Objective:
The objective of this agreement is to assess the role of different regions of Foot-and-Mouth Disease Virus (FMDV) genome in virulence during the infection in the natural host. Amendment 2 expands the objectives to include: Understanding of the mechanisms of FMDV persistence and the viral carrier state in animals.

Approach:
Collaborators from the University of Connecticut (UConn) have expertise in mapping and evaluating the role of viral genetic determinants associated with virulence in natural hosts. UConn will provide ARS-PIADC with expertise in developing a library of genetically modified cDNA infectious clones to produce FMDV mutated viruses at PIADC. UConn will also provide expertise in the evaluation of pathology aspects of animals infected with wild type and mutated viruses. Persistent infections will be established with wild type and mutant FMDV in relevant cells of bovine origin. Cells and viruses will be characterized during persistence and the mechanisms mediating the establishment and maintenance of persistence will be studied. Cellular and molecular biology techniques such as the use of fluorescence confocal microscopy and similar techniques will be utilized by UConn and ARS scientist in collaborative studies. ARS-PIADC will provide the FMDV infectious clones, assist in the development of the mutated viruses and perform the in vitro and in vivo characterization of those viruses both during acute and persistent infections. ARS-PIADC will also conduct virus isolation, RT-PCR sequencing and phylogenetic analysis of the samples. These collaborative efforts will allow for a better understanding of molecular mechanisms of FMDV pathogenesis. Amendment 2 will include genetic and phenotypic characterization of FMDV isolates from persistently infected cells. Methods to determine the role of autopahagy will be developed. Characterization studies will be continued to develop an in vitro system to examine the mechanisms of FMDV persistence.