Analysis of the genome sequence of an alpaca coronavirus
L. Jina,⁎, C.K. Cebrab, R.J. Bakera, D.E. Mattsona, S.A. Cohena,
D.E. Alvaradoa, G.F. Rohrmannc
aDepartment of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA
bDepartment of Clinical Sciences, Oregon State University, Corvallis, OR 97331, USA
cDepartment of Microbiology, Oregon State University, Corvallis, OR 97331, USA
Received 2 February 2007; returned to author for revision 20 February 2007; accepted 15 March 2007
Available online 24 April 2007
Coronaviral infection of New World camelids was first identified in 1998 in llamas and alpacas with severe diarrhea. In order to understand this
infection, one of the coronavirus isolates was sequenced and analyzed. It has a genome of 31,076 nt including the poly A tail at the 3′ end. This
virus designated as ACoV-00-1381 (ACoV) encodes all 10 open reading frames (ORFs) characteristic of Group 2 bovine coronavirus (BCoV).
Phylogenetic analysis showed that the ACoV genome is clustered closely (>99.5% identity) with two BCoV strains, ENTand LUN, and was also
closely related to other BCoV strains (Mebus, Quebec, DB2), a human corona virus (strain 043) (>96%), and porcine hemagglutinating
encephalomyelitis virus (>93% identity). A total of 145 point mutations and one nucleotide deletion were found relative to the BCoV ENT. Most
of the ORFs were highly conserved; however, the predicted spike protein (S) has 9 and 12 amino acid differences from BCoV LUN and ENT,
respectively, and shows a higher relative number of changes than the other proteins. Phylogenetic analysis suggests that ACoV shares the same
ancestor as BCoV ENT and LUN.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Coronavirus; Alpaca; Bovine coronavirus; Alpaca coronavirus
Coronaviruses are a genus in the family Coronaviridae,
which are enveloped viruses with large, positive sense RNA
genomes of 29 to 32 kb. They are important causes of human
and animal diseases that include respiratory infections, gastro-
enteritis, hepatic and neurological disorders, as well as immune-
mediated diseasessuch asSARSandfelineinfectious peritonitis
(reviewed in de Groot-Mijnes et al., 2005; Kahn, 2006; Spaan
et al., 1988; Wege et al., 1982). The coronaviruses possess a
characteristic genome composition. The 5′ two-thirds of the
genome encodes two polyproteins (1a and 1ab) that contain
proteins necessary for RNA replication. The 3′ one-third
encodes two non-structural proteins (NS1 and 2) and several
structural proteins, including a nucleocapsid protein (N) and
three or four envelope proteins: the membrane (M), spike (S),
hemagglutinin-esterase (HE), and/or a small membrane (E)
Originally,coronaviruseswereclassified into threegroups on
the basis of their antigenic cross reactivity (Cavanagh et al.,
1993; Cavanagh and Davis, 1993; Tsunemitsu et al., 1995).
When coronavirus genome sequence data became available, the
original antigenic groups were converted into three genetic
groups based on the similarity of their nucleotide sequences
(Gonzalez et al., 2003; Lai, 2003; Vijgen et al., 2006). Group 2
coronaviruses include murine hepatitis virus (MHV), bovine
coronaviruses (BCoV), human coronavirus OC43 (HCoV-
OC43), rat sialodacryoadenitis virus, porcine hemagglutinating
encephalomyelitis virus(PHEV), caninerespiratorycoronavirus
and equine coronavirus. Fifteen strains of BCoV have been
sequenced and have been implicated in a variety of diseases
including respiratory and enteric infections (Chouljenko et al.,
2001a; Dea et al., 1995; Han et al., 2006; King and Brian, 1982;
Park et al., 2006; Woloszyn et al., 1990). For example, the ENT
and LUN strains were isolated from animals with fatal shipping
fever pneumonia (Chouljenko et al., 2001a). The former was
Virology 365 (2007) 198–203
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E-mail address: firstname.lastname@example.org (L. Jin).
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associated with enteritis; whereas the latter is associated with
respiratory infection (pneumonia). Mebus, Quebec and DB2 are
virulent strains associated with neonatal calf diarrhea and winter
dysentery in adult dairy cattle (Dea et al., 1995; Han et al., 2006;
Park et al., 2006).
Coronavirus associated with outbreaks of diarrhea in all age
groups of New World camelids (llamas and alpacas) was first
identified in 1998, in Oregon. The symptoms were similar to
cattle infected with bovine coronavirus. Sick camelids showed
varying degrees of severity of clinical disease, with some
animals dying and others requiring intensive medical care.
Coronavirus-like viruses were frequently isolated from the
diseased animals (Cebra et al., 2003). Because of the severity of
the infection and the ability of coronaviruses to cross the species
barriers, including human–animal barriers, we undertook an
investigation to further characterize this virus. In this report we
describe the sequence of the ACoV and compare it to other
members of the coronavirus genus. We found that it is closely
related to Group 2 bovine coronaviruses.
