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Journal of Virology, January 1999, p. 667-675, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Recombination in Tula Hantavirus Evolution:
Analysis of Genetic Lineages from Slovakia
Claus
Sibold,1
Helga
Meisel,1
Detlev H.
Krüger,1,*
Milan
Labuda,2
Jan
Lysy,2
Oto
Kozuch,3
Milan
Pejcoch,4
Antti
Vaheri,5 and
Alexander
Plyusnin5
Institute of Medical Virology, Charité
School of Medicine, Humboldt University, D-10098 Berlin,
Germany1;
Institute of Zoology,
Slovak Academy of Sciences, 842 06 Bratislava,2
and
Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava,3
Slovak Republic;
Regional Hygienic Institute of South Moravia, Brno, Czech
Republic4; and
Haartman Institute,
Department of Virology, University of Helsinki, FIN-00014 Helsinki,
Finland5
Received 11 August 1998/Accepted 18 September 1998
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ABSTRACT |
To examine the evolution of Tula hantavirus (TUL), carried by the
European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from
Slovakia, the country where the virus is endemic. Phylogenetic analysis
(PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or
complete N-protein-encoding sequences divided Slovakian TUL variants
into two main lineages: (i) strains from eastern Slovakia, which
clustered with Russian strains, and (ii) strains from western Slovakia
situated closer to those from the Czech Republic. We found genetic
diversity of 19% between the two groups and 4% within the western
Slovakian TUL strains. Phylogenetic analysis of the 3' noncoding region (3'-NCR), however, placed the eastern Slovakian strains closer to those
from western Slovakia and the Czech Republic, with a greater distance
to the Russian strains, suggesting a recombinant nature of the S
segment in the eastern Slovakian TUL lineage. A bootscan search of the
S-segment sequences of TUL strains revealed at least two recombination
points in the S sequences of eastern Slovakian TUL strains (nt 400 to
415 and around 1200) which agreed well with the pattern of amino acid
substitutions in the N protein and deletions/insertions in the 3'-NCR
of the S segment. These data suggest that homologous recombination
events occurred in the evolution of hantaviruses.
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INTRODUCTION |
Hantaviruses (genus
Hantavirus, family Bunyaviridae) are enveloped
viruses with a tripartite, negative-sense RNA genome, consisting of S,
M, and L segments, which encode the nucleocapsid (N) protein, the
surface glycoproteins (G1 and G2), and the polymerase of the virus
(11). Currently at least 16 hantavirus types have been described (45). Some of the virus types (Hantaan [HTN],
Dobrava [DOB], Seoul [SEO], and Puumala [PUU]) are the etiologic
agents of hemorrhagic fever with renal syndrome (HFRS), whereas others (Sin Nombre, Bayou, Black Creek Canal, and the recently described Andes
[30]) are the causative agents of human pulmonary
syndrome (HPS), which was demonstrated for the first time in 1993 in
North America (35). Each of the hantavirus serotypes is
primarily associated with a single rodent host species (27,
45). Transmission of the virus to humans is assumed to occur via
aerosolized excreta of persistently infected rodents (26).
The virus types most relevant for Europe, PUU, DOB, and perhaps SEO and
HTN, are thought to be associated with the bank vole
(Clethrionomys glareolus), the yellow-necked mouse
(Apodemus flavicollis), rats (Rattus norvegicus and R. rattus), and the striped field mouse (A. agrarius), respectively (45).
Another serotype present in Europe, Tula (TUL), was found by screening
of lung tissues from rodent species present in regions where HFRS is
known to be endemic: new viral nucleic acid sequences could be detected
in European common voles (Microtus arvalis and M. rossiaemeridionalis) in Russia (Tula region) (41),
Slovakia (Malacky region) (57), and the Czech Republic
(southern Moravia) (40). Recently TUL was isolated in cell
culture from voles trapped in Moravia (64). The
pathogenicity of TUL for humans is not known; however,
hantavirus-specific antibodies preferentially reacting with TUL antigen
were found in the serum of a healthy forest worker (64).
The territory of Slovakia is one of the geographic centers of TUL
distribution identified so far. In addition, this is a region where
hantaviruses carried by members of the Muridae subfamilies Arvicolinae (PUU-TUL-like viruses) and Murinae
(HTN-like viruses) cocirculate and where HFRS is endemic (13, 39,
55). In the present study, we addressed the genetic variability
of members of the TUL genetic group found in Slovakia and their
phylogenetic clustering relative to other known TUL strains in Europe.
Tissue samples of small rodents, trapped in various geographical
regions in central Europe, were examined for hantavirus antibodies and antigen and subsequently subjected to reverse transcription-PCR (RT-PCR) and sequencing. Phylogenetic analysis divided all currently known TUL strains into two main branches. Analysis of either partial or
complete N-protein-encoding sequences or 3' noncoding region (3'-NCR)
sequences, as well as bootscanning revealed that the TUL lineage from
eastern Slovakia has characteristics of a recombinant hantavirus.
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MATERIALS AND METHODS |
Rodents.
