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.

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

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.

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

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

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

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.

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).

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.

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.

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.  

XI. Literature Cited

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