viernes, 22 de septiembre de 2017

Identification of a divergent genotype of equine arteritis virus from South American donkeys J. Rivas1 | V. Neira2* | J. Mena2 | B. Brito2 | A. Garcia3 | C. Gutierrez3 | D. Sandoval1 | R. Ortega1* 2017

Identification of a divergent genotype of equine arteritis virus from South  American  donkeys


J. Rivas1    |  V. Neira2*       |  J. Mena |  B. Brito2    |  A. Garcia3    |  C. Gutierrez |
D. Sandoval1    |  R. Ortega1*




1Facultad de Ciencias Veterinarias, Departamento de patolog ıa y medicina preventiva,  Universidad  de Concepcion, Chill an, Chile
2Facultad de Ciencias Veterinarias  y Pecuarias,  Universidad  de Chile, Santiago, Chile
3Laboratorio y Estacio  n Cuarentenaria Pecuaria,  Complejo  Lo Aguirre, Servicio Agr ıcola y Ganadero,  Santiago,  Chile

Correspondence
V. Neira, Facultad  de Ciencias Veterinarias  y Pecuarias,  Universidad  de Chile, Santiago, Chile and R. Ortega,  Facultad  de Ciencias Veterinarias,  Departamento de patolog ıa y medicina  preventiva,  Universidad  de Concepcion   , Chill an, Chile.

Present address
B. Brito, Foreign Animal Disease  Research Unit, Plum Island Animal Disease  Center, ARS, USDA, NY, USA


Summary


 
A novel  equine  arteritis  virus (EAV) was  isolated  and  sequenced from feral donkeys in Chile. Phylogenetic analysis  indicates  that  the  new  virus  and  South  African asi- nine  strains  diverged  at  least  100  years  from  equine  EAV strains.  The  results  indi- cate  that  asinine  strains  belonged  to a different EAV genotype.

KEY W ORD S
donkey,  equine,  equine  arteritis  virus, equine  viral arteritis







1    |    INTRODUCTI ON


Equine viral arteritis  (EVA) is a viral disease  in equids,  namely horses, donkeys,  mules  and zebras.  The causative  agent  is the  equine  arteri- tis   virus   (EAV), genus   Equartevirus  from   the   Arteriviridae  family (Adams et  al., 2017).  EAV strains  have  been  classified  based  on  the ORF5 phylogeny  into three  genotypes, the  North  American (NA) and the  European 1 (EU1) and 2 (EU2) lineages  (Zhang et  al., 2007).
Clinical disease  is characterized by  fever  and  respiratory symp- toms;   however,   economic   losses   are  mostly   due   to  its  ability  to cause  abortion  in mares  and  severe  disease  or death  in young  foals (Balasuriya, Go, & MacLachlan, 2013).  EAV increased global reporting during more  recent years has been  attributed to more  frequent international horse  movement (Dominguez,  Munstermann, de  Guin- dos,  & Timoney,  2016).  EVA is not  only transmitted through direct


*These authors should  be considered joint senior  authors.


contact during  clinical respiratory disease,  but  it can  also  be  trans- mitted  through the  venereal  route.  Stallions can become persistently infected  (carriers) and  transmit  the  disease  during  breeding  (Guthrie et  al., 2003).


2    |    MATERIAL S  A ND  METHODS


In Chile, the  EAV has  not  been  detected in horses.  In 2013,  during surveillance  activities,  samples  collected  from  feral  donkeys  ranging in small herds  in hills and  plains  nearby  the  Atacama  Desert were positive   to   neutralizin antibodies  against   EAV  (Moreira,   Garc ıa, Valencia,  & Moreno,  2016).  Following  results  from  this  study,  two male  adult  donkeys,  clinically healthy,  were  captured in the  annual rodeo   event   in  October 2013.   The  rodeo   was  conducted at  Car- rizalillo, Freirina,  Chile  (   29.099469,    71.406169).  Donkeys   were
sent  to a slaughterhouse for human  consumption.



