Supplemental Figure S1. Larval hemocyte cultures have relative few contaminating adipocytes. 
(A) Hemocytes were live-imaged at 20x magnification in complete medium on a Nikon Ti-Eclipse 
(Nikon Instruments) inverted microscope equipped with a SpectraX LED (Lumencor) excitation module 
and fast-switching emission filter wheels (Prior Scientific). Scale bar is 100 μm. (B) Hemocytes were 
live imaged at 100x magnification. Scale bar is 10 μm. (C) Cells were fixed in 4% paraformaldehyde 
and then nuclei were stained using Hoechst 33258 stain at 30 mg/mL, and images were captured at 
20x magnification. Fluorescent images were captured using paired excitation/emission filters and 
dichroic mirrors for DAPI. From left to right: combined image, bright field image, and DAPI stain image. 
Scale bar is 100 μm. (D) Fixed images were captured using either bright field or fluorescent imaging at 
100x magnification. Scale bar is 10 μm. Together these images show that the isolation technique 
utilized in this study results in relatively pure cultures of larval hemocytes with relatively little 
contaminating adipocytes.
A B
20x  100 μm 11000x0 DxIC  10 μm
C
D
A B C
rep 1 rep 2 rep 3
4x105 ***
1.4x106 *** 1.2x105 ***
*** ***
3x105
1x106
8x104
2x105
6x105
4x104
1x105
***
2x105 *** ***
0 ***0 0 ***
hpi 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
PBS SBV PBS SBV PBS SBV
Supplemental Figure S2. Sacbrood virus (SBV) replicates in primary honey bee pupal cell cultures. 
SBV abundance was assessed in mock- or SBV-infected pupal cells via qPCR over a time course (i.e., 0 hpi, 24 hpi, 48 hpi, 72 hpi, 96 hpi) in three 
biological replicates. (A) In replicate 1 pupal treated with a filtered larval lysate containing 2.5x106 SBV RNA copies had 4.8x higher SBV by 48 hpi as 
compared to 0 hpi (p < 0.0001), which remained high at 72 hpi (12x, p < 0.0001) and 96 hpi (11.4x, p < 0.0001). (B) In replicate 2, SBV was significantly 
higher than 0 hpi by 48 hpi (59x, p < 0.0001) and remained high at 72 hpi (126x, p < 0.0001) and peaked at 96 hpi (1,047x, p < 0.001). This representative 
biological replicate is shown in Figure 2. (C) In replicate 3, SBV abundance at 24 hpi was lower (0.017x) than at 0 hpi (p < 0.0001). SBV abundance at 48 
hpi was 2.2 x higher at 48 hpi relative to 0 hpi (p = 0.0012), as well as at 72 hpi (14x, p = 0.00013) and 96 hpi (72x, p < 0.0001). Overall (i.e., from the 
lowest at 24 hpi to the highest at 96 hpi) SBV abundance increased by 1,817x (p < 0.0001). Differences in means relative to 0 hpi were assessed by a 
Dunnett’s test. Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005. 
SBV RNA coSBV RNA equivalentsper 20 ng RNA pies
per 20 ng RNA
SBV RNA copies
per 20 ng RNA
SBV RNA copies
per 20 ng RNA
A B
rep1 rep2
***
**
4x106 4x106
***
3x106 ** 3x10
6
2x106 2x106 **
1x106 1x106
0 0
hpi 0 24 48 72 96 0 24 48 72 96 hpi 0 24 48 72 96 0 24 48 72 96
mock DWV mock DWV
Supplemental Figure S3. 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, 96 hpi) in two biological replicates. (A) In replicate 1, pupal cells infected with 4.3x106 DWV 
RNA copies had a 2.7x (p < 0.001) and 3.5x (p < 0.001) increase in mean DWV abundance relative to 0 hpi at 48 
hpi and 72 hpi, respectively. At 96 hpi, mean DWV abundance was 4x higher relative to 0 hpi (p < 0.001). 
(B) In replicate 2, DWV levels remained similar to inoculum levels through 48 hpi, but were 3.2x higher at 72 hpi 
(p = 0.001), and 6.6x higher at 96 hpi, relative to 0 hpi (p = 0.003). One representative biological replicate (i.e., 
rep1) is shown in Figure 3. Differences in means relative to 0 hpi were assessed by a Dunnett’s test. 
Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005. 
DWV RNA copies
 per 20 ng RNA
DWV RNA copies
 per 20 ng RNA
A rep1 B rep2
*** ***
5x108
5x108
4x108
4x108 ***
***
3x108
3x108
8
2x108
2x10 **
***
*
1x108 1x108
0 0
hpi 0 24 48 72 96 0 24 48 72 96 hpi 0 24 48 72 96 0 24 48 72 96
mock FHV mock FHV
Supplemental Figure S4. Flock House virus (FHV) replicates in primary honey bee hemocytes. 
