Iden%ﬁcaon and expression of type I interferons in Meagre ... · Iden%ﬁcaon and expression of...

Iden%fica%onandexpressionoftypeIinterferonsinMeagre(Argyrosomusregius)

Chris Secombes

Acknowledgements

University of Aberdeen, UK Douglas J. Milne Jun Zou

Xinhua Chen, Inst Oceanography, Xiamen, China

Karl Andree Cindy Campoverde IRTA, Spain

Infection can lead to poor growth

As seen in infectious salmon anaemia (ISA) outbreaks in Scotland in 1998-9

and in Chile in 2007-8 which led to large losses of farmed salmon.

Infection can lead to high mortality

Salmonid alpha virus (SAV) causes chronic infection.

Viral diseases: a threat to aquaculture

Virus Abbreviation Genome type

Taxonomic classification

Aquaculture host

Geographic distribution

Infectious salmon anaemia virus

ISAV ssRNA (-)

Orthomyxoviridae Salmonids Europe, North and South America

Viral haemorrhagic septicemia virus

VHSV

ssRNA (-)

Rhabdoviridae

Samonids, Flatfish

Europe, North America, Asia

Viral nervous necrosis virus

VNNV

ssRNA (+)

Nodaviridae

Grouper, Turbot


Salmon alpha virus

SAV

ssRNA (+)

Togaviridae

Salmonids

Europe

Infectious pancreatic necrosis virus

IPNV

dsRNA (-)

Birnaviridae

Salmonids


Singapore grouper iridovirus

SGIV dsDNA Iridoviridae Grouper Europe, Asia, North America, Africa

Koi herpesvirus

KHV dsDNA Alloherpesviridae Cyprinids Europe, Asia, North America, Africa

RIG-I: retinoic acid inducible gene I MDA5: melanoma differentiation-associated gene 5 LGP2: laboratory of genetics and physiology 2 MAVS: Mitochondria antiviral signalling protein IFI16: interferon inducible protein 16 DAI: DNA-dependent activator of IFN STING: stimulator of interferon genes TRIF: Toll/IL-1 receptor domain-containing adaptor inducing IFN-β IRF: interferon regulatory factor

Pattern Recognition Receptors (PRRs)

Virus

IFN production

TLR22

dsRNAssRNACpGDNA

TLR3TLR7/8TLR9

Endosome

TRIF Endoplasmic reticulum STINGMAVS Mitochondria

ViralRNA

RIG-I,MDA5 DAI,IFI16,P204

Bacterial+viralDNA

ISRE GAS

dsRNA

Nucleus

LGP2

CleavagebyViralproteases

TRAF 3 recruits and activates IKK related kinases

TRIM25

IRF3

IRF3

IRF3

IRF7

IRF7

IRF7

P

P P

P

Virus detection and activation of antiviral pathways

OAS = 2’, 5’ oligoadenylate synthetase PKR = protein kinase R – phosphorylates eIF, which becomes inactive eIF = eucaryotic translation initiation factor Mx = GTPase, able to block the traffic of viral proteins Viperin = virus inhibitory protein, down-regulates viral structural proteins needed for virus assembly

PKR Viral protein synthesis eIF

Viral RNA degradation

OAS ribonuclease

Mx, Viperin, etc

IFN

The “antiviral state”

Anti-viral defences in vertebrates

Sadler & Williams, Nature Reviews Immunol 8, 2008, 559-68.

X

In mammals type I IFNs occur as multiple genes and multiple subgroups - α, β, κ, ε, ω, τ, δ, ζ

Chr 9

IFN

K

21067868 21471058 27516496 bp

6.04 mbp 0.40 mbp

IFN

T1

IFN

A2

IFN

A8

IFN

A1

IFN

A4

IFN

A7

IFN

A13

IFN

B1

IFN

Aw

1

IFN

A5

IFN

A6

IFN

A10

IF

NA

16

IFN

A17

IF

NA

14

IFN

A21

13 alpha interferons in humans

Eg Human type I IFNα genes are clustered, on chr 9

Major events in fish interferon research

19752003

20072009

20112009

Immunogenetics (2009) 61:315–325

Original Paper Identification of an additional two-cysteine containing type I interferon in rainbow trout Oncorhynchus mykiss provides evidence of a major gene duplication event within this gene family in teleosts Mingxian Chang1, 2, Pin Nie2, Bertrand Collet3, Christopher J. Secombes1 and Jun Zou1

Trout RTG-2 cells were incubated with rIFN for 4 h prior to virus challenge.

VHSV +

IFN VHSV Trout type I IFNs enhance host resistance against viral infection

Zou et al, J Immunol, 179, 2007, 3859-71.

