ICANCER RESEARCH 48, 1319-1325, March 1, 19881 Geographical Pathology of Duck Livers Infected with Duck from Chiba and Shimane in Japan and Shanghai in China'

Toshikazu Uchida,2 Koyu Suzuki, Masahiro Arli, Toshio Shikata, Ryo Fukuda, and Yixun Tao First Department ofPathology, Nihon University, School ofMedicine, Tokyo,Japan IT U., K. S., M. A., T. 5.1; Department ofMedkine, Shimane Medical &hool, Shimane, Japan IR. F.); and Department ofLaboratory Sciences,Shanghai SecondMedical College,Shanghai, China IT. T.J

ABSTRACF lular carcinomas after a long period of chronic host infection, and viral I@NA sequences are frequently integrated into the In order to evaluate geographicaldifferencesin the liver pathologyof genomes of carcinoma cells (3, 8, 9). The hepatocellular carci ducks Infected with duck (DHBV), ducks in Chiba and nomas are usually associated with chronic hepatitis or cirrhosis Shhnane4Japan,and Shanghai, China, were investigated.The numbers (DHBV positIve/negative)andthe maximumageof the ducksexamined in the noncarcinomatous area oflivers of virus-carrier hosts (3, were 18/10 at 19 IflO, 15/I at 3 yr 4 mo, and 72/27 at 18 mo, respectively. 8, 9). DHBV infectionwas InducedexperImentallyinducks fromChibaand In contrast to mammalian hepadnaviruses, the occurrence of Shimane but was present congenitally in those from Shanghai. Ducks pathological changes due to DHBV infection is controversial. were examined regarding liver function tests, conventional histology, Actually, in our construct of persistent DHBV infection the immunohistology, electron microscopy, and molecular hybridization for ducks did not show any significant hepatitis activity histologi DHBV DNA in the serumandliver. cally (6), which conflicts with the reports of other laboratories There was no significant difference between DHBV-positlve and (10—12).The latter reported that DHBV infection provoked -negativeducks in bilirubin and transaminaseand alkaline phosphatase chronic hepatitis, cirrhosis, and hepatocellular carcinoma. activities in the sen. Histologically, while the livers ofducks from Chiba and Shimane did not show necrolnflammatory(hepatitis) activity, those Interestingly, the most advanced liver disorders were reported fromShanghai frequentlydid (523%). Necroinflammatoryactivityof the in the ducks from Shanghai rather than in those from Japan Shanghaiducks was presentalmost equally in both DHBV-positive and and the United States. Therefore the possibility is raised that -negative livers. The livers ofShnngb*i ducks but not the other two areas the variation in the histological change ofDHBV-positive livers often (8.3%) had ground-glass inclusions which corresponded ultrastruc may reflect the difference in strains of ducks, the presence of turally to numerous virus particles in the dilated clsternae of the prolif some subtype of DHBV, or environmental factors, including erated endoplasmic reticulum. No advanced liver disease, such as clrrho hepatotropic agents and diet. The present study was performed sis or hepatocellular carcinoma, was observed. There was no significant to investigate the putative differences in liver function, liver difference in the amount of DHBV DNA in the sera or in Its pattern in morphology, and virology between ducks from Japan and the liver tissue among ducks of the three areas. In addition, the livers of Shanghai. Chiba ducks frequently had amyloidosis, while those of Shanghai ducks werecontaminatedwith parasites. In conclusion,DHBV infectiondid not appearto provokesignificant MATERIALS AND METhODS hepatitis activity or advancedliver diseaseIn the examinedducks of all three areas, and the DHBV-positivelivers from Shanghai ducks showed Ducks and the Transmission of DHBV a differentmorphologicalappearancefromthoseof the othertwoareas. This variation might reflect the difference in the strain ofducks, subtypes DucksfromChiba and Shimane.The ducks used werewhite-feathered of DHBV, environmentalfactors,or a combinationofthese influences. Pekin group (Anas domesticus) adults weighing ‘@-3kg.The ducklings were purchased immediately after hatching from commercial farms in Chiba Prefecture (Chiba ducks) and Shimane Prefecture (Shimane INTRODUcTION ducks). Chiba is located in central Japan, and Shimane is in the South. The farms showed an absence ofDHBV infections. The ducklings were DHBV3 was discovered in 1980 (1) and constitutes a member inoculated i.v. with 50 @zlor25 @lofDHBV-positive serum (“-4xiO@ of the family of hepadnavirus (2) which also includes the complete virions per ml as estimated from DHBV DNA hybridization hepatitis B virus, woodchuck hepatitis virus (3), and ground assay).This inoculation procedure on the same day as hatching pro squirrel hepatitis virus (4). Infection ofDHBV in the ducks was duced 100% successful persistent viremia. Negative control ducklings achieved by congenital transmission from DHBV-positive were not inoculated with any serum. mother ducks to their offspring (5), or by experimental inocu The DHBV-positive serum for the Chiba and Shimane ducks was lation of DHBV-positive serum into the ducklings within 3 originallyderivedfromPekinducksfrom Philadelphia,PA,andTaipei, days after hatching (6, 7). The experimental inoculation of Taiwan, respectively. The ducks were first kept indoors in cages for 1 DHBV in the duckling 5 days after hatching resulted in tran to 3 mo and then outdoors, and they were fed ad libitum a standard sient viremia associated with mild acute hepatitis features (7). commercial diet. They were sacrificed at ages of from 14 to 18 mo for Chiba ducks and 3 yr to 3 yr 4 mo for Shimane ducks. Mammalian hepadnaviruses are known to cause hepatocel The serawereassayedfor DHBV DNA and liverfunctiontests, and the tissues,forDHBV DNA, conventionalhistochemistry,immunohis Received4/20/87; revised 11/23/87; accepted 12/3/87. tochemistry, and electron microscopy. The costsof publicationof this article were defrayedin part by the payment Shanghai Ducks. With light brown feathers, these 1.5-kg adults are of pagecharges.This article must thereforebe herebymarkedadvertisementin locally known as “eggducks.―Ninety-nine ducklings aged 12 to 18 mo accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. were purchased from a farmer in suburban Shanghai, where they had I This research was supported by Japanese Ministry of Education Grant (C) 59570l60and by the Foundation for the Promotion ofCancer Research,Japanese been kept outdoors. They were Sacrificed, and their sera and tissues Ministry ofHealth and Welfare. were used as described above. 2 To whom requests for reprints should be addressed, at Department of Pathology, Nihon University, School of Medicine, 30-1 Oyaguchi-kamimachi, DHBV DNA Assay Itabashi-ku, Tokyo 173, Japan. 3 The abbreviations used are: DHBV, duck hepatitis B virus SSC, standard saline citrate; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic Serum was used for determining DHBV DNA by spot hybridization transaminase;PBS,phosphate-bufferedsaline. assayusing cloued DHBV DNA (donated by Dr. William Mason) with 1319

