Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses

18 Pages·2017·2.72 MB·English

viruses
Article
Pressure for Pattern-Specific Intertypic
Recombination between Sabin Polioviruses:
Evolutionary Implications
EkaterinaKorotkova1,2,†,MajidLaassri3,†,TatianaZagorodnyaya3,SvetlanaPetrovskaya3,
ElviraRodionova3,ElenaCherkasova4,AnatolyGmyl2,5,OlgaE.Ivanova2,5,
TatyanaP.Eremeeva2,GalinaY.Lipskaya1,VadimI.Agol1,2,* ID andKonstantinChumakov3,*
1 ANBelozerskyInstituteofPhysical-ChemicalBiology,MVLomonosovMoscowStateUniversity,
Moscow119899,Russia;[email protected](E.K.);[email protected](G.Y.L.)
2 InstituteofPoliomyelitisandViralEncephalitidesofMPChumakovFederalScientificCenterforResearch
andDevelopmentofImmune-and-BiologicalProductsofRussianAcademyofSciences,Moscow108819,
Russia;[email protected](A.G.);[email protected](O.E.I.);[email protected](T.P.E.)
3 USFoodandDrugAdministration,SilverSpring,MD20993,USA;[email protected](M.L.);
[email protected](T.Z.);[email protected](S.P.);
[email protected](E.R.)
4 NationalHeart,Lung,andBloodInstitute,NationalInstitutesofHealth,Bethesda,MD20895,USA;
[email protected]
5 IMSechenovFirstMoscowStateMedicalUniversity,Moscow119991,Russia
* Correspondence:[email protected](V.I.A.);[email protected](K.C.);
Tel.:[phone](V.I.A.);[phone](K.C.)
† Theseauthorscontributedequallytothisreport.
AcademicEditor:KenStedman
Received:23October2017;Accepted:20November2017;Published:22November2017
Abstract: Completegenomicsequencesofanon-redundantsetof70recombinantsbetweenthree
serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing) of
crossoversitesof28additionalrecombinantsweredeterminedandcomparedwiththepreviously
publisheddata. ItisdemonstratedthatthegenomesofSabinvirusescontaindistinctstrain-specific
segmentsthatareeliminatedbyrecombination. Thepresumedlowfitnessofthesesegmentscouldbe
linkedtomutationsacquireduponderivationofthevaccinestrainsand/ormayhavebeenpresent
inwild-typeparentsofSabinviruses. These“weak”segmentscontributetothepropensityofthese
virusestorecombinewitheachotherandwithotherenterovirusesaswellasdeterminethechoice
ofcrossoversites. Theknowledgeoflocationofsuchsegmentsopensadditionalpossibilitiesfor
thedesignofmoregeneticallystableand/ormoreattenuatedvariants,i.e.,candidatesforneworal
poliovaccines. Theresultsalsosuggestthatthegenomeofwildpolioviruses,and,bygeneralization,
ofotherRNAviruses,mayharborhiddenlow-fitnesssegmentsthatcanbereadilyeliminatedonly
byrecombination.
Keywords: vaccine-derived polioviruses; attenuation; reversion; virulence; vaccine-induced
adversereactions
1. Introduction
Geneticrecombinationwasdemonstratedtooccurinpracticallyallvirusesinvestigatedinthis
respect[1],althoughitsfrequencyinRNAvirusesvariesmarkedly,dependingontheorganizationof
thereplicativemachinery[2]. Thereismultipleexperimentalevidencethatrecombinationmayserve
differentevolutionarytasks,suchasmaintenanceofthegenomestructurethroughpurgingdeleterious
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mutationsorconverselyacquisitionofbeneficialornovelqualities. Nevertheless,thedrivingforces
andbiologicalconsequencesofspecificinstancesofnaturalrecombinationinRNAvirusesarerarely
wellunderstood. Inthisrespect,thewidespreadoccurrenceofintertypicrecombinantsbetweenthe
poliovirusvaccinestrainsisofspecialinterest.
