Serum anti-WHs (—)
Serum WHsAg (—)
Serum WHV DNA (—)
Innate immunity
Adaptive immunity
Lifetime
HCC risk
Chronic = 60-75%
100%
Resolved = 25-40%
15-20%
0
WHV7P1
(WHV7-11)
4
8
12
16
t (post inoculation)/wk
20
24
88
Median time
of sampling
Supporting Figure 1. Neonatal woodchuck model of experimental WHV infection. Schematic serological profiles for WHV DNA, WHV surface
antigen (WHsAg), and virus-neutralizing antibody to WHsAg (anti-WHs) in chronic and resolved WHV infections in the neonatal woodchuck model.
Woodchucks born to WHV-negative dams during early spring were subcutaneously infected at 3 days of age with 5 x 106 infectious doses of the same
WHV7P1 inoculum containing WHV strain WHV7-11. The proportions of chronic and resolved WHV infection outcomes usually range between 6075% and 25-40%, respectively. Chronic WHV infection is characterized by high blood levels of virus and viral antigens without seroconversion to antiWHs antibody. Resolved WHV infection is characterized by a substantial clearance of virus and viral antigens from the blood and seroconversion to antiWHs antibody. The lifetime risk for the development of HCC in established chronic and resolved WHV infections is 100% and 15-20%, respectively.
HCC in uninfected, WHV-negative woodchucks is not observed. The time point for tissue (liver, spleen and kidney) sampling, at a median age of 88
weeks = 22 months (range 15-28 months), is indicated. Approximate time intervals for the development of innate and adaptive immunity are shown.
Liver
Carrier
Liver
Resolved
Liver
Negative
PBMC
Carrier
PBMC
Negative
All reads (5,741,102) combined (100%)
Map to WHV7-11 genome
(85% id, 80% cov)
Unmapped (99.6%)
Map to Human Refseq (85% id, 80% cov)
Mapped (22.7%)
Chimeric, Repeat, or Unmapped (76.1%)
23,618 Contigs
Map to Human Genome (85% id, 80% cov)
(avg: 749, N50: 1,005)
Unmapped (53.7%)
Mapped (3.3%)
Chimeric (12.6%)
Contigs
De-novo assembly
10,037 Isotigs
(avg:1,022; N50:1,203)
De-novo assembly
25,615
Singletons
Merge to contigs (48,933)
61,039 Contigs, Singletons and WHV
sequences submitted for chip design
33,554 Isotigs
(avg:1,039; N50:1,340)
571,556
Singletons
Singletons longer than 540 bp
excluding repeats (12,101)
5 WHV transcript regions
Supporting Figure 2. Workflow of the woodchuck transcriptome assembly. All reads from the five
sequenced samples (i.e., liver carrier, liver resolved, liver negative, PBMC carrier, and PBMC negative) were
combined and mapped against the WHV7-11 genome (NC_004107) to filter out WHV reads (0.4%). The reads
were then mapped to the human Refseq database and to the human reference genome (hg19) to obtain
transcript contigs conserved in human. The reads chimerically mapped to the human Refseq or reference
genome were likely novel transcript forms and were therefore assembled together using Newbler (454 Life
Sciences, Branford, CT), while other unmapped reads were assembled separately. Finally, all assembled
contigs were merged into 48,933 contigs using Phrap (Phil Green, Genome Sciences, University of
Washington). These contigs, together with 12,101 non-repeat singletons longer than 540 bp, and 5 WHV
transcript regions (WHV polymerase, mature and signal peptide regions of WHsAg, WHx and WHcAg), were
used for microarray design. ID: identified, cov: coverage, avg: average.
PC #2 (12.7%)
U
R
C-N
C-H
PC #3 (8.4%)
Principal Component (PC) #1 (18.4%)
Supporting Figure 3. Persistent infection and HCC induce extensive changes in the liver transcriptome.
Principal component analysis of normalized liver expression data for U (n=10), R (n=11), and paired C-N and
C-H (n=13) samples. Technical repeats (n=3) for each sample are linked.
Leukocyte
extravasation
p=0.0001
Cell cycle control of
chromosomal replication
p<0.0001
Antigen presentation
p<0.0001
Bile acid and steroid
hormone metabolism
p<0.0001
C-N
-2 0
R
U
2
Supporting Figure 4. Persistent WHV infection markedly alters the liver transcriptome. Unsupervised
hierarchical clustering of the top differentially expressed intrahepatic genes for C-N, R and U samples. All
genes had an absolute fold-change >1.5 with a Benjamini-Hochberg corrected FDR<0.05. Heatmap columns
represent samples from individual animals, and rows represent different genes. Red and blue coloring of cells
represents high and low expression levels, respectively, as indicated by the scale bars for normalized values.
Functional annotation of gene clusters was performed by Ingenuity Pathway Analysis, with the top canonical
pathway for each cluster being displayed. Pathway enrichment was calculated with the Fisher’s exact test with
multiple testing correction by the Benjamini and Hochberg method. Gene clusters that were not significantly
enriched for a pathway were not functionally annotated.
p450 metabolism
p=0.0001
Antigen presentation
p<0.0001
GPCR signaling
p=0.0468
TRAIL signaling
FDR=0.0391*
MYC pathway
FDR=0.0447*
C-N
-2 0
C-H
2 4
Supporting Figure 5. WHV-induced HCC extensively modulates intrahepatic gene expression.
Unsupervised hierarchical clustering of the top differentially expressed intrahepatic genes for paired C-H and
C-N samples. All genes had an absolute fold-change >1.5 with a Benjamini-Hochberg corrected FDR<0.05.
Functional annotation of gene clusters was performed by Ingenuity Pathway Analysis, with the top canonical
pathway for each cluster being displayed. Pathway enrichment was calculated with the Fisher’s exact test with
multiple testing correction by the Benjamini and Hochberg method. In cases where Ingenuity did not identify
significant pathways, the annotation was performed by GSEA (indicated by asterisks). Gene clusters that were
not significantly enriched for a pathway were not functionally annotated.
Supporting Figure 6. MYC intrahepatic transcriptional signature in WHV-induced HCC. Network
(“protein synthesis”) created by Ingenuity Pathway Analysis. Color intensity indicates magnitude of
differential expression in C-H relative to C-N. All genes had an absolute fold-change > 1.5 with a BenjaminiHochberg corrected FDR<0.05.
a
p<0.001
p<0.001
20
15
10
p>0.05
5
p<0.001
6
LYZ expression
MPO expression
25
5
p=0.001
4
3
p>0.05
2
1
0
0
C-N
R
U
C-N
R
(n=11)
(n=11)
(n=10)
(n=11)
(n=11)
U
(n=10)
b
8
p<0.001
IGFBP3 expression
IGF2 expression
7
1.5
6
5
4
3
2
1
0
p<0.001
1.0
0.5
0.0
C-N
C-H
C-N
C-H
(n=11)
(n=11)
(n=11)
(n=11)
Supporting Figure 7. qRT-PCR verification of select intrahepatic genes. qRT-PCR data expressed as foldchange relative to (a) the mean of U, and (b) the mean of C-N. The bar height indicates the mean of each group,
and the errors bars represent the standard error of the mean.