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. GEOGRAPHICAL PATHOLOGY OF DHBV the modification of Scott et a!. (13). The entire DHBV genome was RESULTS subclonedfrom Charon 27-RI (14), and then a 32P-labeledprobeof DHBV was prepared by nick (15) with (a-32PJdCTP(800 DHBV DNA Assay Ci/mmol; Amersham, United Kingdom). Among 19 Chiba ducks inoculated with DHBV, 18 showed A sample of 5 @lofserum was mixed with 5 @tlof2 MNaCI and then DHBV DNA in their sera, and all 10 uninoculated ducks did with 10 ,@lof I M NaOH, followed by application to a nitrocellulose not. One duck appeared to have lost evidence of DHBV DNA, filter previously soaked in 20x SSC (lx SSC = 0.15 M NaCl:0.0l5 M sodium citrate, pH 7.0). The nitrocellulose filter was dried and kept at and it was excluded from the DHBV-positive group. Similarly, 80'C under a vacuum for 2 h, prehybridized at 42'C for 4 h, and then all 15 Shimane ducks that had received DHBV inoculation hybridized at 42'C overnight using 2 x l0@cpmof 32P-labeledDHBV revealed DHBV DNA in their sera, while one duck that was DNA (specific activity, 1 x l0@cpm/@g)in 4 ml for 50% formamide, not inoculated was negative for DHBV DNA. Among 99 5x SSC, 5x Denhardt's solution, and 100 @tg/mlsonicated salmon Shanghai ducks, 72 showed DHBV DNA in their sera. The sperm DNA. The filter was then washed 3 times for 10 mm each in amount of DHBV DNA was quite variable among the ducks, 0.lx SCC-0.2% sodium dodecyl sulfate at 50'C. The dried filter was and it ranged from 5 to more than 500 pg/S @lofserum (data autoradiographedat—70'CusingKodak XAR-5 film. The detailed not shown). There was no significant difference in the amount procedure was described previously (6). The results were expressed of DHBV DNA among the ducks of the three different areas. semiquantitatively by comparing the intensity of the standard signals. All the serum samples obtained were subjected to DHBV DNA mess A Southern blot analysis of the livers of 4 ducks (including 3 urement. from Chiba with hyperplastic focal changes described later), 6 Liver tissues were used for a Southern blot hybridization analysis from Shimane, and 6 from Shanghai showed almost the same (16) of DHBV DNA. Total cellular DNA was extracted by treating the pattern of DHBV DNA (data not shown). There were bands tissue overnight with a 20-fold volume of lysis buffer, consisting of 1 corresponding to relaxed circular and linear duplex forms and mM Tris-HC1 buffer (pH 8.0), 50 mM EDTA, 0. 1 M NaCl, 1% sodium smears consistent with replicative intermediates (1). There was dodecyl sulfate, and 100 @ig/mlproteinase K. followed by repeated no smear in the high-molecular region in the lanes with and phenol/chloroform extraction and RNase digestion. The purified DNA without restriction enzyme digestion, and no special band was (10 pig),undigested and digested with restriction enzymes, was separated noted in the lanes after PvuII digestion. The pattern of Southern by electrophoresis in a 1.0% agarose slab gel. X-HindIII DNA fragments blotting was not indicative of the integration of DHBV DNA and cloned DHBV DNA were used to measure the size of DHBV sequences into the host cellular genome. DNA. After electrophoresis the gels were stained with ethydium bro mide. The DNA in the gel was blotted onto nitrocellulose filters by the technique of Southern blot transfer, and they were dried and kept at Liver Function Tests 80C undera vacuumfor 2 h. The nitrocellulosefilters weresubse quently processed as in the spot hybridization. Four Chiba ducks, 6 Data from the ducks of all three areas are given in Table 1. Shimane ducks, and 6 Shanghai ducks with DHBV viremia were used Although statistical analysis was not performed for the Shimane to examine the pattern of DHBV DNA in the tissues. ducks because of the small number that were DHBV negative, Liver FunctionTests no significant difference was recognized between DHBV-posi tive and -negative ducks of the other two areas. There were Sera were examined for bilirubin and GOT (Karmen units), GPT some individual variations found among the ducks themselves. (Karmen units), and alkaline phosphatase (IU) activities.

