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How BVDV escapes and subverts immune defenses
Ernst PeterhansInstitute of Veterinary VirologyUniversity of Bern, Switzerland
1
Acknowlegments
All scientists and farmers who have contributed
to our knowledge
At home: Matthias Schweizer and crew
3
The virus and the host:
A matter of conflicting aims
The virus: to persist in the population
The host: to propagate (humans ??)
For the virus: two options:
„hit and run“ or:„infect and persist
The two options have limitations:
„hit and run“ works if: - probability of transmission to next host host is high- parameters: structure, density and dynamics of host population ?
extent and duration of viral shedding ? dose required for infection ?
„infect and persist“ works if- cost for infected individual is low to moderate- virus must be able to outsmart its host‘s immune system
- for life !
61
Most of these infections can be controlled by vaccination
Vaccines against most of these infections are problematic
BVD: subversion starts with strategy
„hit and run“ : transient infection and
„infect and persist“ : persistent infection
Vaccines ??
immunotolerance
• defined by adaptive immunity: T and B cell• highly specific for infecting viral strain consequence for viral evolution ? • immunotolerance seems to be complete –
but some PI animals show antibodies to BVDV
clinically, PI animals may be „normal“, or show reduced growth and frequent infections
persistent infection may be „perfect“ – or not
persistent infection may be „perfect“ – or less than perfect
By necessity(*), most of us are more interested in „less-than –perfect“ aspects of viral infections than in viral perfection
(*)
„less-than-perfection“
The type of host cell is important: Monocytes and DCs
14
Chris Howard/Bryan Charleston and colleagues:
Antigen-presenting cells from PI calves are „normal“
Monocytes (MO) and DCs susceptible to infection, but onlyMOs killed by cp BVDV. Resistance of DCs not due to IFN
MOs, but not DCs infected with ncp BVDV, show decreased memory or allogeneicT cell stimulation
Detected a „PDC-like –PDC unlike“ cell type that produces IFN-Iin response to ncp BVDV (a myeloid cell type)
The type of host cell is important: Monocytes and DCs
14
Lesya Pinchuk and colleagues: monocytes
cp and ncp BVDV disrupt early antigen uptake in monocytes, this might be related to specific immunotolerance in PI
Early upregulation of TLR3 in ncp BVDV infected monocytes
Upregulation (24hpi) of TLR7 in cp and ncp infected monocytes
Downregulation of TNF-α, IL-1β, IL-6
Downregulation of the receptor of activated C kinase, pyridoxal kinase, Brutons tyrosine kinase in cp BVDV vs ncp infected monocytes
Effect Ncp BVDV Cp BVDV
CPE = + IFN type-I synthesis = + NO synthesis after LPS or S. dublin treatment +
TNF- synthesis after LPS treatment Superoxide production induced by PMA Procoagulant activity induced by S. dublin IL-1 inhibitor activity induced by LPS + + Cytokine-induced chemotaxis
The type of host cell is important: Mφ show functional changes in response to
infection with BVDV: in vitro
(=: no effect compared to mock, +: enhanced, : reduced).
plus factor „X“
14
Roth, Bolin and colleagues: lymphocytes and PMNs
reported defective functions of PMNs and lymphocytes isolated from PI animals
General conclusion from these studies:
BVD viruses cause a wide array of functionalchanges that may explain aspects of disease
Neill, Ridpath and colleagues:
Infection has profound impact on transcription of multiple genes
effects may become apparent „when the cell is asked
to do something“: priming
…back to this at the end !
