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UNIVIA$TY OI Itr IUNJAT
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DOCIIIAII Or tEIIgX[v
(D llv ctllol{lA DESqTATIo}'
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CTRIIFICATE
'IX! E b sdly lh.r thc .xFiE6d *o* d!*Dt d in lt. d6i. lutmied bv Mis
Atit Umhlsb*rffiedoutunlaryd!€1tuFwitid D|h/E$lt!ntorLdi!tlit
M@rat e duly @d.d it dF @L.'t officid mio bo.k. I htrc P@'dv gue
thMsn lne aw d.L dd oqliry ih. co@tsdiulbolioitv of all Gults Fporld h@in
I ftdq @ni& thal th6. &t h.vc @t b.d @d in P[r d tull in ! nnusiDt
ireidy subnin d d in rbe p@!s of flhnisid i! FnilvcmpLt ftmLrdt ord* attd
ofsy otba d€sE ton oy odF itnturdd .1h6. d lbud
I .ls sdt ln.t dF @loi.d .lll]@iPl tli b.@ F.P.nd 8dd 6v sup6visio
.rd I ddo* i6 d.lution fd lt .s.!d oflhD &sE &uC tt offici.l ?ra.{iN 016.
[email protected]!!r,.Lt t@lr Mh@ 2l l,333 G d.cr.t d
6 rE4ad.5la {toc||t@l! tLrl *in t. bla il tt5 cg!t! foi. oinibm of rht v.G tm
tt && of the rb6L d.f.@ miErid,
D{. S. RTAZUDDIN
(DiEtor, CEMB)
lN TEE NAME OT MOST MERCIIUL AIIAEP',/Ea m 8a
'trddA VE Dn rtlqt NOr lNf
'tlt to BxcEpr w4, npu &Br
ylDB KIoNII 10 arS rNnEAD EIOU,4rT rltt tE!' tJtOyE& rW r'D'ar.
n&tD, N InE N/t$ 0Ftnf r&D, fw fllfl c&ED ltL tfi'l,c': |rnEt|flCRAIED MlN AF CONAEAIED NI@D. tBtD, Df IER tllof'r RENEFICEN|IATD,
VHO TtrWI[f t|]E LEE OF fEE PEN; VEO EACEBIE U1N IE tt VHICE HB
&tonau Nqt
a|.{u r N
r arIE cttoll{ltx.Y Dtrt rcattaErS lD tEtals
rO TSD SIUPRXDO STITT OI MY GI|IAT MOTSEB,
i4{rro{En c.alr}MYEUIAAiD&Mf ltaucqlllgE ltUA.talnUa l{
AND IO AII OI MY IAI|TLY.
ACXNOWTJTIGIIIiENIII
I w. ! lot of gDdrurte to hy 3r9cdi6 .!d DiFior ofdE itlritut! CBl"lB, Pnt Dr. S.
nhaddl! fd povidnlg m lhi. .fportDity io wort d lhh pbj4t *ilt dl Me€ry
l.bdtory &cilittu i! clmctid witl lhir rot .rd rivi.r @ htu tult Cdd|G dd
.odinuld c.M i! rh€ posB .nd .!@[id &@rqli.!d6r of Gis poi..a
I rsr.l bL. rolla! Dr. zi..Ivt U@?rnr.2.6. s.h.o, F*,nr, lti x,ue,
Anb4 r....h. d Mr. ^njrd
od r&. Xldid (C.qtr rrb) b.6.n 'im coqcndor
udt lt h.n 6.riiDlid rln fi..ur|nila |t., did.
I@ldlit ro |hdt.n CEMB !D!9. &( rtct coaqrdd hae wituot |nd it mu!
't rEy difidlr fd @ to !.nim ny ldl,
ATIX (MER
TAELA OI CONTINTS
LTSTOF N(;rJRES AND TAILAS
Ch.pl.r r SUMMARY
Ct Dt rl INnoDUCnON
cLltdl LTfERATUREIEVTEW
I
8
ttqoliti! B viru!: @loac.l Dst n & is
St.r.nnldiftrffi t.@ g.t dr.vilE
ClrEidirtica ofHEV: Vid dtucfii, Ari,EFq
ald Offi O'8Adz.lid
GmtyT.r, Hirlory Dd CLaiic.tio!
Murldn R'|. bd eldib !.!.n ofllBv
cliniel Sisrific@ ofHEV Mlt nt
Chloic HBv bferid dd vir.l F i!@
M€lnisn of vinl p6ite!4
lrm@logy of HBV vrccimtid
MAlTf,IAIs IIMEIqOD
B-hin stliE .rd pLoilt
llll
t1
l9
2l
2t
26
27
29
34
38
38
O[go d6ri|.. CoMtnid didrim
Pdy@ chlin Raclid (PCR)
Anifdid of DNA FrgMl ibn A8.l@ G.l
Mirj PreDmtid of Pl4mid DNA by.lt lie LFi! Method
R.siiciion Endouol@ Dig.stior of DNA
[email protected] in Mic&@tiftg. Tut !
Eplcs@ ofbiddL F.r.itPurifc.lio ofFNio tr.{rb crpc!.d b E cd
NESULIS
PErnlore orEBv i. dilE@t ld ofl.*irbr
dd o infenm win gCV
Olieonuol@tid. Pri'@ U!.d For rcR ChGicrialim
OfH€p.titis B vios Surf.s Antign
CldDiq OfTh. PFSI/k+S2/S G@
rito I'ldlml Cldi.8 VeloB
FigE sbowiq ndllir .! difaan
Subcl@iDsI o Elpqn@ V4!or pCEX4Tl
33
38
39
45
43
48
5l
5l
62
6t
1l
W.d.n blot ofFrifed 6sior pot in
DISCUSSION
RETERENCES
15
77
85
NGUru
T.bl. !tr.
LIST OF FIGTJRTS AI{D TAALES
ITI'I,E
SaDLrgic.l MritE .idldaFt ri@
Stuhl difi.re h.rrE HcFddvilw
Dd relaliotuhip b.r*@, dD dia. igdic 6ubryp6 of
HBv 6d Anim Aoid id.nlig {Lt! d A4)d nto po$tiom
020 and 160) in liF S pbr.in.
RcbtioEtip bctwo HBV s@tyFr .nd sbtylq
MrsrrMn Di€?8Eti@ f6rcR.
Us.lim Gins T,{41oirg v6io.
DicstioD of cr!6id Eid ro. fislti@ with &,Rl
PAGE No.
t.DbVItr Ugrd@of €aTtwioa @tor sidi in&n tu cxt@im 4l
42
ll
t4
t1
5l
42
46
T|bL D( Corflmnon of .ll olmr uing rzrHl .Dzyh.
Pepding *qnfuiis tuti@
O@ orHDV irfdtid in dh &d f@lc
p!&rrs in.eord'4 ofrl.i .9..
PErrrce ofHB.A& d.Lrr$. HBV-DNA dxt
HCv in r @hort of 5l I tltiat HBrAg posiriw.
52
Trbl. l.I O ieanucl.otide pride^ u$d for PCRchtuereriz.tioi 5l
or HcpdiG B YiG $rfae &rig...
Figrrc 3 Amplinc.tiM of whol. suif..q g.e ol HBv using
ATUI & ATU5 (F & R) prin.d.
Flgrr. it N.n d Dolytrc@ chain edion (PCR)
Figoru s PCR cnplificrtio ofpr.S2,6g.n Fom smpl6
using vAC F & R piDcts
Flgurc 6 CloninS ofdE PE-SI/P|-SZS g.ft itu
ditr Enl clo.ing velo6.
lilorc 7 MinirEpsRnon oaDNA 6on clon*
FlalE 8 PnybesEtic !E oft*.ty fivc i$l{6 win
rcsp..t |o .n gcno$T.s.
ftrr 9 Mur.tion at ditr@nt nEl@tid. pdirioc ol
lll lhc isolaEs t q!.nc€d.
Flgur.lo rcR confimltion of clone 3A
Ft rcrl Digcsio. of bo.h lA ed pGEX-4Tl wilh E dR I
Figurc 12 Prnficlrion of Pr. S2,5 Cpie (0,8 kb).n rth.
diScationofclon.3A.
FtguE 13 confim.rion of rruf@aE of ,GEx4Tl in
aL-21 ells !ff.r digcsli@ wilh &oRl.
llglr l.h SDS-PACE ol purincd fr&lion lrom GST-S.ph!rce 4S Column, 75
PtsuF r4b w.sLm bloningofpurificd ft!.tio f@ csT-S.Fn.l@ 15
4E Colunn hybndi&d wiih dti-GST ulibodies.
Figure15 w.sLm bbninsofpuified frulion i@o GST-S.phre 76
59
60
62
7l
12
7t
48 Colunn hybndii.d s'rh poltcl.n.l dri-HAs edbodies
AtBf,fvhnoNs
BCIP 5-b|wo-4.n1@linloyl
pboab{. Floluidinim slt
BSA Bovinc Se'm Alblnid
cDs Codinss.q@
cry cryrtdl Prclcit
.,r, cry3i F*in &ftdATP [email protected]
!iPho6p!.r.
DIC Digprigdn
DNA D@xFiho nucldc rcid
dUI? daxy uridire Litlo+b.e
EDTA EtlyLre diuiE r.ta @tic
IcPs [email protected] c.ystr ptDtcia
FTG l$DFpyrFinioc.bcto
LB L!ri. Bdtani
LMP low n lt'sg poht &€@& gel
rnD! ac8. Ddb
hol, n4. Doleculltrq8hi
NBT pliLo Ue r.n@lim
O,D, OttndD.nliq
PBS PldphlE bulfa elnE
PAGE Pob.cryldi& gcl
.LclFphoBis
RN Rrb.nrcLio eid
rPD r.volution pd minDt
SDS !.diM dddyl$lnr.
T.q netuEaquchd
|RNA futu riboNoleic aid
qP CrtlnteslindlhGphoLiDde
fti&Ho f.i.(hdlrrndhyl)
[email protected] lldDotlqid.
llF ai6F.nd.
ELISA Elrrd hl.d
i@ulororbal dylD.Ag lliDdrBdoeda6ID.A8 g.?.ridr B .slop. eli86
UCV ILFtErCdu
NwlI NdllvdtlmtirPdt
ISo\g Eartid. B c@ ertBd
& I@oglohdic
CHAPTER 1
Itarritis B vitu {HBU irfdrior i! | glob.l phlic balth poblc4- n n
cninuted tur thcE @ '@ th.n 300 nillid HBv sri4 h tb€ vo d, ofvlnch I
miuid will di. rmudy non HBv-Elricd liH dise.
Itprrin B vdiril e anElly ocdiog, .!d my t v€ ben rlarcd .@
a lons psiod of 1ift, pobably cdlurio!, wh@r n p.titir B nutlnll aE varidl!
*hich b.w .voltd @ ! shdLr pc.iod of liD. .irtur ty *leliw imun. prcs@
or ty us of thdpeutu, Vdi&ir my lls b. Efod.d to s subg!.s. A wid.
S.ognphicrl vriltid i3 kM 10 4in for Ea venna worldwid., n.king it
.sstiil for .&h Eei@ !o &6rc i6 om vdiots lrd our. s rtrd th.n b& h
cli al oursnq Mutali@af tn. H.prtilb B lurfde intiga (IIBe{g) of heprritis B
vic (IIBD is @ of s. inpodar fa.loE rhrr lwl i. iMW c*.F .!d eusfaihE of [email protected] md @p. n@ immuno<h@tion .st!.
Irr prqr surdy n I p.n or, '!3@h
pqa. rihod to de\ elop ! b€ner dd
E@ imwgdio @im or rt t !tu of infomation obr.ird nm eMina tn.
tutltion in ou! locrl t@ulitid oflcpaliti! B virEl i$lat6. Thce 3tudics [email protected]
rhc h.Dolitis a vir.r irol,Gr 6w difcal loqlitix oflhtilh rnd *ler.d . vdidl
d lh. b$i ot 3.quemin8 lhowing nul|tior in lha r'&r.tuimt of thc s e* ol
HBv. ni. e&iet dy h. eiDdat€<l for produoilg ! bctler rffibimt HBV
DNA vram or fn di€mstic purp.G fd our loql p.prlard.
In lhi! 6tud, tle blood sp€rnDcB of bep itis pttient wcrc t*fl ton
diffdd citi6 of rvo pbvinc Mely N.WF.P. sd Punj.b (irclu.lins c!jl!ML,Foisdobad, L.hoE, Sdsodh., Srhisd), Ands 1270 hcpalilis polierrs, 5t I pdicnb
w sutr€.ils witl HBv inftorid ud ums lncs dly l,{3 h.rl vimia (IIBV
DNA dd.ctd h ldn) Am@8 tlre 148, I 16 wd sde dd 32 *@ fddes. Ii
rhi: tudy,I fould 9 p.liDG {ho rw Egltiw for HB.A3 ed p6idw rd HBV
DNA C@E Duhnt) .!d o& Drtidt *ho *r mgrriw for gBsAs dd p6iliE f6
HBv DNI- Ml)Fvd, t 3 !.ti6l! w lo@d !o tlE @ intedim wi6 Hcv.
To $lq rll of lne 5l I HBv inf*r.d p.iicnE, I d*ig!.d FiDm 6m lhc
core cgion (?rind1&2) n@ [email protected] th& ruf@ 3d. uiDs Prind3 $nw@
ahtuJ/bioinfomdics.r.imn.&.illcci-bitdim.r/orih.r3.cEi) mdrrying . 620bp
poduct. nEn, for tne varialion mlyfi I d6i8rcd two 6eB or lrineN, A-rUl &
ATU5 and ATHI &ATH5 ddliig thc Pe.Sl, Prc-S2 od S spre,
so I eldlert ttrrnry [E cbbdo HBv p.tial' nu lnas PcR p6'!ive c@s
lnd .!DU6€d I@ eiti AlUl & ATU5 s.r ofpridd io idcnt'ry n$.tios within
tb. HkA& Ib. prodrct upli6.d ot bjft..tr peid.6 9@ clon d ir ditrcEnt
cloidg €106 to obl.i! singl. tyF of DNA n|8era i. c4 of aqging vdttirs
urda sled€ FM or i@@ '!9oie
or tlEnpy udasoins.TrF @iring
Mlv. speintu re !.rpli6.d Bing diludon L.lmique (mplirying rhc pI ducr
fron sinejc nol€DL)- TrE .npli6.d prcdkr. (l.2kb) w@ pun6€d {rd scq!.ied.
m d Aal PRISM 3100 Cen dc Arly6 {Applicd Bi6yn@3).TlE s.qum.r
oblqin d v€re aMlytsed fd its lmolo&r !o diff.EDt g.notFs of HBv by Mullalign
prcghm ol NCBI w.hsiL. In dl chelic p.ti6l! sl@Ld lion NWIP dd Phjdb,
seloqT. D w6 fourd 0Dd all lh. iblrlls *E found b hnr EidioD in tne
i.pltitis B sdac sce wlion ntrg. ion 3-t J% Espfttiwly which ws i.a\%d by
Eins NCBI Blasl .t wch6ib: *w.rcbi,nln.iih.tovlblt<-hln. nE nulatiN
iddrifi.d at d'ff€al sib of ncl6tid. eql).!g of HBdg i. !I isohtes of HBV
chonic patidls @ shM i! d6 |alE (FisuE l2). MulbpL s.qu.rc .lisDDal or
mino eid sqfted uilg Cl$r.lw 1.8 thdlgh BCM Iluch.r sil. sbw.d m
dii6@r o{bti@ iD uy of th. .piiopo. ot gt"{ da! I orl IIIA cbs II a$icLd
.pitopd bll @ h.ving 6!i.li6 wilhi! iho libr Aa t' d.!.mimt
'ft. 800br a.smlt of FFS2 & S i.d! 6codi!8 nildl. Foi.in of tlit
vdn* w aqlifi.d ujlg eth.l Ft of Flc 0oli!8 tn. Pr*s2 o|d S 8!D by
VACF .rd VACR Fiid .nd ilFr.d irto tOEX4Tl dpt65i@ @ior aing
EcoN sitc L€piig rhc t o. i! F.itid, Tt r*@bi@t pLeid tling &.
Fmior w r@fotuld inio x,.!n !*idi.l 6[ I!. &! t[. .:F!sid of hiddL
th. tsford.d elL of r..,lr rs bte!.bd ido r cdnl& @.di@, T!.
cdtu de[.reh/t d|oqnrdt Fo!ft. ft. F!oi! u oDi.ird r. tund
Fori! rin Girrdno.-Str!&o.. hd4 s{NlD. odddt tdglt uiq
I'ltrldit .qcri6 d Frif€dn O,6..ltadn bld ub& .lded 6.
dq4!d 3i2. or tt Flt ir ui4 dt€Sr db.6a h[ Dt *ith dli-HBt
rdibodi. .,tic[ .bred qy Fo. tildila 0i.!r 6.!d) d e bidiig rdvity dt&
CHAPTER 2
IN'ITODUCTION
Fcprtiti! B ViN iDfatid oIBV) i! 3till . mid ow of li!.t di&@ .rd of
noftlit, ltNgholt d!. sql4 deFil. Oc cficry of 6e rei*3 (SeutB d a1,
l99l). HEV qu6.s tiaiat dd cb@ic irf*titu of the liv.r. Tlui.nlinf.clioG
mcy prcducc sioN ilffis, ed appoxiMr.ly 0.5% temimlai wiln falll, fDlrni@l
nepdiitir ChMic infFti@ bry lls h.v. siN cdE€qu.n@: @ly 25%
r€miEi. h ut!.h!l. liu ..|E (Ba8l.y 1938). WdldvirL d.dtu nm [Ec.l@ eu&d by HBv inf6tio Fo6.6ly dc€d o@ ninion Fr y..r. (El d ua,
1998, Pl*iD €, al, 1999).ft ha b*! dtiMbd ft!1 nj@ tho 150 milid of the
wo d poDuldie e chbnic dicts of rhc vin. (lE 1997). HBv in&ction is
.si.t d wiih r widc s?c.ltuE of clinidl ruifslltiql5 Ergi.g fiDD &t!. dftlnitut h@!dri! ro vdi* 6d! of chMic iddid imludiq rryspioD.ti
qftia $ar., chMic h.pornis! liE cirn@ir dd n Ft oeluld csil:@ gCC
(ke d 4t.,l991. and d al.,rgsr).
Fbn I.& 1980's, lhft ha b+n ircrsing ldbd oa lclond m hcpriitir B
(HB) pnial! virh .tpic.r lav i@bsicll M*@. ADly.b of HBv in rboe
pstiall d.noBn i.d sut'a|J (c.tqizo d aL, 2r].]0j @, d ol., 198, B@ d
aL,1995, W.irtuA.r et al., 199. Shinji .l al, 1998, S.hori6 er at, 2000), Mutarios
coDld b. fohd within dE c g*, S sd., P !pn6 ud X cee (Brun.lto €' ar, t999,
C,lrm €t dl, l9t). HAdAg qEodcd by rh. S g* di6 th. co@on &tcmiMl
h a rcll a'b m y &d' d ? $btF d.LmiM! md b cLBifi.d inro fou
Eljd $bir?€, i,., ,rp, a4, ory, az. SubDri.s of r.iBV rr.iu i! !!ed for
epiddiologi..l purDos or ro 6rd comLrio t twm dise ldt prnidld
subtlTe (eon tio wilbility Evid 2002), InlaFdinsly, o siDgle point nulati@ in ltF
S gqe cd tudc .idF r onplct. 6wi.h fbn oE 3ublyF to dorhr, or ! ls of
c,gEstm of d .pit'pc .s!i!ir wlicn |h. [email protected] uli-fiBs cAoE ir
diE1.d. Tpo sood eMpLs e poirt nulltiB n @ pcirid 122 (d0) aid )@
(-/d, i.hlvins ly3i* d .4ilirc (Sri* d dt, r92, Ctu d zr, 1990).T[e
nuh.ls my mt t@siv i oEct wiln |n. B @u .pnoF of IlBsAs. of cli.icd
sisdfcae G the facr dat lbc veinio doc! rct.li.r th. lbilig of the virus lo b€
olhon d b the helal@yr. od ftpliclio, Lding lo chMic livd disase. Fd dnDDL,
th. substibtion of dgirirc !t positior l4s by dyoirc ..u!es ! vr@irc .se. mulrln
b derge (C.mm €, dl, 1990, Hrbor d dl,l99l, \{leF el aJ., 1992J. m.
dv!l@ in aol@ubr biolog/ E hriqs! ir th. psl da.de hlv€ l€<t to the
cLrifetim of h.p6tilt B vinl DNA 5.qllffi, Howwr, n&b mlttr !o b.
atiiblirh.d r.3rdiDg llc qragirg h.p.rftir B nurrE. &4nl put lirc fom of
n plritis B viro he ben isl|ig4 blt dFn ctid.niolo6/, et6l oum of
i etio old cliriel sislifcs tlDh sl.r.ht Conpoud;ng 6€e Flblms @
f'croc .@led by hul@ i;&rEntio , slch s H&as ms imuiz.lior',
ch€norhenpy of cbmic IIBV p.tidt! .nd HBIg pbpnyhxb of ordrotopic liE
telPhd (OLT) p.tieft, wlicn Eld io {coung. ** nusnrs to em4q D€rpii.
th. availabili$/ ol HB6Ag vccciG .nd tlF @s imunization $n@es nndddLd
in oler 30 counhi4 woddwid., qBV infarion @nliDu.s !o b€ ! rujor puhlic heaM
poblen. TheE G oq 150 nillim otMic qni6 worldrid., o!1 of which abdl
loelo di. ol cidosis dd h.priocllul.r cei.od (kro ar oa. l99O. A widc
sosnlhiqr veilrid dists for HBv, tu8jDg tom lw, ltruen ftemedi,n, t)
higb '!d vay high ddcoiciry. TIE ld pEvllc@ nt6 id d€[email protected] ru$i.6
sh{ply @tn5t wnh rl'€ hign iDtelion Ers of Soutbe3t A5ir, aaic.! chiM .nd .h.
MidnL E!sr: Perimt0l tllmi!€ion ed childnood norizont l infetid e k om td
h. v€ry co@@ in cnd@ic @t of dE wotld. Ardbusn the emca.t or sBV
imuizslio is wlt D.ovc4 c@ of v@iB nil@k djng lo chdicig, & e slf'
tinii'd syq&a.ic b.p.riG !r4 in r@ e c$.n ftlnimr b.Ffiis, h!rc ba
E9otud (He r zr, 1988). In @. of lh srudia, ud-BBs [email protected]
drtrnll d sMlly Mi.A @F ndrnt! hrrc t or idefiifi.d (C.m.r a ar,
1990, YlMoro d rt, 194).
Th€ nosl inpmtonr S gae hul|tidr e lh@ affstirB tne olttenicily of
ItBr{g ! d€tuimi (s 124 to 144 to *hich lb. mjor [email protected] of
ndtditng polylloDl mriMi.r twtcd, ln $tu w!y, the HBsAa nuhrtr cd
csp. d.r.cti6 by mt ftthod! lnd irtFm th. .fi*t or HBv v@iM. A Ios
of drigoiciry d rh. a d.r.diM4 Ladilg to l&t of a.t .tim of th* tublE by
Mc nomloEl EBsAs iNNy }j!, hE b6r' r@lied (c.M d at,r995,
C.m d zr, 191). Thm, rh.E i! . FE thdr b oE sl@ of l@iBrio
pDsMs ard dE efcty ofblood spply in c.ddic !@ of th. wdrd {h@ tb6.
nur,rE @ prFdol! Br!.d on tL Blu;tdon d.t cudtt vciB cdtainins s
pbLin do nol ms$pd.cr.stinrl $ g@ n!l.d., . madEmn al nddel P&dicts
ln. diepFdae of wild qTa EBV ir .@ eith HE,Ag endcnicity dd lhc
enqsfl@ of S s€ne nullit in appoxjMt.ly 100 y.as d. cd*qrd@ of
uriwl HBv vrccimlior Mullnl vids! 6.y *lp. dctecti@ by cmftirl
HkAg ki6. Tl€E e *Ml rcporb on HBsAg nceativ. strim (HBV-DNA
posiriE) of S€Fm DuhrE vith imrndil.r't infetior or "unutDl" slogic
c6t llltions. Adas! S s@ nuLrb tuw ba loutrd to b. ll$u&dvith a n@
sv@ clioiel @ of HBV irferion &d hlp.ffx.lllL. csimo (ECC),ih.
clidal 3igri6c.@ or rh. gmtic wi.bility of HBV gceo!.s rnd HBSAa ould!
n.€d. io b. tunnor inv.6d!eLd. D.tcction of HBlAg me<t! 1o b. inpovcd bv lhe
inEoduction of Dc{ HB&{g 46.y6 $b ro @gnia e Iu d€dib.d s sdc Eur.nB
Tb. dlironic pdtm of $G pEml h.f.litc B v36jft mv rct b€
ld.qutc to pod@ edbodid c4.bL of @E lizing tb. dasirg celF Dura,6
sld @y rEd io b. nodifieiL It is, $@foE, Fr,moot to 6$bli.h mt onlv
pEr€1fl*s of HBsAg iD ctrddio aM5 but .lso to f@s m dD t tc of nut.a
.n rgiig .rd it! rlc i! lh. dtishi.ity of HD&$ Ilis vill h.lP fihE policv
ridcili@ €bting io @iE co@porat3, 4 vdl e di.S[cric Ny dei8lt
Tt snld, pt4otld in 6i rh6i. i. . avd.d ro; (l) ScGniDg &d blood
mplins of gBV po6itive pttu non difiRnl locslili.s (2) S.bcno. of a wiatn
of HBV frm I@l popDlatio. (3) PCR mpliicaliot sd cloniDg of imunosaic
.1Esic of gBV orne eriSpr (4) Slb.ldits ofdpffi.d ptod@l in atr .xpEssi@
!€tor. (5) E pF6i@ !tdi6 ofFoLitr-
rn $. pllsl sbd, I ha* idd$i6.d thc ablid tusing toD 2-15% in
$.fec stu.non8 0E Iwdty 6rc islic. of HBV in PtLi6nni popuhtid whicb
is quite sisnificDt and lh. subty?ec e.rc .t{ ed adr' Tnc @oobiMt HBsAg
daircd noE . v&i6r @ [email protected] .xpEs.d in lEl.risl 5],ts1d for iinld
']siFrlarion. Th€ Fot in pui6.d lhash PGEX puifieiion llit 3!rc posiivc mlt
uins Dti€ST slibodi* bul ml wilh 0E oli-HBs enbodi6 n dv h. due b
ohbsed s?ecincfty oi lhc nuianl, .nd lh. nuLnt .sp.d ffon innutilv i6 t norel
nur,nt whi.h ! not v&ciE indu.n blbnt ht nay be nubLd un&t eredirc
CHAPTER 3
LITERATURE STJR!,IY
Tk r.rn viral rr.D.rfti., ir !*rvld fd i.fdli@ of ln€ lis bv @ of d.
dktuct h.D.nn, vis6 LA.B). In 196l,5dRhitrC for pol}roDhlc ,.m pbc@
Blunh€rs disted ! pEviously unbdh porcit ir rb! blood nob b Aftti.rib
.bqisim. H. tcm.d l}i! prot iI Aut lie (Au) lDrigd (Blurbelg er al 1965) lt
b.cm. apDMl tlDt this mti8.! wd Gl.t d to tt!. B [email protected] In 1970' De. fomd
lh. vitGlit prnicles in th. @ of lyF B h.4.titb i @lcd t dda (De dzr,
l97O). Thc& D.rtcl6 {@ ooruid€Ed lo b. hepditi! B viru. Othct n*El.tod
h.qtitis viE w@ diso$Ed lar.r, bu lh. h.4.titi. B vitu EiriDd 't!
luG.
