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Dt i t f Determinantsof Pathogenicity for Gumboro Pathogenicity for Gumboro Virus and Epidemiology in Europe Prof. Dr. Vilmos Palya Scientific Support and Investigation Unit Ceva-Phylaxia Veterinary Biologicals Ltd, Budapest, Hungary Ceva-Phylaxia Budapest, 19/01/2012

Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

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Page 1: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

D t i t fDeterminants of Pathogenicity for GumboroPathogenicity for GumboroVirus and Epidemiology in

Europe

Prof. Dr. Vilmos PalyaScientific Support and Investigation Unit

Ceva-Phylaxia Veterinary Biologicals Ltd, Budapest, Hungary

Ceva-PhylaxiaBudapest, 19/01/2012

Page 2: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

IBD virusIBD virusTaxonomy:• Family: Birnaviridae

Genus: Avibirnavirus

Virus properties:Genus: Avibirnavirus

non-enveloped virion, 55-60nm diameter,

Prof. Steward McNulty, Queen’s University of Belfast

single-shell,icosahedral capsidp

2

Page 3: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

IBD virusGenomic organisation

Double stranded RNA genomeTwo segmentsSegment A

3261 nucleotides in length two partially overlapping open reading frames (ORF)two partially overlapping open reading frames (ORF)

Segment B 2827 nucleotides in length2827 nucleotides in lengthone ORF

Segment BSegment A Segment B

VP 1VP2 VP4 VP3

VP5

Segment AORF 1

3

V 5

ORF 2

Page 4: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

IBD virusSegment A

Large ORF encodes a polyprotein (VP2 VP4 VP3)Large ORF encodes a polyprotein (VP2-VP4-VP3)Small ORF encodes VP5

S t BSegment BORF encodes VP1

VP2 VP4 VP3VP5

Segment A Segment B

VP 1NCR NCR NCR NCR5’ 5’ 3’3

Large ORF

VP5

polyprotein VP 1

5 5 33’

transcription & translationSmall ORF

p yp

pVP2 VP4 VP3

VP 1

auto-proteolysis

4VP2

proteolysis

Mature VP2

Page 5: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

IBD virus

Viral proteinsViral proteinsSegment A

VP2 main capsid protein, major host-protective antigen with antigenic regions responsible for induction of g pneutralizing antibodiesVP4 serine protease, self-processing of viral polyproteinVP3 inner capsid protein control the incorporation of polymeraseVP3 inner capsid protein, control the incorporation of polymerase and genome into the nascent particle VP5 nonstructural protein, regulate the release of intracellular virions

Segment BVP1 RNA-dependent RNA polymerase, covalently bound to the

5

genominc dsRNA segments

Page 6: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

IBD virusIBD virusVP2 is folded in three distinct domains:

base (B) inner surface of capsid connection with VP3base (B), inner surface of capsid connection with VP3shell (S) forming the outer surface of the capsidprojection (P), four main loops within the hypervariable region

A B

p j ( ), p yp g

6Reference: Coulibaly et. al. 2005. Cell. 120:761-772.

Page 7: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

IBD virusHypervariable region of VP2 contains two major and two minor hydrophilic regions maching to the four loopsminor hydrophilic regions maching to the four loopsAmino acid changes in these regions can influence:

the virulence of virus (by change in the efficiency of attachment tothe virulence of virus (by change in the efficiency of attachment to the target cell surface)antigenictity (by change in the main neutralizing epitopes)

Hydrophylic regions mach to the four loops

Major hydrophilicpeak A

Major hydrophilicpeak B

Minor hydrophilicpeak A

Minor hydrophilicpeak B

PB PC PD PE PF PG PH PI

PBC loop PDE loop PFG loop PHI loop

210 220 230 240 250 260 270 280 290 300 310 320 330 340. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGUK661/86GB .............A.................................I.....................................I....S.............................................VarE/85US ....N........T...................V......K....S........................N......I...............................D....E.....................2512/Tw ........L........................V...........................T.......................I..................................................

peak A peak Bpeak A peak B

7

D78 .................................V..........H................T........N....T.............................................R..............P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F................................................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................L...N............

