Duck hepatitis B virus infection of Muscovy duck hepatocytes and nature of virus resistance in vivo.

Copyright ©D 1994,AmericanSociety for Microbiology

Duck

Hepatitis B Virus Infection of Muscovy Duck

Hepatocytes

and Nature of Virus Resistance In Vivo

JOHN C. PUGH* AND HEIDI SIMMONS FoxChase Cancer Center, Philadelphia, Pennsylvania 19111

Received 1 December 1993/Accepted 17January 1994

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes,weisolatedhepatocytesfromMuscovy ducklings and chickens, birds that have been shown to be resistant to duckhepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocyteswere resistant to infection, but we were able to infect approximately 1%of Muscovy duckhepatocytes inculture. Infection requires prolonged incubation with virus at37°C. Virusspread occurs in theMuscovy cultures, resulting in 5 to 10%DHBV-infected hepatocytes by 3 weeks afterinfection. Therelativelylowrate ofaccumulationofDHBV DNA in infectedMuscovy hepatocyte cultures is most likely due toinefficient spread of virus infection; in the absence of virus spread, the rates of DHBVreplication in Pekin and Muscovy hepatocyte cultures are similar.5-Azacytidinetreatment can induce susceptibilityto DHBVinfectionin resistantprimary Pekinhepatocytesbut appears to have no similareffect in Muscovycultures. The relatively inefficient infection of Muscovy duckhepatocytesthat wehave described mayaccount for the absence of a detectable viremia in Muscovy ducklingsexperimentallyinfected with DHBV. Animportantfactor indeterminingwhether a cell becomes

infected withagiven virusis theavailabilityofsuitable host cell receptors to facilitate virus binding and uptake. The impor-tanceof virusreceptors indetermining theveryrestrictedhost andtissuerangedisplayed by hepatitisBvirus (HBV) is largely inferred,as areceptorhasnotbeenidentified foranymember ofthehepadnavirus family. Duck hepatitis Bvirus (DHBV) is the bestcharacterized of the avian members of the hepadna-virus family. The hepadna-viruswasoriginally isolated from the domes-tic Pekin duck (Anas domesticus) but can be successfully transmitted to other closely related ducks and to geese (4). Marion and coworkers have shown that DHBV cannot be readilytransmitted to Muscovy(Cairina moschata) ducks or to chickens, as indicated by the absence of detectable DHBV DNA in the sera and livers of birds challenged with virus during the first few days after hatching (4). The Muscovyis a

species of domesticated duck that is distinct from all other domesticated ducks, which were originally derived from the mallard (Anas

platyrhzynchos).

In this study, we have asked whether resistance of Muscovy ducks and chickens to in vivo infection with DHBVcan beattributed solelytoresistance of hepatocytes toinfection.

To determine resistance to DHBV, we isolated primary

hepatocytes from Muscovy ducklings and chickens and in-fected them in vitro with DHBV. We report that primary

hepatocytes isolated from Muscovy ducklings canbe infected with DHBV but that this infection is less efficient and the courseofinfection is retardedascomparedwith thatin Pekin duck hepatocytes. Chicken hepatocytes appear to be

com-pletely resistant to DHBV infection invitro,however. MATERIALSAND METHODS

Experimental animals,virusstocks, and cell culture. Pekin ducks were purchased as 1-day-old ducklings from Metzer Farms, Gonzales, California. Muscovy

ducklings

were

pur-*Correspondingauthor. Mailingaddress: Fox Chase Cancer

Cen-ter, 7701 Burholme Ave., Philadelphia, PA 19111. Phone: (215) 728-4780.Fax:(215)728-3574.

chased as 1-day-old ducklings from Hoffman Hatchery, Gratz, Pa. Ducklings were tested for DHBV infection by dot blot hybridization ofserum, and any infected birdswere isolated.

The sourceofDHBV used for infections ofprimary hepa-tocytes was a pool ofsera isolated from 3-week-old ducklings

from

acongenitallyinfected flockmaintained by the Fox Chase Laboratory Animal Facility. The serum was stored in aliquots at -

700C.

Primary hepatocytes were prepared from 1- to 2-week-old ducklings by collagenaseperfusion of the liver. Theprocedure

is based upon thatdescribed by Berry and Friend (la) and is described in detail elsewhere (6, 13). Approximately5 x 106

cellswereplated oneach60-mm-diameter dish(4mlofa 1%

[vol/vol] suspension of cells). Cellswere maintained in Liebo-vitz-15 (L-15) medium (Gibco BRL) supplementedwith insu-lin (Sigma), hydrocortisone-hemisuccinate (Sigma), HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid), and antibiotics, as previously described (13), but without fetal bovine serum,dimethyl sulfoxide, oradditional sodium bicar-bonateorglucosesupplements unlessotherwise indicated(6).

