Humoral immune responsiveness in duck hepatitis B virus-infected ducks.

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Vol. 61, No. 3

JOURNALOFVIROLOGY,Mar. 1987, p.916-920

0022-538X/87/030916-05$02.00/0

Humoral Immune Responsiveness in Duck Hepatitis B

Virus-Infected Ducks

MICHAEL S.

HALPERN,'*

WILLIAM S. MASON,2 LAURA COATES,2 ANNA P. O'CONNELL,2 AND JAMESM. ENGLAND3

The Wistar Instituteof Anatomy andBiology, Philadelphia, Pennsylvania

191041;

InstituteforCancerResearch, Philadelphia, Pennsylvania191112; andDepartment of Pathology, Hospital ofthe University of Pennsylvania,

Philadelphia, Pennsylvania 191043

Received 18September 1986/Accepted 20November 1986

Immunofluorescenceassays with fixed tissuesectionswereused tocharacterize antibody reactivity insera obtained from duckhepatitisBvirus-infected ducks.Under conditionsofexperimental infection, antibody to coreantigenbut nottosurfaceantigenwasdetectable.Amajorityof theducks infected at 8daysafterhatching andaminorityof those infected at 1dayafterhatchingshowedatransient anti-coreantigenhumoralresponse;

thisresponse was stronger in theantibody-positiveducks infected onday 8 than in those infected onday1. Antiviral antibody was not detected in the sera of ducks congenitally infected with duck hepatitis B virus.

Several of the infected ducks, but none of the uninfected ducks, exhibited autoantibody reactivity for a-islet-cell-associated antigen.

Muchofthehepatocellulardamageassociatedwith hepa-titis B virus infection has been ascribed to the immune response to viral antigen (9). Despite much clinical interest in this pathology, none of the several animal models of hepatitisBinfectionhasbeenwidely used forimmunological analysis. We have, therefore, initiated a study of immune responsiveness in Pekin ducks infectedwith duckhepatitisB virus (DHBV).

Unlike other animal models ofhepatitis B infection, the DHBV system may be used for a comparison of immune responsiveness under conditions ofexperimental infection (postnatal virus inoculation [6]) versus

congenital

infection (virus transmission from the mother [7],resulting in ahigh levelofreplication inearly-stage embryos [4, 8]). Implicitin such acomparison is an assessment oftheimmunogenicity aswellasthepotentialtolerogenicity of viralantigen. As an initial step in thisanalysis,this studyfocusedonthehumoral response toDHBVinfection,examinedatthelevel of serum antibodyreactivity.

Twenty ducks inoculated intravenously with DHBV at 8 daysafterhatchingand 21uninoculated controls were main-tainedfor 16 weeks and monitored at 2-week intervals for serum antibody reactivity to DHBV antigen-positive cells. All ofthe infected ducks became viremic by 2 to 4 weeks postinoculation, asindicatedby a DNAhybridizationassay forviremia (5). Test sera were screened for antibody in an immunofluorescenceassay(described in the legend to Fig. 1) basedontheuseoffixed tissue sections. This form of assay was chosen because the preservation of tissue architecture facilitates testing of whether reactivity is directed to cell-associated antigen. Assays were carried out with sections preparedfrom pancreas tissue of either 2- to week-old or 3-to 4-month-old congenitally DHBV-infected or uninfected ducks. Previous work has shown that numerous scattered pancreasacinar cells in young, congenitally DHBV-infected ducks and a majority of the a cells localized toa islets in older, congenitally infectedducks are positive for both viral coreand surface antigens (1-3).

* Correspondingauthor.

Antibody reactive to viral-antigen-positive acinar cells was detectablein serafrom 16ofthe20 infected ducks but notinserafrom the uninfectedducks(representativeassays areshowninFig. 1).Reactivityfirstappearedat2to6 weeks postinoculation and generally persisted for 4 to 8 weeks before waning. Those serathatshowed strong reactivity to viral-antigen-positive acinar cells also showed reactivity abovebackgroundleveltobothviral-antigen-positiveot-islet cells(Fig. 2)andviral-antigen-positive hepatocytes (datanot shown). Reactivity to acinar cells or hepatocytes from uninfected ducks was not detected in sera from either infectedoruninfected ducks (datanotshown).