Transbound Emerg Dis. 2017;1–6.                                                   wileyonlinelibrary.com/journal/tbed                                         © 2017  Blackwell Verlag GmbH     1






2.1    Sample  collection

Tissue  samples  including  heart,   lung,  kidney,  testes, vas  deferens, epididymis,  prostate and  seminal  vesicle  were  collected.  One  gram


from each  organ  was  scraped  and  homogenized with  10 ml of mini- mum essential  media (MEM). The mix was centrifuged at 2,823  g for
20 min, and  the  supernatant was  used  for  RT-PCR and  virus  isola-
tion.






 
(a)                                                                     (b)









FI GU RE 1    Cytopathogenic effects  of RK-13 cells: (a) RK-13 cells mock-infected at 7 days post-inoculation. (b) RK-13 cells infected  with Atacama-2014 equine arteritis virus (EAV) isolate  at 7 days post-
inoculation






European 1
European 2
North American
Asinine lineage


U38593.1/Horse/AZ87/Arizona-U.S.A/1987
AF099839.1/Horse/S-436/Poland/1988
AF099850.1/Horse/S1512/U.S.A/1995
EF102379.1/Horse/PLP00-1/Lesser_Poland-Poland/2000
EF102382.1/Horse/PLP02-4/Lesser_Poland-Poland/2002
AY453313.1/Horse/I16/Italy/1995
EF492547.1/Horse/F9/Loire-France/2002
AF099830.1/Horse/3308V-96/Italy/1995
AY359193.1/Horse/H1S/Bekes-Hungary/2001
AY359209.1/Horse/H20F/Heves-Hungary/2000
AY359208.1/Horse/H19F/Pest-Hungary/2000
EF102355.1/Horse/PLH05-1/Silesian-Poland/2005
AY359207.1/Horse/H269S/Zala-Hungary/2003
U38592.1/Horse/AUT68/Vienna-Austria/1968
AF099813.1/Horse/S-1128/Canada/1992
U46952.1/Horse/Vienna/Vienna-Austria/1968
AF099811.1/Horse/Vienna/Austria/1964
EF492558.1/Horse/F20/Ile_de_France-France/2001
AF099814.1/Horse/EAV-86-R/France/1986
GQ903863.1/Horse/S4222/California-U.S.A/2008
AY359197.1/Horse/H147S/Heves-Humgary/2001
AF099815.1/Horse/EAV-86-P/France/1986
AF099823.1/Horse/1192VE4-91/Italy/1990
AF099824.1/Horse/135VE2-95/Italy/1994
AF099838.1/Horse/Wroclaw-2/Poland/1978
AY359201.1/Horse/H197S/Fejer-Hungary/2002


