Full-length sequencing and genetic characterization of ...

Journal of General Virology (2009), 90, 2183–2190 DOI 10.1099/vir.0.010165-0

Correspondence Full-length sequencing and genetic
Marcio Roberto Teixeira Nunes characterization of Breu Branco virus (Reoviridae,
[email protected] Orbivirus) and two related strains isolated from
Anopheles mosquitoes

Conceic¸ a˜ o de Maria Almeida Vieira,1 Marcio Roberto Teixeira Nunes,2,3
Eliana Vieira Pinto da Silva,2 Vale´ ria Lima Carvalho,2
Joaquim Pinto Nunes Neto,2 Ana Cec´ılia Ribeiro Cruz,2,3
Samir Mansour Moraes Casseb,2 Helena Baldez Vasconcelos,2
Juarez Antoˆ nio Simo˜ es Quaresma3
and Pedro Fernando da Costa Vasconcelos2,3

1Amazonian Rural Federal University, Bele´ m, Brazil

2Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute, Ananindeua,
Para´ State, Brazil

3Institute of Biological Sciences Federal University of Para´ State, Bele´ m, Brazil

Received 9 January 2009 Breu Branco virus (BE AR 492347) was isolated from Anopheles (Nyssorhynchus) triannulatus
Accepted 10 May 2009 mosquitoes captured in Tucuru´ı, Para´ State, northern Brazil, in 1988. No cross-reactivity by
complement-fixation tests was observed between Breu Branco virus and other known arboviruses.
Results of electron microscopy and physicochemical tests suggested that Breu Branco virus may
be a member of the family Reoviridae. In order to elucidate its taxonomic status, a comprehensive
genetic characterization was conducted for Breu Branco virus and related strains (BE AR 494475
and BE AR 486204) that were also isolated from Anopheles mosquitoes in the same area. This
included full-length genome sequencing, determination of genetic traits and phylogenetic analysis.
Breu Branco virus showed a similar genome organization to members of the genus Orbivirus, family
Reoviridae. Genetically, Breu Branco virus was indistinguishable from strains BE AR 494475 and
BE AR 486204. Phylogenetic analysis suggested that Breu Branco virus BE AR 492347 and its
related strains constitute a novel species of the genus Orbivirus. Breu Branco virus is the first
Brazilian orbivirus and the fifth orbivirus in the world to be sequenced fully.

INTRODUCTION icosahedral symmetry and a diameter of approximately
70 nm. The absence of an envelope structure was
Breu Branco virus (BE AR 492347) was isolated in 1988 confirmed by the persistence of virus infectivity after
from a pool of Anopheles (Nyssorhynchus) triannulatus treatment with sodium deoxycholate, as described else-
mosquitoes collected in the municipality of Tucurui, Para´ where (Theiler, 1957). Two other virus strains, BE AR
State, northern Brazil (03u 469 S 49u 409 W). The virus was 494775 and BE AR 486204, were also isolated in the same
initially tested by the complement-fixation (CF) technique, area in 1984 and 1987 from a mixed pool of Anopheles
and no cross-reactivity was observed against immune sera (Nys.) oswaldoi/Anopheles (Nys.) nuneztovari, and from a
of 195 other arboviruses previously isolated in the Brazilian pool of A. (Nys.) triannulatus, respectively. CF and
Amazon. By electron microscopy, Breu Branco virus was neutralization tests showed that these two isolates were
revealed to be a non-enveloped, spherical particle with indistinguishable from Breu Branco virus, and they are
therefore classified as strains of this virus.
The GenBank/EMBL/DDBJ accession numbers are available in
Supplementary Table S2 (with the online version of this paper). The family Reoviridae contains the genus Orbivirus as well
as other well-established genera (including Orthoreovirus,
Supplementary tables showing primers used for full-length sequencing, Rotavirus, Coltivirus, Aquareovirus, Cypovirus, Fijivirus,
members of the family Reoviridae used for phylogenetic analysis, genetic Phytoreovirus, Oryzavirus, Seadornavirus, Mycoreovirus
relationships of Breu Branco virus and a summary of the nucleotide and and Idnoreovirus). There are currently 22 recognized
amino acid substitutions observed are also available with the online
version of this paper. 2183