Thou shalt not kill is smart: ncp BVDV
Smart viruses make use of Jiv ! - cp BVDV may be viewed as loss-of-function mutants
ncp biotype cp biotypeoverexpression of bcl-2 triggers apoptosis
no apoptosis, no IFN induces interferon-I …
in bovine turbinate cells:
Interferons: the basics:Type I interferons: α, β, δ, έ, τ, ώ: shared receptor
antiviral, immunomodulatory , cytostaticType II interferon: γ: immunomodulatoryType III interferons: λ1, λ2, λ2 (IL29, IL28A, IL28B)
Induction ActionRandall and Goodbourn, J. Gen. Virol. 89, 1, 2008
Effect Ncp BVDV Cp BVDV
CPE = + IFN type-I synthesis = + NO synthesis after LPS or S. dublin treatment +
TNF- synthesis after LPS treatment Superoxide production induced by PMA Procoagulant activity induced by S. dublin IL-1 inhibitor activity induced by LPS + + Cytokine-induced chemotaxis
The type of host cell is important: Mφ show a functional changes in response to
infection with BVDV: in vitro
(=: no effect compared to mock, +: enhanced, : reduced).
plus factor „X“
14
factor „X“: primes cultured cells for apoptosis in response to LPS
Mucosal disease: why are the lesions where they are ?
A contribution of factor „X“?
Cp BVDV
ncp BVDV
but, occasionally, also between the claws and on (thin) skin 15
Activation of innate immunity is essential for triggering adaptive immunity
Pathogen structure (PAMP)
recognized
Innate antiviral immune reactions
Adaptive immunity
Interferon, IL-1, etc.
AntibodiescTx
Pathogen structure not recognized
No immune reaction
Viral infection
Innate Immunity
IFN continued: BVDV is sensitive to IFN-I: but is not eliminated once infection is established
IFN treatment before ncp strain SD-1 (moi = 0.01)
0 0.1 1 10 100[ng/ml]
0 0.1 1 10 100[ng/ml]
IFN treatment after ncp strain SD-1 (moi = 0.01)
0.2 mm
25
Cells remain infected after 10 passages in
presence of interferon
890
Suwa
0.2 mm
0 1 10 100
Does virus persisting in its host cells simply inhibit the action of interferon - like many other viruses do ?
26
The simple approach: test if a virus „newcomer“ is inhibited in BVDV-infected cells after IFN-treatment
rbo IFNα
Mock
ncp BVDV
Degree of apoptosisVSV replication (titer)VSV
ncp BVDV
Mock
27
Bovine turbinate cells infected with ncp BVDV still establish antiviral state: the „newcomer“ VSV is inhibited
01234567
501031
0,5
0,1
0,05
0,01
0,00
5
0,00
1
w/o
rboIFNαI.1 [ng/ml]
VS
V t
ite
r[l
og
(TC
ID5
0/m
l)]
Mock + VSV 0.01 890 + VSV 0.01
28
Cell II: IFN-a/b activityCell I: IFN-a/b induction
PIRF-3
P
IRF-3PP NF-kB
p50
p65 ATF-2c-JUN
P
P
ds RNA /ssRNA
IFN-b gene
IFN-b
IFN-b IFN-b
IFN-b
IFN-b
ISGs
Jak-Statsignal
transduction
Mx PKR
OAS,RNAse L
BVDV
BVDV and IFN-a/β: discrimination between „self“ and „non-self“
BVDV
Npro
1
2self
VSV
EMCV
3non-self
Of the Flaviviridae only the pestiviruses encodethe N-terminal protease Npro and the RNAse Erns.
5´-UTR 3´-UTR
Npro Erns E2E1 NS2 NS3p7C NS
4ANS4B
NS5ANS5B
structural proteinsnon-structural proteins
cellular proteases (signal peptidase; signal peptide peptidase)
viral proteases (NS2, NS3)
Npro Erns
5´-UTR 3´-UTR
Npro Erns E2E1 NS2 NS3p7C NS
4ANS4B
NS5ANS5B
structural proteinsnon-structural proteins
cellular proteases (signal peptidase; signal peptide peptidase)
viral proteases (NS2, NS3)
Npro Erns
Activates proteasomal degradation of interferon regulatory factor 3 (IRF-3)
targets intracellular dsRNA
Of the Flaviviridae only the pestiviruses encodethe N-terminal protease Npro and the RNAse Erns.
