Molecular Evolution and Population Genetics of Tomato spotted wilt virus (TSWV).

Abstract

The overall goal of this dissertation research was to elucidate the molecular evolution and population genetics of Tomato Spotted Wilt virus (TSWV), at the species level and within individual isolates, and to develop a standardized diagnostic system that can be used to assign attribution to initial TSWV infections. Initially, using consensus sequence data from genes encoding five viral proteins we applied a multilocus molecular population genetic framework to characterize the genetic status and recent evolutionary history of the TSWV species. Our analysis provided the first demonstration of population structuring and species-wide population expansions for TSWV, attributed possibly to founder effects. Also, we identified positive selection favoring divergence between Tospovirus species and purifying selection acting at the species level to preserve protein function. In addition, we were able to discover specific amino acid sites subject to positive selection within Bunyaviridae and to estimate the level of genetic heterogeneity of the TSWV species.

Subsequently, in order to characterize the population history and genetic structure of individual wild-type TSWV isolates, thirteen geographically and host-diverse isolates were amplified, cloned and 516 clones were sequenced. Estimation of levels of genetic diversity and haplotype analysis revealed that natural TSWV isolates are highly heterogeneous viral populations that consist of one or more haplotypes with high frequency and an array of closely related rare haplotypes, some of which are defective. These viral populations exhibit a high transitional bias, attributed to the function of RNA-dependent RNA polymerase or an editing enzyme such as dsRAD. Also high levels of among-population differentiation were observed induced by geographic and/or host related factors. Demographic analysis based on tests of neutrality, gene genealogies and the coalescent revealed an excess of rare polymorphism and a shallow population genetic architecture consistent with a model of population growth for all analyzed TSWV isolates.

Finally, data from genes encoding two viral proteins (NSm and L) were used for analysis of optimal informational content and for phylogenetic analysis. Our research has identified partial sequence regions that contain similar phylogenetic information and perform as well as the complete NSm and RdRp genes, for branching points statistically supported (bootstrap value>50%). We propose a new advanced diagnostic system, which will use the NSm and RdRp local regions together with the N gene of TSWV to assign attribution to initial TSWV infections and prevent their spread to an epidemic form.