In 1995, rodents were trapped by using bridge-type
metal traps in selected areas of Slovakia, regularly throughout the
year at three localities in the west (Malacky region and Danube
lowland) and once in the fall at eight localities in the east (Kosice
region). The trapping sites were selected on the basis of reported
human cases of suspected HFRS, where exposure to rodents was considered likely. For serological analysis, the blood was taken from the sinus
orbitalis of deeply anesthesized rodents; then the animals were
sacrificed and dissected for lung and liver tissues. Tissue samples for
antigen detection were stored at 
70°C; samples designated for RNA
extraction were stored in 4 M guanidinium thiocyanate buffer at the
same temperature.
Antibody screening.
The rodent sera were tested by
enzyme-linked immunosorbent assay (ELISA) for the presence of
hantavirus antibodies. For the detection of hantavirus-specific mouse
immunoglobulin G antibodies, an antiglobulin ELISA was established
(7). Briefly, microtiter plates (Nunc, Roskilde, Denmark)
were coated overnight at 4°C with N antigen
(Vranica-Hällnäs strain, amino acids 1 to 213; 100 µl/well) in 0.05 M sodium carbonate buffer, pH 9.0. The optimal antigen concentration was determined to be 20 ng/well. The plates were
washed five times after each step. Following postcoating with blocking
buffer (0.5% Tween 20-1% bovine serum albumin in phosphate-buffered
saline [PBS]) at room temperature for 1 h, rodent serum samples,
diluted 1:200 in PBS with 1% bovine serum albumin, were incubated for
1 h at room temperature and then for 1 h at 37°C with
peroxidase-labeled anti-mouse antibody (DAKO Diagnostica, Hamburg,
Germany) diluted 1:1,000 in 5% calf/sheep serum. Staining was
performed according to standard procedures (0.8 µg of
3,3',5,5'-tetramethylbenzidine hydrochloride/µl of substrate buffer;
Sigma, Munich, Germany). The reaction was stopped by addition of 2 M
sulfuric acid, and optical densities of the reaction products were
measured at 450 and 620 nm. Cutoff values were calculated as the mean
optical density value plus 3 standard deviations for values of negative controls.
Immunoblotting for antigen detection.
Rodent lung tissue
samples (2 to 3 mm3) were homogenized by sonification in
500 µl of Laemmli loading buffer; after denaturation, 15 µl of the
homogenate was loaded on a sodium dodecyl sulfate (SDS)-12%
polyacrylamide gel and separated by electrophoresis. After transfer of
the proteins, the membranes were preadsorbed in 4% nonfat dry milk and
subsequently incubated with rabbit polyclonal antibodies (raised
against TUL/Malacky recombinant N antigen expressed as a His-tagged
protein [55]) diluted in PBS-0.05% Tween 20. The
indicator antibody was a swine anti-rabbit horseradish peroxidase conjugate used at 1:1,000 dilution at 37°C for 1 h. Membranes were washed in PBS-0.05% Tween 20, and the bands were stained with
o-phenylenediamine dihydrochloride.
PCR and sequencing.
RNA of samples hantavirus-positive by
ELISA or immunoblotting was extracted from homogenized lung tissues by
the acid guanidinium thiocyanate-phenol-chloroform method
(6, 56). Hantavirus RNA was detected by RT-PCR with a
genus-reactive S-segment primer pair (S1-S2) as described earlier
(57). For nested PCR, the primers MaS4F (5'-CAT CAC AGG SYT
TGC ACT TGC AAT) and MaS5C (5'-TCC TGA GGC TGC AAG GTC AA), specific
for all known TUL sequences, were used to amplify a 0.5-kb product.
Amplification of the whole S segment was performed either by use of a
single genus-specific primer, complementary to 3' and 5' termini of the
S segment, as described earlier (43) or in two steps by a
combination of this primer with either primer SNMa1
(5'-ATGAGCCAACTCAAAGAAATA; amplifying a 1.8-kb product lacking 63 terminal nucleotides [nt]) or primer MaS4C (5'-CAAGATTATTGCAAT;
amplifying a 448-bp 5'-terminal S-segment region). The amplified
products were cloned into pCRII (TA cloning kit; Invitrogen, Leek, The
Netherlands). Dideoxy sequencing (53) was performed with an
ALF-Express sequencer and Autoread kit (Pharmacia-Biotech, Freiburg,
Germany) as described by the manufacturer.
TUL S-segment sequences from voles trapped in 1995 in the Czech
Republic (Moravia region near the town of Koziky) were also included in
the analysis. Antigen screening and RT-PCR were performed as described
earlier (40).
Sequence comparisons and phylogenetic analysis.