ThesestrainsweregeneratedbyAlbertSabinbyselectionfromthreeserotypesofwild-type(wt)
polioviruses[3]. Upontheirderivation,theyhadaccumulatedvariousserotype-specificmutations,
includingthosethatwereshowntobeneurovirulence-attenuating[4]. Usedastrivalentoralpolio
vaccine (OPV), these strains played a crucial role in the ongoing global campaign to eradicate
poliomyelitislaunchedbytheWorldHealthOrganization(WHO)in1988[5].Thisveryefficientvaccine
hashoweversomedrawbacks. Itisgeneticallyunstableuponreplicationinvaccinerecipients,readily
losingattenuatingmutationsbyreversionsorcompensatorychanges[6–8], leadingto(albeitvery
rarely) paralytic disease (vaccine-associated paralytic poliomyelitis; VAPP) in vaccinees or their
contacts. Virusesexcretedbyhealthyvaccinerecipientsorisolatedfromparalysiscasesoftenturnout
tobeintertypicrecombinants[9–13]. Moreover,thepartiallydeattenuatedvariantscanfurtherevolve
duringlong-termreplicationintheimmunodeficientorganismsorupontransmissionthroughhealthy
populations, giving rise to more diverged, so called vaccine-derived polioviruses (VDPV), able to
inducenotonlysporadiccasesofpoliomyelitisbutalsotocausemoreorlesslargeoutbreaksofthe
disease[14]. TheseVDPVareoftenrecombinantseitherbetweendifferentserotypesofSabinviruses
(orbetweenSabinandwtpolioviruses)orbetweenSabinvirusesandnon-polioenteroviruses[15].
Thespecificreason(s)fortheobviouspropensityofSabinvirusestorecombinewithsuitablepartners
ispoorly,ifatall,understood.
Aimingtounderstandthesereasons,inthisstudywehaveanalyzedsomeaspectsofaunique
naturalmega-experimentwhenamixtureofthreewell-studiedandrelatedviruses(thetrivalentOPV)
wasgiventobillionsofpeopleandaplethoraofprogenyisolateswereavailableforinvestigation.
Specifically,wehavesequenced,fullyorpartially,98genomesofintertypicpoliovirusrecombinants
andanalyzedthesedatatogetherwiththepreviouslypublishedresultstoinfercommonpatternsof
organizationoftherecombinantgenomes. Asaresult,someimportantconclusionshaveemerged
relevant not only for the understanding of the nature of Sabin viruses and their penchant for
recombinationbutalsoofsomegeneralissuesofbiologyofRNAviruses.
2. MaterialsandMethods
Virus strains derived from Sabin polioviruses were mainly collected in Russia and countries
of the former Soviet Union from patients with acute flaccid paralysis (AFP), patients with other
diseasesaswellasfromhealthyindividualsandenvironment. Isolation,identificationandintratypic
characterizationwereperformedaccordingtothestandardWHOpoliosurveillanceprotocol[16].
Nucleotidesequencesweredeterminedbyfull-genomedeepsequencingasfollows. Cellculture
supernatants were treated with micrococcal nuclease to digest free nucleic acids, and RNA was
isolatedbyRNeasykit(Qiagen,Germantown,MD,USA).Itwasthenfragmentedbyusingfocused
ultrasonicator (Covaris, Woburn, MA, USA) to produce 200–400 nt-long molecules. cDNA was
preparedbyreversetranscriptionusingSuperscriptIII(Invitrogen,Waltham,MA,USA).Illumina
adapters with index oligonucleotides (barcodes) were added and the resulting DNA library was
amplifiedbyPCR.SequencingwasdoneusingIlluminaMiseqinmultiplexformatwith10–12barcoded
samplesanalyzedinonerun. Formostsamplestreatedthisway,poliovirus-specificsequencereads
representedthemajorityofallreads(50–95%),andthedepthofcoveragewasbetween10,000and
200,000 reads per genomic position. All new nucleotide sequences were deposited in GenBank
(accessionnumbersMG212427-MG212495).
