32 The Open Vi ology Jou nal, 2008, 2, 32-36
1874-3579/08 2008 Ben ham Science Publishe s L d.
New Gene ic Lineage o Tula Han a i us in Mic o us a alis obscu us in
Eas e n Kazakhs an
Angelina Plyusnina1, Juha Laakkonen2,§, Jukka Niemimaa2, Heikki Hen onen2 and
Alexande Plyusnin*,1
1Depa men o Vi ology, Haa man Ins i u e, Uni e si y o Helsinki, Finland
2Finnish Fo es Resea ch Ins i u e, Van aa esea ch Uni , Finland
§Cu en Add ess: Di ision o Ve e ina y Ana omy Depa men o Basic Ve e ina y Medicine P.O. Box 66, 00014
Uni e si y o Helsinki, Finland
Abs ac : Genomic sequences o Tula (TULV) han a i us we e eco e ed om issue samples o Eu opean common
oles Mic o us a alis (subspecies obscu us) cap u ed in Kazakhs an, Cen al Asia. Phylogene ic analysis o he S ge-
nomic segmen o Kazakh TULV s ains showed ha hey o m dis inc , well suppo ed gene ic lineage and sha e a mo e
ancien common ances o wi h wo Russian lineages o TULV. The deduced sequence o he nucleocapsid (N) p o ein o
Kazakh TULV s ains ca ied speci ic amino acid signa u e: T274Q276T281. The Mic o us a alis g oup includes se e al
sibling species and/o subspecies in Eu asia, indica ing ecen and ongoing e olu iona y adia ion. Ou da a on TULV
lineages in Cen al Asia, he egion no s udied o han a i uses ea lie , highligh he di e si y o bo h Mic o us hos and
he i us and also hei co-e olu ion.
INTRODUCTION
Han a i uses (genus Han a i us, amily Bunya i idae a e
nega i e-s and RNA i uses wi h a ipa i e genome [1].
Cu en ly he genus consis s o a leas 22 dis inc han a i us
species, each hough o be ca ied by a speci ic o closely
ela ed oden o insec i o e hos . Some han a i uses, e. g.
Han aan and Sin Nomb e i uses, a e se e e human pa ho-
gens, while o he s, e. g. Tula i us (TULV), a e apa hogenic
[2]. TULV was disco e ed in ea ly 1990s, i s as a sequence
eco e ed om a issue sample o Eu opean common ole,
Mic o us a alis [3]. La e i has been isola ed in cell cul u e
[4], cha ac e ized, bo h gene ically and an igenically [5-7]
and since hen used as a sa e model o s udy han a i us mo-
lecula o ganiza ion and eplica ion [7-11], han a i us-hos
in e ac ions [7, 11-19] as well as han a i us gene ics and
e olu ion [20-23].
A e i s ini ial disco e y in Cen al Russia (Tula egion,
app oxinma ely 100 km sou h o Moskow [3]), TULV was
epo ed om se e al Eu opean coun ies: Czech Republic
[5, 20], Slo akia [22], Aus ia [24], Belgium [25], Se bia
[26], C oa ia [27]; Ge many [28], Poland [29], F ance and
Swi ze land [23]. The e a e also unpublished da a ( ou S
segmen sequences deposi ed o he GenBank) on TULV
om Omsk (Wes Sibe ia); so a , his has been he only
example o TULV ou side Eu ope. He e we p esen ou ind-
ings on TULV in Eas e n Kazakhs an, Cen al Asia, he e-
gion no s udied o han a i uses ea lie .
*Add ess co espondence o his au ho a he Depa men o Vi ology,
Haa man Ins i u e, Uni e si y o Helsinki, P.O. Box 21 (Haa maninka u 3),
FI-00014 Finland; Tel: +358-9-19126486; Fax: +358 9-19126491; E-mail:
alexande .plyusnin@helsinki. i
MATERIALS AND METHODOLOGY
Roden Tissue Samples
The Mic o us a alis g oup is axonomically a di icul
one, and he names and species s a us o axa ha e o en
changed. He e we ollow Wilson and Reede , 2005 [30].