Nucleotide
and amino acid comparisons were calculated by use of DNAsis 2.1 (Hitachi Software). Sequence alignments for phylogenetic analysis were
generated with PILEUP from the Genetics Computer Group software package
(10). The following programs from the PHYLIP program package
(12) were used to construct phylogenetic trees. Two hundred
bootstrap replicates of the aligned sequences were generated by
SEQBOOT, distance matrixes were computed by DNADIST using Kimura's
two-parameter model (transition/transversion ratio of 2.0), and
phylogenetic trees were calculated by using the additive tree model of
the Fitch-Margoliash (FM) algorithm by FITCH with the global
rearrangement option set. From the obtained data, consensus trees were
constructed by CONSENSE, giving the occurrence ratios at particular
branchings. Bootscanning (51) of the full-length S-segment
nucleotide sequences was done by using the FM algorithm. One hundred
bootstrap replicates were calculated for 300-nt-long regions from the S
segment, with 150-nt overlap. The S-segment sequence of Isla Vista
virus (ILV) was used as an outgroup. Resulting bootstrap probabilities
of joining the Kosice sublineage with one of the other genetic TUL
sublineages (or with the outgroup) were obtained from CONSENSE.
Nucleotide sequence accession numbers.
The sequences of the
complete S segments of TUL strains Kosice/144Ma/95, Kosice/667Ma/95,
Moravia/Koziky/5247Ma/94, and Moravia/Koziky/5276Ma/94 have been
assigned EMBL accession no. Y13979, Y13980, AJ223600, and AJ223601,
respectively. The 458/455-nt partial S-segment sequences of TUL strains
Kosice/676Ma/95, Malacky/715Ma/95, Danube lowland/539Ma/95, and Danube
lowland/540Ma/95 were deposited under EMBL accession no. Y13981,
Y13982, Y13983, and Y13984, respectively.
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RESULTS |
Rodent screening for hantavirus antibodies, antigens, and RNA.
Out of a larger collection of rodents (n = 580)
captured in 1995 regularly throughout the year in western Slovakia and
once in the fall in eastern Slovakia, 106 animals were identified as European common voles of the species M. arvalis. The
proportion of animals of this species among all rodents trapped was
found to be higher in eastern (65/147 [38%]) than western (50/433
[12%]) regions of Slovakia; however, the selected trapping sites had a profound influence on the rodent species composition. The numbers of
M. arvalis are known to fluctuate drastically from year to year (24). Thus, not surprisingly, the frequency of M. arvalis in 1995 was twice as high as found in a previous study,
performed in 1991 to 1992 in the same region in western Slovakia, where about 6% of all rodents trapped were common voles (23).
All M. arvalis specimens from which sera were available
(n = 62) were tested by ELISA for hantavirus
antibodies; the remaining samples (n = 44) were tested
for hantavirus antigen by immunoblotting. A total of seven specimens
were found to be hantavirus antibody (n = 6) or antigen
(n = 1) positive. In six of the seven antibody- or
antigen-positive samples, hantavirus genomes could be detected by
RT-PCR: four samples were positive after first-round PCR, amplifying an
890-bp product (nt 376 to 1265), and two additional RNA-positive samples were detected by the use of nested PCR primers (Table 1).
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TABLE 1.
TUL strains detected by antibody or antigen screening and
RT-PCR in European common voles (M. arvalis) trapped
in Slovakia
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Three of the six RNA-positive samples originated from voles trapped at
two localities (Malacky region and Danube lowland;
distance of about 70 km) in western Slovakia, while the three
others originated from voles
collected at two localities (Kosice
region) in eastern Slovakia (about
50 km away). The distance between
trapping localities in western and
eastern Slovakia was about
350 km (Fig.
1).
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FIG. 1.
Map of Slovakia and neighboring countries in central
Europe. The trapping places of TUL-positive M. arvalis,
including two distinct localities each in Moravia (Tv, Tvrdonice; Ko,
Koziky) and near Kosice (Op, Opinia; Bo, Botany), are marked.
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The trapping site in western Slovakia near Malacky was identical with
the previous one, where two antigen-positive voles (Ma32
and Ma370)
were collected in the spring and late fall of 1994
and identified to
carry TUL/Malacky sequences (
57).
Sequence analysis of the partial S segment (nt 441 to 898).
The nucleotide sequence of the partial S segment was analyzed to
evaluate whether this genomic region could be representative for the
whole N-protein-encoding sequence, which would facilitate primary
characterization of wild TUL strains. For this purpose, we determined
the sequences of the nested PCR products of the six hantavirus
RNA-positive specimens. The analyzed S-segment region comprises
sequences that encode conserved N protein regions (amino acids [aa]
134 to 210), as well as a variable region (aa 244 to 273) that was
found to be most divergent in all hantavirus serotypes (41).
As shown in Fig. 2, the aligned TUL
N-protein sequences display a gap of three amino acids (boxed in the
sequence alignment in Fig. 2) compared with the most closely related
virus types Prospect Hill (PH), ILV, and PUU. Moreover, four amino
acids from the same region are deleted in HTN and SEO, and five are deleted in SN, as described earlier (41). Within the genetic group TUL, strains from central Europe (Slovakia and Czech Republic) can be usually distinguished from Russian strains by an additionally deleted nucleotide triplet, leading to deletion of Ser-252 (see also
references 40 and 57). At
position 252 the N protein of the eastern Slovakian virus variants
(TUL/Kosice) is not deleted; however, there is an amino acid exchange
of Ser-252 to Ala. Interestingly, this substitution is also found in
one of the currently known Russian TUL strains (Fig. 2).
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FIG. 2.
Schematic representation of the hantavirus S segment.
