We will characterize a number of cucumber mosaic virus (CMV) isolates found on the Island of Hawaii. We will also determine the nucleotide sequences of the RNA genomes of these viruses using reverse transcriptase/PCR technology. This first portion of the project will yield information regarding the existing diversity of CMV on the island. Further, by correlating sequence differences with host range and symptom differences, we will identify nucleotides important in determining these viral phenotypes. Our preliminary results show 91% sequence identify between two CMV strains found on different hosts, but less than 10 miles preliminary results show 91% sequence identify between two CMV strains found on different hosts, but less than 10miles part, and 99.1% sequence identify between two strains found on different host species separated by less than 20 feet. The cDNAs for one of the CMV strains will then be cloned for propagation in Escherichia coli. This will serve to provide a source of a homogenous viral population, which can also be targeted for site-directed mutagenesis experiments. The homogeneous viral population will be used to perform experiments in which we infect plants, and then analyze by sequencing the rate at which mutations become fixed during CMV infection. We will infect a single leaf, and after infection becomes systemic, analyze virus in the inoculated leaf as compared to other parts of the plant. We will then passage the virus to other plants of the same as well as different species and similar analyze the viral sequences. This study will reveal the rates of mutations and whether or not they can be accelerated in certain hosts. We will also target the CMV cDNA clones for site-directed mutagenesis. Our earlier analysis will have implicated certain nucleotide positions as candidates for determining certain viral phenotypes, and these mutagenesis studies will narrow the field and eventually identify specific important nucleotides. This analysis will in turn identify important amino acid residues on specific viral proteins. Undergraduate studies will be trained in the handling and manipulating of eucaryotic RNA viruses, without being at risk for infection. During viral characterization and sequence analysis, they will also gain experience in the enzyme linked immunosorbent assay (ELISA), reverse transcription, and the polymerase chain reaction (PCR). The will also become very familiar with nucleotide sequence analysis, cDNA synthesis and cloning, which in turn involves the restriction enzyme treatment and ligation of DNAs, and propagation and transformation of E. coli. Finally, the mutagenesis studies will provide the students with experience in site- directed mutagenesis of DNA and with in vitro mRNA transcription.
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