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GenomeGenome diversitydiversity of of ostreidostreid herpes virus herpes virus (OsHV(OsHV--1): 1): firstfirst resultsresults

T. Renault, T. Renault, P. Moreau, N. P. Moreau, N. FauryFaury, J., J.--F. F. PepinPepin and A. and A. SegarraSegarra

Laboratoire de GLaboratoire de Géénnéétique et Pathologie tique et Pathologie La La TrembladeTremblade

Annual Annual meeting of meeting of the the National National Laboratories Laboratories for for Mollusc DiseasesMollusc Diseases

La Rochelle La Rochelle --

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OstreidOstreid herpes virus 1 (OsHVherpes virus 1 (OsHV--1) has been 1) has been associatedassociated to to outbreaksoutbreaks resultingresulting in in highhigh mortalitiesmortalities in in differentdifferent bivalve bivalve speciesspecies, , includingincluding thethe PacificPacific cuppedcupped oysteroyster, , CrassostreaCrassostrea gigasgigas, , bothboth in in hatcherieshatcheries/nurseries /nurseries andand thethe naturalnatural environmentenvironment worldwideworldwide..

OsHVOsHV--1 has been 1 has been purifiedpurified fromfrom naturallynaturally infectedinfected larvallarval PacificPacific cuppedcupped oysteroyster, , C. gigas,C. gigas, in France (Le in France (Le DeuffDeuff andand Renault, 1999) Renault, 1999) andand itsits genomegenome sequencedsequenced ((DavisonDavison et al., 2005et al., 2005, GenBankGenBank accession naccession n°° AY 509253). AY 509253).

OsHVOsHV--1 has been 1 has been classifiedclassified withinwithin thethe MalacoherpesviridaeMalacoherpesviridae familyfamily in in thethe orderorder HerpesviralesHerpesvirales ((DavisonDavison et al., 2009). et al., 2009). TheThe OsHVOsHV--1 1 genomegenome isis a a doubledouble--strandedstranded DNA of about 207 DNA of about 207 kbpkbp ((DavisonDavison et al., 2005). et al., 2005).

State of State of thethe art: OsHVart: OsHV--11

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OsHVOsHV--1 variants have already been reported in different bivalve 1 variants have already been reported in different bivalve species and in different geographical locations (species and in different geographical locations (ArzulArzul et al., et al., 2001a, 2001a, ArzulArzul et al., 2001b; Renault et al., 2001; Friedman et al., et al., 2001b; Renault et al., 2001; Friedman et al., 2005; Moss et al., 2007).2005; Moss et al., 2007).

Moreover, Moreover, since since 2008, massive mortality outbreaks among 2008, massive mortality outbreaks among Pacific oysters, Pacific oysters, C. C. gigasgigas, were reported in Europe (France, , were reported in Europe (France, Ireland and the UK) and in South Pacific (New Zealand and Ireland and the UK) and in South Pacific (New Zealand and Australia) in relation to the detection of a particular genotypeAustralia) in relation to the detection of a particular genotype called called OsHVOsHV--1 1 µµVarVar ((SegarraSegarra et al., 2010; et al., 2010; GenBankGenBank accession accession nn°° HQ842610). HQ842610).

State of State of thethe art: OsHVart: OsHV--1 1 diversitydiversity

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Several differences in two Several differences in two ORFsORFs (ORF4 and ORF43) (ORF4 and ORF43) including including a deletion of 12 a deletion of 12 bpbp in a in a microsatellitemicrosatellite located uplocated up-- stream of the ORF4stream of the ORF4, are characteristic of this variant , are characteristic of this variant when when compared with the reference compared with the reference OsHVOsHV--1 genome (1 genome (GenBankGenBank accession naccession n°° AY509253). AY509253).

State of State of thethe art: OsHVart: OsHV--1 µvar1 µvar

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erOsHV‐1

OsHVOsHV--1 1 sequencingsequencing

Detection of newly reported genotype (systematic differences in 2 areas: ORF4 and ORF43)

TCGATTGCGAA. . A . . . . . A . .

C2 C6

Non coding zone Coding zone

ATTGCC. . . A . .

AAAACC. . C - . .

TAA--CC. . . GC . .

CCG. G .

GGA. A .

CACT. - - .

Microsatellite zone (repeats of 3 nucleotids ‘CTA’ ) showing a deletion of 12 bp (ORF4) et 2 mutations in the coding area

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OsHVOsHV--1 1 sequencingsequencing

Detection of newly reported genotype (systematic differences in 2 areas: ORF4 and ORF43)

IA2

AAATATC. . - . . . .

TTCGCT. . T . . .

IA1-IA2 fragment (ORF43)

Non coding zone  Coding zone

IA1

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Although both Although both OsHVOsHV--1 genotypes (1 genotypes (GenBankGenBank accession naccession n°° AY509253 and nAY509253 and n°° HQ842610) were detected in association HQ842610) were detected in association with mortality outbreaks in 2008 in France, with mortality outbreaks in 2008 in France, OsHVOsHV--1 1 µµVarVar was was mainly detected in 2009 and 2010. mainly detected in 2009 and 2010.

