RESEARCH ARTICLE Open Access
E olu iona y his o y and molecula epidemiology
o abbi haemo hagic disease i us in he
Ibe ian Peninsula and Wes e n Eu ope
Fe nando Alda
1,2*
, Tania Gai e o
3
, Mónica Suá ez
3
, Tomás Me chán
4
, G ego io Rocha
4
, Ignacio Doad io
1
Abs ac
Backg ound: Rabbi haemo hagic disease i us (RHDV) is a highly i ulen calici i us, i s desc ibed in domes ic
abbi s in China in 1984. RHDV appea s o be a mu an o m o a benign i us ha exis ed in Eu ope long be o e
he i s ou b eak. In he Ibe ian Peninsula, he i s epidemic in 1988 se e ely educed he popula ions o
au och honous Eu opean wild abbi . To examine he e olu iona y his o y o RHDV in he Ibe ian Peninsula, we
collec ed i us samples om wild abbi s and sequenced a agmen o he capsid p o ein gene VP60. These da a
oge he wi h a ailable sequences om o he Wes e n Eu opean coun ies, we e analyzed ollowing Bayesian
Ma ko chain Mon e Ca lo me hods o in e hei phylogene ic ela ionships, e olu iona y a es and demog aphic
his o y.
Resul s: E olu iona y ela ionships o RHDV e ealed h ee main lineages wi h signi ican phylogeog aphic
s uc u e. All lineages seem o ha e eme ged a a common pe iod o ime, be ween ~1875 and ~1976. The Ibe ian
Peninsula showed e idences o gene ic isola ion, p obably due o geog aphic ba ie s o gene low, and was also
he egion wi h he younges MRCA.
O e all, demog aphic analyses showed an ini ial inc ease and s abiliza ion o he ela i e gene ic di e si y o RHDV,
and a subsequen educ ion in gene ic di e si y a e he i s epidemic b eakou in 1984, which is compa ible wi h
a decline in e ec i e popula ion size.
Conclusions: Resul s we e consis en wi h he hypo hesis ha he cu en Ibe ian RHDV a ose om a single
in ec ion be ween 1869 and 1955 (95% HPD), and ende ed a empo al pa e n o appea ance and ex inc ion o
lineages. We p opose ha he ising posi i e selec ion p essu e obse ed h oughou he his o y o RHDV is likely
media ed by he hos immune sys em as a consequence o he gene ic changes ha ende ed he i us i ulen .
Consequen ly, his ela ionship is sugges ed o condi ion RHDV demog aphic his o y.
Backg ound
Vi uses con aining RNA as hei gene ic ma e ial usually
ha e a g ea capaci y o adap . This is so because he
i al polyme ases esponsible o eplica ing he genome
ha e a high e o a e and he e o e, many genomic a -
ian s a e c ea ed a a high gene a ion a e [1,2]. This
high mu a ion a e o RNA i uses makes hem excel-
len models o add essing e olu iona y p ocesses such
as epidemic in asions, since hei ecological and e olu-
iona y dynamics occu a simila ime scales [3-5].
RNA i uses a e he causal agen s o many eme ging
diseases and consequen ly he appea ance, o eappea -
ance o diseases caused by hese i uses is no in e-
quen [2]. Howe e , o he ecological, social, heal h o
beha iou al ac o s besides hei high a e o gene ic
a ia ion can play an impo an ole in he eme gence
o a disease [6].
Rabbi haemo hagic disease (RHD) is a ecen disease
ha was de ec ed o he i s ime in 1984 in China,
and a ibu ed o abbi s (O yc olagus cuniculus)
impo ed om he o me Ge man Democ a ic Repub-
lic. The disease was desc ibed o cause sudden dea h
wi hou appa en de e io a ion o he abbi ’s body con-
di ion, al hough haemo haging in he lungs was a
* Co espondence: [email p o ec ed]m
1
Dp o. Biodi e sidad y Biología E olu i a, Museo Nacional de Ciencias
Na u ales (CSIC). José Gu ié ez Abascal 2, 28006 Mad id, Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Alda e al.BMC E olu iona y Biology 2010, 10:347
h p://www.biomedcen al.com/1471-2148/10/347
© 2010 Alda e al; licensee BioMed Cen al L d. 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 (h p://c ea i ecommons.o g/licenses/by/2.0), 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 wo k is p ope ly ci ed.
equen obse a ion [7,8]. He ea e , he disease apidly
sp ead o o he Asian [9] and Eu opean [10] coun ies.
The i s epo o RHD in Eu ope was in I aly in 1986
[11], and al eady in 1988 i was de ec ed in wild abbi s
in Spain [12] and in domes ic abbi s in Russia, he
Middle Eas , A ica, Ame ica and India [10].
The ae iological agen o RHD is a single s anded
posi i e sense RNA i us belonging o he amily Calici-
i idae [13,14]. Since i s disco e y, many esea ch e o s
ha e been de o ed o i s s udy. Howe e many issues
emain unclea o con o e sial, such as i s e olu iona y
o igin o he causes o he apid succession o epidemics
in such a sho pe iod o ime [15,16].