Page 8: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Criteria for the characterization of IBDV strainsof IBDV strains

PathogenicityAntigenicityAntigenicityGenetic relatedness

88

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Pathotypesyp

Very virulent strainsVery virulent strainsHigh mortality in susceptiblechickens (broilers as well!)

strain classification Mortality rate

F52/70 Cv 36%

Cu‐1wt Cv 54%( )(big differences among strains evenin standardized challenge model)

Cu‐1wt Cv 54%

849VB vv 91%

96108 vv 91%

Harbin vv 58%

HK46 vv 63%

GX vv 57%

Henan1 vv 42%

99

Page 10: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Pathotypesyp

Very virulent strainsSevere bursal lesions, limited regeneration of bursa

3 dpi:Control, haemorrhagic bursa, edematous bursa with gelatinous exudate

Strongest systemic effect (see haemorrhages in muscles)

10

Higher level of MDA and fully developed active immunity is required to protect chickens against clinical signs and bursal lesions

10

Page 11: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Pathotypesyp

Classical virulent strainsT-cell immunhistochemistry (CD3+ cells are stained) in the bursa of SPF chickens; more in vvIBDV bursa

Classical virulent strainsLow-moderate mortality in susceptible chickensSevere bursal lesions, limited bursa regenerationLess severe inflammatory yresponse in the bursa than incase of vvIBDVThymic lymphocyte necrosis Thymic lymphocyte necrosis (during the 1st week post-infection)Transient but significant

Macrophage immunhistochemistry (no difference)

Transient but significant depression of T-cell response tomitogens (spleen).

1111Source: Rauf et al. Veterinary Research 2011, 42:85

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Pathotypesyp

Subclinical classical strainsNo clinical signs or mortalityMild bursal lesions (intensive lymphocyte depletion without inflammation), Good regeneration ability:Good regeneration ability:

„Subclin”28 dpi. 28 dpi

control 

DelE 28 dpi

1212H-E stained and B-lymphocty spec. Immune-stained bursa sections after exp infection of 2 wks old SPF layers

Page 13: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Pathotypesyp

Subclinical antigenic variantSubclinical antigenic variantstrains

Subclinical infectionRapid and severe lymphocyte depletionin the bursa (atrophy)Long regeneration processLong regeneration processWeaker inflammatory response in the bursa compared to cv or vv IBDV strains

Macroscopicall no marked edema or

FR isolate

DelE strain 8 dpi• Macroscopically: no marked edema or peribursal exudate• Microscopically: no plica edema, but thi k d i t f lli l ti

DelE strain

SPF control

8 dpi

thickened intrafollicular septiNo thymic lymphocyte necrosis Transient but significant depression of T- Source: Comparative pathogenicity test of

European variant strains and DelE in 5 wks old

SPF control

13

cells response to mitogens (spleen).13

pSPF layers (Ceva-Phylaxia Scientific Support and Investigation Unit)

Page 14: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Pathotypesyp

Subclinical strains

B:B index IBDV detection by PCRcomparison of „subclinical” variant and classical strains

shows the regeneration process in the „subclin.” groups, but not in the variantDelE group

„Subclinical” IBDV strains are more efficiently eliminated from the bursa than the variant DelE strain

B:B index

than the variant DelE strainNote: this relative rapid clearance in case of subclinical strains has an influence on the results of surveys at slaughter age

1414

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PathotypesypVaccine strainsIntermediate plus/hotIntermediate plus/hot

Highest residual virulenceClinical signs or even mortality can be induced in fully susceptible layer chickens (to be used in broilers and in the presence of MDA)Moderate-severe bursal lesions depending on breed and MDA level

IntermediateIntermediateModerate residual virulenceNo clinical signs or mortality even in fully susceptible chickensModerate bursal lesions, Slower colonization of bursa, than intermediate plus strainsMore efficiently neutralized by MDA than intermediate plusMore efficiently neutralized by MDA, than intermediate plus

MildMost attenuated strains

15

No clinical signs, mild bursal lesions, slow colonization of bursaMost sensitive to MDA 15