AF099812.1/Horse/Fallat/Canada/1986
U46949.1/Horse/19933/Ontario-Canada/1992
U38594.1/Horse/CAN86/Alberta-Canada/1986
U46948.1/Horse/11958/Ontario-Canada/1990
AF099835.1/Horse/NEAV-1/Norway/1988
AF099836.1/Horse/NEAV-2/Norway/1989
AY359199.1/Horse/H172S/Komarom-Esztergom-Hungary/2002
U38607.1/Horse/PA76/Pennsylvania-U.S.A/1976
EF492549.1/Horse/F11/Ile_de_France-France/2004
EF492543.1/Horse/F5/Basse_Normandie-France/2004
EF492562.1/Horse/F24/Basse_Normandie-France/2004
EF492551.1/Horse/F13/Basse_Normandie-France/2004
JX868590.1/Horse/EAV_HSY/China/2011
U38608.1/Horse/PLD76/Wroclaw-Poland/1976
AF099849.1/Horse/TAQ/U.S.A/1994
U38611.1/Horse/VBS53/Bucyrus-Ohio-U.S.A/1953
AF099842.1/Horse/S-2506/Sweden/1989
U38609.1/Horse/SWZ64/Bibuna-Switzerland/1964
AF099843.1/Horse/Bibuna/Switzerland/1964
AF099828.1/Horse/1330VE-95/Italy/1995
AF099825.1/Horse/470VE1-95/Italy/1994
AF099826.1/Horse/73VE2-95/Italy/1994
AY453340.1/Horse/S2/Sweden/1999
AF099832.1/Horse/1908V-97/Italy/1996
AY349167.1/Horse/CW96/U.S.A/1996
AF118783.1/Horse/CA97/California-U.S.A/1997
AY349168.1/Horse/CW01/U.S.A/2001
AF099816.1/Horse/EAV-86-110/France/1986
AF099822.1/Horse/D526/Germany/1996
AY453344.1/Horse/S6/Sweden/2000
AY453278.1/Horse/A4/Austria/1998
* *                                                                                                                                                                               AY359203.1/Horse/H213S/Hungary/1999
AY359202.1/Horse/H204S/Heves-Hungary/1994
AY359204.1/Horse/H215S/Zala-Hugary/2000
AY359210.1/Horse/H72F/Zala-Hungary/1998
AY359200.1/Horse/H189S/Heves-Hungary/2002
AY453335.1/Horse/RSA4/R.S.A/1996
LC000003.1/Horse/GB_Glos_2012/Gloucestershire-U.K/2012
U38599.1/Horse/KY63/Kentucky-U.S.A/1963
AY956598.1/Donkey/J3-931209/R.S.A/1993
AY956601.1/Donkey/J6-940309/R.S.A/1994
AY956597.1/Donkey/J2-931125/R.S.A/1993
AY956600.1/Donkey/J5-940309/R.S.A/1994
AY956599.1/Donkey/J4-931209/R.S.A/1993
*                                                             Donkey/Atacama-2014/Chile/2014

–700                      –600                      –500                      –400                      –300                      –200                      –100                         0

FI GU RE 2    Maximum clade credibility collapsed  tree  of equine  arteritis  virus using 170  ORF5 reference sequences. The Atacama-2014 and South  African Donkey  sequences belong  to a single monophyletic group, the  asinine  cluster  (red). The time to most  recent common  ancestor (tMRCA) of the  asisine cluster  with other  equine  arteritis  virus (EAV) sequences and the  tMRCA of Atacama-2014 with the  closest  reference are indicated  with * and **, respectively






2.2    RT-PCR

RNA was  extracted using  the  commercial  kit MagMAXTM-96  AI/ND Viral RNA Isolation  Kit (Ambion         Cat#  AM1835,  Austin, TX, USA). ORF6 and ORF7 were  amplified by RT-PCR using the  protocols rec- ommended by the  World  Organisation for  Animal Health  (Timoney,
2012).  ORF5  was  amplified  using  the  primers  and  protocols  previ- ously described (Stadejek et  al., 1999).  PCR products were  submitted for Sanger  sequencing. The positive  samples  were  selected for virus isolation.


2.3    Viral isolation

Viral isolation  was attempted in RT-PCR-positive  samples.  First, monolayers of  RK-13  cells  (ATCC     CCL-37TMwere  grown  in  12- well plates  with cell growth  media, which includes minimum essential medium  Eagles (MEM), supplemented with 10% foetal  bovine  serum,
10,000 IU/ml  penicillin  (1%), 10,000 lg/ml  streptomycin  (1%) and
25 lg/ml  amphotericin B (1%). Monolayers  with  80% of cell conflu- ence  were  inoculated with  200  ll  of  filtrated  positive  samples  and
incubated for  1 hr  at 37°C  and  5% CO2.  After  the  incubation,  the


inoculum  was  discarded and  cells were  incubated for 10 days  using cell  growth   medium   previousl described.   The   monolayers were observed daily during 10 days  inspecting  for evidence  of cytopatho- genic  effects.   Positive  cultures  were  tested by  RT-PCR to  confirm the  presence of the  EAV.