Fresh mediumwas added tocellsevery 1 to 2 days. 5-Azacy-tidine (5-aza)(Sigma)wasaddedtoculturesfrom asterile0.1 Mstock inwater. Suramin

(Bayer)

wasaddedtocellsatafinal

concentrationof 100

[Lg/ml.

Hepatocytes were infected for 16 h at 370C with a 1:5 dilution of DHBV duck serum

(approximately

10i

DHBV

DNA-containing particles per ml) in L-15, unless otherwise stated. No attempt was made to remove the great excess of noninfectious surface antigen

particles

from the duck serum

used for infections.

Stainingfor DHBVcoreandenvelope

proteins

by immuno-fluorescence. DHBV core

protein

was detected

by

staining

cells fixed on tissue culture

plastic

with

ethanol-glacial

acetic acid (95:5), by using a

specific

rabbit antiserum

(a

gift

of William Mason)followedbyfluorescein

isothiocyanate-conju-gated goat anti-rabbit

immunoglobulin

G

(Cappell) (13).

For

staining of DHBV

envelope

proteins,

cells were fixed in methanol-acetone (1:1) and stained with a mixture of two

mouse monoclonal

antibodies,

1H.1 and

7C.12,

that are

spe-cific for the

pre-S

and S domains of the DHBV

envelope,

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1:5

DHBV

1:500

DHBV

Day 8p.i.

FIG. 1. Titration ofinfectivityofaDHBVserumstockon Pekin duckhepatocytes. Primary hepatocyteswereplatedon60-mm dishes and infected2daysafterplatingwith 2 ml ofa1:5or1:500dilutionof duckserumin L-15 mediumfor 16 h at37°C.Infected cultures were maintained

in L-15medium for 8 days and then fixed and stained for DHBVenvelope protein (see Materials and Methods).The bar in therightpanel

representsapproximately 200 urm.p.i.,postinfection.

respectively (5a). Antibody binding was detected by using a

fluoresceinisothiocyanate-conjugated goat anti-mouse immu-noglobulinG(Cappell).CellswerephotographedwithaNikon

Diaphot fluorescencemicroscope.

Assayofinfected hepatocytecultures for DHBV DNA. Total cellular nucleic acids were isolated from infected hepatocyte

culturesasfollows. Afterbriefwashingof themonolayerwith phosphate-buffered saline(PBS), 1 mloflysis buffer(20mM Tris-hydrochloride [pH 7.5],10 mMEDTA,0.2 MNaCl,0.2% sodiumdodecyl sulfate (SDS), 0.5mgofpronase perml)was

addedtoeach 60-mmdish,and cellswereincubated for 1 hat

37°C. Thelysatewas extracted oncewith an equalvolume of

phenol, and nucleic acidswereprecipitated with 2 volumes of

100% ethanol. The nucleic acid precipitate was dissolved in 100

RI

of TE (10 mM Tris-hydrochloride [pH 7.5], 1 mM EDTA). A third of each DNA sample was loaded onto individual lanes of a 1.5% (wt/vol) agarose gel, and after electrophoresis DNA was denatured and transferred to a

HybondN(Amersham) nylonfilter. DNAwasimmobilizedby

UVcross-linkingof thefilter inaStratageneStratalinker1800, and DHBVDNAwasdetectedby usinganinvitro-synthesized 32P-labelled RNA complementary to DHBV minus strand DNA(13).

To prepare nucleic acids enriched for DHBV covalently closed circular (CCC) DNA, cells were lysed in the buffer describedabove (excluding pronase)for30 min at37°C, and then KCl was added to a 0.5 M final concentration. The

mixturewasvortexedbrieflyandstored at roomtemperature for 30 min. The protein-detergent complex was removed by

centrifugation, and the supernatant, which contains CCC DNA,wasextracted withanequal volume of phenol. Nucleic

acids were recovered by ethanol precipitation, and DHBV CCC DNA was detected by Southern blot hybridization as

describedabove (10, 13).The hybridization standardonall gels

was10pgoflinear clonedDHBV DNA. RESULTS

Optimal conditions for DHBV infection of Muscovy duck

hepatocytes. In order to determine whether Muscovy duck hepatocytes could be infected in vitrowith DHBV,weinitially

used conditionswhich had

previously

been shownto result in maximalinfection ofprimary Pekin duck hepatocyte cultures

(6). Optimalinfection of

hepatocytes

requires

incubation with ahigh-titervirus inoculum(approximately109 DHBV

particles

per ml) for several hours at 37°C. Staining of Pekin duck cultures for DHBV

envelope

protein 8 days after infection underthese conditions reveals thatalmost all cellsareinfected (Fig. 1, leftpanel [1:5 dilution]).

