (Yamaguchi and

Y. Kobayashi et al. / Infection, Genetics and Evolution 10 (2010) 278–283 279

Gojobori, 1997; Suzuki and Gojobori, 1999). The RABV G protein,which forms trimeric spikes, interacts with the cellular receptorand plays an important role in cell-to-cell spread of the virus, thusaffecting the cell tropism and the pathogenicity of the virus(Dietzschold et al., 2008). In addition, this protein includesantigenic sites and induces the production of RABV-neutralizingantibody (Seif et al., 1985; Macfarlan et al., 1986; Celis et al., 1988).Consequently, it has been proposed that the amino acid changesobserved in the antigenic sites of the G protein may be the source ofvariants that are capable of escaping host defenses and adapting tonew environments (Badrane and Tordo, 2001; Khawplod et al.,2006). However, although genetic variations of the G protein havebeen observed in natural RABV isolates, positive selection forantigenic sites has not been observed, suggesting that RABV maynot be subjected to strong immune selection during evolution(Holmes et al., 2002). In addition, purifying selection has beendetected in nonsynonymous sites of G protein gene, suggestingthat RABV evolution may be affected by strong structural andfunctional constraints (Holmes et al., 2002; Hughes, 2009).

Given the relatively high replication and mutation ratestypically associated with RNA viruses, the populations of theseviruses usually form a genetically heterogeneous populationwithin a single host (Domingo and Holland, 1997). The strongselective pressures conferred by the host immune response caninduce extremely high heterogeneity in protein sequences in viruspopulations, resulting from the production of escape mutants(Yamaguchi and Gojobori, 1997; Nishizawa et al., 1999; Curranet al., 2002; Duffy et al., 2008). The genetic characteristics of theRABV population in vivo can thus be associated with the interactionbetween the host and the virus, and the virus evolution. There areseveral reports that showed the existence of genetic heterogene-ities of RABV populations within hosts (Benmansour et al., 1992;Kissi et al., 1999; Khawplod et al., 2006), suggesting that mutantsin RABV populations contribute to the adaptation to newenvironmental conditions (Morimoto et al., 1998). In these studies,however, only a small number of RABV populations wereexamined, some of which were derived from experimentallyinfected animals. The purpose of the present study was therefore toexamine the genetic heterogeneity of the G protein genes in RABVpopulations within many naturally infected animals in Brazil.

2. Materials and methods

2.1. Viruses and RNA extraction

Twenty-two brain samples were collected from dogs (Canis

lupus familiaris; n = 5), foxes (Dusicyon sp.; n = 5), hematophagousbats (Desmodus rotundus; n = 5), frugivorous bats (Artibeus

lituratus; n = 4), and insectivorous bats (Molossus molossus andM. abrasus; n = 2 and n = 1) in Brazil (Table 1). All samples werediagnosed as rabies positive by the immunofluorescence antibodyassay and mouse inoculation test (Dean et al., 1996; Koprowski,1996).

Total RNAs were directly extracted from the animal brains usingQIAamp Viral RNA Kit (QIAGEN, Hilden, Germany), except forisolates from bat brains, which were isolated after one passage insuckling mice for rabies diagnosis.

2.2. RT-PCR, cloning, and sequencing

The RNA extract was reverse transcribed (RT) using RevertAid Hminus M-MuLV Reverse Transcriptase (Fermentas, Hanover, MD,USA) as specified by the manufacturer. RT1 primer (50-ACT(C/T)TGA(C/T)AAAATGCAGCG-30) was used for synthesizing cDNA.KOD DNA polymerase, which has a proofreading activity (TOYOBO,Osaka, Japan), was used to amplify the full-length G protein gene

(1575nt) by PCR using Ga3222-40 primer (50-CGCTGCATTTT(A/G)TCA(A/G)AGT-30) as the universal sense primer for all sampleswith the following species-specific anti-sense primers: LA6 primer(50-CTCCCGGATCRATCATCATG-30) for hematophagous and frugiv-orous bat isolates, LA7 primer (50-CTCCCGGATCRATCATCATG-30)for dog and fox isolates, and GL-MM primer (50-CTGCCCAAGCT-CAAGCATAG-30) for insectivorous bats. Thermocycler parameterswere 94 8C for 2 min followed by 40 cycles of 98 8C for 10 s, 54 8Cfor 20 s, and 68 8C for 3 min, ending with 10 min at 68 8C.

