2013 International Symposium on Ranaviruses by Jacques Robert

1. THE HOST IMMUNE SYSTEM:
A DOUBLE-EDGED SWORD CONTROLLING
RANAVIRUS INFECTION BUT PROMOTING
VIRAL PERSISTENCE
Jacques Robert,
Francisco DeJesús Andino, Leon Grayfer
Microbiology and Immunology, U. Rochester Medical Center,
Rochester NY
FV3FV3

2. APCAPC
CD8CD8
BB
CD4CD4
Class I
Class II
Co-stim
(B7, CD40
CTLCTL
ThTh
Activation Expansion
Maturation
Effector
phase
Kill class I+
targets
MemoryMemory
CD8CD8
MemoryMemory
CD4CD4
Ags
+ “danger”
signals
Apoptosis
Apoptosis
PlasmaPlasma
cellscells
Memory
Cytokine
release
Cytokine
release
TLRs
Anti-viral immune responses
Innate immunity
Phagocytosis
Antiviral molecules (IFN, iNOS)
Mø
DC

3.  Ranavirus (FV3), large double stranded DNA viruses
infecting with increasing prevalence frogs, fishes and
reptiles
 Xenopus, instrumental laboratory model to study
immunity and pathogenesis of RVs such as FV3
 Adults: critical involvement of CD8 T cells and antibodies
established
 Immunological memory: faster recovery, viral
clearance upon 2nd
infection, protective anti-FV3 Ab,
faster CD8 T cell response
 Larvae: more immature immune system, more susceptible
to FV3 infection. Not not just immature B and T cell
responses, but delayed and poorer innate anti-FV3
response
Xenopus-FV3 model

4. Re-infection
FV3 load
1 3 6 9 1 3 6 9
Days post infection
- 10
- 2
5-
Numbercellsx102
Effective Ab
response
PFU
[x102
]
First infection
Innate
Immune cells1-
Infiltrating
CD8 T cells
Xenopus adult immune response
kinetics in infected kidneys
Chen & Robert, 2011, Viruses, 3:2065

5. 10 µm 22 µµmm
What are the specific roles played by
Xenopus tadpole and adult macrophages
in host immunity against FV3?

6. CD8
T cell
• Antigen processing
and presentation
• Co-stimulation
• Activation of CD8
& CD4 T cells
Mø
Adaptive Immune cell
effector
Innate Immune cell
effector
Permissive cell for viral
quiescence
•Virus transcriptionally inactive
MHC class I
MHC class II
• Phagocytosis
• Pinocytosis
Release of:
• Cytokines (TNFα, IL-1β)
• Chemokines (IL-8)
• Toxins (NO) Infection
Complex role of macrophages in Xenopus
host defenses against RV
Chen & Robert, 2011, Viruses, 3:2065

7. IFNγ
TNFα
Mx1
IL-
1β
Delayed and low innate immune response
in larval kidney during early FV3 infection
* *
*
* *
RQ
RQ
RQRQ
N=3
De Jesús Andino et al. (2012). Virology 432:435

8. Delayed and low inflammatory response by
larval PLs during early FV3 infection
Days post-infection
IL-1βTNFα IFNγ
1 3 6 1 3 6 1 3 6
Heat-killed
E. coli
Foldincrease
IL-1βTNFα IFN-γ
FV3
Foldincrease
Days post-infection
N=3 De Jesús Andino et al. (2012). Virology 432:435

9. Days post-infection
C 2 3C 2 3
*
CellNb/tadpolesx103
53R*
20 µm
%
Increase of infected and total numbers of larval
peritoneal phagocytes during FV3 infection
Days post-infection
15/group

10. The interferon response
IFNs are important antiviral agents produced and released by
all cell types
They allow for communication between cells to trigger the
protective defenses of the immune system
Reptiles, birds and mammals possess IFN, etc.
Fish, amphibians, monotremes and marsupials do not have
IFN,  …
Instead they possess unique type I IFNs
We have identified and cloned a X. laevis type I IFN,
Produced a recombinant X. laevis type I IFN (rXlIFN)

11. Type I IFN gene expression during FV3 infection
Kidney Macrophages
RQ(foldchangeinexpression
0 1 3 6 9
Days post-FV 3 infection
0 1 3 6 9

12. rXlIFN interferes with FV3 infection of A6 cells
(3 day post infection)
10µm
53R composite
10µm
vector
rXlIFN 53R composite
mockA6 cells pretreated with rXlIFN for 8h;
infected with 0.3 MOI FV3

13. FV3copynumber(per50ngDNA)
RQ(foldchangeinexpression)
- FV3 - FV3
+ Vector + rIFN
Kidneys
PLs
Virus load Viral transcription
- FV3 - FV3
+ Vector + rIFN
Pre-treatment of tadpoles with rXlIFN, 8 hrs prior FV3
infection, results in lower viral load and viral activity

14. rXlIFN pre-treatment results in slight delayed
tadpoles’ death following FV3 infection

15. zfMpeg1:GFP and Cherry
transgenic X. laevis larvae
zebrafish macrophage specific promoter mpeg
GFP
Cherry