Antiviral functions of fish IFNs are conserved

IFN

5 Xe

nopu

s IF

N4

Xeno

pus

IFN

3 Xe

nopu

s IF

N1

trou

t IF

Na2

sal

mon

IF

Na1

sal

mon

IF

N2

trou

t IF

Na3

sal

mon

IF

N1

zebr

afis

h IF

N4

catfi

sh

IFN

1 ca

tfish

IF

N5

trou

t IF

N s

tickl

ebac

k IF

N m

edak

a IF

N te

trao

don

IFN

fugu

IF

Nb2

sal

mon

IF

N3

trou

t IF

Nb3

sal

mon

IF

N4

trou

t IF

Nb1

sal

mon

IF

N3

zebr

afis

h IF

N2

zebr

afis

h IF

Nc1

sal

mon

IF

Nc2

sal

mon

IF

Nc3

sal

mon

IF

N2

Xeno

pus

IFN

1 Xe

nopu

s IF

Nα

duck

IF

Nα

goos

e IF

Nα

turk

ey

IFNα

chic

ken

IFN

3 an

olis

IF

N1

anol

is

IFN

8 an

olis

IF

N2

anol

is

IFN

4 an

olis

IF

N6

anol

is

IFN

7 an

olis

IF

N5

anol

is

IFNκ

mou

se

IFNκ

dog

IFNκ

hum

an

IFNβ

cow

IF

Nβ

pig

IFNβ

hors

e IF

Nβ

hum

an

IFNβ

mou

se

IFNε

mou

se

IFNε

cow

IF

Nε

hum

an

IFNα

mou

se

IFNα

cow

IF

Nα

pig

IFNα

hors

e IF

Nα

hum

an

IFNω

hor

se

IFNω

hum

an

IFNτ

goat

IF

Nτ

shee

p IF

Nτ

cow

IF

Nσ

hum

an

IFNω

cow

IF

Nα

shee

p

100

100

100

90

100

100

100

100

100 85

10

0 95

90

100

100

100

95

85

85

75

85

100

80

100

95

100

90

75

90

100

95

80

85

95

85

95

90

90

90

85

75

75

85

2 Cys 4 Cys MammalsRep%les

Birds

TeleostsPhylogenetic tree

of type I IFNs

Tetrapod4CysgroupMammalian2Cysgroup(IFN-βandε)

C2 C4

C1 C2 C3 C4

Vertebrates have three groups of type I IFNs

C1 C3

C1 C2 C3 C4 Fish4CysgroupFish2Cysgroup

At least 44 copies of IFN genes and pseudo genes are

found in trout

IFN-e5 IFN-e2 IFN IFN IFN-e3 IFN-e4 IFN-a3 IFN IFN IFN

732E08 116360 bp

770C03 149654 bp

IFN

IFN

IFN-c1

IFN

IFN-b3

IFN

IFN

IFN-b5

IFN

IFN

IFN-f2

IFN-a4 IFN-c2

IFN-b4 IFN

IFN-c3

IFN-c4

IFN IFN-e6 IFN

IFN IFN IFN

IFN-e1

IFN IFN-e7 IFN

IFN-f1 IFN

816C01 142050 bp

Analysis of 3 trout BAC clones

Full length IFN ORF

Partial IFN ORF Transposon

Zou et al, J Immunol 193, 2014, 2273-86.