Cloning technique is useful to investigate the genetic hetero-geneities of virus populations because it facilitates detection ofminor variants, which may be missed by the direct sequencingtechnique. The PCR products were cloned into pTA vector usingTArget Clone -Plus- (TOYOBO, Osaka, Japan) according to themanufacturer’s instructions. The G protein gene sequences werethen determined for each clone from all virus populations usingABI Prism 3130 genetic analyzer (PE-Applied Biosystems, USA).

The consensus sequences of the RABV G gene analyzed in thisstudy were submitted to the DNA Data Bank of Japan (DDBJ)(Accession numbers: AB247423, AB247426, AB247427, AB247429,AB247430, AB247433–AB247435, AB247437, AB247446, andAB449206–AB449217).

2.3. Sequence analyses

Pairwise comparisons of nucleotide and amino acid sequencesfor all clones were performed using the MEGA computer program(version 3.2) (Kumar et al., 2004) to obtain the mean and range ofthe proportion of difference (p distance). The average number ofsynonymous nucleotide mutations per synonymous site (dS) andthat of nonsynonymous nucleotide mutations per nonsynonymoussite (dN) among G protein gene sequences were computed by themethod of Nei and Gojobori (1986).

2.4. Determination of the error rate introduced by the methodology

To determine the error rate introduced by RT-PCR in ourmethodology, a control experiment was conducted in which arecombinant clone of BR-DR9 was transcribed in vitro with T7 RNApolymerase, followed by RT-PCR, subcloning, and sequencing usingthe same conditions as stated above. Since the nucleotide sequenceof the G protein gene of BR-DR9 is known, the error rate may beestimated by comparing the obtained sequences with the knownone. Of the 14,175 nucleotides that were sequenced for the nineclones collected, only one mutation was observed, correspondingto an error rate of 0.7 � 10�4 per nucleotide and a nucleotidediversity of 0.14 � 10�3.

3. Results

The nucleotide sequences were determined for 7–11 clones of Gprotein genes obtained from RABV populations isolated from thebrain tissue of various hosts, and the mutations observed in thenucleotide and amino acid sequences are summarized in Table 1.The fox BR-Pfx6 population exhibited no mutation. In dog andother fox RABV populations, one or two nucleotide deletions wereobserved, and the diversities of nucleotide and amino acidsequences were 0–0.95 � 10�3 and 0–1.91 � 10�3, respectively(Table 1). Here the nucleotide diversity of 0 corresponds to theoccurrence of deletion mutations, which were excluded from thecomputation of nucleotide and amino acid diversities because theyappeared to destroy the function of G protein. There were alsonucleotide deletions in bat RABV populations, where the diversi-ties of nucleotide and amino acid sequences were 0.12–0.69 � 10�3 and 0–2.08 � 10�3, respectively. Although bat RABVpopulations were isolated after one passage in mouse brains, the

Table 1Sequence diversity of rabies virus glycoprotein gene in different hosts.

Host Population Mutants/no. of clones Mutationsa Sequence diversityb

Nucleotide Amino acid

Canis lupus familiaris BRdg77 3/11 G!A(3329)

C!T(3853)S! L(160)

T!C(3920)

Deletion T(4462)

Deleyion A(4463)

G!A(4837)G!E(363)

0.46 (0–1.91) 0.7 (0–3.82)

BRdg96 1/7 Deletion C(3584) 0 0

BRdg128 3/11 Deletion A(3761)

C!T(3947)

G!T(4273)R! L(300)

0.23 (0–1.27) 0.35 (0–1.91)

BRdg322 3/10 Deletion C(3548)

G!A(3363)G!R(197)

T!C(3999)F! L(209)

0.25 (0–1.27) 0.76 (0–3.82)

