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. 2021 Jul 17;12(7):653. doi: 10.3390/insects12070653

Figure 2.

Figure 2

Sacbrood virus (SBV) replicates in primary honey bee pupal cell cultures. (A) SBV abundance was assessed in mock- or SBV-treated pupal cells by qPCR over a time course (i.e., 0 hpi, 24 hpi, 48 hpi, 72 hpi, and 96 hpi). A representative replicate of SBV-infection in pupal cells is plotted. The addition of larval lysate to pupal cells resulted in a 59× fold increase relative to 0 hpi by 48 hpi (p < 0.0001), with subsequent increases at 72 hpi (126×, p < 0.0001) and 96 hpi (1047×, p < 0.0001). Raw data are included in Supplemental Table S3. See Supplemental Figure S2 for additional replicates. All differences in means relative to 0 hpi were assessed by a Dunnett’s test. Significance levels: *** p < 0.0005. (B) To confirm that SBV was productive in infecting larval hemocytes, the presence of the negative strand (a replicative intermediate) was assessed by negative strand-specific reverse transcription (RT) followed by PCR. Negative strand was detected at all time points after 24 hpi. Additional control reactions were performed using pooled RNA from time points 24–96 hpi (Lanes labeled 1–9). Specifically, RNA isolated from SBV 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. (0, 24, 48, 96 hpi)—RT with random hexamers, PCR with SBV-221-240-For and SBV-478-497-Rev. (1) Negative control: No RT in the presence of (SBV-TAGS-F), PCR with TAGS and SBV-478-497-Rev. (2) Negative control: RT in the presence of (SBV-TAGS-F), PCR with only the SBV-478-497-Rev primer. (3) Negative control: RT with random hexamer primer, PCR with TAGS and SBV-478-497-Rev. (4) Negative control: No RT enzyme in the presence of random hexamers, PCR with SBV-221-240-For and SBV-478-497-Rev. (5) Positive control: RT with random hexamers, PCR with SBV-221-240-For and SBV-478-497-Rev. (6) Negative control: RT with random hexamers, PCR with only SBV-478-497-Rev primer. (7) Self-priming: RT without a primer, PCR with SBV-221-240-For and SBV-478-497-Rev. (8) Negative control: No template, no RT, PCR with TAGS and SBV-478-497-Rev. (9) Negative control: No template, no RT, PCR with SBV-221-240-For and SBV-478-497-Rev. Additional details provided in Supplemental Table S25.