Mic oe olu ion o
Puumala han a i us
du ing a
Comple e Popula ion Cycle o I s Hos , he Bank Vole
(
Myodes gla eolus
)
Ma ia Razzau i
1,2
*, Angelina Plyusnina
1
, Heikki Hen onen
2
, Alexande Plyusnin
1
1Depa men o Vi ology, Haa man Ins i u e, Uni e si y o Helsinki, Helsinki, Finland, 2Finnish Fo es Resea ch Ins i u e, Van aa, Finland
Abs ac
Mic oe olu ion o Puumala han a i us (PUUV) was s udied h oughou a popula ion cycle o i s hos , he bank ole (Myodes
gla eolus). We moni o ed PUUV a ian s ci cula ing in he hos popula ion in Cen al Finland o e a i e-yea pe iod ha
included wo peak-phases and wo popula ion declines. O 1369 bank oles examined, 360 (26.3%) we e ound in ec ed
wi h PUUV. Pa ial sequences o each o he h ee genome segmen s we e eco e ed (app ox. 12% o PUUV genome) om
356 bank oles. Analyses o hese sequences disclosed he ollowing ea u es o PUUV e olu ion: 1) nucleo ide subs i u ions
a e mos ly silen and deduced amino acid changes a e mainly conse a i e, sugges ing s abilizing selec ion a he p o ein
le el; 2) he h ee genome segmen s accumula e mu a ions a a di e en a e; 3) some o he ci cula ing PUUV a ian s a e
equen ly obse ed while o he s a e ansien ; 4) equen ly occu ing PUUV a ian s a e composed o he mos abundan
segmen geno ypes (copious) and new ansien a ian s a e con inually gene a ed; 5) easso men o PUUV genome
segmen s occu s egula ly and ollows a speci ic pa e n o segmen s associa ion; 6) p e alence o easso an a ian s
oscilla es wi h season and is highe in he au umn han in he sp ing; and 7) easso an s a e ansien , i.e., hey a e no
compe i i ely supe io o hei pa en al a ian s. Collec i ely, hese obse a ions suppo a quasi-neu al mode o PUUV
mic oe olu ion wi h a s eady gene a ion o ansien a ian s, including easso an s, and p ese a ion o a ew p e e ed
geno ypes.
Ci a ion: Razzau i M, Plyusnina A, Hen onen H, Plyusnin A (2013) Mic oe olu ion o Puumala han a i us du ing a Comple e Popula ion Cycle o I s Hos , he Bank
Vole (Myodes gla eolus). PLoS ONE 8(5): e64447. doi:10.1371/jou nal.pone.0064447
Edi o : Dhanaseka an Vijayk ishna, Duke-NUS G adu e Medical School, Singapo e
Recei ed Ap il 3, 2012; Accep ed Ap il 15, 2013; Published May 22, 2013
Copy igh : ß2013 Razzau i e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This wo k has been suppo ed by g an s om he Academy o Finland, Sig id Juse
´lius Founda ion, and was pa ially unded by EU-FP6 g an GOCE-
2003-010284 EDEN (h p://www.eden- p6p ojec .ne ) and he EU g an FP7-261504 EDENex (h p://www.edenex .eu) and is ca alogued by he EDENex S ee ing
Commi ee as EDENex 021. The con en s o his publica ion a e he sole esponsibili y o he au ho s and do no necessa ily e lec he iews o he Eu opean
Commission. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: ma ia. azzau i@helsinki. i
In oduc ion
Han a i uses cons i u e a dis inc genus wi hin he Bunya i idae
[1]. This amily is ep esen ed by en eloped nega i e-s anded
RNA i uses ha possess a i-segmen ed genome: he small (S)
segmen encodes he nucleocapsid (N) p o ein and, in some
han a i uses, also a non-s uc u al p o ein (NSs); he medium (M)
segmen encodes wo su ace glycop o eins (Gn and Gc); and he
la ge (L) segmen encodes he L p o ein, he i al RNA-dependen
RNA polyme ase [2].
Puumala i us (PUUV) [3] is he main oden -bo ne pa hogen in
Eu ope, whe e i causes neph opa hia epidemica (NE), a ela i ely mild
o m o haemo hagic e e wi h enal synd ome [4]. The hos o
PUUV is he bank ole (Myodes gla eolus), a woodland oden ha
occu s om he B i ish Isles h ough con inen al Eu ope and
Russia o he Lake Baikal [5]. The occu ence o NE depends
s ongly on he popula ion dynamics o he bank ole; popula ion
peaks a e mi o ed by NE ou b eaks wi h a sho ime-lag [6–8].
Bank oles a e ch onically in ec ed by PUUV [9–10]. Vi al
sec e ion occu s du ing hal a yea a e in ec ion [9], [11] and he
i us ansmission is ho izon al, mainly ia b ea hing o ae osol-
ized exc e a gene a ed by in ec ed oden s [12–14]. Such indi ec
mode o ansmission and i us su i al ou side he hos p omo es
i al pe sis ence in he bank ole popula ion [15–16]. Ma e nal
an ibodies ans e ed by in ec ed emales o hei p ogeny p o ec
hem up o 80 days, and his may shape he dynamics o i us
ansmission du ing b eeding season and sho ly he ea e [17–
18]. Simila o o he RNA i uses, han a i uses exhibi a high
sho - e m subs i u ion a e. Gene ic di e si y in han a i uses is
gene a ed by gene ic d i (i.e., a g adual accumula ion o poin
mu a ions h oughou he genome coupled wi h small dele ions
and inse ions wi hin he non-coding egions o he RNA segmen s
[19]) and easso men o genome segmen s [19–22]. E idence o
homologous ecombina ion has also been epo ed [23–24], bu
his seems o be a a e e en [25]. While he as majo i y o poin
mu a ions obse ed a e silen , e lec ing s abilizing selec ion a he
aa le el [26], he e is some e idence o di ec ional selec ion [27].
