1. Agricultural Biotechnology 399 Research Report
I. Proposal Title: Investigating Sequences of Exon 1 of the CXCL16
Candidate Gene for Equine Arteritis Virus Resistance
Among Equidae, Rhinoceridae, and Tapiridae
II. Name: Brooke Nemec
E-mail: Brooke.Nemec@uky.edu
Graduation Date: May 2015
III. Faculty Advisor: Dr. Ernest Bailey, Department of Veterinary Science
IV. Statement of Career Goals: After completing my Agricultural Biotechnology
degree at the University of Kentucky, I plan to attend veterinary school to become a
wild life or large breed veterinarian.

2. V. Abstract
Equine Viral Arteritis (EVA) is a contagious viral disease of equids caused by
Equine Arteritis Virus, an RNA virus. Prior research identified gene CXCL16 on
horse chromosome 11 (ECA11) with two alleles as having genetic influence on the
resistance of in-vitro EAV infection of T-cells. The two alleles differed by four
mutations in the first exon of CXCL16, each altering an amino acid in the first
domain of the CXCL16 protein. This project is investigates the gene sequences of
exon 1 of CXCL16 present in other families within the Perissodactyla Order
including Equidae, Rhinoceridae, and Tapiridae and identifies phylogenetic
differences in respect to CXCL16 variation.
VI.Introduction and Significance
EVA disease causes a variety of flu like symptoms making it difficult to
distinguish from other viruses. EAV is transmitted by respiratory route or venereally.
Two significant consequences of EVA are abortion in the mare and establishment of
the carrier state in the stallion making this of high interest to the prominent horse
breeding businesses in Kentucky. Mature stallions, but not intact colts, geldings or
mares, can harbor the virus in the accessory sex glands and stallions can shed the
virus in semen.
Recently, graduate student, Dr. Yun Young Go of Dr. Balasuriya’s lab,
identified a polymorphism among horses for in-vitro infection of T-cells. Go found
that horses could be divided into two groups, susceptible or resistant, based on in-
vitro susceptibility of their lymphocytes to EAV infection. Further genome wide

3. association studies showed that a gene on the ECA11 chromosome genetically
influenced this polymorphism. Subsequent work led to identification of the candidate
gene, CXCL16, in this region with two alleles. One allele was associated with
susceptibility and the other with resistance to in vitro infection of T-cells. The 2
alleles differed by 4 mutations in the first exon of CXCL16, each altering an amino
acid in the first domain of the CXCL16 protein. The trait is dominant in the respect
that susceptible horses possessed at least one copy of the variant. CXCL16 has
associated with immune response but not much else is known regarding this gene.
As mentioned, two alleles varying by four mutations have been identified in
Equus caballus (horses) of the Equidae family. Closely related to horses are the non-
horse Equids including zebras, and Asiatic Asses (Figure 1). Distantly related to
horses are Rhinoceridae (rhinos) and Tapiridae (tapirs) who share a Perissodactyla
ancestor classifying them in the same order, Perissodactyla.
Figure 1: The currently accepted phylogenetic tree of Order Perissodactyla.

4. A reference sequence for exon 1 of CXCL16 on ECA 11 has been established.
A successfully annealing primer was already created during prior research for exon 1
of CXCL16 for equines and was used again in this study. The four identified
mutations in the reference sequence and their effects on their corresponding amino
acids are shown in Figure 2. The order of amino acids found for susceptible horses
was established as phenylalanine, histidine, isoleucine, and lysine. In contrast, the
order of amino acids found in resistant horses was established as tyrosine, aspartic
acid, phenylalanine, and glutamic acid.
Figure 2: The reference sequence for CXCL16. Exon 1 is shown in bold and
mutations in question are shown in red. A’s, T’s, G’s, and C’s represent adenine,
thymine, guanine, and cytosine nucleic acids respectively.
Because there are only two alleles shown in horses for CXCL16 of the 256
combinations possible, the null hypothesis of this experiment was that the CXCL16
gene is under strong selection and other members of Perissodactyla Order have the