BRdg659 2/10 G!C(3580)C!S(159)

A!G(4805)

0.25 (0–1.27) 0.38 (0–1.91)

Dusicyon sp. BR-Pfx1 2/10 A!G(3329)

A!G(3678)M!V(102)

0.25 (0–1.27) 0.38 (0–1.91)

BR-Pfx4 5/11 C!T(3934)T! I(187)

Deletion T(4030)

T!C(4301)

A!G(4320)T!A(316)

C!T(4676)

C!A(4780)S!Y(469)

0.58 (0–1.91) 1.04 (0–3.82)

BR-Pfx5 3/8 C!A(3382)P!H(3)

T!C(3487)L!P(38)

G!A(3556)C!Y(61)

C!T(4059)

0.63 (0–1.9) 1.43 (0–3.82)

BR-Pfx6 0/10 0 0 0

BR-Pfx8 5/9 G!A(3377)

C!A(3991)T!N(206)

C!T(4475)

A!G(4483)H!R(370)

T!C(4528)M!T(385)

T!C(4714)V!A(447)

0.95 (0–1.9) 1.91 (0–5.7)

Desmodus rotundus BR-DR3 2/10 Deletion T(3370)

Deletion C(3371)

A!G(3509)

G!A(4448)

G!A(4613)

0.38 (0–1.91) 0

BR-DR6 4/11 C!T(3336)

2�C!T(4568)

T!C(4738)I!T(455)

0.44 (0–1.27) 0.35 (0–1.91)

BR-DR7 3/9 T!A(3535)I!K(54)

T!C(3928)L!P(185)

C!T(4029)

0.42 (0–1.27) 0.85 (0–3.82)

BR-DR9 2/9 T!C(4496)

C!T(4554)P!S(394)

0.28 (0–1.27) 0.42 (0–1.91)

BR-DR21 2/10 C!T(4393)P! L(340)

A!G(4606)E!G(411)

0.25 (0–1.27) 0.76 (0–3.82)

Artibeus litratus BR-AL1 2/12 A!G(3480)T!A(36)

G!A(4878)D!N(502)

0.21 (0–1.27) 0.64 (0–3.82)

BR-AL2 6/11 G!A(3829)R!K(152)

A!G(3961)K!R(196)

2�C!T(4731)

G!A(4769)

Deletion C(4830)

0.55 (0–1.27) 0.7 (0–3.82)

BR-AL3 1/11 A!G(3903)I!V(177) 0.12 (0–0.63) 0.35 (0–1.91)

BR-AL6 3/10 A!G(3565)I!M(54)

C!T(4512)

T!C(4549)V!A(382)

0.38 (0–1.27) 0.76 (0–3.82)

Molossus molossus BR-MM1 4/11 Deletion C(3333)

Deletion T(3334)

G!A(3726)D!N(118)

T!C(4319)

T!C(4591)F! L(406)

0.35 (0–1.27) 0.7 (0–3.82)

BR-MM2 4/11 C!T(3348)L! F(-9)

T!C(3406)I!T(11)

T!C(3495)F! L(41)

A!G(3544)N!S(57)

A!G(4206)K!E(278)

G!A(4713)A!T(447)

0.69 (0–2.54) 2.08 (0–7.63)

Y. Kobayashi et al. / Infection, Genetics and Evolution 10 (2010) 278–283280

Table 1 (Continued )

Host Population Mutants/no. of clones Mutationsa Sequence diversityb

Nucleotide Amino acid

M. abrasus BR-MA1 4/10 A!G(3388)Y!C(5)

C!T(3481)T! I(36)

2�T!C(3517)V!A(48)

T!C(3605)

0.61 (0–1.9) 1.44 (0–3.82)

Transcript RNA (background error control) 1/9 T!C(4026)S!P(218) 0.14 (0–0.63) 0.42 (0–1.91)

a Numbers listed in parentheses indicate the nucleotide positions of mutations according to the numbering in the PV strain sequence (Accession no. M13215) followed by

the amino acid changes and their positions if the mutations are nonsynonymous.b These values were multiplied by 1000. The values in parentheses show the range of p distance.

