STOPBVD

How BVDV escapes and subverts immune defenses

Ernst PeterhansInstitute of Veterinary VirologyUniversity of Bern, Switzerland

1

AcknowlegmentsAll 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

Examples: Hit and run: rabies, mumpsInfect and persist: herpes, lenti

Rabies: the fate of the host

Rabies: the fate of the virus in a closed host population

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 ??

Interaction of BVDV with its natural host: basics

Interaction of BVDV with its natural host: basics

immunotolerance

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: monocytescp and ncp BVDV disrupt early antigen uptake in monocytes, this might be related to specific immunotolerance in PIEarly upregulation of TLR3 in ncp BVDV infected monocytesUpregulation (24hpi) of TLR7 in cp and ncp infected monocytesDownregulation of TNF-α, IL-1β, IL-6Downregulation 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 PMNsreported 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 !

Interaction of BVDV with its natural host: basics

only ncp can do !

Thou shalt not kill is smart: ncp BVDVSmart 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 interferon890

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]

VSV

titer

[log(

TCID

50/m

l)]

Mock + VSV 0.01 890 + VSV 0.01

28

Cell II: IFN-/b activityCell I: IFN-/b induction

PIRF-3

P

IRF-3PP NF-kB

p50p65 ATF-2

c-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“

BVDVNpro

1

2self

VSV

EMCV

3 non-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

PP

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

PP

IFN-b gene

IFN-b

Npro

dsRNA IFN-b

IFN-b

IFN-b IFN-b

IFN-b

IFN-bX

X

BVDV Erns

Erns

Erns

Erns

Erns

Erns

Non-infected cell

PIRF-3

P

IRF-3PP NF-kB

p50p65 ATF-2

c-JUN

PP

IFN-b gene

IFN-b

IFN-bIFN-b

TLR

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“