When necessa y, we also gi e he o me ly used scien i ic
names. Al oge he , 168 Mic o us oles we e apped in ou
main egions in Eas e n Kazakhs an be ween Ap il 15 and
May 3, 2003. These included 128 M. a alis obscu us, 38 M.
socialis, and wo M. oeconomus. The apping was done in
su oundings o he ci ies Taldyko gan and Bakanas, on
oo hills up o he al i ude 1200 m o Dzunga ian Moun ain
ange eas o Taldyko gan, and close su oundings o Ka a al
plague ield s a ion. The de ails o appings, s udy si es,
oden species and numbe s cap u ed, e c, a e gi en in Hen -
onen e al. (in p epa a ion). B ie ly, animals cap u ed wi h
snap aps o e nigh we e placed in a cold box, ans e ed o
he labo a o y and kep cold un il dissec ed in he same day.
Tissue samples om lung, kidney and spleen we e ixed in
RNAla e eagen (Ambion, Applied Biosys ems) Du ing he
apping ime nigh s we e s ill cold, e en os y, and oden s
we e in good shape when dissec ed. The RNAla e - ixed
lung issue samples we e sc eened by immunoblo ing o he
p esence o han a i al nucleocapsid (N) p o ein an igen (Ag)
as desc ibed be o e [20]. B ie ly, he lung issue samples
(app oxima ely 100mg) we e homogenized by sonica ion in
500 mkl o Laemmli sample bu e . Aliquo s o 10 μl we e
sepa a ed by elec opho esis in 10% sodium dodecyl sul-
pha e-polyac ylamide gel and hen blo ed wi h abbi poly-
clonal an ibody aised agains ecombinan Puumala han a i-
us N p o ein. Swine an i- abbi an ibodies conjuga ed wi h
he ho se adish pe oxidase (Dako, Glos up, Denma k) we e
used as seconda y an ibodies.
Tula Han a i us in Kazakhs an The Open Vi ology Jou nal, 2008, Volume 2 33
Re e se T ansc ip ion - Polyme ase Chain Reac ion (RT-
PCR) and Sequencing
RNA was ex ac ed om N-Ag-posi i e lung issue sam-
ples using he T iPu e RNA isola ion sys em (Beh inge
Maannheim) ollowing he manu ac u e 's ins uc ions. PCR-
amplicon co esponding o comple e TULV S segmen se-
quence was p epa ed as desc ibed ea lie [3]. This p oduc
was cloned using pGEM-Teasy cloning sys em (P omega,
Madison, WI) and sequenced au oma ically using he ABI
PRISMTM Dye Te mina o o M13F and M13R Dye P ime
sequencing ki s (Pe kin Elme /ABI, NJ). Pa ial TULV S
segmen sequences we e ob ained by RT-nes ed PCRs. PCR-
amplicons we e gel-pu i ied using QIAquick Gel Ex ac ion
ki (QIAGEN) and sequenced au oma ically using ABI
PRISMTM Dye Te mina o sequencing ki .
Phylogene ic Analysis
Mul iple nucleo ide alignmen s we e p epa ed manually
using he SeqApp 1.9a169 sequence-edi ing p og am. Phylo-
gene ic analysis was pe o med using he PHYLIP p og am
package [31] and T eePUZZLE [32]. In PHYLIP, 500 boo -
s ap eplica es (SEQBOOT p og am) we e ed o he dis-
ance ma ice algo i hm (DNADIST, wi h he ML model o
nucleo ide subs i u ions), dis ance ma ices we e analyzed
wi h he Neighbo -joining (NJ, NEIGHBOR) o Fi ch-
Ma goliash (FM, FITCH) ee- i ing algo i hm; he boo -
s ap suppo alues we e calcula ed wi h he CONSENSE
p og am. In T eePUZZLE, he Hasegawa-Kishino-Yano-85
model was used wi h 10,000 puzzling s eps; base equencies
we e es ima ed om he da ase s. Han a i us sequences used
o compa ison we e eco e ed om he GenBank.