Page 16: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Differences in pathogenesisp g

Differences in the immunpathogenesis(acute phase):

More virulent strains are characterized by:• Higher IBDV load in the bursa• Stronger T cell influx into the bursa• Stronger T-cell influx into the bursa• Stronger induction of innate immune response• Similar level of macrophages, but higher level

Source: Sharma et al. 2000 Dev. Comp. Immunol. 24 223-

of activation• Expression of pro-inflammatory cytokines and chemokines is higherand chemokines is higher• Stronger inflammatory response in the bursa edema, haemorrhages, loss of follicle structure

1616

Page 17: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Differences in pathogenesisp g

Differences in the immunpathogenesisDifferences in the immunpathogenesisMore virulent strains are characterized by (cont.):• Stronger extrabursal response and replication : S g p p

– Thymic T-cell necrosis and atrophy, – Splenic mitogenic response depressed (T-cell blastogenic activity ), – Haemorrhages in the musclesHaemorrhages in the muscles.

Differences in the immunpathogenesis(chronic/recovery phase):( y p )More virulent strains are characterized by:• Delayed lymphocyte repopulation into bursa follicles

f f f• Loss of follicles or non-functional regenerationDifferences in the recovery are the consequences of more severe bursal damage in the acute phase.

17

bu sa da age e acu e p ase

17

Page 18: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Factors influencing the manifestation of strain pathogenicityof strain pathogenicity

Infectious doseInfectious dose

Age of chickens

Breed

Maternally derived immunity

Presence of other immunosuppressive agents

18

pp g

18

Page 19: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Factors influencing the manifestation of strain pathogenicityof strain pathogenicity

Age of chickensAge of chickensYoung birds (< 2wks):• mainly immunsuppressive effect (long-term),y ( g )• less severe inflammatory reaction in the bursa, but longer regeneration

phase (if any functional regeneration takes place)• usually no clinical manifestationusually no clinical manifestation

Older birds (3-6 weeks the most susceptible):• Clinical disease (depending on strain pathogenicity)• stronger inflammatory reaction (cv and vv strains), T-cell accumulation and

T-cell activation in the bursa and activation of macrophages are more pronounced; structure of follicles is destroyed due to the strong cellularimmune response, but usually faster functional regeneration

• Less severe (only transient) immunsuppressive effect due to the presence of extrabursal B-cells and faster regeneration

19

p g

19

Page 20: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Factors influencing the manifestation of strain pathogenicityof strain pathogenicity

BreedLayer-type chickens are more susceptible than broiler typeyp

(long-life broilers show intermediate susceptibility, closer to LT)• No, or less severe clinical signs, lower mortality, less severe bursal lesions

and lower IBDV antigen load in broilers than in layersand lower IBDV antigen load in broilers than in layersBackground:

Stronger T-cell response in the bursa of layersStronger stimulation of innate immune response in the early phase of infection in layers faster and more severe lesion development clinical signs, mortality

Different breeds and even different lines within the same breed can have different susceptibility

E B L h tibl th Whit L h diff

20

Eg.: Brown Leghorn more susceptible than White Leghorn; differences among White Leghorn inbred lines

20

Page 21: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Factors influencing the manifestation of strain pathogenicityof strain pathogenicity

Maternally derived immunityExtent of MDA-effect is different according to the pathogenicity andExtent of MDA effect is different according to the pathogenicity and antigenic characterisitic of the strainHigh level of antibodies can prevent takes of IBDVModerate level can prevent clinical signs but not the development of bursalModerate level can prevent clinical signs, but not the development of bursallesions (although lesions can be less severe, than in fully susceptible birds)

Antigenic differencesDifferences in pathogenicit

DA Classical virulent IBDV

Very virulent IBDV

lof M

DA Classical IBDV

Variant IBDVDifferences in pathogenicity

Leve

lof M

D

Mild vaccinesIntermedier vaccines

Intermedier + vaccinesClassical virulent IBDV

Leve

lAgeL

A

Age

End of rearinghatching

2121

Age

End of rearing

hatching

Page 22: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Factors influencing the manifestation of strain pathogenicityof strain pathogenicity

