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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
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
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
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
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
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.
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............
Criteria for the characterization of IBDV strainsof IBDV strains
PathogenicityAntigenicityAntigenicityGenetic relatedness
88
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
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
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
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
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)
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
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
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
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
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
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
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
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
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
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
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.
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-
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......................
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......................
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
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............
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-)
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-
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-
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-
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
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%
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
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
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
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
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
Thank you for yourattention!