5´-UTR 3´-UTR
Npro Erns E2E1 NS2 NS3p7C NS
4ANS4B
NS5ANS5BNpro Erns
Present on viral particle, lacks typical transmem-brane anchor secreted from infected cells, has RNAse activity with preference for ssRNA
Of the Flaviviridae only the pestiviruses encodethe N-terminal protease Npro and the RNAse Erns.
Erns
Iqbal et al., 2004Baculovirus-produced Erns targets extracellular dsRNA (poly-IC)
Magkouras et al., 2008Free Erns is present in blood of PI animals in concentrationssimilar to those effective in preventing IFN induction in vitro
Authentic Erns prevents IFN induction by poly-IC
Mätzener et al., 2008Authentic Erns prevents IFN induction by BVDV ds and ssRNA
RNAse activity is crucial for this effect
BVDV-infected cell
PIRF-3
P
IRF-3PP NF-kB
p50p65 ATF-2
c-JUN
P
P
IFN-b gene
IFN-b
Npro
dsRNA IFN-b
IFN-b
IFN-b IFN-b
IFN-b
IFN-bX
X
BVDV-infected cell
PIRF-3
P
IRF-3PP NF-kB
p50p65 ATF-2
c-JUN
P
P
IFN-b gene
IFN-b
Npro
dsRNA IFN-b
IFN-b
IFN-b IFN-b
IFN-b
IFN-bX
X
BVDVErns
Erns
Erns
Erns
Erns
Erns
Non-infected cell
PIRF-3
P
IRF-3PP NF-kB
p50p65 ATF-2
c-JUN
P
P
IFN-b gene
IFN-b
IFN-bIFN-b
TL
R
Erns
Erns is a novel type of virus-encoded decoy receptor:
• minimizes danger (and benefit !) of activating innate immune response
• Viral PAMP becomes part of „self“
• efficient: enzymatically inactivates viral PAMP that is constantly produced in PI animals
• possible prime target cell:
natural IFN-producing cell, pDC ?
plasmacytoid dendritic cells other cells recognition of NA in cytoplasm
ssRNA cpG DNA dsRNA 5‘PPP RNA dsRNA
plasmacytoid dendritic cells other cells recognition of NA in cytoplasm
ssRNA cpG DNA dsRNA 5‘PPP RNA dsRNA
General conclusion from these studies:
BVD viruses cause a wide array of functionalchanges that may explain aspects of disease
…and now: back to the „imperfection“ of BVDV
This virus causes disease, in transiently as wellas well as in some PI animals:
And also cytokines, especially IFN-1 can cause disease:
IFN during fetal development and post partum:Cp and ncp BVDV, transient and persistent
infections
14
Bryan Charleston and colleagues: fetus: ncp early: -
cp early: + postnatal tr.I ncp +
Thomas Hansen and colleagues:fetus: ncp early: -/+
ncp late: +postnatal PI ncp +
Yamane and colleagues: postnatal tr. I ncp +postnatal PI +
Metzler and colleaguespostnatal tr.I +
IFN during fetal development and post partum:Cp and ncp BVDV, transient and persistent
infections
14
Conclusion: a general agreement on the activation of the innate immune response to cp and ncpBVDV late in intrauterine development and post partum (transient infection)
more divergent interpretation in persistent infection in (early) fetus and PI animal
WHY ?
(1) PI animals are chimeric with respect to BVDV: how does the innate immune system of the
non-BVDV part of PI animals handle BVDV ?
No final explanations – but here are some moderately educated
guesses:
No final explanations – but here are some moderately educated
guesses:
32
(2) Ontogeny of „natural IFN producing cells“ ?
(3) different virulence of BVDV strains ?
viral load ds and ssRNA (PAMP) levels
different efficiency of viral „attenuation mechanisms“