RESULTS
T apping o Roden s and Sc eening o Roden Tissue
Samples
In he connec ion o a join Eu opean Union suppo ed
esea ch p ojec on plague dynamics [33], also he ma e ial
on oden -bo ne i uses was collec ed in eas e n Kazakhs an
in sp ing 2003. De ails o apping and gene al esul s o
sc eening o se e al oden -bo ne i uses will be published
elsewhe e (Hen onen e al., in p epa a ion). One hund ed
wen y eigh Mic o us a alis oles we e i s sc eened by
immunoblo ing o he p esence o han a i al N-Ag and 20
we e ound posi i e. O hose, 15 we e om he su ound-
ings o Taldyko gan, 1 om oo hills o Dzunga ian Range,
and 4 om Ka a al. In addi ion we had 38 Mic o us socialis
oles, 2 o which we e weakly N-Ag -posi i e. Two M.
oeconomus we e N-Ag-nega i e. Nex , he N-Ag-posi e
samples we e analyzed using RT-PCR and han a i al ge-
nomic sequences (S segmen ) we e eco e ed om ou o
hem (all ou we e M. a alis obscu us).
Gene ic Cha ac e iza ion o Han a i al Sequences
Since he PCR-amplicon co esponding o comple e i al
S segmen sequence (1830 nucleo ides, n ) was success ully
ob ained om only one sample (#322 om Ka a al), a a i-
e y o RT-nes ed PCRs was used o eco e pa ial S seg-
men sequences om o he h ee samples (expe imen al de-
ails a e a ailable upon eques ). Lowe - han-usual e iciency
o RT-PCR in de ec ing TULV sequences among he N-Ag-
posi i e samples could be due o subop imal condi ions o
ield samples anspo a ion and s o age. All eco e ed han-
a i al sequences belonged o TULV geno ype. Co espond-
ing wild- ype TULV s ains we e designa ed as ollowing:
TUL/Ka a al/Ma322/2003, TUL/Ka a al/Ma340/2003, TUL/
Taldyko gan/Ma343/2003 and TUL/Taldyko gan/Ma216/
2003, o Ka a al322, Ka a al340, Taldyko gan343, and
Taldyko gan216, o sho . Fo s ains Ka a al340 and
Taldyko gan343, n 142 o 1296 o n 142 o 1206 o he S
segmen sequence we e eco e ed, espec i ely. The sho es
sequence, n 909 o 1206, was eco e ed o s ain Taldyko -
gan216. The S segmen sequences o ou Kazakh w -TULV
s ains showed di e si y be ween 1.6% and 2.4%. No ably,
all obse ed n subs i u ions appea ed o be silen hus he
deduced amino acid (aa) sequences o he N p o ein we e
iden ical in all ou TULV s ains. This sugges ed a s ong
nega i e (s abilizing) selec ion ope a ing a he N p o ein
le el.
Comple e S segmen sequence o Ka a al322 s ain was
1830 n long ( he i s and he las 22 nucleo ides o he am-
plicon o igina ed om he PCR p ime and hus we e no
de e mined di ec ly). The sequence included: he 5'-
noncoding egion (NCR, n 1 o 42), he open eading ame
o he 430 aa-long N p o ein (n 43 o 1335) and he 3'-NCR
(n 1336 o 1830). The sequence showed he highes le el o
iden i y (87%) o TULV s ains om Russia (Tula) and Eas
Slo akia (Kosice). O he TULV s ains om Slo akia
(Malacky) and Russia (Omsk) and also s ains om Chech
Republic, Ge many, Se bia, C oa ia and Poland we e mo e
dis an ly ela ed: sequence iden i ies 84-86%. Simila ly, he
deduced aa sequence o he N p o ein om Ka a al322 s ain
was mos closely ela ed o he N-sequences o Tula and
Kosice s ains: sequence di e si y was as low as 1.9-2.8%.
The N p o ein sequences o o he TULV s ains showed
highe di e si ies: 3.0-4.7%. Kazakh N p o ein sequence
ca ied speci ic aa signa u e, T274Q276T281, usually an
indica o o a dis inc lineage.
