7 novel insights in the adaptation of avian h9n2 influenza … · 7 novel insights in the...
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Novel insights
in theadaptation
of avianH
9N2 influenza
virusesto sw
ine
José Carlos Mancera Gracia2017
J C Mancera G
racia2017
Novel insights in the adaptation of avian H9N2 influenza viruses
to swine
LABORATORYOFVIROLOGYDEPARTMENT OFVIROLOGY,PARASITOLOGYANDIMMUNOLOGY
FACULTYOFVETERINARYMEDICINEGHENTUNIVERSITY
Novelinsightsintheadaptationofavian
H9N2influenzavirusestoswine
JoséCarlosManceraGracia
DoctorofPhilosophy(Ph.D.)inVeterinarySciences
2017
Promotor:Prof.Dr.KristienVanReeth
Co-Promotor:Prof.Dr.XavierSaelens
LaboratoryofVirology
DepartmentofVirology,ParasitologyandImmunology
FacultyofVeterinaryMedicine
GhentUniversity
MembersoftheExaminationCommittee:Prof.Dr.HansNauwynck(Chairman)DepartmentofParasitology,VirologyandImmunology,FacultyofVeterinaryMedicine,GhentUniversity.Dr.SebastiaanTheuns(Secretary)DepartmentofParasitology,VirologyandImmunology,FacultyofVeterinaryMedicine,GhentUniversity.Prof.Dr.MikhailMatrosovichInstitutfürVirologie,PhilippsUniversität,Marburg.Prof.Dr.DominiekMaesDepartment of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine,GhentUniversity.Dr.AnnebelDeVleeschauwerVeterinaryandAgrochemicalResearchCentre,Brussels.Dr.KarenvanderMeulenPerseusBVBA,BelgiumManceraGraciaJ.C. (2017).Novel insights intheadaptationofavianH9N2 influenzavirusestoswine.PhDthesis,GhentUniversity,Belgium.Theauthorandthepromotorgivetheauthorizationtoconsultandcopypartsofthisworkforpersonaluseonly.Everyotheruseissubjecttocopyrightlaws.Permissiontoreproduceanymaterialcontainedinthisworkshouldbeobtainedfromtheauthor.ThisstudywasfundedbytheresearchprojectFLUPIG“Pathogenesisandtransmissionofinfluenzainpigs”(FP7-EUprojectNo258084).
“Iamfondofpigs.Dogslookuptous.Catslookdowntous.Pigstreatusasequals.”
WinstonChurchillUnitedKingdomPrimeMinister1940-1945
and1951-1955
Amispadres,InmaculadayJoséCarlos.
TABLEOFCONTENTS
LISTOFABBREVIATIONS
1GENERALINTRODUCTION.................................................................................11.1 Introductiontoinfluenzaviruses...................................................................2
1.1.1 Taxonomyandvirionstructure...............................................................21.1.2 Replicationcycle......................................................................................51.1.3 Mechanismsofevolutionandemergenceofnovelinfluenzaviruses.....7
1.2 Ecologyandpathogenesisofavian,humanandswineinfluenzaviruses......8
1.2.1 Avianinfluenzaviruses............................................................................81.2.2 Humaninfluenzaviruses.........................................................................91.2.3 Swineinfluenzaviruses.........................................................................11
1.3 Interspeciestransmissionandthespeciesbarrier.......................................14
1.3.1 Transmissionfrombirdstomammals………………………………………………..141.3.2 Transmissionbetweenhumansandswine.………………………………………..151.3.3 Avianinfluenzaviruseswithpandemicpotential…………..…………………..16
1.4 Molecularmechanismsofhostrangerestriction........................................21
1.4.1 Theroleofthehemagglutinin...............................................................211.4.2 Theroleoftheneuraminidase..............................................................231.4.3 Theroleofthepolymeraseproteinsandtheinternalgenes................24
1.5 Adaptationofavianinfluenzavirusestoswine...........................................25
1.5.1 Naturalinfectionsofpigswithavianinfluenzaviruses.........................251.5.2 Experimentalinfectionsofpigswithavianinfluenzaviruses................271.5.3Theroleofthepigintheadaptationofavianinfluenzavirusesto
mammals................................................................................................291.6 References...................................................................................................30
2 AIMSOFTHETHESIS.......................................................................................43
3 EFFECTOFSERIALPIGPASSAGESONTHEADAPTATIONOFANAVIANH9N2INFLUENZAVIRUSTOSWINE..........................................................................47Abstract................................................................................................................483.1 Introduction.................................................................................................493.2 Materialsandmethods................................................................................513.3 Results..........................................................................................................573.4 Discussion.....................................................................................................663.5 Acknowledgements......................................................................................713.6 References...................................................................................................71
4 A REASSORTANT H9N2 INFLUENZA VIRUS CONTAINING 2009 PANDEMIC H1N1 INTERNAL-‐PROTEIN GENES ACQUIRED ENHANCED PIG-‐TO-‐PIG TRANSMISSION AFTER SERIAL PASSAGES IN SWINE ................................................................. 77 Abstract ................................................................................................................ 78 4.1 Introduction ................................................................................................. 79 4.2 Materials and methods ................................................................................ 81 4.3 Results ......................................................................................................... 85 4.4 Discussion .................................................................................................... 93 4.5 Acknowledgements ..................................................................................... 97 4.6 References ................................................................................................... 97 Supplemental materials……………………………………………………………………..…………..101
5 GENERAL DISCUSSION .................................................................................. 107
6 SUMMARY -‐ SAMENVATTING ....................................................................... 123
7 CURRICULUM VITAE ..................................................................................... 131
8 AKNOWLEDGEMENTS .................................................................................. 135
LISTOFABBREVIATIONSA aa aminoacid
AEC 3-amino-9-ethyl-carbazole
AIV avianinfluenzavirus
AUC areaunderthecurve
B bp basepairs
BSL biosafetylevel
C CDC CentersforDiseaseControlandPrevention
cDNA complementarydeoxyribonucleicacid
CI confidenceinterval
cRNA complementaryribonucleicacid
D dpc dayspost-contact
dpi dayspost-inoculation
E ELISA enzyme-linkedimmunosorbentassay
F FAO FoodandAgricultureOrganization
G GOF gain-of-function
H H1N2v H1N2variant
H3N2v H3N2variant
HA hemagglutinin
HEPA highefficiencyparticlearresting
HI hemagglutinationinhibition
HPAIV highlypathogenicavianinfluenzaviruses
L LPAIV lowpathogenicavianinfluenzaviruses
M M matrixprotein
M1 matrixprotein1
M2 matrixprotein2
MDCK Madin-Darbycaninekidney
MOI multiplicityofinfection
mRNA messengerribonucleicacid
N NA neuraminidase
NEP nuclearexportprotein
Neu5Ac non-O-acetylatedN-acetylneuraminicacid
Neu5Gc N-glycolylneuraminicacid
ng nanogram
NGS nextgenerationsequencing
nM nanoMolar
NP nucleocapsidprotein
NS1 non-structuralprotein1
NS2 non-structuralprotein2
O OIE WorldOrganizationforAnimalHealth
OFFLU OIE-FAOglobalnetworkofexpertiseonanimalinfluenza
P PA polymeraseacidic
PB1 polymerasebasic1
PB2 polymerasebasic2
PBS phosphate-bufferedsaline
pH1N1 2009pandemicH1N1
PRDC porcinerespiratorydiseasecomplex
PRRS porcinereproductiveandrespiratorysyndrome
R R0 basicreproductionratio
RBS receptor-bindingsite
RNA ribonucleicacid
RNP ribonucleoprotein
RT-PCR reversetranscription-polymerasechainreaction
S Sia sialicacid
Siaα2,3Gal α2,3Gal-linkedsialicacids
Siaα2,6Gal α2,6Gal-linkedsialicacids
SIV swineinfluenzavirus
T TCID50 tissuecultureinfectiousdosewitha50%endpoint
TRIG triple-reassortantinternalgenes
U URT upperrespiratorytract
US UnitedStates
V vRNA viralribonucleicacid
W WHO WorldHealthOrganization
Chapter1
1
1
GENERALINTRODUCTION 21
Generalintroduction
2
1.1 Introductiontoinfluenzaviruses 3
1.1.1 Taxonomyandvirionstructure 4
Influenzavirusescauseahighlycontagiousrespiratorydiseaseknownasinfluenzaor 5
“flu”,whichhasposedasignificantthreatforanimalsandhumanssincetheancient 6
times. These viruses are the most important members of the family 7
Orthomyxoviridae.Theirgenomeiscomposedof7to8segmentsofnegative-sense 8
single-stranded ribonucleic acid (RNA). Influenza viruses are divided into 4 9
genera/types:A,B,CandD. This classification (into the4genera) isbasedon the 10
geneticandantigenicdifferencesbetweentheirnucleocapsid(NP)andtheirmatrix1 11
(M1)proteins(Wrightetal.,2013).InfluenzaBandCvirusesarelargelyrestrictedto 12
humansbuthavealsobeensporadicallyisolatedfromothermammals(Kimuraetal., 13
1997, Hause et al., 2013). Since 2011 influenza D viruses have been identified in 14
cattleandpigs(Ducatezetal.,2015,Chiapponietal.,2016,Fergusonetal.,2016). 15
InfluenzaB,CandDvirusesareconsidereda lowerthreattopublicandveterinary 16
health,andtheywillnotbefurtherdiscussedinthisthesis. Incontrast, influenzaA 17
virusesareofgreatimportancebecausetheycaninfectawidevarietyofavianand 18
mammalian species (Fouchier et al., 2003, Harder and Vahlenkamp, 2010). These 19
virusesarefurtherdividedintosubtypesbasedontheantigenicpropertiesoftheir 20
surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). As of today 18 21
differentHAs (H1-H18)and11NAs (N1-N11)havebeen identified (Fouchieretal., 22
2005,Mehle, 2014).With the exception of subtypes H17N10 and H18N11, which 23
wereonly found inbats,allknownsubtypesof influenzavirusesareestablished in 24
birds.Yet justa limitednumberofsubtypeshavebecomeendemic inhumansand 25
pigs(Table1). 26
27
Chapter1
3
Table 1.OverviewofHA andNA subtypes of influenzaA viruses that are or have 28beenendemicinbirds,humans,pigsandotheranimalspecies(adaptedfromtheUS 29CDC:http://www.cdc.gov/flu/about/viruses/transmission.htm). 30 31
HA NA
Subtype Hostspecies Subtype Hostspecies
H1 N1
H2 N2
H3 N3
H4 N4
H5 N5
H6 N6
H7 N7
H8 N8
H9 N9
H10 N10
H11 N11
H12
H13
H14
H15
H16
H17
H18
32
Generalintroduction
4
Thecurrentnomenclaturesystemfor influenzavirusesgives informationaboutthe 33
type,host,placeofisolation,strainnumber,yearofisolationandantigenicsubtype 34
of thevirus. For instance,anH9N2virus isolated fromquail inHongKong in1997 35
wasdesignatedasA/quail/HongKong/G1/1997(H9N2). 36
Influenza A virions are spherical or pleomorphicwith a diameter between 80 and 37
120 nanometers. They are enveloped viruses and encode 10 viral proteins on 8 38
separate segments of negative-sense RNA. A few additional proteins were only 39
recentlyidentified(Shietal.,2014).Theenvelopeisalipidbilayerderivedfromthe 40
hostcellmembraneandcontainstheHAandtheNAthatplayacrucialrole inthe 41
virusbindingand release, respectively (Figure1).Another transmembraneprotein, 42
thematrixprotein2(M2), isatypeIIIprotonselective ion-channelessential inthe 43
release of the uncoated ribonucleoproteins (RNPs) into the cytoplasm of the host 44
cell. Justunderneath thevirionenvelope, theM1 formsa layer that interactswith 45
boththeenvelopeandtheRNPs.ThisM1isresponsibleforthestructuralintegrityof 46
thevirionandencapsidatesthe8RNPs.TheRNPsconsistofaRNAsegment,NPsand 47
thepolymerasecomplex(PA,PB1andPB2)thatisresponsibleforviralRNA(vRNA) 48
replicationand transcription. Thenon-structural (NS) segmentencodes2proteins: 49
NS1, a host antiviral response antagonist and NS2 (also named nuclear export 50
proteinNEP),whichisassociatedwiththeM1andmediatestheexportoftheRNPs 51
fromthenucleustothecytoplasm. 52
53
Chapter1
5
54
Figure 1. (A) The structure of influenza A virion. (B) Non-structural proteins 55
identified,thecolourofeachproteinmatchesthecolourofthegene-segmentthat 56
codesit.(C)RepresentationoftheinfluenzaAribonucleoprotein(adaptedfromShi 57
etal.2014). 58
59
1.1.2 Replicationcycle 60
The infection of a susceptible host cell is initiated by the interaction of the HA 61
receptor-bindingsite(RBS)withterminalsialicacids(Sia) linkedtoglycoproteinsor 62
glycolipidsonthehostcellmembrane(seeFigure2).ThecontactbetweenSiaand 63
the HA is established by low affinity bonds such as hydrogen bonds, hydrophobic 64
interactions and van der Waals contacts (Gamblin and Skehel, 2010). Therefore, 65
bindingofmultiple cell receptors tomultipleHAspikes isneeded toobtainahigh 66
binding avidity. Upon binding to the host cell Sia residues, a clathrin-mediated or 67
clathrin- and caveolin-independent endocytosis occurs and, as a result the virus 68
enters in the host cell in an endosome (Skehel and Wiley, 2000, Sieczkarski and 69
Whittaker, 2002, Lakadamyali et al., 2004). During the migration from the cell 70
surface towards the nucleus, the viral and the endosomal membranes fuse. This 71
fusion is triggered by conformational changes produced in the HA by a low pH 72
environment (Cross et al., 2009).Moreover, the low pH present in the endosome 73
Lipid%envelope%derived%from%host%cell%
HA%
HA%NA%
M2%
M1%
NS2%
Ribonucleoprotein%
PB2%PA%%
PB1%
Ribonucleoprotein%
Polymerase%complex% NP% Viral%genomic%
RNA%
NS1%
PB1CF2%
N40%
PACX%
PACN155%
PACN182%
NonCstructural%and%newly%idenKfied%proteins%
M42%
A" B"
C
Generalintroduction
6
opens the M2 ion-channel creating an additional influx of protons into the virus 74
interior,which causes the disconnection of the RNPs from theM1proteins (Pinto 75
and Lamb, 2006). The RNPs are subsequently released into the cytoplasm, and 76
migratetothenucleus,wheretranscriptionandreplicationoccur.Soastoenterinto 77
thenucleus,theRNPsuseknownnuclearlocalizationsignalsthatbindtothecellular 78
nuclear import machinery (Boulo et al., 2007). As influenza A viruses possess 79
negative-strandedRNA,acomplementarycopyofitmustbemadetoobtaintheviral 80
messenger RNA (mRNA). Furthermore, vRNA does not contain 5´methylated caps 81
that are needed to complete the synthesis of mRNA (Das et al., 2010). Thus, to 82
overcomethatsituation,the5’capsofcellularhostderivedpre-mRNAareboundby 83
thePB2andcleaved-offbytheendonucleaseactivityofPAatapproximately10to13 84
nucleotides fromthecapstructure inaprocesscalled“cap-snatching” (Guilligayet 85
al.,2008,Diasetal.,2009).ThiscellularcappedRNAfragmentissubsequentlyused 86
for priming viral transcription by the viral polymerases (Reich et al., 2014). The 87
production of new vRNA requires the synthesis of an intermediate positive-sense 88
template, called complementary RNA (cRNA), which serves as template for the 89
productionofprogenyRNAsegmentsinthenucleus.AllproteinscomprisingtheRNP 90
migratefromthesiteofsynthesisinthecytoplasmtothenucleuswheretheymerge 91
with vRNA. The interaction of M1, NS2 and cellular nucleoporins prompts the 92
nuclearRNPexportintotheinnersurfaceoftheplasmamembrane(Neumannetal., 93
2000). There, the 8 gene segments are gathered together by packaging the RNP 94
segments,NS2andM1underneaththeapicalcellmembrane.Virusassemblyoccurs 95
whileHA,NAandM2arealreadyembedded in theapicalplasmamembrane. The 96
buddingofthevirusalsoinvolvesM1proteinsandseveralhostfactors(Nayaketal., 97
2009).The laststepconstitutesthereleaseofthenewlyformedvirion.Duringthis 98
process NA acts as a “razor” that enzymatically shaves the Sia residues from the 99
glycoproteinsandglycolipidspresenton thecell surfaceallowinganefficient virus 100
release(GamblinandSkehel,2010). 101
102
103
104
105
Chapter1
7
106Figure2.SchematicoverviewoftheinfluenzaAvirusreplicationcycle(adaptedfrom 107Shietal.,2014). 108 109
1.1.3 Mechanismsofevolutionandemergenceofnovelinfluenzaviruses 110
Because of the nature of their viral genome, influenza A viruses carry their own 111
polymerase genes, which lack exonuclease-proofreading capability. Therefore, 112
influenza A viruses are evolutionarily dynamic and exist as populations of 113
quasispecies(Domingoetal.,1998)withmutationratesrangingfrom1x10-3to8x 114
10-3 substitutions per site per year (Chen and Holmes, 2006). The surface 115
glycoproteins,HAandNA,arethemost importantantigens for inducingprotective 116
immunity in the host, but also the most variable genes. Mutations that change 117
aminoacids in theantigenic sitesof thoseproteinsmayallow influenza viruses to 118
escape from pre-existing immunity. Such selective mutations produced in the HA 119
andNAantigenicdomains are knownas “antigenicdrift”. It canproduce selective 120
advantagesforviralstrains,anditisthereasonwhythevirusstrainspresentinthe 121
humaninfluenzavaccinesneedtobereviewedeveryyear. 122
Due to the presence of 8 independent RNA segments in the influenza A virus 123
genome,simultaneousco-infectionofahostcellwith2ormoredifferentvirusescan 124
result in progeny viruses containing a novel combination of gene segments from 125
mRNA
vRNA (-)
cRNA (+)
Proteinsynthesis
Binding
Endocytosis Assembly
Packaging
Sia-linkedreceptors
Nucleus
FusionandUncoating
Buddingandrelease
H+
Protontransport
Generalintroduction
8
bothparentalviruses.Thisphenomenonisknownasgenetic“reassortment”.When 126
the genetic reassortment results in the emergence of a virus containing novel HA 127
and/or NA proteins it is designated as “antigenic shift” (Taubenberger and Kash, 128
2010). The combination of antigenic drift and shift allows influenza viruses to 129
constantly reinvent themselves posing a continuous threat to animal and human 130
health(LiandChen,2014). 131
132
1.2 Ecologyandpathogenesisofavian,humanandswineinfluenzaviruses 133
1.2.1 Avianinfluenzaviruses 134
Wild aquatic birds (orders Anseriformes and Charadriiformes), especially ducks, 135
shorebirdsandgullsarenaturalhostsformostinfluenzaAvirussubtypes(Sorrellet 136
al.,2007).Asmuchas82differentHAandNAcombinationshavebeenisolatedfrom 137
aquaticbirds(Olsenetal.,2006).Avianinfluenzaviruses(AIVs)primarilyreplicatein 138
thecellsoftheintestinalepitheliumofbirds,andareexcretedinhighconcentrations 139
intheirfaeces.Asaconsequence,wildbirdstransmitAIVsviathefaecal-oralroute. 140
Thus migrating aquatic birds can carry viruses between their summer and winter 141
habitats, spreading the viruses during their feeding stops (Munster et al., 2006). 142
InfluenzaAvirus infections inwildaquaticbirdsaregenerallyasymptomatic(Olsen 143
etal.,2006). 144
Land-based poultry such as chickens, turkeys, quails and pheasants are also 145
susceptible to influenza A viruses (Alexander, 2000). In these species, influenza A 146
viruses are classified into the following 2 categories based on molecular 147
characteristicsandtheabilityofthevirustocausediseaseandmortalityinchickens 148
inalaboratorysetting: 149
Lowpathogenicavianinfluenzaviruses(LPAIV): 150
Most AIVs are LPAIV. Infection of poultry with this pathotype usually causes a 151
subclinicalormilddiseasewithsymptomssuchasruffledfeathersand/oradropin 152
eggproduction.However,inchickensandturkeysLPAIVinfectionmayalsoresultin 153
respiratorydisease,sinusitisanddepression,whichcanbeaggravatedbysecondary 154
bacterialinfections.Themostfrequentlyisolatedsubtypesfrompoultryare:H1,H3, 155
H5,H6,H7andH9(Alexander,2000,PerezanddeWit,2017andSimsetal.,2017). 156
Highlypathogenicavianinfluenzaviruses(HPAIV): 157
Chapter1
9
AIVsofH5andH7subtypesmaybecomeHPAIVuponintroductionfromwildaquatic 158
birds intopoultry.AllHPAIVshave arisenby the insertionofmultiple basic amino 159
acid (aa) residues in theHAcleavagesiteof LPAIVs.Thiscleavagesite isknownto 160
process the HA precursor (HA0) to the functional components HA1 and HA2 161
(AlexanderandBrown,2009).AbasiccleavagesiteallowscleavageofHAbywidely 162
distributed cellular proteases that do not cleave LPAIV HAs. This facilitates 163
replication in a wider range of organs resulting in a more severe illness with 164
morbidityandmortality ratesup to100%.HPAIVshavebeenmainly isolated from 165
chickens and turkeys. The typical signs in those species include decreased egg 166
production, respiratory signs, lacrimation, sinusitis, cyanosisof combsandwattles, 167
oedemaof thehead, ruffled feathers,diarrhoeaandnervoussymptoms.However, 168
thesesignsarevariabledependingonthehostspeciesandage,thevirusstrain,and 169
the possible concurrence of secondary bacterial infections (Webster et al., 1992, 170
Simsetal.2017).ThemechanismsleadingtotheswitchfromLPAIVtoHPAIVviruses 171
inpoultryarenotcompletelyunderstood.Therefore,itisimpossibletopredictwhen 172
HPAIV will emerge. Nevertheless, it is assumed that the odds increase in direct 173
proportionwiththecirculationofH5andH7LPAIVswithinpoultryflocks. 174
1.2.2 Humaninfluenzaviruses 175
Influenza A viruses circulate in humans as an annually recurrent epidemic disease 176
thatcausesmillionsofhumaninfectionsworldwideandhassignificanteconomicand 177
health burdens (Medina andGarcia-Sastre, 2011).Only 3 specific subtypes (H1N1, 178
H2N2andH3N2)havereachedtheendemicstatusinhumanssince1918(Figure3) 179
(Taubenberger and Kash, 2010). The high population immunity forces human 180
influenzavirusestochangecontinuously.Changesoccurmainly intheviralHAand 181
NAandmayleadtoepidemicsandpandemics. 182
Theantigenicevolutionofcirculatingstrainscouldresultinan“antigenicdrift”that 183
causesanepidemicoutbreakaffectingmorethantheexpectednumberofpeoplein 184
acommunityorregionduringagivenperiodoftime.Whenanepidemicvirusspread 185
in human population across a whole continent or even the world it generates a 186
pandemic.Thegenerationofanovelpandemicvirusdependson2mainfactors:the 187
introductionofanantigenicallynovelHAsubtypeinthehumanpopulation,andthe 188
ability of the virus to spread efficiently between humans. Even though the exact 189
Generalintroduction
10
mechanisms bywhich pandemic influenza A viruses are generated are still poorly 190
understood,3differentmechanismshavebeensuggested.Afirstmechanismisthe 191
introduction in humans of a novel virus subtype from a different host. The 1918 192
pandemicH1N1virus,alsoknownas“Spanishflu”,isbelievedtoresultfromsuchan 193
event.ThisH1N1pandemicviruscaused≈50milliondeathsworldwide.Recentlyall 194
genesof thisviruswereshowntobeavian-like (TaubenbergerandMorens,2006). 195
ThesecondmechanismisthegenerationofaviruscontaininganovelHAand/orNA 196
subtypebygeneticreassortmentbetweenanimalandhumaninfluenzaviruses.The 197
1957 H2N2 pandemic, also known as “Asian flu”, was generated by reassortment 198
betweenanavianvirus thatdonated theHA,NAandPB1gene-segments,and the 199
thencirculatinghumanH1N1strain.Anotherexampleisthe1968H3N2“HongKong 200
flu”pandemic.Thisvirusacquiredavian-originHAandPB1gene-segmentsandthe 201
remaining 6 segments from the human H2N2 virus that was circulating in 1968 202
(TaubenbergerandKash,2010).Thethirdmechanismisthere-introductionofaHA 203
that disappeared from the human population but that has been kept in stasis in 204
anotherreservoirspecies.Throughthismechanismanearliercirculatingstrainmay 205
be re-introduced into humans from a non-human reservoir when the human 206
populationimmunityhasbecomeverylow.TheH1N1virusthatgeneratedthe1977 207
pandemic,namedthe“Russianflu”,wasmostlikelytheresultofare-introductionof 208
thehumanH1N1virusthathasbeencirculatingfrom1918upuntiltheemergence 209
ofH2N2pandemicin1957(TaubenbergerandKash,2010).Geneticanalysessupport 210
thebeliefthatthereintroductionwasduetotheaccidentalreleaseofa1950sfrozen 211
virus from a laboratory (Nakajima et al., 1978). A better example of the third 212
mechanism is the 2009 pandemic H1N1 virus (pH1N1). Interestingly, this virus 213
belongstoH1subtype,whichhasbeencirculatinginhumansfordecades.However, 214
pH1N1 HA protein is antigenically very distinct from the human-seasonal H1N1 215
virusesthatwerecirculatingin2008-09.Itcontainsa“classicalswine”H1HA,which 216
is derived from the 1918 pandemic H1N1 influenza viruses and that remained in 217
antigenicstasisinswine(seeFigure4).Consequently,peoplebornafter1950swere 218
immunologicallynaïvetopH1N1(Kashetal.,2010). 219
Chapter1
11
Inhumans, seasonal influenza infectionsare restricted to the respiratory tractand 220
are characterized by a high morbidity and low mortality. Figure 3 depicts the 221
evolutionofthepandemicinfluenzavirusesreportedsince1918. 222
223
224
225
Figure3.Originofthe4knownpandemicinfluenzaAvirusesinhumanssince1918. 226 227
1.2.3 Swineinfluenzaviruses 228
Inpigs,influenzaAvirusesareoneofthemajorcausesofacuterespiratorydisease 229
outbreaks.Although“influenzaAvirusinswine”isthenomenclaturerecommended 230
by the OIE-FAO Global Network of Expertise on Animal Influenza (OFFLU) for 231
influenzaAvirusesthatareendemicinpigs,inthisthesiswewillusethetraditional 232
designation“swineinfluenzavirus(SIV)”(OIE,2017).SIVsmaycontributetotheso- 233
calledporcinerespiratorydiseasecomplex(PRDC)inconcertwithotheragentssuch 234
as porcine reproductive and respiratory syndrome (PRRS) virus andMycoplasma 235
hyopneumniae,generatingmultifactorialdiseaseproblems.Thoughonly3influenza 236
virussubtypes(H1N1,H3N2andH1N2)arecurrentlycirculatinginswineworldwide, 237
the origins and the antigenic characteristics of these SIV subtypes are different in 238
differentregionsoftheworld(Figure4).InEurope,thefirstsignificantSIVoutbreaks 239
1918 1957 1968
H3N2pandemic“HongKongflu”
H2N2pandemic“Asian flu”
?