To compare the relativeamountsof infection in Pekin duck and Muscovy duck hepatocyte cultures following infection under identical conditions, hepatocytes isolated from both birdswereinfectedwithequivalentamountsof DHBVat4and 37°C. The relativeamountsof DHBVDNAincultures 8days

after infection were determined by Southern hybridization (Fig. 2).The much greateramountof DHBV DNA in infected Pekin cultures suggests that many more hepatocytes become

infected in the Pekin than in the Muscovy duck cultures

following incubation with the same amount of virus. The results also indicate that prolonged incubation at 370Cwitha

high-titer virus inoculum isrequired foroptimal infection of

Muscovyduckhepatocytes. Suraminwasaddedtocells

imme-diatelyafter infection in theseexperimentstolimit thespread

of virus infection. However, it appears that uptake of bound virus is sensitive tothe action of suramin and thatincubation for several hoursfollowingvirusadsorptionisrequiredtoallow virustobypass the suramin-sensitive step (5). This accounts for the absence of detectable DHBV DNAafterinfection at40C,

atwhichtemperature nouptake of bound virus should occur, and for the much larger amount of DHBV DNA in cultures incubated with virus for 5 hat37°C(Fig. 2, lanes 3) compared with 1 h (Fig. 2, lanes2) before addition of suramin.

Kinetics ofDHBV replication following infection of Mus-covyduckhepatocytes.The appearance of CCCDHBV DNA was assayed following infection of Muscovy and Pekin duck hepatocytes under the conditions described above (Fig. 3). The number of CCC DNA molecules in DHBV-infected hepato-cytesistightlyregulated (10, 12); thus, CCC DNA serves as an indicator of the relative number of infected cells. CCCDNA could be detected in cells 24 h after addition of the virus inoculum to Pekin duck hepatocyte cultures, indicating that most cells in the culture had taken up andconverted at least

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Pekin Muscovy 1 2 3 1 2 3 hs

RC - t

s

FIG. 2. Relativesusceptibilityof Pekin andMuscovyduck

hepato-cyteculturesto DHBV infection.Primary hepatocytecultures(60-mm dishes) prepared from 1- to 2-week-old ducklings were exposed to

equivalentamountsof DHBV (1:5dilution of stockserum)for 1h at

4°C (lanes 1), 1 h at 37°C (lanes 2), or 5 h at 37°C (lanes 3). After

removal of thevirusinoculum,cellswerewashedbrieflywith PBS and

then maintained in L-15 containing suramin at 100 p.g/ml. Nucleic

acidswerepreparedfrom cultures 8days afterinfection, and equiva-lent amounts of DNA from each culture were loaded on a 1.5%

agarosegel. DHBV DNAwasdetectedbySouthern blothybridization (see Materials and Methods). RC and SS, relaxed circular and

single-stranded DHBVDNA, respectively; hs, hybridizationstandard

of 10pgof cloned linearDHBV DNA.

one molecule of virus DNA. The level of CCC DNA in

infected Pekinduck hepatocytecultures increased until6days after infection, after which time no significant increase was

observed (Fig. 3).This resultwould be expected if all suscep-tible cells in the culture were infected by the original virus

inoculum. CCC DNA could not be detected 24 h after

infection ofMuscovy duckhepatocyteswith the same amount

ofvirus, indicatingthat eitheruptake and conversionof DHBV

DNAwasdelayedorthatarelativelysmall numberof cells had

Muscovy

dayp.i. 1 3 101316 2024hs

RC_ ___Z

ccc -4

Pekin

dayp.i. 1 3 4 6 8 10 1316hs

RC

FIG. 3. Kineticsof DHBVreplication inMuscovyand Pekin duck

hepatocyte cultures following infection with equivalent amounts of virus. Cultureswere infectedwith ahigh titerof DHBV (1:5 dilution

of stockserum)for 16 h at37C.After removalof the virus inoculum,

cells were maintained in L-15 medium. Cultures were harvested for nucleic acids atthe timesindicatedpostinfection (p.i.) byaprocedure

that enriches for CCC DHBV DNA molecules (see Materials and

Methods). Equivalent amounts of DNA were loaded on a 1.5%

agarose gel,and DHBVDNAwasdetectedbySouthernblot

hybrid-ization (see Materials and Methods). RC and hs are defined in the

legend to Fig.2.

been infected. CCC DNAwasnotdetected until 10

days

after

infection,

and thisamountincreasedtenfold between 13 and16

days

after infection.