In tests of a-cell reactivity with sera from uninfected ducks,thelarge majority ofcells in

viral-antigen-positive

a islets showed no fluorescence above the background level observed with the section as a whole (Fig. 2C and D). However, aminority of cells, comprising from5 to20% of thetotal numberofcells in individuala islets, was fluores-cent tolevelsconsiderablyabovebackground(Fig.2C;asin all the assaysof duck serafrom uninfected ducks, fluores-cence was scored with a fluorescein-specific barrier filter because oftheuseofafluorescein-labeledgoatanti-chicken immunoglobulinserum). Asimilar percentage of fluorescent islet-associated cells was observed in assays ofsera from uninfected ducks as tested with pancreas sections from age-matched uninfectedducks(datanotshown). Most,ifnot all, ofthese cellsweredoublyfluorescent, scoring positively with either a rhodamine- or fluorescein-specific filter. Be-causeislet tissue in pancreas sections that were unreacted with any sera showed a minority of autofluorescent cells as scored with either filter, the use of the rhodamine-specific filter served to enumerate autofluorescent cells in tissue reactedwith fluorescein-labeled antisera. Since virtually all positivecells detected in pancreas tissue assayed with sera fromuninfectedducks and with the fluorescein-labeled anti-immunoglobulin serum were doubly fluorescent, it seems likelythat in these assays thefluorescence scored with the fluorescein-specific filterwasdue to autofluorescence rather thantoantibody reactivity.

Thedoubleimmunofluorescence assays(Fig. 1 and 2) did notin themselvesdistinguish between duck antibody recog-916

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FIG. 1. Serum antibody reactivity forviral-antigen-positive pancreas acinar cells. The two panels in each row show double immunoflu-orescenceassays of the same field ofexocrine pancreas cells from a 3-week-old congenitally DHBV-infected duck. (A and C) Serum from a10-week-oldDHBV-infected duck (infection at 8 days after hatching). (B and F) Rabbit anti-surface antigen serum (2). (D) Rabbit anti-core antigenserum(3). (E) Serum from a10-week-old uninfected duck. Tissue sections were prepared as previously described (2). The following order was used for the addition of each reagent (30 to 40 ,ul) to an individual section: (i) the test duck serum (diluted 1:3 with phosphate-buffered saline), (ii) rabbit antivirus serum (diluted 1:40 with serum from an uninfected duck and further diluted to 1:200 with phosphate-buffered saline), (iii) fluorescein-labeled goat antibody directed to chicken immunoglobulin (the affinity-purified goat antibody preparation was purchased from Kirkegaard & Perry, Gaithersburg, Md., and used at a concentration of 200 ,ug/ml; its reactivity in the immunofluorescence assaywasinhibitedbyacompeting preparation of duck immunoglobin), and (iv) arhodamine-labeled immunoglobin fraction of agoat anti-rabbitimmunoglobin serum (diluted 1:320 withphosphate-buffered saline). Bar equals 20 ,um.

nition of viral antigen or recognition of nonviral antigen induced as a consequence of infection. Nevertheless, in termsoftherelative levels of fluorescence associated with individual acinar cells (Fig. 1),thepattern of staining medi-atedbythe duckantibody paralleled that mediated bycore

antigen-reactive

but not by surface antigen-reactive rabbit antibody.These assaystherefore suggested that much, ifnot all, of the reactivity in thepositive duckserawasdirectedto viral core antigen.

To test this inference, preparations of DHBV surface

antigen particles

orimmature viralcores were

purified

from serum orliver,respectively, asdescribed

previously

(3),and wereassayed fortheircapacitytocompetewith the reactiv-ity of the positive duck sera. Whereas competition with surfaceantigen particles hadno discernibleeffect,

competi-tion with immaturecoresreduced the levelof cell-associated fluorescence to that ofthe

background

(representative

as-sayswith

viral-antigen-positive

acinar cellsareshown in

Fig.

3;dataarenotshownforassayswithviral-antigen-positive a cells or hepatocytes). We therefore concluded that, as scored in the immunofluorescence assay, the specificity of theduckantibodywas directedto viral coreantigen. How-ever, the

possibility

is not excluded that the absence of detectable antibody to viral surface antigen reflects the in vivocomplexing of antibodyratherthanthelackofimmune response.

Analysis was then extended toducks experimentally in-fected with DHBV at 1 day after hatching or congenitally infected with DHBV. Astested withseraobtained upto 16 weeks afterhatching,one or morebloodsamplesfrom 13of 30 ducks infected on day 1 exhibited

reactivity

for viral-antigen-positivepancreasacinar cells.Bycontrast,noblood samples from seven

congenitally

infected ducks exhibited suchreactivity (datanotshown),aresultconsistentwith the premise of immune tolerance.