5. same two alleles present. Thus, the null hypothesis was that the CXCL16 gene is
under different selection pressure and other members of the Perissodactyl Order do
not have the same two alleles present.
VII. Methods
Gene sequences from distantly related species were assessed in-silico using
genome sequences available by means of public databases, specifically the Genome
10K Sample Collection Database. Available DNA samples of two horses of known
genotype, rhinos, tapirs, and non-horse equids were collected first. The two horses of
known genotype were the controls for comparison of the experiment. Horse S had the
susceptible allele and Horse R had the resistant allele. Several samples of rhino
species including Black rhinos (n=5), white rhinos (n=6), and Indian rhinos (n=3)
were used as the experimental group for the rhinoceridae family. Several samples of
non-horse equids including onagers (n=7), Hartmann’s Zebras (n=17), Grevy Zebras
(n=6), and Grant Zebras (n=7) were used as the experimental group for the non-horse
equids family. Several samples of tapir species including Baird’s Tapirs (n=5) and
Malayan Tapir (n=5) were used as the experimental group for the tapiridae family.
Exon 1 of CXCL16 was then amplified for the equus family samples using a
previously developed primer designed by graduate student, John Eberth of Dr. Ernest
Bailey’s lab, from previous ECA 11 CXCL16 studies. At first, the rhinoceridae
family was amplified using the equidae family specific primer as well but unspecific
binding occurred. To remedy this, a rhinoceridae family specific primer was then
designed by John Eberth using the reference sequence available for rhinoceridae

6. provided by the Genome 10K Sample Collection Database for use with the
rhinoceridae family in substitution of the equidae specific primer. Unfortunately there
is not currently a publically available tapiridae reference sequence for use in
developing primers so the tapiridae family samples were limited to being amplified
using the distantly related equidae family’s specific primer.
Next, samples were shipped to Eurofins for Sanger Sequencing. Then,
sequences were aligned and compared using the sequence-aligning program,
Sequencher, Nucleotide sequences within species were compared. Then, nucleotide
sequences across species and then across different families were compared to the
known Horse R and Horse S sequences. Lastly, amino acid sequences across species,
then families, were compared as well as compared to Horse R and Horse S. Amino
acid sequences shown in each species were then applied to the established
phylogenetic tree (Figure 1) to compare phylogenetic differences and similarities in
sequence conservation.
VIII. Results and Discussion
Gel electrophoresis of the samples post PCR showed successful amplification
of exon 1 of CXCL16 for the rhinos, horses, and non-horse equids using their
respective family specific primers. Tapirs were excluded from further
experimentation due to unspecific binding and possible amplification of a
contaminant when using the equidae family specific primer. Fortunately, it has been
recently found that two introns surrounding exon 1 of CXCL16 are conserved in both
rhinoceridae and equidae families. Since the sequence is conserved in these two

7. distantly related Perissodactyla, there is a chance that it is conserved in the tapiridae
family as well. This could be useful in the future development of a tapiridae family
specific primer beginning at the site of the preserved introns rather than the sites of
the original equidae and rhinoceridae primers.
Comparing the sequences among species within the rhinoceridae family, it
was found that the nucleotide sequence of exon 1 of each of black rhino was identical,
each white rhino was identical, and each Indian rhino was identical. There were
however species specific differences comparing black rhinos, to white rhinos, to
Indian rhinos confirming that the correct DNA sample was amplified and there was
no cross contamination. This was also true for other groups comparing within species
as well- all onagers were identical, Hartmann Zebra’s were identical, Grevy Zebras
were identical, and so forth but comparing across species showed they were different
due to species specific differences (Figure 3).
Figure 3: The sequence for CXCL16. Exon 1 of each species. Note that among species
the sequences found were the same but species-specific differences (circled) were found
confirming desired amplification. A’s, T’s, G’s, and C’s represent adenine, thymine,
guanine, and cytosine nucleic acids respectively.

8. When comparing nucleic acid sequences across species and families, it was
found that only Horse R had the first thymine to adenine mutation and second
cytosine to guanine mutation. All of the non-horse equids sequenced had an exon 1
sequence identical to Horse S excluding species-specific differences. Rhinos however
did have the third mutation displaying an adenine and the fourth mutation guanine
characteristic of Horse R (Figure 4).
Figure 4: The sequence for CXCL16. Exon 1 of each species. Note that across species the
four mutations (indicated by red arrows) characteristic of Horse R were not present in
entirety. Only the third and fourth mutation were shared with rhinoceridae. A’s, T’s, G’s,
and C’s represent adenine, thymine, guanine, and cytosine nucleic acids respectively.
Finally, when comparing amino acid sequences, it was found that no species
shared a similar amino acid sequence to Horse R. Horse R has an amino acid
sequence of tyrosine, aspartic acid, phenylalanine, and glutamic acid. All non-horse
equids had a sequence similar to Horse S. This sequence was phenylalanine, histidine,
isoleucine, and lysine. Despite similarities with Horse R in respect to the third and
fourth nucleotide mutations, rhinoceridae had an amino acid sequence unlike any of
the equid species. Rhinoceridae had an amino acid sequence of phenylalanine,
glutamine, phenylalanine, and glutamic acid (Figure 5).