The N-protein-encoding sequence (ORF1) and the hypothetical ORF2 are
marked. Black boxes, 5'- and 3'-NCRs; gray box, region of S segment
selected for partial sequence analysis (Table 2; Fig. 3); *, Russian
strains T76, T249, and T175;  , eastern Slovakian strains K144, K667,
and K676;  , western Slovakian strains Ma32, Ma370, and Ma715; §,
Czech Republic strains M86, M93, M94, M02, and Mko76.
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Comparisons of paired partial S-segment nucleotide and deduced amino
acid sequences of hantavirus RNA-positive strains from
Slovakia and all
other known TUL strains (
40,
41,
57) were
performed.
Sequence alignments of partial S segments of TUL strains
from local
rodent populations in western Slovakia and the Czech
Republic show
nucleotide and deduced amino acid divergences of
0 to 2.4% and 0 to
1.3%, respectively. Sequence comparisons between
two strains from a
local rodent population in the Kosice region
show a nucleotide
divergence of 7% (all reflecting silent mutations),
which is as high
as the divergence found between Russian TUL strains,
all originating
from the same local population (1.8 to 8.1%).
However, compared to
TUL/Kosice, TUL/Tula strains display a higher
variability in this
N-protein region (amino acid differences 0
to 3.3%), probably due to
the greater number of strains analyzed
and the larger trapping area
(
41).
Although the Kosice region is geographically closer to western Slovakia
and the Czech Republic than to Russia, the nucleotide
divergences
between TUL/Kosice on the one hand and strains from
western Slovakia
(TUL/Malacky and TUL/Danube lowland) and the
Czech Republic
(TUL/Moravia) on the other were in ranges (18.1
to 19.4% and 18.1 to
19.6%, respectively) similar to that of the
nucleotide divergence
between TUL/Kosice and TUL/Tula strains
(17.0 to 18.8%). The
corresponding amino acid differences between
TUL/Kosice and TUL/Tula as
well as between TUL/Kosice and strains
from western Slovakia and
Moravia range from 2.0 to 3.3%. The
highest amino acid divergence (4.6 to 6.0%) was observed between
strains from Russia on the one side and
those from western Slovakia
and Moravia on the
other.
Analysis of the complete S segment of TUL strains.
Total
S-segment sequences amplified from voles from the two locations in
eastern Slovakia (K144 and K667) were determined to enable comparisons
of the whole N-protein-encoding sequence, as well as of 3'-NCR
sequences (see below). The S segment of each strain from eastern
Slovakia has a total length of 1,833 nt, including 42 nt of 5'-NCR, an
open reading frame (ORF1) encoding an N protein of 430 aa, a second,
internally overlapping ORF2 (+1 frame) encoding a hypothetical 90-aa
nonstructural protein (41), and a 501-nt-long 3'-NCR.
By comparison of nucleotide and amino acid divergences of the N gene
and N protein, respectively, three groups of TUL strains
could be
distinguished: (i) strains from eastern Slovakia (2.2%
nucleotide and
no amino acid differences), (ii) strains from the
Malacky region in
western Slovakia, including those from Moravia
in the Czech Republic
(0.3 to 4.6% nucleotide and 0 to 1.0% amino
acid diversities), and
(iii) strains from the Tula region in Russia
(1.5 to 5.6% nucleotide
and 0.5 to 1.4% amino acid diversities).
The differences between the
first and second groups are in the
ranges of 14.6 to 15.7%
(nucleotide) and 1.4 to 2.2% (amino acid);
the differences between the
first and third groups are in the
same ranges (14.4 to 15.0% and 1.7 to 2.4%, respectively). The
second and third groups differ 15.5 to
16.5% in nucleotide and
2.9 to 4.3% in amino acid
sequences.
Phylogenetic analysis based on N-protein-encoding
sequences.
We have constructed a phylogenetic tree by using
partial S-segment sequences (nt 441 to 898) comprising all known
strains of the TUL genetic group and selected hantaviruses of other
genetic groups (Fig. 3). TUL strains
cluster into two main branches: strains from Russia and eastern
Slovakia (branch I) and strains from western Slovakia and eastern Czech
Republic (branch II). Within branch I, eastern Slovakian strains are
well separated from Russian strains (bootstrap probability of 74%).
Within branch II, strains from the Danube lowland are located closer to
strains from Moravia (Czech Republic) than to those from the Malacky
region; the latter form a well-supported sublineage (bootstrap
probability of 100%). The phylogenetic tree based on complete
N-protein-encoding S-segment sequences (not shown) essentially display
the same branching order for TUL strains as computed for the tree based
on the partial S-segment sequences. The closest relatives of TUL
strains are PH and ILV; both viruses occur in Microtus
species in North America (28, 58).
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FIG. 3.