Gene ic he e ogenei y is compa able among han a i uses species
ound in di e en oden sub amilies. Nucleo ide (n ) di e si y
among han a i us species is a iable and cha ac e ized by he
numbe o s ains ecognized. In e -lineage di e si y o PUUV
may be as high as 38% o he S segmen , and sequence
he e ogenei y is une enly dis ibu ed along he genome [28].
Despi e he g owing da abase o genome sequences and he
inc easing numbe o s udies on gene ic a iabili y o han a i uses,
none o da e ha e conside ed he na u e o i al gene ic di e si y
PLOS ONE | www.plosone.o g 1 May 2013 | Volume 8 | Issue 5 | e64447
on a scale o a hos popula ion and sampled du ing se e al-yea s
pe iod. Howe e , such s udies o i us mic oe olu ion a e essen ial
o a be e unde s anding o he mechanisms shaping han a i us
e olu ion. P e iously [19], we examined PUUV gene ic di e si y
in a bank ole popula ion. This s udy included 31 dis inc i al
s ains ci cula ing du ing 2005 in Cen al Finland. A subs an ial
gene ic di e si y was obse ed be ween ci cula ing PUUV s ains.
Mo eo e , phylogene ic analysis o he S, M and L genome
sequences clus e ed he a ia ion in o wo dis inc genog oups. As
such, ecogni ion o easso an s ains was s aigh o wa d and
20% o he ci cula ing PUUV s ains we e ound o be ca ying
in e -genog oup easso an genomes. This analysis aised se e al
ques ions conce ning empo al luc ua ions o he di e si y o
PUUV s ains, how i is in luenced by he hos popula ion densi y,
and he a e o indi idual PUUV s ains. He e, we expand ou
ea lie s udy and epo he mic oe olu ion o PUUV in i s hos
popula ion du ing a i e-yea pe iod, co e ing bo h peak and
decline phases o he bank ole popula ion cycle.
Ma e ials and Me hods
E hics s a emen
Acco ding o he Finnish Ac on he Use o Animals o
Expe imen al Pu poses (62/2006) and a u he decision by he
Finnish Animal Expe imen Boa d (May 16 h, 2007), he
echniques employed o cap u e oden s, i.e., li e- and snap-
apping a e no conside ed an animal expe imen and he e o e
equi es no animal e hics license om he Finnish Animal
Expe imen Boa d. The species cap u ed o his s udy, Myodes
gla eolus, nei he is p o ec ed no included in he Red Lis o
Finnish Species. Animal apping ook place on p i a e and
Finnish na ional o es by pe mi (1013/204/2002). Landowne s
we e consul ed and he apping was allowed be o e he s udy was
conduc ed.
Sampling o oden s
Roden s we e apped a Konne esi, Cen al Finland (62u349N,
26u249E) wice a yea (May and Oc obe ) om 2005 ill 2009.
Du ing he ea ly 2000s, bank oles we e s ongly cyclical in
Cen al Finland [7]; 2005 was a cyclic peak yea , 2006 decline and
c ash, 2007 a s ong inc ease phase, 2008 a high peak, and 2009
ano he decline. T apping was done a 38 si es wi hin 120 km
2
o
ypical aiga o es , mainly domina ed by No way sp uce (Picea
abies), Sco s pine (Pinus syl es is), downy bi ch (Be ula pubescens) and
sil e bi ch (Be ula pendula). O 38 apping si es, 14 we e sampled
om May 2005 o Oc obe 2009 using g ids o 363 Ugglan
Special li e aps (G ahnab, Hille s o p, Sweden) se 15 m apa .
T aps we e se o h ee nigh s and checked wice pe day o
minimize animal s ess. F om May 2007 onwa ds, 24 addi ional
apping si es we e added o inc ease he s udy ma e ial. These
consis ed o 4 ansec s o 15 s anda d snap- aps a 15 m in e als
o e wo nigh s. The apping si es we e si ua ed 500 o 1000 m
apa om each o he . Bank oles cap u ed ali e we e anes he-
ized wi h iso lua ene (Fo ene, Abbo , UK), bled om he e o-
o bi al sinus and sac i iced by ce ical disloca ion and immedia ely
ozen. Simila ly, snap- apped bank oles we e lash- ozen on
d y ice un il nec opsy. The dissec ion o oden s was pe o med in
a class II lamina low hood in a biosa e y le el 3, animal
anno a ion (weigh , sex, ma u i y, age) was comple ed and issue
samples we e indi idually deep- ozen un il u he analyses.
