Figure 3.

Deformed wing virus (DWV) replicates in primary honey bee pupal cell cultures. DWV abundance was assessed in mock- or DWV-infected pupal cells via qPCR over a time course (i.e., 0 hpi, 24 hpi, 48 hpi, 72 hpi, and 96 hpi). (A) Pupal cells infected with 4.3 × 10⁶ DWV RNA copies had a 2.7× (p < 0.001) and 3.5× (p < 0.001) increase in mean DWV abundance at 48 hpi and 72 hpi, respectively, relative to 0 hpi. At 96 hpi, mean DWV abundance was 4× higher relative to 0 hpi (p < 0.001). Figure 3 includes results from one representative biological replicate. The data for additional replicates are presented in Supplemental Figure S3. All differences in means relative to 0 hpi were assessed by a Dunnett’s test. Raw data are included in Supplemental Table S4. Significance levels: ** p < 0.005; *** p < 0.0005. (B) To verify DWV replication in purple-eyed pupal cells, negative-strand-specific RT-PCR was performed, and the replicative intermediate form (i.e., negative strand) of DWV was detected at 72 hpi. Additional control reactions were performed using pooled RNA from time points 24–96 hpi (Lanes labeled 2–9). Specifically, RNA isolated DWV containing cell lysate was reverse-transcribed with the primer listed below, treated with Exonuclease I to remove excess primer, and amplified using the PCR primers listed for each lane. (L) Molecular weight ladder. (1) 72 hpi RNA template—RT with DWV-TAGS-F, PCR with TAGS and DWV-Rev. (2) Negative control: No RT in the presence of (DWV-TAGS-F), PCR with TAGS and DWV-Rev. (3) Negative control: RT in the presence of (DWV-TAGS-F), PCR with only the DWV-Rev primer. (4) Negative control: RT with random hexamer primer, PCR with TAGS and DWV-Rev. (5) Negative control: No RT enzyme in the presence of random hexamers, PCR with DWV-F and DWV-Rev. (6) Positive control: RT with random hexamers, PCR with DWV-F and DWV-Rev. (7) Negative control: RT with random hexamers, PCR with only DWV-Rev primer. (8) Self-priming: RT without a primer, PCR with DWV-F and DWV-Rev (9) Negative control: No template, no RT, PCR with TAGS and DWV-Rev. (10) Negative control: No template, no RT, PCR with DWV-F and DWV-Rev. Additional details provided in Supplemental Table S25.