Se e al hypo heses ha e been p oposed o explain he
o igins o abbi haemo hagic disease i us (RHDV), o
example: (i) sp ead o b own ha e synd ome i us - a
closely ela ed calici i us - o he abbi [17], (ii)
changes in he p ope ies o a non pa hogenic i us ha
make i i ulen ; o (iii) he i usa ose oma i us
in ec ing ano he species [18]. The de ec ion o RHDV
speci ic an ibodies and RNA agmen s in abbi se um
samples om 1955 o 1980 [19-21], howe e , seem o
suppo he idea ha he i us was al eady ci cula ing in
Eu ope in an a i ulen o m be o e he i s epidemic
was de ec ed in China in 1984 [16].
Se e al causes ha e also been p oposed o explain he
apid expansion o RHD. Ini ially i was assumed ha
China was he o igin om whe e he i us sp ead caus-
ing se e e epidemics [18,22]. Howe e , subsequen phy-
logene ic s udies ha e demons a ed ha he Chinese
s ains o he i us o igina ed in Eu ope and cu en ly
ci cula ing RHDV s ains do no ha e a single o igin.
Ra he , he i us seems o ha e o igina ed a leas wice
in he pas : i s ly in Eu ope, wi hou causing he dis-
ease, and la e in he abbi s expo ed o China [16,23].
These s udies ha e also de ec ed a la ge numbe o
RHDV e olu iona y lineages ha show low gene ic
di e gence bo h wi hin and among phylogene ic g oups
[17,19,24,25]. Some o hese lineages do no p esen a
clea geog aphic s uc u e, bu hey do e lec a em-
po al s uc u e whe eby some lineages become ex inc
o less equen , while o he s de i ed om hem a e
able o pe sis and cause new ou b eaks o he disease
[16,17,24]. Despi e showing no signi ican gene ic di e -
ences in di e en geog aphic egions, RHDV does di e
in i s epidemiology and i ulence [15,26-29].
Some egions, no wi hs anding he d as ic educ ions
su e ed by abbi popula ions a e a i s epidemic,
ha e seen a decline in he ini ially high i ulence o
RHDV and his has enabled popula ions o g adually
eco e [15,26]. In he Ibe ian Peninsula, howe e ,
whe e abbi s a e o g ea ecological and economic sig-
ni icance [30,31], RHD s ill a ec s many egions in
which abbi popula ions ha e been decima ed o e en
ex inguished [28,32].
This he e ogenei y in he way RHDV a ec s abbi s
in di e en geog aphic egions may indica e ha he e
a e ac o s such as he hos , clima e o popula ion size
ha de e mine he epidemiology o he i us [15,28].
Rega ding he hos , in he Ibe ian Peninsula, he
Eu opean abbi is an au och honous species. Analyses
o mi ochond ial and nuclea ma ke s ha e e ealed
wo highly di e gen lineages in he Eu opean abbi
wi hin he Ibe ian Peninsula ha co espond o sub-
species O. c. algi us and O. c. cuniculus [33-36]. These
wo lineages a e he esul o wo di e gen popula-
ions ha e ol ed sepa a ely o a long ime in wo
glacial e ugia, one loca ed in he sou hwes o he
Ibe ian Peninsula and ano he in he no heas . A
pos -glacial expansion migh ha e c ea ed a con ac
zone in he cen e o he Ibe ian Peninsula [37].
Fu he mo e, he no he n O. c. cuniculus expanded
ou side o he Ibe ian Peninsula and o igina ed all
Eu opean abbi s, as well as hose o No h A ica,
Ame ica, Aus alia, New Zealand and all he domes ic
b eeds. This expansion o igina ed an in ense bo le-
neck e ec ha diminished conside ably he gene ic
di e si y in he O. c. cuniculus popula ions ou side o
he Ibe ian Peninsula [34,38-40]. Consequen ly, he
abbi in he Ibe ian Peninsula shows he la ges
gene ic di e si y ac oss he wo ld dis ibu ion o he
species [34,41]. This gene ic dis inc i eness migh pose
di e en selec i e p essu es and e olu iona y con-
s ain s o RHDV compa ed o o he egions. How-
e e , so a , compa a i e s udies including he wo
abbi lineages a e sca ce [16,42].
The objec i es o he p esen s udy we e: o examine
he e olu iona y his o y o RHDV in he Ibe ian Penin-
sula and o in e he i us’demog aphic his o y, wi h
special ocus on i s Wes e n Eu opean dis ibu ion.
Gi en he gene ic cha ac e is ics o his i us, we
would expec o obse e: (i)ahighgene icdi e si y
and (ii) a empo al gene ic s uc u e due o i s high
mu a ion a e. In e ms o he possible o igin o he
i us and disease in he Ibe ian Peninsula, we could
specula e ha (iii)i a i ulen o mo heRHDVwas
in oduced, he age o he mos ecen common ances-
o should co espond o he ime elapsed since he
appea ance o he disease, while i he i us al eady
exis ed in an a i ulen o m, i s age will be olde . Also,
acco ding o he ecen and apid geog aphic expan-
sion o RHDV we would expec (i ) i s demog aphic
his o y o e lec signi ican g ow h o i s popula ions
and ha ( ) i he in ensi y o he epidemics has
dec eased, o a leas he numbe o hos s, a subse-
quen decline should also be obse ed.
