ANNOTATED SEQUENCE RECORD
Complete genome analysis of equine coronavirus isolated in Japan
Received: 19 June 2015 / Accepted: 4 August 2015 / Published online: 14 August 2015
Ó Springer-Verlag Wien 2015
Abstract Equine coronavirus has been responsible for
several outbreaks of disease in the United States and Japan.
Only one complete genome sequence (NC99 isolated in the
US) had been reported for this pathogenic RNA virus.
Here, we report the complete genome sequences of three
equine coronaviruses isolated in 2009 and 2012 in Japan.
The genome sequences of Tokachi09, Obihiro12-1 and
Obihiro12-2 were 30,782, 30,916 and 30,916 nucleotides in
length, respectively, excluding the 3’-poly (A) tails. All
three isolates were genetically similar to NC99
(98.2–98.7 %), but deletions and insertions were observed
in the genes nsp3 of ORF1a, NS2 and p4.7.
Equine coronavirus (ECoV) is an enveloped, positive-
stranded RNA virus belonging to the species
Betacoronavirus 1 in the genus Betacoronavirus. Several
outbreaks of ECoV, characterized by clinical symptoms of
fever, anorexia, lethargy, leucopenia, and digestive prob-
lems, have been reported in the United States  and Japan
[2, 3]. These clinical signs were reproduced in our exper-
imental challenge study . However, to the best of our
knowledge, only four ECoV strains have been isolated in
the United States and Japan, and the complete genome
sequence of this pathogen is only available for one
American strain (NC99) . To improve the molecular
diagnosis of ECoV and to better understand its epidemi-
ology, more genomic sequence data are required. There-
fore, in this study, we determined the complete genome
sequences of the remaining three ECoV strains isolated in
Japan and compared them with that of NC99.
Three ECoVs, Tokachi09, isolated in 2009 , Obihi-
ro12-1 and Obihiro12-2, isolated in 2012 , were ana-
lyzed in this study. These ECoVs were isolated from adult
horses showing symptoms of diarrhea at Obihiro Race-
course, Hokkaido, Japan. Tokachi09, Obihiro12-1 and
Obihiro12-2 were passaged 4 to 5 times in HRT-18G cells
[2, 3]. Viral RNA was extracted using a MagNA Pure LC
Total Nucleic Acid Isolation Kit (Roche Diagnostics,
Mannheim, Germany). RT-PCR was performed with six
primer sets designed based on the complete genome
sequence of NC99 (GenBank accession number:
NC_010327) (Supplementary Table 1). RT-PCR was per-
formed using a PrimeScript II High Fidelity RT-PCR Kit or
PrimeScript II High Fidelity One Step RT-PCR Kit
(Takara, Shiga, Japan).
These PCR amplicons were sequenced using Ion Torrent
technology (Thermo Fisher Scientiﬁc, MA, USA). Librar-
ies were constructed using an Ion Xpress Plus Fragment
Library Kit and an Ion Xpress Barcode Adapters Kit.
Emulsion PCR, enrichment and loading onto an Ion 318
M. Nemoto and Y. Oue contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-015-2565-1) contains supplementary
material, which is available to authorized users.
& Manabu Nemoto
Epizootic Research Center, Equine Research Institute, Japan
Racing Association, 1400-4 Shiba, Shimotsuke,
Tochigi 329-0412, Japan
Hokkaido Kushiro Livestock Hygiene Service Center, 127-1
Otanoshike, Kushiro, Hokkaido 084-0917, Japan
Thermo Fisher Scientiﬁc, Life technologies Japan Ltd.,
Sumitomo Fudosan Mita Twin Bldg., 4-2-8 Shibaura,
Minato-ku, Tokyo 108-0023, Japan
Dairy Hygiene Research Division, Hokkaido Research
Station, National Institute of Animal Health, 4 Hitsujigaoka,
Toyohira, Sapporo, Hokkaido 062-0045, Japan
Arch Virol (2015) 160:2903–2906