Time-course analysis of IAPV infection in honey bees

Based on the results of the preliminary experiments 1 and 2 (Preliminary Experiments S1), a more extensive IAPV inoculation experiment was designed to study the time line of infection and associated gene expression patterns, and to assess bees for variability in IAPV susceptibility.
One microliter of the inoculant (PBS as control or virus solution containing 10⁴ genome equivalents of IAPV) was injected using a NanoJet™ syringe pump (Chemix, USA) with an infusion flow rate of 0.1µl/sec, following manufacturer’s parameters. The needle was inserted in the lateral abdomen between the fourth and fifth tergite of young, white-eye honey bee pupae (Figure 2A).
Two strong, IAPV-free hives were selected from the UNCG research apiary, representing two distinct sources of bees for the experiment. From each hive, 200 white-eye pupae were collected for each of the following treatment groups: without inoculation (W/O), PBS inoculated (PBS), and IAPV inoculated (IAPV). From each treatment group and hive, 50 bees were frozen at 0 h, 5 h, 20 h and 48 h after inoculation and a subset of these samples was individually analyzed for viral titers and gene expression patterns. The first time point directly after inoculation was used as a control of the initial states of the bees in the experimental and control groups. The time point of five hours post-infection was chosen to measure the virus impact before completion of the replication cycle, based on the assumption that IAPV follows the picornavirus family average timing for a replication cycle, of 7–12 hours [32] , [34] (link), [35] (link). Any gene expression changes at this time point represent the bees’ response to inoculation without complications from virus-related tissue damage. The time point of 20 h post-infection was considered representative of events after one complete cycle of virus replication, and the 48 h time point represents the established diseased state, characterized by visual symptoms.
Based on the results of the preliminary experiments (Preliminary Experiments S1), we tested the effect of IAPV injection on gene expression of six commonly used reference genes that have been reported to be constantly expressed across different experimental conditions [4] (link), [7] (link), [41] (link). We studied the transcription of Actin, ribosomal 28S RNA, ribosomal 18S RNA, ribosomal protein RPS5, MGST1, and Histone H2A, under IAPV infection. Histone H2A is not common used in honey bees, but it was added to our experiment because its expression is constitutive and cell-cycle independent, and it is commonly used on other models [42] (link). The sequences of utilized H2A primers are: 5′-AAAGGAAATTACGCAGAACGA-3′ (H2A Forward) and 5′-CGGCTAAATATTCCATAACGG-3′ (H2A Reverse). In addition, the titers of IAPV and DWV were quantified in these samples.