Molecular genetic analyses of the N, NSm and NSs genes of a local population of Orthotospovirus tomatomaculae reveal purifying selection in crops in the southeastern USA.

Orthotospovirus tomatomaculae [tomato spotted wilt virus (TSWV)] is a major pathogen in horticultural and row crops worldwide including the USA. In this study, tomato spotted wilt disease incidence was monitored in Arachis hypogaea (peanut; year 1990 to 2024) and Nicotiana tabacum (tobacco; year 2000 to 2024) in commercial farmers' fields in the Southeastern USA. Furthermore, nucleocapsid (N), nonstructural movement (NSm) and nonstructural silencing suppressor (NSs) protein gene sequences of TSWV global populations from North America, South America, Europe, Asia-Pacific, Africa and Australia were compared with local US population and analysed to understand the genetic variability in the virus genome. In our study, full-length sequences of 94 N, 111 NSm and 78 NSs genes were amplified from TSWV-infected A. hypogaea (peanut), Capsicum annuum (pepper), N. tabacum (tobacco) and Solanum lycopersicum (tomato). nt-based phylogenetic analysis of N, NSm and NSs genes correlated with the geographical location of the TSWV isolates, with notably higher substitution rates in the population of recent years. In addition, the least genetic variability was observed in the N gene of the local population upon comparison with other global TSWV population. The neutrality test of TSWV suggested a non-neutral evolution of the virus genome. Low variation among the selected genes might be attributed to strong purifying selection pressure in the populations. Furthermore, estimation of selection pressure (dN/dS) on small (S) segment-encoded N protein and nonstructural protein showed higher purifying selection than the movement protein encoded by the medium (M) segment of the TSWV isolates. Single-likelihood ancestor counting suggested an overall negative selection pressure on several codons of the selected genes, which indicated that natural selection and population bottleneck events might have influenced the evolution of TSWV. Our study also deciphered high gene flow and low genetic differentiation amid the different TSWV population sets. Additionally, BEAST analysis of TSWV N gene sequences from GA predicted the most common recent ancestor existed ~25 years ago. This data was further correlated with disease incidence data from peanut and tobacco crops obtained in the last three decades. These findings suggest the intermixing of TSWV isolates between peanut, pepper, tobacco and tomato crops, while the virus genome has undergone strong purifying selection.