Alda e al.BMC E olu iona y Biology 2010, 10:347
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Me hods
Sampling and RNA isola ion, ampli ica ion and
sequencing
Rabbi s we e collec ed in Spain be ween 2003 and 2007
(Addi ional File 1). Abou 125 mg o lung issue we e
homogenizedin1.25mlo s e ilePBS(8mM
Na
2
HPO
4
,1.5mMKH
2
PO
4
,2.7mMKCl,137mM
NaCl, pH7.4) and cen i uged a 2500 × g o 15 min.
The supe na an was collec ed and s o ed a -20°C upon
RNA ex ac ion.
Vi al RNA was ex ac ed om 100 μlo helung
homogena e using T iPu e eagen (Roche) ollowing
he manu ac u e ’s ins uc ions. A agmen o he
majo capsid p o ein gene VP60 was ampli ied in all
samples using he p ime s RHDV1, RHDV2, RHDV3
and RHDV4 [19].
The p ime s RHDV1 and RHDV4 we e used o RT-
PCR ampli y a 698 bp agmen co esponding o posi-
ions 6096-6794 o he RHDV [43]. The RT-PCR eac-
ion was conduc ed unde he condi ions indica ed in
he AccessQuick RT-PCR Sys em ki (P omega) and he
he mocycling p og am consis ed o 45 min a 48°C o
cDNA syn hesis, ollowed by 2 min a 95°C, 40 cycles o
1 min a 95°C, 40 s a 64°C, 1 min a 72°C, and a inal
ex ension o 5 min a 72°C. Nex , we pe o med a
nes ed PCR wi h he p ime s RHDV2 and RHDV3 o
ampli y a 573 bp agmen co esponding o posi ions
6135-6719 o he RHDV. The PCR eac ion con ained
200 μMo dNTPs,0.2μM o each p ime , 1 μg/μlo
BSA, 1 U o Taq polyme ase (Eppendo ), 2.5 μlo PCR
bu e 10X (500 mM KCl, 100 mM T is-HCl pH8.3,
15 mM Mg
2+
) and 0.5-2 μl o a 1:100 dilu ion o he
RT-PCR p oduc . The PCR p og am in ol ed an ini ial
dena u ing s ep o 2 min a 95°C, 40 cycles o 1 min a
95°C, 1 min a 64°C and 1 min a 72°C, and a inal
ex ension s ep o 5 min a 72°C. All he RNA ex ac ion
and ampli ica ion p ocesses we e ca ied ou in a labo a-
o y speci ically equipped o his pu pose. All eac ions
included posi i e and nega i e con ols. PCR p oduc s
we e pu i ied using ExoSAP-IT (GE Heal hca e) and
sequenced using he BigDye Te mina o 3.1 ki
(Applied Biosys ems) and he p ime s RHDV2 and
RHDV3 in an au oma ed sequence ABI3730.
Phylogene ic analysis
Ch oma og ams we e checked isually and he consen-
sus sequence was cons uc ed o each sample using
Sequenche 4.6 (Gene Codes Co po a ion). Nucleo ide
sequences we e ansla ed in o amino acids using Mac-
Clade 4.05 [44] and manually aligned wi h o he homo-
logous sequences a ailable in GenBank.
Recombina ion has been p e iously desc ibed in RHDV
[45,46]. Al hough his seems o be a a he a e phenom-
enon ha does no ad e sely a ec a e es ima ions [16],
we did no include sequences ha had been iden i ied as
pu a i e ecombinan s [16,45,46]. The alignmen in e ed
om all o he a ailable da a (AD, n= 151, 563 bp) was
subdi ided in smalle da a se s comp ising sequences o
he main geog aphic a eas p e iously analyzed: Ge many
(GER), F ance (FRA1), Uni ed Kingdom (UK), China
(CHI) and Ibe ian Peninsula (IB) (Addi ional File 2).
Fo a wide compa ison, we included a ailable da a on
non-o e lapping VP60 sequences which could be o
in e es because o hei empo al o geog aphic o igins.
Thus, an alignmen o F ench RHDV isola es, consis ing
o non-o e lapping sequences o 501 bp a he 3’end o
he VP60 gene, was buil (FRA2, n= 21) (Addi ional
File 2) [24,47].
To e alua e he gene ic a iabili y o RHDV o each
o hese da a se s om he main geog aphic a eas we
calcula ed hei gene di e si y (H
d
), nucleo ide di e si y
(π) and numbe o polymo phic si es (S)usingDnaSP
5.10.1 [48].
Phylogene ic ela ionships among all he RHDV
s ains (AD, n= 151) we e in e ed using di e en phy-
logene icme hods.Asanou g oupweused ou abbi
calici i us (RCV) sequences (X96868, GQ166866,
EU871528, NC011704), a seemingly a i ulen , an igeni-
cally and gene ically ela ed o RHDV [49].
The e olu iona y model ha bes i ed ou da a,
acco ding o he Akaike In o ma ion C i e ion, was cal-
cula ed in jModelTes 0.1.1 [50]. Fi s ly, we pe o med a
Maximum Likelihood (ML) analysis, and used he bes
e olu iona y me hod o ou da a as ob ained in jMo-
delTes 0.1.1, and allowed he p og am PhyML 3.0 [51]
o op imize he ee opology and he alues o gamma
and p opo ions o in a ian si es. Suppo o he ML
ees was assessed by 1000 boo s ap eplica es. Sec-
ondly, a Bayesian In e ence (BI) analysis was pe o med
using M Bayes 3.1.2 [52], simula ing ou simul aneous
Ma ko chains (MCMC) o 4 × 10
6
gene a ions each,
and using a sampling equency o 100 gene a ions. The
i s 250,000 gene a ions we e disca ded as bu n-in. The
BI analysis was pe o med conside ing one and h ee
pa i ions o he da a co esponding o he 1
s
,2
nd
and
3
d
codon posi ions each assigned di e en subs i u ions
models. Bayesian pos e io p obabili ies we e ob ained
o assess he obus ness o he BI ees.