ConventionalHistochemistry Conventional Histochemistry Pieces of the liver were fixed in 10% neutral formalin and embedded Chiba Ducks. On gross examination, all livers were found to in paraffin after dehydration. The sections were stained with hematox have a smooth surface with no tumorous or cirrhotic changes. ylin-eosin, orcein (17), and/or Victoria blue (18). Histologically, the livers of 4 among 18 DHBV-positive and 2 Immunostaining for DHBV among 10 -negative ducks on occasion showed minute vague bulging areas that were composed of deeply eosinophilic hepa Formalin-fixed and paraffin-embedded thin sections were incubated tocytes (Fig. 1). The changes were not accompanied by distor with anti-DHBV (donated by Dr. William Mason), produced in rabbits, tion of the lobular architecture, and they closely resembled the in a 1:200 dilution in 0.01 M PBS (pH 7.2) at 4C overnight, and then hyperplastic foci seen in rat livers in the course of chemical with anti-rabbit immunoglobulin goat IgG conjugated with horseradish peroxidase (Miles Yeda, Israel) at room temperature for 1 h in a 1:500 hepatocarcinogenesis (19). dilution in PBS. Anti-DHBV has been well characterized previously Neither DHBV-positive nor -negative ducks revealed nec (6). As a control, anti-DHBV was previously absorbed with DHBV roinflammatory activity of the parenchyma or fibrosis, but a positive or -negative serum. The slides were treated with 0.05% 3,3- portal lymphocytic reaction occurred in 6 of the former and 4 diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in of the latter groups. Necroinflammatory activity indicates the PBS for visualization of the reaction product, and they were subse histological feature of hepatitis activity characterized by hepa quently counterstained with hematoxylin. Immunostaining was per tocellular necrosis with lymphocytic infiltration of variable formed for 6 ducks (4/2, DHBV positive/negative) from Chiba, 4 (3/ intensity. 1) from Shimane, and I2 (5/7) from Shanghai. Amyloidosis was present in 7 DHBV-positive and 3 DHBV Electron Microscopy negative ducks. Otherwise the livers revealed nonspecific changes. Neither DHBV-positive nor -negative ducks were pos Pieces of the liver (-“1mm3)were fixed in cold 1% osmium tetroxide in Millonig's solution (jH 7.5) and embedded in Epon 812 after itively stained by orcein and/or Victoria blue. Table 2 shows a dehydration. Ultrathin sections were double stained by uranyl acetate summary of the liver pathology. and lead citrate and then observed with a JEM-100C electron micro Shimane Ducks. Gross and microscopic examinations of the scope. The ultrastructural study involved 4 (3/1, DHBV positive! livers of all ducks revealed no significant changes, including negative)Chiba ducks and 7 (6/1) Shanghai ducks. tumors, cirrhosis, necroinflammatory activity, hyperplastic fo cus-like alterations, fibrosis, or portal inflammatory reactions. Statistical Analysis Unexpectedly, one DHBV-positive duck had amyloidosis. All Data were analyzed statistically using Student's I test. livers were negative for orcein and/or Victoria blue staining. 1320 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. GEOGRAPHICAL PATHOLOGY OF DHBV