Aq inporto d€tlopn@t in h.Fctitit B Es@h wls lna dieoverv tEt
h@d HBv viru is 6. plolotyF fd . Gnnv of vilws, Gf6.d to 6
Hepodtu*ide.'f6.e notr'.1@ly EbLd 6 HEV brc b4! rdnd in *sdchoc*r
(S'Jlmw €t or, r9?3) Dd gond t$imb (M{id .t al, rgao' I.$nn et ar'
l9r, T6tll.r,t, l990. Th.* viB bt rtq, ?096 !.q@ b.Dolo8/ bsBv
6ut @ lot kno{n to i.Iool hu!@ or ollH Fimt si it contul HBv ir infetioue
for dl. gt.t .F' B@@ of siniLiti6 in DNA s€qu.M sd s@m o4&i?,tctr\
lh. viN.s inf*lins msn ls e eslp.d h the gru Oul,gdd@tld M@
di6t! ry rhLd vire,, sitb onwhnl tiDil! lemdc olganizrion but almo6t m
s.quqe lFmto8y, e foud i! dutt.d sd (M.d ,, zr', 1980' sFasel a
dl, rgss). The !E goupcd h nr. A6!t litetadvti@ Dlak h.Ftilis B vios
(DSBV) h.t bcn u$d pd@ril, s . aod.l sy3llo b clwict'rt' ho*
hcp!&'!vi@s Gplic.L, Elhq ln6n s . dilee Dodcl. riE chMic DHBV
inGolion na3 g...6lly b..D tludi€d aft€r in orc t&lni$ion l. ovo tr'M]s'M,
*it! .xposE to DHBV IFn tlE b.SiditS of mtt di. detlopndl tid inf*rio
oflbe d.wlopins liwt rulno et ot, 19et, .tpeDtlv pH.nrs ih' d'vdopmDt ofm
.fi*liv. i@d sPoN io iln Ed h.ptiocvtF 0lrhd et al l98?) woodthutt
nepsliti! viro (wltv) inc.liont h!@ b*n studi.d i cnelv 6 r nodcl fd thdio
liE di&& &it 6c .tt .ddr li€ c'Ig Bolb \r4lv Dd DHBV l|.vc ba u!'d io
ddelop stivir.l 4.nts $<t lhdpiB Th. HBv lrhsg@ic ndre hls al& been
intodu.cd ld 6n FltF6., r sll s fd cl|tDraizin8lhE setPilililv of HBv'
rct jst to alPha inLrfmtr but rlm 10 vtrious oihd ot&lincs produed dDting e
i'W. €iF.s ro s inf..rion,
seloglol Mrrd !d llrdPr.r.do!
Krowledsc of tne vint 8pllfu hs allo{od u* b fomutale @@l
slogic.l nur&6 of HBv in&ctid Tl, i dpt&lid of HBv mlogi*l DrtG
is desnbcd iD Ttbl. I si@ b.low:
;;-cr4.dd"u-t .ed-"r-
T.ma.*.o!!
ffir &!.oib^!b-
hidror d r6*ry I'on br.drorf|i||o.ofdd4obl.ollxl.
tn clinicd D@ric. ch.nic gBV do6 dv b. ditid'd inb rtu 6 v
idcrlifiable sotosi@l typ.s: ds. who aE Dosiliv. for gE AS (wild lr"e) ed tboe
*ho e HB.Agmg.tirc &dpolitiw fd.trli-gBe- Cl..t@ of n p.dris B. Drigd
(ltBoAs) indic.cr ElMitior to . *.te of low-lcv.l vinl t?licarion lhal ir
.aonDonicd by hiehdidl mierid of lis diee ltd poldgpd 'Wirl (cba
1993),
An nptd al${tne !l ohMio id@tioc h!rc b@n onlv liDi&d !uc@s
Atphr inLrf.6! dEipy ic e6@lirc i! ildelins vi$ .liFidIim i' r $bst of EBv
c!ri6, eQ*ially tnoe eith th. Dod 4li€ di&e, bll is irc,f4riv. it ll@ ths
50% or .did (H@nsgl. d a1., 1997). T1E cfi*dw$ of @iddon in
F.t@ridg blood-hom l:misid frcn u infoLd Dolnq !o hd wbon b lnour
9oplo (sl!v6s d, at, 1985), h Asi., pqiulll ttasi$ion nt b hbh ond 30-40e4
tuiln nodi.B e ffi6 .td 5()e,4 of dtt ceid nothqs @ lieEv inletiN
IIARC nonosdphl. In Asia wncF pdimLl r.lmis6ion p€domiml€s. wdn'lim
cfic-y i! otny ?e, s mDFtld io >85% in od.r alw of dE wld IiARC
Monosnph s9, 19941, Th. etrs0y oiv!4in.5 in pEvdting p'riElll inloction m
b. inpbrc<t by acrirc-PGsiw imui?,rion reliEdk i. rh' tdDitLE{im or
b.prlitis B immos)obuln aldg wi6 HBV vecin .l bi.$ (St'vtu €t al 1985'
Th. Clobal pEv.l.M of ChMic HBv inl€lion Ei6 widclv (6grE 12)
iiom nish .rdcnicny (>8% ..g, in afic! Asi4 $d tllc wdtem Plcinc) io
inr.mediaic (2-Tl. c.g,, in euth.n rtd crd€s EuFp.) dd low (4% 's in
w.stcn Eusp., Norlh Arqict tnd Ausr..1ir) IrE plldooiat out t of
hn6missid vtrt .ccordins to tho cnddioily oi lhc HBv infeolio!' In @$ of liish
e lenicit, p€rin.i.l tr.@iei@ is tne tui! 6uL
oI lov .DiLoicig, s!,16i&t ums bigFi.r
hl.lIof9|
of lemisioD, *h.re4 ir .Ea
.dulr! i. F€dmiirlr {SaElE?
T.bldu: Stuctnl Ditr d*et twdn E r.dEviNs
CSARIC'TERICrICS OF Tf,E SEPATTNS B VTRUS
^" v|nr 3rru.rur., A!dS.q' od CeloD. Orarlll ttor
Te fo6r of vi6l pdti.t6 6 h. f;ud in tbd @n of D6!.ols iDlcLd
with tlc h@Iitir B vis. IIE infcd@ h.!.litis E vtui6 (D!e pddcL) is 0 42'6
pdrrjcr. th.t cdsists of t lipolroLin dwlotc cMLining lh@ drcloF polt?eprit !
!d a Mtd4dd - foEred by ttE h@riti! @ dri8.', tlF FBv polv!@s - tld
th. HBV eqon . N@inf4lious 22-M sphcrial ard fil@alous subvinl pani.l.r
@ .l$ El€rd durilg i!f*ti@ ..n $.y dn!@hd dE i!t@rio$ Frticl6 io tu
Th. vint sd@e @Bis& of . cituld, pnisllv doubl+fidn€d DNA
nol6ub, appDritut ly 32oo bs pliE in ldsq dlt conllits fou oFn edns
fBm3 (ORD; dEy .tcodc ttE neldlp3r4 d, 6Elop., ud lne X $d poltftte
prcLiN,Tbe lucl.@prid olu hlE two in ph.Fpiae lut codoB tbr allov lhc
llsLtid of r*o cl@ry elrt d toryFpt'ldd- Tb. fiBr sn codd dtfn6 th.
p@E pob?.plid€, Il! fint 29 mim Did! tumd@ a r risdl p.?nd'e thd
turoiG dE tolyFPti& iDro lh. ddoplrsic ldiculM (ER) Ah'r t'r N'Emin'l
l€dd is Foorcd ty ! sigEl p.pidi!. in th. ER bd rlE oidoxv t'mi@ i!
tri!!.d to mow lhc lSinift-rin Ncld lo.dian@ ltd @Fid.ri6 dotuitrr
th. tminirS I?kDa ,olt!.ptnb is seoEtcd d n€pttilis B € 3 i8en (HB.&). Tl&
t oE FlFin its.li L not .eqritld fd vinl litn€d@ (ch€D ,r a, l9t)' h foo!
n.lurlly (ouning Nlrnt lEpltitis B virules hv. hen d'*tib'n lhd do rcl
sFrhdize HB.A8 ituc !o ! tft.* .toP ood@ (Er@tio ?, ar, l99o).
Tn Fod sun c.don h the nuol@46id ORF tbin6 rLt 183 mi6
fid! of tk h.lotids B vins dlis6 (HBcAs) (oD .r da, 1985) HBc& 'u
b'
da.oe<t Fnicks, vhich pukSc lb. vinl polvhcrf,e lDd pEsFD@ic RNA'
Nucl@Fid pdticl6 t v@ two diddl psth*.t5 i.lidt lhlt ccll, m of vhich
tdlpG lha viEl god. to tlF c.[ nul.us *hile lh. oiher rtDspotu it !o the E&
wh.F tlE n@lcalptid p.tlil* bild b vi.rl 6EloF pFLiB tDd mluE irio
conplote virions to bc &fticd frcn th. c.I. Apln non dis inportort rcl' in t'o
vinl li6 cda, HE A8 My dcn d indnd lrolinl tuNrid bv inhihitirE lt'
hiD$npbor of ln€ anrivid oltot(it. a intertcon in 6c inleoted c'll (Tn 'r 'r"
1933'
A&itioD,lv bo$ ptEoc Dd c@ poEil @ rl$ pEdomiet !4p15 olthc
T-o.ll tudi .d imuN rc5pos. (ctj$i ., 4r, (.dc) 1995) d di&uss'd hclow
Tt cmlopc OrJ 6uis ihFt i._phs$ !t lrcodos thd eFra rht PESI, pES2
dd s s.qetuca. The pFducr of6. s sd i! lte slldveloF prciein dEt is als
d.sicror€d rli. s.jor dveloF pbEin, de !o ils absd!re i! !'ron Th' PDdul of
rhc 6nbi'Ed pES2 dd S 8prc5 ir th. niddlc .Mlopo Drcr.in !d th. sqeDe oI
t[. Fqas l, pes2, snd S4rd. co<t . fd lb. Lre. dwlope Proi'in lxtik rt' sndl-
and DiddtcmElopc proLiN rE pFMl it aU vi6l md subvntl ovolop panicls'
dc r.rg.{nreloP pror.in is pEf.mrbtty pllml in ib. tubvjhl 6bM13 .td
viri6 (hs p.nicr6) (P!rs .t e1,,r936,P18 a al l994,bL et al. r9a1 ' Ebr.
A'l HBsfu rmt @nhh 6. cmo! 'd utiStric &ittuiM! pt6 'itlEr
rhc 'd" or 't" d.t roiNt &d eitha dE ", or "/ d.icmi@! s tid four vinl
subtyT.!, nm.ly, EBV .dw, .t{, adr tDd .rr !E de6n'd Tn€ first EFn or
rd;bility itr HBv cda fi@ (L Bdvid l97l) sho d.e!b.d t*o sunllv
oxclusi& 6ubir?. &remimq d ind y Th* E5id. in lhe 6urlrce prol€in iogctrid
with IE Eain disenjc dccmiMl. (Irtn. & Dl[st€4, 1969). Two adtit'on'l
Thc D.m t nicL it Im.d by bdding ilto the 16.! of dt ddopleoic
Fticnlm. lt is $c, adtpdied to th. Colsj tppdlu6 tnd .tur elvcotvbtim of
dEloF Gidud, sci.d nd lh. @u h conir.d to ln. @jd ed Didlc
.iv.lop. Foheplides, q@s lary.{wloD. pob!€pti& ctuot b. srct'd (Chiui
& Fded@ 192).
DeEsultl.d oreas<tEdon oI lne ldsd!.lop€ poltFpddc in inf"lcd
h.pltocyr6 6lg d Munulalion of lo4. @s{drbL il'ffi in lh' ER tb'l
lsd !o s goD,rd-sla$ .tp@. of dF infoded @U!, @d.r tFn dquisirclv
sruiri{c lo dF oylo!.6i0 afat of itL.rmr $sm (Gi[6 d dt, 1992)' 'nd
tt rav
hish @Mtdiod My.v6nDl|y €r tld b ili. (chiei.r,?, 1937)
dccmituB w & r vd ddibcd by Bll@ti "t dl, (19?2). rn ! Itrs. .bE,
lddniodl3lbryp€ wE [email protected] by (Co@e-P6uty €r zr, 1933) Tbe ni*
subty?6 dc!6b.d w@ !)sl{tt4, atl' ldwa.dw4..drq' ftd.dq+. I ir
irDonot io ,ot 6!1 tnis cLsifiqti@ i.!o sbtyF. shicn b. b€. vid.lt G€n fa
etid.niolosio.l strdi.s, is b€sd on lntibody @osnitio. of on site of lh€ vi.al
ptDbir v/hiL the $brtF cLsiftlti@ it m[',.lt *lsit for tbn Pdt of lh.
env.lop€ prot in, n dcs rct excb& honolosie. or did.En@3 iD otlFr DtrB ofth.
T.bl. Ift Dict EldjmtiD belir@ the ni. eriglnjc $bVFd of HBv
ud Anino.oid id€ntii{ Lrs or A4) al t*o PoliliN 020 td 160) in lt S protein
Dv
Dlnng the l98O!, it h€ctn imsitSlv clod dDl dF sub$T' detcmrmE
.E speitu by @ sindc dino &id , or F.ilim 122 (d d v)Dd | 60 G or w) b 6'
S pbteia [email protected] Ot noto .! al, 1987b, c, Ashon_Rrictard@ MuEdv, 1939''
b, Nodq ,r zl., l99l). Subtr?. d.r.mimDn d lDd { hrv. a bEiN at boln p6irim'
h.E 6 d dsiniE at bor! posiion ildnd. sbq?e d.L@imts v di r (TadeID'
Addilional su!tt?. d.l!mi@t rcelivitiG i6v. b€m tuPp.d to tDim &id posdo4
127, I44, I45, 153, 159, 17? tld I?3 (obboto d al l989'Ndd.( ?r al r99&)'
ThG polynro oRF qrod6 th. l68psr fiBV tolr!.pti{h. Tle 3r2-eim
&id HBv polrme pmt irL *iich is ttsld€d by inla!,I dbosoml inili*i@ of
thc 3,trrb (m $RNA (KNooro.t dl.,lg$, h.c!6 et al., 1989)' @ lins
m tlleipr@, DNAi.!a& DNA polyi.re, .rd RNA$ H divilv.
Pdyhe@ m1 dly pbrs . ble it viEl @p.id.tion bd d$ conbiro both lhc
pri@r ud tne dzy4€ fo see E!ffiiptior R€plietim of HBv, wliot involws
tlE !!rEe tMiprion of ! RNA iftddli.tc' is sioilf L drt of t lDvi@s .d
is s@.|Plib!e to nuhtio4 drc dE €nzrms i.volr€d in HBv Eplic.lid do lot
p.!ss peaadi.g lbilitic! fd cdEctim ol.@E duins r.plic.tid (Metdi a uI.
1994). This ul@rl Epliotio 3!.t!5, hli l.d 10 lhe brPlhsn $!t IIBV ir
cvoludmnly Ellbd lo Et@id6c! in d! !€* dut bolh viruld mv hav€ a
co@@ dccslor (Mind e Ro6iM 1936. Molld 1988' Mtla 1988' MiUq l99D
M@8, cmi.t Eble squ.E .irilditid h4s*n ttE Fltffi p(t'is of bolt
vitls qni T*o 82- ad 94nirc rcid lcqu@s !1 the N lminu! or lh' HBV
pobdc@ proLh 3h@ 45 ud 4t6 bonololv io t!. ffi ftffiiptr* of
EFoviru..Snnihily, lh. C iemitu or lb. HEv Flr!.@ dilplrvs honoloaid to
lh. rib.nu.leE li dmin o, rhc Etovi@ polvi.@ Thc sinnditi" b'tw@
th* scq!.B @ sjgnifid'l rub lh.t dE Pobabilirt or! @iEi'tcrc is l4r lhtt
Io{(Toh.,al,1983).Mo lnreB{ngly, cvtobnc T @Is E@gt' 'pitot4
wilhin
lt.s biologicalty sieni6{t lrd hishv mstd N!5 inPty'ng that tlF CIa
cpilopa h.ve Doi bd cn ng.d fd oillioB of )€s.
lte Eo6t .oNdd tgrd of oll ttl!ffitliu H@!dEvid&' n ! 62-
nucl@tiL ddin of dE HBv cc a.G lt@, 25 b.s e contleLlv idenlic'l '!dsinilor lo rhe U5 egim of fie lTR of wdd rclroYitu$ Tnis FCid is biologiollv
rirrifmr for p.cr,giry of r.tDviM. Firally, the 8.nc oida il*tf sd tt
oxisLM. ol o ovdlappinr oRF sulg€dt . clos .volutionay relolioDsbip bcts@
HBV !d rhc R€loYidd&,
Tn.E e no M.codins E8im in $e gdome, e lh.r all esul.iory lisnrb
.E al$ pIt of pbt i. @odilg sqm6i HBv d q8od. ry!rcx. teld mpbbitr lh,r euld be c&eiod doD ib g@tu l.ngd (c@d & V|nus, r 937). h
Th. x oRF etuod.! , l54fiiDo 6cid pbi.in. TIE x pbrcin is fouDd in i[.
.yloplas of inf@t d @ll! .d nlEtim s ! E@ripli@l t64tiv.to. of odd
vi..I ul elull (e.g., ..'Ja FJu) [email protected] (Hl d al., 1990). ft .x.rts thi!
6licti@ ii D indi&i {ry by hndhg lnd aorivrting @lluhr tnscriptior leio'!,
ehicn noilifi6 rh.ir rhility lo bi.{t to regqlrbry .ld.r&. su. of dE celule 8.Giubs€q@ndy rrNcrivlr.d G HrA-DR (Hu d zI. r 990), MHc cLs l (zFu d ar.,
190), .Dd ICAMI (llu .r dl, 1992), Ii i! inir.lting dE1lh. qposion of theso
gd6 ni8llr sicdfiudy.nb@ rh. hosrl r@E iri@ olvinlly irfdt d ell. by T
IrrtrPt&yLs. wlil. tb. x pobin ds sr €E b h. Da€ty fd viEl r€pliqtid
in yito, it Dy contiibuto to h€palocsinog!rcsrs, !iDc. ismotuli%d c.n lir.6 lhr!
& c@feted wnn lh. x g@ c& h. Eurm.d Gtolw d ar., l99o). Sin dy,
hbn-nbh+v€l qpEsio of rb. X potei. in raaldio Ei* ill:€s tne inciddE
of [email protected]. (Koit .,al., l99r). I.thi.E Fct, ili!,orewonhydler
't ha tMlly bcD $M rblt rh. X poEin f@. coqLrG with rlF pt, rllMppesr plot in dd hlibiti i6 $qE e eeifE DNA bi.dilg c.peiry,
rdsclivrtion fDnctioa cnd, pdn+6, its indircd DNA E?!ir tunction in vitro (wdg
l.mr of HEV €brorioq lbb lad! io tr! o@Gitg i..d.*t!: th" E of €rce
r.sdiprrs wnn ili l@t of ptuf r.ditr8 tE& !o MiDiiin I €l.liv.lv tigh
nurltidrl 6rc, wh@. lh. dtw. cmp.ct6 of tll. ge|@c vill pfftn a rtr8.
{t gE of s*tic di.lility n@ @ui.g.
C.ntrp.a ELaort ..d ClrsEqrbr
Tn g.r.lio lEthsqity of dE HBV gcma hr! b€n .lllbliihed (C'trM 3l
al, 1993), !,i s€m !@rr!a (A to C) { bG cL$ifi.4 b4.d on @pes of
comprer. HBV s@@ Oordd .1 a1.,1994, Okturt et zl., 1988, Stuwt! €r ,t,
2000) vith diEdry ol>3% h.lwen rh sDlp{Ki&-Lju4era,,2r,2002)
In 1988, otlmro ar at, 0989 tun ogg€t d dat th. r.ditidl siblyF
cold h. mdelmEd d EpL..d by ! cbsiftii@ of diftMt r]Bv !r6i6 irb
gcrciic $b8rcup. Conp6rins lh. tul Ml@lid. 6.qua@ of 18 IIBV 3mis, rh.y
found rt te clut.ld inio ftu gMF. ftis d.gE of diverspe. h!5 3i@
b@n€ th. dcinilioD fd EBv sfrryp., Iho o@ltondm h.twd subgpee {dgrertT6 i! 3nom i! T.!le Iv.
T.bl.Iv: R.Ltio6hir bcr@r IBV smqp6 {d ob$T.3
U!i!g thc pdync@ gm irurad of rb. wlFL s.'rofu, dto.r,r, 0989)
olb falrd fou sp.Ei. subsFltr difcring slishdy fion thc grdF ddrib.d by
olDoto e, ar, (l988). CoEo.rioN of s s@ s€qu@ rcF &e by N6.ta a al,
(1992b). In .ddilim 10 q!li. rinil[ ro tlDe by Oklhoro '' 44, i1933), h{o d@
go!r,, E .rd F ffi deib.d. In r l,tEe .$dy, 6.y @palEd dF S g.e t qtrM
n@ 122 st .ir. and [email protected] {E .n!l.nE ol dE t*o Dew grorps (Nordd ?t zr.,
l9$b). A $oit Esion of the pI.St gd. wd sudi.d by Uy .r ol, (1992). ri'hd
so)dyiD3 ln pbLin squ.G ot 33 HBv rhina O3!a er ar, (1993) roud .
edoDTic paucn dut 4r.ed wilh th. Esl& ofokmoio ., al, (1938).
An .xiended 3idy, wh@ $c mplcr. nucL.lnk sequoD@s of swftl stliru
v@ ooE .D4 snorld lhlt lt ptlviouly &$!H 8rcup E fdnd in lvsr Anic.,
rlllolgh cloely d!r.! !o goup D, @titur.d a s.notFe or ii! M (Noftt . a ,1.
1994)- nEFlrb.foE,Inis y div.rsdt (l Jvo) rt in f&n BEil b.d ta Epo.l.d
(N.DM .r al, 1993). I. dpEs.d lnc .ds4 Dlcstyp. ard mdtucs gtut}Tc F.
I h.! oh.. b€n u$d a m our 8rouD in phyloE!rctic ltDdid ol HBV. as il i thc
bct dilEglnt humni.drd &tuit?e trpofied. R*rdX Sdta.r 4 aL, (20.0)
d.erib.d d [email protected] gdoty?c, C. scwnl ld,Citioral sludie suppo rhc iheory oa
qdbiErio hetw..r gdolyp.r dDt @i&drr. ir m 848Ephic.l Eaioo!,
such e g.not ?e Bi/C lwitobilg lnd s@tyt. Ar'D sit hilg (Boqd & SinL 2000,
Md@v ,, al, 2000, O,ir.dr .r ar, 20Ol). Tw 5tudi€5 mltsrg viet'@st!.id found eombielio bet*d s@tyFs C .!d A (ILmun er ,1, 2000b) ud
B .Dd c (Yure d ar, 2000), FQ@tiwly, nr@ is cyiderue ro suscFl rh.r s@otT.
C of HBV i! lb. prd@imt gcslype of eue !.p.titit B in Nag.&ri egi@ in
Japan. HBv.iD p€sblin lto eDD rd nore lhh @ y.d.n r mpl.ic clinisl
ud ffiIogiql l@vty t b euE li6l h.o.riik (Lin d zl, 2@l).
Mrt do! IUt Dd {olrlior D.ttm ol IIEV
An &cmtc tm €ne. of dF eund@ nte of dr nepaduvi@s @ld h. of
rsGtinc. i! .5rnn.dng wha nc difreEDr vi!1& dd dEn emtr?€s b@b€d otr
Iiom @h othq, nD firlr sfidy ltsnptins to 6lidt dE Nbtid hL of HBV 6E?otad by olMoto a ar., O98?r). By conp{ils ditraat gBv clo@! hoLr.d
flw dE 'dm of . dppos.ly @nlly i.lelod I y€a old aomn, tn€
..tid!d mbdd c v6 fourd io bG tcw@ t-4 nd 3-2xl0r dbstitutioE pa
s't p€r y{i Tbis nurrtih d. i ldsd tn$ fiat fd DNA yi@r dd nm limild
10 catcin RNA vi@!- wl.n utaing lh. Eic of .ub3lit]lioE in ftnhd d.pd,
Oriro e, dt, 0939) f@d th. nre of rynonynour (rilo9 substilutios to b.
lpgdio.t ly 5rloj F siic pd y€r, wnion ph@ the dbijon Eic ol HBV
betw*r dEs of DNA vilws dd tuvi@. ft is i.r.Etitrg ro DorG in tht cdl.n
lbrl S.y fourd a highq nE of 6rn6rDd thr! of @!{trmyDouc aubsttrliod in
cI fow ORF8, sisrirying. coBmift of rhc sBv soes by.iino &id chang6.
Conicdns eulr, w.E pq.n&d by ll.@ .r ar, (2000.), wlE mn-
rylorynous slb€titutioc coturitut d 88 % ofdr ch486 [email protected] in p.tidt! ql$,
pEsumbly, hdb.tn iiGcLd vcnic.lt.
Tli. 6te of sut€tuniw 1' }'w d.r.rtu on r lMbcr of &.16, wtic[ eboln s€pmte ed in!4de!.n<t nt of @t othd. Th$, dF oonflicd.g vi$ slr$.gi6
of o @np!d remn. .rd Epli{rion rtdgl Fw @iplioD conbiE win h..t
tadm, such rs iohure Esrow, and lho win th€ rist of mur.nioro siliD8 fbD
.nlivirEl tqhali Thc lb6r GtoE !!r.c to 6c e iDlorr.lt f4tu, s th@ is
.viddc. lh.t lhc nutidd an ovr n.ny d€lds is resligiblc in sil@t or @ult
HBv inlctioq wl*r ltr is ni.ioir no6r rEspds (BL.tb6s & I<jd(l-Ljulssa
2(XlOb)- b a ds.r of Aubliu HBvslr,ing sn@bhi|ltEn, €@ of wh@ t d
be.. tollored for 2
'r!s, no s€qw. wi.dotu vlE foEd ov.r tise (Mclft@n .r
dr, 198), shicn inplis ttltrhd. tuy er !.Du! ch.4c in balhv cei6 eilbr'
Prliftin:ry E lls non . fadily wh@ . diar aothd tsloitted dr itl4tion io
aI of nd fiv. childr.., lnoeld t r dcE ss m 3.qutu. vri.ti@ b.rth dF
HBv 5td! oflh.nothaald Ihoeoflt otil6r@, t"o ofvbm r@ id.oliod twitu
ig.n 8 yelE (urFublilhld d.la). TtE srudy of ttF *olution ry nL of i vi6l Cplbn
in !.ijatr wi6 [email protected] n ntl| i|||m E*olB @y Flrlt dinbt w of
dE conioudiDg fe1oE pEse!| whd alsmg mullnon ELs
The cvolurion ry 6les of 0ln6 n D.dovid!, n,fuly W1ry ed DIIBV,
h.w h€n studicd qrdincnblly (Gim5 & Milld 1939, A4lnlini e, .t, 1999, Pdt
€, zl. 2001.)- By @uirs t[. !uh6 of E qtr t oft ctrop.dlic DiIBV snai!
injcct d i o ducklingq . ddB.ti6l6od.l @ ulcd lo .slio.le lh. nunbd of
$h6dnltiqi, !o 6.r*en 0,8 ald 4,5 0! F.iE Fr Smdo (Pult,,,.i, 2(roIi),
which ogpoximtes d& nutotid nt for HBV sgg..0rd by otnds (Obhob a dt,
Th.r. h.v. b€n !.v.d eor .tEnpl! io adllu l[. doluti@ry history of
qBV, horh h i.m of$c cisin of EBV.!d lh. dn. toinl wla n divlr8ld filo
llro oirFr n p.dmvie. (Nord.r et oL, 1996, \Nffia et al,, 1999, tanrod a al.,
2000, Tdr!.!hi e, al, 20@) dd ds lion rh. Doinr of diwr@ bcr*&n HBv
s@t?cs (Mi"ot?$i 4 al., 1997, Bollyty & ltoln€!, 1999). MicDomld er ol,
(2000 nls $miud ln. atrdpts t'y s@l ruth6 to drt@im 0E hirrdi..l
ElaiioNhip of h|l,@ .rd n@+M HBV sFaia dd slg8eLd tblt ! nuch I!186
Mbd ol torh prnnaic rtliB od numn HBv stoiB, nom p@ilv inv.stig.lcd
.gr wirb tish .8!€nicity, ,€.d lo b. dly*d bcf@ dv 6m @lGic cd t
Hirll8htins 6c siglfic.e ofi[. daltppi.S OPJS fu the nutrtim Elc or
HBV, Mizot ni ,i al,, (1997) teposd tbe t m .,st.ked Nlutiu f6 tIft
mludor of HBv. Du. io li6 wi.hility of obditrtion nt6 obs@d in & i._d.!dt
pbylogo.lic .nrlr€is of ! h!$ nmhd of@Dple€ 8cM lrd S &@ !.q8rc.6,
ro Eli.blo dol*urd clek for dF ddclornat ol lhe HBv 3anm. cdld he
obr.i'Ed .d t!. dgin of HBv lmiG otauE (Bollyly & Holn6 1999)-
gBVV.r|ld. & M . r
HBV crddic !@ in Ft Eastco cdttbs haE rcdlc.d th.ir ca!@ bits
by nmr ol univ.el vaeination pbsnmt. How.v.r, lh€r is evid.t@ of 4urint
iDfdriG mons HEsls vMi!.r.d ildirid'Bl (On ., zr, 195). Tbe cliniol
dii.D€ of @h aut nts ceot b€ i8mF4 .sp€ci.lly siM $.s. suhnG
€pli6E *itn n@l .6cicey rn<t @ irf.d eoiu, Ir '!diN .gtid fd HEV
cddic 's io deicmirc lt DL ot na v@i!.lio &d th. .Fe.t of l@l
nutarB wnich tuy not he poiccted by [email protected] dlibodies. Policy detidB
in tbc tuh E8lrdinr vein ti6 .!d HE.Aa E iie sL!d io h@6t fi@ tut
TL Gl45R nut !t b rcll dogli6d aqldeidt It n i @nno.ly ber
lound mons viccim( Hfl d dl, 1988, Klnhis.s dr al, 1994, luji d ot.,r992,I 'qr.r!pL'* p.rim|l on HBk F.phyrdi{cm €r dl, 1996 GLdy er ar, 195)
chMjc HBv patidb wirh d wiihoul inmscwp|*im (call@ d al., 1996,
Modyer .l,r,199D md ,6tiots infeokd with h.p.titis B viros slo8jc.lly non-
EDrirc ro lHiLbL clfu@irl tir{cdn d ur,1997, Kidc-Lju4cr er z!,
1992, rEi6lcrAl,|is €t ,t, l99l). Ir lus 0&o b€a lorD4 io r l.sg d&nq in n.@l
[email protected]. TniB sub5titutio of glycin by qinire D$itio! 145 r.dr lo l.rd ro.
mul,nt with cmplcL ."tig6ic wnd@ Th. Fsitio i! *ithi. 0E @ llgl,t?
cftid €pit f€ ro ehi:[ veie.ildu@d enaliring stib.dy binds, Il b dro lhc
tarset for dete{tion by spdifrc a,'rit dy of nost cmrE ially availabl. HB&{8 hib.
