Bovine herpesvirus 1 (BoHV-1) is a ubiquitous pathogen of cattle and is implicated in many production limiting diseases. This virus is responsible for flu like infections in cattle worldwide. This infection can then, in turn, lead to secondary bacterial infections which are responsible for the development of severe disease complexes such as bovine respiratory disease (BRD). The virus has been divided into three genotypes; BoHV-1.1, BoHV-1.2a and BoHV-1.2b. Between and within these genotypes there is considerable variation in the capacity of BoHV-1 strains to cause disease, referred to as virulence. Currently, the molecular basis of the variation in virulence capacity between BoHV-1 strains is poorly understood. Additionally, it has been reported that the current Australian vaccine against BoHV-1 infection (Rhinogard™) has shown signs of loss of efficacy. Due to these factors, it is important to understand the interactions of the virus and cells during infection including miRNA production and, exosome interactions.
Exosomes are extra-cellular organelles believed to be involved with cell-to-cell communication and establishing anti-viral responses between cells. This anti-viral response is believed to be due to the transmission of viral miRNA between cells which, in turn, leads to the development of specific RNAi molecules.
Exosomes were isolated from bovine kidney cells after infection with Australia’s oldest BoHV-1 isolate; V155. Isolation was performed via ultra-high-speed centrifugation methodology. The virus and exosome were pelleted and separated using a self-forming 60% optiprep gradient. The location of virus was confirmed via: reinfection, detection of BoHV-1 miRNA (miR-6), detection of glycoprotein C DNA and, detection of glycoproteins B and C. The location of exosomes was found via the detection of TSG101 and bovine CD63 proteins. Initial experimentation suggests the presence of viral miRNA or DNA encapsulated in the exosomes. To confirm this, cells were treated with exosomes before infection with a clonal virus of V155 (pBHVGFP) containing a GFP cassette. Infection was observed to determine the impact this treatment had on infection over time.