Results and discussion
Cloning and sequence analysis
RT-PCR cDNA cloning to encompass the entire RNA genome.
listed in Table 1. The 5′ and 3′ end of the genome were
determined by 5′ and 3′RACE, respectively. This resulted in a
genome sequence of 31,076 nt including a poly A tail of 38 nt.
The genome encodes 10 ORFs characteristic of Group 2 BCoV
and 5′ and 3′ untranslated sequences of 210 and 298 nts (which
do not include the poly A tail), respectively (Fig. 1). The ORF
coordinates are shown in Fig. 1. The predicted ORF 1a and ORF
1ab contained 13,152 and 21,282 nt, respectively (Fig. 1). ORF
1ab contains a 26 nt region that overlapped with ORF 1a and
included a predicted ‘slippery’ sequence UUUAAAC. Based on
evidence from other coronaviruses (Chouljenko et al., 2001b),
this sequence causes a −1 frameshift during the translation of
avoiding translation termination and containing an additional
2711 amino acids. Upstream of five of the genes, there is a
repeated intergenic sequence, UCUAAAC, that is predicted to
interact with the viral transcriptase along with cellular factors to
‘splice’ the leader sequence onto the start of each ORF. ORF-
NS1, HE, S, M and N were all preceded by this sequence (Table
2), which would be predicted to give rise to a nested set of
mRNAs characteristic of the order Nidovirales.
Sequence comparisons with bovine coronaviruses
The sequence of ACoV was closely related to bovine
coronaviruses (Table 3). It has 99.54% to 99.55% identity to the
BCoV ENT and LUN strains, respectively. It was slightly less
related to the Mebus and Quebec strains at about 98.5% (Table
3). ACoVis also closely related to a human corona virus (strain
0C43) (>96%) and porcine hemagglutinating encephalomye-
litis virus (>93% identity) (Fig. 2 and Table 3). Phylogenetic
analysis of the entire genome of ACoV and other selected
coronaviruses was carried out and the results are summarized in
Fig. 2 and Table 3.
Comparison of nine predicted BCoV and ACoV proteins
Analysis of the predicted ORF 1a and ORF 1b shows 14 aa
and 1 aa changes, respectively, compared to the corresponding
ORFs in the most closely related BCoV ENTstrain. The ACoV
HE protein is phylogenetically similar to those of BCoV ENT
and LUN, with one and three amino acid differences,
respectively (Fig. 3B). In addition, the following differences
were noted between ACoVand the most closely related BCoV
strains for the structural proteins: membrane (M), one difference
out of 229 aa; internal orf (I), two differences out of 207 aa; and
N, one to five differences out of 448 aa. The nucleocapsid (N)
protein of coronavirus has been used as an early diagnostic
Oligonucleotide primer sets used for RT-PCR, 5′RACE and 3′RACE
199 L. Jin et al. / Virology 365 (2007) 198–203
marker for coronaviruses such as the SARS virus (Che et al.,
2004). The highly conserved small membrane envelope (E)
protein is unique to BCoV Group 2 and is identical between
ACoVand the closely related BCoV strains. A similar pattern of
relatedness was evident for the non-structural (NS) proteins.
There were one to two aa differences in the predicted 32 kDa
NS1 protein of ACoV compared to the most closely related
BCoV ENT and LUN strains. This protein is not essential for
virus replication in vitro, but has been implicated in virus
mutations between the predicted 12.7 kDa NS2 and those from
the most closely related BCoV isolates.
Comparison of the BCoV and ACoV predicted spike protein
Because the S protein has been implicated in tissue tropism
(Gallagher and Buchmeier, 2001; Godet et al., 1994; Schultze
et al., 1991; Schultze and Herrler, 1994), its affinity may be
reflected in the type of diseases caused. The spike protein of
ACoV and BCoV is 1363 aa long. The predicted proteolytic
cleavage site (KRRSRR) was conserved between all strains.
et al., 2006). S1 and S2 of ACoV were predicted to be 760 and
603 amino acids, respectively. The neighbor-joining method of
molecular evolutionary analysis revealed that the ACoV S
protein appears to evolve at a somewhat accelerated rate
compared to other proteins, e.g., the HE (Fig. 3). The predicted
ACoV S protein had 9 and 12 amino acid differences from the
predicted S proteins of BCoV strains LUN and ENT,
respectively. Most of the mutations occurred in the S1 subunit.
Three ACoV S amino acid changes are particularly striking,
including: serine at aa #174; proline at 565; and serine at 702. In
all the other S proteins analyzed (Fig. 3A and Table 4), these
one proline (#174) and the gain of another (#565) could
from the closely related viruses. It has been shown that S2 is not
directly involved in receptor binding, indicating that changes in
S1 could be involved in host specificity (de Haan et al., 2006).