9. Key Amino Acids
Phenylalanine F Glutamine Q
Tyrosine Y Isoleucine I
Histidine H Lysine K
Aspartic Acid D Glutamic Acid E
Figure 5: The amino acids in question produced by exon 1 for each species (red
arrows). Note that all equids share similar amino acids as Horse S and rhinoceridae
are completely different.
The amino acid sequences can then be applied to the currently established
phylogenetic tree to compare differences and similarities between species (Figure 6).
It can be interpreted that the equidae family share a common allele characteristic of
the susceptible horse but only horses, equus caballas, have the resistant allele. The
rhinoceridae family has a completely different sequence of amino acids compared to
equidae indicating that this allele is under different selective pressure. This pressure
could include different pathogens causing the genetic selection for stronger immunity
or simply random variation in the alleles from speciation. However, the exact cause
for the difference cannot be determined at this time. The tapiridae sequences were
undetermined at this time.

10. Figure 6: The amino acid sequences (indicated in red) as they appear across families.
Note that the equids share the S sequence across species within the family but outside
the family, neither the R nor S sequence appear.
In further research it will be beneficial to further investigate if the sequence is
present in this family to determine if this allele is newly evolved to the equids,
specifically the horse, or present in tapirs as well. It would also be beneficial to
continue testing more samples as only a few rhinoceridae (n=14) and tapiridae (n=10)
were available. The allele could possibly be conserved but not as prominently thus
our available samples may not correctly reflect the entire population. More samples
of non-horse equids should also be analyzed to confirm that the samples accurately
reflect the population.

11. IX. Conclusion
In conclusion, the null hypothesis stating that the CXCL16 gene is under
strong selection and other members of Perissodactyla Order have the same two alleles
present is rejected. Thus, the null hypothesis was accepted stating that the CXCL16
gene is under different selection pressure and other members of the Perissodactyl
Order do not have the same two alleles present. It was found that there was
susceptible allele conservation within equidae and the resistant allele only appeared in
equus caballus. This could indicate that the allele is newly evolved in horses.
Rhinoceridae sequences were unlike the susceptible and resistant genotypes
incidating that this family could be under different selective pressure for the CXCL16
gene.
X. Acknowledgements
Special thanks to Dr. Ernie Bailey for supervising this project and John Eberth
for designing primers and teaching necessary skills to complete research. A
special thanks also to Allison Sparling for providing test samples from associate
laboratories.

12. XI. Literature Cited
Balasuriya,
U.
B.,
Patton,
J.
F.,
Rossitto,
P.
V.,
Timoney,
P.
J.,
McCollum,
W.
J.,
&
MacLachlan,
N.
J.
(1997).
Neutralization
Determinants
of
Laboratory
Strains
and
Field
Isolates
of
Equine
Arteritis
Virus:
Identification
of
Four
Neutralization
Sites
in
the
Amino-­‐terminal
Ectodomain
of
the
G(L)
Envelope
Glycoprotein.
Virology
,
232,
114-­‐128.
Balasuriya,
U.
(2013).
Identification
of
Host
Genetic
Factors
Responsible
for
Establishment
of
EAV
Carrier
State.
In
E.
I.
Diseases.
Lexington,
Kentucky.
Eurofins
MWG
Operon
Kentucky.
(2014).
DNA
Sequencing
Services.
Louisville,
Kentucky.
Genome
British
Columbia.
(2013).
Sanger
Sequencing:
How
DNA
is
Read.
Vancouver,
British
Columbia,
Canada.
Go,
Y.,
Cook,
R.,
Fulgencio,
J.,
Campos,
J.,
Henney,
P.,
Timoney,
P.,
et
al.
(2012).
Assessment
of
correlation
between
in
vitro
CD3+
T
cell
susceptibility
to
EAV
infection
and
clinical
outcome
following
experimental
infection.
Veterinary
Microbiology
(157),
220-­‐225.
Life
Technologies.
(2013).
PCR
Buffers.
Retrieved
from
Life
Technologies
Product
Information:
http://www.lifetechnologies.com/au/en/home.html
New
England
BioLabs
Incorperated.
(2013).
PCR
Protocol
for
Taq
DNA
Polymerase
with
Standard
Taq
Buffer.
Retrieved
from
New
England
BioLabs
Incorperated
Protocol:
https://www.neb.com/
Purcell,
S.,
Neale,
B.,
Todd-­‐Brown,
K.,
Thomas,
L.,
Ferreira,
M.,
Bender,
D.,
et
al.
(2007).
gPLINK:
a
toolset
for
whole-­‐genome
association
and
population-­‐based
linkage
analysis.
American
Journal
of
Human
Genetics
(81).
Timoney,
P.
J.,
Cordes,
T.
R.,
&
McCollum,
W.
H.
(2001).
EVA,
Equine
Viral
Arteritis:
A
Manageable
Problem.
Washington,
D.C.:
U.S.
Dept.
of
Agriculture,
Animal
and
Plant
Health
Inspection
Service.