Figure 1.
Detection of IAPV infection in a representative honey bee colony.
(A) Gel electrophoresis of RT-PCR amplification for specific detection of IAPV from samples of worker eggs, worker larvae, worker pupae, adult workers, drones, queens and parasitic mites, Varroa destructor collected from the same colony. (B) Gel electrophoresis of RT-PCR amplification for specific detection of IAPV from samples of colony foods, queen feces, and drone semen. For both A and B, a PCR band of 586 bp indicating the IAPV infection is observed in examined samples.
Figure 2.
Relative abundance of negative strand RNA of IAPV genome copies in different tissues of honey bees and in situ hybridization analysis of queen somatic and germ tissues.
(A) The hemolymph harbored the minimal level of IAPV and therefore was chosen as a calibrator. The concentration of negative strand RNA of IAPV in other tissues was compared with the calibrator and expressed as n-fold change. The y-axis depicts fold change relative to the calibrator. (B) The slides were not hybridized with DIG-labeled IAPV probe (top row, negative control) and the slides were hybridized with DIG-labeled IAPV probe (bottom row). Positive signal is dark blue to purple and the negative areas are pink in color. The infected tissues of queen gut, ovary, spermatheca and queen eggs are indicated by a dark blue/purple color.
Figure 3.
Average prevalence of IAPV infection in a single month.
(A) Strong colonies. (B) Weak colonies. For both strong and weak colonies, the prevalence of IAPV infection in the brood was significantly higher than in adult bees. While strong colonies did not exhibit significant seasonal variation in IAPV infection, the infection rate of IAPV in adult bees in weak colonies increased from Spring to Summer and Fall and peaked in the Winter. All strong colonies survived through the cold winter months while the weak colonies collapsed before February.
Figure 4.
Genome-wide sequence diversity and phylogenetic relationship of IAPV isolates.
(A) A graphical representation of the pair-wise global alignments of the reference sequence of IAPV (NC_009025), the first complete sequence of IAPV, with other IAPV genome sequences individually. This figure is retrieved from GenBank and modified. The alignments were pre-computed using the “band” version of the Needleman-Wunsch algorithm. The top histogram shows the average density of nucleotide changes (excluding gaps, insertions and undetermined nucleotides) in all additional sequences per a reference sequence segment. The length of the segment is equal to the length of the reference sequence divided by the width of its graphical representation (in pixels). The deletions, insertions and differences among the sequences are highlighted in blue, green and red-violet, respectively. If no significant alignment could be obtained for a particular sequence, no horizontal bar is shown. (B) Phylogenetic tree showing the relationship of IAPV strains from different geographic locations globally. Numbers at each node represent bootstrap values as percentages of 500. Individual sequences are labeled with their GenBank accession numbers.
Figure 5.
An overview of gene expression profiles in IAPV infected adults and brood.
(A) Venn graph compares regulated genes between adult and brood. The intersecting circles indicate overlapping genes between adult and brood. Of 4615 genes with altered expression in IAPV-positive adult and 1350 genes with altered expression in IAPV-positive brood, the number of overlapping genes between adults and brood was 336. (B) A heat map illustrates differential expression profiles of up- and down- regulated genes for adults and brood. The number of genes with altered expression was significantly higher in IAPV infected adult than in IAPV infected brood. The relative levels of gene expression are depicted using a color scale where blue indicates the lowest and red indicates the highest level of expression. Significantly enriched Gene Ontology (GO) terms of up- and down regulated gene clusters inducted by IAPV infection (ρ≤0.05) appear on the right side of the heat map.
Figure 6.
Regulated molecules that are involved in host metabolism and immunity.
The figure illustrates a network predicted by Ingenuity Pathway Analysis that is centered by viral infection and associated with molecules involved in host energy metabolism and immunity. Solid and dashed connecting lines indicate the presence of direct and indirect interactions, respectively. Nodes indicate input of genes into the pathway analysis and the different symbols indicate gene functions (Legend in bottom left). The intensity of the node color-(red) indicates the degree of up-regulation while the intensity of the node color-(green) indicates the degree of down-regulation. The numbers shown in each node indicates the fold change in response to IAPV infection.
Figure 7.
Expression levels of immune-related transcripts in IAPV infected adults.
The expression levels of genes that were assigned to JAK-STAT, mTOR, MAPK and Endocytosis pathways were measured by microarray analysis and further confirmed using TaqMan RT-qPCR. The expression results obtained from microarray and qRT-PCR analyses showed good alignment.
Figure 8.
IAPV-encoded putative suppressor of RNAi.
(A) Highly conserved octamer sequences identified in dicistroviruses. A putative viral suppressor of RNAi (VSR) is presumably located upstream of DvExNPGP. The cleavage site between the glycine and proline is marked by an arrow. (B) Quantitative analysis of the effects of silencing putative VSR on IAPV replication. The amount of negative stranded RNA of IAPV was measured by RT-qPCR, normalized to the corresponding β-actin in the same sample. The data shown represent the mean value for three separate experiments. Error bars represent the range of fold change.
Table 1.
IAPV primers used in the study.
Table 2.
Primers of immune genes in the study.