The major research interest of my laboratory is to understand host-pathogen interactions and the molecular basis for pathogenesis of two human infectious agents Mycobacterium tuberculosis and Japanese Encephalitis Virus. Mycobacterium tuberculosis perhaps achieves this by specific expression of certain genes while growing within the macrophage. To identify such 'differentially expressed genes' we took both immunological and molecular biological approaches. A genomic DNA expression library of a local field strain of Mycobacterium tuberculosis was constructed and screened with sera from TB patients which yielded several genes that are encountered by the host's immune system during an active infection. We have also attempted to identify differential genes of M. tuberculosis using techniques such as DDRT-PCR and subtractive hybridisation. We showed successful use of a novel technique to efficiently polyadenylate in vitro the 3' ends of messenger RNAs on polysomes of prokaryotic organisms and used it to identify host specific genes of M. tuberculosis. A new promoter in the M. tuberculosis genome that functioned efficiently in E. coli was identified, characterized and used to construct a vector that allowed its use to drive expression of heterologous genes in M. tuberculosis and in BCG, the latter holding promise for the development of BCG-based multivalent vaccines. The immunology of M. tuberculosis infections in humans and development of TB vaccines superior to the currently used BCG is another major area of study in our laboratory. We are using GFP-tagged genes inserted into the M. tuberculosis genome to follow the movement of these proteins within the infected macrophage using confocal microscopy. Interaction of M. tuberculosis with the innate immune cells is a current focus and glycosylated proteins of the organism have been found to down regulate production of interleukin 12 and interleukin 2 from mouse bone marrow-derived as well as human monocyte-derived dendritic cells. Knock out and knock in mutants of BCG have been constructed to study the mechanisms of suppressing host innate responses. Japanese encephalitis virus (JEV), a member of the mosquito-borne encephalitis complex of the family Flaviviridae is the principal cause of viral encephalitis over a large part of southern and eastern Asia resulting in 35,000 cases of encephalitis and 10,000 deaths annually. The genome of JEV is a single stranded, positive-sense RNA of approximately 11 kb in length that codes for a single large polyprotein, which in the infected cells is proteolytically processed into at least ten mature structural and non-structural proteins. We characterized for the first time the biochemical and kinetic properties of the flaviviral replicase obtained in a membrane-bound form from virus-infected cells and delineated the architecture of the viral replication complex within host membranes that serve as sites for viral RNA synthesis. The orientation of viral replicase proteins that carry out this major function within the double membrane vesicles have also been deciphered. We have also demonstrated the unexpected presence of viral RNA synthesizing activity within the nucleus of 3 flaviviruses, the human pathogens JEV, Dengue and West Nile viruses. This along with the dependence of the virus on the nucleus of the infected host cells opens up novel avenues for ati-viral interventions.