areasChiba Table 1 Serum liverfunction testsofducksfrom three mo oldStatistical 10)Total ducks14—19 analysisDHBVpositive(n = 18) DHBV negative(n = (0.1—0.9)NSCGOTbilirubin(mg/dl)0.23 ±0.12―(0•o@o•5)b 0.34 ±0.23 (13—98)NSGPT(Karmen units)33.7 ±26.8(11—100) 34.7 ±33.3 (8—30)NSAlkaline(Karinen units)21.1 ±12.7(9—59) 16.4 ±6.6 phosphatase(IU)135.8(80—281)NSShimane ±100.2(55—479) 133.3 ±59.3

oldDHBV yr—3yr4 mo 1)GOT47.0 ducks3 positive (n = 15) DHBV negative(n = 39GPT23.4±6.4(15—40) ±21.0(24—96) 19Shanghai

oldDHBV mo 27)Total ducks12—18 positive (n = 72) DHBV negative(n = (0.0—0.9)NSGOT33.3bilirubin0.21 ±0.21(0.0—1.3) 0.23 ±0.22 (10—56)NSGPT33.3 ±I 1.8 (15—72) 28.7 ±10.0 (7—58)NSAlkaline ±11.4 (13—69) 32.7 ±13.0 phosphatase268.4 ±217.9(23—1119) 236.6 ±216.7(41—911)NS

a Mean ±SD. b Numbers in parentheses, range. C NS, not significant.

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Fig. I. Hyperplastic focus-like changecomposedofrelatively large hepatocytes Fig. 2. The liver of this Shanghai duck (5-53) has a well-defined tumorous with deeplystainedeosinophiliccytoplasm.(Chibaduck,BP-18; H & E, x 200). lesion(arrows),whichshowshistologicallygranulomatousinflammation.

Table 2 Summa,y ofliver histopathologyforChibaducks Table 3 SummaryofliverducksDHBVhistopathologyforShanghai DHBV positive DHBV negative Statistical negativeStatistical(npositiveDHBV (n = 18) (n = 10) analysis 27)analysisParenchyama = 72)(n = Hyperplastic(20.0)NSbchangesLymphocytic focus-like4 (22.2)a2 (48.1)NS―tivityGround-glassNecroinflammatory ac 39 (54.2)a13 (40.0)NSportalreaction of6 (33.3)4 tractsAmyloidosis6 (8.3)0(0)Positivecells7 (33.3)3 (30.0)NS (12.5)0(0)Victoriaorcein/9 aNumbersinparentheses,percentage. bluestainCoagulative b NS, not significant. (0)Granuloma3necrosis1 (1.4)0 (18.5)Parasites6 (4.2)5 (22.2)Portal (8.3)6

