Genetic Characterization and Phylogeny of Sabiá Virus, an Emergent Pathogen in Brazil

Abstract

Sabiá virus, one of five arenaviruses from South America known to cause hemorrhagic fever in humans, emerged in 1990 when it was isolated from a fatal case in Sao Paulo, Brazil. Subsequently, it has caused two laboratory-acquired infections. Its natural distribution and host are still unknown. Using viral RNA and multiple polymerase chain reaction products as templates, the nucleotide sequence of the small (S) RNA segment of Sabiá virus, which codes for the nucleocapsid (N) and glycoprotein precursor, was determined. This virus shares an ambisense genome in common with other arenaviruses, although it has a unique predicted three stem--loop structure in the S RNA intergenic region. Phylogenetic analysis of a portion of the N gene sequence confirmed that Sabiá virus is distinct from all other members of the Arenaviridae and shares a progenitor with Junin, Machupo, Tacaribe, and Guanarito viruses.

Full text

VIROLOGY
221, 318–324 (1996)
ARTICLE NO.
0381
Genetic Characterization and Phylogeny of Sabia
´Virus, an Emergent Pathogen in Brazil
JEAN PAUL J. GONZALEZ,*
,
† MICHAEL D. BOWEN,‡ STUART T. NICHOL,‡ and REBECA RICO-HESSE†
,1
*Institut Franc
¸ais de Recherche Scientifique pour le De
´veloppement en Coope
´ration, ORSTOM, 213 rue LaFayette, 75480, Paris, France;
‡Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia 30333; and †Department of Epidemiology and Public Health,
Yale University School of Medicine, P.O. Box 208034, New Haven, Connecticut 06520
Received April 3, 1996; accepted May 8, 1996
Sabia
´virus, one of five arenaviruses from South America known to cause hemorrhagic fever in humans, emerged in 1990
when it was isolated from a fatal case in Sao Paulo, Brazil. Subsequently, it has caused two laboratory-acquired infections.
Its natural distribution and host are still unknown. Using viral RNA and multiple polymerase chain reaction products as
templates, the nucleotide sequence of the small (S) RNA segment of Sabia
´virus, which codes for the nucleocapsid (N) and
glycoprotein precursor, was determined. This virus shares an ambisense genome in common with other arenaviruses,
although it has a unique predicted three stem–loop structure in the S RNA intergenic region. Phylogenetic analysis of a
portion of the N gene sequence confirmed that Sabia
´virus is distinct from all other members of the Arenaviridae and
shares a progenitor with Junin, Machupo, Tacaribe, and Guanarito viruses.
q1996 Academic Press, Inc.
INTRODUCTION ver outbreaks with high mortality in South America and
Africa since the 1950s and 1960s, respectively (Parodi
The members of the family Arenaviridae have a single- et al., 1961; Frame et al., 1970). Sabia
´virus is a new
stranded RNA genome composed of two segments, L member of the South American arenaviruses known to
(large) and S (small), with an average length of 7100 and cause disease in humans (Coimbra et al., 1994); others
3400 nucleotides, respectively. Two other RNA mole- are Junin, Machupo, Flexal, and Guanarito (Parodi et al.,
cules have been detected in virions and are presumed 1961; Johnson, 1965; Pinheiro, 1986; Salas et al., 1991).
to be ribosomal RNAs since they comigrate with 28 S Sabia
´virus emerged in 1990 when it was isolated from
and 18 S ribosomal RNA (Carter et al., 1973; Southern a fatal case of hemorrhagic fever in Sao Paulo, Brazil
and Bishop, 1987; Iapalucci et al., 1989). Arenaviruses (Coimbra et al., 1994); subsequently, two nonfatal labora-
utilize an ambisense coding strategy to express their tory infections have occurred (Vasconcelos et al., 1993;
genes. The genomic S RNA segment encodes the nu- Barry et al., 1995). Further studies on the epidemiology,
cleocapsid (N) gene at the 3*end and the glycoprotein ecology, and diagnosis of this hemorrhagic fever have
precursor (GPC) gene at the 5*end (Southern and been severely restricted due to the biohazards associ-
Bishop, 1987). The N protein is expressed through tran- ated with handling this agent and the lack of specific
scription of a subgenomic mRNA from the genomic S diagnostic reagents. Thus, the origin, geographic distri-
RNA template. The GPC is expressed via transcription bution, natural maintenance cycle, and epidemiology of
of a subgenomic mRNA from the antigenomic S RNA this virus are unknown.
