Analysis of quasispecies variation in single and mixed viral infection

Population diversity was examined in individual and natural mixed infections of Cowpea chlorotic mottle virus (CCMV) and Cucumber mosaic virus (CMV) isolates in two systemic hosts, cowpea and Nicotiana benthamiana. Isolates of CCMV and CMV obtained from a cowpea field in Arkansas were separated biologically in cowpea and tobacco plants, respectively. After separation, individual and mixed cultures of both viruses were serially passaged ten times by mechanical inoculation in cowpea and N. benthamiana. High-fidelity reverse transcriptase-polymerase chain reaction (HiFi RT-PCR) of RNA 3, followed by cDNA cloning and sequence analysis was used to assess the quasispecies cloud size of CCMV and CMV populations in passages zero and ten of each host species. The levels of population variation were generally higher in individual infections of CCMV-Car1 and-Car2 isolates, and the CMV-Car2 isolate compared with mixed infections, in both host species, although the significance of the differences varied depending on how mutations were counted. There were no significant differences in the levels of population variation in individual and mixed infections of the CMV-Car1 isolate. Partially fixed mutations were observed in both individual and mixed infections of the CCMV-Car2 isolate in N. benthamiana and CMV-Car1 and-Car2 isolates in both cowpea and N. benthamiana.