Staining

forDHBVcore

antigen (DHBcAg)

confirmedthat

only

approximately

1%

of cells in the

Muscovy

duck

hepato-cyteculturewereinfected

(Fig. 4)

underconditions in which80

to

100%

of Pekin duck

hepatocytes

become infected

(Fig.

1,

left

panel).

Staining

also revealed that the increase inDHBV DNAin

Muscovy

duck

hepatocyte

cultures thatoccurredat16

days

postinfection

was due to

spread

of virus infection in the culture, as foci of

DHBcAg-positive

cells

appeared

concomi-tant with the increase inDHBV DNA

(Fig. 4).

Fociwere not

detected when suramin was maintained in the medium after

infection

(Fig.

4).

Suramin is known to

prevent

the

spread

of DHBV infection in vitro

(5).

Comparison

of the rateof DHBV

replication

in Pekin and

Muscovy

duck

hepatocyte

cultures when

equal

numbers of cells are infected. The

highly

efficient initial infection of

primary

Pekin duck

hepatocytes

made itdifficulttodetermine whether virus

spread

was in fact any more efficient than in

Muscovy

duck cultures. In order to compare the relative

capacity

of the twoculture

systems

to

support

DHBV

replica-tion,

the virus titer in the inoculum was

adjusted

such that

approximately

equal

numbers ofcellswere infected. Titration

of virus

infectivity

on Pekin duck

hepatocytes

showed that a

1:500 dilution of a

DHBV-positive

duck serum resulted in

approximately

1%infected cells after 8

days

(Fig.

1,

right panel

[1:500

dilution]).

Infection of

Muscovy

duck

hepatocytes

witha

1:5 dilution of the same serum also resulted in

approximately

1% infected cells

(Fig. 4).

Thus,

having

established conditions

for

infecting equivalent

numbers of cells in Pekin and

Muscovy

duck

hepatocyte

cultures,

we

compared

the kinetics ofDHBV

DNA

replication

in thesecultures

(Fig. 5).

The appearance of

single-stranded,

minus strand DHBV DNA in both cultures

confirms that de novo DHBV DNA

replication

takes

place

following

infection. A

proportion

of the relaxed circular

DHBV DNA

signal

iscontributed

by

infecting

virus

particles

that remain associated with

cells; therefore,

single-stranded

DNA,

which is not

present

in maturevirus

particles, provides

a morereliable marker ofDHBV DNA

replication.

Theresults

indicate that the rate ofaccumulation of DHBV DNA

repli-cationintermediates in

Muscovy

duck

hepatocytes

was

slightly

slow

compared

with that in Pekin

hepatocyte

cultures

(Fig. 5).

Single-stranded

DNAcanbedetectedat

day

6

postinfection

in

the infected Pekin

hepatocyte

culture but not until 15

days

after infection in the

Muscovy

duck culture

(Fig. 5).

We also

compared

the kinetics ofDHBV DNA

replication

inPekin and

Muscovy

duck

hepatocyte

culturesinthe absence of virus

spread (Fig. 6).

Approximately

1% of cells in both

Pekin and

Muscovy

duck

hepatocyte

cultures were infected

with

DHBV,

asdescribed

above,

andsuraminwasaddedtothe cultures I

day

after infection

(5).

The cultureswere

prepared

independently

from those

represented

in

Fig.

5,

which may

account for the small differences in infection

efficiency.

Suraminwasmaintainedinthe culturemedium until cellswere

harvested for DHBV DNA

analysis

at the times indicated.

Maximum levels of intracellular DHBV DNA were attained

approximately

14

days

after infection in both Pekin and

Muscovy

duck

hepatocyte

cultures.

Hence,

when

only

a

single

round of infection was allowed to take

place,

it was not

apparent

that the kinetics of DHBV DNA

replication

were

significantly

differentin

Muscovy

versusPekinduck

hepatocyte

cultures. Themore

rapid

accumulationofDHBV DNA

inter-mediatesininfected Pekin duck

hepatocytes

inthe absence of suramin

(Fig.

5)

may,

therefore,

be due to more efficient

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plus

suramin

minus

suramin

Day

15

p..

Day

26

p.i.

I

FIG. 4. Spreadof DHBV infection inMuscovyduckhepatocytesinfected in vitro.Muscovyduck cultureswereinfected with DHBV andstained

for DHBVcoreproteinat 15 or26daysafterinfection (seeMaterials and Methods). Following infection,cellsweremaintainedin L-15plus

suramin(100,ug/ml)orin L-15 mediumalone,asindicated. The barrepresentsapproximately 120 pum. p.i., postinfection.

spread of DHBV in Pekin duck

hepatocyte

cultures rather thanto anincreasedrate of DHBV DNAreplication.