Bioinformaticanalysiswasperformedusingtwoin-housesoftwarepackages: ‘swarm’program
running on MacPro (Apple, Cuppertino, CA, USA) computers in UNIX (Darwin) environment,
andcloud-basedHIVE(highly-integratedvirtualenvironment)system[17]. Theanalysispipeline
includedthefollowingsteps. First,rawsequencedatawerepre-screenedtoeliminatelow-quality
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reads and to remove adapter and index sequences. Next, sequence reads were aligned (mapped)
to a curated database of 500 full-length reference sequences representing the entire spectrum of
humanenteroviruses. RecombinationpatternswereanalyzedusingSimplotalgorithm[18]. Theexact
positionsofcrossoversweredeterminedbycomparisonsofthesequencesofrecombinantRNAswith
therelevantregionsoftheirparentalgenomesofSabinviruseswiththehelpofClustalW[19].
The location of crossovers in some recombinants investigated years ago and reported for the
firsttimehereweredeterminedasfollows. ViralRNAwasextractedfromtheculturalmediumof
virus-infectedcellswiththeTrizolreagent(LifeTechnologies,Washington,DistrictofColumbia,USA)
andreversetranscribedwithavianmyeloblastosisvirusreversetranscriptase(Promega)usingrandom
hexamerprimers(BoehringerIngelheim,IngelheimamRhein,Germany).Approximatepositionsofthe
crossoverweremappedbyusingtherestrictionfragmentlengthpolymorphism(RFLP)assay[20,21].
Tothisend,PCRamplificationofseveralregionsofthereversetranscriptswasperformed,andDNA
products thus obtained were treated with restriction endonucleases chosen to generate unique
electrophoreticprofilesforeachSabinserotype. Theinheritanceofgenomicregionsfromdifferent
parents(i.e.,therecombinantnature)andtheapproximatepositionsofthecrossoverscouldbethus
deduced. To determine their precise location, the regions suspected to contain crossovers were
PCR-amplifiedbyusingappropriateprimersandsequencedmanually[22]orbyusingABIPrism310
geneticanalyzer(AppliedBiosystems,Carlsbad,CA,USA).
3. ResultsandDiscussion
3.1. SetoftheVirusesInvestigated
The set of intertypic recombinants between the Sabin vaccine polioviruses analyzed here,
189samples,included98virusescollectedmostlyinRussiaandcountriesoftheformerSovietUnion
sincethe1980s,whosegenomiccrossoversitesweredeterminedinthisstudyeitherbyfullgenome
deepsequencing(70samples,TableS1)orpartialgenomesequencing(28samples,TableS2),aswell
as 91 isolates whose full (Table S3) or partial (Table S4) sequences were previously reported by
us and other investigators. Over half of the samples (55%) were isolated from the acute flaccid
paralysis(AFP)cases(Table1). Inafewcases,differentrecombinantswereisolatedfromthesame
patient. Thecollectionalsoincludedisolatesfrompatientswithotherdiagnoses,fromhealthypersons,
andfromsewage;theoriginof14isolatesisunknown. Fourteen,78,and97viruseswereofserotype1,
2,and3,respectively. Noeffortwasmadetoselectparticularserotypes;therefore,relativenumbersof
recombinantsofdistinctserotypesinthisdatasetroughlyreflectsthefrequencyoftheirisolationby
otherauthors,withtype2and3recombinantsbeingthemostabundant[12,23].
Asjudgedbythelevelofdivergenceoftherecombinant’scapsidproteinVP1-codingsequences
fromthoseoftheirvaccineparents,recombinationgenerallyoccurredearlyintheevolutionofthe
vaccinestrains. Indeed,outof107genomesinvestigatedinthisrespect,87couldbeclassified,byusing
officialcriteria[24],asSabin-like,i.e.,“young”viruses(Table1). Ontheotherhand,therewereseveral
examples,whenthe“ages”ofsegmentsofagivenvirusoriginatingfromdifferentparentsappeared
tomarkedlydiffer, suggestingthepossibilityoflaterecombinationaswell. Thus, themultipartite
recombinant13688(TableS1)ofstructureS1-S3-S2-S1-S2-S3,havingonly2replacements(0.05%)in
the proximal 3840 nucleotides derived from serotype 1, exhibited 2.2%, 1.3%, 4%, 0.8%, and 1.2%
mutationsinthesuccessivesegments,respectively.
