Post on 20-Feb-2021
949 1765 2480 3833 5372 5987743 4124 5438 7370
3386 5110 7441
capsid
P1 P2 P3
non-capsid
poly (A)60 ± n
VPg-pU
initiation oftranslation
5’termination oftranslation
polyprotein(247)
●P1(97) P2(65) P3(84)
●VP0(37) VP3(26) VP1(34)
2A2BC(48) 3AB(12) 3CD(72)
●VP4(7) VP2(30) 2C(38) VPg(2) 3Cpro(20) 3Dpol(52)
3C’(36.4) 3D’(35.6)2B
3’
IRES
digestive tract
Replication of polio virus and disseminationReplication of polio virus and dissemination
Oral infection
barrier (tonsils, Peyer’s patch)
blood stream
barrier(BBB)
central nervous system
motor nerve cell
Skeletal muscle
axon pathway
Research on Research on pathogenicitypathogenicity
1. Mechanisms for determining species-specificity
2. Mechanisms of dissemination
3. Mechanisms for determining tissue-specificity
4. Ability to cause damage to the target cell
Research on Research on pathogenicitypathogenicity
1. Mechanisms for determining species-specificity
2. Mechanisms of dissemination
3. Mechanisms for determining tissue-specificity
4. Ability to cause damage to the target cell
Role of poliovirus receptor during polio virus infectionRole of poliovirus receptor during polio virus infection
human, monkey cells
inside cell
outside cell
polio virus
Can infect Cannot infect
extraction
polio virus gene
transfection
mouse cell
Cannot infect
transfectionCan infect
outside cell
polio virus receptor
inside cell
exon 1 2 3 4 5 6 7 8
HC 3
HC 510 Kb
ATG TGA
PVR αPVR β
PVR γ
BamHI
Hindlll
Structure of Poliovirus Receptor (PVR; CD155)
C
N
SS V
SS C2
SS C2
Domain 1
Domain 2
Domain 3
Cell membrane
Cytoplasm
Principles of viral tropismPrinciples of viral tropism
1. Receptor-dependent tropism2. Protease-dependent tropism3. IRES-dependent tropism4. Natural immunity-dependent tropism5. Others
Research on pathogenicity
1. Mechanisms determining species-specificity
2. Mechanisms of dissemination
3. Mechanisms determining tissue-specificity
4. Ability to cause damage to the target cell
digestive tract
Replication of polio virus and transmission in the bodyReplication of polio virus and transmission in the body
Oral infection
barrier (tonsils, Peyer’s patch)
blood stream
barrier(BBB)
central nervous system
motor nerve cell
Skeletal muscle
axon pathway
NeurotropicNeurotropic Polio virus InfectionPolio virus Infection
hPVR ExpressingMouse (Tg21)
TissueDistribution
BBBPermeation
CNSToxicity
Mahoney
Sabin 1 ?
?
?
? Strong
Very Weak
In BBB percolation of PV in the mouse, In BBB percolation of PV in the mouse, proactive import occurs in the brain proactive import occurs in the brain independent of CD155.independent of CD155.
Speed of PV accumulation in the blood of a mouse cerebrumSpeed of PV accumulation in the blood of a mouse cerebrum
speed of accumulation ( µl / min / g tissue )
virus and control substances mouse with CD155 expression
mouse without CD155 expression
PV ( Mahoney )
Negative control ( Albumin )
Positive control( Cationized rat serum albumin )
0.164 0.123
― 0.001
0.123 ( Rat )
( Yang et al.,1997, Virology 229 , 421- 428 )
Percolation examination using thePercolation examination using theBBB BBB in vitroin vitro modelmodel
mouse cerebrovascularendothelial cell
+
fluorescent dye
( Alexa Fluor )
fluorescent dyed PV ( 126.55 µg / ml )
Periodic measurement of fluorescence after PV addition
TimeCle
aran
ce
volu
me
( µl )
PS
( PS : Permeability-surface area product )
Clearance volume
( µl )
amount of PV moved to basolateral surface ( µg )
Primary concentration of PV at the luminal surface ( µg / µl )
==
purified PV
PV percolation in the BBB PV percolation in the BBB in vitro in vitro model is extremely highmodel is extremely high
05
10152025303540
0 10 20 30 40
PV ( mw 9800K )
Dextran ( mw 2000K )
Transferrin ( mw 80K )
Dextran ( mw 70K )
0
5
10
15
20
25
30
0 10 20 30 40Time ( min )
PS = 0.7953
PS = 0.3103
PS = 1.0877
PS = 0.2112
Cle
aran
ce v
olum
e ( µ
l )
Cle
aran
ce v
olum
e ( µ
l )
Time ( min )
Examination of PV intake into a mouse Examination of PV intake into a mouse cerebrovascularcerebrovascular--endothelial cellendothelial cell
TransferrinDextranDextran
(( mw 3K mw 3K )) raft
Cholera Toxin
TransferrinTransferrinreceptorreceptor
DextranDextran(( mw mw >> 15K 15K ))
fluorescent dyed PV
path between intercellular gap
??
Pathway into Pathway into endosomeendosome
??????
PVPV ++ DextranDextran ((mw. 3K mw. 3K ))
PVPV ++ Cholera ToxinCholera Toxin
_
Dextran( mw15K - 20K )
667μg / ml 1mg / ml
_ m.o.i. 5000Purified PV
EndocytosisEndocytosis of of transferrintransferrin ( ( TfTf ) is inhibited by PV ) is inhibited by PV
20 µm
Alexa 488 - Tf10μg / ml
( MBEC4 cell )
PV percolation in BBB PV percolation in BBB in vitro in vitro model is lowered by model is lowered by TfTf
0
5
10
15
20
25
30
35
0 5 10 15 20
Time (min)
Cle
aran
ce v
olum
e (µ
l)
PV
Tf + PV
FITC-Dextran(2000K)
PS = 1.89
PS = 1.02
digestive tract
Replication of polio virus and transmission in the bodyReplication of polio virus and transmission in the body
Oral infection
barrier (tonsils, Peyer’s patch)
blood stream
barrier(BBB)
central nervous system
motor nerve cell
Skeletal muscle
axon pathway
Sampling time PV antigen
1.5h - - +
3h + + +
6h + + +
12h - ± ±
Polio virus antigen inside an Polio virus antigen inside an ischiadicischiadic nerve axon nerve axon 2cm away from administration2cm away from administration
Tg +PV
Merge α-bungarotoxin PV
20μm
PV localized at neuromuscular junctions in PV localized at neuromuscular junctions in TgTg micemice
Neuromuscular junction of a Neuromuscular junction of a TgTg mousemouse1.5 hrs after PV injection1.5 hrs after PV injection
Immunity electronmicroscope(detects PV )
Transmissionelectronmicroscope
100nm
100nm100nm
Principles of viral tropismPercolation examination using the� BBB in vitro modelPV percolation in the BBB in vitro model is extremely highExamination of PV intake into a mouse cerebrovascular-endothelial cellPV percolation in BBB in vitro model is lowered by Tf