Passage ofDHBVfrom infected Muscovy duckhepatocyte

cultures. Analternative explanation for the

apparently

more

rapid rate ofaccumulation of DHBV DNAintermediates in Pekin duckhepatocytes mightbe that virus is released more

efficientlyfromMuscovy duckhepatocytes.To testthis hypoth-esis, we monitored the release of infectious DHBV from

Muscovyduck cellsbytransferring virustohighly susceptible

cultures of Pekin duckhepatocytes. Figure7Ashows therate atwhich DHBV DNAaccumulated in the infected Muscovy duckhepatocytecultures. DHBVwasrecovered from medium harvested from the Muscovy duck cells and used to infect

freshlyprepared Pekin duckhepatocytes. The Pekin duck cells wereharvested 8 days after infection, and the relative amount of DHBV DNA in infected cultures was determined by Southern hybridization (Fig. 7B). The results show that the

maximum amount ofinfectious DHBV is released from the

Muscovy duck cells between 16 and 19 days after infection

(Fig. 7B). For reasons that are not clear, single-stranded DHBV DNAwas not recovered efficiently from the infected Pekin duck cultures in this experiment. Maximal release of

infectiousvirus coincides withamarkedincrease in the amount

Muscovy Pekin

dayp.L. 2 4 6 810 13 15 171820hs 2 4 6 8 10

RC

-

SS-FIG. 5. KineticsofDHBVreplicationinMuscovyandPekinduck hepatocyteculturesfollowing infectionofequivalentnumbers ofcells. Primary Muscovy andPekin duck hepatocyte cultureswere infected for 16 h at 370Cwith a virus inoculum determined to infect equal numbers(approximately 1%)ofcells in both cultures(a1:5dilutionof DHBVduckserumforMuscovyduckhepatocytesanda1:500dilution for Pekin duck hepatocytes). Cells were maintained in L-15, and infectedcultureswere harvested fortotal nucleic acids atthe times shownpostinfection(p.i.).DHBV DNAwasdetectedbySouthernblot hybridization(seeMaterials and Methods andFig.2). RC, SS,andhs aredefined in thelegendtoFig.2.

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Muscovy dayp.i. hs 6 9 12 15 20 23 27

Pekin

dayp.i. hs 2 4 6 8 10 12 14 16

RC- _

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SsS

FIG. 6. Kinetics ofDHBVreplicationinMuscovyand Pekin duck

hepatocytes when virus spread is inhibited by suramin. Primary Muscovy and Pekin duck hepatocyte cultures were infected as de-scribed in the legend to Fig. 5 in order to infect approximately equivalentnumbers of cells in both cultures. After removalof the virus inoculum, cells were maintained in L-15 containing suramin at 100

p.g/ml

to inhibit spread of virus infection. Total nucleic acidswere

prepared from cultures at the times shown postinfection (p.i.), and DHBV DNAwasdetectedbySouthernblothybridization (see Mate-rials and Methods andFig. 2).RC, SS,and hsaredefined in thelegend

toFig. 2.

of intracellular DHBVDNA

(Fig. 7A),

whichwe have shown is associated with the appearance of virus

spread

in the cultures

(Fig. 4).

Maximal release ofDHBVfrom Pekin duck

hepatocytes

is also coincident withanincrease inintracellular DHBV DNAand

spread

of virus infection

(data

not

shown).

Thus, it appears that DHBV is released from

hepatocytes

isolated from both Pekin and

Muscovy

ducksat similarstages

during

the virus infectious

cycle.

The results also indicate that

a

single

round of

replication

in

Muscovy

duck cells does not

appear to reduce the

infectivity

of DHBV on cells isolated from the normal

permissive

host.

When virus released into the medium from a DHBV-infected

Muscovy

duck

hepatocyte

culture was

passaged

toa

second

Muscovy

culture, a time course similar to that in the

original

infected culture was observed

(results

not

shown).

Very

few cellswereinfected, however,

presumably

because of both the

relatively

low amounts of virus released from the

original

infected culture and the small number of

susceptible

cells.Hence, it appears that in vitro infection ofMuscovyduck hepatocytes hasnotselectedfor rare DHBV variantswiththe

capacity

to infect these cells, as infection at the second pass

might

thenbe

expected

tobe moreefficient.