1977 2009Re-emergedSeasonal H1N1“Russian flu”
2009pandemicH1N1
Generalintroduction
12
occurred in1979after the transmissionofanavianH1N1virus fromwildducks to 240
pigs in Belgium and Germany (Pensaert et al., 1981). This European “avian-like” 241
H1N1virusrapidlyspreadthroughoutEurope,andnowadaysitisstillthedominant 242
subtype in many European regions (Simon et al., 2014). Later, in the 1980s, 243
reassortant viruses containing human-seasonal H3N2 surface protein genes in the 244
Europeanavian-likeH1N1backboneemergedandbecameendemicinEuropeanpigs 245
(Simonetal.,2014).Sincethemid-1990s,thoseEuropeanH3N2SIVsreassortedwith 246
a human-seasonal H1N1 virus HA generating the H1N2 virus lineage that became 247
established throughout Europe (Brown et al., 1998, Van Reeth et al., 2000). For 248
many years, those 3 SIV lineages have been co-circulating in the European swine 249
populationkeeping theepidemiological situation rather stable (Simonet al., 2014, 250
Watsonetal.,2015).However,thissituationchangedwithemergenceofthepH1N1 251
virus (Watson et al., 2015). This virus has become endemic in European pigs 252
complicating the swine influenza epidemiology in many cases due to its 253
reassortment with pre-existing H1N1, H3N2 and H1N2 SIVs (Harder et al., 2013, 254
Langeetal.,2013,Morenoetal.,2013,Roseetal.,2013,Simonetal.,2014,Lewiset 255
al.,2016). 256
InNorthAmerica theepidemiological situationwasstable fornearly80years.The 257
“classicalswine”H1N1wasthedominantsubtypeintheUSsinceitsfirstobservation 258
in 1918. However, this situation dramatically changed in 1998, when “triple- 259
reassortant” H3N2 viruses containing gene segments from the “classical swine” 260
H1N1viruses(NP,M,NS),human-seasonalH3N2viruses(PB1,HAandNA)andAIVs 261
(PB2 and PA) became successfully established in the pig populations (Zhou et al., 262
1999, Webby et al., 2000). These “triple-reassortant” H3N2 viruses co-circulated 263
with “classical swine” H1N1 viruses and subsequent reassortments led to the 264
generationandspreadof“triple-reassortant”H1N1orH1N2viruses(Karasinetal., 265
2002, Webby et al., 2004, Vincent et al., 2008). In addition, these H1 “triple- 266
reassortant” viruses also reassorted with human-seasonal H1 viruses generating 267
H1N1andH1N2reassortantscontaininghuman-originH1HA(Lewisetal.,2016).As 268
seen in Europe, the introduction of the pH1N1 virus and its reassortment with 269
endemicNorthAmericanSIVshasalsodeeplychangedswineinfluenzaepidemiology 270
(Kitikoonetal.,2013,Rajaoetal.,2016).ThepH1N1isareassortantvirusoriginated 271
Chapter1
13
inMexican swine by the combination of 2 different swine influenza viruses (SIVs) 272
circulatingintheNorthAmericaandEurope,seefigure4(Smithetal.,2009,Menaet 273
al., 2016). Indeed,novelH3N2,H1N1andH1N2viruses containingpH1N1 internal 274
geneshave spread in theUS swinepopulation andhavebeen recurrently isolated 275
from humans since 2011, raising public health concerns. These viruses with the 276
abilitytoinfecthumansweredenominatedas“variant”viruses(Kitikoonetal.,2013, 277
CentersforDiseaseControlandPrevention,2016). 278
279
280
Figure 4.Origin of the 2009 pandemic H1N1 virus. The NA andM protein-genes 281derivedfromtheEuropean“avian-like”H1N1virusandtheremainingprotein-genes 282derivedfromthoseoftheNorthAmerican“Triplereassortant”H1N2virus(adapted 283fromNeumannetal.,2009). 284 285
InSouthAmericaandAsiatheepidemiologyofSIVs isdifferentwhencomparedto 286
Europe and North America (Pereda et al., 2010, Cappuccio et al., 2011, ). For 287
instance,inAsiavirusesfromEuropeandNorthAmericahavebeenintroducedand 288
co-circulatewith local strains (Yuet al., 2009,Vijaykrishnaet al., 2010,Ngoet al., 289
2012).Inconsequence,theco-circulationofsomanygeneticallydiverseSIVshasled 290
toaverycomplexcollectionofreassortantviruses. 291
292
293
1918 1968 1977 2009European “avian-like”H1N1
2009pandemic H1N1
1984
1994 1998
“Classical”swine H1N1
NorthAmericanavian
Humanseasonal H3N2
PB1PB2
PAHANPNA
NSM
PB1PB2
PAHANPNA
NSM
PB1PB2
PAHANPNA
NSM
PB1PB2
PAHANPNA
NSM
PB1PB2
PAHANPNA
NSM
NorthAmerican“Triplereassortant”H1N2
PB1PB2
PAHANPNA
NSM
Generalintroduction
14
1.3 Interspeciestransmissionandthespeciesbarrier 294
1.3.1 Transmissionfrombirdstomammals 295
InfluenzaAvirusesinfectionsaregenerallyspecies-specific.Thereisastrongspecies 296
barrierthatrestrictsthetransmissiontonovelhosts.Actually,wildaquaticbirdsare 297
considereda reservoir for influenzaA viruses inmammals.However,AIVshave to 298
overcome that strong species barrier to become efficiently adapted tomammals. 299
The likelihoodof theseviruses tosuccessfullyovercomethatbarrierdependson2 300
maintypesofinteractions. 301
Host-hostinteractions: 302
Infections of mammals with AIVs are in most of the cases dead-end events. 303
However, if the interactions between the donor and the recipient hosts become 304
commonitwillincreasethelikelihoodofthevirusestoovercomethespeciesbarrier. 305
Forexample, the live-poultrymarkets (wet-markets)wherechickens,ducks,geese, 306
quails and other species of birds are sold for human consumption have been 307
suggestedasariskfortheintroductionofAIVsintomammals(Offedduetal.,2016). 308
Then, assuming that the virus can be transmitted between individuals of the new 309
hostspecies,infectionswilldependoninteractionsbetweenrecipienthosts. 310
Virus-hostinteractions: 311
Virus-host interactions are the steps that influenza viruses have to accomplish to 312
efficientlyinfectanewhost.First,theseviruseshavetodealwiththemucinspresent 313
in the respiratory tract secretions. Those mucins can specifically bind them 314
interfering their binding to the airway epithelial cells (Yang et al., 2014). The 315
following interaction is the successful attachment to the receptors present on the 316
host cell surface.Once theyhaveentered thecell thevirusesmust takeoverhost 317
cell processes to replicate there. Finally, theyneed to be efficiently released from 318
thecellinordertobeabletoinfectnewcellsandtospread(Kuikenetal.,2014). 319
Themechanisms for adaptation of AIVs tomammals are still largely unknown. To 320
increase the insight in influenza virus transmission 2 contrary experimental 321
approaches are mainly used. The “loss-of-function” approach consists of 322
modifications toviruseswhichhavealreadybeenadapted toanovelhost species. 323
When they do not longer transmit between different subjects, the proteins or aa 324
motifs that are essential for transmission can be identified. In contrast, “gain-of- 325
Chapter1
15
function”(GOF)experimentsaimtoachievetransmissionwithanon-adaptedvirus 326
tobetterunderstandthemechanismsforadaptation(Imaietal.,2013).Whereasthe 327
firstapproachmayincuralowerbiosafetyrisk,thesecondgivesmoreinsightsinthe 328
exactmechanismsthatruletheadaptation. 329
1.3.2 Transmissionbetweenhumansandswine 330
ZoonoticinfectionswithSIVshavebeenreportedworldwidesince1958.In1976,the 331
first large-scale case of transmission of SIV among humans occurred on amilitary 332
base in Fort Dix, New Jersey. Up to 230 soldiers were diagnosed with “classical” 333
H1N1 SIV infection using serological investigation (Gaydos et al., 2006). Later on, 334
swine-to-humantransmissioneventsweresporadic.Until2009,withtheexception 335
oftheSIVoutbreakwhichhadoccurredatFortDix,therewas limitedevidencefor 336
person-to-personspreadofswine-originviruses(KruegerandGray,2013).Thenthe 337
2009 pH1N1 virus appeared and spread worldwide, being the first pandemic 338
influenzavirusofprovenswineorigin(Menaetal.,2016).Furthermore,thepH1N1 339
reassorted with other endemic SIVs generating viruses with the ability to infect 340
humanssuchastheH3N2vthathasbeenisolatedfrom402humansintheUSsince 341
2011 (World Health Organization, 2017a). The emergence of the pH1N1 virus 342
dramatically changed the understanding of SIV ecology and cross-species 343
transmissionbecauseitwasefficientlytransmittedfromswine-to-humans,butalso 344
fromhumans-to-swine. 345
A recent studydemonstrated that transmission fromhumans-to-swine is farmore 346
frequent than the other way around (Nelson and Vincent, 2015). Actually, 347
reassortant viruses containing human-origin gene segments have become 348
widespread in swine worldwide (Watson et al., 2015, Lewis et al., 2016). For 349
instance, the European H3N2 SIVs were generated upon the spread and 350
reassortment of a human-seasonal H3N2 “Port Chalmers/75-like” virus (Brown, 351
2000).However,thelackofsurveillancemakesitdifficulttoaccuratelyestimatethe 352
extentoftransmissionofhuman-seasonalvirusestoswineinthefield.Experimental 353
studies also confirm the susceptibility of pigs to human viruses. In fact, the 354
inoculationofpigswithahumanH3N2orareconstituted1918pandemicH1N1virus 355
resultedintransientfever,mildrespiratorysignsandnasalvirusexcretion.However, 356
thoseviruseswerenotable toachieveefficientpig-to-pig transmission (Landoltet 357
Generalintroduction
16
al., 2006, Weingartl et al., 2009). Although it has been demonstrated that 358
transmissionofinfluenzaAvirusesbetweenpigsandhumansoccurs,untilnowonly 359
thepH1N1hasbeendemonstratedtobeabletoefficiently transmitandspread in 360
bothspecies. 361
1.3.3 Avianinfluenzaviruseswithpandemicpotential 362
Between1959and1996only3casesofAIVsinfectinghumanshavebeenreported. 363
Consequently,AIVswereconsideredasalowriskforhumanhealth.However,since 364
thefirst isolationofanH5N1HPAIVinHongKongin1997,AIVsbecameacauseof 365
concern. Furthermore, the number of zoonotic AIV infections has dramatically 366
increased during the last decade. This section will highlight the avian influenza 367
subtypes with the highest pandemic potential according to the World Health 368
Organization(WHO)(WorldHealthOrganization,2017a). 369
H5Subtype 370
Sofar,onlyinfluenzaAH5N6andH5N1virussubtypeshavebeenreportedtoinfect 371
humans (Yang et al. 2015, World Health Organization, 2017a). Since 2014, H5N6 372
HPAIVs havebeen sporadically isolated fromhumans in China, but the number of 373
cases and their geographical spread remain very limited. In contrast, the H5N1 374
HPAIVsareontopofthelistofavianinfluenzasubtypeswiththepandemicpotential 375
(World Health Organization, 2017a). As mentioned, the first human outbreak 376
producedbyH5N1HPAIVsoccurred inHongKong in1997, causing severedisease 377
withhighfatalityrates(around60%)(Claasetal.,1998,Subbaraoetal.,1998).This 378
H5N1viruswas indeeda reassortantviruscontainingaH5HAderived fromgoose 379
H5N1virusandtheremaininggenesfromH9N2andH6N1virusesprevalentinquail, 380
as shown in Figure 5 (Guan et al., 1999, Hoffmann et al., 2000). Wet markets 381
appearedtobethesourceofthehumaninfection(Shortridge,1999).Althoughthe 382
outbreakwasabortedwiththecullingof1.5millionpoultryinfarmsandmarketsof 383
HongKong,H5N1HPAIVwasagainreportedinhumansinHongKongin2003(Peiris 384
etal.,2004). 385
386
Chapter1
17
387
Figure5.OriginandsourcesfortheH5N1HPAIV. 388
389
Later, human cases have also been reported in Vietnam, Thailand, Cambodia, 390
Indonesia,Turkey, Iraq,Azerbaijan,Egypt,DjiboutiandNigeria (Peirisetal.,2007). 391
As ofMay 2017 a total of 859 human cases have been reported worldwide, 453 392
(52.8%) of whom died (World Health Organization, 2017b).Most of those human 393
cases were associated with direct contact with infected poultry and efficient 394
transmissionwithin humanswas never reported.However, the evolution ofH5N1 395
viruses is quick and its direction is still unclear. Significant genetic and antigenic 396
evolution has occurred (Guan et al., 2004), involving antigenic drift in the HA, 397
mutations inothergenes,andreassortmentwithotherAIVs (Chenetal.,2006,de 398
Vriesetal.,2015).Eventhoughmammalianadaptationmarkershavebeendetected 399
insomehumanandavianHPAIVH5N1isolates,andhuman-to-humantransmission 400
betweenfamilymembershasbeenreported,thereisnosustainedhuman-to-human 401
transmission(Ungchusaketal.,2005,Wangetal.,2008). 402
In order tounveil the requirements for transmissionofH5N1HPAIV in humans, 2 403
differentGOF approacheswere tested in ferrets, as they are considered themost 404
important animal model for influenza transmission studies (Belser and Tumpey, 405
2017). In the first approach, Herfst et al. demonstrated efficient airborne 406
transmission between ferrets of an avian H5N1 HPAIV containing only 5 aa 407
substitutions.ThosemutationswerepresentintheHAandthePB2gene-proteins,3 408
outof5wereartificiallyintroducedinthevirusandtheremaining2emergedafter 409
Quail& H9N2&virus&
Quail& H6N1&virus&
H5N1&virus&Goose&
Domes6c&chicken& H5N1&HPAIV&virus&
Se@ng:&Habitats'shared'by'wild'and'domes2c'birds'and/or'live'bird/poultry'markets'
Generalintroduction
18
10passagesand3 transmissionexperiments (all aamutationsmentioned in those 410
adaptationexperimentswill bedescribed inChapter1.4) (Herfst et al., 2012). The 411
secondapproachevaluatedifH5N1virusescouldbecomeairbornetransmissiblein 412
ferrets upon reassortment with contemporary human influenza viruses, with or 413
without mammalian adaptation substitutions. On one hand, Imai et al. achieved 414
efficientairbornetransmissionto4outof6contactferretsaftertheintroductionof 415
4 aa mutations in the HA of a reassortant virus containing H5 HA in the pH1N1 416
backbone(Imaietal.,2012).Ontheotherhand,Chenetal.reachedpartialairborne 417
transmission in ferretsby introducingkeypointmutations in theH5HARBSanda 418
human-seasonal H3N2 NA gene (Chen et al. 2012). In summary, these studies 419
demonstrate that H5N1 viruses can become adapted to ferrets in the laboratory 420
eitheruponreassortmentwithhuman-adaptedviruses(withtheneedforadditional 421
substitutions)oronlybytheintroductionofcertainkeyaamutations. 422
H7subtype 423
Morethan100humaninfectionswithH7N1,H7N2,H7N3andH7N7subtypeshave 424
been reported in Australia, Canada, Italy, Mexico, the Netherlands, the United 425
Kingdom and the US. Most of these infections were associated with poultry 426
outbreaksandresulted inconjunctivitisandmildupperrespiratorysymptomswith 427
the exception of 1 death that occurred in the Netherlands (Fouchier et al., 2004, 428
Stegemanetal.,2004,Nguyen-Van-Tametal.,2006,Ostrowskyetal.,2012). 429
On March 2013, a novel influenza H7N9 LPAIV crossed the species barrier and 430
caused for the first time human infections in China (Gao et al., 2013). Until May 431
2017, 1486 laboratory-confirmed human infections, including at least 571 deaths 432
(40.1%),havebeenreportedinChina(WorldHealthOrganization,2017a).Thisvirus 433
waslikelytransferredfromdomesticduckstochickensbefore“jumping”tohumans 434
anditwastheresultofthereassortmentofatleast4originviruses.Thisresultedin 435
anH7N9viruswithaduckoriginH7HA,aduckorigin(probablyalsowildbird)N9NA 436
and2differentchickenoriginH9N2virusesinternalgenes,seeFigure6(Lametal., 437
2013,Liuetal.,2013). 438
439
Chapter1
19
440
Figure 6. Origin and sources for the H7N9 influenza virus. (Adapted from Dan 441Higgins/PHILCDC). 442
443
Epidemiologicalevidencehaslinkedhumancaseswithexposuretolive-birdmarkets 444
(Shi et al., 2013). The closure of these markets, with the likely assistance of 445
environmental factors, helped to control the first wave of the outbreak from 446
FebruarytoMay2013(Yuetal.,2014).Nevertheless,publichealtheffortsfailedto 447
completely stop the spread of humanH7N9 infections and up to now 5 epidemic 448
waves have been recorded. Although sporadic H7N9 virus transmission among 449
family members has been reported, the likelihood of sustained human-to-human 450
transmissionislow(Qietal.,2013,WorldHealthOrganization,2017a). 451
Soonafteritsemergenceinhumans,severalstudiesevaluatedairbornetransmission 452
ofavian-originH7N9virusesamongferrets(Richardetal.,2013,Belseretal.,2013, 453
Zhuetal.,2013,Kuetal.,2014,Siegersetal.,2014). Interestingly,despitethefact 454
thatmutations associatedwith increasedhumanadaptationwerepresent inmost 455
humanH7N9virusisolates,efficienttransmissioninferretswasneverachieved(Luk 456
etal.,2015,NeumannandKawaoka,2015).Hence, it isstillunclearwhetheravian 457
H7N9virusescouldbecomeadaptedtomammals.However,aslongasthoseviruses 458
continuecirculatinginpoultry,humancasescanbeexpected. 459
H9N2subtype 460
H9N2 AIVs are endemic in wild waterfowl worldwide. This subtype has become 461
endemic in different land-basedpoultry species acrossmultiple Eurasian countries 462
Se#ng:'Habitats'shared'by'wild'and'domes2c'birds'and/or'live'bird/poultry'markets'
Generalintroduction
20
(Guanet al., 1999, Sun et al., 2010, Sun and Liu, 2015,VanBormet al., 2016). In 463
1999, H9N2 viruses were first isolated from humans in Hong Kong (Peiris et al., 464
1999).Since then,only29humancaseshavebeenreportedandhuman-to-human 465
transmission has never been described (WorldHealthOrganization, 2017a). These 466
infectionswereassociatedwithpoultryoutbreaksandmainlyresultedinmildupper 467
respiratory symptoms. Strikingly, a seroprevalence for H9N2 antibodies between 468
2.1%and13.7%hasrecentlybeenidentifiedamongpoultryslaughterhouseworkers 469
inChina(Huangetal.,2013).This findingsuggeststhatH9N2infections inhumans 470
gofrequentlyunreported.Asamatteroffact,mostoftheH9N2AIVscirculatingin 471
poultrypossessanaasubstitutionintheHARBSthatwasdescribedasamolecular 472
markerforhumanadaptation,therebyincreasingthepossibilityofthesevirusesto 473
infecthumans(Matrosovichetal.,2001,WanandPerez,2007).Interestingly,these 474
viruseswerealsorecurrentlyisolatedfrompigsinChinasince1998. 475
As in H5 and H7 experimental studies, different approaches were also tested to 476
evaluatepotentialofH9N2virusestobecomeadaptedtomammals.In2008,Wanet 477
al.evaluatedvirusreplicationandtransmissioninferretsofdifferentH9N2wild-type 478
virusestogetherwithareassortantcontainingH9N2HAandNAinahuman-seasonal 479
H3N2backbone.Althoughthereassortantvirusreplicatedbetterthanthewild-type 480
viruses,efficient transmissionwasnot reached (Wanetal., 2008). Later,upon ten 481
passagesof this reassortant in ferrets thevirusacquired2aamutations in theHA 482
and1intheNAthatwererelatedwithefficientairbornetransmission(Sorrelletal., 483
2009).Inalaterexperimentthesamegroupdemonstratedtestedthecompatibility 484
ofthepreviouslyusedwild-typeH9N2withpH1N1internalgenes.Interestingly,this 485
reassortantvirusshowedefficientairbornetransmission in ferretswithnoneedof 486
adaptive mutations. Moreover, this experiment also suggested that an optimal 487
balancebetweenHAandNAactivity is required forefficientairborne transmission 488
(Kimble et al., 2011). In addition, in 2013 different H9N2 strains were tested for 489
replicationandtransmission inducks,pigs,miceandferrets inordertoestablisha 490
rankingofH9N2virusesthathavethehighestpandemicpotential.Attheendofthe 491
experiment, thehumanH9N2 virus isolateswereon topof the list, but still those 492
isolateswerenotasfitasthemammalian-adaptedstrains(TheSJCEIRSH9working 493
group,2013).Insummary,efficientH9N2adaptationtoferretshasbeenachievedby 494
Chapter1
21
reassortment with human-adapted viruses (with or without adaptive mutations). 495
However, unlike in the H5N1 subtype, aa mutations that could confer efficient 496
adaptationtothosevirusesarestillunknown. 497
498
1.4 Molecularmechanismsofhostrangerestriction 499
1.4.1 Theroleofthehemagglutinin 500
ThemainSiasrecognizedbytheHAaretheN-acetylneuraminicacid(Neu5Ac)andN- 501
glycolylneuraminicacid(Neu5Gc)(Matrosovichetal.,2015).InfluenzaAvirusescan 502
recognize the Sias bound to galactose in either anα2,3- orα2,6-glycosidic linkage 503
(Connoretal.,1994,Matrosovichetal.,2009).Theselinkagesareveryimportantfor 504
thespecificityof theHAmolecules in thebinding tocell surfacesialicacids.While 505
AIVs demonstrated highest preference for the α2,3Gal-linked Sias (Siaα2,3Gal), 506
humanandswinevirusespreferentiallybindα2,6Gal-linkedSias(Siaα2,6Gal)(Skehel 507
and Wiley, 2000). The RBS is a shallow pocket placed in the HA1 that mediates 508
bindingtoSiaspresentonhostcellglycoproteinsandglycolipids(Matrosovichetal., 509
2015).TheaaresiduesmakingupthisRBSshowedtobeveryconservedandsimilar 510
regardlessofthevirussubtypestudied(Figure7). 511
512Figure7.Surface representationof theRBS in theHA1monomerofA/quail/Hong 513Kong/G1/1997 (H9N2) bearing an α2,6-linked sialic acid. The major structural 514components of the RBS are represented with the following colours: the central 515bottominblue(aminoacids(aa)98,153,183and195),therearinyellow(aa155, 516190and194),therightsideinpurple(aa134until138)andtheleftsideinred(aa 517224until228)(SkehelandWiley,2000). 518
Α2,6Gal(linked.Sialic.acid..
Generalintroduction
22
Therefore, aa changes in and around the RBS can dramatically alter the receptor 519
binding preference of influenza viruses. Thus, a change in the receptor-binding 520
preference of an influenza virus owing tomutations in the RBS is inmost cases a 521
prerequisiteforovercomingthespeciesbarrier.ForH2andH3HAs,thesubstitutions 522
HA-Q226LandHA-G228S(allaapositionsarereferredtoH3numbering)havebeen 523
describedtoconferacompleteswitchfromSiaα2,3GaltoSiaα2,6Galbinding(Vines 524
et al., 1998). Furthermore, in H9 and H7 subtypes HA-Q226L substitution also 525
increasedSiaα2,6Galbinding invitro(WanandPerez,2007,Liuetal.,2014).Inthe 526
caseofH1HAs, theHA-E190Dand theHA-D225G substitutionsare critical for the 527
shift fromavian tohuman receptor recognition (Glaseretal.,2005,Stevensetal., 528
2006).However,themechanismsthatrulethespecificaathatdeterminereceptor- 529
binding specificity within the RBS are not completely understood as described 530
mutations vary among the different HA subtypes or even virus strains. Table 2 531
summarizes the main RBS aa mutations described as important for mammalian 532
adaptationofdifferentHAsubtypes. 533
TheHAproteinalsomediates the fusionof theviralenvelopewith theendosomal 534
membrane derived from the host cell. This fusion is influenced by the HA heat 535
stabilityandthepHatwhichHAexperimenttheconformationalchangeswithinthe 536
endosome.Different influenzaviruseshavedifferentactivationpHvalueranges, in 537
avianvirusesitrangesfrom5.5to6.0whileinhumanvirusespHvaluesrangesvary 538
from5.0to5.5(Russieretal.,2016).RecentstudiesdemonstratedthatHAstability 539
played a role in H5N1 virus adaptation in ferrets (Imai et al., 2012, Herfst et al., 540
2012). Inparticulartheauthorsfoundthatmutations intheHARBSthat improved 541
human-type receptor binding had an unfavourable effect in HA stability that 542
required compensatory mutations to offset the virus fitness. Those results were 543
further supported by the description of 8 aa substitutions that emerged after 544
adaptationofEuropeanavianlikeH1N1virusintoswineandthatinducedchangesin 545
HA stability (Baumann et al., 2015). Collectively, these findings suggest that the 546
optimumpHforfusionandHAstabilitymaybecriticaltoensurevirustransmission 547
inanewhost. 548
549
550 551
Chapter1
23
Table2.VirusmutationsintheHARBSthathavebeendescribedaskeymarkersfor 552theadaptationofavianinfluenzaAvirusestomammals. 553
554
1.4.2 Theroleoftheneuraminidase 555
TheNAenzymaticactivityisessentialfortheoptimalreleaseofthevirusesfromhost 556
cells, aswell as for the escape fromextracellular inhibitors and the prevention of 557
self-aggregation(Colman,1994,Yangetal.,2014).Manystudiessuggestedthatan 558
optimalbalancebetweentheHAandtheNAactivitiesisrequiredforefficientvirus 559
replicationandtransmission(Wagneretal.,2002).Thisbalancecanbedisturbedby 560
reassortment or transmission to a new hostwith a different set of receptors. For 561
instance, theNAproteins ofmost influenza viruses havehigher enzymatic activity 562
MutationVirus
subtype
Modelused
inthestudy
Effectoftheaasubstitutiononthevirusreceptor
preferenceReferences
A138S H1N1H5N1
MutationpresentinearliestH1N1humanisolatesandinH5N1subtypeaftertenpassagesinferrets.IncreasedaffinityforSiaα2,6Gallinkages.
Matrosovichetal.,1997Herfstetal.,2014
E190D/N/V H1/H9N2
CrucialforadaptationofH1subtypetomammals(togetherwithG225D).Importantmarkerfor
mammalianadaptationofH9N2(togetherwithQ226L).
Matrosovichetal.,2000Cauldwelletal.,2014Lakdawalaetal.,2015
N224K H5N1
IncreasedtheaffinityofH5N1forSiaα2,6Gal(togetherwithQ226L). Imaietal.,2012
D225G H1/H9N2
CrucialforadaptationofH1subtypetomammals(togetherwithE190D).Relatedwithhighervirulencein
humanpatientsandwithenhancedaffinityforSiaα2,3Gal.
Matrosovichetal.,2001Liuetal.,2010Lakdawalaetal.,2015Iovineetal.,2015
Q226L
H3N2H2/H9N2H7N9H5N1
CrucialforadaptationofH3N2subtypetomammals(togetherwithG228S).IncreasedtheaffinityofH5N1,
H7N9andH9N2forSiaα2,6Gal.
Connoretal.,1994Matrosovichetal.,2000WanandPerez,2007Herfstetal.,2012Imaietal.,2012Chenetal.,2012Lametal.,2013Liuetal.,2014
S227N H5N1
IncreasedSiaα2,6Galaffinity. Yamadaetal.,2006
G228SH3N2H2/H5N1
EnhancedSiaα2,6Galaffinity(togetherwithQ226L).
Connoretal.,1994Suzukietal.,2000Herfstetal.,2012Chenetal.,2012
*Nonpolarhydrophobicaa:glycine(G),alanine(A),valine(V),leucine(L),phenylalanine(P),tyrosine(Y)andisoleucine(I).Polarhydrophilicaa:serine(S),threonine(T),asparagine(N),glutamine(Q).Negativelychargedaa:asparticacid(D),glutamicacid(E).Positivelychargedaa:lysine(K),arginine(R),histidine(H).