2.4    Phylogeny

ORF5  was  used   to  reconstruct the   EAV phylogeny.   ORF5  is  the most  variable  region  of the  virus and  commonly  used  for EAV phy- logeny.   ORF5  sequence  generated  for   this   study   and   reference sequences covering  the  known  spectrum of  ORF5  genetic  diversity were  aligned  using  MUSCLE (Edgar, 2004).The  codon  partition  and nucleotide  substitution  model   was  selected  using  partition   finder based  on  the  Bayesian  informatio criterion  (BIC) (Lanfear, Calcott, Ho,  & Guindon,  2012).  The  best  scheme  consisted in one  partition for  each  codon  position:  HKY+I+G  for  codon  position  1,  TrN+I+G for codon  position  2 and  GTR+I+G for codon  position  3. We  used  a Bayesian  approach for  time  divergence estimation  implemented  in BEAST 1.8.2  (Drummond  & Rambaut,  2007).  Initially, a  strict  clock
model  and  an  uncorrelated relaxed  lognormal  clock, in combination







GQ903859/2007/USA/S3901
GQ903809/2005/USA/S3583
GQ903901/2005/USA/M405
GQ903811/2008/USA/S4216
EF492559/2002/France/F21
AF118780/1998/USA/G4
AF118777/1995/USA/G1
AF118779/1997/USA/G3
AF118778/1997/USA/G2
AF118782/1996/USA/RQ
AF118781/1996/USA/BT-PA96
AF118776/1997/USA/P2
AF118775/1996/USA/P1
AF118774/1998/USA/R2
AF118773/1996/USA/R1
AF118769/1995/USA/A1
AF118770/1996/USA/A2
AF118771/1996/USA/A3
AF118772/1997/USA/A4
EF492556/2001/France/F18
EF492557/2001/France/F19
EF492555/2001/France/F17
EF492560/2002/France/F22
EF492554/2001/France/F16
EF492561/2002/France/F23
EF492548/2003/France/F10
EF492553/2001/France/F15
JN211317/2000/France/F60
JN211316/2007/France/F27
AY349167/1996/USA/CW96
AY349168/2001/USA/CW01


European 1
European 2
North American
Atacama-2014


HK25
Hela-EAVP35
EF492564/2004/France/F26
EF492563/2004/France/F25
EF492562/2004/France/F24
U81013/1953/USA/VBS53
U81020/NA/USA/ATCC
EF492552/2003/France/F14
EF492550/2004/France/F12
EF492549/2004/France/F11
EF492545/2004/France/F7
EF492544/2004/France/F6
U81019/NA/USA/ARVAC
pEAVrMLV
EF492543/2004/France/F5
EF492546/2004/France/F8
EF492551/2004/France/F13
DQ846750/1953/USA/Bucyrus
U81021/1986/Canada/CAN86
U81018/1976/USA/PA76
AH007128/1999/USA/CA95G
GQ903862/2007/USA/S3955
AF107267/1999/USA/D85
AF107271/1999/USA/D89
AF107269/1999/USA/D87
AF107270/1999/USA/D88
AF107273/1999/USA/D92
AF107274/1999/USA/D94
AF107272/1999/USA/D91
AF107276/1999/USA/E86
AF107268/1999/USA/D86
AF107277/1999/USA/E88
AF107278/1999/USA/E89
AH007129/1999/USA/CA95I
U81015/1977/USA/KY77
U81017/1993/USA/KY93
AF107275/1999/USA/E85
AF107279/1984/USA/KY84
AF107266/1999/USA/D84



















Donkey/Atacama-2014/Chile/2014



0.08

FI GU RE 3    Maximum likelihood ORF6 phylogenetic tree  using 93 equine  arteritis  virus reference sequences. Atacama-2014 isolate  is a singleton  genetically  distant  from reference sequences coloured  in red