Las ly, o es o he p esence o s a iscally signi ican
geog aphical clus e ing in RHDV, we pe o med ando-
miza ion es s on h ee ee-shaped s a is ics: he pa si-
mony sco e (PS), he associa ion index (AI) and he
monophyle ic clade s a is ic (MC) using BaTS 1.0 [53].
Randomiza ions we e pe o med ac oss he pos e io
dis ibu ion o ees ob ained om M Bayes 3.1.2, hence
accoun ing o phylogene ic unce ain y.
Alda e al.BMC E olu iona y Biology 2010, 10:347
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Es ima ion o e olu iona y a es, da es and pas
popula ion demog aphic his o y
The o e all popula ion dynamics o RHDV (AD) was
in e ed in BEAST 1.5.3 [54] using he coalescen Baye-
sian Skyline Model [55] and an unco ela ed logno mal
elaxed clock. Fou independen analyses we e pe -
o med unde he bes - i subs i u ion model and he
SRD06 pa i ion model, ecommended o p o ein cod-
ing genes [54,56], and un o 5 × 10
7
gene a ions and a
sampling equency o 1000 s eps. All ou pu gene a ed
we e analyzed in T ace 1.5 o es o con e gence and
mixing, and we e used o es ima e he subs i u ion a e
and ime o he mos ecen common ances o
( MRCA) o RHDV o each o he majo lineages ound
and all he geog aphic egions.
We explo ed he changes h ough ime o he ela i e
gene ic di e si y (N
e
τ,whe eτis he a e age gene a ion
ime) o RHDV using he Bayesian Skyline Plo (BSP)
me hod [55]. Median and 95% highes pos e io densi y
in e als (HPD) we e ob ained wi h T ace 1.5.
We also es ed o demog aphic changes using sum-
ma y s a is ic based me hods. The e ec i e popula ion
size pa ame e (θ
0
)unde ag ow h-declinepopula ion
model (θ
1
ixed a 1,000,000) was calcula ed o RHDV
in DnaSP 5.10.1. Th ee ime pe iods we e de ined based
on he esul s ob ained in he BSP analysis: a i ulen
pe iod (be o e 1984), epidemic b eakou (a e 1984)
and ecen pe iod (a e 2000). Because samples sizes
di e ed among ime pe iods and, consequen ly, e ec i e
popula ion size es ima es migh be biased, we con-
s uc ed o he “a e 1984”and “a e 2000”pe iods 10
da a se s wi h 9 andom samples. These esul s we e
a e aged and compa ed wi h he minimum sample size
o RHDV be o e 1984.
Selec ion analyses
To es ima e selec ion p essu es be o e and a e he i s
epidemic o RHDV in 1984, we calcula ed he a io
be ween non-synonymous (d
N
) and synonymous (d
S
)
subs i u ions o each indi idual codon using he ixed-
e ec s likelihood (FEL) and andom e ec s likelihood
(REL) me hods [57]. These analyses we e pe o med in
he online package Da amonkey [58]. Absence o ecom-
bina ion poin s was checked using GARD [59]. We
es ablished a alue o a= 0.1 o FEL and BF = 20 o
REL. In bo h cases, he d
N
/d
S
a io was calcula ed using
a neighbou -joining ee based on he bes - i e olu ion-
a y model.
As indica ed abo e, o accoun o he e ec o di e -
en sample sizes in he analysis, we analyzed 10 da a
se s wi h 9 andom samples isola ed a e 1984, and
compa ed wi h he esul s om RHDV isola ed be o e
he i s epidemic.
Resul s
RHDV was de ec ed in 47 wild abbi s om Spain and i
was possible o ob ain a 563 bp sequence om each
sample. All he sequences we e analyzed unambiguously
and we de ec ed no e idence o in ec ion by mo e han
one i al s ain in he same indi idual (GenBank acces-
sion numbe s: HQ198325-HQ198371, Addi ional File 1).
Along wi h he homologous sequences a ailable in
GenBank, we cons uc ed an alignmen o 151 sequences
(AD). F om his alignmen we ex ac ed he da a se s
o Ibe ian Peninsula (IB, n= 71), F ance (FRA1, n=8),
Uni ed Kingdom (UK, n= 49), Ge many (GER, n=7)and
China (CHI, n= 10). The alignmen o non o e lapping
sequences om F ance (FRA2) con ained 21 sequences
isola ed be ween 1988 and 2003 (Addi ional File 2).
The 151 RHDV sequences analyzed cons i u ed 122
unique haplo ypes (H
d
= 0.993 ± 0.003). The geog aphic
egions ha showed he highes gene ic di e si y we e
F ance and Ge many. In con as , among he 71 Ibe ian
samples we ound 45 haplo ypes, which ep esen ed he
lowes RHDV di e si y (H
d
= 0.968 ± 0.010) (Addi ional
File 3).