Generalintroduction
24
againstSiaα2,3Gal,whichmaybeexplainedbytheneedtoovercometheSiaα2,3Gal 563
richmucinsintheupperrespiratorytract(URT)(Richardetal.,2014).However,the 564
human derived N2 and the pH1N1 N1 NAs showed enhanced activity against 565
Siaα2,6Gal that balances the marked Siaα2,6Gal HA activity (Kobasa et al., 1999, 566
Garciaetal.,2014).Additionally,bypassagingreassortantviruseswithavianHAand 567
humanNAitwasshownthatreplicationwasreconstitutedwhenmutationsoccurred 568
in HA and/orNA,which led to a functional balance (Shtyrya et al., 2009). On the 569
otherhand,Herfstetal.andKimbleetal.describedefficienttransmissioninferrets 570
ofH5N1andH9N2virusescontainingpurelyavianNAs(Herfstetal.,2012,Kimbleet 571
al.,2013).ThesefindingssuggestthatadaptationofNAisnotmandatorytoachieve 572
mammalianadaptationofAIVs.However,theexactnatureofthechangesintheNA 573
that are needed to achieve efficient adaptation of AIVs to humans is not fully 574
understood. 575
1.4.3 Theroleofthepolymeraseproteinsandtheinternalgenes 576
The PB2 gene, in particular the aa residue 627, has been appointed as the main 577
polymerase determinant for influenza adaptation to a new host (Subbarao et al., 578
1993, Cauldwell et al., 2014). Mammalian viruses typically contain PB2-627K 579
whereas avian viruses possess PB2-627E. The presence of PB2-627E restricts 580
replication at 33o C, the temperature of the humanURT. (Cauldwell et al., 2014). 581
Sculletal.(2010)foundthatthePB2-K627Eresultedinrestrictedreplicationofthe 582
virusinhumanrespiratoryepithelialcells,regardlessofthetemperatureconditions 583
(32oCversus37oC).Interestingly,thepH1N1didnotcontainthismutation.However, 584
tocompensateforthelackofPB2-627K,pH1N1containsthePB2-Q591Ksubstitution 585
(Moncorge et al., 2013). In addition to those mutations, PB2-D701N was 586
demonstratedtoenhancethebindingofPB2toimportinα(Moncorgeetal.,2010). 587
The importinα is responsibleprotein for themigrationof thePB2 to thenucleus, 588
thusthePB2-D701Nsubstitutionimprovesnuclearlocalizationofthepolymerasein 589
mammaliancells. 590
Thecompleteinternalgenecassettehasbeenalsodemonstratedtoberelevantfor 591
the adaptation of avian viruses to mammals. In 2009, Sorrell et al. obtained an 592
efficient ferret-to-ferret droplet transmission of the reassortant H9N2 virus 593
containinghuman-seasonalH3N2 internalgenesaftertenserialpassages in ferrets 594
Chapter1
25
(Sorrelletal.,2009).Additionally,since2011,H3N2vandH1N2vvirusescontaininga 595
pH1N1-origin M gene have been recurrently isolated from humans (Centers for 596
Disease Control and Prevention, 2016). This finding put the attention on those 597
viruses and their “quadruple-reassortant” internal genes. Moreover, while pH1N1 598
viruses efficiently transmit among ferrets, a reassortant possessing the North 599
American“triple-reassortant”originNAandMgenesvirusisolatedfromhumansdid 600
nottransmittoallexposedferrets(Lakdawalaetal.,2011).Italsosuggestedthatthe 601
European“avian-like”swineNAandMgenescontributedtotheimprovementofthe 602
transmissionof theseviruses,at least intheferretmodel.This isconsistentwitha 603
study that demonstrated the importance of the pH1N1 NA and M genes for 604
replicationandtransmissionofH9N2reassortantvirusesinpigs(Maetal.,2012).In 605
more recent studies performed in pigs and ferrets reassortant viruses containing 606
avianH9N2orH5N1HA andNA in the pH1N1backbone, an improved replication 607
andtransmissionwasdemonstratedwhencomparedtotheparentalviruses(Qiaoet 608
al.,2012,Abenteetal.,2016,Imaietal.,2012).Tosumup,thepeculiarcombination 609
ofinternalgenesthatispresentinthepH1N1maybeanadvantagefornovelHAand 610
NAgenestoovercomethespeciesbarrier. 611
612
1.5 Adaptationofavianinfluenzavirusestoswine 613
1.5.1 Naturalinfectionofpigswithavianinfluenzaviruses 614
Infections of pigswith AIVs in nature have been recurrently documented. In fact, 615
“avian-like”H1N1virusesemergedfrombirdsandbecameendemicinpigsinEurope 616
and Asia (Lewis et al., 2016). Since 1998, H9N2 influenza viruses have been 617
recurrentlyisolatedfrompigsinHongKongandChina.Thissubtypeis,togetherwith 618
“avian-like” H1N1, the AIV subtypemost frequently reported in pigs (Peiris et al., 619
2001,Yuetal.,2008b,Yuetal.,2011).Inaddition,recentserologicalstudiesinpigs 620
revealedaprevalencefrom4.9%upto15.6%forantibodiesagainstH9N2virusesin 621
slaughterhousesinChina(Lietal.,2015,Wangetal.,2016).H1N1,H3N3andH4N6 622
waterfowl-origin influenzaviruseswere recovered inpig farms inCanadabetween 623
1999 and 2002 (Karasin et al., 2000a, Karasin et al., 2000b, Karasin et al., 2004). 624
Although serological data suggested transmission of H4N6 between pigs, the 625
infection was not sustained (Karasin et al., 2000b). In China, an H10N5, probably 626
Generalintroduction
26
originatingfromduckswasisolatedin1pigattheslaughterhousein2010(Wanget 627
al.,2012).HPAIVhavebeenalsoreportedtoinfectpigsinnature.In2003,whilean 628
H7N7 caused outbreaks in poultry in the Netherlands, anti-H7N7 antibodies were 629
detected inpigshoused close to infectedpoultry farms (Loeffenet al., 2004). The 630
source appeared to be the feeding of the pigs with broken eggs from infected 631
poultry. Furthermore, during the H5N1 outbreaks in South-Eastern Asia, these 632
viruseswere isolated frompigs in IndonesiaandChina (Zhuetal.,2008,Shietal., 633
2008, Takano et al., 2009). In addition, serological investigations in Vietnamese 634
slaughterhouses located in the neighbourhood of H5N1 HPAIV reported areas, 635
confirmedalimitednumberofanimals(8outof3175)tobeseropositive(Choietal., 636
2005). 637
Reassortantvirusescontainingavian-origingeneshavebeenalsoisolatedfrompigs 638
worldwide. Actually, European H3N2 and H1N2 subtypes, North American “triple- 639
reassortant” and pH1N1 viruses contain avian-origin genes (Watson et al., 2015, 640
Lewis et al., 2016). In addition, other reassortant viruses containing avian-origin 641
genes have been isolated from swineworldwide but only as dead-end infections. 642
H3N1 viruses containing human-originH1N1 (NA), turkey-originH3N2 (HA andM) 643
and the remaining genes of swine-origin were isolated in North America, 2003 644
(Lekcharoensuk et al., 2006). Later, in 2006,H2N3 reassortantswere also isolated 645
(Maetal.,2007).In2008,2H5N2influenzaviruseswereisolatedfrompigsinSouth 646
Korea.Thegenomeof1 isolatewasentirelyofavianorigin,while theotherwasa 647
reassortant virus containing swine-origin H3N1 PB2, PA, NP andM genes and the 648
remaininggenesfromanavianH5N2virus(Leeetal.,2009).Thesourceofinfection 649
ofpigswiththeAIVsremainedunclearinmostcases.Onlyin2casessurfacewater 650
usedforwateringoftheanimalswasthesuspectedroutefortheintroductionofthe 651
avianvirusesintothepigs(Karasinetal.,2000a,Maetal.,2007). 652
InmostcasestheinfectionofpigswithAIVsresultedsubclinicalorverymilddisease 653
(Karasinetal.,2000a,Karasinetal.,2004,Xuetal.,2004,Maetal.,2007,Congetal., 654
2008,Leeetal.,2009).However,aspointedbytheserologicalstudiessomenatural 655
infectionswithAIVmaybeclinicallyunnoticedinpigs(Peirisetal.,2001,Choietal., 656
2005,Wangetal.,2016).So far,mostof thewhollyAIVs isolated inpigswerenot 657
abletoestablishthemselvesinswinepopulations(Guanetal.,2009,Yuetal.,2009), 658
Chapter1
27
buttherecurrenttransmissionofthosevirusestopigsmayincreasetheriskforthe 659
adaptation. 660
1.5.2 Experimentalinfectionsofpigswithavianinfluenzaviruses 661
ExperimentalstudiesalsodemonstratedthesusceptibilityofpigstoAIVS.Infact,the 662
first experimental evidence of the susceptibility of pigs to LPAIV was provided in 663
1981. After intranasal inoculationwith several H1N1,H2N3,H3N2 andH7N7AIVs 664
pigsdidnotshowclinicaldiseasesymptoms,butnasalvirussheddingwasreported 665
for up to 7 days post-inoculation (dpi) (Hinshaw et al., 1981). Those results were 666
confirmedlater,in1994byKidaandcolleagues.Twentynine(includingatleast1of 667
eachHA subtype, fromH1 toH13)outof 38 LPAIV fromvariouswildbird species 668
were shed up to 7 dpi and induced a serological response in pigs after intranasal 669
inoculation. Again, the infectionwas subclinical. These results suggested that pigs 670
could be susceptible to AIVswith non-mammalianHA subtypes.Moreover, itwas 671
evidencedthatcombinedinfectionofpigswithH1N1SIVandH3N8AIVresultedin 672
thegenerationofanH3N1reassortantwithefficientreplicationinswine(Kidaetal., 673
1994). 674
Later, since the isolation of H5 andH7HPAIVs from humans, the susceptibility of 675
swinetothoseviruseswasexaminedbyevaluatingwhetherthosevirusesmaypose 676
ariskforthepigpopulation.For instance,rightaftertheH7N7HPAIVwas isolated 677
from poultry in Belgium and the Netherlands in 2003, Loeffen and colleagues 678
inoculatedpigsintranasallywiththevirusleadingtovirusreplicationandshedding. 679
However,pigsdidnotget sickand thevirusdidnot transmitatall (Loeffenetal., 680
2004). Similar resultswere obtained in experimental infections of pigswith H5N1 681
HPAIVs isolated fromVietnamandThailand (Choietal.,2005).Finally,Lipatovand 682
colleagues infected pigswith 4H5N1HPAIV isolates fromhumans and birds from 683
Asia in 2003-2005 (Lipatov et al., 2005). The intranasal inoculation provokedmild 684
weightlossandnasalsheddingwith3outof4viruses.Ontheotherhand,intranasal 685
inoculation of pigs with the wholly avian-origin and the avian-swine reassortant 686
H5N2viruses isolated frompigs inKorea in2008 resulted inmild symptoms.Both 687
viruseswereshedforatleast5days,butonlythereassortantviruswastransmitted 688
to contact pigs. This suggests that swine-origin internal genes may give LPAIVs a 689
selectiveadvantagethatfacilitatespig-to-pigtransmission(Leeetal.,2009). 690
Generalintroduction
28
In2009,theemergenceofpH1N1virusboostedresearchontheadaptationofAIVs 691
to swine. That year, De Vleeschauwer et al. demonstrated pig susceptibility to 692
differentAIVsubtypes.However,thosevirusesdidnotinducediseaseandwerenot 693
transmitted (De Vleeschauwer et al., 2009). In addition, due to their recurrent 694
isolationsfromhumansandpigs,avianH9N2viruseswerealsoproposedtohavethe 695
potentialtobecomeadaptedtopigs.Forinstance,severalinvivoandinvitrostudies 696
demonstratedthatthosevirusespossessinternalandsurface-geneproteinsthatare 697
compatible with other subtypes genes. This increases the possibility of the 698
generationofa transmissibleviruswithanovelHAandNA, towhichhumansand 699
swine are immunologically naïve. In experimental conditions pigs were also 700
susceptibletoH9N2viruses.UnlikeinferretstheHA-Q226Lmutationdidnotseem 701
tobe restrictive (TheSJCEIRSH9WorkingGroup,2013,Wangetal.,2016). Like in 702
other hosts the wild-type H9N2 influenza viruses showed very limited pig-to-pig 703
transmission. Though other reassortment experiments demonstrated that pH1N1 704
internalgenesincreasedtransmission, itwasmuchlessefficientthanthatofswine 705
adaptedH1N1viruses.Interestingly,reassortantvirusescontainingH9N2HAandNA 706
in the backbone of pH1N1 demonstrated higher transmission efficiency when 707
comparedtotheoriginalH9N2(Qiaoetal.,2012,Obadanetal.,2015).Thisfinding 708
suggeststhatpH1N1internalgenesmayposeanadvantageintheadaptationofAIVs 709
to pigs. This hypothesis was also tested in pigs infected with reassortant viruses 710
containing H5N1 HA and NA and pH1N1 internal genes. This reassortant also 711
presentedenhancedreplicationwhencomparedtotheparentalH5N1.However,the 712
transmission efficiency was still lower when compared to that of endemic SIVs 713
(Abenteetal.,2016). 714
WhileH7N9viruseshadneverbeenisolatedfrompigsinnature,thoseviruseswere 715
able to replicate in the swine URT after experimental inoculations. After their 716
introduction in pigs those viruses readily changed, but efficient transmission was 717
neverachieved(Zhuetal.,2013,Liuetal.,2014,Siegersetal.,2014,Xuetal.,2014). 718
Insummary,asseeninfield,pigsaresusceptibletoabroadrangeofAIVsalsounder 719
experimentalconditions.Nevertheless,thechangesthatarerequiredtomakethose 720
virusestransmitefficientlybetweenpigsarenotknown. 721
722 723
Chapter1
29
1.5.3 Theroleofthepigintheadaptationofavianinfluenzavirusestomammals 724
Fora longtime,swinehavebeenconsideredtoplayaroleasa“mixingvessel”for 725
avianandhuman influenzavirusesoras intermediatehost for the transmissionof 726
AIVstohumans.ThishypothesiswasfirstenunciatedbyScholtissekandcolleagues. 727
ItwasbasedontherescueofatemperaturesensitiveNPmutantofanavianH7N1 728
virus by co-infections of chicken embryo fibroblasts with either avian, human or 729
swineH3N2isolates.TheyfoundthattheviruscouldberescuedbyallAIVs,bynone 730
of thehumanvirusesandby2outof10SIVs.Thus, theyproposed that theNPof 731
SIVshadabroaderhostrangeregardingitscompatibilityinreassortantvirusesand 732
thatpigswereapotential “mixingvessel” for thegenerationof thosereassortants 733
(Scholtisseketal.,1985).This“mixingvessel”hypothesiswasfurthersupportedby 734
thesubtypesimilaritiesbetweenswineandhumaninfluenzaviruses,thefindingthat 735
pigscouldbesimultaneouslyinfectedwithSIV,AIVand/orhumaninfluenzaviruses 736
and the frequent isolation of reassortant viruses from pigs (Kida et al., 1994). In 737
1998, Ito et al. gave molecular support to this hypothesis demonstrating the 738
presence of both Siaα2,3Gal and Siaα2,6Gal receptors in the pig trachea. 739
Furthermore, they demonstrated that some “avian-like” SIV acquired molecular 740
traitsofhumanadaptationbycontinuousreplicationinpigtrachealexplants(Itoet 741
al., 1998). Therefore, the pigmay also act as an intermediate host in which AIVs 742
mightgainmammalianadaptationtraits.However, laterstudiesdemonstratedthat 743
thepresenceofbothSiareceptorswasrestrictedtothebronchiolesandalveoliand 744
that the distribution of Sia receptors in swine resembles the one of human and 745
ferrets(vanRieletal.,2007,VanPouckeetal.,2010,Nellietal.,2010,Trebbienet 746
al.,2011). 747
The emergenceof the 2009pH1N1 virus in swine increased the concern again on 748
pigs as source for pandemic viruses (Smith et al., 2009, Mena et al., 2016). 749
Nowadays,thishypothesishasstillnotbeenprovenandtheexactroleofthepigin 750
the generation of pandemic viruses remains unknown.Nevertheless, the pig is an 751
importantnaturalhostforinfluenzaAvirusesandduetoitssimilaritieswithhumans 752
in terms of anatomy, physiology and immunology they may be an incomparable 753
animalmodeltobetterunderstandtheadaptationofAIVstomammals. 754
755
Generalintroduction
30
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AvianH9N2InfluenzaVirusesinPigs.JVirol,90(7),3506-14. 1350 1351Wang,N.,Zou,W.,Yang,Y.,Guo,X.,Hua,Y.etal.(2012).Completegenomesequenceofan 1352
H10N5avianinfluenzavirusisolatedfrompigsincentralChina.JVirol,86,13865-6. 1353 1354Watson, S. J., Langat, P., Reid, S. M., Lam, T. T., Cotten, M. et al. (2015). Molecular 1355
epidemiology and evolution of influenza viruses circulating within European swine 1356between2009and2013.JVirol,89,9920-31. 1357
1358Webby,R.J.,Swenson,S.L.,Krauss,S.L.,Gerrish,P.J.,Goyal,S.M.etal.(2000).Evolutionof 1359
swineH3N2influenzavirusesintheUnitedStates.JVirol,74,8243-51. 1360 1361Webby,R.J.,Rossow,K.,Erickson,G.,Sims,Y.andWebster,R.(2004).Multiplelineagesof 1362
antigenically and genetically diverse influenza A virus co-circulate in the United States 1363swinepopulation.VirusRes,103,67-73. 1364
1365Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. and Kawaoka, Y. (1992). 1366
EvolutionandecologyofinfluenzaAviruses.MicrobiolRev,56,152-79. 1367 1368Weingartl, H. M., Albrecht, R. A., Lager, K. M., Babiuk, S., Marszal, P. et al. (2009). 1369
Experimentalinfectionofpigswiththehuman1918pandemicinfluenzavirus.JVirol,83, 13704287-96. 1371
1372Wise, H. M., Foeglein, A., Sun, J., Dalton, R. M., Patel, S. et al. (2009). A complicated 1373
message: IdentificationofanovelPB1-relatedprotein translated from influenzaAvirus 1374segment2mRNA.JVirol,83,8021-31. 1375
1376Wise, H. M., Hutchinson, E. C., Jagger, B. W., Stuart, A. D., Kang, Z. H. et al. (2012). 1377
IdentificationofanovelsplicevariantformoftheinfluenzaAvirusM2ionchannelwith 1378anantigenicallydistinctectodomain.PLoSPathog,8,e1002998. 1379
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World Health Organization. (2017a). Influenza at the human-animal interface. Available: 1381http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_in 1382terface_05_06_2017.pdf?ua=1. 1383
1384WorldHealthOrganization.(2017b).Cumulativenumberofconfirmedhumancasesfor 1385
avianinfluenzaA(H5N1)reportedtoWHO,2003-2017.Available: 1386http://www.who.int/influenza/human_animal_interface/2017_04_20_tableH5N1.pdf?ua 1387=1 1388
1389Wright, P. F.,Neumann,G. andKawaoka, Y. (2013).Orthomyxoviruses. In: Knipe,D.M.& 1390
Howley,P.M. (Eds.)FieldsVirology.6thed.LippincottWilliams&Wilkins,Philadelphia, 1391Pensylvania,USA,pp1186-243. 1392
1393Xu,C.,Fan,W.,Wei,R.andZhao,H. (2004). Isolationand identificationofswine influenza 1394
recombinantA/Swine/Shandong/1/2003(H9N2)virus.MicrobesInfect,6,919-25. 1395 1396Xu,L.,Bao,L.,Deng,W.,Zhu,H.,Li,F.etal.(2014).RapidadaptationofavianH7N9virusin 1397
pigs.Virology,452-453,231-6. 1398 1399Yamada, S., Suzuki, Y., Suzuki, T., Le, M. Q., Nidom, C. A. et al. (2006). Haemagglutinin 1400
mutations responsible for the binding of H5N1 influenza A viruses to human-type 1401receptors.Nature,444,378-82. 1402
1403Yang,X.,Steukers,L.,Forier,K.,Xiong,R.,Braeckmans,K.etal.(2014).Abeneficiaryrolefor 1404
neuraminidaseininfluenzaviruspenetrationthroughtherespiratorymucus.PLoSOne,9, 1405e110026. 1406
1407Yang,Z.F.,Mok,C.K.,Peiris,J.S.&Zhong,N.S.(2015).HumanInfectionwithaNovelAvian 1408
InfluenzaA(H5N6)Virus.NEnglJMed,373,487-9. 1409 1410Yu, H., Hua, R. H., Wei, T. C., Zhou, Y. J., Tian, Z. J. et al. (2008a). Isolation and genetic 1411
characterizationofavianoriginH9N2influenzavirusesfrompigsinChina.VetMicrobiol, 1412131,82-92. 1413
1414Yu,H.,Hua,R.H.,Zhang,Q.,Liu,T.Q.,Liu,H.L.etal. (2008b).Geneticevolutionofswine 1415
influenzaA(H3N2)virusesinChinafrom1970to2006.JClinMicrobiol,46,1067-75. 1416 1417Yu, H., Zhang, P. C., Zhou, Y. J., Li, G. X., Pan, J. et al. (2009). Isolation and genetic 1418
characterizationofavian-likeH1N1andnovelressortantH1N2influenzavirusesfrompigs 1419inChina.BiochemBiophysResCommun,386,278-83. 1420
1421Yu, H., Zhou, Y. J., Li, G. X., Ma, J. H., Yan, L. P. et al. (2011). Genetic diversity of H9N2 1422
influenzavirusesfrompigs inChina:apotential threattohumanhealth?VetMicrobiol, 1423149,254-61. 1424
1425Yu, H.,Wu, J. T., Cowling, B. J., Liao, Q., Fang, V. J. et al (2014). Effect of closure of live 1426
poultrymarkets on poultry-to-person transmission of avian influenza AH7N9 virus: an 1427ecologicalstudy.Lancet,383,541-8. 1428
1429Zhu,H.,Wang,D., Kelvin,D. J., Li, L., Zheng, Z. et al. (2013). Infectivity, transmission, and 1430
pathologyofhuman-isolatedH7N9influenzavirusinferretsandpigs.Science,341,183-6. 1431 1432
Chapter1
43
Zhu,Q.,Yang,H.,Chen,W.,Cao,W.,Zhong,G.etal.(2008).Anaturallyoccurringdeletionin 1433itsNSgenecontributestotheattenuationofanH5N1swineinfluenzavirusinchickens.J 1434Virol,82,220-8. 1435
1436
45
1437
AIMSOFTHETHESIS 1438
14392
46
1440
1441
1442
1443
Aimsofthethesis
47
InfluenzaAvirusescauseinfluenzaor“flu” inhumansandfewanimalspeciessuch 1444
asbirdsorpigs.Thosevirusesareusuallyspecies-specificandtheyhavetoovercome 1445
a strong species barrier to infect new hosts. However, when a virus successfully 1446
overcomes thatbarrier itmayposea significant threat for thenovel host species. 1447
Birdsareconsideredasasourceforinfluenzavirusesthatcouldbecomeadaptedto 1448
mammals. On the other hand, pigs have been proposed for a long time as 1449
intermediate hosts where AIVs can adapt to mammals and become pandemic. 1450
However,themechanismsthatruleadaptationofinfluenzavirusesandthepossible 1451
roleofthepiginthegenerationofnovelpandemicsarenotelucidatedyet. 1452
1453
1. ToassesswhetheranavianH9N2viruscouldbecomeadaptedtopigsbyserial 1454
passagingandtodescribethegeneticchangesinducedbythatprocess.Since 1455
1998,H9N2AIVswithenhancedbindingtohuman-typereceptorshavebeen 1456
recurrently isolated from pigs and humans. These isolations increased the 1457
public health concern about those viruses. Other research groups already 1458
evaluated thepossibilityof adaptationby serial passagingofH9N2AIVs into 1459
mammalianspeciessuchas ferretsand/orguineapigs.However, thegenetic 1460
changes requiredbyavianH9N2 influenzaviruses toevolve intomammalian 1461
transmissibleformarenotelucidatedyet. 1462
1463
2. ToevaluateiftheintroductionofpH1N1internalgenesledtoanadvantagein 1464
theadaptationofH9N2AIVstopigsandifthisreassortantcouldreachefficient 1465
pig-to-pigtransmission.In2009thepH1N1influenzavirusemergedandspread 1466
worldwide inhumanandswinepopulations.Thisviruswasgenerated inpigs 1467
and has unique gene constellation containing genes from avian, human and 1468
swine origin. Interestingly, pH1N1 thoroughly reassorted with human and 1469
swine endemic viruses generating novel viruses containing pH1N1 internal 1470
genes.Furthermore,previousexperimentsdemonstratedthatbyreassortment 1471
with pH1N1 internal genes,H5N1 andH9N2AIVs enhanced their replication 1472
andtransmissioninferretsandpigs.However,inpigsthosereassortantviruses 1473
did not transmit with the same efficiency as the endemic swine influenza 1474
viruses. 1475
48
1476
1477
1478
49
1479
EFFECTOFSERIALPIGPASSAGES 1480
ONTHEADAPTATIONOFAN 1481
AVIANH9N2INFLUENZAVIRUS 1482
TOSWINE 1483
1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500
1501
1502
1503
1504
1505
1506
Adaptedfrom: 1507
ManceraGracia,J.C.,VandenHoecke,S.,Saelens,X., 1508
VanReeth,K. 1509
PLoSONE(2017)12(4),e0175267 1510
3
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
50
Abstract 1511
H9N2 avian influenza viruses are endemic in poultry in Asia and theMiddle East. 1512
These viruses sporadically cause dead-end infections in pigs and humans raising 1513
concerns about their potential to adapt to mammals or reassort with human or 1514
swineinfluenzaviruses.WeperformedtenserialpassageswithanavianH9N2virus 1515
(A/quail/HongKong/G1/1997) in influenzanaïvepigstoassessthepotentialofthis 1516
virus to adapt to swine. Virus replication in the entire respiratory tract and nasal 1517
virus excretion were examined after each passage and we deep sequenced viral 1518
genomic RNA of the parental and passage 4 H9N2 virus isolated from the nasal 1519
mucosaandlung.TheparentalH9N2viruscausedaproductiveinfectioninpigswith 1520
apredominanttropismforthenasalmucosa,whereasonly50%lungsampleswere 1521
virus-positive. In contrast, inoculationofpigswithpassage4 virus resulted in viral 1522
replicationintheentirerespiratorytract.Subsequentpassageswereassociatedwith 1523
reducedvirusreplicationinthelungsandinfectiousviruswasnolongerdetectable 1524
in the upper and lower respiratory tract of inoculated pigs at passage ten. The 1525
broadertissuetropismafter4passageswasassociatedwithanaminoacidresidue 1526
substitutionatposition225,within the receptor-bindingsiteof thehemagglutinin. 1527
We also compared the parental H9N2, passage 4 H9N2 and the 2009 pandemic 1528
H1N1(pH1N1)virusinadirectcontacttransmissionexperiment.Whereasonly1out 1529
of6contactpigsshowednasalvirusexcretionofthewild-typeH9N2formorethan4 1530
days,all6contactanimalsshedthepassage4H9N2virus.Nevertheless,theamount 1531
of excreted virus was significantly lower when compared to that of the pH1N1, 1532
which readily transmitted and replicated in all 6 contact animals. Our data 1533
demonstratethatserialpassagingofH9N2virusinpigsenhancesitsreplicationand 1534
transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely 1535
requiresanextensivesetofmutations. 1536
1537
Chapter3
51
3.1 Introduction 1538
AIVsof theH9N2subtypeemergedfromthenaturalwaterfowlreservoirandhave 1539
becomeendemicinpoultryinAsiaandtheMiddleEastsincetheearly1990s(Sunet 1540
al., 2010). Genetic characterization and phylogenetic analysis revealed that most 1541
poultryH9N2virusesbelongto1of3differentlineages:G1-like,Y280-likeandY439- 1542
like,alsoknownasKorean-like(Guoetal.,2000,Slomkaetal.,2013).In1999,H9N2 1543
viruses were for the first time isolated from 2 patients with mild respiratory 1544
symptoms (Peiris et al., 1999). These human isolates were genetically and 1545
antigenically similar to theG1-like lineage (Linet al., 2000). Later, from2002until 1546
2016, H9N2 viruses belonging to the G1-like and Y280-like lineages have been 1547
occasionallyreportedinhumans(Peirisetal.,1999,Buttetal.,2010,WorldHealth 1548
Organization,2016).ThefirstH9N2virusthatwasisolatedfromswinebelongedto 1549
the Y280-like lineage andwas reported inHongKong in 1998 (Peiris et al., 2001). 1550
Sincethen,differentlineagesofH9N2virusesweresporadicallydetectedinswinein 1551
MainlandChina(Congetal.,2007,Yuetal.,2008,Yuetal.,2011).Althoughthese 1552
infectionsofhumansandpigsindicatethatH9N2virusescanovercomethespecies 1553
barrierwithoutprioradaptation,nohuman-to-humanorpig-to-pigtransmissionhas 1554
beenreportedsofar(SunandLiu,2015). 1555
Influenza A virus infection ismediated via the attachment of the viral HA RBS to 1556
sialyloligosaccharides present on the host cell surface. Avian influenza viruses 1557
preferentially bind sialic acids linked to galactose by anα2,3 linkage (Siaα2,3Gal). 1558
Conversely,mosthumanandswineinfluenzavirusesmorereadilybindtoreceptors 1559
thatcontainterminalα2,6-linkedsialicacid (Siaα2,6Gal) (Matrosovichetal.,1997). 1560
BecauseofthepredominantexpressionofSiaα2,6Galinthehumanandswineupper 1561
respiratory tract (VanPouckeet al., 2010), a switch fromSiaα2,3Gal to Siaα2,6Gal 1562
receptor binding preference is considered an important factor for avian influenza 1563
virusadaptationtomammals(Matrosovichetal.,2015).Specificaasubstitutionsin 1564
theHARBShavebeenassociatedwithenhancedSiaα2,6Galbinding(Taubenberger 1565
and Kash, 2010). For H1 subtype viruses, for example, a crucial role has been 1566
assigned to the substitution of glutamic acid by aspartic acid at position 190 (HA- 1567
E190D)(H3numbering),andglycinebyasparticacidatposition225(HA-G225D).For 1568
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
52
H2andH3subtypes,thecrucialchangesweresubstitutionofglutaminebyleucineat 1569
position226(HA-Q226L)andglycinebyserineatposition228(HA-G228S)(Connoret 1570
al.,1994,Vinesetal.,1998,Suzukietal.,2000,).Asignificantproportionofpoultry 1571
H9N2 field isolates contain the HA-Q226L mutation, which has been shown to 1572
enhance binding to Siaα2,6Gal and replication of these viruses in human airway 1573
epithelialcellsinvitro(Matrosovichetal.,2001,WanandPerez,2007).Inaddition, 1574
Wan et al. reported that HA-Q226L increased virus replication and direct contact 1575
transmissionofH9N2inferrets,whichareconsideredthe“goldstandard”modelfor 1576
human influenza pathogenesis and transmissibility studies (Wan et al., 2008, 1577
Matsuokaetal.,2009).However,virustransmissionoftheseH9N2viruseswasstill 1578
less efficient compared to that of human adapted viruses (Wan et al., 2008). This 1579
raises thecriticalquestion:whichotherchangesare required tomakeavianH9N2 1580
virusesfullyadaptedtoamammalianhost? 1581
PigsareimportantnaturalhostsforinfluenzaAviruses.Theseanimalshaveasimilar 1582
Sia receptor distribution as humans (Van Poucke et al., 2010), and they are 1583
susceptible to both avian and human influenza viruses (Kida et al., 1994, De 1584
Vleeschauweretal.,2009b).Since1985,pigshavebeenproposedas intermediate 1585
hosts for the adaptation and transmission of avian influenza viruses frombirds to 1586
humans(Scholtisseketal.,1985,Kahnetal.,2014).However,theexactmechanisms 1587
by which AIVs may fully adapt to pigs and cause a pandemic are not completely 1588
understood. Previous studies reported on the pathogenicity, infectivity and 1589
transmissibilityofdifferentH9N2viruses in swinebasedondifferentexperimental 1590
approaches(Kidaetal.,1994,DeVleeschauweretal.,2009b,Qiaoetal.,2012,The 1591
SJCEIRSH9WorkingGroup,2013,Wangetal.,2016).Furthermore,theinfectionwas 1592
subclinicalinallcases.IntranasalinoculationofpigswithH9N2virusesisolatedfrom 1593
humansorchickenssince the late1990s resulted innasalvirusexcretion formore 1594
than1day,buttransmissionwaseitherundetectableorfarlessefficientthanthatof 1595
endemicswineinfluenzaviruses(Qiaoetal.,2012,TheSJCEIRSH9WorkingGroup, 1596
2013,Wangetal.,2016).SerialpassagingofAIVsinmammalsisaprovenstrategyto 1597
promotetheselectionofvirusvariantsthatarebetteradaptedtoreplicate in,and 1598
transmit between this non-natural host (Sorrell et al., 2009, Herfst et al., 2012, 1599
Shichinoheetal.,2013).Herewesought to improve theadaptationofawild-type 1600
Chapter3
53
H9N2AIVbyperformingtenblindserialpassages in influenzanaïvepigs.Tomimic 1601
the natural situation as much as possible, we used virus isolated from the nasal 1602
mucosaasinoculumforeverysubsequentpassageinanattempttoselectforviruses 1603
with improvednasalvirussheddingandtransmission.Bydoingso,weobtainedan 1604
H9N2viruswithapredominantmutationatposition225 inHAthatreplicatedand 1605
transmittedbetterinpigsthantheparentalvirus. 1606
1607
3.2 Materialsandmethods 1608
Ethicsstatement 1609
TheexperimentswereauthorizedandsupervisedbytheEthicalandAnimalWelfare 1610
Committee of the Faculty of Veterinary Medicine of Ghent University, with the 1611
EC2013/63number. 1612
Influenzaviruses 1613
A/quail/Hong Kong/G1/1997 (H9N2) was isolated from quail at a live birdmarket 1614
andunderwent7passages in11-day-oldembryonatedchickeneggs,3passages in 1615
Madin-Darby Canine Kidney cells (MDCK), and 2 passages in pigs. After each pig 1616
passagetheviruswasalsopassagedonceinMDCKcellstogrowalargestocktobe 1617
usedintheexperiment.Thevirusstockusedintheexperimentsharesatleast99% 1618
identity at the nucleotide and protein levels with the original A/quail/Hong 1619
Kong/G1/1997 virus (GenBank accession numbers: AF156435.1, AF156421.1, 1620
AF156449.1, AF156378.1, AF156407.1, AF156396.1, AF156463.1 and AF156477.2). 1621
This strain was selected because it is representative for the G1-like lineage and 1622