These results raise questions about the emergence, These results raise questions about the emergence, the virulence and the origin of the virulence and the origin of OsHVOsHV--1 1 µµVar.Var.

State of State of thethe art: OsHVart: OsHV--1 µvar1 µvar

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In this context, more data are needed in order to In this context, more data are needed in order to describe describe OsHVOsHV--1 diversity1 diversity in relation to time in relation to time

collection and geographical location. collection and geographical location.

State of State of thethe artart

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For this purpose, we have analysed around 80 For this purpose, we have analysed around 80 OsHVOsHV--1 1 virus isolates mainly collected in France, but also in the virus isolates mainly collected in France, but also in the USA and Asian countries including Japan, for a period USA and Asian countries including Japan, for a period covering 18 years, between 1993 and 2010.covering 18 years, between 1993 and 2010.

The main aim of the present study is to describe The main aim of the present study is to describe genetic diversity among virus isolates.genetic diversity among virus isolates.

ObjectivesObjectives

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In a approach, In a approach, 8 8 ORFsORFs were targeted by PCRwere targeted by PCR ((ORFsORFs 30, 30, 37, 61, 64, 79, 103, 117 and 121) and 37, 61, 64, 79, 103, 117 and 121) and 5 isolates tested 5 isolates tested (2008).(2008).

In a second approach, In a second approach, 3 3 ORFsORFs (ORF4, ORF37 and (ORF4, ORF37 and ORF43) were selected for analysis of ORF43) were selected for analysis of 79 isolates.79 isolates.

-- Existing primers (ORF4 and ORF43) and specific primers Existing primers (ORF4 and ORF43) and specific primers designed to target specifically designed to target specifically OsHVOsHV--1 ORF 37 were used.1 ORF 37 were used.

-- PCR products were then directly PCR products were then directly sequencedsequenced. . -- Finally, genome diversity was analysed based on the Finally, genome diversity was analysed based on the

comparison of the sequencescomparison of the sequences obtained from the virus isolates obtained from the virus isolates (ongoing research).(ongoing research).

MolecularMolecular characterisationcharacterisation

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ORF 37 ORF 61 ORF 64

R µ µ µ R R µ µ µ R R µ µ µ RRR : OsHV: OsHV--1 non 1 non µµVarVar

µµ : OsHV: OsHV--1 1 µµVarVar

1

2

FirstFirst approachapproach -- PCR PCR analysisanalysis of 8 ORFsof 8 ORFs

OsHVOsHV--1 PCR 1 PCR productproduct sizessizes werewere similarsimilar for all ORF for all ORF testedtested exceptexcept for ORF37 (no amplification for OsHVfor ORF37 (no amplification for OsHV--1 µVar)1 µVar)

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OsHVOsHV--1 µVar 1 µVar demonstrateddemonstrated polymorphismpolymorphism for for severalseveral ORFsORFs

Substitution Amino acid modification Comparison

ORF 61

1 substitution TAT (Ref) replaced by TGT (µVar) Y replaced by C

(AY509253= Ref ) ≠ µ VAR

ORF 64

1 substitution GAA (Ref) replaced by AAA (µVar) E replaced by K

(AY509253= Ref) ≠ µ VAR

ORF 79

1 substitution GAT (Ref) replaced by AAT (µVar) D replaced by N

(AY509253= Ref) ≠ µ VAR

ORF 103

1 substitution GAG (Ref) replaced by AAG (µVar) E replaced by K

(AY509253= Ref) ≠ µ VAR

ORF 117

2 substitutions

CTG (AY509253) replaced by ATG (Ref) L replaced by M

AAA (AY509253) replaced by GAA(Ref) K replaced by E

(AY509253= µ VAR ) ≠ Ref

AY509253≠ (Ref = µVar)

ORF 121

Séquences identiques AY509253= Ref = µVAR

ORF 30

1 substitution AAA (AY509253) replaced by GAA (Ref) K replaced by E

AY509253≠ (Ref = µVar)

FirstFirst approachapproach -- PCR PCR productproduct sequencessequences

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µ µ µ

FirstFirst approachapproach -- ORF37 ORF37 deletiondeletion characterisationcharacterisation

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µ µ µ

FirstFirst approachapproach -- ORF37 ORF37 deletiondeletion characterisationcharacterisation

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erFirstFirst approachapproach -- OsHVOsHV--1 µVar 1 µVar characterizedcharacterized by a by a

605 605 bpbp deletiondeletion

OsHVOsHV--1 µVar 1 µVar isolatesisolates demonstrateddemonstrated a a systematicsystematic 605 605 bpbp deletiondeletion correspondingcorresponding to to thethe total total lacklack of 2 ORFs of 2 ORFs andand thethe

partial partial lacklack of a of a thirdthird ORFORF

OsHVOsHV--1 : AY5092531 : AY509253

OsHVOsHV--1 µVar1 µVar

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erSecond Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolates

All DNAs All DNAs extractedextracted werewere analysedanalysed by by classicalclassical PCR PCR targetingtargeting 3 3 selectedselected ORFs (ORF4, ORF37 ORFs (ORF4, ORF37 andand ORF43)ORF43) usingusing existing primers (ORF4 existing primers (ORF4 and ORF43) and specific and ORF43) and specific primers designed to primers designed to target specifically ORF target specifically ORF 37.37.

