Establishment of reverse genetics system for NBV strain MB.

<p>(A) Viral titers in cell lysate following plasmid transfection. L929 cells were infected with rDIs-T7pol for 1 h and cotransfected with 10 plasmids encoding each of the 10 gene segments of strain MB or nine plasmids that did not include a plasmid encoding the S3 segment. The cells were collected at 24 and 48 h post transfection. The viral titers were determined via a plaque assay. Results are represented as the mean for triplicate samples. Error bars indicate standard deviations. n.d: not detected. (B) rsMB contains a signature mutation in the S3 segment as a genetic marker. A nucleotide substitution from A to C was introduced at position 640 to create the EcoRV site in the S3 segment, which is underlined. (C) Digestion of the amplified S3 gene fragments of MB and rsMB using EcoRV to confirm the introduction of a silent point mutation. Size markers are indicated. (D) Sequence analysis of S3 gene fragments from recombinant viruses. The S3 gene fragment was amplified using RT-PCR with viral dsRNA extracted from the purified native MB and rsMB virions. The amplified fragments were subjected to direct sequence analysis. The sequence chromatograms show the A to C substitution at position 640 of rsMB. (E) BHK/T7-9 cells were cotransfected with the 10 MB plasmids. Following 48 h of incubation, viral titers from transfected cells were determined using a plaque assay. Results are presented as the mean for triplicate samples, and error bars indicate standard deviations. n.d: not detected.