010165 G 2009 SGM Printed in Great Britain

C. de M. A. Vieira and others infected with the viruses and observed daily for detection of
cytopathic effect (CPE). When cells displayed 75 % CPE, the
species in the genus Orbivirus that are vectored by different supernatants of infected cell cultures were collected, centrifuged at
insects, including mosquitoes (Anophelinae and 3000 r.p.m. at 4 uC for cell-debris precipitation, aliquotted and either
Culicinae), Ceratopognidae (Culicoides midges), ticks and stored at 270 uC or used immediately for RNA extraction. This was
Phlebotominae flies. Orbiviruses have a genome character- performed as follows: 6 ml infected supernatant was transferred to a
ized by a ten-segmented, double-stranded (ds) RNA 50 ml polypropylene tube containing a solution of 50 % polyethylene
(Mertens et al., 2005). glycol (PEG) 8000 (Invitrogen) and 23 % NaCl for virus precipitation.
Subsequently, supernatants were removed by centrifugation at
The most well-known species of the genus Orbivirus is 3000 r.p.m. at 4 uC for 10 min and the virus pellets were eluted into
Bluetongue virus (BTV). This virus has been found in 250 ml HPLC RNase-free water (Invitrogen). RNA extraction was
different countries and continents, such as Australia, USA, carried out by using TRIzol LS reagent (Invitrogen) or by using a
Africa, the Middle East, Asia and Europe, where it is commercial kit (QIAmp Viral RNA mini kit; Qiagen), following the
considered an important veterinary problem due to serious manufacturer’s instructions.
disease in livestock (sheep, goats, cattle and deer). Its
occurrence is seasonal in affected Mediterranean countries Electrophoretic profile. For evaluation of the RNA electrophoretic
(Purse et al., 2005). BTV, African horse sickness virus profiles of Breu Branco virus and related strains, PAGE was
(AHSV) and epizootic hemorrhagic disease virus (EHDV) performed. A 10 % PAGE gel (2 M Bis acrylamide, pH 8.8; 10 %
are vectored by Culicoides midges. These viruses are among ammonium persulfate; TEMED) was run as described elsewhere
the most economically important orbiviruses that infect (Shapiro et al., 1967). The RNA was run with Irituia virus (IRIV), an
vertebrates (Mertens et al., 2005). Amazonian orbivirus isolated in 1961 from Oryzomys sp. on the
Bele´m–Bras´ılia highway km-92 (Travassos da Rosa et al., 1984), which
Genetic studies have been conducted for some of the was used as a positive control. RNA extracted from uninfected C6/36
insect-borne orbiviruses, including the recent descriptions of cells was used as a negative control.
the full-length sequences of Yunnan orbivirus (YOV) – a
novel orbivirus isolated from Culex tritaeniorhynchus mos- Isolation and purification of virus RNA. Polysegmented Breu
quitoes in China (Attoui et al., 2005), Middle Point orbivirus, Branco virus RNA extracted from supernatant of infected C6/36 cells
isolated from sentinel cattle in Australia (Cowled et al., 2007), was segregated by conventional electrophoresis in 1.2 % agarose gel
Stretch Lagoon orbivirus, isolated from Culex and Aedes stained with ethidium bromide (0.5 mg ml21). After that, each RNA
mosquitoes (Cowled et al., 2009), and Toggenburg orbivirus segment was removed from the gel by using a sterile stylus and
(Chaignat et al., 2009). In contrast, little attention has been purified by using a commercial Qiaquick Gel Extraction kit (Qiagen)
paid to tick-borne orbiviruses. There is currently a lack of according to the manufacturer’s instructions.
genetic data available for orbiviruses in terms of entire
genome sequencing, which is mainly represented by nucleot- Genome amplification. The RNA segments of Breu Branco virus
ide sequences obtained for BTV serotype 17 (Mertens et al., and related strains were amplified by using the single-primer
2005), Broadhaven virus (BRDV; Moss et al., 1992) and St amplification technique (SPAT) as described by Attoui et al.
Croix River virus (SCRV; Attoui et al., 2001). (2000a, b).