Such fiadilgr G bo'dd !o $6ti6 rtr cffi@y oa @iDrio ptolr@! Dd rh.
sdiliviry of HBs& slmin8 lib rd bl6d .lomE, B€sid6 i6 Dlc ib esp€, th.
signidaf,t dclct or lh. cl4tR nuent for emd6 of inf.ctids vinm Md dF
diddrH st'ilit, of @nrt viriB ory libit glob.l r?!ad of dr tutilt Grlidie
Ot|lq nut.ra, sch s T4 l26N ud Kt4lE, b.v. ter iold€d in J.!o(Wltib ., al,r994) and Gubn K.rdDgm d 4t,1994) rs?rrivcly. Mooddd
lndbody binding rc6ts wilh tne RoFl IG hepdritis B sur&@ (FHB' pee! of
.ntb.di.r Bdlcd d@tqt i@mEeriviq in 6 rcEl wiets of IIB3AS (H@ er
aL,2001),
h Papu Nw GuiR doft,6 @y 6 liE wimE *w lound .!Ms 13
PcR-posilirc sntl6 tLrt g!rc disrdat &sult wirh aotulout ed polyclml
HBsAs EUSA kit6 (C.ftm ,, al, ,997). In South Aiii.., subrlTc drry wd no&
diFcult io d.rcd wirh &iihble @irl hE, s @mpdlcd io ebtyF aAr. Thd
D.lHl or @ciM&n h6lh ce wott6 io S.dinir verc sliu foud io b. .rti-HB.
pGiriw, d indidli@ of p@bL inf@rid virl ou|'rls, L no.r of 6d o!.n
dil9orrio f.ilu6 uy @u d.F ils m 6e EUSA tit u!.d ( Cmin 199?),
IIE lslE h.E witr ir0li6iitu ir dr cli't..| .rd bLod tutu i6 *nilg,
(Hr\*hs.r dl,l996). A! tta inhuiation br.s .lft.1in s.udi Ahbiq ii will bo
.xFFn tn.t i.s.p. bunlrs' wi0 ce4q It b eir.l b d.6!. dd dllblisb llF
pEvrl.rc of och db s h ou s@ml poputitio. .!d .slrblii! rhe clini..l Dl.
d$y pLy iD our coMuniry.
EAII lAd.py
tdr{.m rba.ty eitb HBIg rDtb.di6 ory dcn i|MMloeicd prsE
@ th. HB viroq induins rh€ S sd !o n[br.. Thi! b @lly DoE ps.ldl uong
oinobprc IiK tu+lln (OLT) Frie6 wn D.od to.mbid iMuGutF*id*ili los:lab itm@lneEpy 6 i.de tlBv i.f6rio (C!@ .t dl., 1996,
Hiwtjns ., ,1, 1996). A 3t@ng c@Lri@hi9 t tw .mas6@ ol s.p.6uthts, highnF pE n/.ri., .dl ddri@ of 6.np, bs b4n t toi.d er.r2d-
kolle d dl, 1998, Tong et al, 1993). ln most oMd, tle €noigine dut nl! tend ro
t w i d@birdirg aF.i9 fo.ltBI& ladi.8 ro €$.p. of uttlianid.nd
b@tlhour! irGcli@. Th. ,$ri6lo 4 oI @dbituiid thdpy with utivirl
mmpo'D& io cd'@ hur.d6 ofth. s !p!o FoaiE 1o b..rtloEd.
CXllcrl Slgd0c.m. oIEBV M{trnlt
Thft ir eyiddc thd HBV nutlnb cm intlem. lol oily lhc co@ of
iltedo but .l- th clinidl miGnltioo of ln. di5e M6r of u lowlds. of
th! clinical .i8ni6@ of HBV muirnE is bed d paliall eith niatid in rhe ,6.
crc gda. By &r, rn€ nqr cM rurdiD i! rL. p€oE s.ne is. siqle br&
cbogc of C to .4 at nuLoti& 1396. Thjs !.inr nui.tior har pbvid.d tE st bilizing
efret '&.d.d
fo. rn @rg.@ of ! Ey 6!td (C.M d dr., 1996). TtF sin8le
be clEn8o d oodoD 28 i5 trow !o F.v.rt tlDlclptron or E DE{oE 8cnc,
@lrins in rh. fi E b.ncod.0E [email protected] @n io Fr.io HB.fu: Thq. Ftnnt'.
rh.{C L.kirs HBe&, *in ibN Fsirt Dl HBV-DNA p@itivity sd .clive vidl
Elli*tid (T1E Ircid.tr1 iMdg id b.nB md od@, 199?). R€rly, fou
HEsaA .eia ,rsoft re fou.d io trw tdsin d th. viru 1o llEir palidtt
dcaiE tb€ir {ri-EBe 3rr!E (c{pr!tr ,, al, 1995).
A wid. g.qFphicd vdndon sD t e@g. for th. p@r. NLnIg ,{6
higl s 25% of clmic gBv i.ierios i! p.ti@t iD D.fti, hdir. @ foud to tqu!.d by nubrb (cmd a al. l9l). sibilrnly, hiSh pr.vd@ Eee h.ve bcd
Epdld in HBv{rdmic @d ir rIE Mcditem.m b.siD (BM.no €, dl, 1989)
idcludirs lr.D ed Cre {z.&dr ,, al. 1988) ..d F& I€lbm 6uti.., $cb aChiri and Jipe (luji ., at, l992,tta)&n.t o1., l99l). In coDrr.st rhc nur.nt i!
l€ dmi@l in ma&nic @!5 sn c l!rcF.!d North An6d (lius 1991,
Om!l. er ar, l99l ). A higi Mntion of tulsjmr n pqtitb h.! be€n r.Dor@d wirh
th'3 surad i. Iapd (F|rtoyioi., ar, 1936). In s!n@I, p.ri.nr! inft.Ld witn lhis
4ulhr c nN lit ly io pbg@ ro cinn6is ud hcplritic ineffici@t @rp!!!d
10 thoe infci.d by llrc wild viEs {A$ki .r d, 19q). rn a stldy or chrenic hepniris
l i{rs i! Fns., z.sli d d, foE<1. !i9|i66r ie@ in cinnolii rl@g PI+
ooE Nlanl inf4iio6, Thi! q6 atEibuied io plti.nis hrvils edly onndnood
iDf..tioB (Ro@,r.t, r99l). Tn@ e Epork tbi 6* F.{oE mudll coutd
irdu.c Einae ro inEd.m o.r4.r etns et at,, t993) iftEe ircidac. of
fitolirg chobrn.i t t ririr in liE bftpLrt p6dc!6 (Rrifondo a al-, 1991\
inc@e ds.pribiury io h.Ftiti. D co-infanm 6o tr d or,l99l) Dd e!&
tulni@t b.p.ric frl,ft (MiDd d ,r, 1996)- D.qi& lh. hjs! Isv ..dcni€ity in
P*kl4 ad th. rFrr.d hid F.eld. of rh. F.'@ Dut nt in rh. M.!Lt@!@
Egidl' very litrlc is krM .bour ltBV pr4ore nnbnll iD this oountlt,
HBv rurfie 6ut rr3 hrrc be. i&dli6.d a@ @only ooD3 fiBsAg-
n.8ni* p.tiau d@ b di.Brc.ric tit f'ilu6, in wciMtd inf.n$ boo io HB!\g
DositiE noln6 du. ,o vlooiE 6ilmq d iD OLT Ehidts du. io 6iluE of
imo.prphylai' Tba. ir .ridee !o .hoe |h.r d. trat ofdebcrjd of HBsAg
(@cult idec$n) ii the pFsce of low vinl l*ls of Fplirqnd oay be on .d by
ln. djli.G of viEI sm6 brnotua.Ll.rim iI tt prc-Slqrd th.I.fcct tlE
S plmod (C.bqi,o d ot, 2000). Th. .nrt@ of p8tidls widr o@h I.tsV
inlerion 6u.d by HBsAg vddt4 ln.Ef@, t ! iaplh.li@ fdr lh.n posiblc
lrMisi@ thblsh s{41 [email protected] ald ty blood tuturi@ (H@ et dt, 2001).
Urlikc th. pEsE Nbdr, vay liltL ie lmq rb@t rh. clinic.l
dgnifidc. of lhc HBv slrftsc nuritui cxc.pl rhat lho dil@ t&y c.@ sfus ro
&v.lop elicr, md tlE iDccrim i5 quiio otun .rrlptorntic lrd t s slrong
isistio wirh diE xBv Edicrrion. b 6ni! cls of ntulEht d
h.p.L..lluld c.rcinoba only HBV-DNA pcitivity is obd€4 HB.A8 6ai.ing
w8.tirc (AlFrLn Fa 1988). In . [email protected] 6trdy, PDr4tskD[. a .4., tond 449,4
of OLT p.ti.nts wilh mbnl infccti@ to h.vc a wso clini@l oulom., ompaEd io
23% of@dob (ToDs ?r.1., 1993).
CENOME RDPLICATION
Th. lif. clcL of lEFrlD.virus i! o|ldldizd by th. 3tdtb63 of r -3 t!
p.rlialy doublestanM, Eb.cd{iol.r DNA (EDNA) Cdon by rc@.
r.sonption of m RNA intqr.dn&, r!. Fcg.lM.- Tn Epln .id c-tct. of dE
h.ladtir B vios drr3 wnn dr rft \nlat of rne virio! b lt e! [email protected] ia
upr.t{. iDio tne cyl9lm of veio$ Ell ty?.!, bui minly lh. n@atocyte, Tloug! the
squiEscnls and whrniG of{ft ilird pl@ !E sl not cldly &6rcd, n n
t M th.t se6l r.ploE iftludil! lnc tuFdrs d.pld (FM a ,r.,
1992), lh. Flyrlbumin rld lpolipoFoiein H (M.!di ., al, 1994) on the h.pqtoorlr
ndbtu.nd dF pEsqot i6 @ th6 si& of d. viru n.y phy. olc (N@th.,
aL, 1986, Ponti.o .r ul, 1939)- I|ui& 6. el, gAV pl6 turd DNA s)arb4is is
oonpl.Ld *ilhir the nu.l@rpdd partioL, vhich t'nQon6 rbo vnal 8.tune
tovltd! lh. @I nuol*, Si!€ Nl.qFid Fnicld clml .nEr lhssn 0E ;dlln@ld por6, it h$ lE@!y bccn ssse,r.d rld 0F op.! ciEuld vidl senoe ir
Gleds.d by nucl.mllsid db$$hbt d dr ctrod.mic fac. of rhc rucld
nmb@, vh.E,pon il ah dF nrcld doD (Guidotri ., al, 199q. tsid. dE
Ducleu5 DNA t!D!n dztm6 6rrr thc oF!.i!.uLr vn l DNA into coslddy
clos.d cioukr DNA (€CDNA) aoms, vhich s.ft a rh. r.opral. for ltnrhcsie oI
lEstuic rldr@sn RN^! (Smd,,ar, 1982) fqlw@,.nElq.,.d
lol)rbce pdliB. Th. RNA ghnc is lnq ninsponcd ifto n.w nuclc@qFid
Darticl*, Inrft!. thc pdtiold,lh. DEg€lMic RNA i! l€Weinteft.djaE eprcbts
! miq!. Dohmin dudnA rh. eplic.rim of rte HEv vd3 (5u@6 a ,4, 1932)-
As . lst $ep, pluFsbld DNA i! pr.dlcd, s dat dE partially doubl.{bndc<t vinl
DNA e!.M. is corplci?d.g.in, Nlcldlp6ids @6iDi4 virn lgb|G q do
b. Etupott€d inio th. Dulea 1o dpliry ln€ HAV copy nmbd i. ll'. ell
(Tultlm ct dl , 1986) d, .fta &quirirg dE oura cNelop. 5h.i, larc dE ccll d
TIE nechlnim of RNAdieEd DNA srlthdi. h4 h.a rcll clEtzcLtiz.d
olnush rcr€{ic s ftn ! bio.h.oicrl $di.., *nich bE b*n ddsib€d in crcbl
Fview6 (Gll@ dr da, 1994, Seeg.r & M.6 1996). In conrnsr, .mly .v6r! of rlE
vi6l Ii& cycle, ifthding dty, N@li!€" .d &liv.ry of 0E vinl gEmc irt tb.
el nuLlr, G mr ml uddltood Tlln ix in p.4 d& !o d. abs.e of @ll liG
0'it e d@eptibl. to HepldEviru! i!fe.ti@,
Chm c EDV tdctro! .!d vlr.l Fsrrt ne'nE Liv.r Di&a& in Haititi! B lrf6orim is Inmmlogically Medid.dl
ft. f.c1o6 rnd @n ri@ rh.t @s th. v.rtiry fme of chDri
infl,@tory liv.r di!.& ard Mno]ly i@dible liva injury mltns in
cirnFsir .Dd d i@!ed nsk of HCC h!rc b6 difiioult to *Ddy, siDce HBV hls a
liniEd t6t Bns. .rd ritu alrF s)4r@ io pbF$c n do ror qis!
rldevq, it is ol.d rhat th. viru it llis not diEcdy criop.niio (Chiei d
d, 1939) rnd rb.t lb. ettllir iM tnDoE is iMlvrd io rh. p.ltogmh of
dis.e, Irl€€d, inltalldlory &llulr inll!.tes Mk rhe 6sr podDlosial chss€s
in [E biop!i.! ofpatiat sidr b.p.rfti. a inf€tion- Duiag & e irf-lio'r, Mhnl
liller (NK) elk tute up. oF6ft of .ll liE-ir6lE{ins ell!' wh@5lt y dr@
b.low l0% in tlE ctronic 5$!o (Di@ e, "r,198?),
Thcs oell6 cmrtiru& o
inport arnisd.tuEF.ific &ur. d.fe@ nF.rqid (Mi@ & Wnil. 1984) thtr
euld omioblc lad lo ooeenn viEl dc.l!@ of iL oE e{d.
T|E mjodly of li6-iD6l[.liDs &U. in dE .!d clMic i.feriot, hoewr
ft T lymphocra4. cD&F6itiw T @ls d fo@<t in thc ryee of tiBtue in cl@
mr.cl wiln li6 @!ls (Dift! .r 4t, 198?), dd ln. mjqig of rh@ ens di9hy
&tirdi6 mtt n, qniL md CD4 lyrpheyb e fod withb rhc porid tlols
ind silusidr (Dic!6 .l 4l, 193?. Bggi4t ,r dl, 1982, Hulby dr ,r, I 982, Moiano .t
al,l983). B cclL !€.ls pEsrl in tne p.t6l t!&t .!td sinuids *h.E ltnlnoid
fonich! my fm !"d et ! FidEd lyDpnoid dte (Bllbxtin &, Dcs€t 1934).
Tl& d€wlopr!@r of m HBv-nusaic E@ 6od.l bl! n d. i pcliblc b
slin diEl imidt inio i.ltletdn p{dosd.dc ie$.ils. duin3 HBv irfeti@
rnjcctioD or HBv*paific C[ inio ta!8oic si@ dpE$ing gBsAs m
hcp.rcyrd daLd thrl iriti.rior or ljE di* in &ut i.p.tilir i. t s! @
Drigd-dpeiffo Eos.ilid by CD8-losiriv. lyntborl€s, qhich lri|ch to HBrAg-
pcirirc h.par.ct^6 &d l.isser rhd !o u&rso .popid€i5 (Ardo d oL, 1994).
Apodoric h.pdtocyb .!'!tu s Eidophjri: @ilD& bodi.6. Dlnng tb. f6r fow
houa.tu o@t of &ur. b,.prtiti!, CI.I- @roir dtisFD-mtupeiic irflmmbory
hosl €115, seh & rr.opt g6 rnd NK @lL, inro th. liH, @tin8 e etig6,
hd.F .Lnt &pli6@tion r-.<td fti. s{fud !r.p 66 nod of tthtiopdlholosical mf,ifcalltion of livd dis@ ud cd b€ pEvated by ncuE tizins
uttodid r. grlrm ilr.rfdtr (IFN 1) or bt tldiv.rid of @r!fhag6 MGI
comon!, nep.lilir i! lhir nod.l is trEidt &d mntauli it d@s not Eull i.
olIdic di!{e, pEM.bly ba.e lne.<lopN.ly lrssfred CII- hlvc a linit d
Iif. d!.n dd cml b. cllr€d
Mdir.id ol vi..l l6l*.d'Ihc mjd qqlioi l€mining io b. sw@d to md6t|.d tne paihoscesi!
of chmjc XBv iIt.lid L tow 6. vir. Fsid. a! l[. fe of tat d.ftnt
m.oheisd. In eoeEl iom, Dohfti$r of vnd DasisnM my involvo vihl dd
Hon ftcrors drt contr_b{t! to vid !.6isL@ n.y .l@ irclude .xn utid of
{E CTCL tblr eosDt dshly imunogaic HBv cpiiop.r duir8 lh. euc
infetion. T cell th.t ocouDr.r . ltron8 !.d lolgllnin8 intiSoic srimulu! ot .
Ar fs $ hon fotoB G .dd.4 rh. dli6t iroisl i @ fiom $.
clinic.l oheriri@ dlt ohrclic iDfcrid ii h@ n!q@. i. p.lials wilh iDpaiEd
T{.ll tumtion, i.e. P.rim|3 wirh IlIv idGcti@ d q:lignncy d |}orc d
imunNDpt*irc ltd cytoroxi. ln.E9y. Fudt rioE, ionuolosic.lly nmilp.tieDts witt olmnio HBv infdrid uMlly lhov a wk q undct cbbL p.riph@l
blood cTL EDoE to HBv, which didiirrbh.s dEn nom p.li6ti wirh auE
hoprtitt wno psdscc a 6ttdB CTL MDoe !o HBV and cld dE vio!.
N@ddar, trrisd-Feific CDE poritiva T c.lk en b. iloLr.d .rd *Drn d n D
thc livm (BaMbr et .t, 1989) of p6rt6tc wilh clmi. iDfelioa bur il .p!eN dEt
lh.y G p'!$t.r nuch bw ftqwiq l||.n ir t||. li!ft of p.li.trlr wi6 &ut!
h.pltilis. Ovchll, thc dl@ erivity rffi 10 @re|.l. wirh th. de3re oflhc Taell
supemtigd e ond dsgemd ro unde8o ipoPldis (w.bb e,,r, 1990) sin c in
@. iNb6 lbi pb.mM ble ale hai nFriod [email protected] b inf@tim witt
low dos.s ofvitu .nd ee.k .!*igpni. rtinli (Mmch er al.,1993), ldditionil fa.lor
sch s d. specifc viEl i$lrc rldtb.IIL b&l-eDuid ofd. holstould ils b.
oqsid.rcd, Al!4iircly, !mr6/ of HBv-lpoific CIa misnt te iaduc.d irrhe T
elc EEiw s@. blt mt all of tle sigd! .6de<l for lctiv.iid. Gffilly, two
sis!.b aE Eqlir.d ld corpLi. .clivltiotr of t T ell: d. fBr ft is otigd-
deD.nd.nt.nd elioiLd wnen lbo Tsn e.pro! onplex @sri4! . p.Plid. in tlE
MIIC bindins sNv. of e illig+?Ds,riig €[. TlE ae4 .!ti8d itd.Fldd
sigBl cd he povidcd in tne f@ of! *totjm d,g, ILI (C.rdar 3t 41, 199r, Vd
Kdi.n er zr, l99l) d ir rt ldn of c.ll*ll d|!ct This @U @h.1 n od.
b.N@, CD23, pr.mr m 90% of cBl- dd 500/6 of CD3-Fositiv€..ri:els, antt iE
urnl lis..4 tb. 87 ml@ul. @ s. oqg*preemilg @[ (ctul €r ar., 1986,
riNky et .t, 1990). In cotut6t lo sp€idiad !ofesi@|" adig+pEenry colk,
sct rs &driti. ccus in th. lyDD[ !odd, leprloctro do mt qp6s thc 87
nohcuL, tnousn dDy @ ds $L io F.@r viEl p.fli&s in rh. con|.n of MHC
cla$ I nolauld Ii ooy CTL @ogdz. HBv-i.leEd hepat@t'L! withoul lh.e
ostinuhrdysisdl3,dE eiShl t€@ 0ol@t to UBv bt 6. indEriu of lM&/.
Il i. .lso posibL d[1 HBv ojs||| di6 ill ll irvisiblo fd CIL by.lovo
Eguhlion or coe.pto6 (R@h! & vor B&lmd l9l), HLA .xp6ion, dd
expBior olvinl udgtr {Guido[i ?r,r, t 994, ciuo .r ar., 1992). ftis ilvinbilry
could Bdt in irclfrcicnt pdnils of cTL is wll a irclfici€nt cffetor rsction.
siM dowsd.nm of lb. apEsion ol vi6l !r!8pa ba .lso E*Ddy t€@ shoM
b b. .r effer oacTl. ln. i oncdm b.r*6 dnE ad c-rL (cuidotti ?t zr, 1994)
is a cdplq ,(@$ lhit d@ t[€ vi& A€tuD of HBv-EI.r.d liE di&s
hom to lbe oliniciD Tt Dolccal& b$i.6r CTLitduc€d dol@[email protected] ltBv
sere express@ @@ io be ! poltt!@ripto0l neebrnin lhal eelmLr th€
de8.(tirid of .ll viEl gft ptoduob, idcluditrg rtlic.tirc iDcrD€di.l4t. I1. .tret
isu o lrd fd v{i!!L.d Foldg.d riDd. tlowd, it ffis !o b. a dolble
cdsed s*oidi by slppr.sing tnl rcplidtior, it nay @ HBv-iniecd hepllo.ttt
ritb.ur hlling dFn .!d dEEby cdtribll. t vinl clc@- olr th. olnd h.D4
paii,l dorNguhtio! otvinl &d$D would @lribt . to viEl FrsisllM.
An 6f.@blc in!.dio bcr'd vitu dd ldl my d$ cult 6@ vi|w
inrlk.d Fletiv. ill4n6upp@id. Ia vnal 6titE6 e [email protected] on HLA
ml.c!16 of T .nd B lrnpn@ybr, rh* ells 6.odm nish b€drc b!8 i' qHBv{peift clotoric ells. Whjb n b @t poh dlt rL* .v.ob pby !dienifi61 olc duing HBV iifcctim, ln. nanMnns wn@by lhoy niSh @d
b!rcb€ncla.lydcscdt d.Fin!HBvsdrerrt clBUFcshsp.thMyofT
oelr vi. !h. hsfdrins Epror! whict !6dcF dc ells @.ptible io dBtructi@ by
HLA .le n-6lrict it CTI (!B@ er al., 1992, F6Ei .r al, t 992). Ssond, $tubL
HBsAg cd efficiatly ord tu cl,B I plE6irs D.ti*!y in . vei.q or eI rr!.s,
whi.h baonc rrger for ol6s l-rcsriciod CTL (S.himbet a zl., t994), Inftd.
HBs-qEihc B cels cD .l$ bte !p lolubl. HBIAB ru rh.ir sD.citr
imEo8lobulin t@prdi rld $blcq@ity bem ir4.r.d for {*6rncd@ by CII-
(Broabi sr ai., 1990). tf ihes renhiu! @cu in vivo, lhc werr HBv$pecifio T-
cen cstoDe iD lh. F iphdil bl6d of Fdarr with ctmni HBv cd di L.<l
Eltcol . Edrclio ir tlE nunb€r oIFBV-61ci6. T @U'
Tho vins night de repliqc ir inmunologjcilly prililesFd tisse, lhat aE
inGibl. io rnc CIL. Tis6 swh s rh. lwl t]bubr epilh.liu !E [email protected]
by a c@Iinuos @dorhclid e[ rlFr bd . lhjct b.a6t 'ffibdc, Tnen tumrid
6 rnal B.Fon nu boo denonlband in r$gpnio mic. (Ando,r 4l, Igq)
inirvsoulirly injeLd HBv s!.ci6c CT! w. oDly !bl. ro llz. HBV irf€&d
b.p.brr6 bur @r ri$c litc bdnl ddr.y, el'. rhr lrs.rqEss.d HBv mrjg@,
whrn tbc CTL wft inj@ld diE-dy inb rh.& ti$uo i.e, boroth r.hc h n y €psule
oi inta@6Flly, hoM, th.y *.rc rLo.blr 10.r.rt dEn cytoplthic n@do. In
huM, n b ds ldDu di.r qBV Epliocs i! qdrncFdc tis6. R.tliqlivc
foms or lhc viru hov. b..r d.L.t d in bilc drcr .pitbeliun nd smooth nurle cell!
{BIM d al, 1983), in prnc6., kidrey, and slin (Dcjcd er al, 1984), b6in,
.tdarirc lisld,.nd trth Eod6 (Y.fic d al., 1990, a*|cy., zr, 1993) add
Dnr.pli@live foEs h!v. b.d foud in @lls of rhe imu. ryrlen th.@lve!.
Mitoseoic srisulation ofdE* eth indMd viml Eplioatior (Korba er ar, t989). lt i6
u@roE pBiblc lhat duing er hl*tio4 HBv oisnt @n qtl.!.p.ric
Eemi4, wheE il@t hc €nrli{Ed by sp6i6c CI!, Ftm t[.E, il biSnr e.d
tho p.riph.ry virh inpuniry, occadiomly rcl.ain8 i ldgc qu$tity of vinl p.iticlas
rhlr lwh rh. liv6.nd infd teFiocy6, whih e rts MogDiz.d by CTL. Thi,
Und of ploB nishl a@ rlE rclt-boq n@ duing chre o h.t.liri4 yhich,
how.vd, Eely le.ds !o vihl clffime
a! sboq ror olna viM (K@p 1994) HBv nishr de .li.r $e pDdudid
and €trel of cylokires, lh. n6t inrorrnl $.duhbor oi u! nmrc Esp@E,
Iorcrfercr peducrion hs b.a reponed io br dcf*rir in . p€Edtlg€ of parimls
*ilh cbMic b€p.titis (Chog & Titrs 1939).d $i! ba lcd to dE irn idc. ot
tsting clmlic HBv illdrioD *i$ rr.tnirc niri dtg or idrtd. In 6is
E e! it is inicEsti4 dEl tb. HBv @E !*i8eD d inhibit s@ roMripti@ ol
bcb i .rf.bn (c.nry er 4t, 1995) lnd rh. Lrn'dl poeid of r5. HBv poirm.lE,
n r4'p!!s @llubr '!*oMro irEGlw.lpb.d s'lrd.
FiDll, th. pGsibiliv ih.t vial nut d6 m cliaiDtc Tall cpir)p6 dmL Tall @ph-.rn8@in @ibD6 rld riG pobnliiUy au* vinl FEi.r.M
h6 bccr di.ndvely disused norr lw.dy. The hcporid. B vitu6 i! chrn tdi4d
by o nuldim EL of La 10 32 x lo_r @lcotid. NL.thnioD F y.tr (O*uoro d
da, !987). vniL it is cld lnst mut rio6 m {w in HEV dEt rbesale .pil@a
l@srilion by CTL (BlnoUfti er ,t, l9q), rhis .pp.G to bc a FE *nt I'dc.d, rc
.9iioreir&tisrirs nulldm '@ fo@d i! os rw@g ldditidd HIA-A2 poriri*
pltiall wir! chenic 6cp.tiris witli! SLA-A2 E6ti.tcd epitop.! in thc n&le@psi4
awlop., and polynete pbteia, This coftsFDd6 witn tu failN of th.s t tiors
ro munt ! yigM C IL !E?o!e to .b6. .pii.'p6. b S.mI, rr@g CTI *letidpEssE nd cn$ to sl-1 fdr viEl nutrtids, ud dc vi6l nutitiod 6urt b€
cmpatiblc *i{r thc viability of dE viru, Botn oondirids s.d mt to b. filq@{y
frrlfilled duiiig HBV ioJ4ricd. Ar ddrib.d adid, tuy of ll|. tM CIL
.piioF6 e l@!€d in A@ vilh o.itical biologic.l tuncrio$, lnd somc ofrh.h ft@Ercd st o y 6.t*a Hcpld.virid.. but .l$ h.twd rh.ir pcsun d
.wlDliordy ucel.G TIE Cn- FlpoM ir polFlord.d oi i+cift i! Drdab
w'lh eule hep|titis md b wa\ sdeleobbl., or @ly olisorp.cinc in pari.nk with
clmdc [email protected], Fd el*tio! pEM !o @r, rh. CTL Gspon* husr b. tug! l 'rryly f@ued. Th! .pi(rpc in whicl r!. nui.rid @6 snodd als 6. ithost imnDmdohi@l epiroF within tne whol. viru!, so d io lllow for rID *Lctiv.
dtgofi! of lh. Ntotd vitu. Fd dl thcs l€!l@, C.lr .e.F nutltts niShl bc
sondlry evcn$ in . cnmio itrfootioD, hn n is vory unliL.lv thit $c, .E th.
pdn&y @e of viEl FEirtcM.
