The major research focus of Dr. Balasuriya’s laboratory is to characterize the molecular epidemiology and pathogenesis of equine arteritis virus (EAV) and equine herpesvirus-1 (EHV-1) infections of horses and develop improved recombinant vaccines to prevent infection of horses with these viruses, as well as to develop improved tests to diagnose the infections. Also, some of the recent studies in his laboratory are focused on characterizing the virus-host interactions by combining contemporary molecular biology techniques, host genomic analysis using genome-wide association study (GWAS), transcriptome, and miRNA profiling.

Identification of Host Genetic Factors Responsible for Establishment of EAV Carrier State

Recently, we demonstrated an association between establishment of the long-term equine arteritis virus (EAV) carrier status in stallions and in vitro susceptibility of a subpopulation of lymphocytes (CD3+ T cells) to EAV infection. A genome-wide association study (GWAS) has shown that CD3+ T cell susceptibility is associated with a dominant haplotype on equine chromosome 11 (ECA11) in different horse breeds. These studies have established a correlation between the in vitro susceptibility of CD3+ T lymphocytes to EAV and establishment of persistent infection in stallions. Specifically, we have identified a network of cellular factors that are responsible for the establishment and maintenance of EAV carrier state in the stallion. Identification of the molecular events leading to the development of persistent carrier status should lead to treatments for the disease and development of a genetic test to identify stallions that are predisposed to persistent infection with EAV, which will prevent further spread of the virus. In summary, in these studies we used equine viral arteritis (EVA), an infectious disease of horses, as a model system to understand the genetic basis of establishment and maintenance of persistent viral infection in the stallion reproductive tract. This research will open up a new frontier of equine infectious disease research by combining contemporary genetic research tools that can be applied to other equine infectious diseases.

Mechanisms of EAV Pathogenesis and Persistent Infection of Stallions

The clinical manifestations of EVA reflect endothelial injury and increased vascular permeability. However, the relative roles and importance of direct virus-mediated endothelial cell injury and virus-induced macrophage-derived vasoactive and inflammatory cytokines in the pathogenesis of EAV-induced vascular injury are not yet clearly defined.

Our ongoing studies are focused on the development of in vitro assay systems to study the mechanisms of EAV pathogenesis using cultured equine endothelial cells, lung derived macrophages, and peripheral blood derived macrophages. Studies are also focused on identification of specific cell type(s) infected by EAV and characterization of the immunological signatures associated with EAV persistence in the male reproductive tract of the carrier stallion using contemporary molecular biology techniques including LCM, mRNA ISH, transcriptomics, miRNA profiling, among others.

Characterize the reproductive tract microflora and study its dynamics during EAV persistence

We are currently investigating the potential influence of EAV infection on the microflora (microbiome) present in the reproductive tract of carrier stallions. For this purpose, we performed high-throughput sequencing of the 16S rRNA and data being analyzed.

Characterization of Equine Immune Response to EAV Infection

There is little information on cell mediated immune (CMI) response to EAV, and we are interested in identifying the specific viral protein(s) that is targeted by the CTL response of EAV-infected horses.

Recently, we have characterized the systemic and mucosal antibody responses in persistently infected stallions, and future studies will be directed at characterizing B-cell repertoire and isotype switch using state-of-the-art transcriptomics. Furthermore, we are working on further characterizing the immunopathogenesis of EAV in the male reproductive tract by analyzing specific immunological pathways by transcriptomics and miRNA profiling of seminal exosomes.

Molecular Epidemiology of EAV Infection and Evolution of EAV

Previously we have undertaken extensive studies to characterize the molecular epidemiology and evolution of EAV. We will continue these studies to better understand the evolution of EAV during persistent infection in the stallion.

The current studies involve sequencing of a large number of sequential EAV isolates directly from the semen of persistently infected carrier stallions collected over a period of 2-10 years. Sequencing of the entire genome of EAV strains present in each stallion over a long period will give us an insight into the temporal evolution of the virus in the male reproductive tract.