template, a replicative intermediate. The GPC undergoes Initial characterization of Sabia
´virus by complement-
posttranslational cleavage to generate the envelope gly- fixation, immunofluorescence, and neutralization assays
coproteins G1 and G2 (Southern and Bishop, 1987). The indicated that Sabia
´is a new member of the Tacaribe
L RNA segment encodes two proteins. The viral polymer- complex (Coimbra et al., 1994). A limited sequence analy-
ase gene is located at the 3*end of the segment and a sis using 250 nt of the S RNA indicated that is genetically
zinc-binding protein gene at the 5*end (Salvato, 1993); distinct from Junin, Machupo, Pichinde, Tacaribe, and
these are also expressed by an ambisense coding strat- Guanarito viruses (Coimbra et al., 1994). To further char-
egy (Salvato, 1993). In both segments, the intergenic re- acterize Sabia
´virus, the S RNA was sequenced and com-
gion separating the two genes has been predicted to pared to other arenaviruses, both genetically and phylo-
form hairpin structures which may play an important role genetically.
in transcription termination during mRNA synthesis
(Franze-Fernandez et al., 1993). MATERIALS AND METHODS
Arenaviruses have caused sporadic, hemorrhagic fe- The Sabia
´prototype strain (SPH114202) was iso-
lated from serum of a human fatal case by infecting
1
To whom correspondence and reprint requests should be ad-
dressed. Fax: (203) 785-4782.
newborn mice by intracerebral inoculation (Coimbra et
318
0042-6822/96 $18.00
Copyright q1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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319SABIA
´GENETICS AND PHYLOGENY
al., 1994). This virus was then passaged twice in Vero 19 nt at the 3*end of the segment which was inferred
from the primer sequence.E6 cells to produce working stocks of virus. Virus RNA
was purified as previously described (Gonzalez et al., The majority of the remaining S RNA sequence was
obtained by sequencing products obtained by reverse1995). The 3*end of the Sabia
´S RNA was sequenced
initially by dideoxynucleotide chain termination se- transcription–polymerase chain reaction (RT–PCR). For
this purpose, RNA was extracted from infected cell cul-quencing of virus RNA (Rico-Hesse et al., 1987) using
the oligonucleotide ARE/3*END (5*-CGCACAGTG- ture supernatant by acid guanidium thiocyanate–phe-
nol–chloroform extraction (Chomczynsky and Sacchi,GATCCTAGGC-3*) to prime reverse transcription. This
primer is complementary to the 19 nt at the 3*end of 1987), treated with 10 mMmethyl mercury hydroxide, and
then reverse transcribed using primer ARE/3*END. Theall known arenavirus S RNAs (Wilson and Clegg, 1991).
The sequence of the 369 nt at the 3*end of the Sabia
´Sabia
´S RNA was amplified by RT–PCR using primers
designed from the partial sequence determined by directS RNA was obtained by this method, exclusive of the
FIG. 1. Comparison of the amino acid sequence of the GPC protein encoded by the S RNA of Sabia
´virus with those of other arenaviruses (LCM
arm, lymphocytic choriomeningitis Armstrong strain; LCM we, lymphocytic choriomeningitis WE strain; LAS jos, Lassa Josiah strain from Sierra
Leone; LAS nig, Lassa strain from Nigeria; MOP, Mopeia; JUN, Junin; MAC, Machupo; TAC, Tacaribe; SAB, Sabia
´; GUA, Guanarito; PIC, Pichinde).
Dot indicates deletion. Putative cleavage site, arenavirus conserved antigenic site, and probable CTL epitopesare inboldface. Conserved residues
among the New World arenaviruses are shown in the top consensus line; conserved residues among New World and Old World arenaviruses are
shown in the bottom consensus line. Dash indicates absence of conserved residue.
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320 GONZALEZ ET AL.
RNAsequencingandprimers constructed from aconsen- it has been pointed out, CTL epitopes present several
advantages in eliciting protective immunity: CTL re-sus sequence of other New World arenavirus S RNAs.
PCR products of predicted size were excised from agar- sponses may be encoded by structural or nonstructural
viral proteins, may be more important in virus clearanceose gels and then purified from gel slices using a Seph-
aglas Bandprep kit (Pharmacia Biotech, Inc.). Purified than humoral responses (Oldstone and Dixon, 1970), and
are commonly cross-reactive against serologically dis-PCR products were sequenced directly by the dye termi-
nation cycle sequencing technique (Applied Biosystems, tinct viral strains (Whitton et al., 1989). It remains to be
seen whether these regions serve as CTL epitopes inInc.) with the same primers as used for PCR product
amplification. To obtain the sequence at the 5*terminus Sabia
´virus.