16. B
Heterogeneity of X. laevis larval
myeloid cell populations
Double transgenic X. laevis
xLurp:GFP/xfMpeg1:Cherry
Green: Granulocytes (lurp+/Mpeg-)
Orange/yellow: macrophage (lurp+/Mpeg+)
Red: Macrophages? (lurp-/Mpeg+)

17. Increased numbers of
total and GFP+
PLs
6 days post-FV3
infection
Total PLs GFP+
PLs
C 6dpi C 6dpi
50 µm
FV3 53R Xlurp:GFP Overlay
xLurp:GFP transgenic Xenopus tadpoles

18. Increased susceptibility to FV3 infection
of F0 Tg tadpoles with b2m KD
%survival
Days post-infection
5 x 106
PFU FV3 in 5ml water for 1 hr
Median survival
time
C = 38.5 days
Tg = 19.5 days
p> 8x10-5
Median survival
time
Tg-Scr = 28.0 days
Tg-B2M = 13.5 days
p> 0.002
Tg-Sc
Tg-B2M
N=20/group
Nedelkovska et al., Biol. Open, 2013

19. Pre-metamorphic (st 57) OB tadpoles
Effective and specific KD of the
class Ib gene XNC10 by transgenesis
Vα6 Jα39 C domain
Edholm et al., 2013, Proc Natl Acad Sci USA (In press)

20. %survival
Days post-FV3 infection
FV3 i.p 10,000 pfu (Trial 1)
Increased susceptibility to FV3 infection
of LG-15 F0 Tg tadpoles with XNC10 KD
Edholm et al., 2013, Proc Natl Acad Sci USA (In press)

21. 6 dpi6 dpiCC
15 dpi15 dpi 21 dpi21 dpi
MCPMCP
ββ2-M2-M
LMPXLMPX
MCPMCP
-RT-RT
ββ-actin-actin
IEIE
ββ2-M2-M
MCPMCP
MCPMCP
PCRPCR
RT-PCRRT-PCR
RT-PCRRT-PCR
PCRPCR
6 dpi6 dpiCC
15 dpi15 dpi 21 dpi21 dpi
In vivo FV3 infection and transcription in adult PLs
Viral DNA
Viral
transcription
6 dpi 15-21 dpi
Morales et al., 2010, J. Virol. 84:4912

22. Mø infected in vitro for 2 days with FV3
1 µm 1 µm
100 µm100 µm
Morales et al.,
2010, J. Virol.
84:4912

23. Experimental Method
FV3 infection
(5 x106
PFU)
0 30
PL cells collection
RNA and DNA isolation
Virus load, viral transcription
Bacterial
stimulation
32 35 days

24. vDNA-Pol
FV3 reactivation
+ C (3dpi)
- C (uninfected)
DW
30 dpi
35 dpi FV3
+
HK E. coli
DNA
cDNA
EF-1α
1 2 3 4 5 6 7 8 9 10
35 cycles
1 2 3 4 5 6 7 8 9 10
35 dpi FV3
+
HK E.coli
vDNA-Pol
vDNA-Pol
EF-1α

25. Survival curve in Adult frogs after FV3
infection, followed by Bacterial Stimulation
C n=22
FV3 n=23
C Exp
DNA 0/23 5/23
cDNA 0/23 8/23At 35 dpi

26. SummarySummary
 Although FV3 triggers poor type I IFN response in tadpoles, rXlIFN
pre-treatment is protective, suggesting that larval antiviral effector
system is functional and capable of controlling FV3 infection.
 Thus tadpole susceptibility likely stems from poor recognition
system and/or highly efficient FV3 immune evasion
 Although the adult frog immune system is efficient in controlling
FV3 infection, FV3 persists in a fraction of otherwise healthy
asymptomatic frogs
 This persistence involves macrophages harboring quiescent,
transcriptionally inactive virus
 Preliminary evidence suggests that bacterial stimulation-mediated
inflammatory signals can reactivate quiescent FV3 infections
 Loss-of-function approach by Tg suggests that besides class Ia-
restricted conventional CD8 T cells, class Ib-restricted invariant or
innate T cells are critical for Xenopus anti-FV3 immunity, especially
at larval stages

27. Model
FV3 targets adult macrophages to escape host
immunity and persist in asymptomatic host, but
targets larval macrophages to overcome immune
defenses and rapidly disseminate

28. Jumping Frog Lab
Leon Grayfer, PhD
Eva-Stina Edholm, PhD
Nikesha Haynes
Maureen Banach
Fransisco De Jesús Andino
Jason Sifkarovski
Tina Martin
Collaborators
Brian Ward (U. Rochester)
Paige Lawrence
V.G. Chinchar (U. Mississippi)
NIH R24-AI-0598R24-AI-059830

30. MetamorphosisMetamorphosis
Accessible to experimental manipulation (e.g., thymus)
Absence of maternal influences on the embryo
Immune system develop early (10 days post-fertilization)
Larvae are immunocompetent but immature
B and T cell receptor repertoires distinct from adult
Presence of CD8 T cells but not NK cells, weak T cell