Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR.

This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1 g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1 year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.

Ensemble of secondary structures for encapsidated satellite tobacco mosaic virus RNA consistent with chemical probing and crystallography constraints.

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated. The high-resolution crystallographic structure of the satellite tobacco mosaic virus (STMV) particle reveals 30 helices of well-ordered RNA. The crystallographic data provide global constraints on the possible secondary structures for the encapsidated RNA. Traditional free energy minimization methods of RNA secondary structure prediction do not generate structures consistent with the crystallographic data, and to date no complete STMV RNA basepaired secondary structure has been generated. RNA-protein interactions and tertiary interactions may contribute a significant degree of stability, and the kinetics of viral assembly may dominate the folding process. The computational tools, Helix Find & Combine, Crumple, and Sliding Windows and Assembly, evaluate and explore the possible secondary structures for encapsidated STMV RNA. All possible hairpins consistent with the experimental data and a cotranscriptional folding and assembly hypothesis were generated, and the combination of hairpins that was most consistent with experimental data is presented as the best representative structure of the ensemble. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold.

Natural Incidence of Mixed Infections and Experimental Cross Protection Between Two Genotypes of Tobacco mild green mosaic virus.

ABSTRACT Isolates of Tobacco mild green mosaic virus (TMGMV), a member of the genus Tobamovirus, from Nicotiana glauca in southern California fall into two major genotypes, large (TMGMV-L) and small (TMGMV-S), distinguishable by the size of the coat protein (CP) subgenomic RNA. Mixed infections in the field were rare (1.6%), even at sites where both genotypes were common in single infections (62% for TMGMV-S; 37% for TMGMV-L). When plants experimentally protected by TMGMV-L were challenged by TMGMV-S, almost complete cross protection (90% of total plants challenged) was observed regardless of the protective time period (minimum 12 h and maximum 14 days). When plants protected by TMGMV-S were challenged with TMGMV-L, complete cross protection was observed when the protective time was 5 to 14 days. However, when the protective time was 3 days or less, protection by TMGMV-S was greatly reduced (11%), with mixed infections of TMGMV-S and -L predominating (69%), and single infections of the challenge virus TMGMVL were frequently observed (20%). When TMGMV-S and -L virions were co-inoculated, the virus progeny from individual plants most often contained only the TMGMV-L genome (61%) or, less frequently (39%), both genotypes. Therefore, TMGMV-L was more competitive than TMGMV-S and was able to displace TMGMV-S in experimental situations. The results obtained from cross-protection experiments in the greenhouse would explain the low frequency of natural mixed infections. It is possible that the experimental superior competitiveness of the novel L genotype has already or will play a role in its abundance in southern California.