The noncoding region which separates the stop co-of the S RNA, a poly(A) tail was added onto the Sabia
´S
cDNA using terminal deoxynucleotidyl transferase (Loh dons of the GPC and N genes spans nt 1526 to 1619.
The predicted secondary structure of this region consistset al., 1989) and the tailed template was PCR-amplified
using the Sabia
´S RNA complementary primer 3108C (5*- of two large stem–loop structures and a third smaller
stem–loop (Fig. 2). All three structures are predicted toCCAAGTGGCATTTTTGTTTTCATGG-3*) and aprimerthat
annealedtopoly(A)tails(5*-CGGGATCCCGTTTTTTTTTT- form regardless of whether the intergenic region is ana-
lyzed in the genomic or antigenomic sense. This feature3*). PCR products were then cloned into the TA cloning
vector pCRII (Invitrogen Corp.) and individual clones se- distinguishes the Sabia
´S RNA from the S RNAs of other
arenaviruses whose intergenic regions are predicted toquenced.
Nucleotide and amino acid sequence compilation, form either one (LCM, Pichinde, Lassa) or two (Mopeia,
Junin, Tacaribe) stem–loop structures (Auperin et al.,analysis, and alignment were performed using the Wis-
consin Sequence Analysis Package version 8.0 (Genet- 1984, 1986; Romanowsky et al., 1985; Franze-Fernandez
et al., 1987; Ghiringhelli et al., 1991; Wilson and Clegg,ics Computer Group, Inc.). The Sabia
´virus S RNA was
determined to be 3366 nucleotides in length (GenBank 1991). The stem–loop structures of the intergenic region
are thought to play an important role in termination ofAccession No. U41071). The S RNA contained two open
reading frames in an ambisense orientation as found in transcription during mRNA synthesis (Auperin et al.,
1984; Franze-Fernandez et al., 1993). Alternatively, theseother arenavirus S RNAs (Auperin et al., 1984). The 5*
noncoding region is 58 nt long and contains an extra structures may be involved in translational initiation of a
second gene product (Auperin et al., 1984). The signifi-nontemplated G residue at the terminus as described
previously for other arenaviruses (Raju et al., 1990). cance of the variability in number of potential secondary
structures in different arenaviruses is unknown.
The putative N protein gene is located at nt 1620 to
RESULTS AND DISCUSSION 3308. Its 562-aa gene product is 33.6 to 41.5% divergent
from the N proteins of the New World arenaviruses andThe putative GPC gene, which begins at nt 59, is 1464
nucleotides long and encodes a 488-aa gene product.
The putative GPC protein precursor is 46.1–64.7% diver-
gent from other arenavirus GPCs (Fig. 1). The putative
GPC cleavage site is located at an R– R motif, at residues
251–252, as determined by analogy with the cleavage
site for the GPC of lymphocytic choriomeningitis virus
(LCM) (Buchmeier et al., 1987). The amino acid sequence
of the G2 region, located at the C terminus of the GPC,
is generally more conserved than the N-terminal G1 re-
gion. The most hydrophilic domain is located in the G2
region at residues 347 to 352. Eleven potential N-linked
glycosylation sites are present: seven in the G1 region
and four in the G2 region. An antigenic site conserved
among arenavirus GPCs, KFWYL (Weber and Buchmeier,
1988), was present in the Sabia
´GPC sequence at aa
359–363 (KFWYV) with only one amino acid change (L to
V). CTL epitopes identified within the LCMV glycoprotein,
residues 31–42, KAVYNFATCG (Pircher et al., 1990), and
residues 278–286, VENPGGYCL (Joly et al., 1989) have
FIG. 2. Potential base-paired hairpin structures in the intergenic re-
corresponding regions in Sabia
´, residues KGMINLWKSG
gion of Sabia
´virus S RNA (nucleotides: upper case, intergenic region;
and NDMPGGYCL, respectively (Fig. 1). Potential anti-
lower case, stop codons) as predicted by the MFOLD program of the
genic sites and CTL epitopes shared by several human
Wisconsin Sequence Analysis Package, version 8.0. The free energy
pathogenic strains are presumed to play an important
value for the Sabia
´intergenic region folded in GPC coding (genomic)
sense is 064.2 kcal/mol.
role in mechanisms of infection (Whitton et al., 1989). As
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321SABIA
´GENETICS AND PHYLOGENY
FIG. 3. Comparison of the amino acid sequence of the N protein encoded by the S RNA of Sabia
´virus with those of other arenaviruses (LCM
arm, lymphocytic choriomeningitis Armstrong strain; LCM we, lymphocytic choriomeningitis WE strain; LAS jos, Lassa Josiah strain from Sierra
Leone; LAS nig, Lassa strain from Nigeria; MOP, Mopeia; JUN, Junin; MAC, Machupo; TAC, Tacaribe; SAB, Sabia
´; GUA, Guanarito; PIC, Pichinde).