tractsLymphocytic Shanghai Ducks. Gross examination revealed an extensive, (100.0)NSFibrosis43reaction72 (100.0)27 anemic coagulative necrosis in one duck's liver and granuloma (66.7)NSDuctal (59.7)18 tous inflammation (Fig. 2) in another. There were no tumorous proliferation38 (52.7)14 (51.9)NS or cirrhotic changes. aNumbersinparentheses,percentage. b NS, not significant. As summarized in Table 3, about half of both the DHBV positive and -negative livers histologically exhibited necroin The parasitic infections appeared to be related to granuloma flammatory activity in the parenchyma (Fig. 3). All livers had and coagulative necrosis. lymphocytic exudation in the portal tracts, half of which were Approximately one-tenth of the livers of DHBV-positive associated with slight fibrosis and proliferation of the bile ducks had ground-glass cells (20) uniformly scattered through ductules, irrespective of DHBV infection. The overall changes out the lobules, without conspicuous clustering (Fig. 5). These somewhat resembled chronic active hepatitis in the human liver. cells were positive for orcein and/or Victoria blue staining. The In addition, many livers had parasites (Clonorchis or Acantho DHBV-negative ducks showed no such cells, and they were cephala) in the portal veins or interlobular bile ducts (Fig. 4). negative for orcein and/or Victoria blue. 1321 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. @@@@ .@ ‘S@@ S ‘ :@

GEOGRAPHICAL PATHOLOGY OF DHBV

of interlobular bile ducts (Fig. 6). The hyperplastic focus-like r.@1@t.:@•‘;‘‘S@‘@changes were occasionally stained more strongly (Fig. 7); how ever, the staining was uniform overall without conspicuous focal @,S@- : or patchy clustering or specially accentuated regions of the @M,:.:@•S. cytoplasm. The nuclei were consistently free of staining. Prior absorption of antisera with DHBV-positive but not with -negative sera removed the staining; thus staining was con firmed to be specific. Shanghai Ducks. All 5 ducks which were DHBV DNA posi tive in the sera showed positive staining, whereas 7 DHBV DNA-negative ducks did not. Staining was present in the cy toplasm of all hepatocytes and many biliary epithelial cells, .@ a@•• similar to the Chiba and Shimane ducks. The staining was diffuse and uniform without an irregular distribution from area @ a ‘ -S , -@ ‘ , •@5) .,- to area (Fig. 8). Distinct from the Chiba and Shimane ducks, the livers of the Shanghai ducks showed stronger staining in Fig. 3. There are a focal necrosis and lymphocytic infiltration in the paren general, with an occasional accentuation of a localized portion chyma. The left corner reveals a portal tract. (Shanghai duck, S-88, DHBV of hepatocyte cytoplasm, which corresponded to the ground negative;H & E, x 200). glass appearance (Fig. 8, inset). There was no amyloidosis. Electron Microscopy The livers of 3 Chiba and 6 Shanghai ducks that were positive for DHBV DNA ultrastructurally had virus particles in the .;@g hepatocytes. The hepatocytes contained some envelope (surface antigen) particles and/or empty virions, 35 to 60 nm in diameter

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Fig. 4. A parasite consistent with Clonorchis is present in the large bile duct. S@ (Shanghai duck, S-56; H & E, x 100).

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Fig. 6. Indirect immunoperoxidase method for DHBV revealsstrong staining in the interlobular bile ducts(arrow)and weaker staining in all hepatocytes(Chiba duck, BP-35; counterstained by hematoxylin, x 400).

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@ ‘ Fig. 5. Scattered hepatocytes with a ground-glass appearance (arrows). (Shang hal duck, S-62; H & E, x 400).

