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*
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.
Emails: victorneira@u.uchile.cl;
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,
Mu€nstermann, 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
neutralizing 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/2000AY359210.1/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-37TM) were grown in 12- well plates
with cell growth media,
which includes minimum essential
medium Eagle’s (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
previously 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 information 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
samples (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
V. Neira http://orcid.org/0000-0001-9062-9969
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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.