Dot indicates deletion. Continuous dots indicate information not available for GUA. CTL epitope and probable antigenic site are in boldface.
Conserved residues among the New World arenaviruses are shown in the top consensus line; conserved residues among New World and Old
World arenaviruses are shown in the bottom consensus line. Dash indicates absence of conserved residue.
46.5 to 52.1% divergent from the N proteins of Old World as one of the highest points of hydrophilicity (residues
56 to 62: RKSKRND). A CTL epitope described for LCMarenaviruses (Fig. 3). A probable antigenic site that was
previously described around a conserved pair of amino virus (Whitton et al., 1989) identified by the first 5 amino
acids of the sequence GVYMGNL was found at residuesacid residues (K–R) (Gonzalez et al., 1995) was identified
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322 GONZALEZ ET AL.
123 to 129 (GVYLGNL) on Sabia
´virus, where an M to L function of this segment. Although this region is pre-
sumed to be involved in transcription (Meyer and South-change occurs between the anchor residues of the CTL
epitope (Falk et al., 1991). It remains to be seen if the ern, 1994), there is no obvious structural similarity be-
tween the arenaviruses studied to date.potential CTL site on Sabia
´virus is immunogenic.
The 3*noncoding region of the Sabia
´S RNA is 58 nt To determine the evolutionary relationship of Sabia
´to
other arenaviruses, phylogeny was inferred by maximumlong and includes, at the 3*terminus, the 19 nucleotides
conservedamongallarenavirusesstudied todate.These parsimony analysis of available N gene sequences. The
S RNA sequences of 10 arenaviruses [Junin (GenBank19 nt represent the only portion of the Sabia
´S RNA
not actually sequenced in this study. Other arenaviruses Accession No. D10072), Tacaribe (M20304),Lassa Nige-
ria (X52400, K03362), Lassa Josiah (J04324), Guanaritohave 3*noncoding regions of varying lengths (range, 52
to 96 nt), thus demonstrating plasticity in the encoded (L42001),Machupo(X62616),Mopeia(M33879),Pichinde
FIG. 4. Phylogenetic relationship of arenaviruses based on nucleotide sequence differences in the N gene 3*terminus. Comparisons were done
by the maximum parsimony method using the PAUP software program (Swofford, 1993) run on a Power Macintosh 8100. LCM, LAS, and MOP
viruses from the Old World were used as an outgroup for rooting the tree. Horizontal branch lengths are proportional to nucleotide step differences
between viruses and are indicated above the lines. Bootstrap confidence limits were calculated by 1000 replications of the analysis and limits in
excess of 50% are indicated in parentheses below the branch (LCMarm, lymphocytic choriomeningitis Armstrong strain; LCMwe, lymphocytic
choriomeningitis WE strain; LASjos, Lassa Josiah strain from Sierra Leone; LASnig, Lassa strain from Nigeria; MOP, Mopeia; JUN, Junin; MAC,
Machupo; TAC, Tacaribe; SAB, Sabia
´; GUA, Guanarito; PIC, Pichinde).
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323SABIA
´GENETICS AND PHYLOGENY
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ACKNOWLEDGMENTS
Trop. Med. Hyg. 14, 816–818.
This work was supported in part by Grants A1-10984 and A1-01124 Joly, E., Salvato, M., Whitton, J.L., and Oldstone, M. B. A. (1989). Polymor-
from the National Institutes of Health, and DES-AB8004 from the Institut phism of cytotoxic T-lymphocyte clones that recognize a defined
Franc
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´veloppementen Coope
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tion. We thank our Brazilian colleagues for providing the Sabia
´proto- meningitis virus glycoprotein. J. Virol. 63, 1845–1851.
type virus. Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davies, M. M. (1989).
Polymerase chain reaction with single-sided specificity: Analysis of
T cell receptor delta chain. Science 243, 217–220.
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AID VY 8006 / 6a1a$$$$43 06-16-96 19:10:30 viral AP: Virology