On phylogene ic ee calcula ed o he S segmen se-
quences, TULV s ains om Kazakhs an o med dis inc ,
well-suppo ed gene ic lineage indeed (Fig. (1); FM- and
PUZZLE- ees e ealed same b anching pa e n, no
shown). Wi hin he lineage, TULV s ains show geog aphic
clus e ing: wo s ains om Ka a al we e loca ed close o
each o he . The monophily o hese wo s ains was appa en
on he NJ- and FM- ees and ecei ed easonably high boo -
s ap suppo : 65% and 63%, espec i ely. Fi e o he linea-
ges o TULV we e seen on he phylogene ic ee (Fig. 1).
Two Russian lineages included s ains om Tula (Cen al
Russia) and Omsk (Wes Sibe ia), espec i ely. The ou h
lineage consis ed o s ains om Ge many and Poland, he
i h lineage o s ains om Eas Slo akia and Se bia, and
he six h - om C oa ia and Cen al Eu ope (Ge many, Swi -
ze land, Wes Slo akia, and Czech Republic). In e es ingly,
he Kazakh lineage sha ed a mo e ancien common ances o
wi h wo Russian lineages. Simila ly, he i h and he six h
lineages sha ed a common ances o and, mos likely, an-
o he , e en mo e ancien common ances o wi h he ou h
lineage. The boo s ap suppo o he monophyly o hese
h ee lineages was 68%, o he NJ ee (Fig. 1), 63%, o he
FM- ee (no shown), and 60%, o he PUZZLE- ee (no
shown).
34 The Open Vi ology Jou nal, 2008, Volume 2 Plyusnina e al.
Fig. (1). Phylogene ic ee (Neighbo -joining) o TULV based on he coding egion o he S segmen (n 141-1206). Only boo s ap suppo
alues g ea e han 70% a e shown. PHV, P ospec Hill i us, s ain PH-1 (GenBank accession numbe Z49098); BLLV, Bloodland Lake
i us, s ain MO46 (U19303); ISLAV, Isla Vis a i us, s ain MC-SB-47 (U19302). Tula i us (TULV) s ains: Taldyk343,
Taldyko gan/Ma343/2003 (AM945879); Ka a al340, Ka a al/Ma340/2003 (AM945878); Ka a al322, Ka a al/Ma322/2003 (AM945877);
Omsk23, MG23/Omsk (AF442621); Tula 249, Tula/249M /87 (Z30944); Tula76, Tula/76Ma/87 (Z30941); Tula175, Tula/175Ma/87
(Z30943); Tula53, Tula/53Ma/87 (Z30942); Tula23, Tula/23Ma/87 (Z30945); Ge mD5-98, Ge many/D5-98 (AF289819); Ge mD63-98,
Ge many/D63-98 (AF289821); Ge mD17-98, Ge many/D17-98 (AF289820); Lodz1 (AF063892); Lodz2 (AF063897); Se bia (AF017659);
Kosice667, Kosice/667Ma/95 (Y13980); Kosice144, Kosice/144Ma/95 (Y13979); Ge m20, Ge many/g20-s (AF164093); Swi z91,
Swi ze land/91Ma; C oa ia, C oa ia/c109-s (AF164094); Malacky370, Malacky/370Ma/94 (U31534); Malacky32, Malacky/32Ma/94
(Z48234); Koziky47, Koziky/5247Ma/94 (AJ223600); Koziky76, Koziky/5276Ma/94 (AJ223601); Mo a ia02, Mo a ia/5302Ma/94
(Z49915); Mo a ia86, Mo a ia/5286Ma/94 (Z48573); Mo a ia93, Mo a ia/5293Ma/94 (Z48574); Mo a ia94, Mo a ia/5294Ma/94
(Z48741).