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Table1.Overviewofthesetofanalyzedintertypicrecombinants.
OurStudy PublishedData Total
FullGenome PartialGenome FullGenome PartialGenome
MethodsofAnalysis
Sequencing Sequencing Sequencing Sequencing
Numberofisolates 70 28 33 58 189
8(11), 2(7), 3(9), 1(2), 14(8),
Number(%)oftype1,type2,
30(43), 9(32), 13(39), 26(45), 78(41),
andtype3isolates
32(46) 17(61) 17(52) 31(53) 97(51)
Number(%)ofVDPV 5(7) ? 15(45) ? ?
Number(%)of
65(93) ? 18(55) ? ?
Sabin-likestrains
Number(%)ofAFPcases 40(57) 20(71) 24(73) 20(34) 104(55)
Number(%)ofisolatesfrom
6(9) 4(14) 1(3) - 11(6)
patientswithotherdiagnoses
Number(%)ofisolatesfrom
12(17) 1(4) 7(21) 16(28) 36(19)
healthypersons
Number(%)ofisolates
9(13) 3(11) 1(3) 11(19) 24(13)
fromsewage
Number(%)ofstrainsof
3(4) - - 11(19) 14(7)
unknownorigin
VDPV,vaccine-derivedpoliovirus;AFP,acuteflaccidparalysis.
3.2. Serotype-SpecificPatternsofRecombination
The majority of recombinants of serotype 1 had the first crossover points close to the border
betweentheregionsencodingstructuralandnonstructuralproteins,resultinginthereplacementof
significantportionsofthegenomebysequencesderivedfromserotype2inmostcases(Figure1A).
Thesizeofserotype2-derivedfragmentsvariedbetween~600to>3000nt. Alloftheseviruseshadalso
atleastoneadditionalcrossover,predominantlyleadingtotheacquisitionoftheserotype1-derived
3(cid:48)-terminalpartsofthegenome,demonstratingpreferenceforhavinghomotypicgenomeends.
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Figure1.Cont.
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Figure1.Cont.
1
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Figure1.Schematicrepresentationofthegenomestructuresofrecombinantsofserotypes1(A),2(B),
and3(C).Sequencesderivedfromtheseserotypesaredepictedinred,orange,andgreenrespectively.
Ineachpanel,thegenomesareshownthatweresequencedfully(a)andpartially(b)inthisstudy,fully
sequencedfromGenbank(c),andpartiallysequencedpreviously(d). Theoccurrenceofadditional
crossoversinthepartiallysequencedgenomescouldnotberuledout.Thepresenceofforeignsequences
inthegenomesofrecombinantsofagivenserotypeissummarizedin(e).Thefunctionalgeneticmap
ofthepoliovirusgenomeisgivenatthebottomofeachpanel.Heavierandlightershadingrefertothe
proposed“weak”and“provisionallyweak”genomicareas(seetext).UTR,untranslatedregion.
Incontrast,allbutonerecombinantsofserotype2hadheterotypicgenomeendswithacommensurable
representationoftype1-andtype3-derivedsequences(Figure1 1B).Themajorityofrecombinantshad
Viruses2017,9,353 8of18
a single crossover located in many cases downstream of the central P2 genomic region. However,
10outof78recombinantsofthisserotypeappearedtohaveasmanyas4–5crossovers.
Recombinantsofserotype3demonstratedamorevariablepatternoftheinheritanceofheterotypic
sequences (Figure 1C). A sizeable proportion of genomes acquired type 2 sequences around the
structural/nonstructural border but in the majority of cases the crossovers were located more
downstream within the P2 (i.e., 2A-C-encoding) region, resulting in the acquisition of either type
2-ortype1-specificsequences(thelatterpreferablyinthecaseofrelativelymoredistalcrossovers).