Immunostaining for DHBV @. S ,@; . Chiba and Shimane Ducks. All 4 livers from Chiba ducks and 3 livers from Shimane ducks that were DHBV DNA positive in the sera showed positive immunostaining, whereas one Shi mane and 2 Chiba DHBV-negative ducks did not. In the posi Fig. 7. A hyperplastic focus-like change (arrowheads) is somewhat more tive livers, DHBV stained diffusely and relatively weakly in the strongly stained by the immunoperoxidase method for DHBV. (Chiba duck, AP cytoplasm of all hepatocytes, and strongly in the epithelial cells 26; counterstained by hematoxylin, x 100). 1322 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. @@@@@@@@@@@@@@@@@@@@@@@ . —@,@ S. • : \k@ @‘S •@J, @,•S@b ‘SS@5@ @S

GEOGRAPHICAL PATHOLOGY OF DHBV

(Fig. 9), and a few had complete virions, 40 nm in diameter in the DHBV-positive or -negative ducks from the three areas. the cisternae of the endoplasmic reticulum and cores and 27 The Chiba ducks showed multicentric hyperplastic focal nm in diameter around the peroxisomes. Compared with the changes characterized by bulging areas composed of eosino livers of the Chiba ducks, those of the Shanghai ducks showed philic hepatocytes, and some of these were relatively strongly apparently increased numbers of incomplete virus particles in stained for DHBV. These changes resembled the irregular the remarkably dilated cisternae of the endoplasmic reticulum, regeneration ofchronic active viral hepatitis in the human liver; and these were especially conspicuous in the ground-glass however, they were recognized in both the DHBV-positive and containing hepatocytes (Fig. 10). The virus particles sometimes -negative livers. Therefore they seemed unrelated to DHBV appeared to be located in the hyaloplasm outside the endo infection and may be induced by some toxin or food contami plasmic reticulum of the cells that showed the ground-glass nant. appearance. The DHBV-negative livers had no virus particles Both the DHBV-positive and -negative ducks from Shimane seen by electron microscopy. did not show histologically any necroinflammatory activity or hyperplastic focal changes, even though they were older than DISCUSSION the Chiba ducks. It is likely that the Shimane ducks were fed under better nutritional and hygienic conditions, as suggested In the present investigation there were no advanced liver by their lower frequency of amyloidosis. It has been reported disorders such as cirrhosis or hepatocellular carcinoma in either that white Pekins have a genetic predisposition for amyloidosis, which is expressed more frequently under strong social and environmental stress (21). @r The brown-colored Shanghai ducks were of a different strain @ that those from Japan. b Histological differences were also distinct. The Shanghai @ , @4. ducks frequently had hepatitis-like features, characterized by @@@ : @. necroinflammatoryactivityintheparenchymaandlymphocytic :,tS4 . exudation, fibrosis, and ductular proliferation of the portal @5S @ @S tracts. These changes, however, were seen in both DHBV

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‘SI regard, parasitic infections are worthy of mention, since para @ S @- , S • @5 : sites such as Clonorchis sinensis (22) and Schistosomiasisjapon @@ ,@ ica (23) induce fibrosis and cirrhosis, which could possibly lead [email protected] , . eventually to hepatocellular carcinoma. Twelve % ofthe Shang \@ ‘S •@4@t@@\ S@ @ SI hai ducks had infection by parasites which can induce infarction @ and granulomatous inflammation. This parasitic infection pres Fig. 8. Indirect immunoperoxidasestainingfor DHBV. The interlobularbile ence suggests a high probability of the participation of various ducts(armwhead)arestronglystained,while hepatocytesareoverallweakly hepatotropic agents in the environment of the duck liver. Fur stained. There is individual scattering of hepatocytes with strong cytoplasmic staining (arrows). Inset, high-power view of hepatocyteswith ground-glass inclu thermore, many other agents, including aflatoxins, which were sions.(Shanghaiduck,5-62; counterstainedbyhematoxylin,x 200). present in large amounts in Chi-tung County in China (24),