Sinildy, s HBV mllm win 6 pcc stog coddhs on n hdn cLib.rt !o
ElGc1 [email protected]., bcro* lh. nuitlt vitu &o Dor prsde lh. HB. mti8.D-
cliniclll, HB.Asi.f4i6t HBv nnllnl! hsve b*n foutd in dti-HBeposilirc
p.li.d win hisD sndb of Nsoloer..l k iN .!d in Fti.nrs with tutni[ft
netaitiE (xegrwa er al, l99l). on tlE otlrq hnd, it he al.o t€n shoM (Boim
er,l, l98o drr r[. d4r.wth of fiB.-Dg.tiw olll. t'@oFGglo(x,lfu
10 Mti-HB., iftsr@rirc of tbe rrDptod or $8!s of livq iljury rnd iftsPdiYe of
tu @ ofviEl DNA.
HoM, n na bd d@cr!.ad $ tne sut dd n.y oonfd . epliqdw
.dvorc. io rh. yiru. Speif6lt plw outa[@ o@ in w.I-d€fi!.d sicr .rd
i.&.d, iD rddins priE on qon 5i. of th. $emnoop slruct!& of lll. DFgdohio
viEl RNA (O ., al. 1993, tlt.,,i., lt94) $ q io ioDbrc rt 6.& Firitr8..d
lhdby slrbili4 dE 61cs loop gtrctE. Tti6 bichr @Dfq . Eplicrlion advdla$ to
th. viru, sirc. this Esid dni,r irs di8in of E?liad6 (W!nE .t al., t9t) a\d
tb. p.cksng 3isn l (ruDtd-Ni.pDm d,l, l99O),
I@lrdoo, ol EDV Veirrtto!
L 1982, wci@tior lgriDst HBV fs-t be!fre avdl.ble (Smues a 4t,
1980)- E rly @iE l@ &ntql fili dF plns. orchonic ItBv mia ed
wd $hE€qucndy EpLcd by [email protected] EonbiMl v@imr fDn '@r
6
Chin* n.m!k oEry elk thrt conr.i6 rhc m.jd fl.loF por.in .I@
lVt].@d[ d al-, 1982) d rt nnldc ud mjo d'€loF Foteir! cdbi!.d
(Michel d ,1, 1984). Po!..tio! lgiiDn ltBV bfdtio4 a n.aEd by HB! mh_body
Lcls of tte thu loolu/nr. cd t idD.d h i@ rh.! 95% of
inshocmPcrot aduts (Smq.6 ?r ol, i980, Didlttg d al, 1934). In 80% or rho
v@irE ! protccrio h51s fq @ 6d 5 y.s olrdla ?,.t, 1986, Gibd d,r.,
1988) G€vioqd in Rct K.lkov & Dr@ilg l99l). h lhw wilt HBs dtibody liL$
of 163 rh,! l0ru6n|. iMlosic.r :llfury !t IE Bcll L*l *bs !o hc st ns
ctoud to. ! quiol sli-HBs $Fre b bo6t r v@iDlim (Krsf,m A DNid$n ).
Il h.r €@ b€d Epdtod dat .xForE to HBV tulE h rh. @ !i$ of dE
.ntib.dy tiLr 3!frci.nl to pehr otMb inf€cti@, At lL. T4[ lcv.l, r sE68
dwloF-lpeific T-ccll F pon& lEs bd d.c.Ed in the v.cciH (C.lir d.l,
HM6, !p to 5% of in1ll::|M.rD.E t.dulE, a0% otp.li@E virt oh$di.
En l rrilur., .od ?0-80% ofpltidll wifi olge tlnelell do nor nouDt an adcqulc
nlfud iEe E !o.6e ,o 6c.lm r*i6 (Sanng r, ,1. 1980, D@5ns
4t 41,, l9u, Ktr]lof .t nl.. r99r, Gob er al,, 193!). Th. imnunolosic.l b6!i. fd
non'!s?o!$ hu ben abircly sn!di.d, In oth@i& isolrocorla.nt !drt!,
noDEsFD. !o HBgAS oly b. lintcd b 6nin MHC hiploqps, esp€ci.lly to Hi-A-
88, SCOI, DR3, od B|4, DR7, FCll (P.@ e, dl., l99l). A .imih seiltion of.
mm+6@ *itt @rllin Hl-A rll.lc! ha b*n €port ! in nie (Alb.rti.r dr., l9e8),
In lnir mimal rDrLl, i Eaw 6uld b. i.drc€d 6y irchdirg pE-s pmr.im in rlE
reift; in chjn43ll24, imuD. pbE.i@ m prsl.nd inrru,ztior wnn !prc.S polrT.plidc alorc. Th. cmbiMtion of S ard pE,S mtigtu nishl h. clos !o
ola Mn|nl infdtiot! s'e pes2 .rtiboli* & sstaE of viol clesM ed atent
in patiqrs with clirenic hcDitirt (Albcrti dr dt, I933).
A @Dbiutior of ltB$g *iln odE virar podE h!! ben disusd G welt.
HB&{g b of particuLr irGEst b.c.ue of n oipeity io indue a stue .r.6s tr
Esiicled h.lp€r Tqll efi6r 6{ cold @hr@ 4li-tiBs proddid by 6. PIl)@
of i enolauld h.lp <ti&wed .horc. I!i! is a putiodlrly atF*1i!. i&. b..@
,onespordd lo culed HB!A! va@inc! F8lldly nount ! pooactir .nti-HBs
imtu Es?d* duing utlEl inIdio., wi@ rll of rh. vird ed8pns, ielrding
HBCA& a erlr6&d 3d [email protected] ro dF im@ B)rieb.
A v@irc corf,iling @ vir.l prlEi6 Disht d$ pEwn b@t-d@!gh"
infcri@, *nioh h!v. bcn des!.d ir wiFr.d ciildtd (c.mn d dl., 1995,
W.iors e, ar, 1992), nnen ft. HBv seDdo w6 loquenood in fEe childrcn, vno
iDilidry d.}elop.d poiecdrc gBr dtib.dy tiEE upor veirijon, I si4L
lud@tidc cb.!a. E$ltirs ir ! srbsrit]tioD of of dsiniE for glycim .t pGition 145
of lhc !.ti8e.ic i'i'i delomimr w.s laund. Th.,.r'i d€himnr could 'o lons.r b.
E@3nizd by muerlin.S HBs rnrrMi6. r.i.din8v rh€ sc 4irc &id
nbnitutio bor .160 be6 ddecr.d in a s{b&t ofplri68 rqtat wi.h . htgh dG of
HBr anlibodies 10 Fevdi Einfetion of ihc e!.,i ator livd Fanstl.ntArion, It ir
.ltost oqnin lh.r viBl esF edd bc nirimizd if lno leift indu.d ihnwE$o@ or thc rldbdic rDribody pEDdtior'' r.retiv.l, targc|d n@ B+lt
.pnopes in difi@nt niBl DblciB (MisLovd.y a ar, l99l).
A rcw atrd v.ry .ffrcidt vkim dt ie6, &wlopod ncl lendy b DNA-
Eedtucd iduniari@, lntlEuauLr rdninjfiarid of DNA cxpBion wqt@
dcoding sqlcfu 6f dE d.€irld imbgoi Follir ud.r the @Dtol of D
appopiiaE pbroter hft ta shoM b indue ! snons inmuN Es?o& !t tlF T-
and Bsll ldEl in ni6 (Dlvir .r dr. 1991) .!d nG tar dbu io ilnE m.mi
H& Espctrsc in dhwi* rob@t HB&Ag trtue@ic Die (Mmini d dr, r9q), a
nod.l ro. rh. chrdic HBV uid rbic. lnpotuDdn in drs ni@ tu liE i4ury
wa Fi38€Ed ddoitc lh. bsting o{ roLn&.. r thi reiEdo dn !.s/ @uld
.wDtualty b. lppli.d io h!tl@, it would hlyc s@l dtuLg.!: nftq DNA vet6
ft cb.tpq .dl *iq io ptud@ lh& di8d,c lbEiE ud ir tI. cuft i@icdd soDd, DNA imnutidon in<tuoe. . Btong CTL EQonlo a well $ r T-hcl!.r
md utibody eao4 !o llle dpr*.d poi.in (Dlvis ?t 4r, 1993), hd€4 i!di@I., DNA.n diorod imuiztid hc b.!o stoq b idkc a CTL ed oribody
Espone agtil.l ! bE.d vdiety of paltoslr6, sun I irfllEu (,lnq .t al., r99t),
HIv-l (w!qaar, 1913, \valg.r aa, t993), nbi.. vi@ (Xius.r at, l99t)_ rtD.
will r.ll whelh.r thb pmirins.ow i@ui2ati@ sttsr.s will prove lo b. elf.criw
cmdrrolEledrosppleadr|h.cm.lHBvnein .
CHAPTER 4
MAIIRIAI.S ND ME'TqODS
Drl.rlrl rtnh| .!d Dt mld.
Eschqichn col (t c,rt s[!i! DtlS llpha, tMl09, JMl2, wm obtoin d
n@ cnltoE oncdon ,r CEMB.E. @t dnir plsnid Eh DOEM-T *a n6PoDes4 ed erp!66ia !@ior pCEx-4Tl !.d E @/t shdtr BL-21 (DE3) pLFE
wdo non Nov.s.r. Blood @pLs of HBV p6iti@ prticnb w.E olLdcd ndsh.uL.l Kh.nm [email protected] TnEt {SIo':I), Sts}h z.y.d Ho6pilll(Szt, Cnitm
Hospihl, Iqn 6n!La PIM'S IsLD.bd ed F iebbdd lt6pibr.
R6dctim GnzyEe re obi.inod A.D N.q E 8t.nd Biotab!, Bcth6d.
Rcsfth t bo6iori6 G&r), Bo.bjnser Mroh?in ed CEMB .D4me pmductid
llboatory, Cclf int.lrinal oltrlift Phosphrtre, r.r8c ftdgncDls oa DNA polthdsc I
{Kaow) .nd T4 DNA tigr!. *@ obtai!.d a@ N@ E{sland Biotabn Tlq DNA
porrre@ vft olbi!.d non CEMB dzac Foductjon labodo.y. veloB
pBIE Son (CEMB sio.b), PCR 2.t0nvirbs6), pcEM-nlencsa), pCEX-4Tl
(Pbrmrcir) w usn. IUSA (.n4mo tiDtc.t im@$6€nt asy) iwitrbrc
@nl'thi.lly ws u!.d fd HB$g &Erio4 dni,HCV a,it HBeAg,
HBv DNA .xihction n rhod *s srrndldiz.d by usins difr@r ldtaiqu6,
Sort $.d!od $ler.d i! fottowine:
. T.ke 100!l of P.ncrt'r Uood pl8nq ldd l2!r of Pbi.iM$ K
{2ooglbl 0.! U/O, l2od of Foioire K diluri!8 bof.r 5x KDB (368/ of
dcioized utd in d cF.ndorf iicl@ftif@d fibc),
. PlieGt6O0d&ldC6iii ryviil|l56 c fd 2 hM.
. Now add ?ndolchlobrom (ll) inr. dis loln in equl volum€ od
in vigotuly, lb6 s?ir rr lat fd 5bi.ui...
. T@Er .qu@r pbe 10 n{n tub€ dd Ete.i th@l I chlmlom
. Now tlb OE qU4E pde !o a n tub. lid.&t VIOU volm. of
dltosetr (o.lognl), < 0-2M N.CI (fo! 6m.).d dd 2,5 rclu. of 100'4
chilled clb.nol. Mil il rd pld rh. trh6 |t - 20C orEighl d - 70C for 15-30
. Cdtinl8cd !4l< fd 30 DinEs.
. R€mv. sup.hrt r! Mh it eil! ?0% .lhaml, spin for 5 ni lc!.
Rtuc $p.rDralt rrd !n &y.
. ResGD.nd Fller in locr dcionied si.r.
OlA.ru.l{dd6 Colc.lf .do! Edo:do!
hrifi.d olisoNdcdi&s F&s€nd.d i! 20oei of d.riliz.4 d.iooi&d *!t r,
'Ir! ondbatjon of olisoNlodd. @ .lritu&d A€rophotoftctidlly by tatiry
O,D 260m, Pi@t6 (paoL.) of [email protected] Fr bioF lit.r w@ cdcutr&d
qing 6. foloviry fmul4
Pnol.tFl= (6@. h u/[t r lCP, n (330)
wbcE, ntu. of nwLotide in tbe dis@
PdyE.@ Ct.i! R.xctlo! (ICR)
Ioljm.n* chlin @tion {.re done by 0E optimiation uinA difrftrn s.r
of prim6. A @l volm. of 50d conniftd 25mM Mgor 50oM Kcl, ed lornM
TrilHO pH 8.0, IOOIM of @n &orynhe*l6id. uiph6?hlte (dNI"), 50
pel6 of @h fddd rnd p@ pri@. I Dil of Taq DNA polrr.@ od 5G
l00ng ol lenplal€ DNA. TIE Md@ nixtus w@ ovo aid witl 50ri of ligllt
nindl oil. Tl* nixrN. *@ &rplitu ir 35 oy.l6 by pbg:@ing lne PCR
drchirc (lu Rcwn) @ordinS io th! qFt d ldgln of lh. P€R F.de&, To
lDplit 1.2tb DNA fflgDdr toD l[. DNA or HBv cxt:ccd non p.ri..t . tuple,
lho PCR progran vas as follows;
94c 5 bidcs
94c 20 s.cond!
56 C 20 sonds
72C I nirulB
72C t0 nim&.
Onc. rh. PCR rs mhplet d. ir ws t.pr .l 4t n tunhd p@$jns.
T.bl.V MasldMn PEFrtion For pCR
Mrll.r ML Fo. rcR (5o Ro.don)
rMKCI
1i4l(Pr' 3 0) !!?sdlMMrcl,
fu!!L.'{roxl 5q/
J?l 69d (qFrrylo dd DN^ d rlq tr ro!4
P!.i[..tion oa DNA Frrg!6r fiu A!ro C.l
DNA tus@s EE irllEd fton lo* n.lliDe poid (LMP) rg3l@ s.ls
follo*ing ilc Eelhod def,l€d by Smbot er al, (1989) wilt sdd6l
modifiotids, Bri.lly, lp.cifc DNA dis€Btld with ippoFiaL E fticlion
dd\rnd@se N6 dr m loyo low Elnin8 r.np.EtuE (l-MP) d sead,iqE.s.rue
td. Th. 'quiEd
ilsnal rc cur ou oftt spl uldq Uv lie! .nd hmrcftd to oEppado.I t]b.. ft. g.l slie corrrilila DNA n:gnat {s nc[.n sl 65 C in ml€.
bad lor.pproxnmrcly 30 ninutes. olr bics libr RNA (6to.h: lng/nl) Nr! add.d
B @ftid DNA od s.aDl. Ms qxt!.td tbe limes wilh rn .qul voluha of
brfald p[aol rtat h.rl bd plem.n io 3ot. Thc sup€dud ws .djuc0cd bo.lMNlCl rnd €ntitugld !t 14 aD for tclsnituies. Cldsup.dlr *s nkd
in t$h rppardod tub. abd DNA pEipinted wirh 2,5 wlm.s of cold rbsorur.
.ihmol, Pell.l wa6 washcd hrioe wid) 80% icHold €thonol lnd ai.,rhicd t toe E-
$5pqdins in rul6e t4 w.ld. Altedliwty rh. DNA fi.bn Dt of inr.€sr w4
pEifiad nm LMP .gr@ s.l ui.g B,Asrc azyr. (Nq Enslard Biotibt fdlhe dig.slior of a$l@ e tbrcu,h prpA Ctu DNA puifiotid lit (Bio-&n)
acoordjnA b $pDid'6 i6Eucrior
Aiol])cr De.d@ whicb w.! adoDhd !o.xrlcr puE DNA nnSrenr qa! 6e
DNA .xt:cdd rit ofFmd|ls (in rpFndi\).
Lieiiion of PCR mplined thsnats (sith A{vqian$ !t j end, vs dore
in pGEM-T v*tor PCR 2.1 (with T{vqh.ns5 !t 3 ods) &cording ro insrucri@s
suppli.d vilh (he Lit (Plmcs! rd lDi[osen). To sub ctons rcR lioduct fim
PcR2.l v.cror 10 pGEX-4Tl dp4sion vcclor, pksmid Nas digest d with E aRI
eozyme and dephoehoryl.l€d using olf int *iol dlkrlin Phosph.ta* (CIP-NEB)
io pffir slfiiSdion of lnc rdrori insn DNA ea de dtFd.d {irh E@Rl
.nu}nc fiw PCR 2.1 cloc. Di8c.&d nl3,ndls cki..d up br ph.tuvcnltufom
.xhclion rnd lig.t d to d@hosphoryla|d rdld in . I i ools Etio ol @tor io
iNrr It. lisarion letim v6 *icd olr in . lorll lolMc of lopl .1 loc 14 C
T.bl.vl Lisalio. Usin! TAaloni.g v*t .
Llg|lio! U.hgTA-Oo !s VEror
Us thc fohuL b.Iw ro qsrioc thc mounr ofpcR prcducr rccd.d ro ticlr.
*im mg (20 f mot6) otrcR 2. I telorl(Y hr PcR Edldr rso. !cR'2. r \ gfut
sirbt oreFR9r lEe-rmUg.do! UNhE Etpdiotr veto.
Bcfore lig.lin, inErt ed vector w6 disesl.d wirh EcoRr Eslrictjon cnzyDc
ud thd th. veror E t aad with CI!.
T.bl.l'Ir Disdion of cxpBim varor fo. ligltio wi.h t oRl,
33!l 2!t!l!E! n(rox) Jd tulcE@tu 120U/d) tu
2tt ZFrtur rK@ir rrc rn ) hort
c.IP Trsh.!( Io dc+ldDhorybt ah. v*ror for lla.lto! rd.tlon)
coMtElion (ralpl) x lo!/n (M.W of he* in dolble sftnd)
l pelpl x r ot 5400 (650) = o3oPdoL
So lu/|rl of CIP c &idcd irto ttr @lion of 50,! io dibre n io 0.3pmL.
rcd it at 37 C fd @ nu.rxl dE scl dehphoBir ws Frlolmd $d
puific<t t[c b8n<l usitrg Bel dterid tit.
Trbl.Vm Ligaiion of Expellid vootor widi lDsrr for E4e$ion Studies,
TrulorDtlon oa E,coI
t.o[ seiDs.uch !s DH5.lpha ed BL2l w* us.d lor vrious pl!mi{,s
lnd lislr.d oiroE i@fo@rios. CdllErdt celk vw tEpr&d for hirt
effidmy t rEforDllio' by .l-troDorEri@ s d...nb.d in rhe E cotr elccrbpoElid
hadbook (BRL). A sinslc oolony filD li.ihy glwing ttctes qs incurrted iDlo
2nl of LB n.di@ Gpp€ndixl) md gbM vi|n vjgoDu drki.8 { 3? C omighr,
Fiw ninitcl of ovcmishr cdre w dilur.d in 5oonl of SOB Dcdi@ (E.rD."dix-3) in r 2-lit r n.* ald stM 16 2,1 houE with visoos slulins .r l7C
unlil OD s.r 0.3 .r 550m. Cetb *@ hdelled by oanjtusa on,nd pcll.ls weE
wahc.I hric. by E liledins in ieaold loelo n.rire 8tycmt. The c.ll pell€t w4
lla E€s!.nded iD tbt of loelo dyvot to td.g d. nMI en daEily lo 20O.2jO
o.D.sso uit3. c.lb w llqDlod in toopj @t@ ltd sL'€d .t-?OC.
COMPONENTS OTLIGATION REACIION CONCENTRATIONS
loPi ar4 i n r6 c fd r3 hoh4
Elero?oBrim rs &E uirg BicRrd [email protected]{mrid s}6td, Fq th.
lesfom{io of ligatd DNA, lh. li$tio mi\t'n *6 €.hdol p@ipilllod, w.sh.d
silh 70% .lhlnol, rLied ud E{u.p.d€d ir 5n0!l ofd.idiz€d @ter, It lf of th. F
sus?ndod ligitid nird@ M. D..d in clclroFElion of l00 or DH5! @ll. ntpul* fd cletopontio M Cim .t 2.20Kv sltlg!, 200 ot@ 6ise ud 25|!
c.tlcitrtu . Ana dctDpoEtio cc[! re idn diri.ly lbitod io SOC n.dr@
(s .pp.dnr) .d etw fd 30 riur.. ro I hdr- C.I birtre a th.n pLlld 6
LB .g.r pht . (* apFndn-2) @uinii8 X{.1., IPTG (s .pFdirj. 6) |t
loldil cmflnldon ach dd Anpi.illin rt. [email protected] of I0{) ug/ml fot
PGEM-I tdnsfomonts. LB lsar wnn Anpioillin ai a cocentatid of looudmt ws
urod !o &lel pcrx4Tl, lufomb.
Tru.foddo! t. crct bar.d cdpetcr ccllr:
80ul
Pl&. 6o @ftituep rut cont inirg rhb solutin !t 4 C rd 20-30 miNtcd lh.t
ii wa! hoi sh@k d at 42'C fo. 2 oiluios tur r.pt .t 4 C !s'il. ln SOC 6cdn ( lnl)
th. F!6fomd6 brxtrF wc !dd.d. t?rrr3?tfor |-2r:hour. Iianwc
5!E d o &.I-IPTG pl,a16.rd tha pb!. v@ Lpllt 37C oiEd8!!
Mtll Pr.p.nrlon of Pleid DNA by rlldl Ltdr M.thod
Planid DNAg w@ i$llt€d by tho ilkilinc tlt nelhod iD wnich singlc
brcocricl @lony vs us€<l io in@ht 5rJ oftB n <liun Ge appddl\- l ) oonllininS
|nc lppbD! . lD.ibiolic ad sDm |r 37C with vigru sbiking ror 12-16 ho6
(ovnisnD. l.5d of e€ otEfught culhE wB stitod ilio ! bicrc @ntntuge tub.
!.d mEitued al 12,00&pn ld 3 Eirut , Th. [email protected] B {k $Ld ed rt
Flld m E{s?.n&d in rooul of i.. @lil 4[ $tadd $luud ( 25nM Tn]
HcI pmm, r0du EmA,5onM glugB,2ig/Ml be?@), iFutoEd fd s
nituL., ar.t 6.1 2oool of EBht pr.?.Gd all lr!i! Flulior (l %SDs 0.2M N.OID
wc ldded. And imediably nixitrg by in6in8 tbe tub. *wrd tire ed 5
ni'uEs i*ltal|on of bsE r! @m 6P.ruF, l50El of ic. cold 3M poh$iu
rcct t $lutioD. pg 4 8 wB .d&d .nd lgrin Dir.d by 'nvmid
tollovr'ed ht 15
minnLs 'mbatid or ice, Cdtitugllion wa doF al l2,000rPn lor 15 ninultu ud
ol@r supqmht @ cxlr&Gd *ilh h .qul rclu. of Ph@l eumfm: isoMvl
rlohol (25 : 24 : l) nirt@. E*ltuLd .q!dt plre qc p@ipir,bd in 3 fisrr tub.
by .ddiq 2-5 rcl@d of ie @ld ft$luL c0rel.lrEi'illim *a cd.d out d -
20cfd 15biNLelolld.dbyeeiftsrti{.l12,00019fott5Didi6 Ttcp.Uc
vd rid.d with i@ld ?o% .drml rn rti{ d tHrFnd.d h 25d of rekc
R6rrlctioo Endomcle.re Dhedo! o( DNA
DNA M6 dis.sted ueDg @iric6or o duclo@s obt iNd lion Ne*
Eng!.nd Biolabs, CIBCO BRI. Bo.hitgcr N&d cin &d CEMB Enzlt
rsduclior LboELory 'n
N aproDrisG lX sotioD bufid Dis€stm wa Frfom€d
in M ep!.'dorf th.mosllt .r l?c fo. 60-90 niflt 3 Tb. db.sled DNA Rrs
cheled by 6g4e 8pl eletopbodis 4 d.sib.d try S.Dbsr d ir'' (1939) ODc
iJ ol RN4 A (rrEk loDs/tt) w .dd.d in tb. hst I 5onur6 ol tbe drgdtd
t4rim. Th. r*tid rc ErDi icd hy.r'dilg V5 rclu4 L{dilg dv€ (02
bbnophml blu€, 0 25% ryl@ cyrmL FF, 0.!3% rJycdl b v.ior)
T.Dr.Ir Corlfindio! of^[ Clc Urbg B.al|I.q/oe
DNA (200nt)BD HI (20u/pl)9S,{ 00x)NEB II (IOX)
1.5!l
SNIUENCINC ntACltON
I,NA Qoliny I q{.d'd.d |b d.'d of Pui6.d DNA bv ttEuig
0r .h.dt @ .t 2& r d by W Bhr D.ri.il .lo.| ti|! dd.d. Tn
t@r'.d.DoD! b u b cyEh..qEEi!g E !6 *i6 ih. dcTl Bi@t!
T6irr.. Kir @ dFm i! T.bh h.lor.
TtDbX Pq.ilg S.4trdi!8 ReliD
Fc acb etioc ru tb ftllrnat 6!6L b . 4.oE tuh.l
ffindy@Mn
SEgJa&ed.d DNA
Dorbt6.trodd DNr{
5cr0o !8
2m"t00.1
3G9O trl
20d
GEiG--6ixne 'id!Ly
6. '*d- 6ixne 'idr.o d oflisti.i*nl ol
CycL S.q!.!cl!3 o! th. coq{oD 9600 r 2.r0r,
R.Fir d. buowi.! tu 2J clrl.*
. Rrr,id th€ru| @f (s i3 l'c)to 96t
. Rrpid rlEDl rq ro
'(,"c
. ndrr'turqln@b6l,"C
Prdpit 60. lr lttr@lrh!. Trb€
l) PiFtr. rhc 6tiF oonronr of *n dEEior FDrid itrl4r r.5-Dl
nio|aaEin|gctuba
2) Add 20 ]ll of &ioiail*G &d 60 d of 100% isoprcpel
TIE fild bopED@l colatltid d@ld h. 60 J 5%.
l) Cl@ tlF llls rd hncr bri.[r,
4) Leve lhe |lbd .r @ htEatuE for 15 airull. ro tsipi|lE r[o
qtrrid pr.dEb,
Not Preeihtio tnrB<I5 EiluEs will lwh in lnc bd of vqy shon
cxr.Nid prcduc!. PEipibiid tiE D24 IbE vill iM ti. pEiDitrti@ of
EiEe6 .d dF emiobE.
5) P!@ dF tub!! in ! Diffibitug. @d E.* [Fn oneDlttioD,
Spin 6G i|t6 f6 20 bidr6 d aiou rycd.
q C.EinV rpiob! th. eDq[[d! wir! i lFEro piFt& dp fo. qcb
spl. Dd rlisld, Pcll.b my or dy bt b. ndblc.
7) Add 250 d ol75% isopopanol io llE u$6 ard rcr1ax lld bricny.
3) Pl&e dE tub.s in lh. hicec.nEitus& i. the ld. ori.nlllim !3 in dF
t dd lpin for t riluGs al luih@ !F49) Aspinl. rb. oFmh.i! c&ftly 6 b $+ 6.
l0) Dry lh. $r4l€ h ! v@un dlritugc fd 10-15 nituB or ro
d4nc$. (Al.ndively, pl.€ lhc rub.r wi$ lt lids opor in a hd blel q
rh@rl cyclq rt 90C fo. I rinuc,
Eg.dlo! ol rlddL pmr.i!
Tic PCR .!din.4 mcr..rib *qu@ oodins ror ni{id|. F.r.in *6
clmd in-tEEc in , colj ceEsim rccior pCEX-4Tl (Phmoi.) undd de @ntol
ofTT p6nord. T[. clones wirh rn-huo on.nbrim of coding r.qu.Dc. wi$ 6pr.l
10 fi lrdoci w@ &l@cd bt |ne u& of Gr.h blor of rll 0r. PCR positive.