contains theHA-Q226Lsubstitution that isknowntoenhancehuman-like receptor 1623
specificity. 1624
A/California/04/2009(pH1N1)virusunderwent3passages inMDCKcells,1 ineggs 1625
and a last passage in MDCK cells before use. This virus is representative of the 1626
pH1N1virusesthatcirculateworldwideinswine. 1627
Animals 1628
Forty-seven 3-week-old piglets were purchased from a commercial herd that is 1629
serologicallynegative forswine influenzaandporcinereproductiveandrespiratory 1630
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
54
syndrome virus. Before the start of the experiments, all animals were free of 1631
influenzavirusantibodiesasdemonstratedbyacommercialblockinganti-influenza 1632
A nucleocapsid ELISA (ID-VET) and by virus neutralization (VN) tests using 1633
A/swine/Belgium/1/98 (H1N1), A/swine/Flanders/1/98 (H3N2), 1634
A/swine/Gent/7625/99 (H1N2) swine influenza viruses (SIVs), as well as 1635
A/California/04/09 (pH1N1). Upon arrival, the animalswere housed in a biosafety 1636
level-2 (BSL-2)highefficiencyparticle arresting (HEPA) filtered isolationunit for at 1637
least2daystoallowtheiracclimatization. 1638
BlindserialpassagesofavianH9N2influenzavirusinpigs 1639
Forthefirstpassage,2pigswerehousedinabiosafetylevel-3(BSL-3)HEPAfiltered 1640
isolator and inoculated intranasally with 3 ml of phosphate-buffered saline (PBS) 1641
containing 106.2 TCID50 of A/quail/Hong Kong/G1/1997 (H9N2). For inoculation 1642
unanesthesized pigs were held in a vertical positionwith the neck stretched. The 1643
inoculum(1.5mlpernostril)wasgraduallyinstilledintothenasalcavitybyinsertion 1644
ofa fifteenmmcannulaattachedtoasyringe.Todeterminevirusexcretion in the 1645
nose,individualpigswereswabbeddailyfrom0to4dayspost-inoculation(dpi).At4 1646
dpibothanimalswerehumanelyeuthanizedbyslowinjectionofanoverdose(³100 1647
mg/kg)ofpentobarbital in the jugularvein.At the timeofnecropsy, the following 1648
tissuesampleswerecollectedforvirustitrations:nasalmucosarespiratorypart(i.e. 1649
nasal turbinates), nasal mucosa olfactory part (i.e. ethmoid labyrinth), trachea 1650
(upper and lower half) and 5 different samples representative of the entire lung 1651
(apical+cardiaclobesleftandright,diaphragmaticlobesleftandright,andaccessory 1652
lobe). The nasal mucosae (respiratory part) were pooled and a 20% (w/v) tissue 1653
homogenatewas prepared and used to inoculate 2 pigswith this passage 1 virus 1654
inoculum. Ten blind (meaning that the viral load in the inoculumwas not known 1655
priortotheinfection)serialpassageswereperformedinthisway.Nasalswabsfrom 1656
all pigs, tissue samples and the inocula used for the subsequent passages were 1657
titratedinMDCKcells. 1658
Swinetransmissionstudies 1659
Theinoculumgivingthehighestvirusisolationratesandvirustitersthroughoutthe 1660
respiratorytractandinnasalswabswasexaminedforitstransmissionbetweenpigs. 1661
Chapter3
55
Wecomparedthetransmissibilityofmentionedvirus to thatof theparentalH9N2 1662
andthatofthepH1N1.Nine3-week-oldpigswereused.At0dpi(=2daysbeforethe 1663
startofcontacttransmission),3pigswerehousedin3separateBSL-3HEPAfiltered 1664
isolators(1indexpigperisolator)andintranasallyinoculatedwith106.5TCID50ofthe 1665
respective virus. Forty-eight hours pi, 2 pigs were introduced in each isolator 1666
allowingdirectcontactwiththeinoculatedpig.Allanimalswereclinicallymonitored 1667
for general (depression and anorexia) and respiratory (coughing, dyspnoea, 1668
abdominalthumping,tachypnoea)diseasesigns,andnasalswabsforvirusisolation 1669
were collected daily fromall pigs during 11 days after cohousing. Sixteen dpi, the 1670
animalswererelocatedtoaBSL-2HEPAfilteredisolationunit.Serumsampleswere 1671
collectedat0,16,23and30dpiand0,14,21and28dayspost-contact(dpc). 1672
Growthkineticsofparentalandpassagedvirus 1673
Tocomparethesingle-stepgrowthcurvesoftheparental,passagedvirusandpH1N1 1674
confluentmonolayersofMDCKcellswereinoculatedwithanidenticalmultiplicityof 1675
infectionof5MOIpercellat37oC.After1hourofincubationthecellswerewashed 1676
with phosphate-buffered saline (PBS) containing 10 IU/ml penicillin and 10 µg/ml 1677
streptomycintoremoveunboundvirusparticlesandoverlaidwithMinimalEssential 1678
Medium(MEM)containingsupplements(1mg/mllactalbumin,100IU/mlpenicillin, 1679
10 µg/ml streptomycin, 50 µg/ml gentamycin and 2 µg/ml trypsin). The 3 viruses 1680
weretestedintriplicate,supernatantswerecollectedat0,4,6,12and24hourspost 1681
inoculationandtitratedinMDCKcellsasdescribedbelow. 1682
Virustitrations 1683
Cottonswabswereweighedbeforeandaftercollectiontodeterminevirustitersper 1684
100 milligram nasal secretions. Nasal swab samples from both nostrils were 1685
suspended in 1 ml PBS supplemented with 10% foetal bovine serum, 100 IU/ml 1686
penicillinand100μg/mlstreptomycinandmixedvigorouslyat4°Cfor1hour.Tissue 1687
sampleswereweighedandgrounded inPBSwith10 IU/mlpenicillinand10µg/ml 1688
streptomycin to obtain 20% (w/v) tissue homogenates. Nasal swab samples and 1689
tissue homogenates were clarified by centrifugation and stored at -70°C until 1690
titrationonMDCKcells.Briefly,confluentmonolayersofcellswereinoculatedwith 1691
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
56
10-foldserialdilutionsofthesample.After5daysofincubationat37oCwith5%CO2, 1692
virus positiveMDCK cellswere visualized by immunoperoxidase staining. The cells 1693
were fixed with 4% paraformaldehyde for 10 minutes at room temperature and 1694
subsequently incubated with mouse anti-NP monoclonal HB-65 antibody (1:50, 1695
ATCC) for 2 hours. Incubation with horseradish peroxidase-conjugated goat anti- 1696
mousepolyclonalantibody(1:200,Dako)for1hourwasfollowedbyadevelopment 1697
step with H2O2 as substrate and 3-amino-9-ethyl-carbazole (AEC) as precipitating 1698
agent.VirustiterswerecalculatedbythemethodofReedandMuench(1938)and 1699
expressed as log10 50% tissue culture infective doses (TCID50) per 100 milligram 1700
(nasalswabs)orpergram(tissues). 1701
Virusneutralizationassays 1702
VirusneutralizationtestswereperformedonMDCKcellsin96-wellplates,using100 1703
TCID50ofvirusperwellaspreviouslydescribed (VanReethetal.,2003).Briefly,2- 1704
foldserumdilutionswereincubated(1h,37oC)with100TCID50ofMDCKcell-grown 1705
virus.MDCKcells(800.000cellsperml)wereincubatedwiththevirus-serummixture 1706
for 24h, after which virus positive cells were visualized by immunoperoxidase 1707
staining. 1708
Dataanalysis 1709
Becausethedetection limitwas1.7 log10TCID50per100milligramnasalsecreteor 1710
gramtissues,samplesthat testednegative forvirusweregivenanumericvalueof 1711
1.6log10TCID50per100milligramnasalsecreteorgramtissues.Samplesthattested 1712
negative in the virus neutralization assay were assigned a value corresponding to 1713
half of theminimumdetectable titer. For each transmissionexperiment, thebasic 1714
reproductionratio(R0)wasestimatedonthebasisoftheoutcomeoftheexperiment 1715
(final-sizemethod)with use of themaximum likelihood estimator. Thus, themost 1716
likely reproduction ratio was estimated using the total number of animals in the 1717
experiment,thetotalnumberofsusceptibleandinfectiousanimalsatthebeginning 1718
of the experiment, and the number of animals that became infected during the 1719
experiment. A pig was considered to be infected when it experienced 1720
seroconversion.The95%confidence intervals (CIs)wereconstructedsymmetrically 1721
aroundtheestimatedvalueofR0(DeVleeschauweretal.,2009b). 1722
Chapter3
57
Inaddition,nasal virus sheddingof individualpigswasquantifiedbycalculationof 1723
theareaunder the curve (AUC). Themeansof theAUCswere comparedbetween 1724
different viruses using standard two–sample Mann-Whitney U tests. Differences 1725
wereconsideredsignificantwhenp<0.05.GraphPadPrismSoftware,version5,was 1726
usedforstatisticalanalyses. 1727
Aminoacidsequencesalignment 1728
Amino acid sequences of HA proteins fromH9N2 viruses isolated from swine and 1729
humansinthefieldwereobtainedfromGenBank.Differenthumanandswineamino 1730
acid sequences were compared with the HA of the A/quail/Hong Kong/G1/1997 1731
(H9N2)strain,withspecialattentiontoaminoacidsatpositions190,225,226and 1732
228 because these contribute to transmission betweenmammals (Matrosovich et 1733
al.,1997,TaubenbergerandKash,2010).Aminoaciddifferenceswereidentifiedby 1734
alignmentusingMEGA6.06software. 1735
RT-PCR 1736
We compared the genetic diversity of the parental wild-type H9N2 virus and 2 1737
samples from the passage 4 virus (which was also selected for the transmission 1738
experiments):1 fromthenasalmucosa(upperrespiratorytract,duetothe limited 1739
amountoftissueobtainedtheviruswasamplifiedinMDCKcells)andanotherfrom 1740
the lungs(lowerrespiratorytract).TheRNAfromthesesampleswas isolatedusing 1741
theQIAampViralRNAMiniKit(Qiagen).cDNAwassynthesizedwiththeTranscriptor 1742
First Strand Synthesis kit (Roche), as described previously (Van denHoecke et al., 1743
2015). Two separate reactions were performed, using primers specific for the 1744
influenza A vRNAs: CommonUni12G (GCCGGAGCTCTGCAGATATCAGCGAAAGCAGG) 1745
and CommonUni12A (GCCGGAGCTCTGCAGATATCAGCAAAAGCAGG). Subsequently, 1746
all8genomicsegmentswereamplifiedin1PCRreactionusingamixcontainingan 1747
1:1 mix of CommonUni12G and CommonUni12A cDNA, primer CommonUni13 1748
(GCCGGAGCTCTGCAGATATCAGTAGAAACAAGG) (200 nM) and the Phusion High 1749
Fidelitypolymerase(ThermoScientific)(VandenHoeckeetal.,2015,Watsonetal., 1750
2013,Zhouetal.,2009).RT-PCRwasperformedasdescribed(VandenHoeckeetal., 1751
2015),withthefirst5PCRcyclesperformedwithanannealingtemperatureof45°C 1752
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
58
(instead of 72°C). PCR products were purified using the High Pure PCR Product 1753
PurificationKit(Roche)andelutionoftheDNAwasinsterileultrapurewater. 1754
IlluminaMiSeqlibrarypreparationandsequencing 1755
150 ng of each purified RT-PCR sample was sheared with an M220 focused- 1756
ultrasonicator(Covaris)settoobtainpeakfragmentlengthsof400bp.Thefragment 1757
endsof50ngoftheseDNAfragmentswererepairedusingtheNEBNextUltraDNA 1758
LibraryPreparationkit(NewEnglandBiolabs),followedbyadditionoftheadaptors 1759
byusingtheNEBNextMultiplexOligosforIlluminakit(DualIndexPrimersSet1,New 1760
EnglandBiolabs).Theresultingfragmentsweresize-selectedat300to400bpusing 1761
Agencourt AMPure XP bead sizing (Beckman Coulter). Afterwards, indexes were 1762
added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt 1763
AMPureXPbeads.FragmentswereanalysedonaHighSensitivityDNAChiponthe 1764
Bioanalyzer(AgilentTechnologies)beforeloadingonthesequencingchip.Afterthe 1765
2×250 bp MiSeq paired-end sequencing run, the data were base called and 1766
convertedtoIlluminaFASTQfiles(Phred+64encoding). 1767
NextGenerationSequencingdataanalysis 1768
SequencedataanalyseswereperformedontheresultingIlluminaFASTQfiles(Phred 1769
+64encoding)usingCLCGenomicsWorkbench(Version7.0.3)followingtheanalysis 1770
pipelineasdescribed(VandenHoeckeetal.,2015).Theprocessedsequencingreads 1771
weremapped to the consensus sequence obtained afterde novoassembly of the 1772
sequencing reads for the A/quail/Hong Kong/G1/1997 (H9N2) virus stock sample, 1773
sincethisvirusstockwasusedtostartthevirusadaptation.Thisconsensussequence 1774
is available through GenBank accession number (KY785896-KY785903). The raw 1775
sequencingdataweresubmittedtotheNCBISequenceReadArchivewheretheycan 1776
befoundunderprojectnumberSRP078326. 1777
Geneticcharacterization 1778
Toevaluateifthemutationsdescribedinpassage4weremaintainedthroughoutthe 1779
subsequent passages, the partial coding sequences ofHA,M and PB1 geneswere 1780
determined forall inocula frompassage5onwardsbydirectSangersequencingof 1781
overlappingRT-PCRproducts.RNAwasextractedfromviruswiththeQIAampViral 1782
Chapter3
59
RNAMiniKit (Qiagen,Valencia,CA).Reverse transcriptionandamplificationof the 1783
genes was done by one-step RT-PCR (One-step reverse-transcription polymerase 1784
reactionkit,Qiagen,Valencia,CA)usingcustomspecificprimers(primersequences 1785
availableuponrequest).Afteramplification,RT-PCRproductswerepurifiedusingthe 1786
Nucleo Spin Gel and PCR clean up (MACHEREY-NA-GEL GmbH&CoKG, Duren, 1787
Germany) DNA purification kit. Samples were sequenced by GATC Biotech AG 1788
(Constance,Germany)usingtheSangerABI3730xlplatform.Sequencingwasdone 1789
with custom designed primers (primer sequences available upon request). HA,M 1790
andPB1genesegmentswerecomparedatthenucleotideandaminoacid(aa)level 1791
usingMEGALIGN programwithin the DNASTAR 5.01 software package (DNASTAR, 1792
Inc., Madison, WI, USA). The complete genome sequences of A/quail/Hong 1793
Kong/G1/1997 (H9N2) were already available from GenBank (accession numbers: 1794
AF156378.1 (HA),AF156463.1 (M),AF156421.1 (PB1). The sequences generated in 1795
thisstudyareavailablethroughGenBankaccessionnumbers(KY785881-KY785895) 1796
1797
3.3 Results 1798
SerialpassagesofavianH9N2virusinswineisassociatedwithatransientincrease 1799
invirusreplication 1800
Theprimegoal of this studywas to assess the capacity of an avianH9N2 virus to 1801
adapttoswine.Forthis,tenblindserialpassageswereperformedininfluenzanaïve 1802
pigs. Animals were clinically scored and samples from the upper and lower 1803
respiratory tract were isolated to monitor viral loads. None of the pigs showed 1804
clinicalsigns.Anoverviewofthevirustitersinnasalswabsofindividualpigsthrough 1805
thedifferentpassagesisshowninFigure1.H9N2viruswasdetectedinnasalswabs 1806
ofallpigs,except for1pigduringpassage9andbothpigsduringpassage10.The 1807
highestvirustiterswerereachedduringpassages4and5(7.3log10TCID50/100mg). 1808
1809
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
60
1810
Figure1.NasalvirusexcretionofA/quail/HongKong/G1/1997influenzavirusduring 1811tenblindserialpigpassages.Twopigs(solid lineanddashedline)were involvedin 1812each passage, each line represents the individual virus titers in nose swabs. The 1813detectionlimitofthetestisindicatedwithadottedlineat1.7log10TCID50/100mgof 1814secrete. 1815
Chapter3
61
Onday4after inoculation,pigswereeuthanizedandsamples fromtheupperand 1816
lowerrespiratory tractwerecollectedtodeterminetheviral load. InfectiousH9N2 1817
viruswas found in the respiratory tractof all pigsexcept for thoseofpassage ten 1818
(Table1).Thenumberofviruspositivesamplesandamountofvirusinthedifferent 1819
tissue samples were in concordance with nasal virus excretion results, with 1820
considerablevariationbetween individualpigsandpassages (compareTable1and 1821
Figure1). Interestingly, replication rateswerehigher in theupper respiratory tract 1822
(nasal mucosa: respiratory and olfactory parts) with 36 out of 40 (90%) samples 1823
testingpositive,thaninthelowerrespiratorytract(tracheaandlungs)with65outof 1824
134 (49%) samples positive. In the upper respiratory tract, virus was isolated at 1825
similarfrequencyfromtheolfactoryandtherespiratorypartsofthenasalmucosa. 1826
Afterthefirst3passagesviruswasisolatedfromsomeofthelowerrespiratorytract 1827
samples,indicatingthatatearlypassagesthevirusdidnotreplicateuniformlyinthe 1828
entirelung.Onlyduringpassage5,afterinoculationwithpassage4virus,infectious 1829
virus was clearly detected in all samples, suggesting replication in the entire 1830
respiratory tract. Remarkably, following passage 5, the virus seemed to gradually 1831
have lost its ability to replicate in the lower respiratory tract and eventually, at 1832
passage10,infectivitywascompletelylost. 1833
1834
Table1.DistributionofA/quail/HongKong/G1/1997avianinfluenzavirusintherespiratorytractduringtenserialpassagesinpigs. 1835 1836
Numberofpassage Pignumber
Virustiter(log10TCID50/gramoftissue)atday4post-inoculationaNasalmucosa
respiratorypartNasalmucosaolfactorypart Proximaltrachea Distaltrachea Apical+cardiac
loberightApical+cardiac
lobeleftDiaphragmatic
loberightDiaphragmatic
lobeleftAccessory
lobe
1#1 4 3 2
b 4.7 <
c 4.5 < <
#2 5.2 6.5 2.5 4.5 < < < 1.7
2#3 5.5 4.5 2 < < < < <
#4 6.5 4.2 < < < < < 2
3#5 6.5 6.5 < 2 < < < <
#6 5 5.3 1.7 < < < 1.7 1.7
4#7 7.2 6.2 < < < 1.7 < 1.7 <
#8 5.7 3.3 4.5 4.2 5 1.7 1.7 < 4.3
5#9 5 5.5 3.3 4.3 5.3 5.2 3.7 5.2 2.3
#10 7 7.7 5.8 5.3 3.8 2.2 2 5 4.7
6#11 4.7 5.5 3.7 2.7 4.5 2.6 < < 5.7
#12 5.7 4.5 3.5 5.3 5.5 2 4.4 4.4 5.5
7#13 7 2.8 5.5 < < < < < 1.7
#14 5.3 4 2.7 5 6.4 4.7 4.5 3.8 5.7
8#15 3.7 5 < < < 2.3 < < <
#16 5.3 1.7 3.5 5.5 2 < 4.5 3.3 4.7
9#17 2 1.7 < < < < < < 1.7
#18 4.2 2.3 < < < < < < 2.3
10#19 < < < < < < < < <
#20 < < < < < < < < <aVirustitersareshownforeachindividualpig(#). 1837bProximalanddistaltracheasampleswerecombinedduringpassages1to3. 1838
c<detectionlimit(1.7log10TCID50/gramoftissue). 1839 1840
Chapter3
63
H9N2virusbecomescontacttransmissibleafter4serialpassagesinpigs 1841
To assess the level of adaptation of the virus after serial blind passages in pigs, 3 1842
different viruses were tested in 3 independent direct contact transmission 1843
experiments. As a positive control, we used the A/California/04/2009 (A/Cal/04/09) 1844
pH1N1virus,whichisrepresentativeofswine-adaptedinfluenzaviruses(Vincentetal., 1845
2014, Watson et al., 2015, Lewis et al., 2016). The parental A/quail/Hong 1846
Kong/G1/1997 (A/Qa/HK/P0) H9N2 virus served as starting material for the serial 1847
passages.Thepassage4H9N2virussample(A/Qa/HK/P4)thatwasusedtoinoculate 1848
animalsinpassage5wasalsoselectedforthetransmissionexperiment,becausebased 1849
onvirustitersinnasalswabsandrespiratorytractsamplesthisvirusseemedtohave 1850
gainedthehighestswine-adaptation.All3viruseswereamplifiedinMDCKcellsbefore 1851
inoculation of the index pigs and the transmission experiment was performed in 1852
triplicateforeachvirus. 1853
Figure2 shows thenasal virus sheddingof the inoculatedand theco-houseddirect- 1854
contact animals. All piglets remained clinically healthy, basedon clinical observation 1855
scores.All inoculatedanimalsexcretedvirusbetweendays1and8post-inoculation. 1856
Comparison of the AUC revealed lower virus excretion for A/Qa/HK/P0 (18.4) and 1857
A/Qa/HK/P4 (19.1) than forA/Cal/04/09 (26.6), although thesedifferenceswerenot 1858
statisticallysignificant(p<0.05).Ofall9inoculatedanimals5reachedamaximumvirus 1859
titer of ≥ 6.5 log10 TCID50/100 mg of secrete. All inoculated animals developed 1860
antibodies against the homologous virus (Table 2). Higher antibody titers were 1861
observedinA/Cal/04/09inoculatedpigsthaninH9N2infectedanimals. 1862
1863
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
64
1864
1865
Figure 2. Nasal virus excretion and direct contact transmission of A/Qa/HK/P0, 1866A/Qa/HK/P4 and A/Cal/04/09 influenza viruses in pigs. Three pigs were intranasally 1867inoculatedwith6.5log10TCID50oftheindicatedvirusperpigandindividuallyhousedin 1868different isolators. Forty-eight hours later, 2 direct contact animals were co-housed 1869with the inoculatedpigs.Eachgraphic is identifiedwitha2digitsnumber: the first1 1870correspondstotheisolatornumberandthesecondtothevirustested.Therefore,each 1871columnrepresentsadifferentvirusandeach rowrepresentsadifferent isolator.The 1872detectionlimit(dottedline)ofthetestwas1.7log10TCID50/100mgofsecrete. 1873 1874
Chapter3
65
Table 2. Antibody responses in inoculated and direct contact pigs involved in 1875transmissionexperimentsmeasuredbyVNtest. 1876
1877
VirusstrainNumberofantibodypositivepigs(rangeofantibodytiters)
Inoculatedpigs(n=3) Directcontactpigs(n=6)
0 16 23 30dpia 0 14 21 28dpcb
A/Qa/HK/P0 0(<2) 3(2-12) 3(6-24) 3(6-12) 0(<2) 2(24-32) 2(8-16) 2(16-24)
A/Qa/HK/P4 0(<2) 3(32-384) 3(64-128) 3(48-128) 0(<2) 1(4) 5(2-64) 5(2-12)
A/Cal/04/09 0(<2) 3(128-192) 3(192-384) 3(512-768) 0(<2) 6(192-1024)6(256-1536) 6(192-1024)adpi:dayspost-inoculation. 1878bdpc:dayspost-contact. 1879
1880
Only1outof6directcontactanimalsintheA/Qa/HK/P0groupshedvirusduringmore 1881
than5 days resulting in ameanAUCof 2.8. In contrast, all 6 animals thatwere co- 1882
housedwithanA/Qa/HK/P4virus inoculatedpig, excretedvirus for5daysormore. 1883
Thevirusexcretionwasnothomogeneousovertime(meanAUCof5.9)andpeakviral 1884
loads in contact animals were approximately 100-fold lower compared to the viral 1885
titers in thenasalexcretionsof theA/Qa/HK/P4 indexpigs (Figure2). Incontrast,all 1886
directcontactpigsintheA/Cal/04/09groupshedvirusinapatternthatisverysimilar 1887
tothatofthe inoculatedpigs,withameanAUCof19.7,andsimilarpeakviraltiters. 1888
ThedifferencebetweentheAUCvaluesoftheA/Qa/HK/P4direct-contactpigsandthe 1889
A/Qa/HK/P0 direct contact pigs was not statistically significant (p<0.05). However, 1890
bothgroupsshedsignificantly lessvirus thanA/Cal/04/09contactpigs (p<0.05).Five 1891
out of 6 A/Qa/HK/P4 contact animals had antibodies against the homologous virus, 1892
while only 2 animals that had been exposed to A/Qa/HK/P0 infected index animals 1893
seroconverted. As expected, all 6 A/Cal/04/09 contact animals seroconverted, with 1894
higher titers than theonesdetected in theH9N2direct contact animals. Thesedata 1895
allowed us to determine the R0 value, a measure for the transmissibility of an 1896
infectious agent. R0was 0.76 (95%CI: 0.10-5.49) for A/Qa/HK/P0 and 2.27 (95%CI: 1897
0.54-9.29)forA/Qa/HK/P4.BecauseallA/Cal/04/09contactpigsbecameinfected,the 1898
estimatedR0valuewas∞(95%CI:0.83-∞)forthisexperimentalsetting.Insummary, 1899
A/Qa/HK/P4 showed enhanced transmission compared to A/Qa/HK/P0, but both 1900
virusestransmittedlessefficiencythanA/Cal/04/09. 1901
In addition, we evaluated the replication kinetics of the viruses tested in the 1902
transmissionexperimentsbydeterminingthesingle-stepgrowthcurve inMDCKcells 1903
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
66
foreachvirus.Asdemonstratedinfigure3,thegrowthkineticsofthe3viruseswere 1904
verysimilar.Therefore,theresultsshowthattheenhancedinvivotransmissionofthe 1905
A/Qa/HK/P4virusdidnothaveanimpactontheinvitroreplicationkineticsofthevirus 1906
inacontinuouscellline. 1907
1908
1909
Figure3.Theeffectoftheserialpassagingontheamountofvirusproducedoverthe 1910course of the experiment using a single-step growth assay inMDCK cells. Each data 1911pointonthecurveisthemean±SDofthe3independentexperiments. 1912 1913
Mutationsassociatedwithpigadaptation 1914
WecomparedtheviraldiversitypresentintheH9N2virusstockthatwasusedtostart 1915
theserialpassageswiththeviraldiversitypresentinthevirussampledafterpassage4 1916
fromthenasalmucosa (afterasingle roundofamplificationonMDCKcells)andthe 1917
lung. The viral diversity was analysed in virus (pooled from 2 pigs) sampled after 1918
passage 4, since higher virus shedding and higher viral replication in the upper and 1919
lower respiratory tract was obtained upon inoculation with this virus. It was 1920
anticipatedthatacomparisonoftheviraldiversitypresentinthevirusfromtheupper 1921
and lower respiratory tract would enable us to determine if these 2 airway 1922
compartments favour the acquirement of a different set of mutations for optimal 1923
adaptation. In addition, sequencing of the virus stock is needed to determine if the 1924
detectedpigmutationswerealreadypresentinthevirusinoculumatthestartofthe 1925
Chapter3
67
serial passages or if they arose de novo. Illumina MiSeq deep sequencing was 1926
performedon full genomeRT-PCRproductsof the samples.Nucleotidevariants that 1927
differ fromtheconsensussequenceof theA/quail/HongKong/G1/1997 (H9N2)virus 1928
stocksamplethatwasusedtostarttheserialpassagesandthatoccuratafrequency 1929
of 5% or more are shown in Table 3. Two mutations were present in the starting 1930
material. These arenon-synonymousmutations that alter 2 adjacent aminoacidsof 1931
the polymerase acidic protein (PA). Interestingly, after 4 passages a substitution of 1932
aspartic acid by glycine at position 225 of the HA RBS (HA-D225G) was present in 1933
80.1%ofthesequencesderivedfromtheviralpopulationofthenasalmucosaandin 1934
99.6%oftheviralpopulationofthelungtissuesample.Anothersubstitutionwasclose 1935
to 100% present in lung homogenate virus, resulting in an alanine to threonine 1936
substitution at position 29 (M2-A29T) inmatrix protein 2 (M2). In addition, 2more 1937
substitutionsappeared in thenasalmucosaafter4passages:1 inPAandanother in 1938
non-structuralprotein1(NS1).These2mutationswerepresentatafrequencybelow 1939
10%.Inthelungvirussamples,3mutationsnewlyappearedwithfrequenciesranging 1940
from23.37to44.19%.Thesemutationswerepresentinpolymerasebasic1(PB1),HA 1941
andnucleoprotein(NP). 1942
1943
Table 3.Mutations present in parental and passage 4 H9N2 virus isolated from the 1944nasalrespiratorymucosaorfromthelung.Theviralgenesegmentisindicated,along 1945with thenucleotidepositionandsubstitutions, thepredictedaminoacidchangeand 1946itspositionandfinallythefrequency(percentage)ofsequencereadswiththedetected 1947mutations. Only nucleotide substitutions that resulted in amino acid changes and 1948appearedatafrequency≥5%inthereadsareshown.Thevirus inthenasalmucosa 1949samplewasamplifiedonMDCKcellsbeforesequencing. 1950
1951
Segment Nucleotideposition Reference Mutation Aminoacid
changeFrequency
A/Qa/HK/P0
FrequencyA/Qa/HK/P4(nasalmucosa)
FrequencyA/Qa/HK/P4
(lung)PB1 559 A G Glu172Gly <a < 42.34
PA1239 G A Glu399Lys < 5.00 <1506 A G Lys488Glu 10.25 11.21 7.841509 T A Cys489Ser 21.37 21.79 10.27
HA207 A G His44Arg < < 23.37750 A G Asp225Gly < 80.88 99.96959 T C Phe295Leu < < 44.19
NP 1347 G A Ala428Thr < 7.29 10.99M 818 G A M2:Ala29Thr < 23.26 98.33NS 191 A G NS1:Thr49Ala < 9.58 <
a<:mutationnotdetectedusing5%asvariantcallingthreshold 1952
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
68
Toevaluateifthesubstitutionspresentatthehighestfrequencyinthepassage4virus 1953
(HA-D225G, HA-F295L, M2-A29T and PB1-E172G) were maintained during the 1954
subsequent passages we sequenced the HA, M and PB1 coding-genes of the virus 1955
present in the inocula used to infect pigs from passages 6 to 10. Interestingly, the 1956
substitutions observed in the A/Qa/HK/P4 nasalmucosaweremaintained during all 1957
passages. However, the substitutions detected in the lung sample were no longer 1958
foundinfurtherpassages. 1959
1960
3.4 Discussion 1961
AvianH9N2virusesplayapivotalroleintheecologyofinfluenzainpoultryinEurasian 1962
countries.Sincethelate1990’s,recurrentdead-endinfectionswithH9N2viruseshave 1963
beenreportedinpigsandhumans(WorldHealthOrganization,2015,Buttetal.,2010, 1964
Guetal.,2014).Moreover,serologicalstudiesinAsianpoultryworkersandinChinese 1965
pigsrevealedsignificantexposuretoH9N2(Blairetal.,2013,Zhouetal.,2014,Wang 1966
etal.,2016).ThecontinuousexposuretoavianH9N2virusesmightposearealthreat 1967
forpublichealthiftheseviruseswouldacquiremutationsthatallowthemtotransmit 1968
efficientlybetweenmammals.Pigsareconsidered intermediatehosts inwhichavian 1969
H9N2 influenza viruses may acquire such mutations. We have therefore examined 1970
whetherblindserialpassagesofanavianH9N2virus inswinewouldresult inswine- 1971
adaptivemutations.Weconsideraninfluenzavirusasswine-adaptedifitsucceedsto 1972
transmitbetweenpigsinanexperimentalsettingwithasimilarefficiencyasendemic 1973
swineinfluenzaviruses.Therefore,wealsoperformedatransmissionexperimentwith 1974
theparentalandatleastpartiallyswineadaptedH9N2virus. 1975
OurresultsconfirmthesusceptibilityofpigstointranasalinoculationwithavianH9N2 1976
influenzavirusesreportedinpreviousexperimentalstudiesandinnature(TheSJCEIRS 1977
H9 Working Group, 2013, Wang et al., 2016, Kida et al., 1994, Sun et al., 2010). 1978
However, they are not in agreement with 2 previous studies in which avian H9N2 1979
virusesfailedtoreplicateinpigs.DeVleeschauweretal.(2009)intranasallyinoculated 1980
pigs with A/chicken/Belgium/818/1979 (H9N2) and found that this strain was not 1981
excretedatallbytheinoculatedpigs(DeVleeschauweretal.,2009b).Thoughgenetic 1982
information of this specific isolate is not available, the lack of replication in the 1983
Chapter3
69
inoculatedpigswas likelyduetothe fact thatEuropeanH9N2 isolatesbelongtothe 1984
Y439-likelineage,whichlikelylacktheabilitytoreplicateinpigs(Slomkaetal.,2013, 1985
Wangetal.,2016).Inthesecondstudy,Qiaoetal.(2012)usedthesameA/quail/Hong 1986
Kong/G1/1997(H9N2)strain,toinoculatepigsintratracheallyandtheydetectednasal 1987
virusexcretioninonly1outof12inoculatedanimals,albeitatlowvirustiters(Qiaoet 1988
al.,2012).Theverypoorcapacitytoreplicateinswinecomparedtoourfindingswith 1989
A/quail/Hong Kong/G1/1997 (H9N2) can be explained by the different inoculation 1990
routesusedinourandQiao’sstudy,whichwillresultindifferentsitesofprimaryvirus 1991
replication. Ithasbeendemonstrated that replication in thenasalmucosaandnasal 1992
virusexcretionissecondarytoanddependentonreplicationinthelowerairwaysafter 1993
intratrachealexposure. Intratracheal inoculation thususually results indelayednasal 1994
virusexcretionand lowernasalvirustitersascomparedtothe intranasal inoculation 1995
(DeVleeschauweretal.,2009a).Moreover,weconfirmedpreviousreportsdescribing 1996
the susceptibility of pigs to AIVs of different subtypes (Kida et al., 1994, De 1997
Vleeschauweretal.,2009b,Liuetal.,2014).TheparentalH9N2virusreplicatedinthe 1998
upper respiratory tract at low to moderate titers while most samples of the lower 1999
respiratory tract tested virus negative. The relative lack of Siaα2,3Gal receptors in 2000
humanandswineupperrespiratorytractisthoughttorestricttheefficientreplication 2001
ofAIVsinmammals.Therefore,ashiftfromSiaα2,3GaltoSiaα2,6Galreceptor-binding 2002
preference has been appointed as a critical step in the adaptation of AIVs to 2003
mammalianhosts(ImaiandKawaoka,2012).ForH9,aswellasforH3,H5andH7avian 2004
subtypes, the HA-Q226L substitution contributes to increased Siaα2,6Gal receptor 2005
preferenceand isassociatedwithhigher replication ratesand transmissibility inpigs 2006
andferrets(WanandPerez,2007,Wanetal.,2008,Liuetal.,2014,VanPouckeetal., 2007
2013,Herfst et al., 2012). ThoughourparentalH9N2 virus already contains theHA- 2008
Q226Lsubstitution,thevirusdistributionandreplicationratesdescribedinourstudy 2009