For 12oftheducks infectedon

day

1whose serashowed

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918 NOTES

FIG. 2. Serumantibody reactivityforviral-antigen-positivea cells. Thetwopanels in each row show double immunofluorescence assays

ofthe samefield ofpancreas froma4-month-oldcongenitallyDHBV-infected duck. (A) Serum froma 10-week-old DHBV-infectedduck

(infectionat 8 daysafter hatching).(BandD)Rabbit anti-surfaceantigen serum.(C) Serum froma10-week-old uninfected duck. The large structures resolvedin panels B and Dcorrespondtoaislets,asshownbythereactivityofantiglucagonserumfor thesestructuresinadjacent

sections. Barequals 20 p.m.

C I

FIG. 3. Effectof competition with purified viral antigen on serum antibody reactivity for viral-antigen-positive pancreas acinar cells. The twopanels ineach row show doubleimmunofluorescence assays of the same field of exocrine pancreas cells from a 3-week-old congenitally

DHBV-infected duck. (A) Serum from a10-week-old DHBV-infected duck (infection at 8 days after hatching) in competition with purified

surface antigen particles(40

K.g/ml).

(B)Rabbit anti-surface antigen serum in competition with purified surface antigen particles. (C) Same serumasin panel A in competition withpurified immature viral cores (20 ,ug/ml). (D) Rabbit anti-surface antigen serum in competition with

purified immature viral cores. Bar equals 20p.m.

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A"*

FIG. 4. Serum antibody reactivity for pancreas cells. The two

panelsineachrow show doubleimmunofluorescence assays ofthe

samefield ofpancreascells.(A, C,E,G,and I)Serum from thefinal

blood sample (14 weeks postinfection) ofa DHBV-infected duck (infection at 1 day after hatching). (B, D, F, and H) Rabbit

anti-surface antigen serum and pancreas cells from a4-month-old

congenitally DHBV-infected duck(anaislet isshown) (B),exocrine

pancreasfroma3-week-old congenitallyDHBV-infected duck (D), pancreas cells from a 4-month-old uninfected duck (an a islet is

shown) (F), and pancreas cells from the duck that was used to

provide the test serum (an a islet is shown) (H). (J) Rabbit

antiglucagon serumandpancreascellsfrom the duckthatwasused

to provide the test serum. The antiglucagon serum has previously

been described (1) and, as used here, was diluted 1:5 with

phos-phate-buffered saline. Barequals 20 p.m.

demonstrable reactivity, antibodywasdetectable at2weeks

postinfection, and for the remaining duck, antibody was

detectable at 4 weeks postinfection. As with the ducks

infected at 8 days afterhatching, reactivity was blocked by

competition with purified preparations of immature viral

cores but not by competition with preparations of surface

antigen particles. In addition to the lower percentage of antibody-positive ducks among the group infected at 1 day

after

hatching,

two sets ofobservations indicated that the anti-core

antigen

humoral response was

considerably

weakerintheseducks than in theducksinfectedonday 8:

(i)

a more

rapid

waning of antibody production (8 of the 12 ducksinfectedonday1thatexhibited antibody reactivityat 2 weeks postinfection had lost detectable reactivity by 4 weeks postinfection) and (ii) a 2- to 3-fold-lower titer of

serum

antibody

at2weeks

postinfection

(ontheorder of1:3 forthe

antibody-positive

ducksinfected onday 1versus 1:6 or 1:9forthe

antibody-positive

ducks infected onday

8).

Assays fora-islet cellreactivitywerethencarriedoutwith sera obtained from the

ducks

infected on day 1 at times

subsequent

to the

waning

ofdetectable reactivity for

viral-antigen-positive

acinar cells. As tested with

viral-antigen-positive

islet

tissue,

mostofthese sera yielded apatternof a-islet-associated fluorescence indistinguishable from that obtained with sera from uninfected ducks. However, sera

obtained from one duck bothat 12 weeks postinfection and when theduckwaskilledat14weeks postinfectionexhibited reactivity to virtually all a-islet cells, including those not

detectably

viral

antigen

positive(Fig.4Aand B show results ofthe assay ofthefinal blood sample from this duck). This serumdid not exhibit detectable reactivity for

viral-antigen-positive

or

-negative

acinarcells

(Fig.

4C and D) or

hepato-cytes (datanot

shown).

The recognition by this duck serum of viral-antigen-negative a-islet cells suggested that reactivity was directed

to

antigen

expressed in uninfected a-islet cells. This

possi-bility was confirmed in assays of the serum with pancreas tissuefrom age-matcheduninfectedducks, with virtuallyall a-islet cells showing fluorescence above background level (Fig. 4E and F; positive fluorescence was detectable at serum dilutions of up to 1:81). The recognition of viral-antigen-negative a-islet cells in pancreas sections from the autologous duck

(Fig.

