Mechanism of the inhibitory effect of ethacrynic acid on vaccinia virus growth

Vaccinia virus (VACV) is globally known for its use in the eradication campaign against variola virus (VARV), the causative agent of smallpox. In 1980, WHO declared VARV as eradicated and the worldwide immunization has been stopped. After 2001, vaccination of selected groups of population was re-introduced because of concerns about variola misuse for bioterrorist purposes. Therefore, drugs effective against variola, post-vaccination complications and consequences of VACV transmission in the population, are intensively studied again. In 2009, laboratory of Dr. Mělková demonstrated that ethacrynic acid (EA) inhibited growth of VACV at the level of late gene expression. The goal of my diploma thesis was to perform a detailed study of the effects of EA on VACV late protein synthesis and on other stages of VACV replication cycle. To this end, the following methods were used: infection of tissue culture cells, plaque assay, northern blot, slot blot, western blot, direct and indirect immunofluorescence of fixed cells and fluorescence of EGFP expressed by VACV in infected live cells. First, we wanted to determine whether EA affects VACV late gene transcription or translation. Our results indicate that EA does not decrease levels of VACV late gene mRNA's and therefore, it probably affects late gene..