3(cid:48)-proximal parts of the gained serotype 3-specific sequences might or might not be replaced
through additional recombination events by type 1 or type 2 sequences, giving rise to more or
lesscomparableproportionsofthegenomeswitheitherhomotypicorheterotypic(mostlyserotype
1-derived) 3(cid:48)-terminal genome segments. Some recombinants of this serotype also had more than
2crossoverpoints.
3.3. PreferentialType-SpecificLocationoftheLostGenomicRegions
Toascertainthebiologicalrelevanceofrecombinationevents,itisimportanttoanalyzenotonly
the acquired but also the lost genetic information. In the recombinants of serotype 1, the deleted
type-specificsequencesmappedpredominantlytotheregionspanningbetweenthecapsidgenesand
internalpartsofthe3Dpolgene,withanapparenttrendtoretain,insomecases,theirown3ABCand
surroundingsequences,suggestingthepossibilitythattherearetwomostlikelyregionsundergoing
removallocatedaroundthe2B/2Cand3C/3Dbordersrespectively(Figure1A).Itshouldbeadmitted
howeverthatthenumberofrecombinantsofthisserotypewastoosmalltomakefirmconclusions.
In serotype 2 recombinants, distal portions of the 3Dpol-coding and 3(cid:48)-untranslated region (UTR)
sequenceswereregularlylost,insomecasestogetherwithmoreorlessextendedpartsoftheP3region
andsometimesalsodistalportionsoftheP2region(Figure1B).Serotype3recombinantsdemonstrated,
asnotedabove,alessregularpatternoflosingtheirowninformation,withtheremovalof(roughly)
P2- or P3-related type-cognate sequences or both (Figure 1C). The absence of short type-specific
sequenceswithinthe3A-codingregionwasacommonfeatureofallbutacoupleofmembersofthis
group. Thecommonabsenceofthisgenomepiececouldbeduetoeitheritsinherentpropertiesorto
theoverlappingofthetwojustmentionedlargersegments.
TheaboveresultsindicatedthatthepatternsofintertypicrecombinationofSabinvirusesarenot
haphazardbutratheraremarkedlyregular. Althoughsomecontributionofchanceevents,forexample,
bottleneckingeffectsduringviraltransmission,cannotbeignored,thesepatternsarelikelydetermined
bymoreprofoundcircumstances. Inprinciple,beingserotype-specific,theycouldbeduetotwonot
mutuallyexclusivereasons: thelostsegmentsmightbeeitherintrinsicallyweak(low-fit)orpoorly
compatiblewithothergenomeparts(epistasis). Somegroundforchoosingthemorelikelyexplanation
canprovideacomparisonofdataofFigure1withthoseofFigure2,inwhichtheabundanceofdifferent
type-specificgenomicsegmentsinthenormalizedcommonsetofrecombinantsofallthreeserotypes
ispresented.
Viruses 2017, 9, 353 7 of 16
should be admitted however that the number of recombinants of this serotype was too small to
make firm conclusions. In serotype 2 recombinants, distal portions of the 3Dpol-coding and
3′-untranslated region (UTR) sequences were regularly lost, in some cases together with more or less
extended parts of the P3 region and sometimes also distal portions of the P2 region (Figure 1B).
Serotype 3 recombinants demonstrated, as noted above, a less regular pattern of losing their own
information, with the removal of (roughly) P2- or P3-related type-cognate sequences or both (Figure
1C). The absence of short type-specific sequences within the 3A-coding region was a common
feature of all but a couple of members of this group. The common absence of this genome piece
could be due to either its inherent properties or to the overlapping of the two just mentioned larger
segments.
The above results indicated that the patterns of intertypic recombination of Sabin viruses are
not haphazard but rather are markedly regular. Although some contribution of chance events, for
example, bottlenecking effects during viral transmission, cannot be ignored, these patterns are likely
determined by more profound circumstances. In principle, being serotype-specific, they could be
due to two not mutually exclusive reasons: the lost segments might be either intrinsically weak
(low-fit) or poorly compatible with other genome parts (epistasis). Some ground for choosing the
more likely explanation can provide a comparison of data of Figure 1 with those of Figure 2, in
which the abundance of different type-specific genomic segments in the normalized common set of
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recombinants of all three serotypes is presented.