PHV
BLLV
ISLAV
Taldyk343
Ka a al340
Ka a al322
Omsk23
Tula249
Tula76
Tula175
Tula53
Tula23
Ge mD5-98
Ge mD63-98
Ge mD17-98
Lodz1
Lodz2
Se bia
Kosice667
Kosice144
Ge m20
Swi z91
C oa ia
Malacky370
Malacky32
Koziky47
Koziky76
Mo a ia02
Mo a ia86
Mo a ia93
Mo a ia94
100
100
100
100
100
100
100
100
100
100 100
100
100
100
83
99
72
68
86
85
99
75
77
KAZAKHSTAN
CENTRAL
RUSSIA
TULA)
RUSSIA
(OMSK)
CENTRAL
EUROPE
and
CROATIA
GERMANY
and
POLAND
EAST
SLOVAKIA
and
SERBIA
TULV
Tula Han a i us in Kazakhs an The Open Vi ology Jou nal, 2008, Volume 2 35
Wi h h ee excep ions, namely: (1) s ain Tula249 om
M. le is ( o me ossiaeme idionalis o epi o icus), a sibling
species o M. a alis, (2) s ain Se bia om M. sub e aneus
( o me Pi ymus sub e aneus), and (3) s ain Omsk23 om
M. g egalis, TULV s ains p esen ed on Fig. (1) o igina e
om M. a alis. The i s s ain belongs o Cen al Russian
lineage and shows minimal gene ic di e si y om M. a -
alis- ca ied s ains ha ci cula ed wi hin he same locali y
[3]. The second s ain [26] is closely ela ed o Kosice
TULV s ains om Eas Slo akia [22], wi h which i o ms
he i h gene ic lineage. Omsk23 s ain p esen s a dis inc
lineage, bu ce ainly belongs o TULV geno ype.
DISCUSSION
Da a p esen ed in his pape desc ibe he i s TULV
s ains om Cen al Asia. Kazakh s ains om wo locali ies,
Taldyko gan and Ka a al, cons i u e no el, dis inc gene ic
lineage o TULV. This lineage is well-suppo ed on phylo-
gene ic ees and possesses a unique aa signa u e:
T274Q276T281. Kazakh TULV lineage appea s o be mos
closely ela ed o lineages om Cen al Russia and Sibe ia.
These h ee lineages sha e a common ances o (boo s ap
suppo alue 83%, on he NJ- ee) and also se e al signa u e
aa esidues. Kazakh and Cen al Russian lineage sha e h ee
signa u e aa esidues: N42, E60, and I269, while Kazakh and
Sibe ian lineage sha e one signa u e aa esidue D291. These
obse a ions sugges common e olu ion his o y o Kazakh,
Cen al Russian and Sibe ian lineages o TULV, which is
somewha di e en om he his o y o o he h ee lineages
ha include s ains om Cen al Eu ope (Ge many, Swi ze -
land, Slo akia, and Czech Republic), Poland and he Balkans
and sha e ano he common ances o . I should be no ed ha
wo g oups o TULV lineages we e seen in he ecen ly pub-
lished phylogeny based on pa ial S segmen sequences [23]
(excep ha Kazakh lineage was no p esen ). The cu en
phylogeny, based on longe sequences (almos comple e cod-
ing egion o he S segmen ), is mo e obus : mos o he
boo s ap suppo alues a e abo e he widely accep ed con-
iden ial limi o 70% [34] - and hence mo e con incing.
G ouping o TULV lineages migh had been oo ed o
glacial dis ibu ion pa e ns o Mic o us oles ac oss Eu ope
and Cen al Asia. The ice age e olu ion o Eu opean small
oden species has been cha ac e ized by isola ed e ugia, no
only in sou he n peninsulas, bu also in mo e no he n e-
gions [35-37]. This is due o he agmen ing/isola ing im-
pac o se e al moun ain anges. In con as , he las ice age
and subsequen o ma ion o as s eppe a eas om Eas e n
Eu ope o Cen al Asia ha e p obably suppo ed mo e con-
inuous Mic o us popula ions.