The sequence analyses presented in this report demonstrate
that ACoV-00-1381, which is associated with diarrhea in
camelids (Cebra et al., 2003), is closely related to bovine
coronaviruses. It appears to have been derived from the same
ancestor as the LUN strain isolated from cattle with fatal
shipping fever pneumonia and the ENT strain isolated from
cattle with either pneumonia or enteritis (Chouljenko et al.,
2001a; Storz et al., 1996). New World camelids have been in
contact with cattle for over 500 years in South America,
compared to their relatively recent and small-scale introduction
to North America. Although the identification of coronaviral
infection and epidemic diarrhea in all age groups of llamas and
alpacas occurred only recently, it is possible that the virus
crossed between species during earlier interspecies contact or is
a BCoV strain that is pathogenic for both bovids and camelids,
although BCoV infection in camelids had not been described
previous to the Cebra et al. (2003) report.
The most significant difference that we observed between
ACoVand BCoV strains was in the spike protein. The S protein
forms distinctive surface projections on the virions and is
responsible for the primary attachment of the virus to cell
surface receptors (Schultze et al., 1991). It is a glycosylated and
acetylated polypeptide with a molecular weight of 170 kDa to
220 kDa and is the major hemagglutinin of bovine corona-
viruses (Schultze and Herrler, 1994). It has been suggested that
the high degree of variation in host range and tissue tropism of
coronaviruses is largely attributable to variations in the S
glycoprotein (Gallagher, 2001). There are a number of distinct
Intergenic sequences upstream of ACoV genes
Gene name Coding sequence Intergenic sequence upstream of the AUG
Fig. 1. Map of the alpaca coronavirus genome (GenBank accession no. DQ915164) and ORFs of the viral coding sequence. NS1, non-structural protein gene 1; HE,
hemagglutinin-esterase gene; S, spike gene;NS2, non-structural proteingene 2; E, envelopeprotein gene; M, membrane protein gene;N, nucleocapsidgene; I, internal
ORF. The numbers above and below each ORF correspond to the nt coordinates of each gene.
200 L. Jin et al. / Virology 365 (2007) 198–203
differences in S proteins between the respiratory isolates and
diarrhea isolates (of bovine coronaviruses, human and porcine).
Although the importance of such variability in the virulence
and tropism of BCoV is unknown, some amino acid changes
could have significant effects on the conformation, charge,
hydrophobicity and antigenic regions of the protein. These
mutations could change either the protein folding or physico-
chemical characteristics and could be involved in altering the
host specificity of the virus.
Material and methods
Cells and virus
An alpaca coronavirus isolate designated ACoV-00-1381
was obtained from a diarrhea sample by the Veterinary
Diagnostic Lab at Oregon State University (Cebra et al.,
2003). The isolate was grown on human rectal tumor (HRT-
18G) cells, which were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum
(Invitrogen), penicillin (100 U/ml) and streptomycin (100 μg/ml)
(Sigma-Aldrich, Inc.) at 37 °C with 5% CO2in a humidified
incubator. The virus was propagated in Dulbecco's modified
Eagle's medium supplemented with 2.5 μg/ml trypsin and
2.5 μg/ml pancreatin, 1× insulin–transferrin–selenium (Cat No.
Viral RNA preparation
Cells were infected at a multiplicity of infection of 1 to 3 and
were incubated for 3 to 5 days. The supernatant was collected
and clarified with a bench-top Beckman Coulter Allegra 64R
centrifuged at 9000×g for 30 min. Viruses were isolated from
the supernatant by centrifuging through a 30% sucrose cushion
with an ultracentrifuge (Beckman model XL-70) at 25,000 rpm
for 2 h in an SW28 rotor. The pellets were re-suspended in
100 μl TE buffer (10 mM Tris–HCl, 1 mM EDTA [pH 8.0]),
and viral RNA was extracted with Trizol (Invitrogen) as
described in the manufacturer's instructions.
A one-step reverse transcriptase (RT)-PCR kit (Invitrogen)
was used to generate viral DNA sequences for analysis. Four
microliters of the RNA extract (0.5 μg/μl) was added to the RT-
PCR mixture (in total 20-μl reaction) containing final
concentrations of 1.25 μM of each forward and reverse primer,
1× buffer for RT-PCR, 0.1 mM MgSO4and 1 U of RT/Taq. The
RTreaction was performed at 50 °C for 45 min, then the reverse
transcriptase was inactivated and Taq was activated at 94 °C for
2 min. PCR was performed for 30 cycles as the following: 94 °C
for 30 s, 50 °C for 45 s, 72 °C for 2.5 min, followed by a 7 min
elongation reaction at 72 °C after the final cycle.