Group I (2 Cys) IFNs

Group II (4 Cys) IFNs

IFN-a

IFN-e

IFN-d

IFN-b

IFN-f

IFN-c

IFNa1 onmy IFN1 onmy

IFN2 onmy IFNa1 sasa

IFNa2 sasa IFNa3 sasa IFNa3 onmy

IFN icpu IFN cyca

IFN1 dare IFN caau

IFNe6 onmy IFNe7 onmy

IFNe5 onmy IFNe5 sasa

IFNe1 sasa FNe4 onmy


IFNe2 onmy IFN4 dare

IFNd1 onmy IFNd1 sasa

IFN Taru IFN teni

IFN orla IFN gaac

IFN dila IFN4 onmy

IFNb2 onmy IFN3 onmy

IFNb3 onmy IFNb1 sasa IFNb3 sasa

IFNb2 sasa IFNf1 onmy IFNf1 sasa

IFNf2 onmy IFNf2 sasa

IFN2 dare IFN3 dare

IFNc4 onmy IFNc2 onmy IFNc3 onmy IFNc1 onmy

IFNc1 sasa IFNc3 sasa IFNc2 sasa 98 83

59

48 68

100

100

68 99

98

92 79 68 97 100

100

96 51 31 21

30 100

99

71 66

48 48

100

55 99

99

82 56 95

50 92

63

34 58

45

35

80

64 87 52

100

18-33%sequenceiden%tybetweensubgroups>81%sequenceiden%tywithinasubgroup

IFN-a

IFN-e

IFN-d

IFN-b

IFN-f

IFN-c




IFN icpu IFN cyca

IFN1 dare IFN caau







IFN Taru IFN teni

IFN orla IFN gaac

IFN dila IFN4 onmy





IFN2 dare IFN3 dare



59

48 68

100

100

68 99

98

92 79 68 97 100

100

96 51 31 21

30 100

99

71 66

48 48

100

55 99

99

82 56 95

50 92

63

34 58

45

35

80

64 87 52

100




IFN-a

IFN-e

IFN-d

IFN-b

IFN-f

IFN-c




IFN icpu IFN cyca

IFN1 dare IFN caau







IFN taru IFN teni

IFN orla IFN gaac

IFN dila IFN4 onmy





IFN2 dare IFN3 dare



59

48 68

100

100

68 99

98

92 79 68 97 100

100

96 51 31 21

30 100

99

71 66

48 48

100

55 99

99

82 56 95

50 92

63

34 58

45

35

80

64 87 52

100




Interferonexpressioninkidneyandspleenoftroutfollowingviralchallenge(withVHSV).

Diversificationsof type I interferon subfamilies in teleosts

d

100

99

100

72

99

72

96

68

58

59

96

e

a

h

f

b

c

C1 C3C3 C4

C1 C3

GroupItypeIIFN

GroupIItypeIIFN

Type I IFN repertoire of Acanthopterygian fish

d

100

99

100

72

99

72 96

68

58

59

h

c

Seabream Sea bass Barred knifejaw Large yellow croaker

Stickleback orange spotted grouper

convict grouper Flounder

Fugu Tetraodon

Tilapia Tilapia

Medaka

Turbot

Killifish Barramundi

Turbot Stickleback Large yellow croaker

Spiny chromis Tilapia

Tilapia burtoni Zebra mbuna

P. nyererei

IFN gene cloning strategy

IFNgenes

Forward

ReverseLarge yellow

croaker genome

Vol11(4),2015,e1005118et al.

3 IFN genes have been cloned in meagre

IFN1 IFN2 IFN3

4 Cysteines 2 Cysteines 2 Cysteines

Large yellow croaker Meagre

Large yellow croaker Meagre

Turbot Meagre

100

100 99

100

100

73

100

60

68

36

24 72

56

100

60 98 58

21 59

80 100

100

99

40

99 52 59

71 43

80 63

IFNdIFNhIFNc

GroupIIFN

GroupIIIFN

Meagre type I IFN genes

Days Post Hatch

815294043608596120

Larva Metamorphosis Juvenile Adult

WholeanimalTissuesSampling

Ro%fers Artemiatoday31Commercialfeedfromday21

Feedingregime

Interferon expression during development

81529404360


00,0010,0020,0030,0040,0050,0060,0070,008

Day8 Day15 Day29 Day40 Day47 Day60

IFNh

0

0,5

1

1,5

2

Day8 Day15 Day29 Day40 Day47 Day60

IFNc

00,00050,0010,00150,0020,00250,0030,00350,004

Day8 Day15Day29Day40Day47Day60

IFNd


8596120


00,0010,0020,0030,0040,0050,0060,007

Gill gut hk sp

IFNh Day85Day96Day120

00,20,40,60,81

1,21,4

Gill gut hk sp

IFNc Day85Day96Day120

00,0010,0020,0030,0040,0050,0060,007

Gill gut hk sp

IFNd

Day85Day96Day120

Highexpressionofall3inspleenIFNcexpressionisthehighest


Meagre tissue stimulation setup

In Vivo

In Vitro

24h

Ip Injection of fish •  PBS •  Poly I:C •  LPS •  β-glucan

Primary cell culture stimulated by: •  PBS •  Poly I:C •  LPS •  β-glucan Samples taken after 4, 12 and 24 h

4h

12h

24h

S%mula%on

Interferon expression in different tissues following PAMP stimulation in vivo

β-glucan: 50 µg/mL PBS Poly I:C: 100 µg/mL LPS: 50 µg/mL

HK = head kidney; SP = spleen

IFNc IFNd IFNh

PBS

Poly I:C: 100 µg/mL

LPS: 50 µg/mL

β-glucan: 50 µg/mL

N/B Similar results with HK cells

Interferon expression in cultured splenocytes

0

5

10

15

20

4 12 24

*

*

*

0 0,5

1 1,5

2 2,5

4 12 24

* *

0 1 2 3 4

4 12 24

* *

*

*

IFNhIFNd

IFNc

Time(h)

Time(h)

Time(h)

Conclusions Ø Meagre have a complex IFN system, with multiple IFN subgroups present

Ø Meagre IFNs are transiently up-regulated when fed a commercial diet

Ø Meagre IFNc is relatively highly expressed in embryos and spleen

Ø Meagre IFNs are induced by poly I:C in vivo and in vitro, indicating an

involvement in antiviral defence

Ø  In cultured meagre splenocyte IFNh is the most highly induced IFN

Ø Submitted to Dev. Comp. Immunol. – and the reviews are in!!

Thanks for your attention.

King’s College: founded in 1495.

Iden%ﬁcaon and expression of type I interferons in Meagre ... · Iden%ﬁcaon and expression of...

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