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UNIVIA$TY OI Itr IUNJAT

Ilth.ldrid tuhlbdoae lqli|ffin ftd.geof

DOCIIIAII Or tEIIgX[v

(D llv ctllol{lA DESqTATIo}'

r(,[email protected]

Ey

I!

altr uulctool -206)

: s"F"i.tAdLthdq.:

TE. & IIAZIDDTNlD6dd &tl Dl$lor

lScl Cd dar..b. b lr.l-rh Ebbs/Urndit of b lqdr lelss Bdr,hl Edr nr4 L.lGst?dt

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), &lt 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

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l40:1808n815.rO. Ccr{d C, M.riin Y, Bnily H, et d.l. ILI .lpb8 tu Prcdeld by T l}bPheyla

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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

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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

,,,. ?S;,f ifil; ,, "-rro

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

of lttuarolo* 19941 20:63G640l:1. zl"i! i, fr'a"ke" KM. C.M. & I-egas. M. Hilkd G' Kdps T XU Y

ioe_D, titg w. ioggodod M & Lu V Mu@l h'patts B vns surree

-tiel* tHi.e.d ;e- im@sai. bur mav hrve a 'hsg€d s!d6'irv

vtrolosy 2004, l:9(,2) 454_464

le. aoo _bX

T&dulos ,A' ou J}L ,r 4/ Acrvaiiotr of ds I mjot_- fio.o.putt ily --pt"" gd. expresion bv helaiitie B vins lomal ofvirclosY l90i 64:'1025_4028

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