Sc eening o samples
All oden s we e i s sc eened o he p esence o PUUV N-
an igen (Ag) using immunoblo ing. B ie ly, lung issue samples
(app ox. 20 mg) in 500 ml o Laemmli bu e we e incuba ed a
oom empe a u e o e nigh , hen homogenized by sonica ion and
hea ed o 80–100uC o 5 minu es. Aliquo s o 25 ml we e
sepa a ed by elec opho esis in 10% sodium dodecyl sulpha e
polyac ylamide gels and p o eins we e subsequen ly ans e ed
on o ni ocellulose memb anes. To e i y he blo ing e icacy,
memb anes we e s ained wi h 16Ponceau S s aining solu ion o
1 minu e and des ained in dis illed wa e un il he backg ound was
clea . Memb anes we e blocked wi h 1% bo ine se um albumin
solu ion o e nigh a +4uC. Memb anes we e incuba ed wi h
abbi polyclonal an ise um made agains ecombinan N-Ag o
wo hou s a oom empe a u e, washed wi h 0.1% Ten-Tween20
solu ion, and incuba ed o one hou wi h Odyssey IRDye 800CW
goa an i- abbi seconda y Ab (LI-COR), dilu ed a 1:10000 in
PBS. Odyssey In a ed Imaging sys em was used o de ec blo ed
p o eins.
RT-PCR and sequencing o he PUUV genome
Vi al RNA was ex ac ed om lung issue samples o he N-Ag-
posi i e bank oles using he TRIsu e eagen (Bioline, UK)
acco ding o he manu ac u e ’s ins uc ions. Re e se ansc ip ion
was pe o med wi h Re e Aid
TM
H Minus M-MuLV Re e se
T ansc ip ase (Fe men as, Li huania) and AmpliTaqHDNA
polyme ase (Applied Biosys ems, Fos e Ci y, CA, USA) was used
o ampli y i al cDNA. PUUV S- (n 631–1085), M- (n 2162–
2613) and L-segmen (n 505–1040) sequences we e ampli ied as
desc ibed ea lie [19]. PCR amplicons we e pu i ied wi h
ExoSAP-IT
TM
PCR clean-up eagen (USB Co po a ion, Miles
Road, Cle eland, USA). Au oma ed sequencing was pe o med
using he ABI PRISM
TM
Dye Te mina o sequencing ki (Pe kin
Elme /ABI). Newly eco e ed S-, M- and L-segmen sequences
(455, 452 and 536 n in leng h espec i ely) we e deposi ed in
GenBank unde accession numbe s JQ319161–JQ319319.
Gene ic a ia ion and phylogene ic analysis
Nucleo ide sequence alignmen s we e gene a ed wi h BioEdi
7.0.9 [29]. Gene ic dis ances we e es ima ed wi hin and be ween
i al popula ions wi h DnaSP [30]. PHYLIP p og am package
[31] was employed o es ima e phylogeny o he PUUV sequences.
Gene ic dis ances we e calcula ed wi h he F84 subs i u ion model
(Dnadis ) and boo s ap suppo s we e ob ained gene a ing 1000
eplica es o he da ase s (Seqboo ) and o he econs uc ion o
PUUV phylogenies he Neighbou -joining (NJ) algo i hm (Neigh-
bo ) was used. Sequence alignmen s we e submi ed o phyloge-
ne ic Ne wo k 4.600 so wa e (Fluxus-Enginee ing) o gene a e
gene ic and e olu iona y ela ionships among he segmen
geno ypes using Median-Joining algo i hm [32]. An examina ion
o ecombina ion among comple e sequences was pe o med using
he ecombina ion de ec ion p og am (RDP) [33] and SimPlo
3.5.1 [34].
Resul s
PUUV p e alence and gene ic di e si y in he bank ole
popula ion a Konne esi
The incidence o PUUV in ec ion and gene ic a ia ion o he
i us we e moni o ed h ough a comple e bank ole popula ion
cycle a Konne esi om May 2005 un il Oc obe 2009. The s udy
pe iod co e ed wo peak-phases in 2005 and 2008, and wo
popula ion declines in 2006 and 2009 (p esumably ollowed by
i us bo lenecks). A o al o 1369 bank oles we e cap u ed, o
which 360 (26.3%) we e PUUV-N-Ag-posi i e. PUUV p e alence
a Konne esi was highe in sp ing (a mean o 39%) when mos
Mic oe olu ion o Puumala han a i us
PLOS ONE | www.plosone.o g 2 May 2013 | Volume 8 | Issue 5 | e64447
oles in he popula ion ha e o e -win e ed a he han in au umn
(20.7%) when younge animals domina e (Table 1).
Pa ial genome sequences we e eco e ed om 356 PUUV N-
Ag-posi i e bank oles: 455 n o he S segmen , 452 n o he M
segmen and 536 n o he L segmen . Di ec sequencing o PCR-
p oduc s was employed; his way he mas e sequences we e
eco e ed and he ‘‘noise’’ o quasispecies igno ed. Taken
oge he , pa ial S-, M- and L-sequences (a o al o 1443 n )
ep esen ed 12% o he PUUV genome. O 356 PUUV genomes,
M-segmen sequences we e eco e ed o 353 genomes (in e es -
ingly, all he emaining we e easso an ). Pai wise sequence
analysis e ealed 182 n di e ences in he segmen s analyzed: 53
mu a ions in he S, 50 in he M, and 79 in he L. The majo i y o
mu a ions we e silen and occasionally non-synonymous subs i u-
ions we e encoun e ed (Table 2). Nume ous gene ic ma ke s we e
ound along he s udied segmen s: 10 o he S (T699C, T750C,
T789A, A810G, G843A, T876C, G936A, T945C, C1014T,
T1044C), 7 o he M (C2180T, C2207T, A2216G, G2471A,
A2483G, G2543A, C2567T) and 29 o he L (G514T, G523A,
T532C, C538T, G656A, C593T, G601A, G679A, C682T,
A742G, A754G, G769A, C829T, A832G, T838C, G904A,
T910C, G916A, A922G, C928T, A964T, C967T, A982T/G,
A988T/C, A1003G, A1006G, C1009T, T1025C, C1034T)
(Table 2). Such gene ic ma ke s allowed he ecogni ion and
disc imina ion o wo genog oups (i.e., gene ically ela ed s ains
wi hin a i us lineage) e e ed as ‘‘A’’ and ‘‘B’’ and ep esen ed in
he igu es in ed and blue colo s espec i ely. These g oups we e
also in e ed om phylogene ic analyses o sequence da a (Fig. 1).
