Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage

During a series of mechanical transfers of tomato spotted wilt virus, two distinct types of mutants were generated. Firstly, a morphologically defective isolate was obtained which had lost the ability to produce the membrane glycoproteins and, as a consequence, was not able to form enveloped particles. Analysis of the genomic RNAs of this isolate suggested that this defect was caused by either point mutations or very small deletions in the medium genomic RNA segment. Secondly, isolates were obtained which had accumulated truncated forms of the large (L) RNA segment. These shortened L RNA molecules most likely represented defective interfering RNAs, since they replicated more rapidly than full-length L RNA and their appearance was often associated with symptom attenuation. Defective L RNAs of different sizes were generated after repeated transfers, and hybridization analysis using L RNA-specific cDNA probes showed that the internal regions deleted varied in length. The presence of defective L RNAs in nucleocapsid fractions as well as in enveloped virus particles indicates that all defective molecules retained the sequences required for replication, encapsidation by nucleocapsid proteins and packaging of the nucleocapsid into virus particles.