FIG 4.
Molecular and in vitro characterization of R6HA RRV. (A) Comparative genome hybridization was used to directly compare viral DNA from the R6HA RRV recombinant to that from WTBACRRV. Alterations within the R6HA RRV genome resulted in incomplete hybridization to the array, depicted by the ratio of R6HA to WTBACRRV, and signaled a potential nucleotide mismatch between the two viral genomes. This comparison identified the HA tag located at the C-terminal end of R6. A second mismatch, indicated with an asterisk, was incorrectly identified, and the identified sequence was confirmed to be similar to the WT via PCR and DNA sequence analysis. (B) RFs were infected with either WTBACRRV or R6HA RRV at an MOI of 2.5 for single-step growth curve analysis. Infected RFs were harvested at the specified time points and subjected to a serial-dilution plaque assay on RFs to determine viral titers. The data from 4 separate experiments were averaged (±SEM). (C) RFs were infected with WTBACRRV or R6HA RRV or left uninfected, and at 48 h postinfection, total cellular RNA was harvested. Equivalent amounts of total RNA were analyzed by RT-PCR for ORF57, R7, and R6 transcripts, with cellular GAPDH as a loading control. Samples were run simultaneously with Taq polymerase only (−RT) to control for input and purity. Numbers at top indicate nucleotides.