Gene ic di e si y o PUUV segmen s wi hin genog oups was
simila o all segmen s (1.5–3.2%). In e -genog oup di e si y
a ied om 4.8 o 10.1%; su p isingly, he highes alues we e
a ibu ed o he L segmen . Fu he mo e, a non-synonymous
subs i u ion was ound in he L p o ein ha could be used as a
molecula ma ke o genog ouping; in he genog oup A, he aa
esidue a posi ion 83 was isoleucine while in he genog oup B his
esidue was aline (Table 2).
Geno yping o Konne esi PUUV s ains
Gene ic di e si y o ci cula ing PUUV s ains was e alua ed,
and he numbe o ci cula ing geno ypes (i.e., gene ically unique
ep esen a i es o each o he i us segmen s) es ima ed. A o al o
45 S-segmen geno ypes, 38 M-segmen geno ypes and 61 L-
segmen geno ypes we e ecognized. Genog oup A included 11 S-,
18 M-, and 26 L-segmen geno ypes e e ed espec i ely as S1A
o S11A, M1A o M18A and L1A o L26A. Co esponding
numbe s o genog oup B we e 34, 20 and 35 and espec i ely
e e ed as S1B o S34B, M1B o M20A and L1B o L35B (Table 2
and Fig. 1). Va ian s o genog oup B we e gene ally mo e
abundan and di e se (Table 1 and Fig. 2). The 356 PUUV
sequences comp ised 184 dis inc gene ic a ian s. O hose, 46
belonged o genog oup A (A
S
A
M
A
L
), 82 o genog oup B
(B
S
B
M
B
L
), and 56 we e easso an s be ween hese g oups
(Fig. 1E). In e es ingly, only 39 o hese 184 dis inc a ian s we e
ound epea edly o e he obse a ion pe iod; o he a ian s we e
egis e ed only once (Table 3). No accumula ion o ce ain a ian s
a he gi en geog aphic si es was obse ed. Nei he subs an ial
de ia ion in di e si y be ween si es no any bias linked o he
e ain opology was egis e ed.
An independen analysis o each segmen o PUUV genomes
allowed he dis inc ion o he mos abundan geno ypes ( om he e
on e e ed o as ‘‘copious’’): 6A, 7A, 8A, 5B, 20B, 21B, 22B and
28B o he S segmen ; 1A, 8A, 14A, 15A, 3B, 8B and 15B o he
M segmen ; and 3A, 9A, 14A, 4B, 8B, 11B, 19B, 21B and 23B o
he L segmen . Figu e 2 shows he equency o occu ence o he
segmen geno ypes as well as hei ela ionships. Clus e s o se e al
geno ypes can be seen o he S and M segmen s o bo h
genog oups. These clus e s consis o a leas one copious geno ype
and se e al geno ypes ha we e seldom obse ed ( om he e on
e e ed o as ‘‘spo adic’’). Fo example, he copious S geno ype 28B
and spo adic geno ypes 27B, 30B, 31B, 32B, 33B, 34B o m such a
clus e . Di e en ly, he L segmen was ep esen ed by mo e
geno ypes, p obably e lec ing a highe n subs i u ions a e in his
segmen . Thus high numbe o d i ed n esul ed in abundan
gene ically- ela ed a ian s illus a ed in in e connec ed ci cles o a
smalle size in he g aphical ep esen a ion o he segmen
geno ypes (Fig. 2).
Table 1. Numbe o bank oles cap u ed du ing he s udy, PUUV p e alence and p opo ions o A and B genog oups and
easso an s a Konne esi.