Fig. 1. Distribution of mutations in the amino acid sequences of the cloned G

protein. The locations of mutations in cloned populations are shown as bars. An

asterisk above a bar indicates that the same mutation was observed in two clones.

Boxes on the G protein line indicate the antigenic sites.

Y. Kobayashi et al. / Infection, Genetics and Evolution 10 (2010) 278–283 281

nucleotide and amino acid diversities were not different fromthose of RABV populations in naturally infected animals indicatedabove (P = 0.77 and P = 0.81, in Student’s t-test). The nucleotidediversity introduced by our methods (0.14 � 10�3) was lower thanthose of all RABV populations observed in our study, except for theBR-AL3 population (0.12 � 10�3).

The RABV G protein is comprised of four distinct domains; thesignal peptide domain (SP; 19 aa), the ectodomain (ECTO; 439 aa),the transmembrane domain (TM; 22 aa), and the endodomain(ENDO; 44 aa) (Tordo et al., 1988). Five antigenic sites (I, II, III, IV,and ‘‘a’’) have been identified in the ECTO domain. Antigenic sites I,III, IV, and ‘‘a’’ are located at amino acid positions 231, 330–338,264, and 342–343, respectively, and antigenic site II, a discontinu-ous antigenic site, is located at positions 34–42 and 198–200(Benmansour et al., 1991). However, mutations observed in thisstudy were dispersed throughout the amino acid sequence of the Gprotein and no universal hypervariable region was apparent(Fig. 1).

In order to elucidate natural selection operating on the Gprotein, dN/dS ratios were compared among the four domains. ThedN/dS ratio for the combined dog and fox RABV populations was0.62 for both the ECTO and ENDO domains (Table 2). The dN/dS ratiowas incalculable for the TM domain because no synonymousmutation was observed, and was 0 for the SP domain. For thecombined bat RABV populations, the dN/dS ratio was 0.37 for the SPdomain, 0.59 for the ECTO domain, 0.43 for the TM domain, and0.31 for the ENDO domain. Although the dN/dS ratios could not bedirectly compared between antigenic and non-antigenic sites ofthe ECTO domain because no synonymous mutation was observedat the antigenic sites, dN values did not differ significantly betweenantigenic and non-antigenic sites in the dog and fox RABVpopulations, as well as in the bat RABV populations (P = 0.68and P = 0.28, in Fisher’s exact test).

4. Discussion

In the present study, the sequence variability of the G proteingene was examined in RABV populations isolated from the braintissue of reservoir animals in the field of Brazil. The nucleotidediversities of RABV populations within naturally infected hostsobserved in this study (0–0.95 � 10�3) were considerably lowerthan those reported for other RNA viruses, such as humanimmuno-deficiency virus (HIV), hepatitis C virus (HCV), denguevirus, and hepatitis E virus (1.1–48.7 � 10�3) (Curran et al., 2002;Nowak et al., 2002; Grandadam et al., 2004; Lin et al., 2004).Interestingly, the RABV populations in the present study appearedto be highly homogeneous even in comparison with the RABVpopulations in previous studies (Benmansour et al., 1992; Kissiet al., 1999; Khawplod et al., 2006). The average number ofmutations per site per clone obtained in the present study (0–0.42 � 10�3) was much smaller than those obtained for rabid dogsin Thailand and foxes in Europe in the previous studies (1.13–2.19 � 10�3 per nucleotide) (Benmansour et al., 1992; Kissi et al.,1999; Khawplod et al., 2006). It should be noted that Taq DNA

polymerases, which appear to have a higher error rate thanproofreading DNA polymerases (Bracho et al., 1998; Malet et al.,2003), were used to amplify cDNA in the previous RABV populationstudies (Benmansour et al., 1992; Kissi et al., 1999), whileproofreading DNA polymerases were used in the present study.Indeed, the error rate associated with our methodology (0.7 � 10�4

per nucleotide) was markedly lower than that reported by Kissiet al. (1999) (9.3 � 10�4 per nucleotide). The low geneticdiversities were also observed for bat RABV populations isolated

Table 2Synonymous (dS) and nonsynonymous (dN) nucleotide diversities in RABV

populations.