In this study, we have determined the full-length sequence Sequencing. The cDNA products generated by SPAT were partially
and genetic traits of Breu Branco virus and performed sequenced by the dideoxy chain-termination method using an ABI
phylogenetic analysis. These data were useful in the PRISM Dye Terminator kit (Applied Biosystems), and analysed in an
determination of the taxonomic status of Breu Branco virus. ABI 377 DNA sequencer. The complementary primer (B; 59-
GGTGCACGGTCTACGAGACCT-39) to the anchor 39-amino-
METHODS blocked primer A (59-PO4-AGGTCTCGTAGACCGTGCACC-NH2-
39) (Attoui et al., 2000b) was used for cDNA sequencing in both
Virus strains. Breu Branco virus (BE AR 492347) and related strains directions. Specific primers were designed for each segment by using
(BE AR 494475 and BE AR 486204) used in this study are listed in the overlapping strategy as far as the sequences were obtained (see
Table 1 and represent relatively low-passage isolates obtained from Supplementary Table S1, available in JGV Online).
the WHO Collaborating Centre for Research, Diagnostic Reference
and Training in Arbovirus, Arbovirus Section and Hemorrhagic Assembly of overlapping sequences. Partial nucleotide sequences
Fevers, Evandro Chagas Institute, Ananindeua, Para´ State, Brazil. obtained for Breu Branco virus and related strains were first inspected
for quality and then assembled by using the SeqMan II program v.
Virus culture and RNA extraction. Viruses were grown in 5.03 (LaserGene DNASTAR software package) in order to produce
monolayer cultures of Aedes albopictus (C6/36) cells. Cells were contiguous full-length sequences. This software was also used to
predict the entire open reading frame (ORF) for the polyprotein, as
well as for both 59 and 39 non-coding regions (NCRs).

Table 1. Hosts and geographical origins of Breu Branco virus and strains BE AR 494475 and BE AR 486204

Virus/strain Mosquito species Place of isolation Date of isolation

Breu Branco virus (BE AR 492347) Anopheles (Nys.) triannulatus Tucuruı´/PA, Base 4 15 August 1988
BE AR 494475 Anopheles (Nys.) oswaldoi/nuneztovari Tucuruı´/PA, Arumateua 20 October 1984
BE AR 486204 Anopheles (Nys.) triannulatus Tucuruı´/PA, Base 4 10 October 1987

2184 Journal of General Virology 90

Scanning of Breu Branco virus ORFs and identification of Full-length sequencing of Breu Branco virus
related proteins. For the identification of related Breu Branco virus
proteins, the full-length predicted ORFs for each of the ten segments Fig. 1. (a) Electrophoretic profile of Breu Branco virus (I) using
(S1–S10) of Breu Branco virus and related strains were scanned PAGE, showing the ten RNA segments. (b) Electrophoretic profile
against other orbivirus amino acid sequences available in GenBank by of the low-molecular-mass RNA segments of Breu Branco (I-1),
the protein–protein BLAST program, available on the NCBI home page BE AR 494475 (I-2) and BE AR 486204 (I-3) viruses. II, Fluid of
(http://www.ncbi.nlm.nih.gov/Blast.cgi), using standard default para- non-infected C6/36 cells (negative control); III, non-infected
meters. Sequences represented by the lowest E value and highest P C6/36 cells (negative control); IV, positive control (IRIV).
score were selected to be used in multiple sequencing alignments
(gene by gene) using CLUSTAL W software (Thompson et al., 1997). to 30 nt for S6. The 39 terminus was longer, with NCRs
The percentage identity for nucleotide and amino acid sequences, as ranging from 33 nt (S6) to 106 nt (S 7). Conserved
well as the mutations along the entire genome, were assessed with the sequences were observed in both 59 and 39 termini of all
MegAlign software (LaserGene DNASTAR package). genomic segments and corresponded to GUUAA and UAC,
respectively. It is important to note that size variability in
Phylogeny. For phylogenetic analysis of the sequences obtained for the 39 NCR region was also observed in S10 of Breu Branco
Breu Branco virus and the related strains BE AR 494475 and BE AR virus and its related strains (Table 2), due to an insertion or
486204, amino acid sequences corresponding to the viral polymerase deletion of the nucleotide sequences CGCGCGGTT/
(VP1, S1), T2 protein [which is encoded by S2 (VP2) or S3 (VP3)] and TTCGCGCGGTTCGCGCGGTT.
T13 protein (S7, VP7) were compared with sequences of represent- The genome scanning implemented in the protein–protein
ative members belonging to different genera within the family BLAST using the entire ORFs of each RNA segment of Breu
Reoviridae (Orthoreovirus, Aquareovirus, Phytovirus, Cypovirus, Branco virus and its related strains revealed protein
Rotavirus, Seadornavirus, Coltivirus, Oryzavirus and Fijivirus) (see identity with other members of the genus Orbivirus (E
Supplementary Table S2, available in JGV Online). Neighbour-joining values50.001; P scores ranging from 1226 to 4071). The
(NJ) (Saitou & Nei, 1987) and maximum-parsimony (MP) highest identity was observed with AHSV S1 (P
(Swofford, 1999) methods implemented in the MEGA 3.0 software score54071; 49.1 %). On the other hand, the lowest
(Kumar et al., 2004) and PAUP 4.0 beta version (Swofford, 1999) were identity was observed with S6 of SCRV (P score51226;
performed. For NJ analysis, the distance matrix was calculated from 19.1 %). As shown in Table 3, the function of each RNA
the aligned sequences by using the Kimura two-parameter formula segment is referred to in comparison with homologous
(Kimura, 1980), and a weight of four transitions versus one segments of BTV, AHSV and SCRV.
transversion was selected. In order to obtain a tree with MP, the Multiple sequence alignment carried out between ORFs of
heuristic algorithm was used in MP analysis. For determination of the Breu Branco virus and its related strains BE AR 49475 and
reliability of the tree topology, bootstrap analysis (Felsenstein, 1985) BE AR 486204 revealed a high degree of nucleotide
was carried out on 2000 replicates. The bootstrap-resampling sequence identity, ranging from 91.4 % for S9 to 99.6 %
technique (Efron, 1982) was then used to further evaluate the for S6. For amino acid sequences, the identities were
reliability of bootstrap analysis with a confidence value of 95 %. estimated to be in the range 95 % (S9) to 100 % (except for
Furthermore, confidence values used as criteria for group inclusion or
exclusion were calculated based on the mean of the amino acid 2185
sequence identities within and among selected members of the
different genera of the family Reoviridae.