To qFls rh. pmLin, ovniehr culfiE *s diluLd l:l0o in LB cd|linins
lo{Ug/ml mpioiuin ind rrcm .r al C ro O,D5$ - 0,6. E.coli ..lk Bl--2I(DFl)
DL'€E olrininB dE r.cmbimnt plsEi& w.F indu*n by adding IPTG (rdvr)
,!d ircuh6ri6 i! . rhrtlr !t 3?C ior t ho&, Cclls w@ h!ruELd !l 7000 rpn, Tt€
p.I€t w.. w.llicd or wirh IM C b!trr.Jd F${qded i! tha s@ buffei
Ff,pErio. wB idl'zd tlllueh SDS-PAGE follo"€d by w..h blonins uins
rnti-GST mtibodid ed et-HBs-
Puifl€do! of furor pmtdr e+€.cd b E erJ
Th. cxpEsion Eror pCEX-4Tl (Pb.mmi.) pbvi.Ld witn ftsiotr ptoi.i! of
cST wa, u&d io Duri& lh. pFtlin witlFu! lsins i6 p6p.iti.5 and ol vrccine
d.velopncnl grad.. Th. furn middl. prollin ot HBV wirh glrljlhimes-
[email protected]) .,qE6.d h f,. .,lt ltrir\ BL.2l@ErhLysE, 9@ puifi.d bt
$i4 Glultlim s.pb@ 4B .firity Ern Fcrd h dryEbb bri6lt@.
An oeciignt cul@ of A .o/ b.rbouiry F@mbidr .xpEsion Pl,Mid E
dilut d 1:100 in 2o0nl of LB @iliring NDiciltin iloo[s/nl) hd Sroq 3t ]7 C
with vi8oDu $lkirs till O,[email protected],6, CultuF wd Ddued vi$ losM lPtO for '
tdn follo"€d by h.rvdr6g tu elL thEllh @ftitu8irid. Ccll FUo! wd F
$Fflhn in PBS buficr (rrDdrdn.2{) .!d $4adoD si,..d lo \e dE ellt
Llsc q6 dilutqd *irh 4 bluftd of PES bufid !trd el@ sp.eL.t oblliEd .n!r
mri6l&tion. Fd puificatioD oa Monb'dr GsT DFr.'4 GST Mic@pir
Purificlrion Eodule ehm@i., 27-4s7o43) Dbvidd GlutolhioD€ Sephlos 48
prcp&Lcd in micrcspin colu@ (GsTnp FF 5ml) equilibdttd wilh lxPBs lle
Fovid.d wirh IOXIBS, IPTG, rqnrcld Gbnl[i@ .!d dilutor butra. Ly$E (:!0 Dl
ctlopl{nic dhr nm E C,?i qD@ing . CST n 5i@ Plotin) qG lo.d.d mlo lh.
@lur Dd ircutai.d for 2G30 ni.. .l RT wirh god. lgiLtd followed by coluu
washins wnh 4CV (col@ rcroq) PBS bufi.r OIr 7.1), locv of PBS b t t
con|liDiDs 5CV clurion buner dd fiD.Uy wi$ 5CV bindi'e b!fid (PBS) PFllir
{6 .lur.d wirh.lutim bfaa (50mM TnFHCI (9H 3.0) with IonM Fduo.d
glut rhiorc) lrd lbl n&doD w@ rrl€n, Frcii@ oniibins cluLd pFLin wp@lcd nd @EdhEd uiiS c.rri@ FGIiE of rh* For.ir wF &dF.d 6
SDS.PACE & &tcctd 6r Gtan blor d.l'6i!.
PFbin in spls folloq.d by SDS-PACE va bloiEd on io
[email protected] (Hybond4 An6ho) 6'ng . T@bloL Sfli-Dry TEnlftr @ll
(Brdtud) .nd w6llm blot dlyc v@ doE 6 &sdib.d by Towbi( .r zl,
(199),TlF b@ble & U@bil fd @ bou i! bloclmg mluti@ (TBST
@ninjDg t% [email protected] rilL .rd 0,05% 6ol|iu AriiL) 6oud.d ty @ ldu
idh.dd d m rdFdnE i! lb Filat .dlody(Gied .dia$ d ldi-UD )
dildd b rlo,ml in lh. UcLiq &b|ic AIE fiE wdirsE, 15 .iflrh. *[
rrt!. rin TBSI, |n. D@6rE w idlild .l M t rFrhE sith llE
m&y !Ldy (!tr||n! Dbfbo. dioged rin di{bDi rd_tcy,
si!r\ 4.1687) dilnr.d d l:l0,0oo b blooE4 bl$iod TIF E o6m @ eal
w&b.d (h!e tiE ! 05 iiui.. @! w!t) &d @lou d€reloFd by uilg
NBT/BCI!.!dkdincpho{bbeoh.! ., T[[email protected]
ddil.dqE dd.nd.i..L
CHAPTER 5
RESTJLTE
PrE LcotEV h a&d .tEolP.&..ioltffi. tinECV
h dri! !fidy, rn bbod ecill6 oa H€tdi& p.IF s *@ btq ftlm
diftHl citi.. ..s. P.dow, Oujd*rJa F.ieLbd rr[c, s&Eodh! .!d
Ft4ed 16 of turjd,..d NvF?. A|Mr 1270 [4nlL Fi...r 5ll da!*4 $tui!8 rid HDv ir6did .rd lMr b* dly lal h.d YimL (6 .Ltud
by HBv DNA d.bc.d h w} Arong dE la& 116 r@ m.le & 12 l@
Aode Ir lhir ddy, I fMd 9 pdi@ }!o hd IAcAS smgltiw dd HBV
I'NA Fiie (t'!.6. drdrl Mffi, ll Ddid re food D !.v. 6
Ee
C'z
a0
.0
30
Dl0
'lor.!o ltoo OlotoAgeOfM.6
O€lItqE of HBY itrfrEtr (in Er. of Edi.ritri vnE nr tu) i. n L
ed Grtuk pdac in eotilda io {Fn.rp.
alffl.la&a ltll^r.lg+.Ha{|$||lFarl|*r.|la"
tl.r!M.a Eria, dlt FIIA d 15{ b. N or lrl
!-I|r.Ar!di. CaqnvnA!a- ll.rtrdF 2ta
r. rtv n$* Dallb r.i!'l -
rrv I A !d'.fiLAtilrq.tt-EvFa d.Srtt-r
TABLE XT OLIGONUCLDOTTDE PRIMERS USED TOR PCRCIIARAC'IERIZAITON 09 HEPATITIS B VIRUS SURFACE AMIGEN,
I d6isn€d moE rhan lo pdh.s lmDli0ing d'iTeEnr rhstnc.Ls ofHBsAl lo
scqucnce th pbpsly- Among thos. I ch@ thc$ pdn u af,q optiti?,tion
Prin.rl and prim.r2 wE d6i8r.d fom pE{oE ad ce cFne of HBv ro
dirsn@ th. infeli@ b.eus of t inSmoE@NFd $Ds eprc
2l 5' TTA CICG TTT AAA TGT ATA CCC ]' 56t
2 ATHz l3 5' TTC CTA TGC GAG TCC CCC 3' 53C
], ATlII 22 5' CATCCT CTT CTT @T TCT TCT C 3'
t3 5' CTC CTA CCA ATC CTG ATG 3' J{C
5 AIH5 20 5' CGC TCA CCA TAT TCT TGG GA 3' 60c
6, ATUI t1 GCA CCA AAC CCC AAA AC 52-C
1. l3 TAT TCT TCG GAA CAA O46 50c
3. 22 J' CGO GAT CCC AAT OCA GTG GAA CTC
2l a ccc GAT ccr rAo ccr rra AAT GTATAC CC I
t6c
t0 t5 5 aTC C,CA GGA GTT CCC CGA @A GAT 655C
II2
25'.{@
.AC GGA OTT CTT CTI CTA GC,C
G.165.5c
4.15.d93
Ardllr€nor ol P.6Sr/PcsrA 3@ !d IrcSl/S gd. Frer vi.d DNA
Irhtd FmD P.d6l'r Blood:
Ator dc d€tElim of gBV fior ldm Dcilr c@ pn64 (Finql&2), rte
sftpl.s wc€ ue<l @ mdiry A. S gtu *hich g.v. podti!. bdrb orly in 30ol. or@s. H@
@ 60@ ieht€, sivinr p$iliv. brnd ol wholc S gcno of HBV Mplified ui,s ATUI rnd
^TU5 pdb* in the fisr dnd ofPCR 4 rhoM i! FiguE 3. TIF rrrFlihc.tid silg aTUl
ed ATU5 Finm b lb. Fin ry rt p rowrld! s.qutuing .rd Euhrin @lysis.
Tle splc! whjcl d'd nd giv. bDd ot L3 kb in tb. lr d.{t of PC& w@
turlh.. analyE€d with Gbd rcR (f, rcud) uiDg tu FimE or.ring dE prbdrct DNA
obroiD€d t m lr brd. I1 glE n. '@ fir. dt pu! fdD of FqdEd DNA. Th. hd sjz
M or l2b $ shM iI FisuE 4. Thi! pfrdui ol EEd PCR n rko uFd b !.qHe rtt
Tbl etu c.rting lor nid{. Fo..ir *tich w! slcEd ro .*pEs for @i&&rclopnd Nd dpliftiluilg VAC F&R ir 6toM ir Fig@ 5 o{ing b.nd ofo.8t!.
or*hole sDrto.. 8cn. or HBV usiDg ATU I & ATU5 G a R) pnmeB
Lmbda DNA narker dig.sntd by .|//dttl
DrflcEnt smpl6 of viEl DNA dDplili.i srng ATU l& ATU5
rk!rca: N.dd rcR
roa: I , 2: PcR ptoduct ( l,3tb) dplif.d in fitg ,Ud. (ATU I &aTUt)
l: PCR prcducl ( | .2k!) hplili.d 'n
sond Eund. (Afi | &ATH5)
PCR hDlifdion of pEsz/sgd noo $pt.s uini vAC F a R FidlF,
], Mdkd di&sr.d with rlrdu
B&d ofo.Skb showing PESZS gs. ofdifiwnt i$ht s.
CT'NING OF TEE }RE.SI/PRE-S2/S GENE INIO DTFTf,RENT CI'NINC
vEcTons:
Fd s.qlldin& 6. yholc or6e gc ru clo.d b de thr ItF of clqine
EbB shoM in Ficr 6. Cel pictuB h dr Fis@ 6 dlb* clG @.filmtion ana
dig.rti@ wirh ,,l'//vhich grw rll .rrEG! bed.i
So I el*Ed tYdy 6E chMic HBV Friall fio6 lh* PCR positiv. .&r !:danplif.n tuF qith ATUI & ATU5 5.1 of Dnn.r to idonrify nubtiore *ilhiD rhe HBrAg,
Thc PrDrhcl lhplifi€d of rhirlq spaiDm w@ cloncd iD diffwnt clmins vetoB ro
obtiD singl. ty?€ of DNA Eienmt in @. ol d.'8ing wisE ud.r $l*tiw pE6sN of
tmut. EEpoE or lbmpy. IL mDilg trclw sp@inoa @ mplified uiDg dilulid
t,hiqrc (rqrirying $e plodeL nm siislc @l6Dl.). In rlir cs I peifi.d rhc ta oa
(l ,2kb) of s se uinS DNA ErF.di6 hl f(05 | 3 (FmB) ..d rhl Flder O20O b)w diedy s.qu.rc.d cing d€ Big,Dt! Tmirtor cy.L s.q||@ing Edy Grtid KIT
(Appli.d Bidydn9. S€q@ing wG ddE 6 s AtI PRISM 3tm Gffic -^alyar
(Applid Bidrir.n Lc,, 850 LiMID Cdtq Driv., F6rer Ciry, CA9{404) }CR pr.tucts
w. equdced fron bodr s[!n& to 8cl c.@DN equdo6, Thc equaes fron pGitivc
dnnds w@ 6ka for mlsis
A3.E !.1 .l€tqiG.rr tu ftdldoofdffdddcE6l$&.41.rrE r. vtildlll, ! PadF + o.trh'
I
o!*^i|re!e',hsli8dldnofPcrt|ccdEn'i,{|?bhgiarolenobsF.disrd lqaftl.4 PCr 2.n.9tb) +rs ({br'd tad
Miri-pFpardim oIDNA fion cloE.
L
2
3.
5.
3,
I Ma*s digdEd wit! hfi dll
l0A cld.d n ICR 2.I velor uing AIU I & 5 prin4
I4A clmd in PCR 2,I Ebr Dsing ATU I & 5 prim6
FAI 8 cloned in PCR 2.1 Ector uins ATH I & 5 DiimoE
SZI I cloDed i' PCR 2 I vooror usir! ATH I & 5 prin.6
Ll7 clorcd r PBSSK v@br wit[ blut end liglrion u6iDg
2b cloEd in PGEM-T E{or uDa VAC F & R FimE
4e v€d ring vac F & R pmm
35 clo..d in PCR 2.I Ecror uing ATH I & 5 pnn E
SZl2 v.cbr usiru VAC I & R prih.b
I lA clon.d i! PCR 2.I B!0. using ATU I & 5 prin6
EoboloAl .ftd16 of vlrd DNA bdr6 ud phy'olcmtic Ar.rtdr:
P.rhrd iELB 6.quaccd in rh. P'!gl sdy w .lig..d *ith r!.
Epr4lbdw nmb.r of s€q@s Id ah mjd gmr}?. dd obryF Er-ed
ftm {!. Ghtunk dlbbe dmugh l|lnddn rucl@tid€,nul@tid. md ptucin
(SM$ PFl) BLAST (B6ic local llig@nr !@h lool) shwG (Nbchul, €r zl,
197) lv.ibbL .t NCBI wb sir. .rd with rhc hlb o{ tne MnttrtisD Foslu prn
eie cdF ilod fc % Nlor c aiEiltuity rDd mh,tio ry disnDe *w E!&fte phylos4dc ldFtu of Hsv
'bl.c. w@ F.foded *irb clwhlw $hqc
uliDe.TlF phylogneho !B w@ ooDfuot d by rh€ PHyU! rnye jt
6njmiio! qiLh lbc web eflice AI nnp/ww.b'.4ul(/clus@1 .
ln .ll chMic pllidE *l-bd nqri N1MFP ud Pujrb, g.!ot$. D lsfoud rd dl rhc islic
'@ ford b h.w wnrd in 6. b.p.riri! B &.h.. s@
*hich rn!. fr!f, !.15% E4€.tircly (Tig6 9) *fic! G @bz.rl !y u.iDs NCBI
A16l it e.b5l. f/w.ncbi.nto..ih.eov/bhn.hh Tne nutatio$ idan6.d n
difi@nt lila of nuLotid. s.qufus of tlDsAs in atl isoloLs ol HBv chrDio
pa6@a E chom i. rhe edE (lisE 9).
Tb. E!l6t tn. p[ylo3a.l* &drEi! of tt *T.-.a Mpl. .lio,ho*d
lh eo.ric disbm of dE gnpLr fr@ kloM fljd e.dyF. B.!.d otr rbs
ciErioll 0E HM. (O 32351 disllE) w.! etccrcd for expcno lrudi.s
EEEEEE$EHEESHEHEggEHSS gsHg:gsF
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Anlno Acld CoDpr.i$tr:
Th. Dim &id &gI.E o@LLd i! tx E@6 by uirg tbe eftwE ECM
huchd wr! llF bla5Ld iD NCBI Gmbak rb E d rh! @ nd. of tust icd
u.qum3 ud so ro sd dE DultbL E qsE ,rt@l oa Fprid4 of S gdc
orEin d ftm B6ty fiE i$lrB of e|[qi! @ia of tlBv, Mulripk i.quE@
dismat usirg Clulblw 1,8 thFuglr BCM IrmlE sE dhow.d no &gninolnL
mulatio in Dy ore..pitoF! d uiD &id Lwl (HtA cl^$ I Bdced .piloF:
PESI 10-17, Pr€s2 109.123, HBs 251!253, H8.260-269, HEs 315-341, HBs 143,
r57 ud HIA cl$sll FsEicBd epiiop€s: lFSl 10.19, PES2l09'l23, H& 132-191,
HBs 182-196), bu @ 6r lh. !"Tl* GA) 5ho{rd rubrio wiihin 6. Hri As !,
Clod4 ud ErpBco! ol tr. pto(d!
To aMlyb if rh€ 50kDr niddL porein is c.Dcbte of biDdi4 to rnii-
HBl dbodis, &. Irt-S2/S gft uplifi.d sing priD6 VACF & VACR 6clored iDio lh. Pfr2.1, ertlz.d by ldiciim digdtid (Iig.ll) .nd br PCR (Fir-
l0). Ttcn EcoRl discsted filgMt was oldcd i! tcoli oxpEsion Etd pcEX.
411 (Ph|ft.ci! Bioret), cl tu 66!ud eB dddom.d inro BLtl (DB) @nr
Tnc idarity of the selead old E 6.6o.d by EstsictioD dieenid (Firl3) ed
by ?CR And IrIG- irdlctor\ rufMll ww eEn d fd.xp@ion of he
ared nlior pFbiD by SDs-P^cE (Fig.l4!),
Thc puntr.d dpEied prckir by usins clutolhbr. S€phfts 4 Frlt Flow
colllm w obEin d vhich grv. !si6v. r$lts itr {4h blodins A ,0 frDr 6$id
Fftin (GsT-niddle pror.ii) lppar.d h GEn blor whd .ni4sT turt dy w
$.d (Fie l4b) lrd did mr sirc Equi,Ed bud vh.. dri,HEs dribodic rere u..d
(Fis-,s) *h.rs oo.[!l wls posirivc,
Sub.lolllg Irro EtPrdtolvdror pGEX-4T,
The vdi. lA was sl6ted lor lhe qDr6eon. u wos 6d cldcd inio PCR2- I veld.
l0: PCR cobfiftdbn ofoloe 3A
L r phrsc DN,t liidnl d,8cs€d us.d s Ef.En€ slrdld
3- 0-8tb Flsndr olIhSZSSqe
Fto.e rlr Digestion of both 3A ed pOEX4Tt with &,Rl.
Lebda diScn iw hfirdltl.
2: &otu + CIP lr.otei VEbr.
r: 34 clone iD PCR2 I disesl€d widl n oRI
Pmficalion ofPE s2^8.n. (0.3 kb) .ta 0F dircstion ofclon 34
ljnes: hn l: t n&kq digc$.d with,rrrdlll .Eymc
3: P@S2/S sere (0,8kb) purifi.d Lo clonc in cxpBion valor.
#41
confirndio ot cristomtuB of pCEX-4Tl i. BL,2t c.lh anlr dlg.stioD
I-!n 7 shows politiw flcfomdr
WESIERN BLOT OT PI]RIIIED FI]SION PROTEIN
Fig!Fl.|l \D\.P4Cl ofpdern p6fite oftPIu induc.d porirn. tu\fomets
wcs6n blodng of puificd fr&tim lrcm
bybddiad witi oti-GSTanribodiesusing
Fdios of plrificd pmt.in an r€l ion.
Bl--21 ell Ly$a Cv€ sbol).
Prc-sinc! ma*e (l.lon lnYibogen).
GST-Sephrc 48 Colmn
6 ta I 2 r
tF r* v.-Udadd.dbn0CT{.dcaACtuti.ill.drr!.6ddddagt|&d&.Ihr.d ra r.L* Rtr d d (8c r.
R@ti6 Il A8 (co"rdol+ rflL !!cch4 rtC. tll d_IlB.
c.ll tf-loa FX{lrfi2l b.dtEo&d.t- -dd ft. tCar&d + hga U !ffi.
2.
a,t6
CHAPTER 6
DISCUSSTON
vidl lE oritir ir d ilrpdtlrt puuic heilt llld *@nic pDbld. Ilortu, infoDtiq &i.f.cliN disdg h d.v.loti.g 6rbtia, dd FtricuLdy i! b.l.r.d muid6, is
sse. h P.kirb. d .d.qu.!. I.€l 6f ffom.tio 6 th€ .prddiology of HBV infetiola
chlmcteriatiq ot lhc g.Ma, vdialility ir lh. S ga€ (€s?€ci.lly in cpiiop.o anil iE
.tr.t on tb. astielnilicig hn Dd bd .qihbb & ni od e inpon&t ioo. be@ iveiMiid pran.8|i!ll tlBV h Pltid& i. io b..dutrccd i! Ih. f!ne.ln thi! !!dy wpdfom€d EoLculr chleLrillieta..d &@Diddiolos/ of lnb vinl interid i.P.$.Ln and loud th tli! h.ldin r/B6 is w .!Fsd iD P,Li.tu rnd ha I.d io a lign
imidcno€ of eute od olmnio dis* in thb egjon.
In 3!n rd, thc el of vihl idstioD in tDpixl .ls i! ml .!a. In ficl th. ets dd
fr.idr iNoiv€d l@ @r i&iritu i! lnc FE or 5n dy. ID lddidoq rtporidt ly d csof HBv idecdoo @ur in Fllm wno {to not b.loa 0o a eoeliud hidFriik soq6
0@ll}l{@ wdk6, inlnwlnoG drug lffi, hdopnili&s and individrk duasei ir
pronisuN sxul b.h.viok), Ore of IF l!cb6 whion nry be inrclrcd io s.h a nisrt
imidde of HBV infadM il nubrion
It fidelily of 8.rEli .d.rid i. [email protected] upon llc '..scy of thc eplicltiw
t&hiDery od dE DrcofE dirg $ili9 or th. polylFe GporoibL lor EplielinS rhi!
nol.iicl. Rd6e liascjpn* b* pd pDof pding abiliry, sddtin8 a hiSn Ere of
n@leotid€ Disincoaorrion duing h!@riDrim, T!u, nutlr'rs s inhode.d into tlF viml
p@1. gBv stu io dlibit . orr.dm r.,r. !@ tba lGfold nth.r rhr otlla DNA
vitlg. Tbc 6t.s for nucl@ti{i. $hditurioN nry d.Fiilg @ l[. 'l.e.
of dii@. Tb6
naluBl @ludomry Dr. for $. HBV g@n. iD cl[6ic hcpatitis B is appoximlely 1.4 ro
!.2 x t0i $htitrtion ditc/yor, esp@idly in lh. coding Esim ofths scDon. and cis-a.ting
Fsulrtorr .ld6ls teae otit! hjs! 6q..t .s (Ornoro €, al, 198?).(14) *ldas in
liwr lr&Ahnrition senine, it is llncl loGrold hidd {Stebsot ., al,r997). Mutstions
tcrd io clut t inlo nutatiml pitEiE h Fniul.t lb. blsd o@ ponor.r (BCD), rh. tFce resio! lDd rh. '!" &tcmi@i of rb. vi.l drl@ Gurb.r .r or, 1999). Th4 @
two ty?es of mdrnll of HBv. NrtrEuy occMi!8 tuials @ tb. @l!r of bndm chdg.s
el*i.d orcr yce of popuhtio tMsE. Thc& vrianrs isludc HBv 3mtyp4 dd
uusrl scqu.lE, whicn my bc ledr &&cr.d by ittlttlltll(,@y. Th. $ldr.d Eiub,
@ mbnlr rhrt oris iI indivi.h.k uder a.di6lly or mtuElly (chbnic h.prdris B)
inbc.d innurc p$sw. ftey ircld. vrein. rnd h.patilis B innmc globulir rh.ra{ry
(IBIg) d+c nul.lrs (C,m., dl, 1990, Chid €, or, 1997, knhig@ d.r, l99q It
d aL, r99a,C@ftmD et ol.,2OO I, C1E d 44, 2000). In livd h.splrr Ecipidr, higl tir.r
lntib.dy i! . silu0lion *ilt low licr viru6 .nd i l!rs. nunbq of Busceptible 6U! (liva
F|ryldlt) i d idal bEdi!! gtud fq Dut trh In cnMi. XBv inletioa Mlntb
@urila .qF nullnrs my h. &Lchn by rhc di-HB. E !@& of rhc IIBV ceid
(Y@anoto €, dl, 199.4, Y6n ?, al, 199r. As ! omsoquoe! ,oD. chonio sEiaB hlve
borh HEsAs d.l ed-HBs, rh. .lribody b€irs diecd.girsl HB B.pibpd m. sb.rcd by
thG ciEu&tiDg utigd dd th6 rcr FolacriE (Y.mnblo d ar., 1994, Shi.L d ar., 1987),
Itd*4 svddl inv.slisdds hlvc ob@ftd oo-cncuktior ofa ruhbd of difreml seq!.@
ir pariente with cbbnic hepniti! (K.Eto t939). Murid Hepatiti B vi@ {ith .ubniudm
vnnh thc cdinS EBid rq HBV srf.@ dd8o o{tc^g) t{ b.6 forod sidtly ircbmic dic6, lt is th*foE importrl to ckriry wn rh€r dF idcntifod substitulions within
rhc HBsAs h.v. inpad 6 dE dtig.nici9 rld [email protected] of HbsAE Qt& g .t zl ,
20c{).
Th.E 's
d$ €vid.tr@ d'al s Fsior is bT*lrnbl..rd erade hort imue Eaos Md
inhlrcd h d. dctemiDlior of ScnoDp€s od 5u6g?.r ofHBv in . populrnm. ftc 8.ndio
Ebbiliiy of hcp.tili3 B yiro 0{B\) EpE* r I oh,llenge for dE s.rriliviry of
itu,a&gn^.n, c6tenly for $e d.&crior ol srt e dlisr (HBsAd. Tr'. Ddtid in
rhe driSdic <t tmi6$ of "S" gq, n qD@!.! to !f*t lle md:.y sturld of tlF
As !d so dE chdg. ir dE sDsifioity ofth. @iLp.! whion Day l€.d 10 4pc. So pE5€nt
rludy v$ ioculcd on ilF mtisenic deffiimnb of dF surfo@ s€re in 06 popuhtion,
Mordur.r Cho.ct rir.dor ofEBV Loltro
b 6i8 study. I.lBv inf.cr.il Ft@!d ,@ lrdy?d rd Hta& &d HBV DNA.
Arn@g tle ody 33% sho*rd vimn u &f!.d !y nBV DNA d.rccled in blood hn br ir
6?% c.6 pb.c ol, HbsAs oDd nor HBV DNA va da.crsd i. blood. IF& @uld b. t$o
expl@ri@, fintly virrl DNA is intosnt d inlo h.p eync cbmosmal DNA 6nd k Dt dEl6itrs it6 dilfarcnl dtis.rs inlo thc blo.d, S@ondly ii ft and.ftd inside lllc c.lls dd is
not iloscd i o dE ciruhtid I brd piclr.d up dl tb. ctenic.Iy inlet d p.ri.rt3 io hlrbig! imidc@ ot hutnion s ol w€E Et6As Dositirc m tb{ d. hsr sn nonthi .!d 13%
@tofrn nwFdtircff HBv DN - T!* 33% cB w@ ple6s.d 6fiha.
Iddtilt rdon otG.noqT6trlrtrirn poDd.do!,
llBV $notlpcs !E .smiar.d silh dilfcMt cliliol di6e& (cilmr 1997, Klo er
a!-, 2000, Kb et al , 2oo0, Otitt .t ,1, 200t ). Pdimas EspoNs to ilterfdn ildpy snal$ ro bc @iatcd wnn !p@i6c I$V smrrlc. (K!o ., dr., 2000, Kio ., at, 2000). n wa
$$6t d dEl HBV edotlF C is lsiiicd {irh . nG ffi liw [email protected] ri.h.low Bre of Es?o* io inlcrftrd t@W g&.t a1.,2(M,KN et at, 2000. TrE !tutyFI found in ny snldic! s s.DtyTc D wlich G @.1widely distributed s.notr?. dd h&
bm fourd univ.srlly, wnn it! hisned pEmlmo in r b.lt srr.tchiqnm Soulhoh Euope
ud Notrh Atri€ (Nodf er zr,r993b, Borch&i{blbchoub er at, 2000) io lldir in v*tid souln Afriq (Bosa.r e, ,1., 197) !d .tMg ihvd$ rklg !5d d lll cofricnl5
(xidrlLjMsem .r zl, 1999; arlcrlers e1 al.,[email protected] a1.,2}od).c@rrF E n 6c
ncl dniLr !o 8.eqF D smdcaly (Noidd d dl, 1993i,b, 199!t) ed hd b*n illaAFr.d
ar . srblct of 8.mqF D wna udng lb. X s* lor phylos.n tio Mlrsis (Kidd-Ljunssa
d dr, 1995). It i6 foDd in wesl od soD{r Afriq. ind oft ofrhe min diffcM@ adg.noty?. D ir tut it d@s ml brw 0E 33 !r dcl.rioD al th. h.ginning of dF pFSt Egim
which id @|llM ro dl eaot}?c D 3E!iE {Bor?.r d al, t997, Nor&r e, !t, 1994,
Odmulm d, ar, 2001) dd no Mn ddctid !.! b6 gn i! odEr gmtyp6. TL
m.4!E of Bis.s. ro ativiil .b.r.py wnn lMiwdiE ffi !o b. iodcp.dllr ot tlE
sdt}?. bur m.y b. elated to de ptls.e ot HB.Ag.