May Oc . May
+
Oc . May Oc . May Oc . May Oc . To al
2005 2006 2007 2008 2009
no. o apped
bank oles
47 100 8 54 132 237 625 78 88 1369
no. o PUUV
+
22 22 0 7 15 106 155 28 5 360
(%) (46.8) (22) (13) (11.4) (44.7) (24.8) (35.9) (5.7) (26.3)
no. o a ian s
o genog oup A
5 4 0 25 4051 135 125
(%) (27.8) (18.2) (28.6) (33.3) (37.7) (32.9) (46.4) (100) (35.1)
no. o a ian s
o genog oup B
10 13 0 4 7 53 67 9 0 163
(%) (55.6) (59.1) (57.1) (46.7) (50) (43.2) (32.1) (45.8)
no. o Reasso an
a ian s
350 13 1337 6068
(%) (16.7) (22.7) (14.3) (20) (12.3) (23.9) (21.4) (19.1)
doi:10.1371/jou nal.pone.0064484. 001
Mic oe olu ion o Puumala han a i us
PLOS ONE | www.plosone.o g 3 May 2013 | Volume 8 | Issue 5 | e64447
Mic oe olu ion o Puumala han a i us
PLOS ONE | www.plosone.o g 4 May 2013 | Volume 8 | Issue 5 | e64447
Dynamics o PUUV a ian s du ing a bank ole
popula ion cycle
Rela i ely la ge collec ion o PUUV sequences eco e ed o e a
5-yea pe iod ga e a unique oppo uni y o ollow indi idual
gene ic a ian s o he i us h ough phases o high and low
popula ion densi y. Analysis o segmen geno ypes dynamics
e ealed ha he copious geno ypes we e p e alen h oughou
he obse a ion window whe eas mos o spo adic geno ypes we e
ansien , only a ew o hem we e obse ed on mo e han one
occasion. New geno ypes we e con inually de ec ed du ing he
s udy, excep in 2006 when no PUUV-in ec ed oles we e ound
(Fig. 3). In o he wo ds, he PUUV genes pool was unique a e e y
ime-poin . Table 4 shows a ian s ha ha e de i ed om one o a
ew n subs i u ions, e lec ing he con ibu ion o gene ic d i o
PUUV di e si y. Impo an ly, no e idence o a ounde e ec ( he
loss o gene ic a ia ion ha occu s when a subpopula ion o a
small numbe o indi iduals is es ablished [35]) was obse ed o
he segmen geno ypes a e a decline in he bank ole popula ion
(p esumably a i us bo leneck). To analyze PUUV s ains
dynamics, we classi ied ci cula ing a ian s in o ou ca ego ies:
(i) he mos equen ly occu ing a ian s, each de ec ed i e o
mo e imes; (ii) epea edly obse ed a ian s, de ec ed 2–4 imes;
(iii) ansien a ian s, de ec ed only once; and (i ) easso an
a ian s, which we e analyzed sepa a ely due o hei peculia
gene ic na u e (Table 5). The mos equen ly occu ing a ian s
($5 imes) included only copious geno ypes o each genome
segmen . Two o hese a ian s we e clea ly dominan : A13 was
obse ed 53 imes om May 2007 o Oc obe 2009, and B3 was
obse ed 21 imes om Oc obe 2007 o Oc obe 2008.
Repea edly obse ed a ian s (2–4 imes) we e de ec ed a no
mo e han wo s udy poin s (Table 4). O hose epea edly
obse ed a ian s, 74% o S segmen s, 67% o M segmen s and
app ox. 30% o L segmen s we e o copious geno ypes. Fo he
ansien a ian s (1 ime), all h ee segmen s we e ep esen ed by
copious and spo adic segmen geno ypes in app oxima ely equal
p opo ions.
Reasso an a ian s
Co-ci cula ion o wo dis inc genog oups o PUUV allowed o
s aigh o wa d ecogni ion and subsequen s udy o easso an s
a ian s. In o al, 68 o 356 (19.1%) PUUV genomes we e ound
o be easso an . They we e ep esen ed by 56 dis inc a ian s
and only h ee o hose we e obse ed mo e han once h oughou
he s udy pe iod (Table 3). All six possible combina ions o PUUV
genome segmen s we e ound among easso an s ains and,
in e es ingly, a ce ain pa e n o segmen combina ion was
obse ed. In he as majo i y o cases (95.6%) only he S o M
segmen s we e exchanged (Table 6). In o he wo ds, he L segmen
p e e ably emained pai ed wi h S- o M-segmen s o he same
genog oup. Reasso an a ian s consis ed o copious S- and M-
segmen geno ypes in 75% and 66% o cases espec i ely, whe eas
he L genome segmen was equally composed o copious and spo adic
geno ypes (Table 4). The composi ion o copious and spo adic
geno ypes was simila o bo h epea edly obse ed and easso an
a ian s. Howe e , he epea edly obse ed a ian s could be seen
a wo di e en ime-poin s, while easso an s we e ansien wi h
he excep ion o h ee a ian s de ailed in Table 5. The p opo ion
o easso an s in he i al popula ion a ied om sp ing o
au umn and was in e sely ela ed o PUUV p e alence, i.e. was
Figu e 1. Phylogene ic ees. Phylogene ic ees (Neighbo -Joining) calcula ed o he S, M and L segmen sequences o PUUV, and con ings o
he h ee segmen sequences: (A) pa ial S (n 631–1085); (B) pa ial M (n 2162–2613); (C) pa ial L (n 505–1040); (D) a con ig o he h ee genome
segmen s omi ing easso an a ian s, and (E) a con ig o he h ee genome segmen s including easso an a ian s. A maximum clade c edibili y
ee wi h an a bi a y oo is shown wi h mean b anch leng hs (subs i u ions pe si e), and non-pa ame ic boo s ap pe cen ages a e shown o each
node. DOBV, ANDV, SNV and TULV we e used as ou g oups and omi ed o he g aphical ep esen a ion.
doi:10.1371/jou nal.pone.0064447.g001
Table 2. Gene ic di e si y o PUUV a ian s a Konne esi.