Host Domain dS dN dN/dS

Dog, fox SP 0.0266 0 0

ECTO 0.00481 0.003 0.624

TM 0 0.00507 –

ENDO 0.00597 0.00371 0.621

Bats SP 0.0117 0.00438 0.374

ECTO 0.00642 0.00379 0.590

TM 0.0177 0.00758 0.428

ENDO 0.00551 0.00168 0.305

Y. Kobayashi et al. / Infection, Genetics and Evolution 10 (2010) 278–283282

after one passage in mice. These findings suggest that RABVpopulations in infected hosts are genetically highly homogeneous.

In other RNA viruses such as HIV and HCV, the amino acidsequences of antigenic sites have been reported to be highlyvariable because of the generation of escape mutants from hostimmune system (Bonhoeffer et al., 1995; Yamaguchi and Gojobori,1997; Curran et al., 2002; Duffy et al., 2008). Although the ECTOdomain of the RABV G protein is located at the viral surface andincludes B and T cell antigenic sites (Macfarlan et al., 1986; Celiset al., 1988), the amino acid mutations in RABV populationsobserved in our study appeared to be randomly distributed overthe G protein. In addition, the distribution of nonsynonymousmutations was not different between antigenic and non-antigenicsites, and the dN/dS ratio for the G protein genes was generally lessthan 1. Taken together, these results indicate that positiveselection did not act significantly on the antigenic sites of RABV.

Strong evolutionary constraints have frequently been observedin vector-borne RNA viruses, such as bluetongue and dengueviruses, where the need to replicate in both vertebrate andinvertebrate hosts is considered to impose significant selectiveconstraint upon the virus (Bonneau et al., 2001; Wang et al., 2002;Woelk and Holmes, 2002). It has been suggested that the geneticconstraint imposed on RABV is the need to replicate in different celltypes, such as muscle, nervous, and salivary gland tissues (Holmeset al., 2002). Indeed, strong purifying selection has been observedto act on RABV genomes (Hughes, 2009). Consequently, the low dN/dS ratios of the G protein gene observed in RABV populations inBrazil suggest that the population dynamics of RABV is dominatedby the stabilizing selection acting on structural and functionalattributes of the virus (Holmes et al., 2002; Hughes, 2009).

Defective viruses have been demonstrated to modulate viralreplication in vitro, which can affect the clinical course of diseaseand lead to the establishment of persistent infection (Weaver et al.,1999; Tsai et al., 2007). The present study showed the existence ofdefective RABV in naturally infected hosts. However, althoughdefective viruses have been reported to be readily generated instandard cell cultures infected with laboratory-adapted strains ofRABV, no apparent correlation between the presence of defectiveviruses and the pathogenic potential of RABV has been observed inexperimental studies using mice and cell cultures (Wunner andClark, 1980; Clark et al., 1981). In addition, DNA polymerases caninduce deletion mutation during PCR amplification (Bracho et al.,1998). Further experimental studies are required to elucidate theexistence and the role of defective viruses in RABV infection.

This study showed that RABV populations in the brain tissue ofinfected animals were genetically homogeneous and that the Gprotein was evolutionarily constrained, which were consistentwith previously reported evolutionary patterns of the RABV Gprotein (Holmes et al., 2002; Hughes, 2009). Consequently, thestructure of RABV populations in vivo may reflect the infectionstrategies employed by the virus, the way in which stabilizingselection operates, and the evolutionary pattern of the virus.

Acknowledgments

This work was supported partly by the Academic FrontierProject for Private Universities from the Ministry of Education,Culture, Sports, Science and Technology (MEXT) of Japan, a Grant-in-Aid for Scientific Research B from the Japan Society for thePromotion of Science (JSPS), and a grant for Research on Emergingand Re-emerging Infectious Diseases, from Ministry of Health,Labour and Welfare, Japan. Y.K. was supported by JSPS ResearchFellowship for Young Scientists.

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