RESULTS

Electrophoretic profile

Analysis of the RNA electrophoretic profile of Breu Branco
virus by using PAGE revealed the presence of a ten-
segmented genome, which was classified according to its
molecular mass into three distinct groups: group 1, high
molecular mass (S1–S3); group 2, intermediate molecular
mass (S4–S6); and group 3, low molecular mass (S7–S10)
(Fig. 1).

Genome analyses

The complete genome sequences of Breu Branco virus and
strains BE AR 494475 and BE AR 486204 were obtained for
all ten RNA segments by assembling nucleotide sequences
obtained for each segment. The segment sizes ranged from
4965 nt for S1 to 773 nt for S10. The predicted proteins
ranged from 1630 aa (Pol, viral polymerase; S1) to 237 aa
(NS3a, S10) in length. Regarding NCRs, the 59 termini
were composed of short sequences ranging from 8 nt for S4

http://vir.sgmjournals.org

C. de M. A. Vieira and others

Table 2. Genome organization of Breu Branco virus and strains BE AR 494475 and BE AR 486204 according to RNA segment,
size, ORFs, protein and 59 and 39 NCRs

RNA segment Size (nt) ORF (nucleotide Predicted protein 5§ NCR 3§ NCR
Size (nt) Terminal sequence
position) size (aa)

S1 4965 14–4903 1630 13 GUUAAAU- --- Terminal Size (nt)
S2 3221 16–3186 1057 15 GUUAAAU- --- sequence
S3 2789 21–2738 906 20 GUUAAAU- --- 62
S4 1999 9–1952 648 8 GUUAAUA- --- - - - -UUAC 35
S5 1707 36–1649 538 35 GUUAAAA- --- - - - -UUAC 51
S6 1670 39–1637 533 38 GUUAAAA- --- - - - -UUAC 47
S7 1281 18–1175 386 17 GUUAAAA- --- - - - -UUAC 58
S8 1125 20–1078 353 19 GUUAAAA- --- - - - -AUAC 33
S9 920 22–855 278 22 GUUAAAA- --- - - - -UUAC 106
43–855 271 - - - -UUAC 47
S10 773/762/782* 21–740 240 20 GUUAAAA- --- - - - -UUAC 65
30–740 237 - - - -AUAC
Consensus sequenced GUUAAww- ---
----UUAC 33/22/42D

- - - -wUAC

*Breu Branco virus, 773 nt; BE AR 494475, 762 nt; BE AR 486204, 782 nt.
DBreu Branco virus, 33 nt; BE AR 494475, 22 nt; BE AR 486204, 42 nt.
dw5A or U.