FHV abundance in mock- or FHV- infected (1x106 FHV RNA copies) hemocytes was assessed over time (i.e., 0 hpi, 24 hpi, 48 hpi, 72 hpi, 
96 hpi). (A) In replicate one, FHV abundance was higher at 24 hpi (3.7x, p = 0.026), 48 hpi (10.7x, p < 0.001) and 72 hpi (37x, p < 0.001) 
relative to 0 hpi. FHV abundance decreased from 72 hpi to 96 hpi (p < 0.05), but was significantly higher relative to 0 hpi (p < 0.001). 
(B) In replicate two, FHV abundance at 24 hpi was similar to levels at 0 hpi. At 48 hpi FHV abundance was 16x higher than the 0 hpi (p = 
0.001). FHV abundance peaked at 72 hpi, with 39.8x higher FHV levels compared to 0 hpi (p < 0.001). At 96 hpi, the abundance of FHV 
abundance was less than the level at 72 hpi, but was still 12.6x higher that levels at 0 hpi (p < 0.001). One representative biological repli-
cate (i.e., rep1) is also shown in Figure 4. Differences in means relative to 0 hpi were assessed by a Dunnett’s test. 
Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005.
FHV RNA copies
 per 20 ng RNA
FHV RNA copies
 per 20 ng RNA
A rep1 B rep2
*** ***
8x109 8x109
**
***
***
6x109 6x109
4x109 4x109
***
*
2x109 2x109
*
0 0
hpi 0 24 48 72 96 0 24 48 72 96 hpi 0 24 48 72 96 0 24 48 72 96
mock FHV mock FHV
Supplemental Figure S5. Flock House virus (FHV) replicates in primary honey bee pupal cells. 
Honey bee pupal cells were either mock- or FHV-infected (2x108 FHV RNA copies) and FHV abundance was quantified over a course of time 
(i.e., 0 hpi, 24 hpi, 48 hpi, 72 hpi, 96 hpi). (A) In replicate one, FHV abundance increased at 24 hpi (1.7x, p = 0.024) and 48 hpi (3.4x p < 0.001), 
with a peak in FHV infection at 72 hpi (5x, p < 0.001) relative to 0 hpi. FHV abundance did not increase from 72 hpi to 96 hpi, but remained 4x higher 
than the levels at 0 hpi (p < 0.001). (B) In replicate two, FHV abundance was higher at 24 hpi (2.6x, p = 0.013) and 48 hpi (7.5x, p < 0.001) relative 
to levels at 0 hpi. FHV abundance increased 20x at 72 hpi, which was the peak of virus infection (p < 0.001). FHV levels in pupal cells were lower 
at 96 hpi relative to 72 hpi (p < 0.01), but were still 15.8x higher at 96 hpi compared to 0 hpi (p < 0.001). One representative biological replicate 
(i.e., rep1) is shown in Figure 5. Differences in means relative to 0 hpi were assessed by a Dunnett’s test. 
Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005. 
FHV RNA copies
 per 20 ng RNA
FHV RNA copies
 per 20 ng RNA
A B
rep1 rep2
4x109 4x109
***
3x109 3x109
2x109 2x109 ***
1x109 ** 1x109
0 0
hpi 0 48 72 0 48 72 hpi 0 48 72 0 48 72
mock FHV mock FHV
C rep1 D rep2
***
2x105
2x105
1.5x105
1.5x105 **
1x105
1x105
***
0.5x105 *** 0.5x105
0 0
hpi 0 48 72 0 48 72 hpi 0 48 72 0 48 72
mock DWV mock DWV
Supplemental Figure S6. DWV and FHV infection transferred from infected to naïve honey bee pupal cells. 
Mock- or virus-infected honey bee pupal cell cultures at 72 hpi were utilized to transfer virus infection to naïve pupal cells. Virus 
abundance was assessed over a time course (i.e., 0 hpi, 48 hpi, 72 hpi) using qPCR. (A) In replicate one, naïve honey bee pupal cells 
were treated with either 10 µL of mock- or DWV-infected cells, which was an equivalent of 1.04x105 DWV RNA copies. DWV abun-
dance was 2.7x higher at 48 hpi compared to 0 hpi and 4.1x higher  at 72 hpi relative to 0 hpi (p < 0.001). In replicate two, naïve pupal 
cells were treated with 10 µL of mock- or DWV-infected pupal cell culture, which contained 9x104 DWV RNA copies. DWV abundance 
increased by 2.3x at 48 hpi (p = 0.002) and was 10.4x greater at 72 hpi, relative to 0 hpi (p < 0.001). (B) In replicate one, 10 µL of 
mock- or FHV-infected pupal cell culture with 2.6 x 108 FHV RNA copies were utilized to infect naïve pupal cells. FHV abundance 
increased 15.8x at 48 hpi (p = 0.003) with a subsequent 28x increase at 72 hpi (p < 0.001) relative to 0 hpi. In replicate two, mock- or 
FHV-infected pupal cells (i.e., 10 µL infected cell culture with 2.9x108 FHV RNA copies) from first round of infection were transferred to 
naïve pupal cells and FHV abundance remained constant from 0 hpi to 48 hpi but was 10.4x at 72 hpi (p < 0.001). One representative 
biological replicate (i.e., rep1) is shown in Figure 6. Differences in means relative to 0 hpi were assessed by a Dunnett’s test. 
Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005. 
DWV RNA copies
 per 20 ng RNA FHV RNA copies per 20 ng RNA
DWV RNA copies
 per 20 ng RNA FHV RNA copies
 per 20 ng RNA
A ago2
rep 1 rep 2 rep 3
1 1 1 *
0 0 0
-1 -1 -1
mock SBV mock SBV mock SBV
B
dcr-like
1 1 *
0 0
-1 -1
mock SBV mock SBV
C
bap-1/mf116383
* * *
1 1 1
0 0 0
-1 -1 -1
mock SBV mock SBV 72 96 72 96
mock SBV
Supplemental Figure S7. Infection of primary pupal cells with SBV results in higher 
bap-1/mf116383 expression. The relative expression of three immune genes (i.e., ago2, dcr-like, and 
bap1/mf116383) in SBV-infected pupal cells was assessed by qPCR at 72 hpi relative to mock-infected 
cells. (A) In replicates 1 and 2, ago2 expression levels were similar in SBV-infected and mock-infected 
cells, but in the third replicate ago2 expression is significantly lower (-0.76 log2-fold) in SBV-infected 
cells than mock at 72 hpi (p = 0.032). (B) While we were unable to collect dcr-like expression data in 
replicate 1, dcr-like expression was similar in SBV-infected and mock-infected cells in replicate 2. 
Similar to ago2, dcr-like expression in SBV-infected cells was less (-0.67 log2-fold) than levels in 
mock-infected cells at 72 hpi (p = 0.032). (C) The relative expression of bap1/mf116383 was higher in 
SBV-infected cells compared to mock-infected cells at 72 hpi (replicate 1 = 0.67 log2-fold, replicate 
2 = 0.95 log2-fold, p = 0.032). While levels of bap1/mf116383 expression in SBV-infected and mock-
infected cells were similar at 72 hpi in the third replicate, at 96 hpi, bap1/mf116383 expression was 
higher (0.73 log2-fold, p = 0.032) in SBV-infected cells than in mock-infected. Together, these data
 indicate SBV infection of honey bee pupal cells has little or no impact on ago2 or dcr-like expression 
at 72 hpi, but does result in higher bap1/mf116383 expression.
log2 fold change log2 fold change log2 fold change
A ago2
rep 1 rep 2
1 1
*
0 * 0
-1 -1
mock DWV mock DWV
B
dcr-like
1 1
0 * 0
-1 -1
mock DWV mock DWV
C
bap1/mf116383
1 1
*
0 0
-1 -1
mock DWV mock DWV
Supplemental Figure S8. Infection of primary honey bee pupal cells with 
DWV had modest impact on the expression of select immune genes. 
The expression of three honey bee immune genes (i.e., ago2, dcr-like, and 
bap1/mf116383) in DWV-infected pupal cells relative to mock-infected cells at 72 
hpi was assessed by qPCR. (A) In both biological replicates (replicate one and 
two), ago2 expression was lower in DWV-infected cells at 72 hpi relative to 
mock-infected cells (p = 0.032). (B) In replicate 1, the expression for dcr-like was 
lower in DWV-infected cells relative to mock-infected cells at 72 hpi (p = 0.032), 
but in replicate two, dcr-like expression was not impacted. (C) In replicate one, 
bap1/mf116383 expression in DWV-infected and mock-infected cells at 72 hpi 
was similar, but in replicate two, the expression of bap1/mf116383 was slightly 
higher in DWV-infected pupal cells compared to mock-infected cells (p = 0.032). 
One representative biological replicate (rep2) for each gene is shown in Figure 7. 
Differences in means relative to mock-infected cells were assessed by a one-sided 
Wilcoxon Rank Sums test. 
Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005. 
log2 fold change log2 fold change log2 fold change
A ago-2
rep 1 rep 2
* *
1 1
0 0
-1 -1
mock FHV mock FHV
B
dcr-like
1 * 1 *
0 0
-1 -1
mock FHV mock FHV
C
bap1/mf116383
1 1
0 * 0
-1 -1
mock FHV mock FHV
Supplemental Figure S9. Infection of primary honey bee pupal cells with FHV 
results in differential regulation of immune genes. 
The expression of three immune genes (i.e., ago2, dcr-like, and bap1/mf116383) in 
FHV-infected pupal cells relative to mock-infected cells was assessed by qPCR at 72 hpi. 
(A) In both biological replicates (replicate one and two), ago2 expression was higher in 
FHV-infected cells at 72 hpi relative to mock-infected cells (p = 0.032). (B) In replicate 
one and two, dcr-like expression was higher (p = 0.032) in FHV-infected cells relative 
to mock-infected cells at 72 hpi. (C) In replicate one, bap1/mf116383 expression was 
slightly reduced in FHV-infected cells relative to mock-infected cells at 72 hpi (p = 0.032), 
but in replicate two, the levels of bap1/mf116383 expression were similar in FHV-infected 
pupal cells compared to mock-infected cells at 72 hpi. One representative biological 
replicate (rep2) for each gene is shown in Figure 7. Differences in means relative to 
mock-infected cells were assessed by a one-sided Wilcoxon Rank Sums test. 
Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005. 
log2 fold change log2 fold change log2 fold change
A ago2
rep 1 rep 2
*
1 1 *
0 0
-1 -1
mock FHV mock FHV
B
dcr-like
1 1 *
*
0 0
-1 -1
mock FHV mock FHV
C
bap1/mf116383
1 1
0 0
-1 -1
mock FHV mock FHV
Supplemental Figure S10. Infection of honey bee hemocytes with FHV 
results in differential regulation of immune genes. 
The expression of three immune genes (i.e., ago2, dcr-like, and bap1/mf116383) 
in FHV-infected hemocytes relative to mock-infected cells was assessed by 
qPCR at 72 hpi. (A) In both biological replicates (replicate one and two), ago2 
expression was higher in FHV-infected hemocytes at 72 hpi relative to mock-
infected cells (i.e., 1.1 log2-fold in rep1 and 0.54 log2-fold in rep2, p = 0.032). 
(B) Likewise, dcr-like expression in FHV-infected hemocytes was higher 
compared to expression levels mock-infected cells at 72 hpi (i.e., 0.45 log2-fold 
in rep1 and 0.63 log2-fold in rep2, p = 0.032). (C) FHV infection did not have 
appreciable impact on bap1/mf116383 expression at 72 hpi. Differences in 
means relative to mock-infected cells were assessed by a one-sided Wilcoxon 
Rank Sums test. Significance levels: * p < 0.05; ** p < 0.005; *** p < 0.0005.  
log2 fold change log2 fold change log2 fold change
ABPV BQCV CBPV DWV
I + - I + - I + - I + - 400
400 300
300 200
200 100
100
IAPV KBV LSV-U SBV
I + - I + - I + - I + - 400
400 300
300 200
200 100
100
Supplemental Figure S11. Sacbrood virus (SBV) inoculum was free of several other common honey 
bee infecting viruses. 
The SBV preparation that was used as inoculum in this study was tested for the presence of contaminating 
viruses using PCR. RNA was isolated from 100 µL SBV stock and reverse transcribed to synthesize cDNA. 
Pathogen specific PCR was carried out to test for the presence of acute bee paralysis virus (ABPV), black 
queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute 
paralysis virus (IAPV), Kashmir bee virus (KBV), Lake Sinai viruses (i.e., LSV1 and LSV2), and SBV, using 
primers listed in Supplemental Table S1. The inoculum was confirmed to have SBV and did not have 
detectable levels any of the other viruses tested. 
ABPV BQCV CBPV DWV
400
300 I + - I + - I + - I + -
400
300
200 200
100 100
IAPV KBV LSV-U SBV
500
400 + - 400
300 I + - I + - I + - I 300
200 200100
100
Supplemental Figure S12. Deformed wing virus (DWV) inoculum was free of several other common 
honey bee infecting viruses. 
The DWV preparation that was used as inoculum in this study was tested for the presence of contaminating 
viruses using PCR. RNA was isolated from 100 µL DWV stock and reverse transcribed to synthesize cDNA. 
Pathogen specific PCR was carried out to test for the presence of acute bee paralysis virus (ABPV), black 
queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Kashmir bee 
virus (KBV), Lake Sinai viruses (i.e., LSV1 and LSV2), sacbrood virus (SBV), and DWV, using primers listed in 
Supplemental Table S1. The inoculum was confirmed to have DWV and did not have detectable levels any of 
the other viruses tested.