were similar to those observed in pigs that had been inoculated with H5N2 AIV 2010
subtypeviruswitha“pure”aviangenome,i.e.withHA-226Q(DeVleeschauweretal., 2011
2009a). Our data therefore question the role of the HA-Q226L substitution in 2012
replication efficiency in pigs but are in line with more recent studies, which 2013
demonstratethatresidue226inHAisnotstrictlydeterminativeforthereplicationof 2014
H9N2 viruses in pigs or ferrets (The SJCEIRSH9WorkingGroup, 2013). Comparative 2015
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
70
studieswithHA-226LandHA-226QwouldberequiredtoevaluatetheeffectoftheHA- 2016
Q226L substitution inpigs. In contrast to theparentalH9N2virus, the virus isolated 2017
after4pigpassagesreplicatedhomogeneouslythroughouttheentirerespiratorytract. 2018
Wecomparedpig-to-pigtransmissionofthisviruswiththatoftheparentalH9N2and 2019
pH1N1.WhiletheparentalH9N2viruslargelyfailedtotransmittodirectcontactpigs, 2020
the fourth passage virus showed enhanced transmissibility as demonstrated by the 2021
AUCandtheR0.Thisvirusshowedalsohighertransmissionefficiencywhencompared 2022
withpreviousstudieswithH9N2aswellasotheravianinfluenzavirussubtypesinpigs. 2023
Inallofthesestudiesthedirectcontactpigsexcretednoorminimalamountofvirus 2024
(De Vleeschauwer et al., 2009b,Wang et al., 2016, The SJCEIRS H9WorkingGroup, 2025
2013).However, transmissionof the fourth passage virus remained far less efficient 2026
thanthatofendemicswineinfluenzaviruses.Althoughthe3virusesshoweddifferent 2027
phenotypesinpigs,theirgrowthkineticsinMDCKcellsweresimilar,indicatingapoor 2028
correlationbetweentheinvivophenotypeandinvitroreplicationratesoncontinuous 2029
cell lines. Furthermore, from passage 7 onwards the virus replication progressively 2030
decreaseduntilitwaslost.Thisresultmaybeexplainedbythegenerationofanarrow 2031
bottleneck after passage 4 due to the strong genetic pressure posed by the 2032
experimental setup,whichmaydecrease thediversityof theviralpopulationwhich 2033
canhampertheadaptationprocess(Varbleetal.,2014,Zaraketetal.,2015). 2034
GeneticchangesduringAIVpassageinanimalsarenotpredictable(Dlugolenskietal., 2035
2011).Tobetterunderstandtheeffectoftheserialpassaging intheviruspopulation 2036
andthebehaviourofthevirusinourtransmissionexperimentwecomparedtheviral 2037
diversityintheoriginalwild-typeH9N2viruswiththevirusisolatedfromnasalmucosa 2038
and lung after 4 passages.We observed that the upper and lower respiratory tract 2039
selected fordifferentvirusvariants.Moreover, themutations identified in theupper 2040
respiratorytractweremaintainedduringthesubsequentpassages,whilethosefound 2041
inthe lungvirussampleswerenotmaintainedinthesubsequentpassages.Although 2042
more genetic analyses might be necessary to better understand this discrepancy 2043
between the viral selection pressure in the upper and lower respiratory tract, this 2044
points to a possible role of the mutations detected in the lung samples in the 2045
enhanced replicationand transmissibility.Only1outof the10mutationswe found, 2046
theHA-D225Gsubstitution,hasbeenpreviouslydescribed in the literature (Romero- 2047
Chapter3
71
Tejeda and Capua, 2013, Richard et al., 2014, Taubenberger and Kash, 2010). This 2048
mutationarosedenovoinboththeupperandlowerrespiratorytractafter4passages 2049
and it was associated with a higher replication efficiency during passage 5. 2050
Interestingly,thismutationwasalsopresentinall42swineand17humanH9N2field 2051
isolatesavailableinGenBank(seeFigures4and5).In1918and2009H1N1pandemic 2052
viruses,HA-D225GsubstitutionhasbeenassociatedwithincreasedSiaα2,3Galtropism 2053
conferring those viruses dual Siaα2,6Gal and Siaα2,3Gal receptor binding affinity 2054
(Lakdawalaetal.,2015,Belseretal.,2011,Tumpeyetal.,2007).Inpigs,aswellasin 2055
humans, Siaα2,3Gal receptors are predominantly found in the lungs and not in the 2056
upperrespiratorytract(VanPouckeetal.,2010).Theemergenceofthismutationafter 2057
4 passages could therefore explain the enhanced replication in the lungs. 2058
Nevertheless,ininvitrosialylglycoproteinbindingassaysH9N2virusescontainingHA- 2059
225G and HA-226L, as our fourth passage virus, showed only slightly increased 2060
Siaα2,6Galbindingaffinity (Lakdawalaetal.,2015,Matrosovichetal.,2001).Though 2061
specific binding tests would be needed to clarify the exact role of the HA-D225G 2062
substitution,ourdata suggest that it isan importantmarker foradaptationofH9N2 2063
AIVstopigs. 2064
Insummary,ourresultsdonotrejectthetheoryofthepigasanintermediatehostfor 2065
H9N2 AIVs, but demonstrate that adaptation of these viruses to pigs is a complex 2066
processandthatthemutationsselectedafter4passagesinpigswerenotsufficientto 2067
confer efficient pig-to-pig transmission. It also shows that additional molecular 2068
featuresmayberequiredforefficienttransmissionofavianH9N2virusesinpigs.Qiao 2069
et al. (2012) and Obadan et al. (2015) showed that reassortant H9N2 viruses 2070
containing pH1N1 internal genes improved replication and transmissibility in pigs, 2071
althoughtheywerestill lessefficientthanpH1N1virus. InpreviousH9N2adaptation 2072
studies in mammals, efficient transmission was only reached with ferret-passaged 2073
reassortantH9N2viruses containingeitherhumanseasonalH3N2orpH1N1 internal 2074
genes (Sorrell et al., 2009, Kimble et al., 2011). Therefore, the combination of 2075
mammalianadaptedinternalgeneswithserialpassagingmayberequiredtoselecta 2076
fullytransmissibleH9N2virus. 2077
2078
EffectofserialpigpassagesonadaptationofanavianH9N2influenzavirustoswine
72
2079
Figure4.AlignmentofthededucedaminoacidsequencesintheHAofallswineH9N2 2080isolates (n=42)available inGenBankon22ndAugust2016.Thenameofeachstrain 2081included in the analysis is followed by the accession number between brackets. 2082Residuesatpositions190,225,226and228arehighlightedingray.Aminoacidsthat 2083are different from those in A/quail/Hong Kong/G1/1997 are shown; conserved 2084residuesareshownasdots. 2085 2086
Chapter3
73
2087
Fig 5. Alignment of deduced amino acid sequences in the HA of all human H9N2 2088isolates (n=17)available inGenBankon22ndAugust2016.Thenameofeachstrain 2089included intheanalysis is followedbytheaccessionnumber inbrackets.Residuesat 2090positions190,225,226and228arehighlightedingray.Aminoacidsthataredifferent 2091fromthoseinA/quail/HongKong/G1/1997areshown;conservedresiduesareshown 2092asdots. 2093 2094
3.5 Acknowledgements 2095
TheauthorswouldliketoexpresstheirgratitudetoDr.JohnNichollsforprovidingthe 2096
H9N2 influenza viruses and Lieve Sys,Melanie Bauwens, Nele Dennequin and Laura 2097
VanDriesscheforexcellent technicalassistance.ThisstudywassupportedbytheEU 2098
FLUPIG project (FP7-GA 258084). Silvie Van denHoecke is supported by Fonds voor 2099
WetenschappelijkOnderzoek(3G052412). 2100
2101
3.6 References 2102
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2106Blair,P. J.,Putnam,S.D.,Krueger,W.S.,Chum,C.,Wierzba,T.F.etal. (2013).Evidence for 2107
avian H9N2 influenza virus infections among rural villagers in Cambodia. J Infect Public 2108Health,6,69-79. 2109
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2114Cong, Y. L., Pu, J., Liu, Q. F., Wang, S., Zhang, G. Z. et al. (2007). Antigenic and genetic 2115
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2124DeVleeschauwer,A.,VanPoucke,S.,Braeckmans,D.,VanDoorsselaere,J.andVanReeth,K. 2125
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adaptation of influenza A(H7N9) virus is limited by a narrow genetic bottleneck. Nat 2307Commun,6,6553. 2308
2309Zhou,B.,Donnelly,M.E.,Scholes,D.T.,StGeorge,K.,Hatta,M.etal.(2009).Single-reaction 2310
genomic amplification accelerates sequencing and vaccine production for classical and 2311Swineoriginhumaninfluenzaaviruses.JVirol,83,10309-13. 2312
2313Zhou,P.,Zhu,W.,Gu,H.,Fu,X.,Wang,L.etal. (2014).AvianinfluenzaH9N2seroprevalence 2314
amongswinefarmresidentsinChina.JMedVirol,86,597-600. 2315 2316 2317
2318 2319
79
2320
AREASSORTANTH9N2 2321
INFLUENZAVIRUSCONTAINING 2322
2009PANDEMICH1N1 2323
INTERNAL-PROTEINGENES 2324
ACQUIREDENHANCEDPIG-TO- 2325
PIGTRANSMISSIONAFTER 2326
SERIALPASSAGESINSWINE 2327
2328
2329
2330
2331
2332
2333
2334
2335
2336
2337
Adaptedfrom: 2338
ManceraGracia,J.C.,VandenHoecke,S.,RichtJ.A.,Ma,W., 2339
SaelensX.,VanReethK. 2340
ScientificReports(2017)7:1323 2341
2342
4
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
80
Abstract 2343
AvianH9N2and2009pandemicH1N1 (pH1N1) influenza viruses can infectpigs and 2344
humans,raisingtheconcernthatH9N2:pH1N1reassortantvirusescouldemerge.Such 2345
reassortant viruses demonstrated increased replication and transmissibility in pig 2346
experiments,butwerestillinefficientwhencomparedtopH1N1.Here,weevaluatedif 2347
a reassortantviruscontaining thehemagglutininandneuraminidaseofA/quail/Hong 2348
Kong/G1/1997 (H9N2) in theA/California/04/2009 (pH1N1) backbone could become 2349
better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 2350
(P0)viruswasrestrictedtothenasalmucosa,withnovirusdetectedinthetracheaor 2351
thelungs.Nevertheless,after7passages(H9N2:pH1N1(P7))thevirusreplicatedinthe 2352
entire respiratory tract.We also compared the transmissibility ofH9N2:pH1N1 (P0), 2353
H9N2:pH1N1 (P7)andpH1N1.Whileonly2/6direct-contactpigs showednasal virus 2354
excretionofH9N2:pH1N1(P0)formorethan5days,4/6direct-contactanimalsshed 2355
theH9N2:pH1N1(P7).Interestingly,virustitersinnasalsecretionsofthose4animals 2356
were similar to those obtained with the pH1N1, which readily transmitted to all 6 2357
contact animals. The broader tissue tropism and the increased post-transmission 2358
replicationafter7passageswereassociatedwiththeHA-D225Gsubstitution.Ourdata 2359
demonstratethatthepH1N1internal-proteingenestogetherwiththeserialpassages 2360
favouravianH9N2virusadaptationtopigs. 2361
2362
Chapter4
81
4.1 Introduction 2363
H9N2avian influenzavirusesarecurrentlyendemic inpoultry inSouthEastAsiaand 2364
the Middle East (Sun et al., 2010). Since the late 1990s H9N2 viruses have been 2365
occasionally isolated from in humans and swine in China, leading to increasing 2366
concernsabouttheirpandemicpotential(Peirisetal.,1999,Linetal.,2000,Peiriset 2367
al., 2001, Yu et al., 2008, Butt et al., 2010, Wang et al., 2016, World Health 2368
Organization, 2016a). However, all reported human or swine H9N2 virus infections 2369
were dead-end events and the virus failed to spread within the human or swine 2370
population(SunandLiu,2015). 2371
A significant proportion of poultry H9N2 field isolates contains a leucine instead of 2372
glutamine residue at position 226 in the HA RBS (HA-Q226L, H3 numbering used 2373
throughout). This mutation has been shown to enhance HA binding to terminal 2374
Siaα2,6GalandtoimprovethereplicationofH9N2virusesinhumanairwayepithelial 2375
cells invitro,supportingconcernsabouttheirpandemicpotential(Matrosovichetal., 2376
2001, Wan and Perez, 2007, Wan et al., 2008). The binding of the viral HA to 2377
sialyloligosacharides, present on the host cell surface, is the first step for influenza 2378
virus infection. Because of the predominant expression of Siaα2,6Gal in human and 2379
swineupperrespiratorytract(VanPouckeetal.,2010,Nellietal.,2010),mosthuman 2380
and swine influenza virusesmorewillingly bind to receptors that contain Siaα2,6Gal 2381
(Matrosovichetal.,1997).Incontrast,avianinfluenzavirusespreferentiallybindsialic 2382
acids linked to galactose by an α2,3 linkage (Siaα2,3Gal). Therefore, a switch from 2383
Siaα2,3GaltoSiaα2,6Galreceptor-bindingpreferenceisconsideredanimportantstep 2384
for avian influenza virus adaptation to mammals (Matrosovich et al., 2015). 2385
Nevertheless, enhanced Siaα2,6Gal binding is not entirely sufficient to make H9N2 2386
viruses transmissible between mammals (Wan et al., 2008, Qiao et al., 2012, The 2387
SJCEIRSH9WorkingGroup, 2013). This suggests that other amino acid substitutions 2388
and/or an adequate constellation of internal genes is also required for efficient 2389
transmissionofH9N2virusesbetweenmammals. 2390
Due to the segmented nature of the influenza viruses, the RNA segments of 2 (or 2391
more)differentinfluenzavirusescan“reassort”whenco-infectingthesamehostcell, 2392
therebygeneratingprogenyviruseswithpropertiesdifferentfromtheparentalviruses 2393
(Taubenberger and Kash, 2010). In that sense, the sporadic isolation of avian H9N2 2394
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
82
influenza viruses from pigs and humans in China increases the possibility of co- 2395
infectionswithendemic swineorhuman influenza viruses,which could result in the 2396
generationofnovelreassortantviruseswithenhancedtransmissibilityinmammals. 2397
The2009pandemicH1N1virus(pH1N1)isaswine-originreassortantviruscontaining 2398
human, swine and avian influenza virus genes (Smith et al., 2009). After its first 2399
appearancein2009,thevirushasspreadaroundtheworldinbothhumanandswine 2400
populations (Brookes et al., 2010). Moreover, novel reassortant viruses containing 2401
pH1N1 internal-protein genes and surface-protein genes from other endemic swine 2402
influenzavirushavebeenisolatedfrequentlyfrompigsworldwide(Nelsonetal.,2012, 2403
Liang et al., 2014, Simonet al., 2014,Vincent et al., 2014). In addition,H3N2 swine 2404
influenza viruses containing the pH1N1-derived matrix protein gene (H3N2v) were 2405
isolatedfrom364humancasesintheUSsince2011(CentersforDiseaseControland 2406
Prevention, 2016). This suggests that pH1N1 internal-protein genes are compatible 2407
with several surface-protein gene combinations and allow for robust replication in 2408
mammals. 2409
Intransmissionstudiesinferrets,areassortantH9N2viruscontainingpH1N1internal- 2410
protein geneswas readily transmitted to all contact animals by respiratory droplets 2411
(Kimble et al., 2011). Although similar reassortant viruses showed increased 2412
mammaliantransmissioncomparedtotheparentalH9N2,theirtransmissionwasstill 2413
notasefficientasswine-adaptedviruseslikepH1N1(Qiaoetal.,2012,Obadanetal., 2414
2015). We previously demonstrated that serial passages of A/quail/Hong 2415
Kong/G1/1997 (H9N2) virus in pigs mildly enhanced replication and transmission in 2416
swine(ManceraGraciaetal.,2017).Althoughtheresultingpig-adaptedviruscarrieda 2417
number of mutations compared to the parental virus, transmission was still less 2418
efficientthanthatofnaturallycirculatingviruses(ManceraGraciaetal.,2017).Inthe 2419
present study,wehaveexamined the replicationand transmissibility after ten serial 2420
pig passages of a reassortant virus containing A/quail/Hong Kong/G1/1997 (H9N2) 2421
surface-proteingenesandNPwithintheA/California/04/2009(pH1N1)backgroundto 2422
evaluateifthepH1N1internalgenesconfersanadvantageinH9N2adaptationtopigs. 2423
ThisserialpassagingoftheH9N2:pH1N1reassortantresultedinavirusthatreplicated 2424
in and transmitted between pigs at high rates. The predominant mutation in the 2425
passaged reassortant viruswas an aspartic acid to glycine at position 225 in theHA 2426
Chapter4
83
RBS. Therefore, our results showed that the combination of reassortment and 2427
mutations induced by the serial passages generated a virus with a predominant 2428
mutation at position 225 inHARBS that replicated and transmitted at high rates in 2429
pigs. 2430
2431
4.2 Materialsandmethods 2432
Viruses 2433
The reassortant virus containing A/California/04/2009 (pH1N1) internal genes and 2434
A/quail/HongKong/G1/1997(H9N2)HAandNAwasproducedbyreversegeneticsat 2435
theDepartmentofDiagnostic/PathobiologyatKansasStateUniversity.Theviruswas 2436
plaque purified, passaged 3 times in MDCK cells, characterized and experimentally 2437
validated(Qiaoetal.,2012).AnotherpassageinMDCKcellswasmadeatthearrivalof 2438
thevirustotheLaboratoryofVirologyatGhentUniversitytogenerateastocktobe 2439
usedinthisstudy. 2440
A/California/04/2009(pH1N1)waskindlyprovidedbytheCentersforDiseaseControl 2441
andPrevention(USCDC).Thevirusunderwent3passagesinMDCKcellsattheUSCDC. 2442
Later 1 passage in eggs and 1 in MDCK cells were performed in the Laboratory of 2443
VirologyatGhentUniversitybeforeuse. 2444
Pigpassagesandtransmissionstudies 2445
Forty-seven3-week-oldpigletswerepurchasedfromacommercialherdfreeofswine 2446
influenzaandporcinereproductiveandrespiratorysyndromevirus.Uponarrival,pigs 2447
were confirmed seronegative to influenza virus with a commercial blocking anti- 2448
influenza A nucleocapsid ELISA (ID-VET) and by virus neutralization (VN) tests. 2449
Consecutivepassagesstartedwiththehousingof2pigs inabiosafety level-3(BSL-3) 2450
HEPAfilteredisolator,andtheirintranasalinoculationwith3mlofphosphate-buffered 2451
saline (PBS) containing 106.5 TCID50 of virus. Pigs were clinicallymonitored daily for 2452
general (depression, anorexia) and respiratory (coughing, dyspnoea, abdominal 2453
thumping,tachypnoea)symptoms.Nasalswabswerealsocollecteddaily from0to4 2454
dpi.At4dpibothpigswereeuthanizedwithalethaldoseofpentobarbitalsodium(³ 2455
100mg/kg) in the jugularvein.The following tissuesampleswerecollected forvirus 2456
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
84
titrations:nasalmucosarespiratorypart(i.e.nasalturbinates),nasalmucosaolfactory 2457
part(i.e.ethmoidlabyrinth),trachea(distalandproximalhalf)and5differentsamples 2458
representativeof the entire lung. Thenasalmucosae (respiratory part)werepooled 2459
anda20%(w/v) tissuehomogenatewaspreparedandusedto inoculate2pigswith 2460
thispassage1virus inoculum.Tenblind(meaningthattheviral load inthe inoculum 2461
was not known prior to the infection) serial passages were performed in this way. 2462
Nasal swabs from all pigs, tissue samples and the inocula used for the subsequent 2463
passagesweretitratedonMDCKcells. 2464
Foreachtransmissionexperimentnine3-week-oldpigswereused.At0dpi3pigswere 2465
housed in 3 separate BSL-3 HEPA filtered isolators (1 index pig per isolator) and 2466
intranasally inoculatedwith106.5TCID50of therespectivevirus.Twodpi,2pigswere 2467
introducedineachisolatorallowingdirectcontactwiththeinoculatedpig.Allanimals 2468
wereclinicallymonitoredandnasalswabsforvirusisolationwerecollecteddailyfrom 2469
allpigsduringelevendaysaftercohousing.Sixteendpi,theanimalswererelocatedto 2470
aBSL-2HEPAfilteredisolationunit.Serumsampleswerecollectedat0,16,23and30 2471
dpiand0,14,21and28dayspost-contact. 2472
Ethicalstatement 2473
AllexperimentalprocedureswereconductedinaccordancewithE.U.AnimalWelfare 2474
Directives, and authorized and supervised by the Local Ethical and Animal Welfare 2475
Committee of the Faculty of Veterinary Medicine of Ghent University (EC2013/63 2476
number). 2477
Virustitrations 2478
Nasalswabsfrombothnostrilsweresuspended in1mlPBSsupplementedwith10% 2479
foetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin andmixed 2480
vigorouslyat4°Cfor1hour.TissuesampleswereweighedandgroundedinPBSwith 2481
10 IU/ml penicillin and 10 µg/ml streptomycin to obtain 20% (w/v) tissue 2482
homogenates. Briefly, confluent monolayers of cells were inoculated with 10-fold 2483
serial dilutionsof the sample.After 5 days of incubation at 37oCwith 5%CO2, virus 2484
positive MDCK cells were visualized by immunoperoxidase staining. The cells were 2485
fixed with 4% paraformaldehyde for 10 minutes at room temperature and 2486
subsequentlyincubatedwithmouseanti-NPmonoclonalHB-65antibody(1:50,ATCC) 2487
Chapter4
85
for 2 hours. Incubation with horseradish peroxidase-conjugated goat anti-mouse 2488
polyclonalantibody(1:200,Dako)for1hourwasfollowedbyadevelopmentstepwith 2489
H2O2 as substrate and 3-amino-9-ethyl-carbazole (AEC) as precipitating agent. Virus 2490
titerswere calculatedby themethodof Reed andMuench (1938) andexpressed as 2491
log1050%tissuecultureinfectivedoses(TCID50)per100milligram(nasalswabs)orper 2492
gram(tissues). 2493
Virusneutralizationassays 2494
VNtestswereperformedonMDCKcellsin96-wellplates,using100TCID50ofvirusper 2495
well as previously described (Van Reeth et al., 2003). Briefly, 2-fold serum dilutions 2496
were incubated (1h, 37oC) with 100 TCID50 of MDCK cell-grown virus. MDCK cells 2497
suspension(800.000cellsperml)wasincubatedwiththevirus-serummixturefor24h, 2498
after which the virus positive cells were visualized by immunoperoxidase staining. 2499
Antibody titers were expressed as the reciprocal of the highest serum dilution that 2500
completelyinhibitedvirusreplicationinMDCKcells. 2501
Dataanalysis 2502
Nasalvirussheddingof individualpigswasquantifiedbycalculationof theAUC.The 2503
means of the AUCs of different viruses were compared using standard two–sample 2504
Mann-Whitney U tests. Differences were considered significant when p<0.05. 2505
GraphPadPrismSoftware,version5,wasusedforstatisticalanalyses. 2506
RT-PCR 2507
The RNA from the virus samples was isolated using the QIAamp Viral RNAMini Kit 2508
(Qiagen, Valencia, CA). cDNA was synthesized with the Transcriptor First Strand 2509
Synthesiskit (Roche),alreadydescribed(VandenHoeckeetal.,2015).Twoseparate 2510
reactions were performed, using primers specific for the influenza A vRNAs: 2511
CommonUni12G (GCCGGAGCTCTGCAGATATCAGCGAAAGCAGG) and CommonUni12A 2512
(GCCGGAGCTCTGCAGATATCAGCAAAAGCAGG).Subsequently,all8genomic segments 2513
were amplified in 1 PCR reaction containing an 1:1 mix of CommonUni12G and 2514
CommonUni12A cDNA, primer CommonUni13 2515
(GCCGGAGCTCTGCAGATATCAGTAGAAACAAGG) (200 nM) and the Phusion High 2516
Fidelity polymerase (Thermo Scientific) (VandenHoecke et al., 2015,Watson et al., 2517
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
86
2013,Zhouetal.,2009).RT-PCRwasperformedasdescribed(VandenHoeckeetal., 2518
2015),with the first 5PCR cyclesperformedwithanannealing temperatureof45°C 2519
(instead of 72°C). PCR products were purified using the High Pure PCR Product 2520
PurificationKit(Roche)andelutionoftheDNAinsterileultrapurewater. 2521
IlluminaMiSeqlibrarypreparationandsequencing 2522
500 ng of each purified RT-PCR sample was sheared with an M220 focused- 2523
ultrasonicator (Covaris) set to obtain peak fragment lengths of 400 bp. Next the 2524
NEBNextUltraDNALibraryPreparationkit (NewEnglandBiolabs)wasusedtorepair 2525
the end and to add the Illumina MiSeq-compatible barcode adapters to 100 ng 2526
fragmentedDNA.The resulting fragmentswere size-selectedat300 to400bpusing 2527
AgencourtAMPureXPbeadsizing(BeckmanCoulter).Then, indexeswereaddedina 2528
limited-cyclePCR(10cycles),followedbypurificationonAgencourtAMPureXPbeads. 2529
The fragments were analysed on a High Sensitivity DNA Chip on the Bioanalyzer 2530
(Agilent Technologies) before loading on the sequencing chip. After the 2×250 bp 2531
MiSeqpaired-endsequencingrun,thedatawerebase-calledandconvertedtoIllumina 2532
FASTQfiles(Phred+64encoding). 2533
NextGenerationSequencingdataanalysis 2534
Sequencedataanalyseswereperformedontheresulting IlluminaFASTQfiles (Phred 2535
64+encoding)usingCLCGenomicsWorkbench (Version7.0.3) following theanalysis 2536
pipelineasdescribed(VandenHoeckeetal.,2015).Theprocessedsequencingreads 2537
were subsequently mapped to the de novo assembled consensus sequence of the 2538
H9N2:pH1N1(p7)stockvirussequence.Thisconsensussequenceisavailablethrough 2539
GenBank accession number (KY785904-KY785911). The processed sequencing reads 2540
fortheH9N2:pH1N1(P7)stockvirusweremappedtoareferencegenomecomposed 2541
of the H9N2:pH1N1 (P7) stock virus sequence and the A/quail/Hong 2542
Kong/G1/1997(H9N2)virusgenome. Therawsequencingdataweresubmittedtothe 2543
NCBI Sequence Read Archive where they can be found under project number: 2544
SRP095510. 2545
2546
2547
Chapter4
87
4.3 Results 2548
SerialpassagesofareassortantH9N2virusresultedinincreasedvirusreplication 2549
Toevaluate if serialpig infectionsmade reassortantH9N2virusesbetter adapted to 2550
swine, 10 blind serial passages were performed in influenza naïve pigs. For each 2551
passage 2 co-housed animals were intranasally inoculated with 106,5 TCID50 of 2552
H9N2:pH1N1 (P0). Todeterminenasal virus excretion, individual pigswere swabbed 2553
dailyfrom0to4dayspost-inoculation(dpi).Noneofthepigsshowedclinicalsignsof 2554
disease.ThereassortantH9N2viruswasdetectedinnasalswabsofallexperimentally 2555
infected pigs. The highest amount of virus was excreted during passages 3 and 8 2556
(Figure1).At4daysafterinfection,pigswereeuthanizedandsamplesfromtheentire 2557
respiratory tract were collected to determine the viral load. Infectious virus was 2558
recoveredfromtherespiratorytractofallpigsduringall10serialpassages(Table1). 2559
Replicationrateswerehigherintheupperrespiratorytract(nasalmucosa:respiratory 2560
and olfactory parts) with all 40 out of 40 (100%) samples testing positive, when 2561
comparedtothelowerrespiratorytract(tracheaandlungs)with81outof140(58%) 2562
samplestestingpositive.Inthefirstpassage,noviruscouldbeisolatedfromthelower 2563
respiratorytract.Incontrast,duringpassages2,3and8,theviruswasdetectedin17 2564
outof18(93%)samples,indicatingreplicationintheentirerespiratorytract(Table1). 2565
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
88
2566
Figure 1.Nasal virus shedding of the reassortantH9N2 virus containing pH1N1 internal- 2567proteingenes (H9N2:pH1N1)during10blindserialpigpassages.Twopigs (solid lineand 2568dashed line)wereused for ineachpassage,each line represents thevirus titers innasal 2569swabsofanindividualpig.Thedetectionlimitofthetestisindicatedwithadottedlineat 25701.7log10TCID50/100mgofsecretion. 2571
Days post-inoculation
Vir
us ti
ter
log 1
0 TC
ID50
/100
mg
of n
asal
sec
retio
n Passage 1
0 1 2 3 40
2
4
6
8
Passage 2
0 1 2 3 40
2
4
6
8
Passage 3
0 1 2 3 40
2
4
6
8
Passage 4
0 1 2 3 40
2
4
6
8
Passage 5
0 1 2 3 40
2
4
6
8
Passage 6
0 1 2 3 40
2
4
6
8
Passage 7
0 1 2 3 40
2
4
6
8
Passage 8
0 1 2 3 40
2
4
6
8
Passage 9
0 1 2 3 40
2
4
6
8
Passage 10
0 1 2 3 40
2
4
6
8
2572Table 1. Distribution of reassortant H9N2 virus containing pH1N1 internal-protein genes in the respiratory tract during 10 serial 2573passagesinpigs. 2574
2575
Numberofpassage Pignumber
Virustiter(log10TCID50/gramoftissue)atday4post-inoculationaNasalmucosarespiratorypart
Nasalmucosaolfactorypart Proximaltrachea Distaltrachea Apical+cardiac
loberightApical+cardiac
lobeleftDiaphragmatic
loberightDiaphragmatic
lobeleftAccessory
lobe
1#1 5.3 5.5 <
b < < < < < <
#2 4.5 5 < < < < < < <
2#3 5.7 5.7 2 4.2 < 3.3 2 3.5 2.3#4 7 5.7 4.3 4 4.3 2.5 2.5 3.8 2.5
3#5 5.7 5.5 5.7 4.7 2.3 3.7 2.3 3 1.7#6 5.7 7.3 2.7 2 2.3 4.5 1.7 2.5 <
4#7 5.5 6.3 2.3 3.8 1.7 < 3.7 < 1.7#8 6.3 5.5 5 4.7 5.7 4 4.5 4.5 2.7
5#9 6 1.7 1.7 < < < < < <#10 4.7 2.5 4.2 2.7 6 2,8 3.2 < 5.5
6#11 5.5 3.5 1.7 < < < < < <#12 6.3 4 < < < < < < <
7#13 5.7 5.5 3.3 2.5 < 1.7 2.8 < <#14 5.5 4.3 1.7 < < < < < <
8#15 6.3 7 4.3 5.5 5.3 5.5 5.5 5.5 4.5#16 6.7 6.3 2.5 2.8 < 3.2 4,8 3,3 4,3
9 #17 6.5 6.7 1.7 < < < 1.7 < <#18 6 5.5 2.5 3.5 < 5.5 4.3 3.5 2,7
10#19 7 5.3 1.7 < 2 < 3.5 2.5 <#20 6.8 3.7 1.7 5 5.5 2.2 < 4.7 <
aVirustitersareshownforeachindividualpig(#). 2576b<detectionlimit(1,7log10TCID50/gramoftissue 2577
2578
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
90
After7serialpassagesinpigs,thereassortantH9N2:pH1N1virusshowedenhanced 2579
contacttransmission 2580
To compare the level of adaptation of the reassortant virus before and after serial 2581
passages in pigs, we performed direct-contact transmission experiments with the 2582
H9N2:pH1N1 (P0) virus and its descendant isolated after 7 passages in pigs 2583
(H9N2:pH1N1(P7)).Thelatterviruswaschosenbecausevirustitersinnasalswabsand 2584
the respiratory tract samples were highest at this passage, suggesting a substantial 2585
degree of swine-adaptation. As a positive control, we used a representative swine- 2586
adapted virus, the A/California/04/2009 (pH1N1) virus (A/Cal/04/09). All 3 viruses 2587
wereamplifiedinMDCKcellsbeforepiginoculation,andthetransmissionexperiment 2588
wasperformedintriplicateforeachvirus.Figure2illustratesthenasalvirusshedding 2589
of the inoculated and the co-housed direct-contact animals. All animals remained 2590
clinically healthy, based on clinical observation. All inoculated pigs excreted virus 2591
between days 1 and 7 post-inoculation. A comparison of the averages of the areas 2592
under the curve (AUC) revealeda lower virusexcretion forH9N2:pH1N1 (P0) (mean 2593
AUC19.4)andH9N2:pH1N1(P7)(24.0)thanforA/Cal/04/09(26.6),eventhoughthese 2594
differenceswerenotstatisticallysignificant(p>0.05).All3virusesreachedamaximum 2595
virus titer ≥ 6.5 log10 TCID50/100 mg of secrete. All inoculated animals developed 2596
neutralizingantibodiesagainst thehomologousvirusand thesewerehighest for the 2597
A/Cal/04/09infectedpigs(Table2). 2598
Chapter4
91
2599
2600
2601
Figure 2. Nasal virus shedding and direct-contact transmission of H9N2:pH1N1 (P0), 2602H9N2:pH1N1(P7) and A/Cal/04/09 influenza viruses in pigs. Three pigs were 2603intranasally inoculated with 6,5 log10 TCID50 of the indicated virus and housed in 3 2604separate isolators. Forty-eight hours later, 2 direct contact animals were co-housed 2605witheachinoculatedpigs.Eachgraphisidentifiedwitha2digitsnumber:thefirstone 2606corresponds to the isolator number and the second to the virus tested. Therefore, 2607eachcolumnrepresentsadifferentvirusandeachrowrepresentsadifferentisolator. 2608Thedetectionlimit(dottedline)ofthetestwas1.7log10TCID50/100mgofsecretion. 2609 2610
A/Cal/04/09 isolator 1.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8H9N2:pH1N1 (P7)
isolator 1.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8 isolator 2.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8 isolator 3.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
H9N2:pH1N1 (P0) isolator 1.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8 isolator 2.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8 isolator 3.1
A/Qa/HK/P0 isolator 1.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
A/Cal/04/09 isolator 1.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
A/Qa/HK/P4 isolator 1.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
Days post-contact inoculated pigs direct contact pigs detection limit!