Ttq. & nultiple poiDt Nr.tions dcioctcd iD dF $rfaoe eere 4p€oiruy ,a'
d.t.mi6t jD f6us i.. M.rl33ntr e.2%); Cbl29Hi., M..133tr , Phe/rtrt34As O ,8%);
Ph./ryrll4t u, Gly l45Alr (1-5%), ald Plol2oTltr (l.l7o) *@ fi. md eqe.t in parionlr
wilh frilue of wain or Ia ilEr'y d sF flm t4,r. Grcnon & Echcrcria 2006).
Mubiios h rh. r' d€enid (bd mr ct45R) ds hrE bed Elon d in EeFdprtia!! with clmnic HBv i.ferion vho nrrc not E.i$:d HBv r@ift or HBIG.
How.v.r, il !pr.G 6!t scn rutadoG & only Fspdsibl. lor a wll proponio! of
"@oulii' o. !'3ilcnl" HBV iDGonons, wnicn @ oh.tucir.d by the presn@ of HBV DNA in
..lm in lha iberc. of deblrble HBr{s. Howcvq, !@c of rh* nuhr fo@ of IiBV in
c€s oa@ult llBV @y rhordicdly e.p. d.bctid !!d 6dd te!. rtuk ro btood
Uling dc Dde.lff l-hniq&s of PCR ud DNA s4!tuidg I b4c cbercriz.d
dE S rc8ion of h.pltitis B vihl eoone lhlt dcoonshc tui3bitiry ar dif@r polirios in
the pi. S/S s.n nnsiDe from 3,15% ir dilliNr prridl poputrtior (FisuE t2).s rhoM in
sulls dd all of dr scnpl.s (25 in no.) h.d sisnifiodt nu|ltion with rsper 10 lti.€rioD in
AI sqhc.6 which I rmd wirnt h.d tu vailliliiy in the €pnopc6 dm.in oI bod'
pE-gS s4 b orc nm.d 3,{ 'ti,t lld wnbilry in tlE'a" &LmiM! | elat d fq
6. *p!si@ !o srdt ir3 inpdl 6 .di8@iciq.
TL 8@li. di.t e. of tbi! ioldr ss 0.32!51 !'d Gdd 6 ! didel bEmh ir dE
pnybeerctic t'ec mplriDs d ln. isoktd agaiul dl rh. smt}?€r lnM. Thir j!*if€d it
h.ing pictcd up a !.dor fq cxpEsion 3t]di6 a de&dbed in lh. ainB and objectire
Cloli.g.'d Ellrsdor olN,'S G.& C.!t lrLt M[. !t fptroF:
Tt h.pdfti! a vi@ b.s lhe dwloFc protcins dd tb. tujor drcloD. pFi.in i S
teleia millilB of 226 .nie &idr A hydrophilic Egion in S tsr.ir (ar 124 10 147) is
dosislooed as '! d.LmiMnt, { dliS@ic d.i.mim! ! coifmd@l €pilope .otmon 1o
ol HBv $6tyD$ TIE sut tio.r, sinlr.d clo&ly t<tjsl !o Ih. a delomiMt @ld.l$
chrna. lhc diE imuodoii@t ECi@ 3ntxt,c &d tLEfdE rE L. rh. alt niiry
4r tuucn rc Eulltim w@ foud winj! lnir'lr-147 Esid (Ktoury B*.t at.,2@lr,
Hm€, rh. cl.$iql <Lfinitid of tk'a imuodonidt 6si@ @y m.d to b..xb.ded lo
EqDiE rdj4.nl sho ..'d! to $ppon i! coDfomalion (Roaov6ky €t ,r, 2000, B.Bjee
d oL, 1999, lju et al,, 2OOr). nE 6fiicn)E poj*b our 6m rh. $ifE. of {r HBv
Fnicl (c.|i@ ,r dr. l99O). C!6rid Ut b.long {@ic rbt @iri! o .&litioD to
HkaS; th€ pESl .rd F.s2 viir pbt.id & ,.!on d lo b. n@ imulbsojc 6rn tr
s.mtion uccitur co ririlg qiy @@biunl HB dcoEt , 2007).
chs I ond Cri$ U Gticl.d TsU epilop.! in dE llBV ovelop€ p$i.h conllinins
P@S I/PESZS &gi6 @ followilg:
Epltop. r.tdua a. st@c
I P€Sl lGlT ?LCFFPDII
1
3
PES2109-123
H& 250-258
H8.260- 9
g& 335-3,t3
HBr 348-35?
MQWNSTALHQALQDP
LLCUIrLV
LLDYQGMLDV
clsPT!1i.LSV
286 NLHDSC 29
's I ltlsc
AI'H PBL
AI'H PBL
lt Sl lcl9 PICFFmHQL Da5
PcS2 109-123 MQ\INSTATHQALQDP DR2
HB3 I82.I9I FPLLTTITI
4 H& t82-196 FFLLTRILIiQSLD
A chrD8. of a Gly to Ar8 .t pcitid l4t (Cm ,r .t,2001, Hetnlt ., ,1,
2001) rld thc ebstitutim of alpGi. &id !t @ 144 by Nldinc (Ni,r 4l, 1995) of HBrAg
snow rot scdaiiy or Hb6Ag wilt !trti .' norloMl ertihody, I is ols r€pon d t!.t
llj@ is i Id Ftul mbd@ .t rt 551 of thr HBv (arr) saorc Ircn A ro O, kldiry to Ilub61itution of n lhimim (Mct) to ulire (Val) !t posilion ll3 in lho '! d€tesiMt of
HBsAe. Tt@ i. e did.G conpdrg Eur6r b rhr wnd-qF HA s,lne lindils &riviry
of 6e rutur H&Ag to n]{t6 (A6, Al I !d Sl7) 8d ro veiE-Eied hu|M eli.lEplriri3
a Nrf,ce altih.dy (hti-sBs) {b.se ltcniftandy (Chen ?r ar, 2003),
b dft study, I clEEcliE<l ! rcwl EBv Dub navirg Duiirion !r @ 28ed 289 of
lb. I{gc dEloF protcin or ul22{125 of rt dll .Mldp. pot in which @sr fd"r" d.r.@iMt of HBV. Tna 5ubltiMon of rh€* 0Nc e
(A Ol2598)cab6d NCBI
in $. !.i deremiM.t of HB3Ag reol|<l h rtre d*q& of Eacriviry of tn.
@onbiMl HbsAg b vrccirc-nisd nm.. diHB5. silce thl ndd B{ell .lligenic
.pibp. ci.l€! in {€ 8ro!p {.cific '!' detemiMr Egto, yhich b t polt d to b€
onromtioEl (KordSi €r,r, 2000), l!. dlt I pFsn&<l cLely denm!!&d rhi .IE
nurarid afi.cb ti. conlomtid of thc ! d€t miur &d !lr.n rlF urig.nicirr or EB.A&
loding io HBsAe esp. Iion th. d.G.rid by .r.dt!d H&As sys.Also,Osio*7
$8sa{ed tb.t lo$ of dcletio D.y @u drc lo mEti6 wnnir ddq oDlsi& of dc.d.hin 'x th.t ctr@l oodomd@l epnopc @gldor ft H&Ag smtion or
dFesid(Osiowy, 2006). Mcr of dF mnrid .si0t d wn imum sF @B
wid)in thc &cond loop of rh. . dot mitull dd tlrt bft b..n the ft.$ of n.ny E6r1
Our obs.aitimr tur$q iDdic.lo th rhe nubrim in dE HBv surfe. sd. nay lqd
lo ! 6id.Eble det:Ile of pFp.rly fold.d $rfuc dig6 *bich Ey Enda (hc yiro
p.nicle l€$ insDbo8aic in Fbdncntg c.f9crivc n.utdizing sti-ltBs b cld. 0E vins Ttr€
subd im of pblire al di!@id poddd 125 .l!o irdioc dE cb.ns. in cdrrmrti@ ol
DFreif,. Thi erigmic.lly divergdl HBv huhrt i5 inpornd fd bodr cliniql .rd
diasD6lic t@m, lh@forE" tunh.r nrdi* Gins qp6sld nutqnt HBc pbt im rnd
MEuLtion oa.ddih@l css vill bc Equied for .lEidition of $. b@huis or th. los
of diigaicity m! pEvd.rce or tLis nulltion h ou Dopulation. To obldc th. pEElcroe
ol this panicuh nuratid in 'a &r.rsjMr ot HBsAg e .tranariw lppdc[ cn 6.
adopted whioh is s lollows:
|. D6ign lnd €rpE$ . p€fii& libB.y o.r.inirs ltcs tnE D clDru6 i,tdividdty iD
.rch p€ptitc usins pnasc diQlay l.chniqu.. Eeh p.ptid. dpr.sed on phage head eill be
6iqE md @ld ditrcr non tl'c othd pepriL. by @ or boE @im .ci&. So rb! libEry
would *rry ,+ddes dkplaing s.vcn oonhimlim for a solelld p.nion of $c etiSmic
d.tcmimt'!' of HBsA&
2. Iorobilia rb.IgG noLcuLr ton d. p.ti.'nb ffi @lLcr.d nd wids Fnt
of Pdlis$n onoo wells of 24- or 6- vell dilhes md pobc sirh ph.gc diQlay libary. E.ch
rcI an h!v. p6l.d m& iofr dc cily imobili,.d in n (aI l0 Fftrb fron Mutt r ir
@ rcl, .ll l0 n@ LL@ i! etbs wll d.). T|. pn.ar rbin .r!.n !o th. wlL 6hould
b. .luen otr !.p.d€ly, .Iow.d to irf..l tc,n ed .I clon6 iol.r.rt {rrlnA phagc DNA
du'g with '.' d.Lmjffit podi6 of S [email protected]* cloFs choul,C be s€qu.@d ro
the pEwlo@ oi ach nulArt in Plti*ari popll.tid. On cd w t[is inll)@tid io
@slruot 6 d*i8n . mbi4todd .,risa crryiD8 e. wild tyF .nd Eid of !'d.r.miMnt Ecognird i! ay stuly.
3. OE My s.r 1b. pFvaldt ouLrr frw 4h ciry 6d om @$p!E dEs to dcller
4. EXDE$ the cobbimloiial ddsc4 pui6/ n od ue n il cllimto@ tiaL.
ln @*lusio4 I h.F it Dtified , nR rurfr. buhd oi HBv. nis bubt Biddti6.d lrd kohed lion . chMic liE di'e Friot 5.6l nut ri@ w@ obsand
in rh. surfec &risd seDe rnon8 arl rIE islat$ &qlesed out of whio[ $is multnr w$
sroctait d th. b65is of oturs. ir rb. rdisaic &crniDlr "!". 11 i! DEdictad ro h.E Id€stbilizirS cf.ct @ rne stucrEd hi.griry of rlF '." &E@imr !d ar$ .tr.r {E
intiSoioity prolilc or th. nubnt Ilt€A& Th.!c ob*pltioB tbu! indi€te .bnt nut rioDr it6e tur&. g@ coulrl Ld 1o . cmi{t€EhL .LcFe 6 coi.qLL .!&r@ ofl()}.dy foldd
uea dt8.n snich i! tm @ld .-lPlri! dE s&@ of BcdE HBIA8 ir dE *M of
APPENDIX
MIDTA AND SOLUITONS
l- lar!.[si-Edb!,0.!-&d!4r[ry1Yarrdt&t 59
N.C1 los
Tt!!o@ loc
Dilohed in I liEr ofdinild &ioircd w!rq, pA .djuiod b ?,5 .rd luloc|v€d.
t LtAnLB cod.i.ing l5g lgd/l lit r.
Bero!^roE Ne
B&iorlrrtdlrr.l 5g
NtCl O534a
KCl 0.1869
Dimnrcrl in I [bt of di.dld &i@izql scr. prl .djui.d !o ?.0 wirt N!OI| .!d
4 socnt dt@
2M Mr$ sbcr
l|,garnllfi
MgSO.THrO
Aural8.d fd d.tiliaiio
?o338
63.049
Ad€d rd of2M M!* rro.& ud rol of2M 8l!G ro 98bl ofsoB M.di@.
5. X-Grl l$rcE.{tlon3 lrdohl FD-di-t'o.lde) Sto.& r|||pdEl
Adtcd ls of -lil x4rl !o 25rl ofDim.dryrMi|a ML.d .rd doEd .t-20!C in a foil *!Dp..l lub..
Ad.Ld lnl of XG.l slocl blltid .nd lnl of IPIG sr&l $tuiin ro I litd or LB
n <tim to givc fiMl cmdt tim of 4ooslit r.
6. lMgtollrltrl ]D lntorlnio.ld.l St .h,,(tds/ol
Added Is oflolid IPTC t 25Dl ofsiedli!.d IirO. Mixod.nd dedia! by fihhrioL
7- A!qbqE!.9&4-&&bt
Acryl@ide 2928
Bi&Acryldide 0,8!
Dilslwd in l00nl of dHrO .dt 6lt.r lciiliz<L
& aX lr.E {slr.6p Gcr B|ltrTria. be 6.069
SDS 0-4s
tu 87oret
Diel'ed i! (lliro, pg .djur.d io 5.8 wirl Hcl EtB D& qro llI,El ein
! axtmlnnhcdrriLTri6.bs l3.l7i
sDS 0,4t
Dn ohEd i! dIIO, pII !dr'dr.d r,6.8 ritilICl voh@ E d. 'pio
l00nt ritt
lG aXSr-LBG.
4XUPpdbutr€r
sDai
B,M.r.p!odh@l
r% b.!o4tdl U@
dgp
1l- lox Ptutd! nurtu lrtrdTii@ b.r
sD6
Di!.ohrd indtlp dd rd. bl|@qN. I lita.
It Lrd Rsolvln! Cd
r{syt@i& d@t &lldib
,ll|0FI
o.8g
d&o
lo9a A'fui@ FslitIEMED
30.05a
'\42.59
5.og
5qd
2Otl
KCI
l5r TBST bdd
lM Tri&gcl pfi 8
,M N.Cl
lM TTiGHCI pH 9.5
tM NaCl
lM M8cl
675d
l6Fl
25Nl
L5EM
llle-EgEtssrsd
&ryLnid. 5r@t rcbri@
lCr.,6 Antuiu Fsulfir.
TEMED
1,L E!p!pial!_E4tr{!!!!&.ll8sl
3og
0208
o2n8
1.158
Dnsolvld i, 90onl &[email protected] wr!.r, pH rdjusrd
Dishd in alt4Lvd dcioli4d wier !o arte 6{t bt@ 10 uN nEr.
Allr|lp.ltdpttt brta..Ior oL. s.dorIG
Bhldnr b!fid
Skim.d 6ilk 5g
Dit olvld i! IES-T bulf.r lo E*. 6d.l bluE 50D1,
lg
IF
C. uB&.rTri&HCl pH 7.t
NrcI
Cql!!..Egrr2
TrigfiCl tH 7.5
NrcI
l('!r,i ou 8.0)
t1-
G.du Brllir 3
TrilHCl plt 9.5
MSCl:
DNA D|r[do! Bnr.r
Tiilllcl
!DT,{
ApLllli! &ock lolbg/rl
Disehld I g of !odi@ Dlt ol s$pi.ilh in loDl of lEriliuil diltiled qi&
rlrl fltardailied- Addd rd of .roctdticilin in @ liEof E diu fq rt fio.t
cot@t[.tio of loolr8/nl.
M.l..ldr L.qulftd For SDS-PAGE:
6x S$d. Elatu (rioF rlh!.t8 .r- 20'C):
q.f.4ry!d!A!, @1tu&)6lticbrchl 6.0D1l0or6ot6ol
30rxni0468) 3.odlxlll68l2nllDIA ZI)IIOJI.BDIA
l2tasDa lrt$86arx2{rytu1 6@d2a-+ao&a1.05%h6.!telt&& ,!dbo.d6l
For Dolt|cqLDld. Cilxlr!41(pll8,8) lox&crnddarEdft.
I MT!i<t(r46.8) 30srilnisDg la5rj &.aCl,Adrlei& 10g86(!?.5t.dyLdileti|" t&iar4a.ll,etbttro.
lota Atudi@ pdlir
rBiED
0.01% sDs
lorrt rgld.t |rtrfg*C.d8isrinr-n6trir D..,&rot[lr&0.lx6a-4tihtb.tt5o :totaa6etzlot h.l t('l A..lic Adt
O.l A-niLldd &|G
(di!$lvl C@!.d. ilErhel6.fti. 20.,6 Brbml
rddiq sr.. dd edc -i!*in Eq|nE llPrClt6l
!,$d..@IA-G!l
BDTA l86.la
N.olI GrFrLb
gro brt l litr.
l!a-@
OjM EDIA
A$.th- Xrni.t
l&*L l8illooatHp (lods/'nt)
srt for *vErl h. tdrtbodL)
lirlC@.
l0A8
(.na ddrolPtdld!)
[email protected] &DNA rDNAt-5 ng 5.S
tru.G!&idd
T.idB.- 5.&a
Clycie 2.93a
M.tlel ,oDl
sro dt l ltut
DNA EXIRACIION KII PROCEDURE rr..n.nt !\
r, Ercrre ihe go| Nrh. olt lnlnSDNA
2,
Run tlE DNA cffiining $lurion 6 a .gdo5. &l .long wnn . DNA
mkcu|f rcight bdta of rrom !iz. .t'd co|@!'idd a. |rming b!6d.
E .irb.r rx cMb&d T E 6!frd (,r0 DM Tris,..d.q InM Em-A pH
8.0) or lx @@E!iod TBE b{fia (89bl,l TrL-b6q lDM f,.DTA pg
8,0), AAq scl elelDpn@rir, lbin L\. r.l wit! cdridjm bbnjd.-
Esrimor. dE concmeadon ol DNA by oompeilg it8 ilbBity *irh tur of iDNA slldtaid of r 3imibr da od IrM mcatltim.
Excie dE DNA bed tw thc lilind .g!I@ gel with r lar bLdc. Cll B
nuh agrc di * L nd@tt !o Borcr tte p@dncr bdnd. To ninini..
d.uc! to DNA sqlc, w lons nE Uv liShl fd 4 snon drc 6 prctic.l.
D.i'mirc 4pbriflc h!@c of sll 3li* !y ftighins Os .q!d.
rpponmi.ly I t 1) rld lno r.EGr io i tL6tic bb. (do mt us sls *|s
Add 3 volm6 of Bhdbg olldor to I rhn. of g€l
The volEe or ad<led Birdine $lutior should be appDxihalely 3 rin6 th.
veisht of sFl slicq It is M.sry ro l@cp $. nnrl ooMDtltior o{ odiu
ro rgsF wi|n TBE r.d l4 sluDc 6f ABE .t)l|6i@ hrrr dd 4.5
rclmB of Bird;D8 $lutim !o ! Sjvm 6l6c or.g@e.
Pbce tn i]b€ h a 55'C wlEi blth Incubdl. dE t$. fc 5 ro l0 ninuros,
lorlenng the bbe bn€fly !v.ry 2-3 iiNicd duins iDcubalion utl !8loF
g.l 6nould be cmplecly dislrd, Il ir hB not dis6olrd bt 3-5 niruler, .dd
5.
L
lbout 20% Dft birdirg $lutior .rd EnlE th. tob. b E|!r bdh util gct i!
Wla DNA b rcl n .gre .nd . sllm of DNA 6.hinjng $htion is 16
lhan l00d w. tffis.nd to rdd enough TB bull€r o. wltcr to tho tub€ ro
bdng |!. alue to s toLl of 100$l be.e ir is M @voicot ro work win
tnc hlmc6 > 100d. Add I voluns ofbindiry slution !o th€ DNA slulim.
Mir ind go io lh. Hr 5cp vino ircub6l6n in*rt rbror
Add Sillcr Powdd Sup.trrrotr ..d !rc6.t lor 5 ntlut N !r s9c
R6$F!d 6. siliq porda flspdbn to pFdu@ . ho6og.ms
s$F$ion by vigons sixine. .
Aiid .h. dli6 porda s.pasior i! .dd.d r. tlF Flulion, nt dd pL&. in t55-C wal€r br& for 5 DituEs io itlow birdinS or DNA !o rhe silix turn,ruDg €e.ry l -2 ninu&c io lrep lilia Fw&r in .spdsjon,
Spltr Silic. Pffid*/DNA oDpt.r lor 5 s!d!. ReDov. ih. Sup.h.trnt
Spin ilie por&r willl rb€ bNd DNA. ta tu susFrsion ir h !nicreniitugc n$e, !pi, i! r nic@en[itug. fd rpprdxidr.ty 5 sonds.
T6sGr 1h. binding suFuerr bturion !o turh€r tub. dd src. Ille
rupmrbfi ciD b. r6ov.d vilh I pip.tte or pouEd @r, spin tb. tubc ,sair
ror 2-3 sonds !d @norc minirg bindi.s &ttrri@ wjlh pip€tE.
W..h DoI.t3 ilDer O Prprr.d W$n Bulld
B.foE uiD8 $is tit Dt evElh h!trd hy .ddins r5nl of Co!6rr.rted
w$hi4 Bulfer to 285r of distjllcd wior .Dd nix. Add 300 6l of 95y.
.lhrDl b...uc ln..bility ofersb buE6 ro t @ DNA on th. ritid Fqrd
dininish.s il the 6dl .mcar.tion of et@l is l6s ihd 50yo. lle p.€pxld
{sh bufcr s}ould b. BroEd al 20C bctr@ ug.
> Ad\.i 500d of i@ cold *!rn buf.r to th p.tl.t. R6u+.nd rlE Dctlct jr llE
w6n buf.r by rr€ins d pip.nine bDt ald tomi c dissnB irio p.trct
wih rb. piFrie ri9. vqt xilg d oue sn€in8 of r.4! DNA fir8mft
> After n ir €su{€n|.d stin fd 3-5 md! in 0E l:dif!g. .nd dj$atd ti.,uFmdnr Rep€cr $. wsh prs.nG dre or noF tjnes.
> Aner dE l.n rutL m@ mdr b{'Id uing . piF[6, Il n iqo'r&l io
.linimlr s nuh .&nol s n ! po$ible in ord.r ro ninini& E pGibiliry
of enzlm. iDhibnid itr nbsquq 6t p. Do et dty the F .t md* vllucsince d.rdryins Mr.. Fdue dE .o@r of DNA eov.Fd fDm dE sitjc!
6, Elote DNA bio W.t.r or T! Bdt r
> R6up.tld a.snod lilior powd.nDNA wirh TE butrr or v.rd by
votuii.s/or pipcftire. Ircubar. dc brb€ .r 55C fd 5 DiDr.s. Cjl.in4c fdlbout 30 6.coDds ud q$uly 60!. rhe suD.mtut @nrlining rh. olutcd
DNA Dd ple i. ! @ rrb..
> AMlya rh. obraincd DNA by eleltopnoEsh sd eslinrE bc cmeDtr.nd
by cdprins *iln a DNA sred.d of i rinitd siz .jd rdlM cdat lrjolP.ept.rior ol C.CL.Ht d cobp.r. ..[r
. lMulm h 5nl ofLB bolh al 3?C fo, 12 l$u! from $o ptrt st@tad for
DHs-F, JM 109, BL,2 t.
. rM N.CIICOO
. s00nl LB bbth in 2L IF!h.
. Dilu& 500n1lt wirh 5li i@utm (l:100).!d r..! {E nqh in lhikd !t 37C
fd.lDo.r 2 ho6iont ltc OD.dDd 0.454.55 rr 6{x)@.
' Now t€p n .t icc for at lo..t 20 ninut . ro g.i it @1. Now rll rh. flrori.ls to tued to( ndE Fo€ils rtdid h. @t,
r CotinrCe tl. h.di. i. fd6 rrbft .t 4t at 4OOO An for 20 oiruo* rikc dF
Fult.tn RdqFid n i! t|'i6d@ hf.r 0O - 20nr), $a dituE it ro 50OEt of
. lEub.t .lio.(49br45bi@r. 6{,dulE.
. Cdrif4! it !t 25OO rps .t 4 C fd lO nitur..
. Disly. lh. ,.ll.r in 4(td Eireri@ bufd m i@, Add 80e,4 glycdt in it io
r*!&.trn l c@. io l5%.
r M*. diqrcr.! of n (100!t d 50d) {rcad N on LB + LB - AMr *n. t5holld Dl gow d LB-AM?,
SDS- PAGE
L Pou Pol}loryl&iL CFl
o l|@bla g.l tudwicn
. wh wU ed ria (6!rl d 95% EOII) pt.i.q A.6, !trd @b,r lay 6et ptrte (qr dr pLL d !!r &6.!)t prr4 [email protected] borM d<t.i&s of t .k pbre
' pl@ top plrle o! iop of6?@ off..r !!6 bon o.d8!by I _2m
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. pon of.qlEu kru dbo.Fr.dig g.l lrd riM vith dlilt O
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REFERENCE
2.
3.
5.
1.
8.
db.d a, Clv.ll.to D, lonrie !, c dl. Antibody rEqot$ io ptes2 ardh.?rtitis B vitu intuc.d [vd ddrgc. L$@t 1988; 331: l42l - 1424.
alFd.n FZ. HcDaritis B iofe[o! ia S.!di Anbia Ad.b of Saudi Medicine1988;8i474480.Aldo K Guidotri t-G, Cdy 4 e, ol. A@6s to eliga r€sti.lt .tnobxio Tbmphocylc funciion in vivo. toumal of lnhaology 1994i 153:482.489Argendrt c, Ld soso, v' BMi, R., D1J8p, E., Guiseppctli, R & RaPketq M.Hepad@viru! .volutior ud molcdd 51raLgy of adlttaton D r ncw host,toldtd of G!!Ml vireloSy 199;80:61?-626,Asnbn-Rjctrrdl IG & M@y K Mdrna of thc letatitis B vitu surf@dtigen th|l d!fir. s@e &ligdic.Iy 6sti.l !6idu6 in dE i@uod@indt aresion. ,oMal of M€dical ViFlogy | 989q29: 196 203.Av€tlotr a & Ecbdria tM. Frquacy of h.patir; B viru 'a ddmimtvdiek in ucl{led Speisn cl@tric cdi6, Joual of M.dical Vitulosy2006:78(l):24-36,Bldrrdln K,{ & Dsmet VJ. Iiterdigihting ed dqdritic rcliculuD celh irchrcnic &tivc h@atilis. Hblop.tholoSy 19841 81657-66?.Do.rcft WH, Mudo4 !- K, & RBcll, P. K Detection oftddilional otigaicddmi@t' of h.palrli! B'ati8pr Joud of Lnmuology 1972J 09r 842_848.Dusjc K Gurt n RC, Bsnt R, Ssr 5K Kr@dctt l. Iddlin@dot of.mvel surf&c tuilnt of h€galitis B viG in a s@neetiv. chenic livd dis.e.atiat. Virus Rdm[ 1999:65: 103'109
10, Bmrb. v, Fmco A Albati A,.r ar S.ktiv€ kjllinS oI hePatiiis B dvelopedrigd-spdinc B cells by cls I rdtrict d, erog@ous dtigcd sPecinc Tbmphocy's. Natur 1990t 3451258-260.
11. Brrub. V, Ftuco A Balsuo AD?ni A. et dl, R4oelition of hcpatitis Bdvelopc prctcinr by livd-inflttating T lynphocles in chsnic ttBV infetior.touEl of lmsolosy 1989: 143:2650.26t5,
12. B€rLy R- H.latiris B vi$: tbe n4d .tioloey of h€r6lo@lluld c@iDomaCd@r 1988: 6l:1942-19t6
13. B.*Lt R'P, Hwss LY, Stev6 CE, d .l E6@y of h.garitis B imu.dobdi. lor prdmlio{ oflaiELl Fxsision of tE n patitb B eitu csiaslrle fral r.polr of a tudonird doublcblitrd pla.cbeconltoll.d tial.Il@dology 1993t3:135-141,
14. E rlollttl A, chh.n w, €r ai. Noh$l veimi3 ofcylotoxic epjtopcs @ [email protected] ets8pnkls for dtiviral cyloloric T ccls, Nane 1994;369:,107-410.
15. B.noudt A, CosraM A,Clisi Fv, er dl. Crtoroxic T ltmphocra. Hpouc to! wild-l}?c hcpatids B vitu epilop. in paliots cnmdcally infdtcd by edidlvim6 cqryi.g subltitutior withh th. cFtopc. ,om.l of Expsimdt l Medicine1994;180:931-943.
