DEVELOPING STRATEGIES TO DETECT ALPHAVIRUS RECOMBINATION

Charles E. McGee, B.S., K.A. Tsetsarkin, M.S., D.L. Vanlandingham, Ph.D., and S. Higgs, Ph.D., FRES

Department of Pathology, UTMB

Background: Positive sense (+) non-segmented single stranded RNA viruses evolve via two mechanisms: 1) clonal accumulation of point mutations via replicative error and/or 2) recombination events occurring between related but disparate VIRAL genomes replicating in the same cell. The alphavirus genome encodes two open reading frames with an inter-genic sequence that is highly susceptible to molecular manipulation. Previous experiments using reverse genetic alphavirus systems, namely Sindbis virus and Venezuelan equine encephalitis virus (VEEV), have identified laboratory generated recombinant genomes with cross over events being identified exclusively in the inter-genic sequences. Objective: to optimize conditions for detection and comparative analysis of the relative efficiencies of inter-genic and intra-genic RNA virus recombination events, using chikungunya virus (CHIKV) and VEEV replicons, that can be translated into heterologous (+) RNA virus systems. Methods: Standard molecular cloning methodologies have been used to generate CHIKV and VEEV replicon/helper systems possessing trans-complementary sequences necessary and sufficient for the generation of full-length viable recombinant viruses, following transfection, replication, and purification in cell culture. The current status of the project including: construct design, analytical methods, and approaches to identify and characterize potential recombinant genomes will be discussed.

Supported by the Department of Pathology.