Viru
s tite
r log
10 T
CID 50
/100
mg o
f sec
rete!
A/Qa/HK/P0 isolator 1.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.1
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
A/Cal/04/09 isolator 1.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.3
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
A/Qa/HK/P4 isolator 1.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 2.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
isolator 3.2
-2 -1 0 1 2 3 4 5 6 7 8 9 10 110
2
4
6
8
Days post-contact inoculated pigs direct contact pigs detection limit!
Viru
s tite
r log
10 T
CID 50
/100
mg o
f sec
rete!
Vir
us ti
ter
log 1
0 TC
ID50
/100
mg
of n
asal
secr
etio
n
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
92
Table 2. Serum VN antibody titers in inoculated and direct contact pigs involved in 2611transmissionexperiments. 2612
2613 Numberofantibodypositivepigs(rangeofantibodytiters) Inoculatedpigs(n=3) Directcontactpigs(n=6)
Virusstrain 0 16 23 29dpia 0 14 21 28dpcb
H9N2:pH1N1(P0) 0(<2) 3(16-128) 3(32-256) 3(32-192) 0(<2) 3(24-192) 6(12-192) 6(48-256)
H9N2:pH1N1(P7) 0(<2) 3(48-96) 3(64-256) 3(48-128) 0(<2) 5(2-256) 4(96-256) 4(64-192)
A/Cal/04/09 0(<2) 3(128-192) 3(192-384) 3(512-768) 0(<2) 6(192-1024) 6(256-1536) 6(192-1024)adpi:dayspost-inoculation.bdpc:dayspost-contact.
2614
Asexpected,all6A/Cal/04/09direct-contactpigsexcretedhighamountsofvirus(AUC 2615
>17)andseroconvertedwithVNtiters≥192.Whileall6direct-contactanimalsinthe 2616
H9N2:pH1N1(P0)groupalsoseroconvertedandexcretedvirus,only2shedviruswith 2617
anAUC>6.IntheH9N2:pH1N1(P7)group,incontrast,just4outof6direct-contact 2618
animalsexcretedvirusbutallofthemshowedanAUC>21,similartotheA/Cal/04/09 2619
group.Theremaining2H9N2:pH1N1(P7)direct-contactpigsdidnotexcreteanyvirus 2620
and failed to seroconvert. VN titers in the 6 H9N2:pH1N1 (P0) and 4 out of 6 2621
H9N2:pH1N1(P7)direct-contactpigswere<256. 2622
Mutationsassociatedwithenhancedtransmission 2623
WenextcomparedthevariantspresentintheH9N2:pH1N1(P0)viruspopulationand 2624
theH9N2:pH1N1(P7)virusisolatedfromthenasalmucosaofadirectlyinoculatedpig 2625
andthedirect-contactpigwiththehighestnasalexcretion inthetransmissionstudy. 2626
Bycomparingtheviruspopulationfromthedirectly-inoculatedandthedirect-contact 2627
pigsweaimedtodetermineifthemutationsselectedduringtheserialpassagingwere 2628
maintainedaftertransmissionorifthetransmissionitselffavouredtheacquirementof 2629
adifferentsetofmutations.Inaddition,theH9N2:pH1N1(P0)virusstockwasincluded 2630
to determine whether the detected variants in the passage 7 virus were already 2631
presentinthevirusstockbeforepigadaptation.IlluminaMiSeqdeepsequencingwas 2632
performed on full genome RT-PCR products of the samples. Sequencing of 2633
H9N2:pH1N1 (P0) virus revealed contamination of theH9N2:pH1N1 virus stockwith 2634
H9N2 internal-protein genes,whichweremost pronounced for the PB1,NS andNP 2635
gene segments (see Table 3). Interestingly, passage of the reassortant H9N2:pH1N1 2636
virus stock in pigs resulted in the selection of a virus that had lost these internal- 2637
Chapter4
93
protein coding H9N2 gene segments, except for the NP segment which was still of 2638
H9N2-origin after 7 pig passages. Sequence analysis of the H9N2:pH1N1 (P7) virus 2639
after contact transmission showed that the geneticmakeup of this viruswas nearly 2640
identicaltotheH9N2:pH1N1(P7)stockvirus.TheselectionforaH9N2viruswiththe 2641
pH1N1-origin internal protein-coding gene segments suggests a strong genetic 2642
bottleneckthatleadstoeliminationofreassortantviruseswithH9N2internalprotein- 2643
codinggenesegmentsuponviralpassaginginpigs.Thenonsynonymousvariantsthat 2644
were detected at a frequency ≥ 5% in the H9N2:pH1N1 (P7) stock virus and the 2645
H9N2:pH1N1 (P7) virus isolated from the direct-contact pig after mapping the 2646
sequencing reads to thedenovo assembledconsensus sequenceof theH9N2:pH1N1 2647
(P7) stock virusare shown in Table 4. As a consequence of the contamination, the 2648
sequencing reads of the H9N2:pH1N1 (P0) stock virus weremapped to a reference 2649
genomecomposedofthedenovoassembledconsensussequenceoftheH9N2:pH1N1 2650
(P7) stock virus and the reference sequence for the A/quail/Hong 2651
Kong/G1/1997(H9N2) virus. The nonsynonymous variants detected at a frequency ≥ 2652
0.5%intheH9N2:pH1N1(P7)genomeareshowninSupplementaryTableS1. 2653
2654
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
94
Table3.MappingofthereadsofthevirusstockofH9N2:pH1N1(P0),H9N2:pH1N1 2655(P7)andH9N2:pH1N1(P7)isolatedafterdirect-contacttransmissiontothereference 2656genome sample obtained from A/quail/Hong Kong/G1/1997 (H9N2) and 2657A/California/04/2009 (pH1N1). The viral gene segment is indicated along with the 2658frequency(percentage)ofsequencereadsoriginatingfromeitheroneoftheparental 2659strains. 2660
2661
Virusstock SegmentPercentageofgenesfrom…virus
A/quail/HongKong/G1/1997(H9N2)
A/California/04/2009(pH1N1)
H9N2:pH1N1(P0)
PB2 6 94PB1 19 81PA 11 89HA 100 0NP 91 9NA 100 0M 26 74NS 91 9
H9N2:pH1N1(P7)
PB2 0 100PB1 0 100PA 0 100HA 100 0NP 100 0NA 100 0M 0 100NS 0 100
H9N2:pH1N1(P7)
direct-contact
PB2 0 100PB1 2 98PA 0 100HA 100 0NP 100 0NA 100 0M 0 100NS 0 100
2662 2663
Chapter4
95
Table4.Variantspresentinthepassage7reassortant(H9N2:pH1N1(P7))virusisolated 2664from the nasal mucosa and from the nasal swab of the direct-contact pig with the 2665highest excretion. The viral gene segment is indicated, along with the nucleotide 2666positionandsubstitutions, thepredictedaminoacidchangeand itspositionand the 2667frequency (percentage) of sequence reads with the detected mutations. Only 2668nonsynonymoussubstitutionswithafrequency≥5%inthereadsareshown.Thevirus 2669inthenasalmucosasamplewasamplifiedonMDCKcellsbeforesequencing. 2670 2671
Segment Nucleotideposition Reference Mutation Aminoacid
change
FrequencyH9N2/pH1N1(P7)(nasalmucosa)
FrequencyH9N2/pH1N1(P7)
(nasalswab)
PA1260 T A Trp406Arg - 5.091990 TA - Leu649- - 5.56
PB2220 C G Thr58Ala - 18.551651 C T Thr535Met 46.74 -1891 T C Ile615Thr 8.09 -
HA 407 C T Leu119Phe 27.52 -750 A G Asp225Gly 47.43 99.70
NS 582 G A NS1:Gly179GlnNS2:Gly22Arg 30.45 -
862 C T NS2:Ala115Val - 25.83 2672
TheH9N2:pH1N1(P7)stockvirusshowedvariationabove5%atpositions535and615 2673
ofthebasicpolymerase2protein(PB2),position119oftheHAandposition179ofthe 2674
non-structural 1 protein (NS1) (Table 4). Nonetheless, those variants were not 2675
maintained after transmission to the contact pig. Interestingly, only 1 mutation 2676
associated with an amino acid substitution was selected after 7 passages and 2677
maintained after transmission, i.e. the substitution of aspartic acid by glycine at 2678
position 225 of the HA RBS (HA-D225G). This mutation was absent (Supplementary 2679
TableS1)intheH9N2:pH1N1(P0)virusstockandpresentin47.4%and99.7%ofthe 2680
sequences derived from the viral population isolated after 7 passages and the 2681
transmitted virus, respectively. Two other mutations appeared de novo after 2682
transmissionatafrequency>15%:asubstitutionofalaninetovalineatposition115of 2683
theNS1andasubstitutionofthreoninetoalanineatposition58ofthePB2. 2684
2685
4.4 Discussion 2686
Sincethelate1990s,avianH9N2viruseshavebeenisolatedfrompigsandhumans,so 2687
far always as dead end events (Butt et al., 2010, Wang et al., 2016, World Health 2688
Organization,2016b).However,thecontinuousexposureofhumansandpigstoavian 2689
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
96
H9N2virusesmightposearealthreatforpublichealthiftheseviruseswouldacquire 2690
thegeneticchangesthatallowthemtotransmitefficientlybetweenmammals.Inthis 2691
study, we evaluated the efficiency of direct-contact transmission between pigs of a 2692
reassortant H9N2 virus containing pH1N1 internal-protein genes, and the effect of 2693
serial pig passages on transmission efficiency.We demonstrated, for the first time, 2694
thatapig-passagedreassortantH9N2:pH1N1viruswasexcretedbymorethanhalfof 2695
thedirect-contactpigsattiterssimilartothoseoftheparentalpH1N1virus. 2696
Ourdataconfirmtheabilityof theH9N2:pH1N1reassortantviruses to infectpigsas 2697
was reported in previous studies (Qiao et al., 2012, The SJCEIRSH9WorkingGroup, 2698
2013,Obadanetal.,2015).Infact,virusexcretionfromthenasalcavityandreplication 2699
intherespiratorytractwerepresentatvariableratesduringthe10serialpigpassages. 2700
This findingcontrastswithapreviousstudy fromourgroupusing thesameparental 2701
H9N2virus(A/quail/HongKong/G1/1997),howeverwithoutreassortmentwithpH1N1 2702
internal-proteingenes(ManceraGraciaetal.,2017).Inthelatterstudy,replicationand 2703
sheddingoftheparentalH9N2viruswasslightlyenhancedafter4pigpassagesbutit 2704
wascompletelylostafter10passagesinpigs.Thisdemonstratesthatthepresenceof 2705
thewellpig-adaptedpH1N1-origin internal-proteingenesmayposeanadvantagefor 2706
theadaptationofH9N2virustopigs.WhiletheH9N2:pH1N1(P0)virusdidnotshow 2707
replication in the lungs during passage 1, it replicated homogeneously in the entire 2708
respiratory tract during passage 2. Strikingly, the virus replication in the lungs then 2709
progressivelydecreasedduringpassages4,5and6toincreaseagainatpassage7.This 2710
suggeststhepresenceofaselectionbottleneckduetothegeneticpressuregenerated 2711
bytheexperimentalsetup(Wilkeretal.,2013).However,adeeperanalysisoftheviral 2712
populationwould be needed to elucidate the possible cause of the decrease in the 2713
replicationrates. 2714
We consider an influenza virus as swine-adapted when it succeeds to transmit 2715
between pigswith a similar efficiency as described for the endemic swine influenza 2716
viruses.Therefore,wecomparedpig-to-pigtransmissionoftheseventhpassagevirus 2717
with that of the original reassortant and pH1N1 viruses.Whereas both reassortant 2718
virusestransmittedtodirect-contactpigs,theseventhpassageviruswasclearlyshed 2719
at higher titers by 4 out of 6 direct-contact pigs. This virus also showed higher 2720
Chapter4
97
transmissionefficiencywhencomparedwithpreviousstudiesdescribingH9N2:pH1N1 2721
reassortant viruses in pigs (Qiao et al., 2012, Obadan et al., 2015). In the 2722
aforementioned studies ≤ 50% of the direct-contact pigs excreted virus. This higher 2723
transmission rate suggests that the serial passaging in pigs contributed to the 2724
adaptation of the reassortantH9N2 virus. Yet of the 6 direct-contact pigs 2 animals 2725
thatwerehoused in thesame isolatordidnotexcretevirusatall.This ismost likely 2726
duetothelowamountofvirusexcretedbythedirect-inoculatedpig(AUC17.1),which 2727
not have reached a critical threshold to allow virus transmission. Further detailed 2728
genetic analyses of the virus excreted by the donor pigwould be needed to better 2729
understand if there is, besides the low titer, also a genetic basis for the lack of 2730
transmission. 2731
Toelucidatetheeffectsoftheserialpassagingandthepig-to-pigtransmissiononthe 2732
viruspopulation,wecomparedtheviraldiversityintheoriginalreassortantviruswith 2733
thevirus isolatedafter7passages inpigsand thevirusexcretedafterdirect-contact 2734
transmission. It should be mentioned here that we detected H9N2 internal-gene 2735
segments in our original reassortant virus stock. Interestingly, the serial passaging 2736
resulted in the purification of the original virus and, after 7 passages only the NP- 2737
codinggenewascompletelyofH9N2-origin.As intheoriginalstocktheH9N2-derive 2738
NP gene segment was present at 91%, this high percentage might have been the 2739
reason for the purification towards the H9N2-origin instead of towards the pH1N1- 2740
originasseenfortheothergenesegments.However,theNS-codinggeneshowedthe 2741
same percentage of contamination and was completely selected upon passaging. 2742
Althoughprevious studies demonstrated that pH1N1-origin internal genes conferred 2743
anadvantageinreplicationandtransmissionofinfluenzavirusesinmammals(Mehle 2744
and Doudna, 2009, Lakdawala et al., 2011, Kimble et al., 2011, Zhang et al., 2012, 2745
Campbelletal.,2014,Abenteetal.,2016),thosestudiesfocusedeitherontheroleof 2746
thePB2andtheM-codinggenesoronthecompleteinternalgenecassette.Notmuch 2747
is knownabout theexact roleofpH1N1-originNP-proteingene in theadaptationof 2748
influenza viruses to mammals. However, it has been described that the interaction 2749
between the NP and importin-α can influence the influenza virus host range. For 2750
example, thepresenceof theNP-N319K substitutionwasdescribed as an important 2751
factorforthereplicationofH7N7viruses inmice(Gabrieletal.,2008).Nevertheless, 2752
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
98
both the H9N2 and the pH1N1 NP-protein genes lack that mutation. Thus, further 2753
analysiswithreversegeneticgeneratedreassortantswouldbenecessarytoelucidateif 2754
thecombinationofH9N2-originNP-proteingeneandpH1N1-origininternalgeneshas 2755
an effect in the replication and transmission of influenza viruses in mammals. 2756
Regarding themutations detected during the analysis, only 1 of themutations that 2757
appeared in the virus after 7 passages andwasmaintained even after transmission, 2758
theHA-D225Gsubstitution in theRBS.Thismutationwas therefore likelyassociated 2759
withthehigherreplicationandtransmissionefficiencyoftheH9N2:pH1N1(P7)virus. 2760
Interestingly, this mutation was present in all 42 swine and 17 human H9N2 field 2761
isolates available in GenBank in November 2016. Thismutationwas also associated 2762
withenhancedreplicationandtransmissionofparentalH9N2after4passagesinpigs 2763
(Mancera Gracia et al., 2017). In the 1918 and 2009 pandemic H1N1 viruses this 2764
mutationhasbeenassociatedwith increasedSiaα2,3Gal tropism(Belseretal.,2011, 2765
Lakdawalaetal.,2015).InpigsandhumansSiaα2,3Galreceptorsaremainlyfoundin 2766
the lungs (VanPouckeetal.,2010).Thepresenceof theHA-D225Gmutationafter7 2767
passagesmaythereforeexplaintheconsistentreplicationinthelungsduringpassage 2768
8.Althoughspecificreceptor-bindingtestswouldbenecessarytoelucidatetheexact 2769
roleoftheHA-D225GmutationwithintheHA-RBS,ourdatasuggestthatitmaybea 2770
markerforadaptationofH9N2virusestoswine. 2771
In line with previous studies (Qiao et al., 2012, Obadan et al., 2015), this report 2772
demonstrated that avianH9N2 influenza viruses could reassortwith pH1N1 internal 2773
genes, resulting in enhanced virus transmission in pigs when compared to the 2774
parental, non-reassorted H9N2 virus. The present study also underscores that 2775
repeated introduction of reassortant H9N2 viruses into the swine or humansmight 2776
resultintheselectionofvirusvariantswithtransmissionefficiencyclosetothatofthe 2777
endemicswineorhumaninfluenzaviruses.However,thelackoftransmissiondetected 2778
in 1 of the contact groupsused for transmissionof passage 7 virus, emphasizes the 2779
complexityoftheadaptationprocessofanavianvirustoamammalianspeciesandthe 2780
need for additional research to better understand this crucial step in cross-species 2781
transmissionofinfluenzaviruses. 2782
2783
Chapter4
99
4.5 Acknowledgements 2784
The authors thank the Centers for Disease Control and Prevention (US CDC) for 2785
providing the pH1N1 influenza viruses and Lieve Sys, Melanie Bauwens and Nele 2786
Dennequin for excellent technical assistance. This study was supported by the EU 2787
FLUPIGproject(FP7-GA258084).SilvieVandenHoeckewassupportedbyFondsvoor 2788
WetenschappelijkOnderzoek(Flanders,Belgium)(3G052412). 2789
2790
Authorcontributions 2791
K.V.R. and J.C.M.G. designed research; J.C.M.G. performed the animal experiments; 2792
J.C.M.G. and S.V.D.H. performed the laboratory and the genetic analysis; K.V.R., 2793
J.C.M.G., S.V.D.H. and X.S. analysed data; K.V.R., J.C.M.G., S.V.D.H., X.S., W.M. and 2794
J.A.R.wrotethepaper. 2795
2796
Competingfinancialinterests 2797
Theauthorsdeclarenocompetingfinancialinterest. 2798
2799
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2966
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103
Supplementary Table S1: Variants present in H9N2:pH1N1 (P0) virus stock. The 2967sequencing reads of the H9N2:pH1N1 (P0) stock virusweremapped to a combined 2968reference genome composed of thede novo assembled consensus sequence of the 2969H9N2:pH1N1 (P7) stock virus and the reference sequence for the A/quail/Hong 2970Kong/G1/1997(H9N2) virus. The nonsynonymous variants detected in the 2971H9N2:pH1N1(P7)genomeabove0.5%areshownpergenomesegment. 2972 2973PB2(H1N1)
ReferencePosition Reference Allele Frequency Aminoacidchange81 T C 0.53 PB2:p.Ser12Pro90 C A 0.61 PB2:p.Arg15Ser141 A - 0.67 PB2:p.Lys32fs145 A G 1.16 PB2:p.Lys33Arg175 C A 0.69 PB2:p.Pro43His196 T A 0.87 PB2:p.Met50Lys199 T - 0.82 PB2:p.Met51fs214 C A 0.57 PB2:p.Pro56Gln237 A T 0.51 PB2:p.Met64Leu242 C A 0.80 PB2:p.Asp65Glu242 C G 0.55 PB2:p.Asp65Glu244 T C 0.55 PB2:p.Met66Thr325 C A 0.53 PB2:p.Pro93His1651 C T 100.00 PB2:p.Thr535Met1933 G A 11.11 PB2:p.Ser629Asn1987 TACTGG - 56.89 PB2:p.Ile647_Val649delinsIle1993 T - 3.36 PB2:p.Val649fs2100 G T 0.64 PB2:p.Gly685Trp2101 G T 0.62 PB2:p.Gly685Val2138 G T 0.51 PB2:p.Leu697Phe2146 A C 0.76 PB2:p.Glu700Ala2157 - AT 7.05 PB2:p.Tyr704fs2232 G T 0.54 PB2:p.Gly729Trp2233 G T 0.67 PB2:p.Gly729Val2263 G T 0.60 PB2:p.Arg739Leu
PB1(H1N1) ReferencePosition Reference Allele Frequency Aminoacidchange
74 AAAAATTCCAGCGCA - 1.25 PB1:p.Leu10_Gln15delinsLeu112 C A 0.67 PB1:p.Pro23His186 C A 0.53 PB1:p.Gln48Lys190 ACT GAA 13.38 PB1:p.Tyr49_Ser50delins*Thr192 T - 4.14 PB1:p.Ser50fs192 T A 12.87 PB1:p.Ser50Thr205 A G 55.27 PB1:p.Lys54Arg269 G T 6.88 PB1:p.Glu75Asp2146 C A 0.60 PB1:p.Pro701His2187 G A 0.87 PB1:p.Val715Met2224 G T 0.63 PB1:p.Arg727Met
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
104
SupplementaryTableS1:VariantspresentinH9N2:pH1N1(P0)stockvirus 2974(Continued). 2975
2976PA(H1N1)
ReferencePosition Reference Allele Frequency Aminoacidchange117 G T 0.52 PA:p.Gly25Trp118 G T 0.60 PA:p.Gly25Val211 A T 5.97 PA:p.Glu56Val214 G T 1.31 PA:p.Arg57Leu247 C A 1.23 PA:p.Pro68Gln340 G T 0.65 PA:p.Gly99Val418 G T 0.60 PA:p.Arg125Met695 A C 70.88 PA:p.Gln217His938 G T 1.82 PA:p.Glu298Asp1260 T A 2.67 PA:p.Trp406Arg1696 G T 0.54 PA:p.Arg551Met1775 G T 0.76 PA:p.Trp577Cys1921 G T 0.54 PA:p.Arg626Met1922 G T 0.68 PA:p.Arg626Ser1944 G T 0.56 PA:p.Gly634Trp1957 G A 1.28 PA:p.Arg638Lys2062 G T 0.68 PA:p.Arg673Met2094 G T 0.55 PA:p.Gly684Trp2095 G T 0.61 PA:p.Gly684Val2095 GG AA 0.69 PA:p.Gly684Glu2097 G T 0.55 PA:p.Gly685Trp2107 A G 0.82 PA:p.Glu688Gly2140 G T 0.59 PA:p.Trp699Leu
HA(H9N2) ReferencePosition Reference Allele Frequency Aminoacidchange
180 C A 0.56 HA:p.Pro43His254 C A 0.62 HA:p.Leu68Ile300 C A 0.76 HA:p.Pro83His314 C A 0.53 HA:p.Leu88Met381 C A 0.59 HA:p.Pro110His383 G T 0.57 HA:p.Gly111Trp459 C A 0.54 HA:p.Pro136Gln575 C A 0.58 HA:p.Gln175Lys591 G T 0.57 HA:p.Arg180Met616 G T 0.51 HA:p.Trp188Cys630 C A 0.67 HA:p.Pro193Gln757 G T 0.75 HA:p.Gln235His821 G T 0.74 HA:p.Gly257Trp899 A G 3.12 HA:p.Ser283Gly996 C A 0.60 HA:p.Pro315His1158 G T 0.63 HA:p.Gly369Val
Chapter4
105
SupplementaryTableS1:VariantspresentinH9N2:pH1N1(P0)stockvirus 2977(Continued). 2978 2979HA(H9N2)
ReferencePosition Reference Allele Frequency Aminoacidchange1437 G T 0.55 HA:p.Arg462Met1589 G T 0.68 HA:p.Gly513Trp1590 G T 0.57 HA:p.Gly513Val1669 G T 0.56 HA:p.Met539Ile
NP(H9N2) ReferencePosition Reference Allele Frequency Aminoacidchange
321 G T 0.62 NP:p.Gly86Trp331 C A 0.54 NP:p.Pro89Gln376 G T 0.62 NP:p.Trp104Leu525 C A 0.51 NP:p.Leu154Ile547 C A 0.54 NP:p.Pro161His579 C A 0.62 NP:p.Leu172Ile583 C A 0.83 NP:p.Pro173Gln617 G T 0.65 NP:p.Lys184Asn624 G T 0.54 NP:p.Gly187Trp632 G T 0.51 NP:p.Met189Ile747 G T 0.70 NP:p.Gly228Trp810 G T 0.56 NP:p.Gly249Trp943 G T 0.73 NP:p.Arg293Met948 G T 0.64 NP:p.Gly295Trp1041 A G 1.50 NP:p.Ser326Gly1110 G T 0.66 NP:p.Gly349Trp1111 G T 0.57 NP:p.Gly349Val1355 G T 0.62 NP:p.Lys430Asn1356 G T 0.53 NP:p.Gly431Trp1442 G T 0.51 NP:p.Gln459His1443 G T 0.73 NP:p.Gly460Trp1447 G T 0.72 NP:p.Arg461Leu
NA(H9N2) ReferencePosition Reference Allele Frequency Aminoacidchange
167 C A 0.69 NA:p.Pro43Gln328 C A 0.75 NA:p.Pro97Thr329 C A 0.61 NA:p.Pro97His367 G T 0.59 NA:p.Gly110Trp368 G T 0.57 NA:p.Gly110Val377 G T 0.90 NA:p.Trp113Leu522 G T 0.51 NA:p.Leu161Phe540 G T 0.69 NA:p.Leu167Phe619 G T 0.53 NA:p.Gly194Trp620 G T 0.71 NA:p.Gly194Val811 G T 0.62 NA:p.Gly258Trp
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
106
SupplementaryTableS1:VariantspresentinH9N2:pH1N1(P0)stockvirus 2980(Continued). 2981 2982NA(H9N2)
ReferencePosition Reference Allele Frequency Aminoacidchange878 C A 0.62 NA:p.Pro280His1046 G T 0.61 NA:p.Arg336Met1066 G T 0.54 NA:p.Gly343Trp1084 G T 0.62 NA:p.Gly349Trp1119 G T 0.56 NA:p.Met360Ile1166 G T 0.61 NA:p.Arg376Met1318 G T 0.59 NA:p.Gly427Trp1415 G T 0.56 NA:p.Gly459Val
M(H1N1) ReferencePosition Reference Allele Frequency Aminoacidchange
92 C A 0.69 M1:p.Pro16Gln103 C A 0.65 M1:p.Leu20Ile301 G T 0.52 M1:p.Gly86Trp307 G T 0.51 M1:p.Gly88Trp308 G T 0.51 M1:p.Gly88Val314 C A 0.57 M1:p.Pro90Gln366 A G 0.77 M1:p.Ile107Met450 G T 0.62 M1:p.Met135Ile514 T G 0.52 M1:p.Ser157Ala670 A G 0.66 M1:p.Thr209Ala687 T G 0.84 M1:p.His214Gln703 G T 0.52 M1:p.Gly220Trp745 C A 1.30 M1:p.Leu234Ile762 C T 1.29 M2:p.[Pro10Leu]771 G A 1.15 M2:p.[Ser13Asn]774 A G 1.20 M2:p.[Glu14Gly]
777 G T 0.70 M1:p.[Met244Ile]M2:p.[Trp15Leu]
780 A G 1.12 M2:p.[Glu16Gly]814 CA TG 1.19 M2:p.Val27_Ile28delinsValVal825 A G 1.64 M2:p.Asn31Ser833 G T 0.53 M2:p.Gly34Trp860 AC CT 1.47 M2:p.Thr43Leu951 G T 0.64 M2:p.Arg73Met963 AA GG 1.02 M2:p.Gln77Arg978 G A 1.13 M2:p.Ser82Asn
NS(H1N1) ReferencePosition Reference Allele Frequency Aminoacidchange
61 CA TG 3.90 NS1:p.[Thr5_Met6delinsThrVal]NS2:p.[Thr5_Met6delinsThrVal]
98 A G 2.75 NS1:p.Ile18Val
Chapter4
107
SupplementaryTableS1:VariantspresentinH9N2:pH1N1(P0)stockvirus 2983(Continued). 2984
2985NS(H1N1)
ReferencePosition Reference Allele Frequency Aminoacidchange119 A C 1.63 NS1:p.Asn25His127 G T 0.75 NS1:p.Leu27Phe138 C A 0.55 NS1:p.Pro31Gln161 G A 0.74 NS1:p.Asp39Asn177 A G 0.82 NS1:p.Lys44Arg186 G A 0.75 NS1:p.Gly47Asp189 A G 0.84 NS1:p.Asn48Ser222 T G 0.77 NS1:p.Leu59Arg235 A T 0.76 NS1:p.Gln63His244 AT GC 0.74 NS1:p.Glu66_Trp67delinsGluArg272 A G 0.58 NS1:p.Thr76Ala329 C A 0.75 NS1:p.Leu95Ile342 C A 0.66 NS1:p.Ser99355 C A 0.73 NS1:p.Phe103Leu364 G T 0.51 NS1:p.Met106Ile477 - C 0.95 NS1:p.Leu144fs502 G T 2.33 NS1:p.Glu152Asp505 G T 0.52 NS1:p.Glu153Asp507 G A 0.56 NS1:p.Gly154Glu557 T G 1.07 NS1:p.[Tyr171Asp]559 T C 1.11 NS2:p.[Met14Thr]578 G A 1.51 NS1:p.[Val178Ile]
582 G T 0.57 NS1:p.[Gly179Val]NS2:p.[Gly22Trp]
612 G A 1.30 NS1:p.[Gly189Asp]NS2:p.[Val32Ile]
619 G A 1.16 NS2:p.[Arg34Gln]
636 A C 1.00 NS1:p.[Asn197Thr]NS2:p.[Ile40Leu]
638 A C 0.93 NS1:p.[Ile198Leu]650 G A 1.23 NS1:p.[Ala202Thr]
660 A G 1.08 NS1:p.[Asn205Ser]NS2:p.[Thr48Ala]
662 T A 0.87 NS1:p.[Cys206Ser]
674 G T 0.78 NS1:p.[Gly210Trp]NS2:p.[Met52Ile]
680 C T 1.48 NS1:p.[Pro212Ser]683 T C 1.40 NS1:p.[Ser213Pro]688 A C 1.04 NS2:p.[Tyr57Ser]695 G A 1.30 NS1:p.[Glu217Lys]697 G A 1.25 NS2:p.[Ser60Asn]704 T C 1.02 NS1:p.[220Arg]
2986
EffectofserialpassagesintheadaptationofareassortantH9N2containingpH1N1internalgenes
108
SupplementaryTableS1:VariantspresentinH9N2:pH1N1(P0)stockvirus 2987(Continued). 2988 2989NS(H1N1)
ReferencePosition Reference Allele Frequency Aminoacidchange
706 A G 1.05 NS1:p.[220Trp]NS2:p.[Glu63Gly]
726 G A 1.02 NS2:p.Gly70Arg742 A G 0.91 NS2:p.Glu75Gly765 A G 1.03 NS2:p.Met83Val861 G A 0.74 NS2:p.Ala115Thr
109
2990
GENERALDISCUSSION 2991
29925
Generaldiscussion
110
PotentialofadaptationofavianH9N2influenzavirusestopigs 2993
AvianH9N2 influenza virusesarewidespread in land-basedpoultry inmostEurasian 2994
countries(SunandLiu,2015).Since1998,thoseviruseshavebeenrepeatedlyisolated 2995
from pigs and humans, but always as dead-end events (Guo et al., 2000, Yu et al., 2996
2011,WorldHealthOrganization,2017).However, thecontinuousexposuretoH9N2 2997
AIVs might pose a threat if those viruses could be transmitted efficiently among 2998
mammals.Althoughpigs are considered toplay a role as intermediatehosts for the 2999
transmissionofAIVs tohumans, it remainsunknownwhetherH9N2AIVs couldever 3000
becomeadaptedtopigs.Sixstudiesbyother researchersalreadydemonstratedthat 3001
pigs are susceptible to different H9N2 virus strains, but they did not answer the 3002
questionastowhetherH9N2virusesmightbecomeestablished inswine(Kidaetal., 3003
1994,Qiaoetal.,2012,TheSCEIRSH9N2workinggroup,2013,Obadanetal.,2015, 3004
Wangetal.,2016).Therefore,thefirstaimofthisthesiswastoevaluatethepotential 3005
ofH9N2virusestobecomeadaptedtopigs. 3006
OurresearchconfirmstheabilityofH9N2AIVsto infectpigsas reported inprevious 3007
experimentalstudiesandinthefield(Kidaetal.,1994,Sunetal.,2010,TheSJCEIRSH9 3008