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contaminated the food, which can also produce hepatitis-like The pathological features of the ducks infected with DHBV changes.4 in our study differed from those reported by other laboratories Although the ducks of all three areas thus did not present (10—12).In fact, no hepatocellular carcinomas were recognized any conspicuous hepatitis activity in relation to DHBV infec in the present investigation. This may be due to age differences tion, the possibility is raised that minimal hepatitis beyond our of the ducks observed, the effect of other hepatotropic agent(s), recognition was present. The slightly elevated levels of trans or differences in the criteria for diagnosing pathological aminase activity in DHBV-positive ducks, as compared to changes. -negative ones, though not statistically significant, may reflect In particular, aging is an important factor in carcinogenesis. this occurrence. Humans present stronger hepatitis activity at Since humans, for example, develop hepatocellular carcinoma the phase of e antigen than that of anti-c in chronic hepatitis B most frequently in the sixth decade after perinatal infection virus infection (25). With DHBV, e antigen/anti-c status is not with the hepatitis B virus, more than 5 yr of observation may well characterized, and this fact makes the histology of the be required to detect possible hepatocarcinogenesis in the DHBV-infected liver more unclear. DHBV-infected ducks, which have a life expectancy of about The livers of Shanghai ducks differed strikingly from those 10 yr. of Japan in the occurrence of the ground-glass cells. The ground-glass appearance corresponded to the inclusion of ACKNOWLEDGMENTS DHBV, especially of its envelope component, and this was confirmed by positive orcein and Victoria blue staining, positive We are grateful to Dr. William S. Mason for providing DHBV immunostaining for DHBV, and the ultrastructurally localized positive sera, antisera against DHBV, and cloned DHBV DNA. We accumulation of abundant surface antigen virus particles in the also wish to thank T. Irikura and K. Tajima for their technical assis dilated cisternae of the endoplasmic reticulum. The ground tance. glass cells of the Shanghai ducks were quite similar to those of the hepatitis B virus (20), although the filamentous structure REFERENCES but no virus particles were seen in the proliferated smooth 1. Mason, W. S., Seal, G., and Summers, J. Virus ofPekin ducks with structural endoplasmic reticulum in the latter (26). The distribution of andbiologicalrelatednesstohumanhepatitisB virus.J. Virol., 36: 829—836, the ground-glass cells was widely scattered and uniform 1980. 2. Robison, W. S. Genetic variation among hepatitis B and related .Ann. throughout the lobules, and it was consistent with that seen in N. Y. Acad. Sci., 354: 371—378,1980. healthy carriers of the hepatitis B virus in an e antigen-positive 3. Summers,J., Smolec,J. M., and Snyder,R. A virus similar to hepatitisB virus associatedwith hepatitis and hepatoma in woodchucks. Proc. Natl. phase.4 Acad.Sci. USA, 57: 4533—4537,1978. The variation in the histological pattern of DHBV-infected 4. Marion, P., Oshiro, L S., Reghery,D. C., Scullard,G. H., and Robinson, livers among the three areas may be due to (a) differences in W. S. A virusof Beecheygroundsquirrelsthatis relatedto hepatitisB virus ofhumans. Proc.NatI. Acad.Sci. USA, 77:2941—2945,1980. the subtype of DHBV involved in the Chiba ducks (originally 5. O'Connel, A. P., Urban, M. K., and London, W. T. Naturally occurring from Philadelphia), Shimane ducks (from Taipei), and Shang infection of Pekin duck embryos by duck hepatitis B virus. Proc. NatI. Acad. hai ducks (in fact, the two complete DHBV DNA sequences Sci. USA, 80: 1703—1706,1983. 6. Uchida, T., Suzuki, K., Esumi, M., Arii, M., Oomura, M., and Shikata, T. that were revealed varied by 5.6%) (27, 28); (b) differences in Occurrence and ultrastructural localization of duck hepatitis B virus in the the strains of the ducks; (c) differences in the animals' exposure liver ofducks after experimental infection. Hepatology, 7: 29—36,1987. 7. Fukuda, R., Fukumoto, S., and Shimada, Y. A sequentialstudy ofviral DNA to hepatotropic toxins; their diet; or a combination of these in the serum in the experimental transmission of duck hepatitis B virus. J. factors. Med. Virol., 21: 311—320,1987. 8. Brechot,C., Pourcel,C., Louise,A., Rain, B., and Tiollais, P. Presenceof 4 Unpublished data. integrated hepatitis B virus DNA sequencesin cellular DNA of human 1324 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. GEOGRAPHICALPATHOLOGYOF DHBV

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1325 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. Geographical Pathology of Duck Livers Infected with Duck Hepatitis B Virus from Chiba and Shimane in Japan and Shanghai in China

Toshikazu Uchida, Koyu Suzuki, Masahiro Arii, et al.

Cancer Res 1988;48:1319-1325.

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