16- Bl.ckb.r8 J & KirlC-Ljugerd, K Oc.ult h.patiG B vitu and &!t slf-limil.d i.fetion paktiry for 30 FG wilholt seq@e veiatiotr. Jomal ofE€patoloBy 2000br 3l; 992-997.
| 7. Blekb.rg J, Beonid JH, Widcll A & Kidd-LjuaggM K Lrng- lm out@mc
of duc h;an ! B ed C h e ourbE* of h.p.titrs itr 1969-?2 Euop6 Joutulof Clinical Micrcbiolosy & Ilf@tios Disc$6 2000; l9r 2l'26.
18. BhD SE, SbwiDs I. Fisu CK a.l Dctlclioa of b.!.dtit B viN DNA ith@atoc!l6. bil. dud @'rhc[u &d ve'nddd6ts bv r uib [email protected];s! ol NdioDl'Aclddy of scicea UsA 198!: 80:6682{685.
19. Bhnn ry BS, AlGr EI, & Vieich S, A bcv etigltl in l.uldi.3@ Ioundof rh. An6cs M.diol Alsidi@ l965il9l:541 t,l6
20- E lvLy PL & Holn6 BC, R@otttuco!8 lh! @bPla 4olutiodry history ofh@|liii! B viru. Jouh.l ol Mol.qtlr Evolution 199; l9r 130-141.
21. E !i!o F, Rcid F, Riz.tio lvl .t al, C!rcnic h.giitb i! HbAg csias wil!s.tu HBV-DNA ald eti-Ht . G!.tldtaotogy 1986;90:1268-1273.
22. Bontulcb!.ro.b I' Gsrpui A & Motdld€.ryouti & GdotvPing ofTutid h.?atili. B viru isLl6 b!s!d d th. 3.qrdirg of F62 .rd S
Egioe. Misb6 .rd rif@ti6 2O00i21607412.23. Bost.r SM & Sin JC. R.l.tiotlliPc *ilhitr &d bdwa g6otvp6 of hcFtitit
I viD6 rr Doinls !.r@ tbc 8p!FE.: foolFi , of tEdbitr li@ ir cdt iti$ltt6, Jounrl oaca.ral Vmlory 2000i El : 379392
24 Lwy.r sM, ve Std6I. Ka MC & Sih lC. A siquc 3.gMt of tlch.D.ritjs B vitu rreup A 8@oqtc id.dificd in isl.td tom Soud AniaJouE l ofoaad violos| t991i1a, l1lyl729
2s. Bme SA & Mutrry K M!r!rjo6.f ldF ciiticd @im &id 6id@ in 6chqDltitir B vitu $dt@ dtiga Joun lnfMcdic.l viologv l99ti 46: l5?- | 61.
26. Bn!.tro M& Rodrig@ U.{ & Bdim F- HlDdilit B vitu nur{t'Irt ditulogy l99i 42: 69-80.
27, Bn!.t!o U, Stdld M. Shodcl r, ., al ld6ti6c.lio of HBv veinr! whichcdnot pode. proE{divcd ltBa{t ud d.y b. rdpodiblc for s€[email protected]. Ih.Itdie JoM.l of G.!n!q @logy l9E9;2lil5lJ.
2E. Crbnrbo M. BdtoloD t, C|mclo C. Buil C & Crmo V. MoldDlas.ly|is of bQ.dlis S viB DNA i. 5tu .!d pqiphdl blood homulcs@ll! t@ h@drds B suf&. mtig6nc8.iiv. c5g fllg.lologv 2000; 32: I lG123.
29. C.brerla M, B.nolon. t. Crlafulo C, Bdil G & C|'mo V Mol-drAn.lrsb of H@atiti! B Viru DNA i! SaW e.l PaiPtd.l Bl@d Mon6!cl{Cclb Frem Lt plritrs B Swfa AntiS+N.gatiE C.g H?llolory20&i32:116121,
30, Crtu|l Wr & IXoM ttc, G6.!cC8tro.nt@logy 1992; 102: 71 l-719.
I l. CrtDrn wF, Fa8rn EA. Hadziyetu S, a al Assialiotr of t PE{oE gdoBicldidt ofh@atiti! B vie! with tulEinet lEp.tilb, H.?rrology t99li l4i 219'222.
l2.C$.u wF, K@1. J, Wdla@ I" MePha R, Mims L & Dekd RFulli@t E*riv.1ion of h.pritis B d@ io 6v.lo!. Pret it mulst oal eaP.dd.letio. by Don@lontl HB.Ag ELISA, ls.r 195; 345:14061'O?
31. c.run WB ThoDs HC & Dmingo E. vi6l g.i.tic adiarior: h.P.iiltu Bvidr $ a cutrcd ddplc L@d l93i 341 :349-353
14. c.t@r wf, Tdtwch C, v& Dc||sr Fr, ColDh K McxintyE G, Moll6 &.r ur Hcpltitb B viM 6v.loF vei.lid .nd t&4LtLtid with dd withoulh.?.riit B ion@ globdi! Flphylub. Hqorolo$, 1996; 24: 4E9493
35. C$or wn ve Dc!'g FJ, Mi'Ds LT, H!di. D, CAPoh R. Dcta R &sod@ R. 'ft. rtud.@ of tue.. .di86 v{i&L of h4.rid6 B viN itP.pu Nq Glin + Sourh Atrie &d S..di!ia H?lrolory l99?i 26:1658- 1666
36. c.t!0 \rB Z$.tti A& I<didis P, .r 4l v@iGilduccd s.p. Dul.ltofb.?.tili! B viM. Lxcs 195; 336:325-329.
37. C.|!! lvF. zdFtti A& Koyi@i! P, w.td J, Muillo G, T@i E .l zr.Veioe ilduccd .s.qc 6utdt ofb.p.tilir B viru. r!4t 1990; 335: 325-329
3E. c|rEd wF. Th. clilicrl siific.e of sutuG .disa v.ti.tl3 of h?!tiri3 Bvitu. Jourd of vir.l Hqcliti! l9?; (Suppl. | ):l | -20
19. Cdl E, O! D & Oos L R@8nition of!.tritit B 3ufr4 4ligd by hufu TlyEDhsyla: Pblif.dirc .rd cytoloxic tBpds to a mjor [email protected] d.6n.d by syDthctic D<tdda Jound of lDr@lo8y r9EE;
l40:1808n815.rO. Ccr{d C, M.riin Y, Bnily H, et d.l. ILI .lpb8 tu Prcdeld by T l}bPheyla
&tiva&d vi! llF CD2 pb, CD2E prlwry!. Joutd of lrMeloa/ l9l;146:56G564,
rl.C.ny A MeHutchjs JG, P.!qui@[i C,.r a/. C]totoxic T lynPh@}{.Gpoe to h?lriris Viruddiv.d pepddcs @!tai ng dtc HLA A2.l biDdingnotit ,oMal ofclidctl hv.$g!ro l95i 95:tl53o
a2. Ctrt I|K & Ting LP, Th. ed-. goc poFot i &il dF hlIM h.t titi! Svitu di6platr . pFfda@ for diffGri.Ld h.P.i4ytd .Vitolo&' | 989J ?0:176183.
43. CL6 DS, Fbn [email protected]! to h.p.tdu: lels 6w lylc B vinl blP.lilitSci@. 193: 262:361370.
44. cb6 r|E, Fds D>q U FQ, Ii!8 BY, rs wG & U SQ. A rcvd h?ditt Bvitu 6uor vith A-lo-G .t !651 i! lh. &rf*. etig@ 86c, World trotrDd ofO!,lrcanqrlos| 2003i 9(2):30+308.
45. Cl.! gS, Ka MC, ttd$etlc wE, a dl ltc Pt!@r. 8rn. of tlt! sood+tuclh.!!dri, viG g6on. is not 6tani.l for vird r.Plicdim i! tb! !.tnl tosls.Joum.lof virolory 1992; 66:5682-5684.
46. Cn.! *N, Oo. CJ & Koh S. Hqizo .l tr&sisio of t tlt.riit B virussrhcc sliA6 muLtrr loud ofclilicd Mi@biololy 2000; 38: 938-919
47. Cnb! EL, lE TS, K@ ,, M.u YC & Ho MS, Altald otiSsii.ity of'.'&t.ditut vaisl! of h.p.titir B eiru. Ioutr! of G66d violo8y 1997;18:2639-2645.
48. Chb.n FV & FGdi C. IEnwbiologt {d Pa$o866i3 of vird hc?.litb, In:Nalh6$n \.t al (.d!). viril Paoosadi5. Nca Yo*: Po!4 PE, 195. I!
49. Chlr.rl Fv, F.rui C & Mond.li MU. filDrliti. B vitu sLlcnF od biolory,Mimb. Prrholo8/ 1989i 6:31 l-325.
50. Ctt .l lV, Filippi P, Bw l, .r dl, StEtuEl &d P.lholo8ici .tr*t8 ofsyrlh6i! of h.p.litir B viu hrgc 6vclop. polyP.ptilc in tisSdic niePd..ding! of Nation l Acaddy of S.idB USA l9E7; 84:6909-6913.
51, Cib.n FV, He?atitis B viu biology ed PAthog@4is I F.dD.!rT,€4Moleuld Go.ti. M€dicin€. Sd Di.go, CAr Acsddic, 1992:67_ 104
52. Clrrk EA, rrdb.tlcr ,A, SoUy Rq .l ,r PolF.pti.t€s on hulM B lyEphocrldeial.d c.ll r.tiElio.. lll|l@ Imuolory 1986; 1610G105.
53. [email protected] MP, lEou-Roels c & PNtij wP, vdilc dd h@alitir Bimwelobutin-induccd sale tuIrd@ of n€ldilis B viu sufu etiSaJourl of Biomcdic.l Sci@ 2001; E:231-247,
54, corn.ctsPrty alv, rhrd A a Soulid JP. Dshibuti@ ofH&Ag lubg?sin rhe world. Vox Sdguinis 1983; 44i 197-21 l.
55. DDe DS, C6.on CH & Bricig! M, vitu$likc panicl€s in sru of Patidtswith Astr lia-dtig@-swiated hcFtiii3. tsc€t 1970i 1:695-698
56. Drvis IIL, Michcl ML & Whalm RC, DNA-ba&d imuiali@ idm*@nthbE scdd of h@atitk s&.. etieo &d hid ld.b of ciEutalngamhody. Hlll@ Molmlr CadiG l99l; 2: | 847- l85l
5?. n jo A Lu8Eisy C, aisi S, d dr. D.r61i@ ofLetadtis B vitu DNA inp.Jl.ls, lidr.y sd stin of rwo h!ru c5i6 of thc viru. JoMal of G€'@lvirlogy l9E4i 65: 651'655,
58. Di.n6 HP. Hlci@th T. H* C & M@d SC, IrnFE@latbn micbs@picobrdaliois oi th. infl.lr1miory inlilFalcs md HLA stigo! in h@6tilir B ednon-A" non-B, Hcp.toloCJ 1987; ?r1l l7-1325,
59. Di.mtrg JL, Wcrcr BG,lo& BF,., a/, Sy I6i! in dimd.clk of h.Patitis Bvein io h.alth .& [email protected]: Saf.ty, i@@Coicit , dd ildic.loB ofc66t. Amtls oa irhal Mcdi@ 1984: | 0l :l1go
60- Ebla BE, Ling@pr VR & G.lm D. H?.ritis B surfle dliAd: An uusutlsdeted pot.in initially 3ydL6iz.d $ a !&!btub@ pob'pcptid. Mol4uleCell Biolod 1986-6;145+1461.
61. Eqsilr Hi, Houihofi IH. Huirh. S. .r a/ Cclldd dd hDoral Umu.e;tiotu in chronic etivc lva dier!.i I Lvmphoctl substs in livs bioD6ies
of patid! wirh bheated idroparhic auioiEhde h.palitis. chslic elivel€patitis B dd piruy bilialy cirho.L, Cliucd ExPqimdial InsNologvl9a2t 50111-24.
62- Ero AA & Lrtd@ wT. Epitcniolog of hcpaflis B, P. h A t. zn&M &H. C. Tt@a (cd), vir.l h.p.lilis. H@urt Bl@ & Co. Lrd.'l,ndo4 UnilcdKinldm.l9E; l0 14
63. Ftug JWS, Tug !YT, Davis GI" d, dl. Fibrcsi4 chol6rdic hc4atiis iD !t@s?lml rccipicni with hepaiiI; B viE Pre{oE nurut Gstrenttulogv1993;105r901-904.
64. F.ttovtch G, Rugg. M, Brcllo L, .t al Clinic6l, vimlosic ed histologic outcon€following sdconv@ion non HBeA8 lo dti lIBe ir chrcnic b.P.titis t}!c B
'{eo obg! | 986i 2. | 67 - 17 2.
65. F.hri c. Pilli M, Pdfu A" ., al Allops4tdion of h€palitn B viN mv'loFutis$ by T c.lb. Joudl ofvirclog! 1992i 66:25tG?540
66.Itodgn! E, Bo8lsen S, Xnutsen M, Sftbkova Ea' Kidd All, Alqcrq OA& Kid,l-Lju8grcn K Reat high incid@. of tulbi@l t?!titi! in Sard€"Rusia: nol*uld aoaD5is ofldailing h.latiii6 B md D viru srric tomal olclinical Micrcbiology 2000; 38: 331 I '3316.
6?. Ftuco A, Pmli M, T61a U, d .1. T@fFin E@to. n€dialcs upl*e ddprmlati@ olhQatitis B dvclop. 61i8d by T lyhph@,r6 J Erp M.d 192;175:1195-1205.
6S. rrji E, Modyet Iq Srrdolo N, Kondo T, YGud. IC Hiaizmi Y, €r al Glvl4j b Ar8 substitltion i! H3! sriSpr of imunc sapc mdrni of nelatilis Bviru. Bi@harcal dd Biophysical R.@h Comuicaiion 192J84:1152-1157.
69. Gd.n D & Vms HE The moleuld biolo$, of thc hepalitis B vitu6,A$ul R.viw ofBiochmidFy 1987; 56,651-693
70. Goem D. Poll&k IR & lavir .1. HoDAriiis B Viru ro6e tr&$npt4 aid ilxmey roles in hepadn*al g.nonic rcllic.iion. Infdtious agflts edDi&ae.1994; 3: 85-93,
?1. Gt.!y MG. Arlla B, Vrllml FG. .t dl S gcn e*aPe nubl e a cak olHBV reinfecrio! in Dalidls who @dv.d HBlg posl-OLT ll€patologv 1995;
221150-151.?2. Gibs A wa&ins E & Dimt!8 JL. t trg-im FEistcncc ofprerdtive stibodv
and h@atitis B v$cjnation of h.altnv adulls. In zwk@ai. AJ, ed. vitlHelatilis &d Uvd Dis.s. Nd Yott: Lis!, 1988: 998-100r '
71. G 6 PN, F.y G & Chisi FV, TMor ndsis &.loFalPh. ncgativelv regulates
n patitis B vitu gene .xp6ion id F&sgmic ni@. , Vircl t92; 66:3955_3960.
?4- Girc!6 R & Mild Rg. Mutatjon 6r. of thc b.padtuviru amome Virlory1989: r?0.595 597.
7t. GoEk P. HmoEl lmwlog.d r*Po@ in labo@rv &imjs .fterydination with laDbiMt r@b6 .gtinn viEl h.?atitis B of drff.Mts@eEri6. ?4gl Epiddiology 2007i61 (2):393-400.
?6. G..tI€ S. Monazahid M. Bohn. I & ThoM R- Ch@tdizalioD of uusuileape Eidrs of hepatitis B vitu ielrt d &oD I h€p.litis B sff@ eligo-negarive subj@r. toumal of Vnolry )998i12: 7692-7696
77. Grcb PJ, Bitw&ser U, Z@ba K d al, Lnnuog€triciiy of a helatitis Bsu6Mir v&cinc iD hdodial'sis md in dal ir.lsplul Mipiots. ArlividR.ffi! 1983t3:43-52.
78, Guidoni LG, Ando K Hobbs MV, a al. Ct'ioioxic T lynphocltcs inhjbithe!6titis B virus g.n€ qp@sior by a .onct'lollic nchaNb in tErEgoicmice. ?rcc Nad Acad sci USA 1994i91t2764-3768
?9. culrber s. lischer L Pult I, Srcdcck M & Will H Nah!4lly o4Eitg ldielsofl€Datitis B vitut, Advm..d ViG R.s@h 1999i 52: 25-l 17
80. Cupl!! RC, Tlaknr v, sdin sK 4 ol lEqudcy ed did€al pofile of FeoFdd surfee b@aii1i3 B Dula s in tuim-lldim P.ti€n$ wilh chrdc liverdisde. Amdicm Jounul of Gslromldlogy 1996; 9l: l3 t2- 1317
3l.Ildler SC, lrMcis DP. Mayndd IE, rt 4i. lrngrem imuogcnicily mdetrc&y of lEpalitis B yucine in hon$.xud hd Nry Engldd touel ofM€dicire 1986; 315:209-214.
82. Edpem MS, MMr WS, Co.i6 L, O'CM.ll A! & ErSldd JM Hboralimue lesposivods in duct n.!.liiis B viru6-infdtcd dwk JoMal ofViology. 1987i 6l:91G920.
83. fl.nnonn C, Horal P & Lindi M. l,ng&@ nutalion 6td in tlF hepatitis B
riN gaomc- Jomal of Gffil viDlo$/ 20004 8l : 75-31
s4. B$i;m C, Nordd H & l,idh M. An alqtut gp.or)T E sdtcd itr
@nbinsi h@atitis B viM slr.i!! fion Vi.hd Jouml of Cflqal ViroloCv
2C{[D:a1.22612272,85. r..iM Tr, Hop6 EA Oo! c,t, e, al hd@addt @gm@ or ' vdine
ir.tu.d .s@. oa n@atitis B vis. Joutul of Hcpaiologv l99l i l3(snppl):105_
10?.
86. tl.tgE. K HMg J, wmd! ,. .l al Als'atim of h'parit'3 B viral pro(mul.;ts q$ tuhn;t hcDatti3 iD Jap{ Virlo6/ l9e|: I85:tl6M6l
87. Urwkrh AE, Cilsn RC,cilbcnN wFghft rO.Gnt X Anl6neBosI Pr
,1. tt@atirir B suf&e viru nulrtios s*iat d with inf-tion 'nd lvdE&$DG' stion. tom.t ofs@rlologv- 1996; 24:8_14
8s H. J\v, rx Q, au QR Dlg SC & wd YV Mudio6 in th' a derditdr or
hdditis B surt&e dDsen dong Chin6. rtunl6 t'c€iving acuv' poslcxposs
h6,liLi5B imuizliotr vd'nc 1998; 16 I?Gl7lSe El$r[ R . vd B..gd P. vs Ro(M! MH" SM@ cNL Pdl'i lvP' Schalm
iW'a O*crt'u* eO-e"ti_tgs aRtr hcPdltrs B immiztion with plem-;dived dd rccombi@t DNA-ddiv.d v&cin6: bindins to nutet HBsAs
vcrfue200r, 19 16?l-1680.SO. tlohn" H- S.i.*f.. S, S.'f- M, a dl Maligrut t'&sfomdion of imorLliz'd_ -
rt-rg*i" lt p"t*''1". uno trdsf€cdon with t?ditis B viru6 DNA Thc EMBO . ,
JoMal 1990: 9: I 137_ l 145.
s r 'f,-*iooL ti A olei*.sli. AM Tt. !6trd of chrcnic vital h€gatiris NoEDEhtJomal of M.drcib. l99?; ll6:347_J56
q.z. n"",il. *-* z, Chds j. lin Y I.u CKL Sr I zhou F \ ates J- [-f*it p] a L* r' p'*d.M or ncuEtlv ccunirs sd&e s6' veidrs
"r-r'i.tJ s 'i; i' *"i-"",t"d surr" ddsm€gariv' chi!6' 6i6'
Hsltolow 20ol: 14:1027_lol4sr ii;, mtrltbfl os. ct"-c cH, Lu cJ. J@ DM. ke cc 't 4/ Fmc&v or a". ;-.;;' n ;i""!.; pos'- in r'iwd roumd of Armcm M'd'cal
A$ociation 1988i 260:2231_2235sa. i;'io. v",ii,'* JM & Siddiqli A TrtuF(ri\'don of HL A-DR 8oe b)
i-","i e "* i e-. p..d* [email protected] or Ndiontl Acad@v or scideUSA 1990i 8?:?1'l&714'r'
ss go rO. yu CH a Vl*l,ne JM UPregrlaliob of rn@elluld adhsion nol4l€_' t r""i"prion ty ttc'a Ulg vr; x prckin Pm4'drnss ofNational Acadmv
olScietc,USA l99i 89:11441_ 11'145.
so. ii-i" C, si".n.n JP. tlsjo K & wiltis SRc Jr' lrnm@histoch@ic'l
.r.mirea-uon ot rt.pa,c l-i$G lvnphocye subpopuldons in li\'r dik6eScadeviu Jolmt ;f Gstro4l@loev 1982: 17:855_860
si. i;ii .";"s@l' - 'l'" "".ludion ;i c@inos@ic ndc ro hms (leer)
.d.VirEl Ho$itis Volme 59:4t-165qa ii"' i. io"i r, r"do" Rs. wrllims R & Nrc@ov Nv Nov'l imuo$av ror-
iiL a.*tion ofi'-",iti" e .utf-c sloc nul'ni! dtd its aPplication in livsbfl-lbl eDim; toutrd of M€dical Virelogv 2001i 63:2lF2l6
L J..!-Ja! O, Weimd T, D.R@ondo AM, rr dl hadal dt y ofnbo$mc ddribosonal s@in! involved in h@atitis B viru P gde exp63ion. Jouhal ofVinloSy 1989j 63: 545l'5454.
100, Jh Y, Shin \vK & Bstffi L Hl|m T @ll 6po@ ro the suf@ dtia@ olhepatili! B viru (HbdA8): Endo$a.l dd nona.loeBal pec$bg PAthwatrde &c.sible Io bolh ddoepnous ed exoSpnous dtigd. Jomal ofl"wrindlal Medi.inc 1988: 168; 293.306.
lol, Jdkr-Nipm! M, Barr@blagd R & Scnd|d HA Snorr cie&tirgsequocc i! r.quir.d fo. ncparitis B virus Pr.gmm. dcopsilation dd sffi.idlfor pekaging of foEiED RNA. The EMBO ,ouFal 1990; 9:3389_3396
102. lGlirb. T, Iwski A, Will H & Slemek M. Defci@y iD viriot ecElion &d.t€cEs.d stability of rh. hQ.rilis B viru i@@ eapc nutllt Gl45R,H@aiology, 2003; 38: l2?+1281 ,
lO3, rre MA Global st tus ofn.ritilb B imuizalion. Ldcet 1996;348:696lO4. xn.lo SMR- ttdt togoeily of th. @E gd. 3.qu6@ ir a p.timl cbrcnic.llv
ideled wirh hepstiis B viru. ,olEal of Inf€tiou dj5s6 t98q 160: 903_904.
105. Kro JII, Ch6 PJ, tli MY & C[o DS. HePstilis B gaoiyP6 coftlatc withclinical oulcones in palimls with chrnic [email protected] B GasLodttuloSv 2000;
118:55+559.106, Ko Jll, wu NH, Chd PJ, Lri MY & Chcn DS H.?atiiis B 8piot)!6 and thc
!€s!o6c io ini.rfmn theEly. ,ouDri ofHo?enologl, 2000; 33: 998_ 1001
lO7. Krrtnl!€lu vD, Allis. LM, Fdgr$on M & HNdd CR. A hcpalitis B vituEvdanr r6md i. th. sda of imuiz€d childm induc.c t confodalioml chdgein d. HbsAg'a d.l@iEt. JoMd of Mcdicd virclogv 199; 58: 3'16352
lo8. K3rdrls6! vtt, ,Allien LMC, lonujr M, Mddy M, wlifil. HC & Howtrd CR
A novei hepatilis B viru vdi6l in th€ *ra or imunised chjldro JoUrul ofGa.6l Vilology 1994; ?5:'143-448.
lO9, XdLov WN & Distag JL ?p.Ddo ed thmlv of viral h@alilis Sdina on
trvdDls66 l99lr ll165l?4,l l0. Krwrhoto s. Ydoolo S, Uc.L K d dl Ttudadon of h@aiitb B virus DNA
polymqs. fron rhc intmal AUG codon, rct noB th. uFlre!,n AUC @do forthe oE potein. Bi@hdicd and Biothtsic.l R@th comuicltid 1990;
1?l:IIl0-l136.lll, Kfoury Bd EM, zhag J, M@uot K vm Le A & Pebtun DL,
Chet.nzarion of . novel nllalitis B viG nutdtr d@mstrdtion of mutdion-indlced hc?rritis B yitu surf@ dieo SrDup sDcific '.' dltmi@r@ntonation chese dd its t{Plicaiior in diagnslic 66.15 TtustusionMedicinc 2001j I lr 355-362
I12. Kidd-Ljuggr€! K & Sinonsdl o. R.4F.ldc of hepltitk B 10 j€d andLid.ey l!&splaltation. N@ E glald iounsl ofM€di.ilc 1999; 341: l2?l2E
ll3. Kid.l'Ljuldgrer K Eldn BG, walm'* E, €t dl Re&tivaiion orbelatiiis Bviros inGction with o uusu.l Dand of smloejcal b&kds Scdde.vidInf€tiou. DGe.g 192; 241657-660.
I 14. Kidd-LjulggE! K Mit*aw. Y & Krdd AH. Gaaic v&ialilitv in Eepatins BviruB.Jomal of G.neral Viblogy 2002i 83:1267_1280.
ll5. Kidd-LjrnggM K ObqC; M & Kidd AH. nF h.patitit B vitu X gae:snal'si! of tutrdional donain 6d gd. phyloeeny usilg nultiPle s€quscBJomd of G.n@l Vimlogy 195i 16:2119-2130.
l16, Koik K Modra K Yeh g, er al, Hid ld.l €4caio! ofn€Patitis B viN I{Bxsae 4d hcpal@eircg€cis in tresgEdic Dc.. H.Patoloey I994; 19:810-819.
ll?, Ko.br BE" Core PJ, wels Fv, .t.l N.turll hislory of wodchuck h@alilis viruidetions duing ih. @UF of dpdincotd vnd inf*iid : Mol*uld vnobgicferlE of the livd dd lymghoid risG. Jouql oa viroloev 1989: 63: l36Gt370,
llE. Kor.k. y, Takai K, Kojim! M, et,/. lulniml h@atilis E: ind@lion bvh€palitis B lids mutets d.fcclive in t!€ pFcorc rc8ron and incapable ofdcoding. dtiSpn- Grltrmrftlo$, l99l;100:1087-1oql-
ll9, Kotrp RA. Virus c&apc fom CTL F@gDition. Jollml of Ex?dimaralMedi.irc | 994; | 80:1 1 9 -i 42,
t2o, Kovu.rl T. Naknuta M, sikat H, Susimolo R Mu4hrka E. Kob LN;od H. l(u|]rgwa | , Mutdde M & Nawar. H AralvsB ol HBs etig6rc€tivc \eMl of hcD&hs B viru: ujqu. sbsriotioB. Glu!29 L Asp and
Clila5 b Alr,n $. s;tee &r'e.n sme Mcd SciMontr2oooi6 llos l169l2l. Knlntr S & David,on M. g€Datitb E @cit: PrcAe6l5 for duotio! or
imuiry, I1E Y.l. Jounal ofBiolog]' McdiciE 60:313-338-
122. Lrrfo.d RE. Chsvcz D. Rrco-H6e R & Mootoi.k A H@ad!.viN infcctioD ircaDU\e crbbo$ Jounal ol Vibloqv 200q 74: 2q55 2959
1,21. L; Bouvhr C. Thc htusddtv ol Ausral'. &ligd .romal ol bfatioaDisea$ l97r; 123: 671-671.