Presence of other immunosuppressive agentsMost important is ICAV

Pathogenicity of both IBDV and ICAV is increasedMore severe bursal lesions and immunsuppressionMore severe bursal lesions and immunsuppressionLonger susceptibility period to ICAV-induced clinical signs

2222

Page 23: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Genetic group based pathotypesg p p yp

Molecular analysis of VP 2 shows the presence of distinct genetic groups, which corresponds to

large extent with the different pathotypes

2323

Page 24: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Phylogenetic relationships of different IBDV groupsIBDV groups

Segment BSegment Ai very virulent IBDV Africai very virulent IBDV Africa

ii very virulent IBDV Europe and Asia

iii classical virulent IBDV Europe

iv classical virulent IBDV US group I

v cell culture adapted IBDVs

vi variant IBDV US

vii classical virulent IBDV US group IIUS group II

viii subclinical strains Europe

ix classical virulent IBDV Australia

Reference: Le Nouën et. al. 2006. J. Gen. Virol. 87:209-216.

Page 25: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Genetic markers of differentpathotype groupspathotype groups

Very virulent strainsvvIBDV signature aa residues: Alanine 222, Isoleucine 256, Isoleucine 294, Serine 299*N d IBDV i l t t thi lNowadays many vvIBDV isolates except this rule:

Alanine 222 Serine in YV Chinese vvIBDV ~90% mortality$ and G336/4 Bolivian, D1211/3 Indonesian vvIBDV isolateG336/ , /3Alanine 222 Threonine in G261/3 Turkish, D892/33 South-African or D1147/3 Malaysian vvIBDV isolate

Major hydrophilic Major hydrophilicMinor hydrophilic Minor hydrophilic

210 220 230 240 250 260 270 280 290 300 310 320. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWUK661/86GB .............A.................................I.....................................I....S............................

j y ppeak A

j y ppeak B

y ppeak A

y ppeak B

256 294 299222

U 66 /86G ............. ................................. ..................................... ....S............................VarE/85US ....N........T...................V......K....S........................N......I...............................D....E....2512/Tw ........L........................V...........................T.......................I.................................D78 .................................V..........H................T........N....T...........................................P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F...............................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................

*Brown & Skinner 1996 Virus Res 40:1-$Liu et al 2002 Virus Genes 24:135-

Page 26: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Genetic markers of differentpathotype groupspathotype groups

V i t t iVariant strainsThreonine or Glutamine 222, Lysine 249, Serine 254, But Threonine or Glutamine 222 Alanine in South-African variant isolates

Major hydrophilic Major hydrophilicMinor hydrophilic Minor hydrophilic

210 220 230 240 250 260 270 280 290 300 310 320. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWUK661/86GB .............A.................................I.....................................I....S............................

j y ppeak A

j y ppeak B

y ppeak A

y ppeak B

254249222

U 66 /86G ............. ................................. ..................................... ....S............................VarE/85US ....N........T...................V......K....S........................N......I...............................D....E....2512/Tw ........L........................V...........................T.......................I.................................D78 .................................V..........H................T........N....T...........................................P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F...............................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................

Page 27: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Genetic markers of differentpathotype groupspathotype groups

S b li i l t iSubclinical strainsSerine 222, Proline 289, Isoleucine 290, Fenilalanine 296

But limited number of sublinical strain tested

Major hydrophilic Major hydrophilicMinor hydrophilic Minor hydrophilic

210 220 230 240 250 260 270 280 290 300 310 320. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWUK661/86GB .............A.................................I.....................................I....S............................

j y ppeak A

j y ppeak B

y ppeak A

y ppeak B

296289 290222

U 66 /86G ............. ................................. ..................................... ....S............................VarE/85US ....N........T...................V......K....S........................N......I...............................D....E....2512/Tw ........L........................V...........................T.......................I.................................D78 .................................V..........H................T........N....T...........................................P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F...............................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................