The bes e olu iona y model es ima ed by jModelTes
0.1.1 o ou da a was T N+I+G. The gamma shape
pa ame e was G = 0.574 and he p opo ion o in a i-
able si es I = 0.172.
The phylogene ic me hods used ende ed cong uen
opologies (Figu e 1). Bo h analyses indica ed h ee main
lineages (Lineage I, II and III) in which mos o he RHDV
samples we e included. Lineage I included RHDV s ains
isola ed in di e en Eu opean egions (Figu e 1), some o
which had been isola ed in he UK be o e he desc ip ion
o he disease in 1984 and o he s isola ed du ing he i s
ou b eaks in F ance, Ge many and Spain (AST/89 and
MC-89). Also, his lineage included all he Ibe ian RHDV
samples isola ed in Spain and Po ugal om 1994 o 2007
(Figu e 1). Six Ibe ian clades we e desc ibed based on p e-
ious indings (Genog oup 1 [24], IB1, IB2 and IB3 [42])
and he new isola es om his s udy (IB4, IB5, IB6). No
clea geog aphic s uc u e was obse ed among he Ibe -
ian samples, al hough mos o he clades we e es ic ed in
ime, wi h he excep ion o IB3 ha was he mos wide-
sp ead clade bo h in ime and space (Figu e 1, Addi ional
File 1). In Lineage II, we exclusi ely ound Eu opean
s ains o RHDV, mainly om he UK, Ge many and
F ance. Con e sely, Lineage III included s ains om di -
e en con inen s. Ge man s ains we e ound a a basal
posi ion wi h espec o all he Chinese s ains, excep a
sample om 1984, which seemed mo e ela ed o hose o
Lineage I and II, bu wi hou boo s ap o pos e io p ob-
abili y suppo . This lineage also con ained samples om
he Uni ed S a es, F ance and Reunion Island in he Indian
Ocean. Con e sely, se e al RHDV s ains om di e en
Alda e al.BMC E olu iona y Biology 2010, 10:347
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Figu e 1 Phylogene ic ee ob ained by Bayesian in e ence o all he RHDV s ains analyzed. Numbe s abo e b anches indica e
boo s ap alues abo e 50 o ML analysis, and pos e io p obabili ies abo e 0.80 o BI a e shown below b anches. Yea and egion o isola ion
is indica ed o all samples. 95% HPD o he MRCA o he main lineages ound a e indica ed. Ibe ian clades wi hin Lineage I a e shown based
on p e ious s udies [24,42] and new da a. Names in i alics indica e a i ulen RHDV s ains.
Alda e al.BMC E olu iona y Biology 2010, 10:347
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egions (F ance, Ge many, Czech Republic, China, Mexico
and New Zealand) and s ains p io o 1989 we e no
included in any o he h ee main lineages iden i ied (Fig-
u e 1).
O e all, he es pe o med o he p esence o phylo-
geog aphic s uc u e using he PS and AI s a is ics,
s ongly ejec ed he hypo hesis o panmixia (obse ed
PS o 21.986, expec ed PS o 71.718 [P< 0.0001];
obse ed AI o 1.133, expec ed AI o 10.175 [P<
0.0001]). Fu he mo e, he MC s a is ic indica ed ha
he co ela ion be ween phylogeny and axa loca ion was
only signi ican o he samples om he Ibe ian Penin-
sula (obse ed MC
IB
o 41.046, expec ed MC
IB
o 3.681
[P= 0.01]), UK (obse ed MC
UK
o 21.040, expec ed
MC
UK
o 2.757 [P= 0.01]) and China (obse ed MC
CHI
o 6.354, expec ed MC
CHI
o 1.175 [P=0.01]), ha
appea ed exclusi ely in Lineages I, II and III, espec-
i ely, wi h he excep ion o he “ancien ”s ains om
he UK ha we e ound in Lineage I. On he o he
hand, he RHDV isola es om F ance and Ge many ha
appea ed in all lineages, and in many cases we e closely
ela ed o s ains om o he egions (Figu e 1), did no
show a signi ican geog aphic co ela ion.
The coalescence based Bayesian analysis implemen ed
in BEAST 1.5.3 e ealed ha he MRCA o all he
RHDV da a da ed back o ~1884 (95% HPD 1941.3-
1730.7) and es ima ed an e olu iona y a e o 5.48 × 10
-
4
subs i u ions/si e/yea (95% HPD 2.79 × 10
-4
-8.10 ×
10
-4
). The h ee main clades ound showed simila 95%
HPD in e als o hei MRCA, om ~1875 o ~1976
(Figu e 1), sugges ing a close ime o eme gence o
hese lineages.
When he main geog aphic egions we e conside ed,
he younges MRCA was eco e ed o he Ibe ian
RHDV (~1936, 95% HPD 1955.7-1869.2) ollowed by
he isola es om he UK (~1898, 95% HPD 1940.3-
1822.5). The emnan egions showed wide 95% HPD
in e als ha we e simila o he age o he comple e
RHDV da a se (Figu e 2).