1
5’-UTR VP2 VP3 VP1 2A 2B 2C 3A 3C 3D

VP4 3B 3’-UTR
FigureFi2g.urTeh 2e. oTchceu rorcecnucrereonfcet yopfe -tysppeec-sipfieccigfiecn ogmeniocmsiecg msegenmtesnitns itnh ethneo rnmoramliazleizdedc ocmommmonons esteto fof
recombreincoamntbsinoafnatsll otfh raelle tsherreoet ysepreost.ypTehs.e Tsheet oseft roecf ormecboimnbainntasnotsf oeaf cehacshe rsoetryoptyepies ins onromrmalaizliezdedt oto1 1..
Sequences derived from these serotypes are depicted in red, orange, and green respectively.
Sequencesderivedfromtheseserotypesaredepictedinred,orange,andgreenrespectively.
Thus, the presence of homotypic 5′- and 3′-adjacent sequences in the type 1 recombinants (Figure
Thus, the presence of homotypic 5(cid:48)- and 3(cid:48)-adjacent sequences in the type 1 recombinants
1A) could be hypothetically due to their preferred mutual compatibility (epistasis) but, as
(Figure 1A) could be hypothetically due to their preferred mutual compatibility (epistasis) but,
demonstrated in Figure 2, this 3′-terminal sequence was evidently good for all serotypes, suggesting
asdemonstratedinFigure2,this3(cid:48)-terminalsequencewasevidentlygoodforallserotypes,suggesting
that it may possess intrinsically higher fitness. Similarly, some other distinct portions of each serotype
thatitamppayeapreodss teos sexihnitbriint esiitchaellry rehliagthiveerlyfi thnigehss (.aS simigniliafirclay,nst opmaret ooft hthere dseisrotitnycptep toyrpteio 2n sseoqfueeancchess eenrocotydpineg
appearneodnsttoruecxthuirbailt peritohteeirnrse, leastpiveecliyallhyi gah 3(′-apsroigxnimifiacla rnetgpioanr toof fththee 2sCe rgoetnyep)e otry ploew2 psreeqfuereennccees (esnercootdyipneg 2
nonstr3u′-cteturmrailnpurso atnedin sse,reostpypecei 3a lsleyqaue3n(cid:48)-cper aorxoiumnadl trheeg bioonrdoefr tbheetw2Ceeng etnhee )Po2r alnodw Pp3 rreefgeiroennsc) eir(rseesrpoetcytipvee 2of
3(cid:48)-termthien unsaatunrde soefr octayppseid3 pseroqtueiennsc. eTahruosu, ntdhet hreelbaotirvdee rinbtreitnwsiece nwtehaeknPe2ssa/nstdrePn3gtrhe goifo ndsis)tiirnrcets pgeenctoimveic
ofthesneagtmuerentos facpappesairdedp troo tpelianys .a Tsihgunsif,icthanet rreollaet iivne thineitrr ilnosssic/awcqeuaiskintieosns /byst rreecnogmthbionfatdioisnt,i nthcotuggehn oefmfeiccts
segmeonft sepaipstpaetiacr eindtetroacptliaoynsa csoiuglndi fincoat nbter doilsecionunthteedir also swse/lal.c quisitionbyrecombination,thougheffects
ofepistaticTihnete nraocttioionn sofc otuheld enxoisttebnecde isocfo purnetseudmaasbwlye lllo.w-fit serotype-specific segments of poliovirus
Tgheenonmoteiso nis oaflsoth seupexpiosrtteendc ebyo tfhpe rsetsruucmtuarbelsy olfo rwec-ofimt bseinraontytsp be-estwpeeceinfi cSasbeignm sternaitnsso afnpdo nlioonv-piroulsio
enteroviruses. Thus, for example, such recombinants of type 1, which triggered polio outbreaks in
genomesisalsosupportedbythestructuresofrecombinantsbetweenSabinstrainsandnon-polio
the Dominican Republic and Philippines in 2000–2001, demonstrated a replacement of a significant
enteroviruses. Thus,forexample,suchrecombinantsoftype1,whichtriggeredpoliooutbreaksinthe
Domin icanRepublicandPhilippinesin2000–2001,demonstratedareplacementofasignificantportion
oftheirP2region[25,26]; thegenomesoftype2recombinantsinvolvedinseveraloutbreakswere
deprivedoftheirhomotypic3(cid:48)-ends[27,28];andpolio-nonpoliorecombinantsoftype3circulatingin
Madagascarpossessthegenomestructuresimilartothoseofintertypicrecombinantsofthisserotype
describedabove[29].