Page 28: Dt i tDeterminantsof Pathogenicity for Gumboro Virus … V... · ncr ncr ncr ncrvp 1 5’ 5’3

Genetic markers of virulence

Cell culture adaptation of IBD virus to the non-lyphoid cellsp ypDelaware E: at position 253 Glutamine→Histidine and in 284Alanine→Threonine → cell culture adaptationGLS: only at position 284 Alanine→Threonine → cell cultureadaptationD78: at position 253 Histidine→Glutamine & 284 Threonine→Alanine8 at pos t o 53 st d e G uta e & 8 eo e a e→ loss of cell culture adaptation

M j h d hili M j h d hiliMi h d hili Mi h d hili

210 220 230 240 250 260 270 280 290 300 310 320 330 340. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGUK661/86GB .............A.................................I.....................................I....S.............................................

/85

Major hydrophilicpeak A

Major hydrophilicpeak B

Minor hydrophilicpeak A

Minor hydrophilicpeak B

253 284

VarE/85US ....N........T...................V......K....S........................N......I...............................D....E.....................2512/Tw ........L........................V...........................T.......................I..................................................D78 .................................V..........H................T........N....T.............................................R..............P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F................................................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................L...N............

28Mundt 1999. J. Gen. Virol. 80:2067Mundt 1999. J. Gen. Virol. 80:2067

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Genetic markers of virulenceCell culture adaptation Site directed mutagenesis of very virulent strain (UK661) L t l 2002 J G Vi lvery virulent strain (UK661) van Loon et. al. 2002. J. Gen. Virol. 83:121-

Construct 1: at position 284 Alanine→Threonine → cell culture padaptation but grows at low titreConstruct 2: at position 253 Glutamine → Histidine and at 284Alanine → Threonine → cell culture adaptation and grows at highAlanine → Threonine → cell culture adaptation and grows at high titreAnimal study with construct 2 (in 14 days-old SPF chickens) →attenuated phenotype

Major hydrophilic Major hydrophilicMinor hydrophilic Minor hydrophilic

210 220 230 240 250 260 270 280 290 300 310 320 330 340. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGUK661/86GB .............A.................................I.....................................I....S.............................................VarE/85US ....N........T...................V......K....S........................N......I...............................D....E.....................

/

peak A peak Bpeak A peak B253 284279

29

2512/Tw ........L........................V...........................T.......................I..................................................D78 .................................V..........H................T........N....T.............................................R..............P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F................................................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................L...N............

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Reversion of virulenceMany times reverted cell culture adapted vaccine strains could be re isolated These viruses:could be re-isolated. These viruses:

Do not cause clinical sings in chickens, but cause more serious lesions in bursa than the parent strain, p ,Only one amino acid change compare to the parent strain in position 253 Glutamine(Q) or Asparagine(N) instead of Histidine (H)

I it d ti f iIn vitro reproduction of reversion Serial passage of cell culture adapted vaccine in SPF eggs H253Q aa change higher pathogenicityaa change, higher pathogenicitySerial passage of reverted vaccine in cell culture Q253H aachange, lower pathogenicity

Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in

30

suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity (Ref.: Jackwood et. al.,. 2008. Virology;

377:110-)

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Genetic markers of virulence

Comparison of the genome of wild and cell culture adaptedIBDV t i (GLS)IBDV strain (GLS)

Reasortant virus: Segment A TC adapted + Segment B bursa derived → Reasortant grows at lower titre in cell culture, but replicates more g , pefficiently in the bursa than TC adapted parent strain → VP1 modulatesvirulenceReasortant virus: Segment A bursa derived + Segment B TC adaptedReasortant virus: Segment A bursa derived + Segment B TC adapted → does not grow in cell cuture → VP2 determines cell tropism

M j h d hili Mi h d hili Mi h d hili

210 220 230 240 250 260 270 280 290 300 310 320 330 340. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .

52/70GB TAADDYQFSSQYQPGGVTITLFSANIDAITSLSIGGELVFQTSVQGLVLGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNLVIPTNEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGUK661/86GB .............A.................................I.....................................I....S.............................................