with  a constant  population and  a Bayesian  skyline  tree  prior,  were run.   We   selected  the   model   based   upon   the   AICm  method-of- moments estimator (Baele et  al., 2012;  Raftery,  Newton, Satagopan,
& Krivitsky, 2007)  implemented  in  Tracer  v1.6  (Rambaut,  Suchard, Xie, & Drummond,  2014).  Based  on  the  lower  AICm, the  uncorre- lated  exponential clock model  and  a coalescent constant population tree  prior were  selected. The analysis was run for 200,000 iterations. Convergence and  mixing of the  simulations  was  assessed using Tra- cer. The maximum clade credibility tree  (MCC) was visualized in Fig- tree    version    1.4.2    (Rambaut,    2014).    Additionally,   phylogenetic analyses  of ORF6 and ORF7, which are more  conserved genes,  were performed  using  MUSCLE for  sequence  alignment   and  maximum likelihood to  reconstruct the  phylogeny  in MEGA7 (Kumar, Stecher,
& Tamura, 2016).


3    |    RESULTS  A ND  DI SCUSSI ON


Viral RNA was  identified  in samples  from  both  animals. From  a vas deferens  sample,   cytopathogenic  effects   (CPE) were   observed at
5 days  post-inoculation. CPE was  characterized by rounding  of cells and  cell  detachment  from  the   monolayer   (Figure 1).  The  isolated virus was named  Atacama-2014.
Viral RNA was  amplified,  and  partial  sequences of  ORF5  (630 nucleotides- nt), ORF6  (193  nt)  and  ORF7  (316  nt)  were  obtained by Sanger sequencing method (Accession numbers MF543058, MF543059 and  MF573786,  respectively).  The  phylogeny  of  ORF5 revealed   that  the  Atacama-2014 belonged   to  a  monophyletic  clus- ter   that   included   viruses   collected   from   donkeys   in  1993–1994
sample (Stadejek,   Mittelholzer,    Oleksiewicz,   Paweska,   &  Belak,








GQ903809/2005/USA/S3583
GQ903811/2008/USA/S4216
GQ903901/2005/USA/M405
GQ903859/2007/USA/S3901
EF492556/2001/France/F18
AF118779/1997/USA/G3
AF118780/1998/USA/G4
AF118778/1997/USA/G2
AF118777/1995/USA/G1
EF492557/2001/France/F19
EF492559/2002/France/F21
EF492555/2001/France/F17
AF118769/1995/USA/A1
AF118773/1996/USA/R1
AF118774/1998/USA/R2
AF118782/1996/USA/RQ
AF118781/1996/USA/BT-PA96
AF118770/1996/USA/A2
AF118771/1996/USA/A3
AF118772/1997/USA/A4
AF118775/1996/USA/P1
AF118776/1997/USA/P2
EF492554/2001/France/F16
EF492561/2002/France/F23
EF492560/2002/France/F22
EF492553/2001/France/F15
JN211317/2000/France/F60
EF492548/2003/France/F10
JN211316/2007/France/F27
AY349167/1996/USA/CW96
AY349168/2001/USA/CW01
GQ903862/2007/USA/S3955
AF107266/1999/USA/D84
AF107267/1999/USA/D85
AF107277/1999/USA/E88
AF107276/1999/USA/E86
AF107275/1999/USA/E85
AF107279/1984/USA/KY84
AF107278/1999/USA/E89
AF107269/1999/USA/D87
AF107270/1999/USA/D88
AF107268/1999/USA/D86
AF107272/1999/USA/D91
AF107273/1999/USA/D92
AF107274/1999/USA/D94
AF107271/1999/USA/D89
U81015/1977/USA/KY77
AH007128/1999/USA/CA95G
AH007129/1999/USA/CA95I
U81018/1976/USA/PA76
U81017/1993/USA/KY93
DQ846750/1953/USA/Bucyrus
U81020/NA/USA/ATCC
EU252113/NA/USA/Hela-EAVP35
U81013/1953/USA/VBS53
EF492564/2004/France/F26


European 1
European 2
North American
Atacama-2014































Donkey/Atacama-2014/Chile/2014




0.03

FI GU RE 4    Maximum likelihood ORF7 phylogenetic tree  using 93 equine  arteritis  virus reference sequences. Atacama-2014 isolate  is a singleton  genetically  distant  from reference sequences coloured  in red





