Figure 4. Generation and Characterization of rLCMV/NJG.

(A) Schematic describing the protocol for generation of rLCMV/NJG.

(B) pS_NJ(−) was designed analogous to pS_IND (also referred to as pSr(−) [36]). It expresses in genomic (−) polarity a recombinant LCMV S segment where the LCMV-GP ORF was substituted for the NJG gene. Transcription is driven by the murine polymerase I promotor (PIP) and is terminated upstream of the murine polymerase I terminator (PIT). UTR, untranslated region; IGR, intergenic region; *noncoding single nucleotide tags.

(C) BHK-21 cells on duplicate coverslips were infected for 24 h with supernatants collected from the experiment outlined in A. Staining with INDG- or NJG-specific mAbs as indicated provided a rough estimate of the relative proportions of rLCMV/INDG and rLCMV/NJG contained in the supernatants tested.

(D) BHK-21 cells (10⁶ per M6 tissue culture well) were infected with different doses of rLCMV/INDG or of rLCMV/NJG or with both viruses in different combinations as indicated in the chart. At 48 h later, the cells as well as the supernatant were collected. Total cellular RNA was extracted and was processed for detection of INDG and NJG RNA by RT-PCR. The PCR products were separated by agarose gel electrophoresis and were visualized by ethidium bromide staining. Specific amplification products of the expected size are indicated with arrows. Viral infectivity in the supernatant (SN) was measured by immunofocus assay [PFU/ml SN (w/o nAb) listed in the chart under the gel picture]. In addition, an aliquot of supernatant was incubated with VSV-NJ neutralizing mAb H6B9D5 prior to testing the remaining infectivity [see PFU/ml SN (+VSV-NJ nAb)] to discriminate rLCMV/INDG from rLCMV/NJG infectivity.