124. tr wM, g@atilis B viru infetim Nry Engldd toumal of M'dicinc 1997;
111:r13t-1745,125. lt!€o. C & Blmb68 BS Ad.litional speiflcitid of Auttzlia etig€tr dd ln'
msibl. iddtlfi@lion ofLeDatitit ffiic6. Nat@ 1969; 22t: 195-196'
t26 Lit!! fJ. hMqawa K Ribe N, el .1 .4 h€ldi$s B virus nubldsiskdwi$; oidhi;of ftlmsdr heDadhs. N EnelJ M.d l99li J24:1705_1709
I 27. Lir zM, YaGulshi !I, Daitoku lv! Hdadt & Nats R' Fnkuda lvl' KoEE M &Yeo M. H@alilis B vitu of 8@otyPc C p6isto@ !ftd @ovcry &m @tcheDalilis B vitu inf@lior in tapm. [email protected] 2003i 25: 2rl4'253
128. Li3l.y PS, Cl&k EA & Irdb.tler JA T .eU mtiSd CD 28 nediatq adhcsion
vi$ Ii c.lis by inr@cting with &livstio! artigm BzBB'l Pceedilss ofNarional Acaddy of S.itu6 USA 1990; E7:5011-5034'
129. Lot ASF, A!@; U & Crtena S Mulatio6 itr lhe F._@r te8ion ofb'pditis Bvins sd. !o enndc€ the sltbility of dc ado {v slru€tue of tne peg'none
6c4si&don signal Prceding! of N.tionrl Acld.Fv of Scim6 USA 1994;
9l:4077-4081.l3O, MlcDon.ld DM' Hotnes EC. Llwis JC & Simonds P Det6tior ofhePttilis B
virus inf6ton i; wiE-bom chinpafu6 (!s toslodt'tes vcrut: phvloSenetic
El,tionships sith hume ud otlE prin6t€ 8dD9€s. Joual of Virclo4/ 2000;
14: 42534251.l3l, Mucinl M, Davis HL, Hadchouel M & TioUais P Anli-Itcpalitis B Imu€
Repon* Induc€d nmush G4.1ic ln@ularion in vivo in nbsAg PFducing
Ttugdic Micc (a!sE). Pdis, Ftuce, O.iobd 3-6, 1994, Mol*ul{ Biolosy ofE patitis B Virus, 1995-
I32, Mrrlor PL, Olhirc Ls, RcF.ry DC, Scull.rd GH, & RobiNn ws. A virus inB.cchey srcud squircls l,hd G rcl.l.d 10 h?.tiih B viru of hllms.r@adings ofN.rroal Acd@y of Sci.ns USA 1980; 77:2941-2945
r33. Mrn.l P, Hugo P, Mcco@k J & Karpl4 JW Dsrh dd T ceU
ImnbologyRcvicq 1993; l33rl19129.134. Me! WS, Scil G & Smm6 J. Viru of P€kin ducts wirh st@t@l ad
biological 6lal.dn.s b nMd h4atitis B viru. rodfl of virclory 1980i 36:
829-836.135. Mclbtosn ED, Cirey & AE8 SS, Cod@ AM, Bug* M & Css.n YE.
Molould epidcoiolog ed vsiatiotr of h.pditG B in @at idi8rdt fmili.sb ABbaIa. Iouml of M.di.dl Virclosy, 1998; 56: 10-17.
116. M.!dl E, Krrbn Mr, Arls FY, .r al g.pariris B viru sutf&. etiSd bi ls tolpolipopmr.in H..towral of viDW! 1994: 6a.ur5-2424
I3?. Mlctcl ML, ?o iss l, Sobczatk E, er dt Si6th6i6 in {imal c.lls of hep.tilisB 6ufee dtigcn p4nicl6 cdyinS . Fc?tor for polvnqired hume 8clm.tbmin. [email protected] of Natiolal Aclddy of S.i.tu6 USA 1984i 81 :7708'
17t2.138. M0l.t RH & Robiner WS. Comon eluli@trv ongin of !€9tili! B viru
ed F1rovirus. P@edings of Nati@l Acad@y of S.ienc* USA 1986;
83:2531'2535,139. MtU.. RIi. Compelive moleular biology of the h.p.litis wirus S€nid on
LivaDs6 l99l: 11:l l3-120,t,10. Mlllct RIl. HMd immod.ficitry viu tuv 4code d novel Pbtcin on thc
emomrc DNA Dlus stud. Scro..l988i 219:141G1422IlL i4lB c & wirh. Do. vnd PdnoEFn5i. tnd lrMuologv orfod Ensl.nd
Bl4kw€lL 19841 120-153.142- Mhui i4 Possin K Ka M, et ai. ?eddc@ DuLtio6 of ll.?ati(is B viu
h heD.tocelluld .@irom6 .lcvcloped in nd{irlDtic liY@ c6tromhlogy1996i ll l:661-6?0.
r43. Mlrlo$ky E, GeBldd K CldaB MI-immuogfiidly of ye55t l@nbinnt le.titis52 + S mtieds, Vuinc l9l i 9:346350
ar al. oDPdaliv. s.fety &dB v@ir6 @ ainitS s dd PF
& Gojobori T. coBtain dviN. Jomal oa Molsul{
l:t4, Mlzoktni M. Orito E, Ohba K n@ K k! JYdlohtion wilh !.s?@t to em. ovalaP ofh.talitis BEvolution 1997: 44. S83-S90,
145. Mollcr RIt- CIo$ €vobrior.ry Ehr€dn* ofthc hep.nris B viru6 dd muinlcul(dia virus polFeEe gdc t quoc.s violoa/ 1988; 164:147'l tt'
146. Motrr.tro L, A;odilel r, Boinl M. cr al An dalvsis ofthe c@potibon ofthrinllmalory infiltrat. in autoimue dd h.palitis B viGinduc€d chonic livddis.&. g?p.lology l98li 3:292-296-
t47. Monyur K Niajim E, Hobjoh H, ,, 4l lbneoslsr€d h@atrris B viMmuiel- Iacet l99l; 337:125.
148 Moroar v. Pis@a M & Grendinin M Honolosous Eobbinalion betwddilt Etrl g6ott!$ ofh.patitG B vitu. Goe 2000; 260: 55 55-
149. N&tu! E, Sch&fd S, YGhida CF, G$re AM, R@p R & Cslich WIl.ldalifrqtion ol a Dq l@atitis B vitu! (HBV) gdog?e fon E@il drtexp&$6 HBv sdfec dtisd slbq"l adw4, Jouoal of Gadl Virclog 1993;14:1621-1632.
150. Nmri A& K@t SA. $ricl N & P.Itd L ldalfctlion.nd cb@icalsltrh6i! of a h6t @ll @@tor binding $t€ on hqatitis B virus. Cell 1986;46.429476,
l5l. Ni F, Fsg DX, Go R, Li Z, Dud S & Xu Z. A na imm. eare tutdt ofh@adtis B vitu with d Asp io Al' $btitution i! 41,14 of th. dwlop. o4orDrctein, Rceeh Vilology 195; 146: 19?40?.
I52. Nord.r H, Courcucc AM & Magntu LO. CodPLt C€nom.s, phylogaeticEhi.dn6, dd srllroel Dbr.i6 of sn !F.ic of th. h@atiti! B viN, fou ofwnich EptMt two nd gtuq?6. Violog/ r94; 198: ,189 501.
153, No.d€r I|, Corcucc AM & Maglius t O, Cmplelc nu€l6tid. s.quoc. of 3nhep.litis B vinl 8.ton€s dcoding the surfa€e dtigm $bl}?6 a)av4q-, eddq- sd th.ir lhyloSddjc clsidstion. AEnivB of virelog/ SupPlm4ll93a;E:189-19.
154, Nord€r lt, Cowuc. AM & Magnius LO, Mol@dd b8i! ofh@atilis B virus
smt". vdialiotrs wiibin fi. fou Eajor subt !6 Jouful of Gmeral Virclogvl92L 1i: il4r-3145-
r55, No .r B, Eb.rr JW, Field. HA Muhdrwd IK & Magniu LO ConpLrcsqudcing of a gibbon h€patitis B viss lenon. r€veals a uDque setot}!€disrsrly elaled to lh. cnimpMa neprlitb B vids virelogv 19 i2rE:214223_
156, No.d€r H, tlamd A & MaEIiu t O. Subt?ilg of HBv al thc g@Dic I*€lby siteiir@ted mplificalion. I! Vi6l HcPaiilis ed Livd Discbe l99l; 195-
. 199.
157. Nordd H, Hamd B, l- SD, BiL K C,@e. Alvl, Mush,hvd IK AMaeniB Lo. Gdctic Flalcdtr$ of tFpatitis B vilal slnic of div6eCBogaphical ongin dd tutur61 vsialions in thc pnidv shuclure of ihe surf&eutigm. Jomal ofcaad Virelogf l93bi ?4: 1341_13"3
158, Oit@uyler SO, Mddd MN, Oycdcl. OL Oi. Sq Odaibo GN, Olal?v. DO &Mulq aP. Phylog@cli€ ealysis of new he?atiii6 B virus ielatF tun NisqiaNppoft mddicny of 8molyp. E il Wed Anic& Jo@al of Mcdical VircloCv200r;65:,163-469,
Lt9. Ost N.M'lle RH.lsn* KC& Pw.[ RH TlE comPl€G nuclotid. scqudeoi pE{oE mrret ofheprrirts B \iru idphcar.d
'n tuLninmr hepaliti! sd rrs
biological chdctrizalior h chinp@d6. Vitulogy 193; l94r 263-276
160. okmto lI, lrnaj M, x@adi M, Nsj@@ T & Mat@i M Goodchetengdcity of h.?aritis B vitu iD a 5+)@-dd wom {ho ortractcd lbcinfc.i;n lboueh nalmcfel ar tnnsmi66ior' Japdce Iomll of ExperimdtalMedicin. 198?: 57r 231-236.
161. Otrnoo {. Imai M. Kdct !i M, N&@m T & Mavumi M G@[email protected] of h.parids B viN i! a 54y@{ld wohd who @ntr.ccd the
i ati;n thtouel datcmr&r ai ddsnilsion Japdc* JoMal ol Expcrincnial
Medicin 19874 57:231 236.
162, OkrDoto II, IJnd M, Mit€laM Y & MrtMi M. SilediFccd mulag@esis ofbdpatitis B surf&. e1i8d sequd@ .t co.lon 160 Aon dgininc io llEine Ioronv@ion of 3ubtypi. de{tui@t tlm r 1o w. Biochdiel dd BiophysicalR6@h Cmm@ic5l6 1987b: l4E: 500-501,
163. OLrNto E, ODi S, Warg Y, ftot Y, T$d. F, Todo T, Al.h!. Y, MiFtar!Y & Matui M. Tt lN ol rubrFic d.t @inds b .]1.16, dy or w/r, @h.?atitG B saflc &dga Moldtlar lom@logy r98q 26: 197-205.
164. Otonroto E, Tsudr F, &tuaaa Ii, Sans@igrjo RI, Lnai M, Mit*rwa Y &Mrtmi M. TypinS h@atitis B vid! by hdology in rucl@tido scq!€@:@nrpdisor of dufee dtiSo subtt?€d, Jolml of Go@l Virelogy 1988i 69:2575J583.
165. Onrh M, Ehai3 T, Yoko$jLr O, a al, Mur.doN ir tn. pecorc Egior ofh@aliris B ritu DNA h parimb with tuloi@t &d s@ hcalriti.. N4EDglad Jo@.| of Mcdicine 191 : 124:1 69+17M.
166- Oo! CJ, Lin GK Goh KT, e, al. Mddle eiddiology of n.p.titis B virov@ine ldiet! in Sitrg+G Veinc 1995; l3t 699-702.
16?. Ontr N, Mill* RH, Ishak KC & Pe.I RH. Ihe coDpl€te nucloti.lc scqua@of a pdoE nutl ofnepalitis B liru implic.tcd i! tulnimt h.Ft'h! ed itsbiological ch@t izaiid ia chinpq@s, Virclogy 1993i 194: 263-2?6,
168. Oriro E, Mizokmi M, tE Y, Mdiyur E\ Kderhim! N, Ymddo M &Gojobori T. gost-ind@ddeDt evol(ion 6d a gtutic cl6sifc.tio! o{ ihehepadtuviM fdily b!s.d m nelon& equ€B. Pmce.dings of thc NationalAcrd@y of Sci@6, USA 1989t A6: 1059-1062.
169. orfto E, Mizokhi r4 Sahsrwa 1l, Micbit*! K Isliklq K Lhida T,O&mole T, YoGuy@ci H,Iino s & Jato EBv Gaotyp. R64h cbur. Ac'6€snuol srudy for cliDic.l ed holduld biological drftffic* betwah@ltitis B virus6 of gerctlq B Md c. H.9&rolog 20ol i 33 | 218-221.
l?0. o3iory C. D.t ction oIHBsAg mul!r6. troual ofMedicil Viblogy 2006;78Suppl I 148-51,
l?1. Ou Js & Ruud wJ. }Iybrid hardrir B vi6lDsr tm&ripG in lhe hnmhepatoma c.ll. ProcerdiDgs ofNation l Ac..l@y ofs.iac6 USA 198t; 82-8?.
U2. Orircd!, ltr, K,, Krevis, A. & Kd, M. C. H@aiiis B viM DN,A in *tm ofh€althy bl&* Alricd adulE F!$vc fq h@atitis B sarfe. dtibody alo.:posible as&irrio! wirh te&birurio! bctw@ gaot,P6 A ed D. Jom.l ofMedical virclo$r 2001; 64i 44t -454,
173. Pukl! D.M., ?iMi t., & Fdlay J. Edihat6 of thc worldwid. incid.Nc of 25maior cdoeB inl990. Irtmrional Jouhsl of Cd6 l999i 80:827-841,
l?4. Prrr€r EJ, Nakqu. GR & YalIe A, lrtuc.lluld tspon dd r@tion ofn palitis B sud&c etiecn iD maba|id @lls, Joumal ofvirlosy l9E4; 5l:3,16-353.
t?5. Ptla E , Nakdd CR, Simonsa CC, .t ar Ifteelluld aobly adpct4rnS of hcaalitb B srrfa.e a igd panicl6 (EN ir lhc @dopbsmicFtiqrlM..lomal ofvnobsy 1986i 58: 8E4892.
176. Po!. a" Chisei FV, Be.tol€tti A a dl. Ctrotoxic T l}nth@)16 r@8nizc etlLA-A2 !.st!idc<l c?itop. within tiie hrpaiitrs B viu [email protected] dtig4Jo@al of Expdindtal M€di.in€ 1991; 1?4r1565.1570.
1??. Polllrs 9., ?ait MA. Baikowtki MI, & Pe@les ME tlum liva pb6ma
nmbtu6 @nui! E.!!oc for t!€ h?lritis B viN pr.Sl rc8ion dd via
rolymdized hme stu albuitr for th. PE 52 rc8ror' Jolml of Vnobgy1989i63:1981-1988.
178. Prci{.r-Ad.B S, S.hl.}tr Itt, P.rd T, .r al scqu@c daltaL of kpatnis Bviru DNA in innuolo8lcaly segativc int@tion. Archivca of viDlo8v 1993;
131:385-396.1?9, PDC.r-XroI. U, NaMtu U, Bdl.ehlagd & .t at- H.patitis B viG tith
dtiqdjcillv alrded bcpatitis B su6.e eri$o is $l6l!d bv hiehice tePditisB i;mune slobuhn atrd ln s Edspldkdon HcprblocJ l o98: 2
_ 254-263
l8O. Pnlt l, Ahbotl N, ZunS Y & Su'|l!6 r. FFqlmv of sloa avid hct.&witu infetior. Johdt ofviFlog 20014 ?5: 9623-9612'
181, RliDondo C, Cupo S, Sm.dil. V, ,r.i EB. sribody uelalcd io ninushelaritk B virus v;dt infction in p.li6ts with cho'ic tvpe D h€pstili5
Jom.l ol t{@atolo$/ l99li l}:S8?-s89ls2. RocbrB& Von Bo;hmd H. Peliph.Bl *l4tioD ofttu T<ellE?trlo'E Scim4
r99l: 25ltl2?5-1224.t$. R4;!. Bl1 G.id M, Soja@ G, e, ai n@a$li. B viru unau' to sm' '{tisd e{t res?oN |o i df@n in ct@nio h.psdft B Gdtro'ntemloly
l99l:105:845-850.I sa Rmo$k! L. gun$o Ir, Fs* Zt- Lins R tehnn L Oegpva I & Plislova
L. Utu3ull h@a$ris B !ue.. dLism vd4@ ir s 'hild 'muizcd
ae4inst
hoatitis B. IoMal of Mcdical viologv 2000i 61 : I l:14185. a;;rot J. Fti&h EF & M&i.d; r. h, Mol6uld Ctonins A lalodory'' ;;;l. c"1d spdns H.r!d L2loralorv, cold sFiis Hdt'or, Nw Yo* 1989
reo. l"".hJ"pl"'t Llii N""-l histo.v;f cluoni; hepdins B In:BudM t3tebe''' Ic,.O"li"a. virat hcpdiris B@l6e Aeio' M€dic'' 2000 21_]l
'rr ibr-l*r n" Melbs
_K Lkulmhd A, a at lDmdizlnon with sllbt'
'- i"pti"i. e '-t sufd. prcrein cliciu muint H_2 '16'! l_Bhoed CD8
cftroxic T lymphdtl. GpotB d vivo JoM'l of Inuuoloey 199'l'
152:1110-lll9.tse. i;I..b*i I v.lt- c Mtu! r & R.iEe '
Anrbodv & c,4o!ori' r{ell-- cpo; ro otuute trcpaids B viru (HB$ s etisd in ruce: Inplicatioo ror
iii'i"'r'.*"Ji' .iirin-.a."a kt;is iomd ;r vibrory ree4; 68:1418-
1425.189. sclorl6 M. ?.ras T &
sigDficmce of b@atidsimunologically n€8atirc81r-
l9o *d c & Men \{ts Rephclrio of tbe h€?'tlus viru SpDon' lr MLO"p'-or"ro rcO r, DNA E?i'csion in 4kelotic cellt Cold SPdnS Hdbor
Ido; n ftc$. Cold sDnns Hehot, N Y 1996:815_831
l9l Shi.lt ltfi, rd€n FF. Caj. AJ. N.leD C & Swatc P Fr'quocv trd'" ii-n.Li".'"r -'.*."i ha;id6 B sud&e arrsd md 4Libodv D rurc ad.ionrc h@atiss B Gsmmrfrlogv 198?io1:075_680
RaseL L lsolttioA chd&t@zation dd bioloSj'al
B viru dutlats Fon seM of a Plridt wil!EBV in&cton. Jorml of H@atologv 2Q001 33: 799'
la2. s rll T. Kord. \. HeafrEa T Had, H okt T. la*"da N shin'ho H
N,l;ua M. Urike r- Yuolo Y & T$r' l Poi BulalG itr rhc S trd PrcS2rd6 obffied in No [email protected] B vNs cMis p@rlive fd strbodv b hepdnis-B sfd. Mdga H@dorshdtftiogv 1998:45:5m_502
l9J. amR.l R i.l.L EF A W H tlol'rjd t d ch@Friadm of tb@lnthB
';tu;dmc inhmns Jomal of v'ology Io88:62:012 937
194. Sa.n..t lt, Gutbd S, Gdl&h J, N&@ov NV, Sdtelonio T' Fishd L'
Rocd X d a/. H.Ddit. B viru *q@c. ch&g4 Nlving in livd @splstdi-.i6k sth tulmdl hcrdrt! lomil of H@atolog 1997:20:754?64
195. StNetr! (T. Toy P]. (otrg MJ. Iavlor PE' VtB GN Nair Pv weGsnd JY &_'' i-*- s. jo-l.t ernolm Mcdictl A3$irrion r98?i 257:2612_2616'
rso. S;;ir CE, rov PT, r.ng MJ, Tlvld PE, vts GN' Ndr Pv'Gud'ali M &K-*d S. to;al of An-icd M.dical Alsocia'tion l98t; 253 i 1?40-l ?45
rsr. idt-r UJ. Tho.." JM A gowstrl CR A topologrcd nodel for h€P'titis E
,utfrc dtso. lnlflimlogv l992: 13: t
'8_ l 58
rct. iower r.bc Gmat s, v- c"tr C zoul' r' lnua v Schwi Rr & R$Mu'_- r- i n* **orvee "r
ncpa B viru!: @plcl' sdome ed phvlogn'ric
FId.&6 JoEd ofcdatl ViDIosv 200q 8l:6? 14
Lgp i".t..l a v*. wS Repli.di; of ihe smone ofa hcpdirrs B lilte viG'__ i' '."*. .*.ti"Li.t
"f t RNA n'm'dide C'll1982:29 'lo3-41t
loo. i'.nin.r, smoi* t a Sntdd R A viru sml- ro hulln hePants B vitu-'' ".*"i"J *trt r.-."ri, -d h@sbma in woodchucb Prccceddc! orNafioml
Acad.dv of Scmcs USA l9?8;7t:4t13_45j7-zor. l[u.i'w,$*oCr & Hd]dsc ttearttisB dolvinseprdsioloeJ' sd--_ *rlcari"* iot c-r.r' Sdind otr LivdDi*66l9ql: I l:84_42
ror i'i"iLifi.-si";* ci, rkhv EJ ., ./ Hepdrt* B t'ccine: Doonsc'don_'- "i"m.*" t " -"*t|€d.|ni;l Er'l ii a bs}_rot poPulaLiotr in rbe djrdsl*. No Ended JoumdofM.dicDe Is80i Jol811_841
zol. [l". s. ltf;.* "v
t'aaLitis B surfs' utsd sft' murtu in Euope ud-'- nr; G.i* l.*i
"rMe<!c'l v@lory 2006:78 sutpl l:41_47
zol. i"i.i.'h ( sDt'- B, Usud. s' Mishjrc s & rrinc' AM F!r-36om'_ skrce ealvFs of hepAtitjs B liru! 3hi6 dovercd tiom chrnpdees
ii*iJ-- 'ijGra,
,'"ti** ro' d disjr or HBv violocy 2000: 267 ,3
20s. irtrr!trl8c. Mrc$\!k] N Mctrm K. colc PJ Korba Bf EnB'' RE Cdn lt'--- wl-'iii. vi.r'-* cn" utt E ., ,./ H.p'loGlluld cdrr'nr rn tuched$n 3
g"fiJ iq"i;r. \sp@ophil8 neho'dsdit): 'virien*
for MirtioD wi$
iourrr rj-r'te'i.' i"tc"ri." Hcpa$logv leeli ll l2l5-1221
t" ili,ii. i-J i.r. i*adirroro. vir-;,6ki rt rekair F D'8ott c Brsk' I'--- s"*StB,-a.Ve e new h.ladraviN od'Dc indric geud squidcB
itr lhska. Jouml ofvnobsv 1996i 10t421o4219
zor. ii" iiJa"'i i"'*tle"ri"i't*m oa oture' romxsion of hcpltitis B ro-'' "*-" F.i r* i'ri
"a sus@s *irhour h€p{iris B ' 4tigm N@ Etr8ldd
iomal of M.tlicine 19971 336:l?8_184.
208, Toh tI, Har@tida H & Miyata T, Seqod.c hmology b€twcd rclroviEl @.6etrdsipi& &d put tive polrnetg of h?atitls B viG ed @Unowa mosicviG. NatuE 1981; 105:827.829,
209. To!8 Sn Li JS, Vitvit*i L .r ar. Evi&d.. for r b.e p.iFd ESjon ofh.patitb Bvitu pEgmm. acT€idatio! ri8lal which iot|!@6 lh. prll.h of pFcoGnul.lioB .bolishirS HB. prcrli! qpBior 'Iodd of Vircloly l99l; 6?:56515655,
210. Tdbln tl, St!€helitr T & Gordo! J. Eletogh@iic treefd of prct ia fmnpoly!.ryleiit aels lo [email protected] sheis: Pd€€duc ard $mc applicatiN.Prccccdinr! olNalioml Acadmy of Scioc* USA.l9?9: 76: 4350-4354.
2l | . fttd.tu T, Pouel C A Sm6 ,. Fmatiotr of lb. pool of @Eldllyclos.d cn uld viDl DNA in h@adnaviru i.L.tcd c.lls, C€ll 1986i 41:451460.
212. Tm JS & Schl@ Rg. Ttilkiprin of lh. tll@ b.L intaferen gac isinlibitcd by nQ.dtis B vi[s. Jowd of ViFloly 198q6:3065-3971.
213, Tvr lS, t@ CH, Lin PM & Scnloa RH. t'l@aliti! B viN sr+P|tsexprc$ion of hl!@ beia.inldfftr P&edin8s of Nati@l Acad4y ofS.idc6 USA l98q 85:252-256.
214, IJ16.r JB, Domely Jl, Prta SE, dr zl. Hetftlogout prcl-tion aga'nstinnu@ hy injetion of DNA d.odins . vir.l pd.in. Sci@c 1993; 259: 1745-
215, Urb.! MK O'Com.ll Al & t ndd WL Sequd.e of .vdls in Darlralirfeton of P.tin duck dbryG with &c! beprti$! B vtu' JouDl of Virclogy1985t55:16-22
216. Uy A Wunderlich C, Olsd DB, Hc€ll)]@ l<H, Gdlich wli & ThmF RGaodic v&iabilily in th. ptsl Fgjon sd dcteminaiion of 6uts oftr.tlMision of h.laftis B viru. .lomst of Goaal Vimlogy 1992t 17:3@5-3009.
217. Vd.tra€l. P, M.diM A Rltta WJ et a?, SFlh6is ad Mbly of h.pad6 Bsuhc. dtigen p..l'cl.! in y.-ast. N$@ 1982; 298:347-350.
218. Vu Kdt n C, Rsinl I. r6c!rl-S&.do D, er zl, Morctin. pbdlctid byhMd T @lls: IL-l slpha produdion rdEict d lo ndory T c.lb Jo@al ofInrmunology 1991 i 146:265+2658.
219. Wtltt. r, Tokushie! K, Mo6@u D, a al Antiscnic all@tion of hcpltili8 Bvins cd€ prorch in tultuindt heplliiis- Hcpaaolory l94r 20r295A.
220, w.!g B, Bolq t, Srilmtar V, e, ar. DNA i!@ullrion ind!6 netalqngimue !€spotu* agtinsl hme imuodclicituy viM typ. I tu mice &dnonhM prioal6, DNA C.ll Biolo8y | 993; t2:799-E05.
l2l. Wlng B, Usm K-E. Snkdrd v, d o/. Cenc in(u dio Cflcrat6 imu.rgpol:s agaist hul@ imuod.fidfty vitu tr?. l. Pc.€dings of NationalAcld@y ofScicnc6 UsA 1993; 980:41564160.
222. W.ng GIt & S€cgd C, Nov.l o-hejm for rcv6c treription in b@a1iti3 Bvi@s Jounrl of Vimlogy 1993i 67:65064512,
221, w.og XW, FoGtr K Yeh H, d ar Hgpatilis B viru X prctci! inbibirs P53seqlc@Aeifi. DNA bindir& tlleipliotul &livity md is 6wiatcd wilhl,aqiption fa.ror ERCC3. Prc@eding! ofNalioMl Ac.d.ny of S.idc6 USA19 :9\12230.2234.
224. w.re! Kg ga.v JL, Swe RA H6Flto & V'eh@r EI A n* greup of- i-ii"-i.i*
""ni."rt" infcctins otuigtd (Ponso pvsn@t tounar of
"'. H::y"ft#Jffi,'i* p,,rar rd3 orrh. .*on "a' dct@'m, or
-- ;-;;'t;;'-'r;* bv a v@iFi'duc'd e"o' mutanr' rNEr ofChcat lnv6tiSdiot! 1992; 90 254!2547
lo. will s, V..ii C e Sp-l , ExhrbvDic totcde or datw T ells: clon'letininarion c a msequire or imuity C.ll l9q0i 63:1249- I 2 t6
227. w.lnb.rid KM, Zoul.k C. Adn T. Bohn S & Jilg W A mv€l d'ldiotr mul8l-- .ri-*ii' e ui*..rr*. drisa Joum.l ofM€dical violosv 1999: 58 105_
I to..Dr. i;q zQ. Sp,bltuck S, Td M, a al v&cMLid wirh a pl@d vator
.*r.._"e 't'.
;b "t
t* 8lv"op.Lb g6. irdu6 Prct*tvc imsilv lgainn
E!'ctviM. vDlosy 1994; l99:112_140
zls. i;;'ob K H.;6" v r'uda F. Iloh K Aklntrc Y Yob@olo s o!ffoto-- n" *""r""t V a r.,rry".i M Na$rlv @Ning 4sPc ml6! o'h?ailis B
Jo' i.i*' mu*i.ni rn tn. s smc in csieE 6mpos'livc lor dribodv to
[email protected] B suf@ $tiga Jomd ofvimlogv 1994: 68: 2671-26?6
zlo. vinc s. e-s or, eh"u Hs & Combd B Exranq$E HePdiris B qrut DNA
sudc n oltials enh mr hettdttu B irf*rtoo H'Paloloe} 1990: 12:187_
rsi.Z:t. i- .nr, ro nS A Kit CY. M-hditu of thc urddetabilitv of EB'Ag it1_'
Dati@L wilh choic livd di!6e lt?€ B Korcd Johal of C4noalerclogv
., iTl;'L'i'*'"1*r' o td Bv. B'n! ^*.
Mon,h,b t, sdo r' Ym'ob N'
rsonm S, vctora r, IshiU* r. uitl'. S & KalMu S I@i6 olhf,aLius B virus iood. r*otqed tom vrcttmc* Pddls w'th tulrniMth@at'trs r mnDa;soD witb rho* of FUtc h.!.t'tb JounEl ol M'dical vireloSv
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c,,, ui H.p.titis B vdiers in Euo!'' Lsc't1988:2:197-205
:rl. /aroiijp. vorri' p, cotuo M, Llr" tM, Bouch. c, "r 'r conpesor of-- *i-rg.p*l,i* -a Iri*-41i86_P6itiv. cbrcDio h'prtitis B in Fr!@ JounDr
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vtrolosy 2004, l:9(,2) 454_464
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