2006);  we  named  this  group  the  asinine  cluster.  The  time  to  most recent common  ancestor (tMRCA) between the  asinine  cluster  and other   EAV genotypes was  estimated at  1695   (95%  highest  poste- rior  density   (HPD)  interval   1424–1892)  (Figure 2,  Appendix   S1). Additionally,  the   tMRCA  of  the   Atacama-2014 sequence  and  the African  donkey   strains   was  estimated  at  1914   (95%  HPD  1779
1983).  The  ORF5  sequence  with  the  highest   identity   to  the  Ata- cama-2014 virus  (78.9%) that  was  public  available  was  J2-931125
#AY9565   (South   African  asinine   cluster).   The  ORF5  genetic   dis- tance   between  groups   (NA,  EU1,  EU2  and   asinine)   was   higher between  the   asinine   group   and   all  other   clusters   (79.5%–81.9%) compared  to  the   remaining  distances between  the   European and North   American   clusters   (90.1%–89.1%).   In  South   America,  only EAV sequences from  Argentina  are  available  from  public  reposito- ries.  These  Argentinean sequences  were  collected  during  the  early
2000s and  in 2011  and  have  been  classified  within  the  EU1 geno- type,  genetically  distant  to  the  asinine  cluster  (Metz,  Serena,  Panei, Nosetto, & Echeverria,  2014).
No  ORF6  and  ORF7  sequences  from  the   donkey   South   Afri- can   isolates   were   available  for  phylogenetic  analysis.  The  ORF6 and  ORF7  phylogeny  shows  the  Atacama-2014 virus  phylogeny  as a  singleton distant   from  all  other   EAVs (Figures 3  and  4).  How- ever,   we   were   able   to   sequence  only  three   ORFs  of  the   virus (10%  genome),  which   we   consider   the   major   limitation   of   this study.
The  phylogeny   indicates   that   the   asinine   cluster   represents  a new  genotype present in South  America and  Africa, both  related  to carrier  donkeys.  The presence of this genotype in two  different con- tinents may  underlie   a  widely  distributed unreported  existence of this  viral  strain,  different  from  the   known   and  well-characterized EAV prevalent in horses.
It is not  clear  how  the  virus was  introduced into  the  feral  don- key  population in Chile. Historical  records  indicate  that  the  original population of  donkeys   arrived  into  the  country   at  least  500  years ago.  However,   it  is  likely  that   subsequent  importations occurred. tMRCA  of  the  Chilean  and  1993   South  African  donkey   was  esti- mated  between 1779  and  1983,  suggesting  that  the  introduction of the  virus may have  occurred during  imports  after  the  original intro- duction  of donkeys  in Chile. However,  because of the  lack of avail- ability  of  viral  sequences  collected   at  earlier  time   points   of  the asinine   cluster,   the   tMRCA  estimates  should   be   carefully   inter- preted.
Although  EAV has  only  been  detected in donkeys  in Chile, the South  African asinine  strains  have  also detected in horses  (Stadejek et  al., 2006);  therefore, the  EAV Chilean  donkey  viruses  may repre- sent  a risk for different equine  populations.
The  characterization  of  this  virus  in  South  America  provides  a novel  perspective of  the  global  distribution  of  EAV. The  isolation and genetic  characterization of this new  virus provides  vital informa- tion for future  EAV surveillance.  It further  contributes to understand the  divergence of  the  virus  and  to  the  proper  design  of  diagnostic test   for  more   accurate detection  in  horses   and  as  well  as  other equids.


AC KN O W L E D G EM E N T S

We  thank  the  staff  of  Chilean  Agricultural  and  Livestock  Services (SAG) for all their support during the  sampling, necropsy  and virology work.


CONFLI CT  OF  INT E RE ST

The authors declare  no conflict of interest.


OR CI D




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How to cite this article:  Rivas J, Neira V, Mena  J, et  al. Identification of a divergent genotype of equine  arteritis  virus from South  American donkeys.  Transbound Emerg Dis.