Chicken

hepatocytes

are resistant to DHBV infection in vitro.Wewereconcernedthatinfectionof thesmall number of cellsinthe

Muscovy

duckculture

might

result from

uptake

of virus by some route that did not involve a

specific

virus-receptor

interaction. Ifthiswere the case, we would

expect

a

similar

proportion

of

primary

chicken

hepatocytes

to be

sus-ceptible

to DHBVinfection. Chickens are resistanttoDHBV

A

dayp.l. 1 3 6 9 1215 202327 hs

RC

-

Ss-B

dayp.i. 3 5 7 9 12 14 1619 21 hs

RC

-

Ss-FIG. 7. Time course to assay release of infectious DHBV from in vitro-infectedMuscovy duck hepatocytes (p.i., postinfection). Primary Muscovyduckhepatocytes were infected with a 1:5 dilution of DHBV duckserumin L-15for 16 h at 37°C. One set of infected cultures (nine 60-mmdishes)wasusedfor nucleic acid analysisto assay the accumu-lation of DHBV DNA intermediates in infected Muscovy duck cells (A). A pair of infected Muscovy duck cultures (100-mm dishes) infected under the same conditions was used to assay the release of virus. Mediumwasharvested from thesecells every 2 to 3 days after infection and stored at -70°C. Virus was concentrated from the medium by precipitation with 10% (wt/vol)PEG 8000(Sigma), and the viruspellet wasdissolvedin 2 ml of L-15 medium and used to infect 60-mm dishes ofPekin duck hepatocytes for 16 h at 37°C. Eight days after infection (p.i.), total nucleic acids were prepared from the infectedPekinduckcultures, equivalentamountsofDNA wereloaded on a 1.5% agarosegel, and DHBV DNA was detectedbySouthern hybridization (B). RC, SS, and hsaredefinedinthelegendtoFig.2.

infection (4),butacell line derivedfrom transformed chicken hepatocytes has been shown to support highly efficient

repli-cation ofDHBV(2). Hence, if DHBV DNAcanbe delivered

tothe nuclei of chicken hepatocytes,replication mightinitiate

efficiently. We prepared chicken hepatocytes and infected

them underconditions that gave rise to maximal infection of

primary Pekin duck hepatocytes. We were unable to detect DHBVreplicationbySouthernhybridizationorbystainingfor DHBcAg in cultures harvestedat48-h intervals between 2 and 20days after infection(Fig.8).Hence,weconclude that fewer than0.001% ofprimarychickenhepatocytesweresusceptible

toDHBV infection. This result suggests that chicken

hepato-cytesare resistant to DHBVbecause ofthe absence of virus receptorsand that thelimited infection weobserved in Mus-covy duck cultureswas indeed receptor mediated. The pres-ence of DHBV relaxed circular DNA indicates that virus remained associated with cells for the 20 days

following

infection. Whether this represents trapping of virus at the plasma membrane or specific binding to a

hepatocyte

cell surface molecule isnotclear.

5-aza cantransformprimaryPekin duckhepatocytesfroma

DHBV-resistant to a DHBV-susceptible phenotype. We have

previously shown that primary Pekin duck

hepatocytes

pro-gressivelylose

susceptibility

toDHBVinfectionin

culture;

this effect is especially

pronounced

in cultures maintained in medium supplemented with fetal bovine serum

(6).

The

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2492 PUGH AND SIMMONS

Chicken

dayp.l.

RC

-

Ss-FIG. 8. Timecourse ofDHBVinfection ofprimarychicken

hepa-tocytes. Primaryhepatocytesprepared fromaweek-old chickenwere plated in 60-mm dishes and infected 2 days afterplatingwitha 1:5 dilutionofDHBVduckserumin L-15. Cellswereincubated with virus for 16 h at 37°C. Total nucleic acids were prepared from infected cultures at the times shown postinfection (p.i.), and equivalent amountsofDNA wereloadedon a1.5%agarosegel.DHBV DNAwas detectedby Southernblothybridization (see Materials andMethods andFig.2). RC, SS, and hsaredefined in thelegend toFig.2.

Pekin

5-azapre- 5-aza

post-Infection Infection

0153060120 0153060120

RC

-Ss

-L15alone

Muscovy

5-aza pre-Infection 0 20

.^-40 hs

-k

_*

___w

_s%ms X

__Xi

_w

_w s

o..w.

sls,ls. v .S.; Q

01530601200153060 120 hS

RC- U

pearance ofresistancetoDHBV infectionprobablycorrelates with the gradual loss of liver-specific gene expression in hepatocytes maintained in culture.

5-aza has been shown to induce expression of avariety of quiescent genes (3, 8). Wewere interested, therefore, to see whether the gradual appearance of virus resistance in Pekin

hepatocytecultures could bereversedbytreatmentwith5-aza. Cultures maintained for 12 days after plating in either L-15 aloneorL-15 plus 5%fetal bovineserum

(Fig.