These studies will definitively identify the variable and conserved regions of the EAV genome during persistent infection. Furthermore, we will continue to characterize EAV isolates from various field outbreaks around the world to monitor the temporal and geographic variation among field strains of the virus. The analysis of sequence data is performed using the commercial and online software.

Genetic Determinants of Virulence and Attenuation of EAV

Geographically and temporally distinct strains of EAV have been identified which differ in the severity of clinical disease they induce and in their abortigenic potential. While the majority of field strains of EAV are associated with subclinical or unapparent infection, some strains give rise to moderately severe signs of EVA. Virulence is intimately intertwined with the pathogenesis of virus infection; however, the genetic determinants of EAV virulence have not been identified.

We are utilizing three full-length infectious cDNA clones of EAV (EAV030, EAVrVBS, and EAVrMLV) and reverse genetic techniques to characterize the virulence determinants of the virus. Furthermore, the sequencing of a series of viruses, ranging from the virulent horse-adapted VBS to the fully attenuated modified live virus (MLV) vaccine (ARVAC®) derived from it, has allowed us to identify the nucleotides/amino acids that likely are responsible for attenuation of the virulent virus. The in vivo virulence (attenuation) phenotype of each virus in this panel has been previously characterized through experimental infection of horses. The significance of individual nucleotide and amino acid differences between virulent EAV VBS strain and avirulent MLV vaccine strain of EAV has been determined by full-length genome sequence analysis. We have developed a panel of recombinant viruses with specific nucleotide/amino acid changes to confirm the putative attenuation mutations of MLV vaccine strain of EAV. This study has clearly identified the attenuation mutation(s) present in the current MLV vaccine strain of EAV.

This information can be used to develop a recombinant plasmid with multiple engineered attenuating mutations, which then might serve as a genetically marked MLV vaccine. Such recombinant plasmids carrying complete copies of attenuated EAV might ultimately also be used as stable validated repositories of seed viruses for live virus vaccine production, which would ensure a genetically homogeneous virus stock. Furthermore, data from these studies will eventually facilitate the rapid identification of new strains of EAV with enhanced virulence, better delineate the role of the carrier stallion in generating them, and guide the logical development of improved vaccines for EAV.

Molecular Characterization of Neurovirulent EHV-1 Strains

The primary objective of this project is to identify additional putative neurovirulence determinants by sequencing genes essential for replication from an extensive panel of archived isolates of EHV-1. Findings from this study will yield important benefits for the equine industry in terms of improved diagnostics and more accurate predictions of the clinical consequences associated with detection of a particular virus genotype.

Molecular Epidemiology of Equine Rotaviruses

The primary objective of this project is to characterize Equine Rotavirus strains using high-throughput sequencing to better identify currently circulating viruses and the potential need for vaccine improvement.

Fishing for Novel Pathogens

Currently, a significant percentage of outbreaks of equine respiratory diseases are undiagnosed, and furthermore, by comparison with the man and other domestic animal species, there are relatively few known viral agents associated with respiratory infections in horses. The objectives of this study are twofold: firstly, to determine if there are viral agents currently undiscovered that comprise part of the normal endogenous flora of the respiratory tract of the horse and, secondly, to establish whether there is a causal relationship between any such agents and occurrence of respiratory disease in the horse using contemporary molecular biology techniques.

Software:

CC+/ Java

FastQC

TrimGalore

STAR

Burrows-Wheeler Aligner (BWA)

TopHat

Bowtie

Cufflinks

miRDeep2

Students:

Zelalem Mekuria (Post-doctoral scholar)

Mariano Carossino (PhD candidate)

Wangisa Dunuwille (PhD candidate)

Bora Nam (MS student)

Collaborators:

Ernest Bailey (Faculty)

Theodore Kalbfleisch (Faculty, University of Louisville, collaborator)

Grants:

Publications:

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