Major hydrophilicpeak A

Major hydrophilicpeak B

Minor hydrophilicpeak A

Minor hydrophilicpeak B

253 284

VarE/85US ....N........T...................V......K....S........................N......I...............................D....E.....................2512/Tw ........L........................V...........................T.......................I..................................................D78 .................................V..........H................T........N....T.............................................R..............P7/78Hu .............S...................V..G........D...............T.T......N.........PI.....F................................................00273/73Au ..............................N..V...............N.....V.....T.T......G...................S.....V......................L...N............

31Liu and Vakharia 2004. Virology 33:62-

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Genetic markers of virulence

Reasortant IBD virus

Segment A vvIBDV + Segment B TC adapted → delayed mortality and morbidity, but same histopathologic lesions as wild-type vvIBDV

Concusion:VP1 does not significantly influence the pathogenicity of vvIBDVVP1 does not significantly influence the pathogenicity of vvIBDVjust delays the course of disease

Boot et. al. 2000. J. Virol. 74:6701-Boot et. al. 2000. J. Virol. 74:6701-

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Genetic markers of virulenceSegment reasortant and mosaic IBDV virus from TC adapted or D78+ wild vvIBDV (D6948) in 21-day-old SPF chickensor D78+ wild vvIBDV (D6948) in 21-day-old SPF chickens

Mosaic TC adapted IBDV with VP2 from vvIBDV → highly increased damage of the bursa compared to TC virus, but no mortalityMosaic D78 with VP2 from cvIBDV→ highly increased damage of the bursa compared to D78 parent virus, 20% mortality (cvIBDV 30%)the bursa compared to D78 parent virus, 20% mortality (cvIBDV 30%)Mosaic TC adapted IBDV with VP3 or/+ VP4 from vvIBDV → no increase in pathogenicity compared to parent TC adapted IBDVMosaic vvIBDV with VP3 C terminal from serotype II IBDV → same histopathological lesions as vvIBDV but no mortality

Conclusion:Conclusion: VP2 has major influence on pathogenicity VP3 and VP4 do not influence pathogenicityVP3 and VP4 do not influence pathogenicityC terminal part of VP3 from serotype II can reduce virulence

Boot et. al. 2005. Arch. Virol. 150:137-; Brant et. al. 2001. J. Virol. 75:11974-

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Naturally reassorted very virulent IBDVsIBDVsSegment BSegment A

Nowadays new, segment reasortantfield viruses

ioccurring:

→ very virulent( )(Segment A) 

→ classical virulentor serotype 2 (Segment B) virusesoccured

Reference: Le Nouën et. al. 2006. J. Gen. Virol. 87:209‐216.

occured

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Naturally reassorted very virulent IBDVs

Chinese segment reassortant IBDV strains → mortality rate significantly lower than European vvIBDV strains but not lower

y y

significantly lower than European vvIBDV strains, but not lower than the other Chinese vvIBDV isolates.

US reassortant IBDV: A seg vvIBDV+ B seg serotype 2→ reducedUS reassortant IBDV: A seg vvIBDV+ B seg serotype 2→ reducedmortality but no changes in morbidity and bursa histopathologycompared to vvIBDV strain

Reference: Jackwood et al 2011 Virology 420 98p

Conclusion:Conclusion:Strain Classification Mortality 

F52/70 Cv 36%

Strain Classification Mortality 

F52/70 Cv 36%

Reference: Jackwood et al. 2011 Virology. 420 98‐

No marked effect of segmentreassortment on the virulence ofNo marked effect of segmentreassortment on the virulence of

Cu‐1wt Cv 54%

849VB/EU vv 91%

96108/EU vv 91%

Cu‐1wt Cv 54%

849VB/EU vv 91%

96108/EU vv 91%

vvIBDV

VP1 only modulate the virulence 

vvIBDV

VP1 only modulate the virulence 

Harbin/China vv 58%

HK46/China vv 63%

GX/China vv–cv reassortant 57%

Harbin/China vv 58%

HK46/China vv 63%

GX/China vv–cv reassortant 57%

Source: van den Berg et al. 2004 Avian pathol. 33(5) 470‐5 weeks old SPF chickens

of IBDVof IBDV Henan1/China vv‐cv reasortant 42%Henan1/China vv‐cv reasortant 42%