Bayesian Skyline Plo analysis o he demog aphic his-
o y o RHDV indica ed ha , as a whole, he ela i e
gene ic di e si y o RHDV inc eased in he 1950’s ol-
lowed by a s a iona y phase un il he mid 1980’s. The e-
a e , RHDV ela i e gene ic di e si y s a ed o decline,
especially in he la e 1990’s when his educ ion was
mo e d as ic (Figu e 3). Because samples we e no
balanced ac oss yea s, he same analysis was pe o med
emo ing hose sequences da ed be o e 1984 and p e-
sumably a i ulen s ains [15]. The plo eco e ed
showed he same demog aphic and empo al pa e n,
al hough he s a iona y phase was no as clea ly di e -
en ia ed (da a no shown).
The e ec i e popula ion size pa ame e θ
0
es ima ed
o he h ee main pe iods iden i ied in he BSP also
showed e idence o change in N
e
o RHDV ac oss ime.
An inc ease o N
e
was obse ed be ween he a i ulen
pe iod, be o e 1984 (θ
0
= 11.597), and he samples a e
he i s epidemic b eakou , a e 1984 (θ
0
= 20.620 ±
2.407, a e age alue based on 10 da a se s o 9 andom
sequences). Subsequen ly, a educ ion o N
e
was
obse ed o he mos ecen isola es a e 2000 (θ
0
=
14.713 ± 5.812).
Adap i e selec ion analyses sugges ed an inc ease in
he selec i e p essu e o RHDV a e he i s epidemic
b eakou (Table 1). Be o e 1984 no codons we e iden i-
ied as posi i ely selec ed by any me hod, and 29 codons
we e conside ed unde nega i e selec ion by FEL, bu
no by he REL me hod. On he o he hand, a e 1984,
codon 137 o he alignmen was iden i ied as posi i ely
selec ed conside ing all he da a a e 1984 and in 9 ou
Figu e 2 Es ima es o he MRCA o he main geog aphical
egions analyzed. Do s indica e median MRCA alues and blue
lines ep esen 95% HPD.
Figu e 3 Bayesian Skyline plo analysis o RHDV.G aphical
ep esen a ion o ela i e gene ic di e si y (N
e
τ) changes ac oss ime
o RHDV. Black line indica es he median and blue lines ep esen
95% HPD.
Alda e al.BMC E olu iona y Biology 2010, 10:347
h p://www.biomedcen al.com/1471-2148/10/347
Page 6 o 10
o 10 andom da a se s analysed. O he codons (e.g.
codon 155) we e iden i ied as posi i ely selec ed, bu
no consis en lyamong he andomda ase s.TheFEL
me hod also de ec ed 100 nega i ely selec ed codons in
all he samples a e 1984 and 49.500 ± 5.126 codons
unde nega i e selec ion conside ing he 10 andom
da a se s (Table 1).
Discussion and Conclusion
Gene ic a iabili y and o igin o RHDV in he Ibe ian
Peninsula
Al hough haplo ype di e si y was high and simila
among egions, he nucleo ide di e si y o he RHDV
s ains a ied among geog aphic egions. In he Ibe ian
Peninsula, UK and China, nucleo ide di e si y was much
lowe han in F ance o Ge many, and was equi alen o
hal he o e all nucleo ide di e si y (Addi ional File 3).
The low gene ic di e si y obse ed in hese egions
could sugges ha he i us o igina ed om a single
in oduc ion (e.g. as i is known o ha e occu ed in
China [7]), o ha i is he ou come o geog aphic isola-
ion, (e.g. he case o he B i ish Isles). Thus, he gene ic
di e si y o he i us in hese egions could ha e been
educed as a consequence o a ounde e en in he o -
me case, o o d i in he la e [60].
To da e in he Ibe ian Peninsula, i was unknown i (i)
RHDV was al eady ci cula ing in an a i ulen o m, (ii)
he ewasasinglein oduc iono RHDVo (iii)subse-
quen con ac wi h o he s ains occu ed. The phyloge-
ne ic da a ob ained he e indica e ha all he ield s ains
o RHDV in he Ibe ian Peninsula ha e a common ances-
o and a e closely ela ed o s ains AST/89 and MC-89
isola ed du ing he i s RHD ou b eaks in Spain (Figu e
1). The age o he MRCA o he Ibe ian s ains is he
younges o all he egions analyzed (Figu e 2), bu s ill
p eda es he i s RHD ou b eak in 1984 (~1936, 95%
HPD: 1955.7-1869.2). Hence, i may be deduced ha he
i us in oduced in he Ibe ian Peninsula came om a
mo e ancien lineage al eady ci cula ing in Eu ope. This
ac was con i med by he samples included in Lineage I
oge he wi h AST/89 and MC-89, isola ed in di e en
egions o Eu ope since 1958 (Figu e 1).
Dispe sal o RHDV is as and e ec i e in sho dis-
ances [61] because i is p ima ily ansmi ed by di ec
con ac be ween sick animals o indi ec ly by dead ani-
mals o con amina ed ood [8]. Howe e , long dis ances
o geog aphic ba ie s, such as hose exis ing in G ea
B i ain o he Ibe ian Peninsula, can a ec i s dispe sal
[42]. The anspo o abbi s o hei p oduc s can also
play an impo an ole in passi e dispe sal o he i us
[18]. Acco ding o he lack o any geog aphic s uc u e
o he Ibe ian s ains (Addi ional File 1), i seems ha
mo emen o abbi s could ha e p omo ed RHDV dis-
pe sal wi hin he Ibe ian Peninsula [32,62]. Howe e ,
conside ing he signi ican global geog aphic associa ion
o all he Ibe ian s ains, his is p obably no he case
o he impo and expo o abbi s ab oad. Only one
i al s ain isola ed in F ance in 2000 was ound in
he Ibe ian RHDV g oup (Figu e 1) [42], which could
ha e c ossed he moun ains anspo ed by insec s o
he wind [63,64]. The e o e, he Py enees migh also
ep esen an e ec i e ba ie o RHDV, as i has been
shown o many o he o ganisms [65].