As men ioned abo e, he as majo i y o TULV genomic
sequences a ailable so a o igina e om M. a alis ha is
conside ed he main na u al hos o he i us [1, 3]. The e
a e also excep ions, like he h ee sequences lis ed in he
Resul s and also, e.g., pa ial S-sequences eco e ed om M.
ag es is ( ield ole) [27] and e en om Lagu us lagu us
(s eppe lemming) (GenBank accession numbe s AF442618-
19). Geog aphical anges o M. le is, M. sub e aneus and
M. ag es is o e lap widely wi h he ange o M. a alis [38,
39] and he e o e he i us spillo e om M. a alis o hese
species can no be excluded. Howe e , some obse a ions,
e.g. highe in ec ion a e in M. ag es is han in M. a alis in
C oa ia, sugges ed, ha M. ag es is in some a eas can se e
as ano he na u al hos o TULV [27]. As o M. g egalis,
i s ange o e laps only wi h he eas e nmos pa s o M a -
alis ange in wes e n-cen al Sibe ia [39] and he spillo e
o TULV om M a alis o M. g egalis is less likely. I hus
seems ha Tula can in ec a numbe o Mic o us species,
e en hose in di e en subgene a [ o subgene a, see 35]: M.
a alis and sibling species in Mic o us g oup, M. ag es is in
Ag icola g oup, M. sub e aneus in Te icola g oup, and
e en M. g egalis in S enoc a ius g oup ha phylogene ically
is mos dis an om he a alis g oup [35]. In connec ion o
his, i migh be o in e es ha he ca ie o Kazakh TULV
s ains, M. a alis obscu us, cu en ly classi ied as one o he
ch omosome o ms in M. a alis [30], has some imes been
conside ed a pa apa ic species o i s own [38]. Bu , e en i
TULV p o en o ha e wo o mo e oden hos species, his
would no be an excep ion. Fo ins ance, Sin Nomb e han a-
i us in some a eas is ha bou ed by dee mouse Pe omyscus
manicula us and in o he s by whi e- oo ed mouse P. leuco-
pus [40].
Mic o us adia ion in many axa is an e olu iona ily e-
cen p ocess, mo e ecen han e.g. in Myodes ( o me
Cle h ionomys), and is s ill ongoing [35]. I seems ha in
Eu asia TULV can in ec se e al Mico us species, also no
closely ela ed ones in di e en subgene a. Mos impo -
an ly, TULV s ains ha bou ed by di e en Mic o us species
om he same egion esemble each o he . I he e o e sa e
o assume ha geog aphic dis ance was so a he majo ac-
o o he di e si ica ion o TULV lineages. Ou da a show-
ing ha Kazakh TULV s ains a e mos closely connec ed o
s ains om Wes Sibe ia and Cen al Russia, be hei hos in
he same o di e en subgenus o Mic o us, suppo his
poin o iew. Unusually high hos di e si y o TULV could
be seen as an indica ion o young s age o i s oden hos
adia ions and he i us-hos co-e olu ion ( he adia ion o
Mic o us began 2 million yea s ago, bu many o he sibling
species ha e de eloped du ing he las 100 000 - 200 000
yea s).
CONCLUSIONS
Ou esul s demons a e he p esence o TULV in com-
mon ole M. a alis in Kazakhs an. Kazakh TULV s ains
o m a dis inc gene ic lineage and sha e a mo e ancien
common ances o wi h TULV s ains cu en ly ci cula ing in
Cen al Russia (Tula egion) and Wes Sibe ia (Omsk e-
gion). The Mic o us a alis g oup includes se e al sis e
species and/o subspecies in Eu asia, indica ing ecen and
ongoing e olu iona y adia ion. Ou da a on TULV lineages
in Cen al Asia, he egion no s udied o han a i uses ea -
lie , highligh he di e si y o Mic o us hos and he i us
and hei co-e olu ion.
ACKNOWLEDGEMENTS
This wo k was suppo ed by g an s om The Academy
o Finland, Sig id Jusélius ounda ion (Finland) and EU
g an ICA2-CT-2000-10048. TULV genome sequences de-
sc ibed in his pape ha e been deposi ed o he GenBank
unde acc. numbe s AM945877-79.
36 The Open Vi ology Jou nal, 2008, Volume 2 Plyusnina e al.
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Recei ed: Ma ch 12, 2008 Re ised: Ma ch 24, 2008 Accep ed: Ma ch 26, 2008