S1, S2, S9 and S10) (see Supplementary Table S3, available Thirteen of these mutations were characterized as non-
in JGV Online). synonymous mutations leading to amino acid changes.
Furthermore, four mutations modified the biochemical
Mutations were observed along all RNA segments when properties of the amino acid involved (see Supplementary
sequences were compared among Breu Branco, BE AR Table S4, available in JGV Online).
494475 and BE AR 486204 viruses. However, S9 was the
most divergent, showing 51 mutations, 45 of which were Phylogeny
identified as transitions and six as transversions. Of 51
mutations, four (7.8 %), six (11.7 %) and 41 (80.5 %) were The phylogenetic analysis based on the amino acid
observed in the first, second and third codons, respectively. sequences (S1, S2 and S3) obtained for Breu Branco, BE

Table 3. Genome comparison and related functions of Breu Branco virus proteins in comparison with selected members of the
genus Orbivirus

Putative protein function Genomic segment Amino acid identity (%) with:

Viral polymerase, Pol S1 BTV AHSV SCRV
Outer shell protein, VP2 S2
Major subcore protein, VP3 (T2) S3 48.3 49.1 38.7
Minor core and capping enzyme, S4 29.0 33.8 29.9 (equivalent to VP3 of Breu Branco virus)
28.2 31.1 20.4 (equivalent to VP2 of Breu Branco virus)
VP4 S5 31.7 30.5 34.2
Tubules, NS1 S6
Outer capside protein, VP5 S7 39.6 29.0 36.8 (equivalent to VP5 of Breu Branco virus)
Major core surface protein, VP7 28.1 30.3 19.1 (equivalent to NS1 of Breu Branco virus)
S8 35.4 38.2 19.4 (equivalent to NS2 of Breu Branco virus)
(T13) S9
Viral inclusion body, NS2 22.2 23.5 21.7 (equivalent to VP7–T13 of Breu Branco virus)
Minor core protein, helicase, S10 27.6 25.2 23.7

VP6 24.8 19.7 22.1
Virus release, NS3

2186 Journal of General Virology 90

AR 494475 and BE AR 486204 viruses and other Full-length sequencing of Breu Branco virus
representative members of the family Reoviridae using the
MP and NJ methods resulted in trees with similar Breu Branco viruses were also related to orbiviruses in
topologies, although bootstrap values in the MP consensus which the T2 protein is encoded on S3 (VP3) (Fig. 3).
tree were slightly lower than those in the NJ tree (data not
shown). Accordingly, the NJ tree was used to represent the DISCUSSION
phylogeny of the selected viruses. By using reliable
bootstrap (.85 %) and confidence (S1, ¡28 %±2SD; S2 Due to the isolation of Breu Branco virus and related
or S3, ¡13 %±2SD) values as criteria for group inclusion, strains from mosquitoes of the genus Anopheles, it was
the phylogram using the entire polymerase (S1) amino acid expected that these viruses would be grouped in the
sequences revealed the genetic relationship of Breu Branco, Anopheles A serogroup of the family Bunyaviridae, genus
BE AR 494475, and BE AR 486204 viruses to other Orthobunyavirus, where several other viruses isolated from
members of the genus Orbivirus (Fig. 2). The phylogeny for these mosquitoes are grouped (Travassos da Rosa et al.,
the T2 protein (S2 or S3) depicted the segregation of two 1992). However, the results obtained by the CF test did not
major groups, hereafter designated the Culicoides- and show cross-reactivity between Breu Branco virus and any
Culicidae-borne group (Cul) and the tick-borne group. In other Anopheles A member, or with other arboviruses
the Cul group, 15 clades (I–XV) were formed and the Breu belonging to different antigenic groups (group A, B, C,
Branco virus and related strains constituted a single Bunyamwera, Simbu and California) that commonly
phylogenetic subclade (I), related more closely to subclade circulate in the Brazilian Amazon region. Breu Branco
II (Kasba virus and D’Aguilar virus). Furthermore, the virus reacted only with its homologous serum and with the
immune sera prepared for BE AR 494475 and BE AR

Fig. 2. Phylogenetic analysis of the viral polymerase S1 of Breu Branco virus and strains BE AR 494475 and BE AR 486204 in
comparison with other members of the family Reoviridae, using the NJ method. Bootstrap values and percentages of divergence
(in parentheses) are given above each main node of the tree. *Amino acid divergence of 0.5 %. Bar, 50 % amino acid sequence
divergence.