Owing o he apid e olu iona y a e o RNA i uses,
hei molecula phylogenies e eal bo h spa ial and em-
po al pa e ns [4]. In F ance, six gene ic g oups ha e
been desc ibed o RHDV ha a e mos ly consis en wi h
hei da es o isola ion bu no hei geog aphical loca ion
[24,47]. In he Ibe ian Peninsula, some clades spanned a
wide ange o ime isola es, bu mos we e es ic ed in
ime (Figu e 1, Addi ional File 1). Fo example, clade IB1
was basal o all he o he Ibe ian clades, including hose
s ains isola ed in 1989. Al hough his ela ionship was
no suppo ed by he ML analysis, i migh indica e ha ,
in he pas , o he ela ed s ains migh ha e occu ed
(e.g. Genog oup 1, IB1, IB4) bu a e cu en ly ex inc .
This empo al pa e n, whe eby lineages successi ely
become ex inc and o he s appea , is ypical o RNA
i uses [66,67], including calici i us [68], and is mainly
condi ioned by posi i e selec ion [69].
Phylogene ic ela ionships o RHDV
In gene al, he phylogene ic ela ionships esol ed he e
a e in line wi h he indings o s udies ha ha e ana-
lyzed di e en po ions o he genome o geog aphic
egions [15-17,19,23,24,42,47].
Th ee di e gen g oups we e eco e ed om ou phy-
logene ic econs uc ion (Figu e 1). These h ee g oups
Table 1 Adap i e selec ion analyses (FEL and REL) o RHDV pe o med in Da amonkey
d
N
/d
S
FEL REL
Posi i e selec ed Nega i e selec ed Posi i e selec ed Nega i e selec ed
Be o e 1984
All da a 0.173 0 29 0 0
A e 1984
All da a 0.162 1 100 N/A N/A
A e age (10 da a se s) 0.158 ± 0.017 1.600 ± 0.843 49.500 ± 5.126 2.400 ± 5.542 24.200 ± 28.389
Alda e al.BMC E olu iona y Biology 2010, 10:347
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(LineagesI,IIandIII)closelyma ched helineages
ob ained in a phylogeny based on 43 comple e
sequences o he VP60 gene, in which wo majo
lineages we e desc ibed, one o which was di ided in
wo well suppo ed g oups [45]. Thus, Lineages I, II and
III desc ibed he e would co espond o g oups Ia, Ib
and II espec i ely eco e ed in he phylogeny based on
he comple e VP60 gene sequence [45]. Howe e , in
o he wo ks in which pa ial sequences o he VP60
gene we e examined, a much la ge numbe o RHDV
g oups we e de ined, many o which a e no suppo ed
[15,19,42,70,71].
As explained abo e, he gene ic s uc u e o RHDV
has usually been desc ibed as being ela ed o i s yea o
isola ion [17,19,24,41] and wi hin empo al g oups, a
link o i s place o o igin eme ges [41]. In ou da a,
howe e , we obse ed a global geog aphical dis ibu ion
o he lineages a he han a empo al s uc u e o
RHDV sequences (Figu e 1).
Theonlylineage ha didno con ainsamples
be o e he i s RHD ou b eak was Lineage III, and we
could conside ha all he RHDV samples in his line-
age a ose om al eady i ulen s ains. On he o he
hand, Lineage I and II included ei he samples iso-
la edbe o e he i s ou b eako a i ulen s ains.
Howe e , hese lineages we e no olde han Lineage
III. All lineages showed simila MRCA es ima es,
sugges ing a common ime o eme gence o all o
hem, be ween ~1875 and ~1976. This obse ed
eme gence p e-da es he documen ed eme gence o
RHD, a possibili y ha Ke and colleagues had
al eady sugges ed [16].
The RHDV s ains om he Ibe ian Peninsula, UK and
China appea ed, almos exclusi ely, in Lineages I, II and
III espec i ely. Howe e , o he s ains such as hose om
F ance and Ge many appea ed in all he main clades (Fig-
u e 1). The p esence o s ains om di e en e olu iona y
lineages in he same egion could be a ibu able o a
g ea e ex en o ances al polymo phism and/o o highe
low o i uses in ce ain a eas o cen al Eu ope [16],
compa ed o mo e isola ed egions such as he Ibe ian
Peninsula and he B i ish Isles. Fu he mo e, hese isola ed
egions we e hose wi h he younges MRCA (Figu e 2),
sugges ing ha RHDV migh ha e o igina ed in con inen-
al Eu asia [16] and subsequen ly colonized he islands
and sou he n peninsulas. Howe e , di e en sampling
sizes migh a ec he accu acy o hese es ima es, and con-
clusions should be d awn wi h cau ion.