9, leftpanels)

were treatedwith various concentrations of 5-aza for

approx-imately 16 h before infection with DHBV. Cultures were maintainedfor6to8days after infectionandthenanalyzedfor the presence of DHBV DNA(Fig. 9; see also Materials and

Methods).Drugtreatmentresulted in a5- to 10-foldincrease

in the amount of virus DNA in the cultures. Staining of cultures for the presence of DHBVcore protein showed that this increase in the amount of DHBVDNA corresponded to anincreasein thenumber ofinfectedcells(Fig. 10).Treatment of cultures with 5-aza after infection did not result in the marked

increase

in DHBVDNAthat was observed when the

drugwasadded beforeinfection(Fig.9).5-azahadnoeffect on thelevel ofDHBV DNAinprimary hepatocytes isolated from

ducklings congenitally infected with DHBV (results not

shown),suggesting that the drug does not affect DHBV DNA

replicationdirectly.

TreatmentofprimaryMuscovy duck hepatocytes with 5-aza does notinduce susceptibility to DHBV infection. The results

described aboveindicate that 5-aza may induce expression of one ormoreproteins required early during DHBV infection of Pekin duck hepatocytes. We hypothesized that the relative resistance of Muscovy duck hepatocytes to infection with DHBVmight be due to repressed expression of host cell genes required for DHBV infection. Hence, we hoped that treatment with 5-aza might remove the block to expression and result in arelativeincrease in the number of cells susceptible to DHBV infection. However, we found that 5-aza added at

concentra-tions that induce susceptibility to infection with DHBV in Pekin duck hepatocyte cultures (Fig. 9, left panels) did not enhance the susceptibilityof Muscovy duck cells, as indicated

by the absence of any detectable increase in the amount of DHBV DNA in treated versus untreated cells (Fig. 9, right

panel).

ss

-FIG. 9. 5-aza induces susceptibility to DHBVinfection in Pekin duck hepatocytes. Pekin duckhepatocytes weremaintained in L-15 medium (top panel) or L-15 plus 5% (vol/vol) fetal bovine serum (bottom panel)for 12days,atwhich time cells wereincubated with0, 15, 30, 60,or 120 p.M5-aza in L-15 mediumfor 16 h at37°C.After removalof5-aza, cultureswereinfected witha1:10 dilution ofDHBV duck serumin L-15 for 2h at37°C.Aduplicatesetof infectedcultures were incubated with 5-aza immediately after infection, asdescribed above.Cultureswereharvested 8 days after infection for nucleicacid preparation,andDHBV DNA wasanalyzed by Southernblot hybrid-ization (see Materials and Methods). Duplicate 60-mm dishes of Muscovy duckhepatocytes (top right panel)weremaintainedinL-15 mediumfor 12days before incubation with 0,20, and 40 ,uM 5-azain L-15 for 16 h at37°C.Cultureswere infectedimmediately following 5-azatreatment, asdescribed above.Tendays afterinfection,cultures wereharvestedfor DHBVDNA analysis (see MaterialsandMethods). RC, SS,andhs aredefined in thelegend toFig.2.

DISCUSSION

Hepadnavirusesarecharacterizedbya

very

limitedhost and tissue range. Experiments involving transfection of liver cell lines with clonedhepadnavirus DNA have shown that hepad-naviruses will replicate efficiently in liver cells isolated from hosts that are not the normal hosts for virus infection (2, 7). However, no cell line has been clearly demonstrated to be susceptible to infection with anyhepadnavirus.Arecent report indicates that HepG2 cells can be infected with HBV under certain conditions, however(1). Resistance to HBV infection exhibitedbycellsthat are competent to support HBV replica-tion suggests thattheprincipal factor limiting host range acts at the level ofvirusuptake.

The present study has investigated the nature of resistance toDHBVinfection in the Muscovy duck and the chicken. Our initial hypothesiswasthat resistance of Muscovy ducklings to infection could be explained by resistance of hepatocytes to virus infection.Wehaveshown, however, that virus resistance in vivo cannot be fully accounted for by the presence of

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A

FIG. 10. 5-aza inducessusceptibility to DHBV infection in Pekin duck hepatocytes. Primary Pekin duck hepatocytes were maintained for 12 days after plating before infection with DHBV. Cells were

incubated for 16 h before infection with 20 jiM 5-aza in L-15 (B)or

with L-15 medium alone (A). Cultures were infected with a 1:10 dilution of DHBV duckserumin L-15for 2 hat370C.Eight daysafter

infection, cellswerestained for the presence ofDHBcAg(see Mate-rials andMethods).The barinpanelArepresentsapproximately 100

m.

resistant hepatocytes, as we are able to infect a significant

proportion of primary hepatocytes isolated from Muscovy ducklings. Virus infection spreads to neighboring cells in the

Muscovy duck cultures, indicating that most cells are suscep-tibletoinfection but that virus in the inoculum binds with low

efficiency. Wewere notabletoinfectprimary chicken

hepato-cytes in vitro, which indicates that resistance of chicken

hepatocytes to DHBV is at the level of virus adsorption and

uptake.Moreimportantly,resistance of chickenhepatocytesto

DHBV infection~indicates that the infection ofMuscovy duck

hepatocytes we have described takes place via specific

virus-receptor interactions.