Phylogenetic consensus trees based on partial (nt 441 to
898) N-protein-encoding sequences. Numbers indicate bootstrap values at
the nodes. HTN, Hantaan virus, strain 76-118 (accession no. M14626)
(54); SEO, Seoul virus, strain SR-11 (M34882)
(1); DOB, Dobrava virus (L41916) (2); BCC, Black
Creek Canal virus (L39943) (47); BAY, Bayoo virus (L36929)
(34); SNcc, Sin Nombre virus, strain Convict Creek 107 (L33683) (29); ELMC, El Moro Canyon virus, strain RM-97
(U11427) (17); KBR, Khabarovsk virus, strain MF-43 (U35255)
(20); PUU/bash, Puumala virus, strain Bashkiria/CG1820
(M32750) (60); PUU/vind, Puumala virus, strain
Vindeln/L20Cg/83 (Z48586) (19); PUU/vran, Puumala virus,
strain Vranica/Hällnäs (U14137) (48); PUU/sot,
Puumala virus, strain Sotkamo (X61035) (62); PUU/9013,
Puumala virus, strain Paris-9013 (U22423) (18); ILV, Isla
Vista virus, strain MC-SB-1 (U31534) (58); PH, Prospect Hill
virus (M34011) (38); TUL/T249, Tula virus, strain
Tula/249Mr/87 (Z30944), (41); TUL/T76, Tula virus, strain
Tula/76Ma/87 (Z30941) (41); TUL/175, Tula virus, strain
Tula/175Ma/87 (Z30943) (41); TUL/T23, Tula virus, strain
Tula/23Ma/87 (Z30945) (41); TUL/T53, Tula virus, strain
Tula/53Ma/87 (Z30942) (41); TUL/K667, Tula virus, strain
Kosice/667Ma/95 (this paper); TUL/K144, Tula virus, strain
Kosice/144Ma/95 (this paper); TUL/K676, Tula virus, strain
Kosice/676Ma/95 (this paper); TUL/Ma32, Tula virus, strain
Malacky/Ma32/94 (Z48234) (57); TUL/Ma370, Tula virus, strain
Malacky/Ma370/94 (U31534) (57); TUL/Ma715, Tula virus,
strain Malacky/715Ma/95 (this paper); TUL/D539, Tula virus, strain
Danube lowland/539/Ma/95 (this paper); TUL/D540, Tula virus, strain
Danube lowland/540/Ma/95 (this paper); TUL/M02, Tula virus, strain
Moravia/5302Ma/94 (Z49915) (40); TUL/M94, Tula virus, strain
Moravia/5294Ma/94 (Z48741) (40); TUL/M93, Tula virus, strain
Moravia/5393Ma/94 (Z48574) (40); TUL/M86, Tula virus, strain
Moravia/5286Ma/94 (Z48573) (40); TUL/MKo47, Tula virus,
strain Moravia/Koziky47/Ma/95 (this paper); TUL/MKo76, Tula virus,
strain Moravia/Koziky76/Ma/95 (this paper).
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Analysis of TUL 3'-NCR sequences.
Comparison of 3'-NCR
sequences of the eastern Slovakian strains with those of other TUL
strains yielded nucleotide identity of about 87 to 94%, which is even
higher than the values for these strains in the coding region (about
85%). In the TUL sequences, only a few deletions had to be introduced
for complete alignment (Fig. 4), and a
consensus sequence was easily derived for the entire 3'-NCR. The
genetic stability of the 3'-NCR within the TUL group is remarkable,
since, for example, the genetic diversity of the 3'-NCR sequences of
the PUU type is as high as 30% (33).
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FIG. 4.
Properties of the 3'-NCR of TUL S segment. Positions of
small deletions/insertions (1 to 3 nt) are shown by vertical lines; the
longest deletion (16 to 18 nt) is shown by a gray rectangle. Black
squares, strains T23, T53, T76, T175, and T249; white squares, strains
T23 and T175; black triangles, TUL/Moravia and TUL/Malacky strains;
diamonds, TUL/Moravia, TUL/Malacky and TUL/Kosice strains; white
triangles, sites of 1-nt insertion/deletion of either T249, T175, T76,
K144, or M86. The bottom part represent a blowup of the longest
deletion. The alignment of nucleotide sequences (minus sense) of all
presently known TUL strains is shown.
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Surprisingly, the 3'-NCR of eastern Slovakian strains exhibit a higher
nucleotide homology to western Slovakian and Czech
strains (~94%)
than to the Russian strains (~89%). Moreover, according
to the
pattern of deletions and insertions in the 3'-NCR, strains
from eastern
Slovakia are closer to those from western Slovakia
and Moravia than to
strains from Russia (Fig.
4). For example,
the longest deletion of 16 to 18 nt occurs in all strains from
central Europe but not in Russian
strains. These findings agree
with the results of the phylogenetic
analysis of the 3'-NCR sequences.
On the related tree, Kosice strains
cluster with strains from
Moravia and western Slovakia (bootstrap
values of 95% at the corresponding
node) and have a common ancestor
(Fig.
5). It should be mentioned
that ILV
was chosen as the outgroup for the phylogenetic analysis
of the 3'-NCR,
because the S-segment 3'-NCRs of different hantavirus
types are so
highly diverged that they cannot be reliably aligned
(
45).
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FIG. 5.
Phylogram showing FITCH consensus tree based on TUL
3'-NCR sequences, using ILV as the outgroup. ILV was chosen as the
outgroup because the S-segment 3'-NCRs of different hantavirus types
are so highly diverged that they cannot be reliably aligned. Bootstrap
values are indicated at the nodes. For abbreviations of strain
designations, see the legend to Fig. 3.