Genome segmen S M L
Leng h n 455 452 536
aa 151 150 178
no. o n subs i u ions G oups Aand B53 50 79
Wi hin g oup A17 16 24
Wi hin g oup B33 32 38
Gene ic di e si y (%) G oups Aand B6.2 4.8 10.1
Wi hin g oup A2.5 1.6 1.5
Wi hin g oup B2.5 2.7 3.2
no. o aa subs i u ions Be ween g oups Aand B-- 1(AI83V B)
Wi hin g oup A3 (K41R, M75I, D79E) 3 (S11N, E43K, C109G) 3 (I43V, N93I, N168K)
Wi hin g oup B9 (F31I, V33E, R39K, P57S,
Q78P, D79N, A87V, A97S,
Q144P)
4 (P71L, S92P, S101A, I110V) 8 (T7S, D46E, R67C, N86D,
R116K, T121I, S141G, V165I)
no. o geno ypes To al 45 38 61
Wi hin g oup A11 18 26
Wi hin g oups B34 20 35
no. o genog oup
disc imina i e ma ke s
Be ween g oups Aand B10 7 29
doi:10.1371/jou nal.pone.0064447. 002
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highe in au umn when he p e alence is lowe , and lowe in
sp ing when he p e alence is highe (Table 1). These oscilla ions
we e no obse ed o pa en al a ian s.
Double in ec ions
A p e equisi e o PUUV easso men is he coexis ence o wo
i al genomes in he same hos , i.e. a double in ec ion. Visual
inspec ion o sequence ch oma og ams e ealed double peaks a
he diagnos ic posi ions o genog oups in wel e sequences: six o
he S segmen , wo o he M segmen and ou o he L segmen .
Double peaks we e dis ibu ed no andomly along he sequences
bu appea ed exclusi ely a he geno ype-speci ic posi ions. Hence,
such double peaks canno esul om simul aneous n subs i u ion
a gi en posi ions e lec a double-in ec ion. De ec ed double
in ec ions wi h wo geno ypes we e no obse ed ci cula ing as
easso an a ian s. Sequencing o indi idual cDNA clones
showed he p esence o wo s ains o he i us (one o each
genog oup) and hence con i med a double in ec ion in he hos .
In e es ingly, all bu one sequence wi h double peaks we e
eco e ed om samples collec ed in Oc obe when condi ions
o double in ec ions seem o be highe (see Discussion).
To sea ch o aces o ecombina ion in PUUV genomes
ci cula ing a Konne esi, ou a ian s, wo o each genog oup (A
and B), we e selec ed and hei comple e S and M segmen
sequences and app ox. 80% o he L segmen sequence we e
eco e ed and analysed. No appa en signs o ecombina ion we e
obse ed. Al hough his esul does no comple ely exclude he
possibili y o ecombina ion, i sugges s ha ecombina ion occu s
much less equen ly han easso men . An al e na i e explana-
ion could be ha ecombinan i uses, e en i gene a ed
equen ly, may no be able o eplica e as e icien ly as pa en al
i uses o easso an s.
Discussion
Fo he i s ime, he mic oe olu ion o PUUV has been s udied
h oughou he hos popula ion cycle. The da a se analyzed he e
(1369 bank oles o which 360 we e PUUV in ec ed) was indeed
la ge han in ou p eceding s udy [19], in which 147 bank oles
cap u ed in 2005 and 40 PUUV genomes we e analyzed. The
p esen esul s suppo and expand ou ea lie conclusions
conce ning PUUV gene ic di e si y and he equency o segmen
easso men . In addi ion, he i e-yea moni o ing o PUUV
genomes allowed he s udy o PUUV mic oe olu ion, pa icula ly,
o ollow he a e o indi idual gene ic a ian s, including
easso an s.
O 1369 bank oles cap u ed, 360 we e ound in ec ed and 356
PUUV genome sequences we e success ully eco e ed. Al hough
no s a is ically signi ican , PUUV p e alence end o be highe in
he sp ing when mos bank oles we e ma u e and had o e -
win e ed, han in he au umn when animals bo n he summe
Figu e 2. Gene ic ela ionships be ween (A) S-segmen , (B) M-
segmen and (C) L-segmen geno ypes. Rela ionships we e
cons uc ed using Ne wo k analysis and he MJ algo i hm.
Geno ypes o g oup A a e ep esen ed by ed ci cles and geno ypes o
g oup B by blue ci cles. The size o he ci cle is p opo ional o he
numbe o ep esen a i es o each geno ype; and numbe s a e shown
inside. Yellow ci cles a e median ec o s sugges ed by Ne wo k. The
numbe s on he link-lines show he numbe o mu a ions be ween
geno ypes; he leng h o link-lines is no p opo ional o he numbe o
mu a ions.
doi:10.1371/jou nal.pone.0064447.g002
Table 3. Gene ic a ian s o PUUV obse ed in he Konne esi
bank ole popula ion.
G oup A G oup B Reasso an To al
To al no. o obse ed
PUUV genomes
125 163 68 356
To al no. o PUUV
gene ic a ian s
46 82 56 184
no. o a ian s
obse ed mo e han
once (%)
14 (30.4) 22 (26.8) 3 (5.4) 39 (21.2)
no. o ansien
a ian s (%)
32 (69.6) 60 (73.2) 53 (94.6) 145 (78.8)
doi:10.1371/jou nal.pone.0064447. 003
Mic oe olu ion o Puumala han a i us
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be o e p edomina e (Table 1). This obse a ion is in conco dance
wi h o he s udies on PUUV p e alence [18], [36–37].