We have shown that 5-aza can transform DHBV-resistant Pekin duckhepatocytes toasusceptible phenotype.The prin-cipalmechanism ofaction of 5-aza istopreventmethylationof

newly replicatedDNAand thus allowexpressionof genes that

arerepressed by methylation (3, 8).Itisnotclear howthedrug

maybeactinginthisinstance, asprimaryduckhepatocytesdo

not actively divide after a few days in culture. However, we

wouldpostulatethat 5-aza isinducingexpressionofa

hepato-cyte protein(s) that is essential for virus infection. The

resis-tance ofMuscovyduckhepatocytes tothisinduction indicates

that the low level of DHBVinfection in these cells is not due to repressed expression of a virus receptor gene that can be induced with5-aza.

The following two models could bothadequately account for the limited infection of Muscovy duck hepatocytes which we have described. According to the first model, Muscovy duck hepatocytes may express the same receptor for DHBV as is present on Pekin duck hepatocytes, though at much reduced levels. Our results would support a model in which very few unoccupied receptors are present on Muscovy duck hepato-cytes and infectious DHBV mustcompete foravailable recep-tors with the huge excess of noninfectious DHBV surface antigen particles. The second model proposes that DHBV infects Muscovy duck hepatocytes via binding to a receptor distinct from that present on Pekin duck hepatocytes. If this receptor had a much lower affinity for binding to DHBV, it might account for the prolonged incubation at 37°C in the presence of virus required to achieve successful infection of Muscovy duck cells.

The rate atwhich replicative DHBV DNA accumulates in infectedMuscovy duckhepatocytes and the period before virus is released from infected cells are delayed compared with the course of virus replication in Pekin duck hepatocytes. How-ever, infection of equivalent numbers of cells in Pekin and Muscovy duck cultures, under conditions in which spread of virus infection was inhibited, indicated that the kinetics of DHBV DNAreplication were similar inhepatocytes from both birds. Hence, itwould appear that areduced rateofspread of virus infection in Muscovy duck hepatocyte cultures may account for the relatively low rateof accumulation of DHBV DNA.

We should consider how we define resistance to in vivo infection forhepadnaviruses. Experimental infection of Pekin ducklings usually leads tomassive infection, withvirus titers as high as1010DNA-containing DHBV particles per ml of serum. If hepadnavirus infection fails to give rise to a detectable viremia, weconclude that infection did notbecome established and that the hostanimal may be consideredresistant. Infection of 1-day-old Muscovy ducklings with DHBV doesnotproduce

a virus titer that can be detected in serum by dot blot hybridization 1 to 3 weeks after infection. It must be

empha-sized that this assay will fail todetectvirus if there are fewer thanabout 106 DHBVparticles per ml ofserum.It is

possible,

inlight of our results, thatinfection of Muscovyducklingswith DHBV does result in infection of a small numberof cells in the liver but that this infection is insufficient to give rise to a

detectable level of viremia. Alternatively, the

relatively

ineffi-cient infection of Muscovy duck hepatocytes may allow the developing immune system in youngducklings to

rapidly

clear DHBV-infected cells from the liver, such that a viremia is never achieved.

In contrast to DHBV infection of Pekin ducklings, in vivo infection of ground squirrelsorchipmunks with

ground

squir-rel hepatitis B virus results in a latency

period

of several months before viremia can be detected(9, 11).Ourresultswith the Muscovy duck may provide apossible

explanation

for how such a long latent period could occur. If

only

a very minor subset of hepatocytes become infected

by

the initial virus inoculum and spread of virus to other cells in the liver is restricted because of inefficient

binding

to available virus receptors, or perhaps because of low receptor

density,

then a

long latent period before detectable viremia

might

be pre-dicted.

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ACKNOWLEDGMENTS

This work was supportedby USPHS grants CA-40737 and CA-06927 and by anappropriationfromthe Commonwealth ofPennsylvania.

We thank Maureen Climaldi for secretarial assistance, the Fox ChaseSpecial Services department forphotographic work, and Jeff Saputelli for helpwith the ducks. Weare gratefultoWilliamMason and Jesse Summers forhelpfulcomments onthemanuscript.

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