FIG 2.

Identification of E1576V in NS2 as a critical determinant for efficient NS2-3-independent virion morphogenesis. (A) Schematic drawing of N7/OsUbi-NS4B*(2/RQ). Cell culture passages of N7/OsUbi-NS4B*(2/RQ) identified the mutation E1576V in NS2 that is critical for NS2-3-independent virion morphogenesis, resulting in increased viral titers of 7.6 × 10⁵ TCID₅₀/ml. Osloss sequences are shown in gray. 2/RQ, cell culture-adapted mutation in NS2 at position 1268 (34). Ubi, ubiquitin insertion derived from BVDV strain Osloss. Viral titers were determined in TCID₅₀/ml. (B) MDBK cells were electroporated with 1 μg of RNA of the chimeric BVDV derivative N7/OsUbi-NS4B*(2/RQ). Cell cultures grown for 24 h and 48 h pe were analyzed by NS3-specific IF. (C) Electroporated cells depicted in panel B were passaged until an increased cytopathic effect was observed (cell passage) and cell culture supernatants were collected. Naive MDBK cells were inoculated with 500 μl of the collected supernatants and analyzed by NS3-specific IF at 72 h pi. Viral RNAs were isolated and the NS2-3-4A-4B* region was sequenced. Analyses identified the mutation E1576V in NS2.