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Thus, the phylogenetic position of the eastern Slovakian (Kosice)
lineage depends on which part of the S segment is selected
for the
analysis. On closer scrutiny, the S sequences from Kosice
strains
possess a combination of markers from both genetically
distinct TUL
lineages: the triplet encoding aa 252 is present
as in all strains from
the Russian lineage (Fig.
2), and the deletion
of nt 1595 to 1610 within the 3'-NCR conforms to all strains from
Moravia and western
Slovakia (Fig.
4). S segments of Kosice strains
could therefore be
considered
recombinant.
Search for recombination sites within the S segment by
bootscanning.
To identify possible recombination sites with higher
accuracy, bootscan analysis (51, 52) of TUL S-segment
sequences was performed. Phylogenetic trees were calculated for 300-nt
regions with 150-nt overlap, using the FM algorithm. The resulting
bootstrap probabilities for placing the Kosice sequences with either of the two genetic lineages (or with the outgroup, ILV) are shown in Fig.
6a. Three peaks exceed the 70% FM
bootstrap value defined as the cutoff level (15, 52). This
suggests at least two recombination points in the S sequences of Kosice
strains. The first point is located around nt 350 to 400 between peak 1 (Malacky/Moravia-like S-segment region) and peak 2 (Tula-like). Since
the region between nt 650 and 1200 is less well resolved, the
localization of corresponding recombination points is more difficult.
However, since the third peak above the FM cutoff level groups Kosice
with Malacky/Moravia-like sequences with high bootstrap values (nt
1200, 72%; nt 1350, 96%; nt 1500, 90%), at least one other
recombination point should be located before nt 1200. Within the less
resolved region, two peaks are under the FM bootstrap cutoff level, the
first (Malacky/Moravia-like) located between nt 670 and 770 and the
second (Tula-like) located around nt 860. Bootstrap probabilities
calculated by using a neighbor-joining algorithm (PHYLIP) are slightly
lower but result in the same pattern (data not shown).
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FIG. 6.
Results of bootscanning of the S segments of Kosice
strains and schematic representation of TUL N-protein sequence
variation. (a) Bootscan searches of 300-nt S-segment regions with
150-nt overlap, using the FM algorithm. Bootstrap probabilities of
joining Kosice strains with the Malacky/Moravia lineage, the Tula
lineage, or the outgroup (ILV) are represented by a solid line, dots,
and a dashed line, respectively. The 70% bootstrap cutoff level
(15, 52) is indicated by a thin dashed line. (b) Amino acid
sequence variation of N proteins of all TUL strains. Sites where TUL
strains differ in the N-protein sequence are indicated under the black
bar, representing the N-protein-encoding sequence in the center of the
figure; vertical numbers denote codon positions. Amino acid residues
identical in Kosice and other TUL strains are boxed; horizontally
hatched rectangles mark residues identical in the N proteins of
Malacky/Moravia and Kosice strains, vertically hatched rectangles
indicate residues identical in N proteins of Tula and Kosice strains.
(c) Locations of Malacky/Moravia-like (horizontally hatched) and
Tula-like (vertically hatched) parts in the TUL/Kosice S segment.
Recombination points identified by bootscanning (above 70% FM values)
are indicated by black arrows; the recombination point deduced from aa
pattern is indicated by a white arrow.
|
|
These observations are in good correlation with the pattern of amino
acid substitutions in the N protein (Fig.
6b), which
are also
consistent with multiple recombination events. Based
on this pattern,
the first recombination point may be placed between
codons 121 and 127 (nt 406 to 421), i.e., close to the region
selected by bootscanning.
The central part of the sequence is
again less resolved, while the end
of the coding region and the
3'-NCR (nt 1200 until the end) obviously
belong to the Moravia
sublineage, suggesting at least one more
recombination event.
Although the central region was not well resolved
by bootscanning,
within the highly variable N-protein region of Kosice
strains,
the Tula-like amino acids (codons 250 to 254; nt 790 to 804)
are
immediately followed by Malacky/Moravia-like residues (codons
258 to 268; nt 814 to 846), indicating the existence of a third
putative
recombination point. Taken together, these data are consistent
with a
mosaic-like structure of Kosice S-segment sequences due
to several
recombination events (Fig.
6c).
| |
DISCUSSION |
Genetic diversity of TUL strains.
This study was performed to
characterize the biodiversity of TUL nucleotide and amino acid
sequences in Slovakia as a defined geographical region. For the
surveillance in such regions, it is useful to monitor wild-type
hantaviruses as potential human pathogens. We have concentrated our
efforts on TUL strains which have recently been shown to circulate in
eastern and central Europe (40, 41, 57). Although a definite
proof of their pathogenicity for humans is lacking, there is some
serological evidence that TUL strains can infect humans
(64). In addition, we were interested in the molecular
biodiversity and evolutionary links between TUL strains as an example
of an RNA virus dependent on a specific animal reservoir.