Ea lie analyses o ci cula ing PUUV in he same hos
popula ion a Konne esi e ealed wo dis inc genog oups, A
and B [19]. Bo h genog oups we e p esen in he i al popula ion
along he moni o ing pe iod wi h a sole excep ion; no a ian s o
he g oup B we e de ec ed in Oc obe 2009 (Table 1 and Fig. 3). I
canno be cla i ied he e i his is a e lec ion o he low numbe s o
ha sample se o ha genog oup suddenly disappea . Genog oup
B was gene ally mo e abundan and di e se (Table 1 and Fig. 3).
The a io o ci cula ing A and B a ian s was ela i ely s able
h ough he hos densi y cycle, excep du ing he decline phases in
2006 and 2009 (Table 1), and no clea signs o compe i ion
be ween genog oups we e de ec ed du ing he s udy. To ou
knowledge, compe i ion be ween han a i us a ian s has only been
Figu e 3. PUUV segmen geno ypes ci cula ing in he bank ole popula ion a Konne esi om 2005 o 2009. Rela ionships be ween S-,
M- and L-segmen geno ypes was cons uc ed using Ne wo k. Geno ypes o g oup A a e ep esen ed by ed ci cles and geno ypes o g oup B by
blue ci cles. The size o he ci cles is p opo ional o he numbe o ep esen a i es o each geno ype; and numbe s a e shown inside. Mos abundan
ci cula ing geno ypes (copious) a e designa ed in ed and blue, o he A and B genog oups, espec i ely. Spo adic geno ypes, de ec ed only once, a e
shown in black and spo adic geno ypes obse ed h ough se e al seasons a e shown in a squa e. Yellow ci cles a e median ec o s sugges ed by
Ne wo k. The numbe s on he link-lines show he numbe o mu a ions be ween geno ypes; he leng h o link-lines is no p opo ional o he numbe
o mu a ions. No e ha he apping e o was inc eased om 2007 hus numbe s om 2005 a e no di ec ly compa able o hose o 2007–09.
doi:10.1371/jou nal.pone.0064447.g003
Mic oe olu ion o Puumala han a i us
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s udied in cell cul u e [38–39] and co esponding da a o o he
RNA i uses a e limi ed (e.g. [40]).
The PUUV popula ion was composed o se e al segmen
geno ypes, L geno ypes being mo e nume ous han S o M
geno ypes. The ange o gene ic di e si y wi hin each genog oup
was compa able o all h ee segmen s and, o he S and M
segmen s also he in e -genog oup di e si y was simila . In
con as , L-segmen sequences showed su p isingly high di e si y
be ween he wo genog oups (Table 2). I should be emphasized
ha he a iabili y o he L segmen egion selec ed o ou analysis
(n 505–1040) is ypical o he segmen among PUUV s ains.
The e o e, a likely explana ion o he high in e -genog oup
di e si y o L segmen could in ol e an independen e olu iona y
his o y o hose genome segmen s o he Konne esi s ains.
Table 4. PUUV a ian s epea edly de ec ed in he bank ole popula ion.
no. o ep esen a i es a ian Geno ypes no. o apping si es apping seasons
SML
2
A4
4 11 24 1 5/05; 10/05
3
A11
6 1 1 2 10/08
53
A13
61317 5/07; 10/07; 5/08; 10/08; 5/09
2
A16
6 1 10 2 5/08; 10/08
2
A24
614 14 2 5/08; 10/08
2
A25
614 16 2 5/05; 10/05
2
A27
614 18 1 10/05
2
A29
615 92 5/08
5
A30
615 14 4 5/08; 10/08
8
A34
714 92 5/08; 10/08
4
A36
714 11 2 5/08; 10/08
2
A40
8131 5/09
4
A41
8 1 4 1 5/09
2
A43
82 4 1 5/09
2
B22
14 319 1 10/08
21
B3
5319 7 10/07; 5/08; 10/08
6
B5
5321 5 10/08; 5/09
2
B8
7321 1 10/08
2
B28
18 6 35 1 5/08
3
B34
20 14 15 1 5/08
2
B35
20 14 16 2 10/08
5
B39
21 15 11 4 10/07; 5/08
2
B40
21 15 14 1 5/07
6
B42
21 15 23 2 10/08
3
B48
22 15 29 3 5/05
3
B59
28 83 1 10/08
10
B60
28 8 4 5 10/08; 5/09
3
B73
28 11 42 10/08
4
B78
31 12 42 10/08
2
B62
28 86 1 10/08
12
B64
28 8 8 5 10/07; 5/08
4
B65
28 89 2 10/05
2
B67
28 811 2 5/05; 10/05
2
B57
28 5 18 1 5/05
4
B68
28 828 2 10/08
2
B72
28 10 30 1 10/05
7
R40
22 811 2 5/07; 5/08
4
R42
22 823 2 10/08; 5/09
4
R51
28 15 14 2 10/08
Mos abundan (copious) segmen geno ypes a e in bold and unde lined. Shadowed blocks con ain a ian s ha migh ha e de i ed om 1–2 poin mu a ions in one
o wo segmen s (e.g., s ains A13 and A16 could ha e o igina ed by accumula ing one o wo mu a ions, espec i ely, in he L segmen o A11 s ains).