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Molecular determinants of pathogenicitypathogenicity

ConclusionAll viral genes can influence the pathogenicity of IBD

virus at different extentsChanges in the cell tropism

VP2 influences the attachment of the virus to the cell → determines cell tropism → has major influence ondetermines cell tropism → has major influence on pathogenicityVP1 influences the efficiency of viral RNA synthesis →

d l t th i lmodulate the virulence

Changes in the efficiency of virus assemblyVP3 influence the efficiency of the assembly of virionsVP4 influence the efficiency of viral polyprotein processingVP5 i fl th ffi i f i l

36

VP5 influence the efficiency of virus release

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Molecular determinants of pathogenicitypathogenicity

C l iConclusionBased on the presence or absence and position of

i i id h h i i f IBDVcertain amino acids the pathogenicity of an IBDV strain cannot be fully determinedP th t i f IBDV t i b d l fPathotyping of IBDV strains based only on a few so-called genetic markers could led to false diagnostic resultsresultsPathotypes of IBDV strains can be more accuratelydetermined by phylogenetic analysisy p y g yPrecise determination of the pathogenicity of an IBDV isolate can be done only by in vivo experiment

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Phylogenetic analysis of IBDV strains in EuropeIBDV strains in Europe

Phylogenetic analysis of IBDV strains in Europe (retrospective and recent) and incidence of different pathotype related genetic groups

3838

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Phylogenetic analysis of IBDV strains in Europe

Early European epidemiologyIBDV strains in Europe

• First appearence of IBDV in the 60’ and 70’s.

• Early isolates belong to the

vv

• Early isolates belong to the classical virulent group.

• Vaccination (mild) was introduced:cv

( )• Prevented or reduced the economical effect of cv IBDV strains. Sub-

clin• Several subclinical strains were isolated during this „silent”period in Hu and Pl

clin.

period in Hu and Pl• Acute IBD, caused by very virulent

IBDV strains appeared in the early 90’ and spread rapidly. Source: Domanska, Mató et al. 2004

Arch. Virol. 149: 465-480

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Phylogenetic analysis of IBDV strains in Europe

Recent European epidemiologyIBDV strains in Europe

D1699/2/1/11PT

D1655/2/11PTD1393/5/10PT

D1575/1/2/11PTD1078/12/08ES

D1699/2/1/11PT

D1655/2/11PTD1393/5/10PT

D1575/1/2/11PTD1078/12/08ES

D1699/2/1/11PT

D1655/2/11PTD1393/5/10PT

D1575/1/2/11PTD1078/12/08ES

Most commonly detected IBDV strains:• Clinical IBD cases are exclusively

caused by vvIBDV strainsDistance 0.02

CS89/89GB

D1751I6PT D1354/13/10PT

D1102I3PT

D1393/17/10PT

D1806/2/11PT

D1078/12/08ES

D1699/4/1/11PTD1393I16p5

D1806/3/11PTD1655/7/11PTD1806/1/11PT

D1575/2/2/11PTTULA94/RU

UK661/86GB

Distance 0.02

CS89/89GB

D1751I6PT D1354/13/10PT

D1102I3PT

D1393/17/10PT

D1806/2/11PT

D1078/12/08ES

D1699/4/1/11PTD1393I16p5

D1806/3/11PTD1655/7/11PTD1806/1/11PT

D1575/2/2/11PTTULA94/RU

UK661/86GB

D1393/17/10PT

D1806/2/11PT

D1078/12/08ES

D1699/4/1/11PTD1393I16p5

D1806/3/11PTD1655/7/11PTD1806/1/11PT

D1575/2/2/11PTTULA94/RU

UK661/86GBcaused by vvIBDV strains (genetically homogenous group).

