Circulating Circulating VDPVsVDPVs DetectedDetected____________________________________Country Number of cases Virus type Duration

------------------------------------------------------------------------Egypt 32 Type 2 1982～1993Hispaniola 17 Type 1 2000～2001

Philippines 3 Type 1 2001________________________________________________

LongLong--term Carriersterm Carriers

Developed CountriesDeveloped Countries

OPV IPV no polio virusno immunization

Many Developing CountriesMany Developing Countries

OPV no immunization

Low cost anti-PV drugsNew OPV with high genetic stability

RATE OF MUTATIONRATE OF MUTATION

Replication of ssRNA genomes ---- 10-3 ~ 10-4

Replication of dsDNA genomes---- 10-8 ~ 10-11

Holland et al. (1982) Science 215:1577

Supporting a SequenceSupporting a Sequence--loop Modelloop Model

Supporting a SequenceSupporting a Sequence--loop Modelloop ModelGeneration of DI particles

ST-RNA+

or

DI-RNA

ST-RNA: RNA of Standard virusDI-RNA: RNA of DI particle

:VPg : polyA : polyU

Generation of recombinants

+A(+)

A(–)+orB(+) B(–)

A(+): + Strand of strain A A(–): ー Strand of strain AB(+): + Strand of strain BB(–): ー Strand of strain B

polio virus

World power relationships of World power relationships of enterovirusesenteroviruses

Modern world After polio virus eradication

echovirus coxsackievirus

other viruses

echovirus coxsackievirus

other viruses

evolution of viruses (invasion）

Pathogenic microbesPathogenic microbes

Effective microbesEffective microbes

Other microbesOther microbes

digestive tract

Replication of polio virus and disseminationReplication of polio virus and disseminationOral infection

barrier （tonsils, Peyer’s patch）

blood stream

barrier(BBB)

central nervous system

motor nerve cell

Skeletal muscle

axon pathway

Schematic Structure of Polio Virus Vector Schematic Structure of Polio Virus Vector for Expression of BDNFfor Expression of BDNF

.. BDNF..M R G R ... … E L G G G G E A L F Q G

Artificial 3Cpro trans-cleavage siteSac INot I

GAG CTC GGA GGA GGA GGA GAG GCT TTG TTT CAA GGT GCTAUA ATG CGC GGC CGC

VP3 VP1 2A 2B 2C 3A 3C 3D5' 3'VP0VPg

P3P1 P2

-1.8 kb

-2.6 kb

5'

VP3 VP1 2A 2C 3A 3C 3D

VP4VPg

non-structural domainstructural domain

OMΔ0.8

PV1(M)OM

OMΔ1.8

OMΔP1

OMΔP1Δ2A

-0.8 kb

-3.1 kb

Non-translated domain

RNA structure of PVRNA structure of PV--deficient mutantdeficient mutant

poly A3B

Non-translated domain

2B

3'

VP2

PV RNA replication activity measuring methodPV RNA replication activity measuring method

in vitro synthesized RNA from defective mutant cDNA

HeLa monolayer cultured cell in 6 well plate

RNA extraction 2hrs/8hrs after transfection

RNA transfection by DEAE-dextran method

The probe is cDNA in a 5’ non-translated regiondetection of PV RNA by slot blot method

non-structure domainstructure domain

P1 P2

-1.8 kb

-2.6 kb

5'

-0.8 kb

-3.1 kb

2C 3A

VP4

3'VP3VP1 2AVP2VPg

3D

OMΔ0.8

PV1(M)OM

OMΔ1.8

OMΔP1

OMΔP1Δ2A

mock

inner standard GAPDH

＋

＋＋

＋＋

＋＋＋

＋

RNA replicationactivity

2 h 8 h 2 h 8 h

−

2B 3C 3D

P33B

RNA structure of PVRNA structure of PV--deficient mutant deficient mutant

VV-P1

RNA packaging of RNA packaging of PVPV--deficient mutantdeficient mutant

amplification of DI particles containing PV- defective mutant RNA

in vitro synthesized PV-defective mutant RNAHeLa monolayer cultured cell

formation of DI particle containing PV-defective mutant RNA

passage of DI particleVV-P1

Passage1

Passage2

Mock

removal VV-P1 by SDS treatment

OMΔ0.8

PV1(M)OM

OMΔ1.8

OMΔP1

OMΔP1Δ2A

mock

＋

＋

＋

＋

＋

inner standard GAPD

2 h 8 h

−

2 h 8 h

RNA replication activities of packaged PVRNA replication activities of packaged PV--defective defective mutant mutant RNAsRNAs

particle formationactivity

Non-structure domainStructure domain

P1 P2

-1.8 kb

-2.6 kb

5'

-0.8 kb

-3.1 kb

2C 3A

VP4

3'VP3VP1 2AVP2VPg 2B 3C 3D

P33B

Function of 2A proteaseFunction of 2A protease

・inhibition of cap-dependent translation

・cytopathic effect ; CPE

2A

Virus RNA replication

・not necessary, but some involvement in RNA replication

・enhance translation-starting efficiency

Effects to infected cell

CPE expressions in CPE expressions in DI DI particle infected cellsparticle infected cells

mock

OMΔ0.8 OMΔ1.8

OMΔP1 OMΔP1Δ2A

PV1(M)OM

EMCV IRES

P3P1 P2

VP3 VP1 2A 2C 3A 3C 3D

VP4pPV1(M)OM

3B

2BVP2

pPVE2A

pPVE2B

PV

IRES

3Cpro cleavage site

2B

Expression vector：pSVA14

poly A

PV

IRES2A

2Apro cleavage site

PV

IRES

EMCV IRES

PVE2APV1（M）OM

Mock PVE2B

(8 hours post infection (h.p.i))

CPE

CPE

CPE

NO CPE

PVE2APV1（M）OM PVE2B

7m008

No Ab

Mock

18h.p.i

Neutralizing assay producing virusesNeutralizing assay producing virusesby I antibodyby I antibody

Polio research in a post genome eraPolio research in a post genome erastructure of whole genome conformation of particles

infectious nucleic acid polio virus receptor

reverse genetics polio-sensitive transgenic mouse

IRES activity and virus tropism receptor and virus tropism（tissue-specificity） （species-specific）

Mechanisms of polio virus replicationand pathogenicity expression in

the body

Circulating VDPVs Detected�____________________________________�Country Number of cases Virus type Duration�--Long-term CarriersDeveloped CountriesMany Developing CountriesSchematic Structure of Polio Virus Vector for Expression of BDNFNeutralizing assay producing viruses� by I antibodyPolio research in a post genome era