The 5′ end of the viral genome was amplified by 5′RACE kit
(Invitrogen), following the manufacturer's instructions. Briefly,
5RACE1 (Table 1). Approximately 1 to 3 μg of total viral RNA
was used as the template in a 20-μl RT reaction containing the
5RACE1 primer.After purification of the first-strand cDNA,the
5′ end was tailed with dCTP using terminal deoxynucleotidyl-
transferase. The oligo(dC) cDNA was then amplified with a
second gene-specific primer (5RACE2) (Table 1) and the
abridged anchor primer (AAP) specific for the 5′ dC tail. The
primary PCR products were then reamplified with the hemi-
Fig. 2. A maximum-likelihood phylogenetic tree of selected coronavirus
genomes. ACoV (GenBank accession no. DQ915164) was compared to other
Group 2 Coronaviruses including porcine hemagglutinating encephalomyelitis
virus (PHEV VW572, GenBank accession no. YP_459958); human coronavirus
OC43 (HCoV-OC43, AY391777); bovine coronavirus Mebus strain (BCoV-
Meb, U00735); bovine coronavirus Quebec strain (BCoV-Q, D00662); bovine
coronavirus DB2 strain (BCoV-DB2, DQ811784); bovine coronavirus ENT
strain (BCoV-ENT, Q91A22); bovine coronavirus LUN strain (BCoV-LUN,
Sequence comparisons of ACoV with selected Group 2 coronaviruses
ACoV-alpacaBCoV-DB2BCoV-ENTBCoV-LUNBCoV-MEB BCoV-QHCoV-OC43 PHEV-VW572
BCoV: bovine coronavirus; HCoV: human coronavirus; PHEV: porcine hemagglutinating encephalomyelitis virus; Q: Quebec; MEB: Mebus.
201 L. Jin et al. / Virology 365 (2007) 198–203
amplification primer (AUAP), under conditions recommended
by the manufacturer. The 5′RACE products were cloned and
sequenced as described for the RT-PCR amplimers.
The 3′ end of the viral genome was amplified by 3′RACE kit
first-strand cDNA was synthesized with an oligo(dT)-containing
adaptor primer (AP) to the end of the viral genome. Approxi-
mately 1 to 3 μg of total viral RNAwas used as the template in a
20-μl RT reaction containing the oligo(dT)-AP primer. After the
cDNA synthesis, 3 μl of the cDNA was used directly in PCR
reaction with a gene-specific primer 3′RACE and abridged
universal amplification primer (AUAP) in a reaction recom-
mended by the manufacturer. The 3′RACE products were then
cloned and sequenced as described for the RT-PCR amplimers.
The oligonucleotide primer sets used for RT-PCR, 5′RACE
to design these primers for RT-PCR of the ACoV genome was
based on the known genomic information of several strains of
coronaviruses BCoV-LUN and BCoV-ENT (GenBank accession
numberAF391541for ENTandAF391542for LUN).Theywere
selected by Primer 3 program available online. The walking
primers (not shown) were selected from the ACoV sequence by
the DNA sequence core facility at Oregon State University.
TOPO TA cloning
The RT-PCR, 5′RACE and 3′RACE products were cloned
into the pCR 2.1-TOPO plasmid vector (vector) following the
manufacturer's instructions (Invitrogen).
DNA sequencing and analysis
Nucleotide sequences were determined by sequencing
cloned plasmid DNA with the universal primers first, then
using primer walking to complete the sequence. In addition,
Fig. 3. A phylogenetic tree of selected Group 2 spike proteins and hemagglutinin-esterase amino acid sequences. (A) The S proteins from the selected strains were
analyzed by the neighbor-joining method. (B) The hemagglutinin-esterase protein of the alpaca strain was compared to other selected Group 2 coronavirus
hemagglutinin-esterase proteins. For information on the viruses analyzed, see legend to Fig. 2.
The amino aciddifferences of the S glycoprotein among ACoVand the two most
related BCoV strains, ENT and LUN
aSequence accession number.
⁎Same as ACoV strain.
202 L. Jin et al. / Virology 365 (2007) 198–203
three independently generated RT-PCR reaction products were Download full-text
combined and processed with a ChargeSwitch PCR Clean-Up
kit (Invitrogen) before sequencing. Selected regions were
reconfirmed by sequencing the RT-PCR products directly. All
the cDNA clones and RT-PCR products were sequenced by the
DNA sequence core facility at Oregon State University. The
nucleotide sequences were assembled and analyzed with the
EMBOSS software. Additional analyses were carried out using
MacVector and programs described by Esteban et al. (2005).
Nucleotide sequence accession number
The sequences reported in this work have been deposited in
the GenBank database under accession number DQ915164.
This study was supported by a grant (F0434A) from the
Alpaca Research Foundation.
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