immune response in fhm cells following infection with frog virus 3
TRANSCRIPT
![Page 1: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/1.jpg)
Differential Transcription of Fathead Minnow Immune-Related Genes
Following Infection with Frog virus 3 (FV3)
Kwang Cheng, B. Lynn Escalon, Natalia Garcia-Reyero, and V. Gregory
Chinchar
![Page 2: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/2.jpg)
Immune Competence Determines Survival in Xenopus
• FV3 infection of Xenopus laevis tadpoles and immunocompromised adults triggers severe systemic disease and high mortality.
• In contrast, FV3 infection of immunocompetent adults is confined to the kidney and associated with high levels of survival.
![Page 3: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/3.jpg)
Cellular vs Viral Responses Determine Survival
• Cellular genes contribute to anti-viral immunity and viral clearance.
– Immunocompetent adults display protective innate (IFN, pro-inflammatory) and acquired (Antibody, CTLs) responses that resolve infection.
• In contrast, viral immune evasion genes antagonize cellular immune responses.
– For example ranavirus vIF2α is thought to block PKR-mediated translational shut-off.
![Page 4: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/4.jpg)
Goal
• Identify cellular immune-related molecules that are differentially expressed in FHM cells after FV3 infection.
![Page 5: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/5.jpg)
Approach
• Monitor the expression of cellular immune-related genes using a 60K feature fathead minnow (FHM) microarray.
![Page 6: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/6.jpg)
Q. When are immune-related genes expressed?
M 4h 8h 16h 24h 48h
MCP MCP
Mx
actin
![Page 7: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/7.jpg)
Methodology
• Sham infect or infect replicate (6) cultures of FHM cells with wt FV3 or the 18K KO mutant, that was shown to be attenuated in vivo.
• Prepare total RNA at 8 hr after infection.
• Monitor cellular gene expression by microarray analysis
• Validate microarray results by qRT-PCR
![Page 8: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/8.jpg)
Confirmation of a Productive Viral Infection
M FV3 ∆18K
MCP
18K
M WT Δ18K
MCP
18K
![Page 9: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/9.jpg)
18
K-1
18
K-2
18
K-3
18
K-5
WT-
2
WT-
4
WT-
5
WT-
1
WT-
3
M-1
M-3
M-2
.1
M-2
.2
M-5
Heat map
analysis of
replicate
cultures of
mock-infected,
wt FV3-
infected, and
∆18K knock
mutant-infected
FHM cells
![Page 10: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/10.jpg)
Gene Name (abbreviation WT ∆18K
TNFRSF14 151 51
Interleukin 8 (IL8) 145 70
EIF2S2 96 24
NFIL3 66 13
ICLP2 62 11
NFKBIA 52 23
GILT 30 9
TRAF3 20 12
CAV1 14 8
POLR2A 14 2
Interferon (IFNα) 11 4
Differential Gene Expression: WT FV3 and ∆18K versus Mock-
infected
![Page 11: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/11.jpg)
Additional Upregulated Genes
Gene Name WT ∆18K
Interleukin 1β (IL1B) 9 7
Mx 9 ND
IRF3 5 2
MHC class I 5 4
RIGI 3 3
IRF1 3 2
CCNT2 3 -2
ADAR 1* 4
![Page 12: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/12.jpg)
Genes down regulated after FV3 infection
Gene Name WT ∆18K
HSPB8 -3 -5
G6PD -3 -3
POLB -2 ND
POLA2 -3 -2
C9 -3 ND
ATG7 -4 -3
![Page 13: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/13.jpg)
Pathways Affected by FV3 Infection
• IFN induction and regulation – IFN, IRF-1, IRF-2, IRF-3, IRF-7, RIGI
• Pro-Inflammatory immune response– IL8, IL3, IL1β
• Immune activation/ anti-viral activity– NFKBIA, TNFRSF14, MxD, ADAR
• Antigen Presentation– MHC class I, ICLP2
• Metabolism– EIF2S2 (eIF-2β), cyclin T2a, HSP70 family members,
POLR2A, CAV1
![Page 14: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/14.jpg)
qRT-PCR Validation of Microarray Results
0
50
100
150
200
250
Mock WT 18K
Rela
tive G
ene E
xpre
ssio
n
IL-8
0
10
20
30
40
50
60
70
80
Mock WT 18K
Rela
tive G
ene E
xpre
ssio
n
INF
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
3
Mock WT 18K
Rela
tive G
ene E
xpre
ssio
n
Cyt2a
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Mock WT 18K
Rela
tive G
ene E
xpre
ssio
n
IRF3
-5
-4.5
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
Mock WT 18K
Rela
tive G
ene E
xpre
ssio
n
HSPB8
![Page 15: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/15.jpg)
Q. Do qualitative/quantitative differences in gene expression
explain why 18K KO mutants were attenuated in vivo?
![Page 16: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/16.jpg)
Pathway – Apoptosis – WT vs. Mock
Red = UPGreen = Down
![Page 17: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/17.jpg)
Pathway – Apoptosis – 18K vs. Mock
Red = UPGreen = Down
![Page 18: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/18.jpg)
Conclusions
• Observation: Numerous immune-related genes, affecting both innate and acquired immunity, were upregulated in FV3-infected cells.
• Hypothesis: Immune-related molecules impair virus replication and lead to long term survival.
![Page 19: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/19.jpg)
Conclusions
• Transcripts induced following infection with wt FV3 and the 18K KO are qualitatively similar.
• However, quantitative differences may reflect the role that the 18K protein plays in viral replication and the modulation of cellular gene expression.
![Page 20: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/20.jpg)
Future Plans
• Use FHM and Xenopus microarrays to analyze global host expression following infection with wt and KO mutants in vivo and in vitro.
• Determine whether specific cellular gene products are critical for a protective immune response.
![Page 21: Immune response in FHM cells following infection with frog virus 3](https://reader036.vdocument.in/reader036/viewer/2022081515/55a0b8fc1a28abf0608b4719/html5/thumbnails/21.jpg)
Acknowledgements
• Kwang Cheng, UMMC (Jackson, MS)
• Lynn Escalon, ERDC (Vicksburg, MS)
• Natalia Garcia-Reyero, Mississippi State University (Starkville, MS)
• Jacques Robert/Guangchun Chen (U. Rochester School of Medicine)
• US ARMY Corps of Engineers
• National Science Foundation