4Gand H) established that

reactivity

was mediated by an autoantibody. The reactivity ofrabbit antiglucagon serum for these target cells (Fig. 41 and

J)

served in turn to directly identify them as a cells. Under conditions ofcompetition with acommercial preparation of bovine glucagon (1), the reactivity for a cells ofthe rabbit antiglucagon serum but not ofthe autoreactive duck serum was blocked (data not shown).

Pancreas sectionsfromthe age-matched uninfected ducks were used to screen a large number of duck sera for a comparable reactivity. In addition to the duck described above,a second duckfrom the group of 30 infected at 1day after hatching exhibited serum antibody reactivity for DHBV-negative a cells; this reactivity was detected in the blood sample obtained at 14 weeks postinfection (serum antibody titer of 1:9) but not in earlier or subsequent samples. Antibody was also detected in the final sample obtained at 16 weeks after hatching from one of the 7 congenitally DHBV-infected ducks examined(antibody titer of 1:27) but in none of the samples obtained from the 20 ducks infectedon day 8. All blood samples fromuninfected ducks wereantibody negative, with the test sample compris-ing the following three groups: (i) 21 uninoculated controls described above, which weremonitored at 2-week intervals for up to 16 weeks after hatching; (ii) 30 ducks from the SPAFAS breeding flock, which were bled at 12, 16, and20 weeks after hatching; and (iii) 60 ducks from the SPAFAS flock, ranging in age from 7 to 12 months, which were bled once.

Thepresence of autoantibodyin a small percentage of the DHBV-infected ducks examined here and its absence inthe uninfected ducks tested raises the possibility that DHBV

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920 NOTES

infection isapredisposing factor for the induction of a-cell autoreactivity. If this suggestionproves correct, the DHBV system may represent a useful model for the study of the induction of autoimmunity in the contextofahepadnavirus infection.

WethankElsa Aglowfor preparations of the tissue sections and

Frank Southwelland Pat Frazerfor technical assistance.

Thsinvestigation wassupported byUnited States Public Health

Service grants AI-22065, CA-10815, AI-18641, CA-06551, and

RR-05539 from the National Institutes of Health andgrant185113

from theJuvenile Diabetes Foundation.

LITERATURE CITED

1. Halpern, M. S., J. Egan, W. S. Mason,andJ. M.England. 1984. Viral antigen in endocrine cells of the pancreatic islets and

adrenal cortex of Pekin ducks infected with duck hepatitis B

virus. VirusRes. 1:213-223.

2. Halpern,M.S., J. M. England, D. T. Deery, D. J. Petcu, W. S. Mason, and K. L. Molnar-Kimber. 1983. Viral nucleicacid

syn-thesis and antigenaccumulation inpancreasandkidneyofPekin

ducks infectedwith duckhepatitisBvirus.Proc.Natl.Acad. Sci.

USA80:4865-4869.

3. Halpern, M. S., J. M. England, L. Flores, J. Egan, J. Newbold, and W. S. Mason. 1984. Individual cells in tissues of

DHBV-infected ducks express antigens cross-reactive with those on

virussurface antigen particlesandimmatureviralcores.Virology

137:408-413.

4. Halpern, M. S., S. B. McMahon, W. S. Mason, and A. P. O'Connell. 1986. Viral antigen expression in the pancreas of

DHBV-infected embryos and young ducks. Virology

150:272-286.

5. Mason, W. S., C. Aldrich,J.Summers, and J. M. Taylor. 1982.

Asymmetric replication of duck hepatitis B virusDNAin liver

cells: free minus strand DNA. Proc. Natl. Acad. Sci. USA 79: 3997-4001.

6. Mason, W. S., M.S.Halpern, J. M. England,G. Seal, J. Egan, L. Coates, C. Aldrich, andJ. Summers. 1983. Experimental

trans-mission of duck hepatitisBvirus. Virology131:375-384.

7. O'Connell, A. P., M. K. Urban, and W. T. London. 1983.

Naturally occurring infection of Pekin duck embryos by duck

hepatitisB virus. Proc. Natl. Acad. Sci. USA80:1703-1706.

8. Urban, M. K., A. P. O'Connell, and W. T. London. 1985.

Sequenceofeventsin natural infection of Pekin duckembryos withduck hepatitis Bvirus. J. Virol. 55:16-22.

9. Vyas, G. N.,J. L.Dienstag,andJ.H.Hoofnagle (ed.).1984. Viral

hepatitisand liver disease. Grune& Stratton, Orlando,Fla. J. VIROL.