In search of animals carrying hantavirus sequences, we used antibody
detection in sera and antigen verification in lung tissues
for the
screening of voles. Subsequently hantavirus genomes were
detected by
RT-PCR specific for a 455- to 458-bp TUL S-segment
region suitable for
fast sequence characterization. Hantavirus
genomes were detected in
84% of the antibody-positive samples.
However, serological screening
of rodent specimens remains the
method of choice to screen for
hantavirus in rodent populations
(
21,
37).
The analyzed partial S-segment sequence (nt 441 to 889) comprises a
highly variable N-protein sequence (aa 245 to 284) flanked
by highly
conserved regions. Serological results support the inclusion
of the
variable part of the sequence into this analysis, since
it carries
important markers. The corresponding region in PUU
was shown to carry
B-cell epitopes recognized by antibodies from
bank voles and humans
(
31,
63). Moreover, two TUL-specific
monoclonal antibodies
(3D3 and 3F10) raised against baculovirus-expressed
TUL N protein were
recently described; the epitopes of both antibodies
were mapped within
aa 226 to 293 (
32).
Analysis of both partial and complete N-protein-encoding S-segment
sequences disclosed an inverse correlation between genetic
similarity
and geographical distance of virus detection, as previously
demonstrated for other hantavirus types (
22,
42,
43,
59).
Essentially three groups of TUL strains could be identified by
sequence
comparisons: (i) the originally described strains from
the Tula region
in the European part of Russia; (ii) strains from
western Slovakia
(Malacky and Danube lowland) and the Czech Republic
(Moravia), and
(iii) Kosice strains from eastern Slovakia. The
genetic differences
found between these groups are in the same
range as those between PUU
strains originating from Finland, Sweden,
and France. Using sequence
data reported by Vapalahti et al. (
62),
Stohwasser et
al. (
60), Reip et al. (
48), Hörling et al.
(
19), and Bowen et al. (
5), we calculated
nucleotide divergences
of 14.1 to 22.9% by paired comparisons of
partial S-segment (nt
441 to 898) and 14.4 to 17.3% for complete
N-protein-encoding
sequences between the PUU strains Sotkamo,
Bashkiria, Vranica-Hällnäs,
Vindeln, and Paris 90-13.
Phylogenetic analyses of the partial S-segment (Fig.
3) and complete
N-protein-encoding (not shown) sequences demonstrate
a clear change
within the TUL genotype from east to west: virus
strains from Russia
and eastern Slovakia form TUL branch I on
the phylogenetic tree,
whereas strains from western Slovakia and
the Czech Republic are
present on TUL branch II. The bootstrap
values for placing Kosice
strains on TUL branch I are 73 and 69%
for the partial and complete
N-protein-encoding sequences, respectively.
Although these values are
not very high, they are still in the
significant range, since bootstrap
values of
70% have been demonstrated
to represent true clades in
over 95% of cases (
15). Based on
these analyses, TUL/Kosice
strains seem to be more closely related
to geographically more
distantly located Russian strains (distance
of about 1,500 km) than to
strains from Malacky/Moravia regions
(distance of about 350
km).
The 3'-NCR sequences of all TUL strains are highly conserved: a
consensus sequence could easily be generated by insertion
or deletion
of only a few nucleotides in the individual sequences
(Fig.
4). The
greater conservation of the S-segment 3'-NCR (87
to 94%) than of the
coding region (85%) is a remarkable feature
that has not been reported
for other hantavirus types studied.
Within the PUU type, for example,
the genetic diversity of the
S-segment coding region is 20% (
5,
45), while that of the
3'-NCR is 30% (
33). This
feature of TUL enables the performance
of phylogenetic analysis of high
accuracy and reproducibility
of this part of the
genome.
While this report was in the final stages of preparation, we learned of
the presence of TUL strains in Austria. Bowen et al.
(
4)
reported two genetically distinct variants of TUL virus
in Austrian
rodents (
M. arvalis) trapped at two locations, Korneuburg
and Wels, 65 and 235 km west of the Malacky region (Slovakia).
The
sequences analyzed by these authors cover and extend (by approximately
100 nt on both sides) the partial S segment analyzed in our study.
Like
strains from western Slovakia and Moravia, the Austrian TUL
strains
display the 3-nt deletion resulting in a loss of alanine
or serine at
position 252 in the N protein of Russian TUL strains.
Phylogenetic
analysis demonstrated that the Austrian Korneuburg
strains are closely
related to Malacky strains, while Wels strains
represent a genetically
distinct variant of TUL in Austria (
4),
presumably located
on TUL branch II, according to our arrangement
(Fig.
3).
Recombination in TUL evolution.
The apparent contradiction in
phylogenies based on the 3'-NCR and on the N-protein-encoding region
(either partial or complete) could be explained by genetic
recombination events during the evolution of the eastern Slovakian
genetic lineage of TUL. This hypothesis was confirmed by bootscanning
and by analyses of amino acid substitutions in the N protein as well as
the pattern of deletions and insertions in the 3'-NCR (Fig. 6).