Working Group, 2013, Wang et al., 2016). The susceptibility of pigs to diverse AIV 3009
subtypes has been demonstrated by several researchers and it is one of the main 3010
argumentstosupporttheroleofthepigasanintermediatehostinthegenerationof 3011
pandemicinfluenzaviruseswithavianHAgenes(Kidaetal.,1994,DeVleeschauweret 3012
al.,2009a,Zhuetal.,2013).Inourstudy,thereplicationratesoftheoriginalH9N2and 3013
itsvirusdistributionintherespiratorytractofpigsweresimilartothosedescribedby 3014
De Vleeschauwer and colleagues in studies with a “purely avian” H5N2 LPAIV (De 3015
Vleeschauwer et al., 2009a). However, as described with other AIVs, infection with 3016
H9N2didnotresultinefficientpig-to-pigtransmission. 3017
Theserialpigpassagesinducedaselectivepressureonthevirusthatledtoabroader 3018
tissue infectionand increasedvirusreplicationafter4passages.Thisconsequenceof 3019
theserialpassagingwasnotonlydescribedbytheincreasedbindingofanavianH3N2 3020
virus toSiaα2,6Gal receptors inpigs (Shichinoheetal.,2013),butalsoshownbythe 3021
adaptationofH5N1andH9N2AIVstoferrets(Sorrelletal.,2009,Herfstetal.,2012).It 3022
is interesting to mention that the broader tissue infection and the increased virus 3023
replication described after 4 passages were not correlated with the in vitro growth 3024
Chapter5
111
kineticsofthevirus.Thisfindingconfirmsthat invitroresults incontinuouscell lines 3025
are often not relevant for the performance of the virus in the animal host. Similar 3026
findings were made in previous H9N2 studies, in which the pathogenicity and the 3027
virulenceofthevirusinpigsdidnotmimicthereplicationkineticsinMDCKandA549 3028
cell lines(Qiaoetal.,2012). Inaddition,ourkineticassaywasperformedwithMDCK 3029
cells that are known to express in both avian-type (Siaα2,3Gal) and human-type 3030
(Siaα2,6Gal)cell receptors.Thus, thiscell line ispermissive tomost influenzaviruses 3031
andthedifferencesduetoadifferentreceptor-bindingaffinityandnot influencethe 3032
celllineinfection. 3033
From pig passage 7 onwards replication of the H9N2 virus progressively decreased 3034
until itwas lost.This loss-of-functionmaybeexplainedby thenegativeeffectof the 3035
selectivepressure.Itwasalreadydemonstratedthattransmissionofinfluenzaviruses 3036
in ferrets incurs in a selective pressure. This pressure is supposed to generate a 3037
bottleneckthatdecreasesviraldiversityallowingminorvariantstobecomedominant 3038
(Wilker et al., 2013). The stringency of those bottlenecks depends in part on the 3039
infection route (aerosol infection versus direct contact infection), and it can change 3040
duringhostadaptation(Varbleetal.,2014,Monclaetal.,2014). InthecaseofH5N1 3041
virusesand1918pandemicH1N1itappearedthattheselectivepressurefavouredthe 3042
viruseswithenhancedfitness(Wilkeretal.,2013,Monclaetal.,2014).However,ina 3043
similarstudydonewiththeH7N9subtypethebottleneckseemedtohavetheopposite 3044
effect and limited the transmissionof the virus (Zaraketet al., 2015). Therefore,we 3045
hypothesized that the serial passages had a positive effect on the selection of virus 3046
variantswithhigher replicationefficiencies.However, at a certainpoint theadditive 3047
effectofsuccessiveselectivepressuresmaybecomenegativeandthismayresultina 3048
progressive decrease of the virus replication and excretion (Figure 1). As our serial 3049
passages were blind this progressive decrease in virus replication resulted in the 3050
inoculation of the subsequent animals with insufficient amounts of virus and a 3051
completelossofthevirusatpassage10.Extensivenextgenerationsequencing(NGS) 3052
analysisofallpassageswouldbeneededtoexaminethegeneticevolutionofthevirus 3053
duringthe10passagesandtoproveourhypothesis. 3054
Generaldiscussion
112
A B 3055
Figure 1. (A) Hypothesis on the effect of serial passaging in the generation of a 3056selective pressure on the virus that selects for different virus variants but also 3057decreasesvirusdiversity.(B)Theselectivepressurecomesmorerestrictive(narrower 3058bottleneck)withmorepassages.Thisresultsinadecreaseinvirusfitnessthatwilllead 3059to the complete loss of the virus at passage 10 (Figure adapted fromMoncla et al. 30602016). 3061 3062ThefourthpassageH9N2virusshowedenhanceddirect-contacttransmissionbetween 3063
pigswhen compared to the original virus. Though transmission of this virus did not 3064
result in high virus excretion by the direct-contact pigs, it was higher than that 3065
observedinpreviousH9N2studiesinpigs,orinotherstudieswithH7N9andotherAIV 3066
subtypes(DeVleeschauweretal.,2009b,TheSJCEIRSH9WorkingGroup,2013,Liuet 3067
al.,2014,Wangetal.,2016).However,transmissionrateswerelowerwhencompared 3068
withH9N2adaptationstudies inmammalsother than thepig.Forexample,after15 3069
passageswithanavianH9N2isolateinguineapigs3outof3direct-contactguineapigs 3070
excretedhighamountsofvirus(Sangetal.2015).Ofcourse it isdifficulttocompare 3071
ourresultsinpigswiththoseobtainedinotheranimalspeciesbecauseofthedifferent 3072
animal models, the different virus strains and experimental conditions used. We 3073
selected swine because it is a natural influenza virus host that may act as an 3074
intermediary in theadaptationofAIVs tomammals,butalsobecausepigs resemble 3075
humans in terms of physiology, anatomy and Sia receptor distribution. In contrast, 3076
other researchers used the ferret as it is considered the “gold standard”model for 3077
research on adaptation of avian influenza viruses tomammals (Belser et al., 2016). 3078
Guineapigsormicearealsobeingusedtoevaluatetransmissionand/orvirulenceof 3079
“adapted”AIVsduetotheireasymanagementinexperimentalconditions.Inaddition 3080
to the evident physiological or physical differences posed by those animal models 3081
therearealsodifferencesinexperimentaldesign.Inpigs,duethelimitationsposedby 3082
their size, we performed direct-contact transmission experiments. However, most 3083
Serial'passages'put'selec.ve'pressure'on'the'virus'that'promote'the'selec.on'of'few'virus'variants'and'decrease'viral'diversity'
The' selec.ve' pressure' turns' to' be' more'restric.ve'“narrower'bo;leneck”'as'passages'are' performed.' This' results' in' a' decrease' in'virus' fitness' that' will' lead' to' the' complete'loss'of'the'virus'at'passage'10''
Passage'1' Passage'2'
...'Passage'7' Passage'8' Passage'9' Passage'10'
...'
Chapter5
113
studies using ferrets or guinea pigs were more restrictive and considered efficient 3084
droplet transmission as a requirement for efficient adaptation (Sorrell. et al., 2009, 3085
Herfst et al., 2012, Imai et al., 2012, Sang et al., 2015). Although we consider that 3086
respiratory droplet transmission restricts virus transmission, it is clear that direct- 3087
contact transmission is one of themain pathways of influenza virus transmission in 3088
nature.Weassumethatavirusthattransmitsamongpigswithasimilarefficiencyas 3089
endemicswineinfluenzaviruses,willbeefficientlytransmittedinnatureaswell. 3090
In conclusion, pigs are susceptible to H9N2 infection under experimental conditions 3091
andserialpassagesinpigsposeaselectivepressurethatenhancesvirusfitness.Thus, 3092
the pig may act as intermediate host for H9N2 AIVs. However, our results 3093
demonstrated that serial passages were not sufficient to confer efficient pig-to-pig 3094
transmission. This is consistent with the fact that H9N2 influenza viruses have 3095
recurrently infected pigs in nature but never became established in swine. 3096
Furthermore, previous experimentswithH9N2 in ferrets also support thesedata. In 3097
those experiments reassortment with human seasonal H3N2 internal genes was 3098
necessary to achieve ferret-to-ferret transmission (Sorrel et al., 2009). However, 3099
introductionof4punctualmutationsintheHAand1inthePB2genesinaH5N1virus 3100
wassufficienttoachieveefficientdroplettransmissioninferrets(Herfstetal.,2012). 3101
ThisrevealsthatadaptationofH9N2AIVstopigsisnotastraightforwardprocessand 3102
thatacombinationofmolecularchangesmaybe required toachieve fulladaptation 3103
(ofH9N2AIVsinpigs). 3104
3105
TheroleofpH1N1internalgenesintheadaptationofavianH9N2virusestopigs 3106
Our results demonstrate that the “pig-adapted”pH1N1-origin internal protein-genes 3107
mayposeanadvantagefortheadaptationofH9N2virustopigs.Thisisconsistentwith 3108
previous studies in which similar reassortant H9N2 viruses showed enhanced 3109
transmission inpigswhencomparedwith theoriginalavianH9N2 (Qiaoetal.,2012, 3110
Obadanet al., 2015).Although those virusesdidnot transmit asefficientlybetween 3111
pigs as the “swine-adapted” viruses, similar reassortant viruses showed efficient 3112
droplet transmission in ferrets (Kimble et al., 2011). These results also highlight the 3113
importance of the pH1N1 internal gene cassette in the adaptation of influenza A 3114
viruses to mammals. This “quadruple-reassortant” internal gene cassette contains 3115
Generaldiscussion
114
avian,human,NorthAmericanandEuropeanswine-origininfluenzavirusgenesandis 3116
widespreadinswineandhumansworldwide(Watsonetal.,2015,Rajaoetal.,2016). 3117
As mentioned in Chapter 4.4.3, the H9N2 reassortant virus used in our study was 3118
contaminated. Its internal genes were a combination of H9N2 and pH1N1-origin, 3119
insteadof only pH1N1-origin. This contaminationwas possibly originatedduring the 3120
reverse genetic generation of the virus due to the unexpected presence of H9N2 3121
internalgenesplasmids.However, theserialpassagingof thesevirusesclearly led to 3122
the selection of a virus possessing mainly pH1N1-origin internal genes, the only 3123
exceptionwasthegeneencodingtheNP,whichoriginatedfromtheavianH9N2.The 3124
NPproteinhasbeenshowntoplayaroleinthehostadaptationbyitsinteractionwith 3125
the host-cell importin-a. This mediates the host-cell protein migration of the viral 3126
ribonucleoprotein to the nucleus and facilitates the evasion from the immune 3127
response in humans (Cauldwell et al., 2014). It was shown that human NP was 3128
necessary forefficient replicationofH7viruses in transgenicmiceexpressinghuman 3129
MxA,whichisaninterferon-stimulatedproteinthatrestrictsvirusreplication(Deeget 3130
al., 2017). Therefore, the fact that avian-originNP remained in our virus and itwas 3131
transmissibleinswinecontrastswiththeseexperimentsintransgenicmice.However, 3132
it raises thequestionhowthis“swine-adapted”viruswouldperform inhumansas it 3133
was demonstrated that avian viruses require a human adapted NP to evade the 3134
negativeeffectoftheMxArestrictionfactorinvirusreplication(Deegetal.,2017). 3135
Unlike the original H9N2, the reassortant H9N2 was not lost after serial passaging. 3136
Moreover, after 7 passages in pigs the reassortant virus showed enhanced 3137
transmission efficiency when compared with the original virus and with previous 3138
studieswithH9N2:pH1N1reassortantvirusesinpigs(Qiaoetal.,2012,Obadanetal., 3139
2015), but also with the “adapted” H9N2 virus tested in Chapter 3. This higher 3140
transmissionratesuggeststhatthecombinationofreassortmentandserialpassaging 3141
in pigs contributed to the adaptation of the reassortant H9N2 virus. This finding is 3142
important because bothH9N2 and pH1N1 viruses are co-circulating in Asia and can 3143
infect pigs and humans increasing the possibilities of reassortment. The repeated 3144
introductionof such reassortantH9N2viruses inhumansor swinemay result in the 3145
selection of virus variants with a transmission efficiency close to that of the 3146
mammalianadaptedinfluenzaviruses. 3147
Chapter5
115
GenetictraitsthatinfluencetheadaptationofavianH9N2influenzavirusestopigs 3148
InfluenzaAvirusadaptationtoanewhostrelieson2mainmechanisms:aamutations 3149
andgeneticreassortment.Sincethemid1990s,fewkeypointaminoacids(i.e.aa190, 3150
225,226and228intheHARBSoraa627and701inthePB2proteingenes)havebeen 3151
identified as important geneticmarkers for AIVs to achieve adaptation tomammals 3152
(Subbarao et al., 1993, Connor et al., 1994, Vines et al., 1998, Glaser et al., 2005). 3153
However,theirroleintheadaptationofAIVstopigsremainsuncertain. 3154
Forinstance,theoriginalH9N2strainusedinourexperimentscontainedaleucineat 3155
position 226 of the HA RBS instead of glutamine that is typical for AIVs. This 3156
substitution,whichwasshowntoincreasetheSiaα2,6Galbindingpreferenceinhuman 3157
respiratory cells, has been also associated with higher replication rates and 3158
transmissibility of H3, H5, H7 and H9 subtype viruses in pigs and ferrets (Wan and 3159
Perez,2007,Wanetal.,2008,Herfstetal.,2012,VanPouckeetal.,2013,Liuetal., 3160
2014).Thus,thepresenceofthismutationinouroriginalviruswassupposedtobean 3161
adaptationadvantage.However,inourexperimentsthissubstitutiondidnotresultin 3162
enhancedvirus replicationandtropismwhencomparedto thatofH5N2AIVs,which 3163
didnothaveleucine,inpigs(DeVleeschauweretal.,2009b).Thus,ourdataquestion 3164
theroleoftheHA-Q226LsubstitutioninreplicationefficiencyofH9N2virusesinpigs. 3165
Thisisconsistentwithotherstudies,whichshowthatresidue226inHARBSwasnot 3166
strictlydeterminativeforthereplicationofH9N2virusesinpigsorferrets(TheSJCEIRS 3167
H9WorkingGroup,2013).AlthoughcomparativestudieswithHA-226LandHA-226Q 3168
would be required to evaluate the effect of the HA-Q226L substitution in pigs, our 3169
results show that the adaptation process of H9N2 viruses to pigs is much more 3170
complexthantheacquisitionofasinglepointmutationintheHARBS. 3171
During the pig passages with the wholly avian H9N2 virus, we observed a new aa 3172
substitution, which was related with a broader tissue tropism and increased 3173
transmission. In addition, we demonstrated that upper and lower respiratory tract 3174
selectedfordifferentvirusvariants.Similarfindingsweremadeinferrets(Lakdawala 3175
etal.2015)andtheymaybeduetothedifferentreceptordistributionpresentinthe 3176
lungscomparedtotheupperrespiratorytract(VanPouckeetal.,2010,Trebbienetal., 3177
2011). During the serial passages with the reassortant virus (Chapter 4), we also 3178
observed the emergence of new substitutions. Only 1 out of the 19 mutations 3179
Generaldiscussion
116
described in both experiment, the HA-D225G substitution, was common to both 3180
experiments.ThissubstitutionislocatedintheHAreceptorbindingsiteandisknown 3181
toresultinadifferenceintheHARBSsurfacestructure,seeFigure2.Furthermore,the 3182
interactionoftheaaontheRBSwiththeSiareceptorisdependentonelectriccharges 3183
andthismutationresultsinthesubstitutionofanegativecharged(asparticacid)aaby 3184
anunchargedone (glycine). Thus, itmayaffect the interactionof the viruswith Sia. 3185
However,asdemonstratedinChapter3,thismutationwasmaintainedwhenthevirus 3186
wasprogressivelygettinglostduringtheserialpassages.Interestingly,all47swineand 3187
12 human H9N2 field isolates available in GenBank also showed this HA-D225G 3188
mutation,whileHA-Q226Lwaslackinginsomeofthem(seeTable3). 3189
3190
Table 1.Numberof swine andhumanH9N2 field isolates available inGenBank that 3191containtheHA-D225Gand/ortheHA-Q226Lsubstitutions. 3192 3193
H9N2virus
isolatedfrom
Numberofisolateswith…substitution
D225G Q226L
Humans(n=17) 17/17(100%) 14/17(82%)
Pigs(n=42) 42/42(100%) 25/42(60%)
3194
The HA-D225G substitution has been previously described in the literature as an 3195
important host specificity marker in H1 viruses (Taubenberger and Kash, 2010, 3196
Romero-Tejeda and Capua, 2013, Richard et al., 2014). In 1918 and 2009 H1N1 3197
pandemic viruses, this substitution has been associated with increased Siaα2,3Gal 3198
tropism conferring those viruses dual Siaα2,6Gal and Siaα2,3Gal receptor binding 3199
affinity(Tumpeyetal.,2007,Belseretal.,2011,Lakdawalaetal.,2015).Inpigsandin 3200
humansSiaα2,3Galreceptorsarepredominantlyfoundinthelungs(VanPouckeetal., 3201
2010). The emergence of this mutation could therefore explain the enhanced virus 3202
replication in the lungs. However, in vitro sialylglycoprotein binding assays H9N2 3203
viruses containing HA-225G and HA-226L, showed only slightly increased Siaα2,6Gal 3204
binding affinity (Matrosovich et al., 2001, Lakdawala et al., 2015). In addition, this 3205
substitution has never been described as an in vivoadaptationmarker of other AIV 3206
subtypessuchasH5,H7orH9(Sorrelletal.,2009,Herfstetal.,2012,Liuetal,2014). 3207
Thoughourdata suggest that theHA-D225Gsubstitution isan importantmarker for 3208
Chapter5
117
adaptation ofH9N2 viruses to pigs, further lectin-binding analysiswill be needed to 3209
confirmtheimportanceofthismutationforthereceptor-bindingaffinity. 3210
3211
3212
3213
Figure2.Comparisonofthesurfacerepresentationofthereceptor-bindingsiteinthe 3214HA1 monotrimer of: (A) A/quail/Hong Kong/G1/1997 (H9N2) virus before serial 3215passagingcontainingD225andL226,and (B) thesamevirusafter4passages inpigs 3216containingG225andL226.Inbothfiguresaa225and226arehighlightedinmagenta 3217andorangerespectively.Thearrowpointstothedifferent3drepresentationproduced 3218bythesubstitutionintheaa225. 3219 3220
Thesecondmechanismofadaptationisthereassortmentofgenesegmentsbetween2 3221
(ormore) different influenza viruses that co-infect the same cell. Thismechanism is 3222
more drastic but genetic reassortment involving AIVs has been involved in the 3223
generationofmostendemicinfluenzavirusesinpigsandhumans.Actually,theH3N2 3224
andH1N2SIVthatareendemic inEuropeanpigsandtheH1N1,H3N2andH1N2SIV 3225
that circulated in North America between 1998 and 2009 were generated by 3226
reassortment of avian, and human viruses. In humans, H2N2 and H3N2 pandemic 3227
viruses were also reassortants containing avian-origin surface genes combined with 3228
internalgenesofhuman-adaptedviruses.Furthermore,the2009pandemicH1N1virus 3229
was a reassortant containing avian, human and swine-origin genes. Its particular 3230
internalgenecassettebecameefficientlyadapted topigsandhumansandhasbeen 3231
describedtoenhancehumantransmissionofotherAIVssuchasH5,and/orH9(Mehle 3232
andDoudna,2009,Kimbleetal.,2011,Lakdawalaetal.,2011,Sunetal.,2011,Imaiet 3233
al.,2012,Zhangetal.,2012,Abenteetal.,2016).Ourexperimentsconfirmedprevious 3234
reportsand,besidestheNPprotein-gene,wedemonstratedthatpH1N1internalgenes 3235
A BA
Generaldiscussion
118
were selected upon adaptation to pigs. After 7 passages the reassortant virus was 3236
transmitted at higher rates to 4 our out of 6 contact-pigs. Those transmission rates 3237
werehigherwhen compared tootherAIVsbutwere still not at the level of pH1N1, 3238
which transmitted to 6 out of 6 contact-pigs. To elucidate the reason why the 3239
reassortant H9N2 virus was not transmitted to all the direct-contact pigs, genetic 3240
analysis of the viruses isolated from the transmission experiments should be 3241
performed. In addition, a subsequent transmission experiment performed with the 3242
virus excreted from the direct-contact pigs may result in transmission to all direct- 3243
contact animals. Thiswould be in agreementwith theH5N1 adaptation experiment 3244
performed byHerfst and colleagues inwhich they obtained efficient ferret-to-ferret 3245
transmission after 10 serial passages and 3 transmission experiments (Herfst et al., 3246
2012) 3247
3248
Conclusionsandimplications 3249
• SerialpassagesofanavianH9N2influenzavirusinpigsresultedinanenhanced 3250
virus replication and transmission. However, it was less efficient when 3251
compared to theendemic swine influenza viruses. This is consistentwith the 3252
naturalscenarioinwhichavianH9N2virusesarerecurrentlyisolatedfrompigs 3253
butneverbecameestablishedinswinepopulations.Similarly, inexperimental 3254
studies in ferrets efficient droplet transmission was only obtained upon 3255
reassortmentwithmammalianadaptedstrains. 3256
• The combination of pH1N1 internal genes and serial pig passages enhanced 3257
H9N2virusreplicationinandtransmissionbetweenpigs. 3258
• TheHA-D225Gsubstitutionemergedinbothexperimentalset-ups.Itispresent 3259
intheRBSoftheHA.Thus,itthatmayhaveanimportantroleintheadaptation 3260
of avian H9N2 to pigs. Nevertheless, further research will be needed to 3261
elucidatetheexactroleofthismutation. 3262
• OurstudiesputlightintheadaptationofH9N2topigs,whichisaninteresting 3263
model to evaluateAIVs transmission tomammals.However, up tonowadays 3264
thepH1N1wastheonlyswine-originvirusthatbecameestablishedinhumans. 3265
So far, the ferret is the reference animal model to evaluate the pandemic 3266
threat of influenza viruses. It could be interesting to perform further 3267
Chapter5
119
transmissionexperiments in ferretswiththevirusesgenerated inourstudies. 3268
Ontheotherhand,neitherpigsnorferretsarehumansandferretsseemtobe 3269
moresusceptibletoinfluenzavirusesthananyothermammalianspecies. 3270
• Toconclude,our studies showed thatadaptationofH9N2viruses topigs isa 3271
complexprocessthatinvolvesmutationsinsingleprotein-genesaswellasthe 3272
interactionsbetweenthosegenes. 3273
3274
References: 3275 3276Abente, E. J., Kitikoon, P., Lager, K.M.,Gauger, P. C., Anderson, T. K. et al. (2016). A highly 3277
pathogenic avian influenza virus H5N1 with 2009 pandemic H1N1 internal genes 3278demonstratesincreasedreplicationandtransmissioninpigs.JGenVirol,98,18-30. 3279
3280Belser, J. A., Jayaraman, A., Raman, R., Pappas, C., Zeng, H. et al. (2011). Effect of D222G 3281
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3284Belser, J. A. and Tumpey, T. (2016). Mammalian experimental models and implications for 3285
understanding zoonotic potential. In: Swayne, D. E. (Ed.)Animal influenza. First ed. John 3286WileyandSons,Ames,Iowa,594-619. 3287
3288Connor, R. J., Kawaoka, Y.,Webster, R. G. and Paulson, J. C. (1994). Receptor specificity in 3289
human,avian,andequineH2andH3influenzavirusisolates.Virology,205,17-23. 3290 3291Deeg,C.M.,Hassan,E.,Mutz,P.,Rheinemann,L.,Gotz,V.etal.(2017).InvivoevasionofMxA 3292
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3299DeVleeschauwer,A.,VanPoucke,S.,Braeckmans,D.,VanDoorsselaere,J.andVanReeth,K. 3300
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3303Glaser,L.,Stevens,J.,Zamarin,D.,Wilson,I.A.,Garcia-Sastre,A.etal.(2005).Asingleamino 3304
acidsubstitutionin1918influenzavirushemagglutininchangesreceptorbindingspecificity. 3305JVirol,79,11533-6. 3306
3307Guo, Y. J., Krauss, S., Senne, D. A., Mo, I. P., Lo, K. S. et al (2000). Characterization of the 3308
pathogenicityofmembersof thenewlyestablishedH9N2 influenzavirus lineages inAsia. 3309Virology,267,279-88. 3310
3311Herfst, S., Schrauwen, E. J., Linster, M., Chutinimitkul, S., deWit, E. et al. (2012). Airborne 3312
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avianinfluenzavirusestopigs.JGenVirol,75(Pt9),2183-8. 3320 3321Kimble,J.B.,Sorrell,E.,Shao,H.,Martin,P.L.andPerez,D.R.(2011).CompatibilityofH9N2 3322
avianinfluenzasurfacegenesand2009pandemicH1N1internalgenesfortransmissionin 3323theferretmodel.ProcNatlAcadSciUSA,108,12084-8. 3324
3325Lakdawala, S. S., Lamirande, E.W., Suguitan,A. L., Jr.,Wang,W., Santos, C. P. et al. (2011). 3326
Eurasian-origin gene segments contribute to the transmissibility, aerosol release, and 3327morphologyofthe2009pandemicH1N1influenzavirus.PLoSPathog,7,e1002443. 3328
3329Lakdawala,S.S., Jayaraman,A.,Halpin,R.A., Lamirande,E.W.,Shih,A.R.etal. (2015).The 3330
soft palate is an important site of adaptation for transmissible influenza viruses.Nature, 3331526,122-5. 3332
3333Liu,Q.,Zhou,B.,Ma,W.,Bawa,B.,Ma,J.etal.(2014).AnalysisofrecombinantH7N9wild-type 3334
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poultryinAsiahavehumanvirus-likereceptorspecificity.Virology,281,156-62. 3339 3340Mehle,A.andDoudna,J.A.(2009).Adaptivestrategiesofthe influenzaviruspolymerasefor 3341
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BottlenecksShapeEvolutionaryPathwaysTakenduringMammalianAdaptationofa1918- 3345likeAvianInfluenzaVirus.CellHostMicrobe,19,169-80. 3346
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betweenswineH3N2and2009pandemicH1N1generateddiversegeneticconstellationsin 3357influenza viruses circulating in United States pigs. In: 24th International Pig Veterinary 3358SocietyCongress.P157,Dublin. 3359
3360Romero-Tejeda, A. and Capua, I. (2013). Virus-specific factors associated with zoonotic and 3361
pandemicpotential.InfluenzaOtherRespirViruses,7Suppl2,4-14. 3362 3363Sang,X.,Wang,A.,Ding,J.,Kong,H.,Gao,X.etal. (2015).AdaptationofH9N2AIV inguinea 3364
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3367Shichinohe, S., Okamatsu,M., Sakoda, Y. & Kida, H. (2013). Selection of H3 avian influenza 3368
viruseswithSAalpha2,6Galreceptorspecificityinpigs.Virology,444,404-8. 3369 3370Sorrell, E. M., Wan, H., Araya, Y., Song, H. and Perez, D. R. (2009). Minimal molecular 3371
constraintsforrespiratorydroplettransmissionofanavian-humanH9N2influenzaAvirus. 3372ProcNatlAcadSciUSA,106,7565-70. 3373