3.4. PeculiaritiesoftheEliminatedGenomicRegions
A plausible assumption is that the observed regularity of losing/gaining distinct genomic
segmentsislinkedtotheirfitness-modulatingcapacities.Aretheysomehowrelatedtothetype-specific
attenuatingmutationsacquireduponderivationofthevaccinestrains?Toapproachthispoint,weused
anarbitraryclassificationofpresumablylow-fitregionsbasedonthefrequencyoftheirremovalupon
intertypic recombination. Those that were lost in 90% or more recombinants of serotypes 2 and 3
andinatleast75%ofserotype1wereconsideredas“weak”(heaviershadinginFigures1and3),
whereasthosethatwereabsentlessoftenbutstillinatleastahalfoftherecombinantswereregarded
“provisionallyweak”. Alessstringentcriterionforserotype1wasduetoarelativelysmallsetofsuch
recombinants. Itgoeswithoutsayingthattheactualbordersoflow-fitregionsmaynotexactlycoincide
withsucharbitraryparameters,whichmerelypointtotheirsupposedapproximatelocations.
Viruses2017,9,353 10of18

Figure3.Locationofknownattenuatingmutations(redtriangles),anoften-revertedserotype-1-specific
mutation(blacktriangle)andmutationsacquireduponthederivationofvaccinestrains(verticallines)
inthegenomesofSabinviruses.Regionsspeculatedtodetermineadecreasedfitnessarehighlighted
asbluerectangles.Theirdarkerandlightershadingrefertotheproposed“weak”and“provisionally
weak” genomic areas (see Figure 1). The schematic representation of poliovirus genome and its
functionalelementsaregivenatthebottom.
As shown in Figure 3, all (for serotypes 2 and 3) or the majority (for serotype 1) of known
attenuating(andtemperature-sensitive(ts)phenotype-modulating)mutationsarelocatedupstream
of the recombination-affected parts of the genomes situated, as demonstrated above, in our set of
virusesdownstreamoftheVP1/2Aborder. MoreoveronlyasingleknownSabin1-specificattenuating
mutation(position6203inthe3D-encodinggene[30,31]mappedtoa“weak”region. Importantly,
asignificantproportionoftheknownattenuatingmutationsofallserotypeshadbeendeattenuated
independentlyoftherecombinationevents(Table2). Thesefactssuggestthatbothrecombinationand
single-basedreversionsoftheknownattenuatingmutations(orcompensatingalterations)couldlead
torestorationofneurovirulence.
Table2. Lossofattenuatingand/ortemperature-sensitive(ts)mutationsinthesetofrecombinants
withthefullysequencedgenomes.
AttenuatingMutationsLost,%
Serotype LocationofMutation
IntheRetainedPartoftheGenome IntheRemovedPartoftheGenome
480 67 -
525 11 -
935 22 -
2438 33 -
1 2741 22 -
2795 67 -
2879 0 -
6203 9 82
7071 8 25
398 49 -
481 100 -
2
2908 5 -
2909 85 -
472 98 -
3 2034 20 -
2493a 100 -
a2493-Uispresentinsomevaccines[32],thereforethismutationdoesnotnecessarilyrepresentreversionduring
severalvirustransmission.
1