Intragenic recombination is one of the well-documented mechanisms of
evolution of positive-strand RNA viruses (for a review,
see reference
25). Double-stranded RNA viruses have been reported
to undergo nonhomologous recombination (
9). In addition,
this
mechanism has been described for a negative-strand segmented RNA
virus, the influenza virus (
3,
36). Our findings raise
several
questions, of which the most important are (i) the necessary
prerequisites
for recombination in hantaviruses and (ii) the impact of
recombination
for hantavirus evolution. The essential prerequisite of
recombination
is the coexistence of two distinct RNA molecules in the
same infected
cell. A classical example is human immunodeficiency
virus, with
its diploid genome highly capable of recombination
(
8). Hantaviruses
cause persistent infection in their rodent
hosts, creating the
opportunity for coinfection with different virus
strains. For
segmented RNA viruses, the most likely result of such an
event
is genomic reassortment, as demonstrated for other members of
the
Bunyaviridae family (
46). For wild-type
hantaviruses, a
few cases of reassortment have also been reported
(
14,
29).
In addition, it was shown recently that during
mixed infection
of cells in culture with two distinct SN virus
isolates, 20% of
the virus plaques contained S or M segments
originating from both
parental viruses (i.e., diploidy)
(
49). Thus, two distinct types
of hantavirus genome segments
can coexist in infected cells enabling
recombination.
Another prerequisite for recombination is the presence of two different
hantavirus lineages in the same geographical region.
For Swedish PUU
strains, a clustering of genetic variants was
shown to be associated
with two different mitochondrial DNA lineages
of bank voles that have
recolonized the territory postglacially
from different directions
(
21). Rodents from northern and southern
Sweden were
associated with genetically distinct PUU lineages.
Within a contact
zone of only 50 km, both the northern and the
southern vole populations
coexisted. Similarly, coexistence in
the same geographical area of
rodents carrying different genetic
subtypes of Sin Nombre virus has
been demonstrated in Nevada and
eastern California (
50). A
study of phylogenetic relationships
within
M. arvalis in
Europe based on phenotypic classification
into different
M. arvalis sublineages postulated a postglacial
remigration of
M. arvalis from three directions into the region
of former
Czechoslovakia:
M. arvalis arvalis from the west,
M. arvalis levis from the south, and
M. arvalis duplicatus
from the
east (
24). To analyze whether the Kosice region
represents a
contact zone for different genetic
Microtus
subspecies, trapped
voles will be genetically characterized. Moreover,
since the prerequisites
for recombination and reassortment are
essentially the same, analyses
of M- and L-segment sequences of the
Kosice lineage may reveal
a correlation between these two evolutionary
processes.
Whether the recombinant S segment of Kosice lineage is of recent
evolutionary origin remains unclear. The first recombinant
point
located within a highly conserved part of the N-protein-encoding
region
(nt 400 to 415) was unequivocally defined by bootscanning,
whereas
recombination point(s) in more variable parts of the coding
region
could not be conclusively mapped. Assuming that recombination
is a rare
event, one could argue that the low resolution of recombination
points
in the divergent region reflects further changes due to
genetic drift
and hence that recombination in Kosice S segment
was not a recent
event. This also leads us to the assumption that
the third peak in the
bootscanning (around nt 800), which corresponds
to the Tula-like
N-protein region (Q-250, A/S-252, G-254), might
originate from either
the Tula or Malacky/Moravia lineage or a
yet unknown TUL lineage (Fig.
6b and
c).
The data obtained for the Kosice sublineage of TUL represent the first
strong case of potential genetic recombination within
the genus
Hantavirus and, in fact, the first case of homologous
recombination in segmented negative-strand viruses. Although this
mechanism was proposed previously for PUU evolution (
44),
limited
sequence data prevented clarification of the issue. The role of
recombination in hantavirus evolution deserves further study.
However,
it seems safe to assume that this mechanism plays a minor
role compared
to the classical genetic drift, i.e., accumulation
of point mutations
and deletions/insertions. Genetic shift, the
reassortment of genomic
segments, seems to be another driving
force. Nevertheless, since the
basic prerequisites, coexistence
of different virus variants in the
same geographical area and
coinfection of the same rodent, are the same
for both reassortment
and recombination, we expect the Kosice lineage
to be a good model
for further studies of hantavirus
evolution.
| |
ACKNOWLEDGMENTS |
The technical assistance of Elsa Nugel and Pia Reiser (Berlin,
Germany) and the technical assistance of Yin Cheng (Helsinki, Finland)
in sequencing are greatly appreciated. We are obliged to Mika Salminen
for advice on the bootscan analysis.
This project was supported by the Deutsche Forschungsgemeinschaft (Kr
1293/2) and Volkswagen Foundation and Fonds der chemischen Industrie,
Germany; Slovak grant agency (VEGA 95/5305/361), Slovakia; and grants
from the Academy of Finland and The Sigrid Jusélius Foundation,
Helsinki, Finland.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Virology, Charité School of Medicine, Humboldt University,
Schumannstr. 20/21, D-10117 Berlin, Germany. Phone: 49-30-2802 2387. Fax: 49-30-2802 2180. E-mail: detlev.kruger{at}charite.de.
| |
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Journal of Virology, January 1999, p. 667-675, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