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C. de M. A. Vieira and others

Fig. 3. Phylogenetic tree of the amino acid sequences of the T2 proteins of Breu Branco virus, strains BE AR 494475 and
BE AR 486204 and other orbivirus species, using the NJ method (analysis was based on partial amino acid sequences between
positions 393 and 548 relative to the BTV sequence). Bootstrap values are given above each main node of the tree. The value in
parentheses represents the amino acid divergence between Breu Branco virus and related strains. GenBank accession
numbers are available in Supplementary Table S2 (in JGV Online). Bar, 10 % amino acid sequence divergence.

486204 viruses (CF titres in reciprocal serum/antigen Genetic characterization using the full-length sequences
reaction: ¢64/¢64±2-fold dilutions). Thus, further obtained for Breu Branco virus and related strains was
analyses were necessary to assess the classification status helpful to better understand the genetic relationship
of this viral agent. among these viruses and with other, previously completely
sequenced orbiviruses. The analysis of the 59 and 39 NCRs
As recently observed for YOV (Attoui et al., 2005), the revealed highly conserved sequences that are similar to
classification of Breu Branco virus and strains BE AR 494475 those identified in other known orbiviruses (Table 2)
and BE AR 486204 as members of the family Reoviridae (Mertens et al., 2005).
(more specifically, as a novel virus species within the genus
Orbivirus) has also been supported by data including the size The genetic diversity of Breu Branco, BE AR 494475 and
of virus particles, morphology, physicochemical properties BE AR 486204 viruses was most evident for S9, which
(sodium deoxycholate sensitivity), electrophoretic profile (10 encodes the VP6 protein – a minor core protein with
RNA segments; Fig. 1) and the genetic relationship with helicase, NTPase and single-stranded RNA/dsRNA-binding
other members of the genus (Figs 2, 3). functions. The analysis of mutations revealed significant

2188 Journal of General Virology 90

differences in terms of amino acid substitutions caused by Full-length sequencing of Breu Branco virus
non-synonymous mutations, some of which lead to
modification of the amino acid’s biochemical properties. agents as a unique species within the genus Orbivirus,
Studies conducted on BTV have associated the helicase isolated from mosquito species of the genus Anopheles.
activity with the separation of the dsRNA and participation
in the viral replication and transcription processes (Sta¨uber Breu Branco virus is the first Brazilian orbivirus and the
et al., 1997; Kar & Roy, 2003). Interestingly, size fifth orbivirus in the world to be sequenced fully. The
heterogeneity was observed in the 39 NCR of S10 among availability of its full-length sequence should be a helpful
the strains used in this study (Table 2). This finding is tool for the development of new PCR-detection strategies,
probably due to major deletions and/or duplications, as as well as for a better understanding of the molecular
demonstrated previously for different strains of yellow epidemiology of this novel orbivirus isolated in the
fever virus isolated in Africa and the Americas Brazilian Amazon region. Finally, we propose the abbre-
(Vasconcelos et al., 2004; Bryant et al., 2005). This could viation BBV for Breu Branco virus.
be of great interest, as the NS3 protein encoded by S10 is
involved in the release mechanism of orbiviruses (Mertens ACKNOWLEDGEMENTS
et al., 2005). Further experimental studies using animal
models (hamsters and/or mice) are necessary to evaluate We thank Geraldo Mendes da Silva, Creuza Lima Carvalho, Luiz
the effective role of the mutations found in the VP6 protein Roberto O. Costa and Basilio S. Buna for their technical assistance.
and of size heterogeneity in the 39 NCR of S10 for strain BE We are also grateful to Andre´ Monteiro Diniz for English revision of
AR 486204 in the virus pathogenicity and viral replication/ the manuscript. This work was supported financially by the Evandro
release processes. Chagas Institute, Ministry of Health and CNPq grants (process no.
300460/2005-08 and 302987/2008-8).
All deduced amino acid sequences showed considerable
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