No abstract

RNA viruses are characterized by high genetic variability resulting in rapid adaptation to new or resistant hosts. Research for plant RNA virus genetic structure and its variability has been relatively scarce compared to abundant research done for human and animal RNA viruses. Here, we utilized a molecular population genetic framework to characterize the evolution of a highly pathogenic plant RNA virus [Tomato spotted wilt virus (TSWV), Tospovirus, Bunyaviridae]. Data from genes encoding five viral proteins were used for phylogenetic analysis, and for estimation of population parameters, subpopulation differentiation, recombination, divergence between Tospovirus species, and selective constraints on the TSWV genome. Our analysis has defined the geographical structure of TSWV, attributed possibly to founder effects. Also, we identify positive selection favouring divergence between Tospovirus species. At the species level, purifying selection has acted to preserve protein function, although certain amino acids appear to be under positive selection. This analysis provides demonstration of population structuring and species-wide population expansions in a multisegmented plant RNA virus, using sequence-based molecular population genetic analyses. It also identifies specific amino acid sites subject to selection within Bunyaviridae and estimates the level of genetic heterogeneity of a highly pathogenic plant RNA virus. The study of the variability of TSWV populations lays the foundation in the development of strategies for the control of other viral diseases in floral crops.

Tomato spotted wilt virus (TSWV) is transmitted exclusively by thrips in nature. A reassortment-based viral genetic system was used to map transmissibility by thrips to the medium (M) RNA of TSWV. To locate determinants of thrips transmission in the M RNA, 30 single-lesion isolates (SLIs) were generated from a single TSWV isolate that was inefficiently transmitted by thrips. Three of the 30 SLIs were transmitted by thrips, and 27 were not. Sequence analysis of the M RNA, thrips transmissibility assays, GC protein analysis, and transmission electron microscopic studies revealed that a specific nonsynonymous mutation (C1375A) in the GN͞GC ORF of the M RNA resulted in the loss of thrips transmissibility without inhibition of virion assembly. This was in contrast to other nontransmissible SLIs, which had frameshift and͞or nonsense mutations in the GN͞GC ORF but were defective in virion assembly. The G C glycoprotein was detectable in the C1375A mutants but not in the frameshift or nonsense mutants. We report a specific viral determinant associated with virus transmission by thrips. In addition, the loss of transmissibility was associated with the accumulation of defective haplotypes in the population, which are not transmissible by thrips, rather than with the presence of a dominant haplotype that is inefficiently transmitted by thrips. These results also indicate that the glycoproteins may not be required for TSWV infection of plant hosts but are required for transmissibility by thrips.

Wild plant species were systematically sampled to characterize reproduction of thrips, the vector of Tomato spotted wilt virus (TSWV), and natural sources TSWV infection. Thrips populations were monitored on 28 common perennial, biennial, and annual plant species over two noncrop seasons at six field locations across North Carolina. Sonchus asper, Stellaria media, and Taraxacum officianale consistently supported the largest populations of immature TSWV vector species. The tobacco thrips, Frankliniella fusca, was the most abundant TSWV vector species collected, comprising over 95% of vector species in each survey season. Perennial plant species (i.e., Plantago rugelii and Taraxacum officianale) were often only locally abundant, and many annual species (Cerastium vulgatum, Sonchus asper, and Stellaria media) were more widely distributed. Perennial species, including P. rugelii and Rumex crispus, remained TSWV infected for 2 years in a small-plot field test. Where these perennial species are locally abundant, they may serve as important and long-lasting TSWV inoculum sources. In random surveys across 12 locations in North Carolina, TSWV infection was documented by double antibody sandwich enzyme-linked immunosorbent assay in 35 of 72 (49%) common perennial (N = 10), biennial (N = 4), and annual (N = 21) plant species across 18 plant families. Estimated rates of TSWV infection were highest in Cerastium vulgatum (4.2%), Lactuca scariola (1.3%), Molluga verticillata (4.3%), Plantago rugelii (3.4%), Ranunculus sardous (3.6%), Sonchus asper (5.1%), Stellaria media (1.4%), and Taraxacum officianale (5.8%). Nine plant species were determined to be new host recordings for TSWV infection, including Cardamine hirsuta, Eupatorium capillifolium, Geranium carolinianum, Gnaphalium purpureum, Linaria canadense, Molluga verticillata, Pyrrhopappus carolinianus, Raphanus raphanistrum, and Triodanis perfoliata. Our findings document the relative potential of a number of common annual, biennial, and perennial plant species to act as important reproductive sites for F. fusca and as acquisition sources of TSWV for spread to susceptible crops.

A serologically distinct member of the tomato spotted wilt virus (TSWV) group was isolated from the hybrid flower crop New Guinea impatiens (Impatiens sp.) and termed TSWV-I. TSVW-I type isolates have frequently been detected in a wide variety of flower crops throughout the United States. TSWV-I~-~i-eg~-many characteristics with TSWV, such as symptomatology and possession of three ssRNA species (L, M and S of 8-3 kb, 5.2 kb and 3-4 kb, respectively) and three structural proteins (G1, G2 and N of 78K, 52K and 28K respectively). The TSWV-I G1 and G2 glycoproteins were serologically related to the respective proteins of TSWV, but the TSWV-I nucleocapsid or N protein was serologically unrelated to that of TSWV. Hybridization analysis under high stringency conditions revealed no hybridization between clones of TSWV-I S and M and the S and M RNAs of TSWV, respectively and in addition, a TSWV S clone hybridized only with TSWV S RNA. The cytopathology of TSWV-I also differed from that of TSWV. TSWV-Iinfected tissue primarily contained filamentous structures arranged in paracrystalline arrays, which were also observed by immunosorbent electron microscopy of tissue extracts. The filamentous structures were only trapped by TSWV-I antibodies. The conserved serological relatedness between TSWV types for G1 and G2, but not N, is consistent with serological analyses of the nairovirus and phlebovirus genera of the Bunyaviridae, the virus family that TSWV most closely resembles.