DNAs DNAs werewere extractedextracted fromfrom frozenfrozen samplessamples usingusing QIAamp DNA QIAamp DNA Kit Kit andand quantifiedquantified usingusing real time quantitative PCR (real time quantitative PCR (PepinPepin et al.et al., , 2008).2008).

Country YearsNumber of analyzed samples

France 1993 3France 1994 4France 1995 4France 1997 2France 2003 6France 2005 5France 2006 8France 2007 9France 2008 18France 2009 5France 2010 9

China 2002 1USA Tomales Bay 2003 1USA Tomales Bay 2005 1

USA California 2006 1USA California 2007 1

Japan 2010 1All years 79

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PCR PCR productsproducts demonstrateddemonstrated a a similarsimilar size for ORF4 size for ORF4 andand ORF43.ORF43.

3 types of 3 types of resultresult werewere obtainedobtained for ORF37: for ORF37: 989 989 bpbp,, 384 384 bpbp andand no amplificationno amplification indicatingindicating thethe existence of existence of atat least 3 least 3 potentialpotential genotypesgenotypes (a 605 (a 605 bpbp deletiondeletion for virus for virus isolatesisolates correspondingcorresponding to to OsHVOsHV--1 1 µµvar).var).

ORF 4 ORF 37 ORF 43

Second Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolates

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SequencingSequencing

PCR PCR productsproducts obtainedobtained for ORF4 for ORF4 andand ORF37 ORF37 werewere directlydirectly sequencedsequenced..

Consensus Consensus sequencessequences comparedcompared usingusing CLUSTALW Multiple CLUSTALW Multiple alignement alignement andand phylogenicphylogenic treestrees constructedconstructed usingusing MEGA 4.MEGA 4.

PCR PCR productsproducts for ORF43 for ORF43 needneed to to bebe sequencedsequenced andand alignedaligned ((ongoingongoing workwork).).

Second Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolates

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-- ORF4 (47 ORF4 (47 sequencessequences, 628 , 628 bpbp): ): polymorphismpolymorphism in in severalseveral areasareas

-- ORF37 (46 ORF37 (46 sequencessequences, 856 , 856 bpbp et 298 et 298 bpbp): ): limitedlimited polymorphismpolymorphism exceptexcept thethe 605 605 bpbp deletiondeletion

Second Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolatesMultiple alignement

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erPhylogenicPhylogenic treetree: ORF4: ORF4

Second Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolates

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PhylogenicPhylogenic treetree: ORF37: ORF37

Second Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolates

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-- «« ReferenceReference » OsHV» OsHV--1 1 ((GenBankGenBank accession naccession n°° AY509253)AY509253): French : French isolatesisolates fromfrom 1994 to 2008 (1994 to 2008 (howeverhowever, a , a polymophismpolymophism reportedreported for ORF37 for ORF37 amongamong French French samplessamples))

-- OsHVOsHV--1 µVar 1 µVar ((GenBankGenBank accession naccession n°° HQ842610)HQ842610): : French French isolatesisolates fromfrom 2008 to 2010/2008 to 2010/JapaneseJapanese andand ChineseChinese isolatesisolates

Second Second approachapproach: 3 ORFs : 3 ORFs andand 79 79 isolatesisolates

Phylogenic analysis: 2 major genogroups based on 2 ORFs (ORF4 and ORF37)

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-- TwoTwo major major genogroupsgenogroups defineddefined on on sequencesequence analysisanalysis (ORF4 (ORF4 andand ORF37) ORF37) groupinggrouping French French isolatesisolates fromfrom 1994 to 2008 1994 to 2008 andand French French isolatesisolates//JapaneseJapanese andand ChineseChinese isolatesisolates, , respectivelyrespectively

-- PendingPending information about ORF43information about ORF43

-- OngoingOngoing studystudy about ORF37 in about ORF37 in orderorder to to definedefine polymorphismpolymorphism detecteddetected amongamong somesome French French isolatesisolates (no PCR amplification)(no PCR amplification)

ConclusionsConclusions

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-- AddAdd more more isolatesisolates (Europe, New (Europe, New ZelandZeland, , AustraliaAustralia, , JapanJapan, , China, China, KoreaKorea, Taiwan, USA, , Taiwan, USA, …….).)

-- SequencingSequencing wholewhole virus virus genomesgenomes fromfrom selectedselected isolatesisolates representingrepresenting genogroupsgenogroups ((ongoingongoing EU EU fundedfunded projectsprojects: : AquaGenetAquaGenet, , BivalifeBivalife andand a a submittedsubmitted ANR projet: DIVE)ANR projet: DIVE)

PerspectivesPerspectives

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er ThankThank youyou veryvery muchmuch to to thethe LGP team for LGP team for valuablevaluable help, help, andand to Carolyn Friedman to Carolyn Friedman andand ColleenColleen BurgeBurge for for providingproviding osyterosyter

samplessamples

AknowledgementAknowledgement

Merci de votre attentionMerci de votre attention