Epithelial Distribution of Foot-and-Mouth Disease Virus RNA Determined Using Laser Micro-Dissection and Quantitative RT-PCR.

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a severe, highly contagious vesiculating disease affecting cloven-hoofed animals. In addition to causing acute disease, this virus can also lead to persistent infection in cattle and ruminants. Initial replication occurs at the site of primary infection and virus then spreads to the lymph nodes and into the circulation, distributing virus through the body to potential sites of secondary viral replication. These sites include the stratified squamous epithelium of many tissues including tongue and skin. Although the pharynx region is the site of primary infection, and the tongue and skin being the sites of main amplification, no data are available on quantification of the specific cell layers within the epithelia involved in harbouring viral RNA. The objective of this study was to develop the system of isolating specific epithelial cell layers from stratified squamous epithelia and to be able to detect and quantify foot-and-mouth disease virus (FMDV). Laser Micro-Dissection (LMD) system combined with quantitative RT-PCR was utilised to isolate specific cell layers from selected squamous epithelia and determine the levels of FMDV RNA. Such observations of distribution and localization of FMDV infection within the different tissues provided insight into the kinetics of FMDV replication and spread of infection within the epithelia. Animal experiments were ongoing within the group and samples were taken from these experiments to evaluate various procedures up- and downstream of LMD. It was important that different protocols be used to test which method would be most suitable to yield highest level of RNA recovery. Such studies would increase the sensitivity of the combined LMD-PCR system to isolate and detect RNA from microdissected samples Once developed, this conjugated system of LMD with qRT-PCR was used to quantitatively analyse FMDV RNA within different cell layers of the epithelia of the foot, tongue and soft palate from experimentally infected pigs and cattle. This would allow the sites of intital infection, the levels of RNA reached and the spread of virus within the epithelia to be determined