doi:10.1371/jou nal.pone.0064447. 004
Mic oe olu ion o Puumala han a i us
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Phylogene ic analysis suppo ed his hypo hesis: he L sequences o
genog oup A sha ed a MRCA wi h PUUV So kamo s ain while
genog oup B sha ed he MCRA wi h PUUV Pallasja¨ i s ain,
bo h s ains (i.e., ce ain gene ic a ian o a i us species) belong
o he Finnish gene ic lineage (Fig. 1A–C). Di e en e olu iona y
his o ies o PUUV genome segmen s we e ea lie sugges ed o
accoun o obse ed a ia ion in he Alpe-Ad ian [41] and
La ian [42] lineages. Inciden ally, 60 km sou h-eas o he
Konne esi s udy a ea, a Pieksa¨ma¨ki, only a ian s o genog oup
A we e obse ed [43]. Whe he his is a consequence o
compe i ion be ween A and B a ian s o peculia i y o hei
geog aphical dis ibu ion emains unclea . Ex ended moni o ing
o he PUUV a ian s in ci cula ion is equi ed o cla i y his issue.
In Finland, PUUV p e alence luc ua es seasonally. Howe e ,
his s udy shows ha gene ic composi ion o he i al popula ion is
no dependen o such seasonali y. A each sampling poin he
PUUV popula ion was oughly enewed; some gene ic a ian s
(i.e., se s o h ee genome segmen s) we e obse ed pe sis ing
h oughou he s udy bu a la ge numbe we e obse ed only
empo a ily (Table 3). In con as , many o he segmen geno ypes
we e de ec ed h oughou he en i e s udy pe iod (Fig. 3). In o he
wo ds, he su i al o segmen geno ypes seems independen o he
ci cula ing a ian s, sugges ing ha genome segmen s pe sis s
independen ly and no in combina ion wi h o he segmen s wi hin
he i al popula ion. A ac o ha could accoun such ou come
may be he equen easso men be ween PUUV a ian s, bo h
closely- and dis an ly- ela ed.
The mos equen ly occu ing PUUV a ian s we e always
composed o copious geno ypes. T ansien a ian s seem o esul
om copious segmen geno ypes by accumula ing n subs i u ions,
sugges ing ha i al genome segmen s a e con inuously d i ing o
p o ide he PUUV popula ion wi h a di e se base om which o
espond o new s esso s. The h ee easso an a ian s ha we e
epea edly de ec ed h oughou he s udy we e composed o copious
segmen geno ypes (Table 4), sugges ing ha hey ha e a highe
chance o co-in ec ing a hos and/o ha e a p onounced abili y o
eplace spo adic segmen geno ypes in a double in ec ion scena io.
Va ian s composed o spo adic segmen geno ypes a e likely o be
eplaced when a co-in ec ion occu ed, hus only he dominan
(i.e., copious) a ian s emain.
A subs an ial po ion o co-ci cula ing PUUV s ains (19.1%)
possessed a easso an genome. Al oge he , 56 dis inc easso an
a ian s we e obse ed. The majo i y o easso an s (53 o 56)
we e ansien , sugges ing ha easso an s did no ou compe e
pa en al a ian s. The analysis o a la ge numbe o PUUV
genomes allowed o he de ec ion o all six possible easso an
ypes (Table 6). This inding p o ed ha all easso an ypes a e
iable in na u al condi ions, ag eeing wi h he in i o easso men
o o hobunya i uses [44]. Di e en PUUV easso an s we e
ound wi h di e en equencies: in he as majo i y o
easso an s (95.6%), ei he L and S segmen s o L and M
segmen s belonged o he same genog oup. Such a pa e n o
segmen s’ combina ion sugges s ha easso an ypes may di e in
hei eplica ion e icacy. Unique easso an a ian s we e
obse ed du ing e e y sampling e en o he s udy (excep 2006,
when no PUUV was de ec ed), and ended o be mo e p e alen in
he au umn, e en- ough he a es di e ences we e no s a is ically
signi ican . Seasonali y o easso men could be explained by
seasonal a ia ions in he age s uc u e o he hos popula ion.
Du ing au umn, mos bank oles a e younge and ecen in ec ions
Table 5. P opo ion o copious segmen geno ypes ha composed he PUUV a ian s de ec ed du ing he i e-yea s udy pe iod.
Va ian s Genog oup no. o a ian s
%o
copious
geno ypes o S
%o
copious
geno ypes o M
%o
copious
geno ypes o L
The mos equen ly
occu ing (
$
5 imes)
A3 100 100 100
B6
Repea edly obse ed (2–4
imes)
A11 74.1 66.7 29.6
B16
T ansien ( = 1 ime) A 32 44.6 58.7 50
B60
Reasso an 56 75 66.1 53.6
doi:10.1371/jou nal.pone.0064447. 005
Table 6. PUUV Reasso an s.
Combina ion o segmen s (S/M/L) no. o genomes Reasso an pa e n
no. o genomes (% o o al numbe
o easso an s)
ABA 6S-L/M 33 (48.5)
BAB 27
ABB 10 M-L/S 32 (47.1)
BAA 22
AAB 2 S-M/L3 (4.4)
BBA 1
Pa e n o combina ion o genome segmen . S, M and L s and o Small, Medium and La ge genome segmen s, espec i ely.
doi:10.1371/jou nal.pone.0064447. 006
Mic oe olu ion o Puumala han a i us
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