Demog aphic his o y and na u al selec ion
The subs i u ion a e es ima ed o he comple e RHDV
da a se (5.48 × 10
-4
subs i u ions/si e/yea ) was e y simi-
la o ha epo ed ecen ly o a simila da a se (7.7 ×
10
-4
subs i u ions/si e/yea , 95% HPD: 3.9 × 10
-4
- 11.0 ×
10
-4
[16]). Howe e , hese a es we e mo e han an o de
o magni ude lowe han he a e es ima ed o he same
gene egion using a ML me hod (1.3 × 10
-3
subs i u ions/
si e/yea , 0.59 × 10
-3
- 2.1 × 10
-3
[72]), and mo e han wo
o de s o magni ude lowe han he a e es ima ed o he
capsid gene o o he calici i us [68]. Al hough he es i-
ma ed subs i u ion a e is wi hin he ange obse ed o
single s anded RNA i uses, i mo e closely esembles he
lowe subs i u ion a es eco ded o i uses ansmi ed
by a h opod ec o s [72,73]. The ela i ely low subs i u-
ion a e o his i us could he e o e explain he long
e m pe sis ence o ce ain s ains in some popula ions
many yea s a e hei in oduc ion [74].
The BSP me hod, which allows he i ing o di e en
demog aphic scena ios ac oss ime [55], dis inguished
h ees agesin hehis o yo RHDV.Fi s ,ag ow h
s age be ween 1950 and 1960; second, a s a iona y s age
be ween 1960 and 1984 and inally a s age o decline
beyond 1984 ha became s eepe a he end o he
1990s (Figu e 3). The di e en demog aphic ends
be o e and a e he desc ip ion o he i s RHD ou -
b eak ma ch he di e en pa e ns o na u al selec ion
o each ime ange. Thus, du ing he g ow h and s a-
iona y s ages, we de ec ed no e idence o signi ican
posi i e selec ion, whe eas a e 1984 e idence did
eme ge o posi i e selec ion, as well as an inc ease in
he numbe o codons unde nega i e selec ion o e he
VP60 agmen analyzed (Table 1).
In ou da a, codon 137, which is loca ed in egion E o
he majo capsid gene VP60, showed he s onges e i-
dence o posi i e selec ion. The p esence o posi i e
selec ionin his egionag eeswi hp e iouss udieso
RHDV and o he calici i us and wi h he ac ha his
egion con ains he main an igenic de e minan s [68,75].
Fu he mo e, he associa ion be ween posi i e selec ion
and an igenici y sugges s ha RHDV e olu ion is mainly
d i en by he hos immune esponse [75].
Thus, he g ea i ulence ha cha ac e ized he i s
ou b eaks o RHD would ha e caused a s ong immune
esponse in he abbi , gene a ing g ea selec i e p essu e.
In his way and as indica ed by he selec ion analyses pe -
o med [75], he e is ongoing selec i e p essu e on ce -
ain RHDV s ains, and consequen ly he ixa ion o
a ou able mu a ions o he pu ging o dele e ious a -
ian s would esul in he s ong dec ease in he ela i e
gene ic di e si y ecen ly obse ed o he i us [76,77].
Addi ional ma e ial
Addi ional ile 1: MCC ee ob ained in Beas o Lineage I and ime
span o each o he Ibe ian clades. Map and lis o he Ibe ian
samples analyzed in his s udy.
Addi ional ile 2: O igin and yea o isola ion o RHDV samples
analyzed in his s udy.
Alda e al.BMC E olu iona y Biology 2010, 10:347
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Page 8 o 10
Addi ional File 3: Desc ip i e s a is ics o he gene ic a iabili y in
RHDV.
Acknowledgemen s
The au ho s wish o hank F. Guil and he membe s o Fundación CBD-
Habi a o hei help p o iding samples. L. Alca az assis ed wi h he
labo a o y wo k and A. Bu on, M. Díez-León and A. Ch is o e son e iewed
he English ex . We also hank R. Za doya o help ul discussions and h ee
anonymous e e ees o hei aluable sugges ions. FA bene i ed om a
FPU p e-doc o al g an om he Spanish Minis y o Educa ion and Science.
This s udy was unded by p ojec s MAM/2484/2002-65/2002 and
010203030003.
Au ho de ails
1
Dp o. Biodi e sidad y Biología E olu i a, Museo Nacional de Ciencias
Na u ales (CSIC). José Gu ié ez Abascal 2, 28006 Mad id, Spain.
2
Ins i u o de
In es igación en Recu sos Cinegé icos (CSIC-UCLM-JCCM). Ronda de Toledo
s/n, 13071 Ciudad Real, Spain.
3
Dp o. Sanidad Animal, Facul ad de
Ve e ina ia, Uni e sidad Complu ense de Mad id. A da. Pue a de Hie o s/n,
28040 Mad id, Spain.
4
Dp o. Ingenie ía del Medio Ag onómico y Fo es al,
Uni e sidad de Ex emadu a. A . Vi gen del Pue o 2, 10600 Plasencia, Spain.
Au ho s’con ibu ions
FA concei ed he s udy, ob ained and analyzed he molecula da a and
w o e he manusc ip . TG and MS designed labo a o y p o ocols and
ob ained molecula da a. TM and GR collec ed samples and molecula da a.
ID p o ided unding and logis ic suppo o he s udy. The inal d a was
ead and app o ed by all he au ho s.
Recei ed: 5 June 2010 Accep ed: 10 No embe 2010
Published: 10 No embe 2010
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