• Vaccine or vaccine-derived

CS89/89GB

G188/04RU D1521/1/11SK

D108/01PL

00/40PL

D691/6/10PL

OKYM/91JP

D1743/1/3/11FR

DV86/86NL OKYMT/95JP

D1304/3/09PLD833/10PL

D1776/3/2/11PLD1487/2/10PL

Very virulentIBDVs

CS89/89GB

G188/04RU D1521/1/11SK

D108/01PL

00/40PL

D691/6/10PL

OKYM/91JP

D1743/1/3/11FR

DV86/86NL OKYMT/95JP

D1304/3/09PLD833/10PL

D1776/3/2/11PLD1487/2/10PL

D691/6/10PL

OKYM/91JP

D1743/1/3/11FR

DV86/86NL OKYMT/95JP

D1304/3/09PLD833/10PL

D1776/3/2/11PLD1487/2/10PL

Very virulentIBDVs

strains are widespreadRearly detected strains with no G92/2/03BR

Br/03/DT BR-3

D1022/7/08ES D991/3/08ES D1078/10/08ESD1067/2/08ES

D1278/8/09ESD1374/2/1/10FR

D1178I7VE D1311I19BR

BR-1 Br/01/BNK

D1420/1/1/10EZ

G92/2/03BR

Br/03/DT BR-3

D1022/7/08ES D991/3/08ES D1078/10/08ESD1067/2/08ES

D1278/8/09ESD1374/2/1/10FR

D1178I7VE D1311I19BR

BR-1 Br/01/BNK

D1420/1/1/10EZ

BR-3

D1022/7/08ES D991/3/08ES D1078/10/08ESD1067/2/08ES

D1278/8/09ESD1374/2/1/10FR

D1178I7VE D1311I19BR

BR-1 Br/01/BNK

D1420/1/1/10EZ

economical impact:• Subclinical strains • Variant strains

849VB/87BE52/70GB

D1588/5/3/11SKD1492/4/10EZ

CEVAC IBD L

0/ / / 0D1420/2/1/10EZ

D1632/1/11SK D1588/3/1/11SKD1540/1/10EZD1463/2/10EZ

Bursine plusBURSINE2

BURSA-VAC STC/67US

849VB/87BE52/70GB

D1588/5/3/11SKD1492/4/10EZ

CEVAC IBD L

0/ / / 0D1420/2/1/10EZ

D1632/1/11SK D1588/3/1/11SKD1540/1/10EZD1463/2/10EZ

Bursine plusBURSINE2

BURSA-VAC STC/67US

D1492/4/10EZ

CEVAC IBD L

0/ / / 0D1420/2/1/10EZ

D1632/1/11SK D1588/3/1/11SKD1540/1/10EZD1463/2/10EZ

Bursine plusBURSINE2

BURSA-VAC STC/67US• Variant strains

Only detected from a restricted area (SP, PO, FR); closely related to

228E

GLS/87US 3212/88US

D1204/9/09FR

VarA/85US

STC/67US

P2/73DE CEVAC Gumbo Lbur706

D78

U28/88US VarE/85US

D814/24/08FR D966/6/08FR

D966/9/08FR European variant

228E

GLS/87US 3212/88US

D1204/9/09FR

VarA/85US

STC/67US

P2/73DE CEVAC Gumbo Lbur706

D78

U28/88US VarE/85US

D814/24/08FR D966/6/08FR

D966/9/08FR

VarA/85US

STC/67US

P2/73DE CEVAC Gumbo Lbur706

D78

U28/88US VarE/85US

D814/24/08FR D966/6/08FR

D966/9/08FR European variant

US variants.00273/73AU

02-30850CA

D966/9/08FR D806/10/07ES

D699/3/1/11PTD806/11/07ES

P7/78Hu H2/Us

D1204/8/09FRD1204/5/09FR

Subclinical IBDVs

pIBDVs

00273/73AU

02-30850CA

D966/9/08FR D806/10/07ES

D699/3/1/11PT

02-30850CA

D966/9/08FR D806/10/07ES

D699/3/1/11PTD806/11/07ES

P7/78Hu H2/Us

D1204/8/09FRD1204/5/09FR

Subclinical IBDVs

pIBDVs

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