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influenzaAvirusisadeterminantofhostrange.JVirol,67,1761-4. 3376 3377Sun,Y.,Pu,J.,Jiang,Z.,Guan,T.,Xia,Y.etal.(2010).Genotypicevolutionandantigenicdriftof 3378
H9N2influenzavirusesinChinafrom1994to2008.VetMicrobiol,146,215-25. 3379 3380Sun,Y.,Qin,K.,Wang,J.,Pu,J.,Tang,Q.etal.(2011).Highgeneticcompatibilityandincreased 3381
pathogenicityofreassortantsderivedfromavianH9N2andpandemicH1N1/2009influenza 3382viruses.ProcNatlAcadSciUSA,108,4164-9. 3383
3384Sun,Y.andLiu,J.(2015).H9N2influenzavirusinChina:acauseofconcern.ProteinCell,6,18- 3385
25. 3386 3387Taubenberger, J. K. and Kash, J. C. (2010). Influenza virus evolution, host adaptation, and 3388
pandemicformation.CellHostMicrobe,7,440-51. 3389 3390TheSJCEIRSH9WorkingGroup (2013).Assessing the fitnessofdistinct cladesof influenzaA 3391
(H9N2)viruses.EmergMicrobesInfect,2,e75. 3392 3393Trebbien, R., Larsen, L. E. and Viuff, B. M. (2011). Distribution of sialic acid receptors and 3394
influenzaAvirusofavianandswineorigininexperimentallyinfectedpigs.VirolJ,8,434. 3395 3396Tumpey, T.M.,Maines, T. R., VanHoeven,N.,Glaser, L., Solorzano,A. et al. (2007).A two- 3397
aminoacidchangeinthehemagglutininofthe1918influenzavirusabolishestransmission. 3398Science,315,655-9. 3399
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avian, humanand swine influenza viruses in porcine respiratory explants and association 3402withsialicaciddistribution.VirolJ,7,38. 3403
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specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and 3406transmissioninpigs.InfluenzaOtherRespirViruses,7,151-9. 3407
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virustransmissionbottlenecksaredefinedby infectionrouteandrecipienthost.CellHost 3410Microbe,16,691-700. 3411
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transmissionofH9N2 influenza viruses in ferrets: evaluationof pandemicpotential.PLoS 3422One,3,e2923. 3423
3424Wang, J.,Wu,M.,Hong,W.,Fan,X.,Chen,R.etal. (2016). InfectivityandTransmissibilityof 3425
AvianH9N2InfluenzaVirusesinPigs.JVirol,90(7),3506-14. 3426 3427Watson, S. J., Langat, P., Reid, S. M., Lam, T. T., Cotten, M. et al. (2015). Molecular 3428
epidemiologyandevolutionofinfluenzavirusescirculatingwithinEuropeanswinebetween 34292009and2013.JVirol,89,9920-31. 3430
3431Wilker,P.R.,Dinis,J.M.,Starrett,G.,Imai,M.,Hatta,M.etal(2013).Selectionon 3432
haemagglutininimposesabottleneckduringmammaliantransmissionofreassortantH5N1 3433influenzaviruses.NatCommun,4,2636. 3434
3435Yu,H.,Zhou,Y.J.,Li,G.X.,Ma,J.H.,Yan,L.P.etal.(2011).GeneticdiversityofH9N2influenza 3436
virusesfrompigsinChina:apotentialthreattohumanhealth?VetMicrobiol,149,254-61. 3437 3438Zaraket, H., Baranovich, T., Kaplan, B. S., Carter, R., Song, M. S. et al. (2015). Mammalian 3439
adaptation of influenza A(H7N9) virus is limited by a narrow genetic bottleneck. Nat 3440Commun,6,6553. 3441
3442Zhang, Y., Zhang, Q., Gao, Y., He, X., Kong, H. et al. (2012). Key molecular factors in 3443
hemagglutinin and PB2 contribute to efficient transmission of the 2009 H1N1 pandemic 3444influenzavirus.JVirol,86,9666-74. 3445
3446Zhu, H., Wang, D., Kelvin, D. J., Li, L., Zheng, Z. et al. (2013). Infectivity, transmission, and 3447
pathologyofhuman-isolatedH7N9influenzavirusinferretsandpigs.Science,341,183-6. 3448 3449
123
3450
SUMMARY-SAMENVATTING 34516
Summary-samenvatting
124
Summary 3452
Avian H9N2 influenza viruses with enhanced human receptor-binding affinity have 3453
been recurrently isolated frompigs andhumans since1998. This isolation increased 3454
theirpublichealthconcern.In2009,theemergenceofthe2009pandemicH1N1virus 3455
in swine put the scope on swine as host where novel pandemic viruses could be 3456
generated. The hypothesis that swinemay play a role as intermediate hosts and/or 3457
“mixingvessels”inwhichAIVsmayadapttomammalsincreasingtheirpotentialthreat 3458
togeneratenovelpandemicswasenunciatedmorethan20yearsago.However,ithas 3459
neverbeenprovedandthereareargumentsforandagainstit. 3460
3461
In Chapter 1, a general introduction to influenza A virus taxonomy, structure, 3462
replicationcycleandvirusevolutionisgiven.Inthesecondsectiontheepidemiologyof 3463
avian, human and swine influenza viruses is summarized. The third and the fourth 3464
sectionsdepictthemolecularmechanismsthatdrivethehostrangerestrictionandthe 3465
avianviruseswithhigherpandemicpotential.Later,thefifthandsixthsectionspresent 3466
asummaryofthehypothesizedroleofthepigintheadaptationofAIVstomammals 3467
andtheuseofthepigasresearchmodel. 3468
3469
Chapter2outlinestheaimsofthethesis.ThefirstaimwastoadaptanentireH9N2 3470
AIV to pigs and to contribute to the general understanding of the mechanisms of 3471
adaptationof theAIVs tomammals.Moreover,our secondaimwas toevaluate the 3472
impactofreassortingthepH1N1backbonewithH9N2AIVonreplicationcapacityand 3473
transmissioninpigs. 3474
3475
Chapter3dealswiththeadaptationofanentireavianH9N2virustopigsbyserialbind 3476
passages. In this chapter,we characterized virus replication in theentire respiratory 3477
tractduringallpassagesandweselectedthebestreplicatingvirustoperformadirect 3478
contact transmission experiment to evaluate if the better performance during the 3479
passages is also correlated with enhanced transmissibility. We also compared 3480
genetically the original material with the virus selected for the transmission 3481
experiment. The original H9N2 virus caused a productive infection in pigs with a 3482
predominant tropism for the nasal mucosa. In contrast, after 4 passages the virus 3483
Chapter6
125
replicated in the entire respiratory tract. Subsequent passageswere associatedwith 3484
reducedvirusreplicationinthelungsandinfectiousviruswaslostatpassage10. The 3485
broader tissue tropism after 4 passages was associatedwith an amino acid residue 3486
substitutionatposition225,withinthereceptor-bindingsiteofthehemagglutinin. In 3487
the direct contact transmission experiment, the passage 4 virus showed enhanced 3488
transmission when compared to the original virus. Nevertheless, it was significantly 3489
lower when compared to that of the pH1N1. Our data demonstrate that serial 3490
passagingofH9N2virusinpigsenhancesitsreplicationandtransmissibility.However, 3491
fulladaptationofanavianH9N2virustopigslikelyrequiresamoredrasticapproach. 3492
3493
InChapter4theeffectofthereassortmentwithpH1N1internalgenescombinedwith 3494
the serial passaging in pigs was examined. In this chapter, we repeated the 3495
experimentalsetupdescribedinChapter3buttheoriginalavianviruswassubstituted 3496
by a reassortant virus containing H9N2 HA and NA protein-genes in the pH1N1 3497
backbone. The aimof this experimentwas to evaluate if the introduction of pH1N1 3498
internal genes offered an advantage for adaptation of H9N2 AIVs in pigs. The viral 3499
replication was increased in contrast to that of the original avian H9N2 virus after 3500
serialpassages.Furthermore,itdidnotloseitsreplicationcapacityafter10passagesin 3501
pigs in comparison to the original avian H9N2 virus. We selected the virus after 7 3502
passages to perform the direct-contact transmission experiment. This virus was 3503
excretedby4outof6animalsat similar rateswhencompared topH1N1.However, 3504
thegeneticanalysisrevealedthattheoriginalviruswascontaminatedandtheinternal 3505
genespresent in theoriginal stockcontainedamixtureofH9N2andpH1N1 internal 3506
genes. Interestingly the serial passaging selected for pH1N1 internal genes and in 3507
passage 7 virus only NP-coding genewas completely of H9N2-origin. The passage 7 3508
virus also contained the HA-D225G substitution described in Chapter 3. Our data 3509
demonstrate that serial passaging of a reassortant H9N2 virus in pigs selected the 3510
samemutationastheoneselectedbythesameexperimentalset-upperformedwith 3511
thewhollyavianvirus.However,itshowsthatadaptationtopigsisacomplexprocess 3512
thatmayrequirethecombinationofreassortmentwithpointmutations. 3513
3514
Summary-samenvatting
126
In Chapter 5, the experimental studies are discussed. In general, our studies 3515
demonstratedthattheadaptationofAIVstopigsisaverycomplexprocessthatneeds 3516
drasticgeneticchangestobeachieved.AdaptationofavianH9N2tomammalscanbe 3517
achievedbythecombinationof reassortmentwithefficientlyadapted internalgenes 3518
andserialpassages.However, it remainsquestionablewhether the resultsdescribed 3519
are extrapolatable to other strains or subtypes and to the possible adaptation to 3520
humans. By setting-up that experimental model we have generated the basis to 3521
generatebetteradaptedvirusandgeneticallycharacterizetheminordertoputlightin 3522
theunderstandingoftheadaptationprocessofAIVs. 3523
3524
Chapter6
127
Samenvatting 3525
Aviaire H9N2-influenzavirussen met verhoogde bindingsaffiniteit voor de menselijke 3526
receptor werden sinds 1998 herhaaldelijk geïsoleerd uit varkens en mensen. Hun 3527
vondstbrengt een verhoogde risico voorde volksgezondheidmet zichmee. In 2009 3528
benadruktedeopkomstvanhetpandemischH1N1-virusuit2009(pH1N1)bijvarkens 3529
het belang van varkens als gastheer waarin nieuwe pandemische virussen zouden 3530
kunnen ontstaan. De hypothese dat varkens een rol kunnen spelen als 3531
tussengastheren en/of "mengvaten" waarin aviaire influenzavirussen (AIV's) zich 3532
kunnenaanpassenaanzoogdierenenzoeenverhoogdepotentiëlebedreigingvormen 3533
voor het ontstaan van nieuwe pandemieën, werd meer dan twintig jaar geleden 3534
geformuleerd. Ditwerd echter nooit bewezen en er zijn argumenten voor en tegen 3535
dezehypothese. 3536
3537
Inhoofdstuk1wordteenalgemeneintroductiegegevenoverdetaxonomie,structuur, 3538
replicatiecyclus en virusontwikkeling van influenza A-virussen. In de tweede sectie 3539
wordtdeepidemiologievanaviaire,humaneenvarkensinfluenzavirussensamengevat. 3540
De derde en de vierde sectie duiden de moleculaire mechanismen die het 3541
gastheerbereik beperken en beschrijven de aviaire virussen met een hoger 3542
pandemisch potentieel. Vervolgens wordt in de vijfde en zesde sectie een 3543
samenvattinggegevenvandeveronderstelderolvanhetvarkenindeaanpassingvan 3544
AIV's aan zoogdieren en van het gebruik van varkens alsmodel voor het onderzoek 3545
naardezeadaptatie. 3546
3547
Hoofdstuk2schetstdedoelstellingenvanhetproefschrift.Hethoofddoelwasomeen 3548
H9N2AIVaantepassenaanvarkensenombijtedragenaanhetalgemenebegripvan 3549
demechanismenvanaanpassingvandeAIV'saanzoogdieren. 3550
3551
Hoofdstuk3behandeltdeaanpassingvaneenvolledigaviairH9N2-virusaanvarkens 3552
dooropeenvolgendeblindepassages.Indithoofdstukhebbenwedevirusreplicatiein 3553
hethelerespiratoirekanaalgekarakteriseerdtijdensallepassagesenhebbenwehet 3554
best aangepaste virus geselecteerd voor het uitvoeren van een direct-contact 3555
transmissie-experiment om te evalueren of de betere prestaties tijdens de passages 3556
Summary-samenvatting
128
ook gecorreleerd zijnmet verhoogde overdraagbaarheid.We hebben ook genetisch 3557
het startmateriaal vergeleken met het virus dat geselecteerd werd voor het 3558
transmissie-experiment.HetoorspronkelijkeH9N2-virusveroorzaakteeenproductieve 3559
infectiebijvarkensmeteenoverheersendtropismevoordeneusslijmvliezen.Navier 3560
passages repliceerdehetvirusdaarentegen inhethele respiratoirekanaal.Volgende 3561
passageswerdengeassocieerdmetgereduceerdevirusreplicatie inde longenenhet 3562
besmettelijkevirusgingverlorenbijpassagetien.Hetuitgebreiderweefseltropismena 3563
vierpassageswerdgeassocieerdmeteenaminozuursubstitutieoppositie225binnen 3564
de receptorbindingsplaats op het hemagglutinine. In het direct-contact transmissie- 3565
experimentvertoondehetvirusvanpassagevierverbeterdetransmissieinvergelijking 3566
met het oorspronkelijke virus. Desondanks was uitscheiding significant lager in 3567
vergelijkingmet die van pH1N1. Onze gegevens tonen aan dat seriële passages van 3568
H9N2-virusbijvarkensdereplicatieenoverdraagbaarheidverbetert.Echter,volledige 3569
aanpassing van een aviair H9N2-virus aan varkens vereist waarschijnlijk een 3570
drastischereaanpak. 3571
3572
Inhoofdstuk4werdheteffect van reassorteringvandeaviaire influenzaH9N2met 3573
internegenenvanpH1N1opdereplicatiecapaciteitinvarkensonderzochtdoormiddel 3574
van seriële passages. In dit hoofdstuk herhaalden we de proefopzet beschreven in 3575
hoofdstuk 3, maar het oorspronkelijke virus werd vervangen door een reassortant 3576
virusdatdeHA-enNA-eiwitgenenvanhetH9N2-virusende internegenenvanhet 3577
pH1N1-virus bevat. De bedoeling van dit experiment was om te evalueren of de 3578
introductievanpH1N1internegeneneenvoordeelbetekendevoordeaanpassingvan 3579
H9N2AIV's aan varkens.Dit virus presteerdebeter danhet oorspronkelijke virus na 3580
tienpassageswerdnogsteedsinfectieusvirusgedetecteerd.Wehebbenhetvirusna 3581
zeven passages geselecteerd om een direct-contact transmissie experiment uit te 3582
voeren.Dit viruswerd uitgescheidendoor vier van de zes dieren aan hoeveelheden 3583
vergelijkbaarmet deze voor pH1N1. Echter, uit de genetische analyse bleek dat het 3584
oorspronkelijke virus gecontamineerd was en dat de interne genen aanwezig in de 3585
oorspronkelijke stock een mengsel van H9N2 en pH1N1 interne genen bevatte. 3586
InteressantisdatdeseriëlepassagesselecteerdenvoorpH1N1internegeneneninhet 3587
virusvanpassagezevenwasenkelhetNP-coderendgenvolledigvanH9N2-oorsprong. 3588
Chapter6
129
Het virus van passage zeven bevat ook de substitutie HA-D225G beschreven in 3589
hoofdstuk 3. Onze gegevens tonen aan dat opeenvolgende passages van een 3590
reassortant H9N2-virus bij varkens dezelfde mutatie geselecteerd heeft als die 3591
geselecteerd door dezelfde experimentele opstelling uitgevoerd met het volledig 3592
aviairevirus.Hetblijktechterdataanpassingaanvarkenseencomplexproces isdat 3593
mogelijkdecombinatievanreassorteringmetpuntmutatiesvereist. 3594
3595
In hoofdstuk 5 worden de experimentele studies besproken. Over het algemeen 3596
hebben onze studies aangetoond dat de aanpassing vanAIV's aan varkens een zeer 3597
complex proces is dat drastische genetische veranderingen vereist. Aanpassing van 3598
aviairH9N2aanzoogdierenkanwordenbereiktdoordecombinatievanreassortering 3599
metefficiëntaangepasteinternegenenenseriëlepassages.Echter,hetblijftonzeker 3600
of de resultaten die hier beschreven worden geëxtrapoleerd kunnen worden naar 3601
andere stammenof subtypesennaardemogelijke adaptatie aanmensen.Doorhet 3602
experimentelemodelop tezetten,hebbenwedebasisgelegdombeteraangepaste 3603
virussen tegenererenenomdezevirussengenetisch tekarakteriseren,metalsdoel 3604
lichttewerpenophetbegrijpenvanhetadaptatieprocesvanAIV’s. 3605
3606
3607
3608
3609
131
3610
CURRICULUMVITAE 36117
CurriculumVitae
132
Personalia 3612JoseCarlosManceraGraciawasborninZaragoza,SpainonJanuary111986.In2004 3613
hecompletedhissecondaryeducationattheInstitutoPabloGargalloinZaragozaand 3614
started the study of veterinary medicine at the Faculty of Veterinary Medicine, 3615
ZaragozaUniversity,fromwhichhegraduatedin2011.InAugust2011hewasgranted 3616
withaLeonardodaVincischolarshipandhestartedaninternshipattheNutrecoB.V. 3617
Swine Research Centre in Boxmeer (theNetherlands). In September 2012 he joined 3618
theLaboratoryofVirology,FacultyofVeterinaryMedicine,GhentUniversitywherehe 3619
studied the possibilities of adaptation of avian H9N2 influenza viruses to swine. His 3620
PhD studieswere funded by the European Commission FLUPIG FP7 project (FP7-GA 3621
258084). 3622
3623
Publications 3624
Publicationsininternationalscientificpeer-reviewedjournals 3625
ManceraGracia, J. C., Van denHoecke, S., Richt, J. A.,Ma,W., Saelens, X. and Van 3626
Reeth,K.(2017).AreassortantH9N2influenzaviruscontaining2009pandemicH1N1 3627
internal-proteingenesacquiredenhancedpig-to-pigtransmissionafterserialpassages 3628
inswine.Scientificreports,7,1323. 3629
3630
VanReeth,K.,ManceraGracia,J.C.,Trus,I.,Sys,L.,Claes,G.,Versnaeyen,H.,Cox,E., 3631
Krammer, F., Qiu, Y. (2017). Hetereologous prime-boost vaccination with H3N2 3632
influenza viruses of swine favors cross-clade antibody responses and protection.npj 3633
Vaccines,2,11. 3634
3635
ManceraGracia,J.C.,VandenHoecke,S.,Saelens,X.andVanReeth,K.(2017).Effect 3636
of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine. 3637
PLoSOne,12,e0175267. 3638
3639
Conferenceabstracts 3640
Mancera Gracia, J. C. and Van Reeth, K. (2016). Could avian H9N2 influenza viruses 3641
becomeathreattoswine?:lessonsfromexperimentalstudiesinthepig.Proceedings 3642
ofthe24thIPVSCongress,Dublin,Ireland,p.158. 3643
Chapter7
133
ManceraGracia,J.C.andVanReeth,K.(2015).Enhancedpig-to-pigtransmissionofa 3644
reassortant H9N2 influenza virus containing 2009 pandemic H1N1 internal genes by 3645
serial passaging in pigs. Third annual meeting of the Belgian Society for Virology, 3646
Brussels,Belgium. 3647
3648
ManceraGracia, J.C.andVanReeth,K. (2015).Aroleforswine intheadaptationof 3649
avian H9N2 influenza viruses to humans?: lessons from serial passages and 3650
transmissionstudiesinpigs.ProceedingsoftheOxfordInfluenzaConference,Influenza 3651
2015:oneinfluenza,oneworld,onehealth,Oxford,UnitedKingdom. 3652
3653
Qiu,Y.,ManceraGracia,J.C.,Li,Y.,Trus,I.,Nguyen,VanUt.,Cox,E.andVanReeth,K. 3654
(2014). Prior infection of pigs with a European H3N2 SIV confers partial protection 3655
againstaNorthAmerican swine-originH3N2v influenzavirus.The5thESWI influenza 3656
conference,Riga,Latvia,p114. 3657
3658
Qiu,Y.,ManceraGracia,J.C.,Li,Y.,Trus,I.,Nguyen.,VanUt,Cox,EandVanReeth,K. 3659
(2014).Cross-protectionstudiesinpigswiththeEuropeanH3N2swineinfluenzavirus 3660
andtheNorthAmericanswine-originH3N2‘variant’,avirusofpublichealthconcern. 3661
TheannualBelgiumImmunologicalSocietymeeting,Ghent,Belgium,p41. 3662
3663
Qiu, Y., Claes, G., Mancera Gracia, J. C. and Van Reeth, K. (2015). Cross-lineage 3664
antibodyresponsesandprotectionuponvaccinationwithinactivatedswineinfluenza 3665
virus vaccines. 3rd International SymposiumonNeglected InfluenzaViruses, Athens, 3666
USA,p46. 3667
3668
Oralpresentations 3669
Mancera Gracia, J. C. and Van Reeth, K. (2016). Could avian H9N2 influenza viruses 3670
becomeathreattoswine?:lessonsfromexperimentalstudiesinthepig.Proceedings 3671
ofthe24thIPVSCongress,Dublin,Ireland,p.158. 3672
3673
3674
CurriculumVitae
134
ManceraGracia,J.C.andVanReeth,K.(2015).Enhancedpig-to-pigtransmissionofa 3675
reassortant H9N2 influenza virus containing 2009 pandemic H1N1 internal genes by 3676
serial passaging in pigs. Third annual meeting of the Belgian Society for Virology, 3677
Brussels,Belgium. 3678
3679
ManceraGracia, J.C.andVanReeth,K. (2015).Aroleforswine intheadaptationof 3680
avian H9N2 influenza viruses to humans?: lessons from serial passages and 3681
transmissionstudiesinpigs.ProceedingsoftheOxfordInfluenzaConference,Influenza 3682
2015:oneinfluenza,oneworld,onehealth,Oxford,UnitedKingdom. 3683
3684
135
3685
AKNOWLEDGEMENTS 36868
Acknowledgements
136
If Iamwriting thesewords it isbecausemyPh.D.degreestudiesare reaching their 3687
end.Atthismoment,Iwouldliketoexpressmysinceregratitudetoallthepeoplewho 3688
accompaniedmeduringthisadventureinFlanders. 3689
My very first thanks go to my promoter Prof. Dr. Kristien Van Reeth. I am really 3690
grateful because you provided me the opportunity to join your research group to 3691
explorethemechanismsofadaptationofavianinfluenzaviruses.Workingunderyour 3692
supervision has been very challenging and allowed me to grow as person and as 3693
researcher.Iwouldalsoliketothankmyco-promoterProf.Dr.XavierSaelens.Thank 3694
youXavierforyoursupportandyourvaluablepointofviewonourresearch. 3695
I highly appreciate the contribution of my Guidance and Examination Committee 3696
members:Prof.Dr.HansNauwynck,Prof.Dr.MikhailMatrosovich,Prof.Dr.Dominiek 3697
Maes, Dr. Karen van derMeulen, Dr. Annebel de Vleeschauwer and Dr. Sebastiaan 3698
Theuns.Thanksforyourtime,interests,andthecarefulreviewofmythesis. 3699
Mydeepestthanksgotoallmycolleaguesatthedepartment,thanksforyourhelpand 3700
your support. Day by day it was very nice toworkwith you. Firstly, I would like to 3701
thank those who left the Lab, Yu, Gerwin, Sjoucke, Amy, Charlie, Kevin, Vishi, Ilias, 3702
Laura,….IwouldalsoliketothankDr.ConstantinosKyriakis,althoughwedidnotmeet 3703
inBelgiumyougavemeverynicetipsaboutpost-PhDlife. 3704
Lieve, Nele and Melanie thanks one thousand times for your patience and your 3705
excellentassistanceintheLab. Ithasbeenapleasuretosharethelaminarflowwith 3706
you,eachdaywasamasterclass.Ofcourse, IwouldalsoliketothankZegerforyour 3707
helpinanimalexperimentsandforthosetripstothefarms.ThanksalsotoGertforhis 3708
valuable help with the administrative work and Dr. Nathalie Vanderheijden for her 3709
helpwiththeprojectissues.IamalsospeciallygratefultoCarolineandAnaforthose 3710
never-endingtalkstryingtofixtheworld.Elien,SharonandAnnaIamhappytohave 3711
metyouandIwishyouthebestforyourfutureresearch. Iwouldalsoliketohavea 3712
wordforDr.SilvieVanDenHoecke,thanksforyourhelpwiththepublicationsandthe 3713
clearexplanationsonthegeneticanalysisoftheviruses. 3714
TambiénquieroagradeceraRobertoBautista,EmilioMagallónylagentedeIngafood. 3715
Vosotrosmemetisteiselgusanillodelporcinoenlacabezaygraciasavosotrostuvela 3716
granoportunidaddedarelsaltoaHolandaparaseguirformándome.Iwouldalsolike 3717
toacknowledgeDr.SandraParedes,HubértvanHees,JanLamersandthepeoplethatI 3718
Chapter7
137
metduringmy timeatBoxmeerbecause theygaveme thevery firstopportunity to 3719
workonresearch.Ireallymissthosegooddays. 3720
AlthoughduringmyPh.D.Iinvestedalotoftimeonresearch,Ialsohadtimetomeet 3721
verynicepeople.Thankstothe“LostinGhent”forthegoodmoments. 3722
To the “Eluners”, that used tomeet every Friday for oneor twoafterworkbeers to 3723
releasethestressoftheweek.Joao,graciasati,aElijayalasniñasporlascervezas, 3724
las catas de vinos, las comilonas con la familia y por ser como sois.Miguelito estoy 3725
muycontentodequehayasvenidodeBrasil sóloparamidefensaJ graciaspor las 3726
nochesenelkaraoke,Lyon,elviajeaBrasilypordescubrirmeelCabraliego.Alfonso, 3727
gracias por tu ayuda siempre que ha hecho falta y por, junto con Evelien,Niklaas y 3728
Fien,hacermesentirpartedevuestrafamilia.Ademásdeporlosbuenosconsejospara 3729
lasentrevistasdetrabajo.Trompi,atiquetevoyadecir,siemprequehetenidouna 3730
dudadecerdos lahasresuelto,conpermisodeAlfonso,eresuncrackysiemprehas 3731
sidoungranreferenteparamí.Además,graciasatiyaVíctorCarreteroporllevarme 3732
devezencuandoa“morir”conlabicicleta. 3733
I would also like to thank Amparo, Minerva, Ana Rafael, Ana (Manu), Michelle, 3734
Gonzalo, Francesco, Mercedes, Alessio, Raffa, Eva, Pedro, Mike, Kevin, Edita, Anna 3735
Kuchmiy,Laura,DomenicoandIamsurethatIforgotsomeone… 3736
Tambiénquierodarlasgraciasamisamigosdel“Insti”:Ari,Lobe,Miri,Perdi,Barneyy 3737
Laura porque 12 años después de nuestra primera visita a Flandes volvisteis a 3738
visitarmeyverquetodoestabaensusitio.AmisamigosRubénGil,Edu,JorgeMaz, 3739
DaniyEnriqueporqueaunqueyallevo5añosfueradeZaragozacadavezqueosveo 3740
escomosinohubierapasadoeltiempoyporelesfuerzoquehacéisporseguirsiendo 3741
misamigos. 3742
Gracias también a mi familia en Gante. Alfonso, Nerea, Dani, Tere y Olga podría 3743
escribirun libroenterodándoos lasgracias,pero lovoyaresumirJ.Graciaspor las 3744
cenas, los viajes, las “Klokkes”, las noches en el video, las risas y las no tan risas. 3745
Durante estos años habéis sido amigos, compañeros de piso, colegas y uno de los 3746
soportesenmiestanciaaquí. 3747
También quiero acordarme demi familia biológica. Demis padres, a vosotros os lo 3748
debotodo.Séquenuncalodigoperosialgunavezsoypadremegustaríaserlamitad 3749
debuenoquevosotros.Demihermana,quesiempreseacuerdaysepreocupapormí, 3750
Acknowledgements
138
de Jesús que lleva ya más de 10 años siendo parte de los Mancera Gracia y de Leyre, la 3751
pequeña de la familia que es una motivación para viajar a casa más a menudo. 3752
También quiero agradecer su apoyo a mis primos y tíos. Especialmente a la tía Mary, 3753
que siempre está pendiente de que todos los sobrinos estemos bien. 3754
L´ultimo ma non meno importante ringraziamento va a Giorgia Rocatello. You have 3755
been my main support during the last 2 years, and since the last year you are dealing 3756
with me every day. I would like to thank you for your patience, for your support, for 3757
your help at any moment, but mostly for your smile in the worst situations. As Antonio 3758
Flores would say: “Eres la flor que siempre quise en mi jardín”. 3759
3760
Gracias/Grazie/Thank you/Dank je wel!!! 3761
3762
José Carlos Mancera Gracia 3763
Ghent, 30/06/2017 3764
3765