Journal of General Virology (1991), 72, 417--420. Printed in Great Britain

417

Natural and experimental infection of wild mallard ducks with duck hepatitis B virus V~ronique Lambert, 1 Lucyna Cova, 1. Philippe Chevallier, ~ Raj Mehrotra 2 and Christian Trepo 1 I l N S E R M Unit4 271, 151 Cours Albert-Thomas, 69424 Lyon Cedex 03, France and 2Department of Pathology, King George's Medical College, Lucknow, India

Wild duck populations were investigated over a 4 year period for duck hepatitis B virus (DHBV) infection and liver disease. It appeared that DHBV is endemic in wild migratory mallards from France and the U.S.A., although neither hepatocellular carcinoma nor viral DNA integration could be detected in liver samples examined. The follow up of natural infection indicated that wild mallards developed significantly higher serum titres to DHBV DNA than Pekin ducks. The results of experimental transmission demonstrated that such differences in viraemia were not related to the breed of ducks but to the virus isolate, since the wild

mallard-isolated D H B V (DHBVwM) induced significantly higher viraemia in both mallard and Pekin ducklings compared to the domestic Pekin D H B V (DHBVP) isolate. The naturally infected mallard and Pekin ducks had only minor histological lesions of the liver compared with experimentally infected birds. There was no correlation between the intensity of viraemia and the severity of liver lesions, suggesting that as for mammalian hepadnaviruses the hepatic injury in DHBV-infected ducks is probably immunologically mediated.

Biological study of human hepatitis B virus (HBV), the prototype member of the hepadnavirus family, is limited by the narrow host range of this virus and failure to infect cell lines in culture. The closely related duck hepatitis B virus (DHBV), commonly found in Pekin ducks (Arias domesticus), has played a major role in the characterization of hepadnaviruses' replication and biology (reviewed by Schfdel et al., 1989). In contrast to mammalian hepadnaviruses, which are known to cause hepatocellular carcinoma (HCC) after a long period of chronic infection (Popper et al., 1987; Marion et al., 1986), the oncogenicity of DHBV is unclear. To date, HCC has been reported only in domestic Pekin and brown ducks from a single region of China (Zhou, 1980; Yokosuka et aL, 1985), whereas DHBV carrier Pekin ducks in other parts of the world do not develop HCC (Marion et al., 1984). These geographical variations in the prevalence of HCC in ducks may reflect the differences in duck breed, age, DHBV strain, and environmental factors such as dietary aflatoxins (Cova et al., 1990). We have previously demonstrated the presence of DHBV in wild mallard (Anas platyrynchos) ducks (Cova et al., 1986). With our interest directed towards defining the oncogenic potential of DHBV, we investigated wild duck populations over a 4 year period for signs of HCC and liver disease with relation to DHBV infection. In

addition, as little is known of the biological properties of hepadnavirus isolates, it was of interest to characterize further DHBV isolated from wild mallard ducks (DHBVwM). In this study, we report that DHBVwM induces unusually high viraemia titres in its natural host and compare its pathogenic potential with that of other DHBV isolates. To investigate the wild duck populations for DHBV infection and liver disease, sera and liver samples originating from wildlife reserves and from ducks shot by hunters were collected in France and U.S.A. Out of the 531 wild ducks' serum samples obtained from French wildlife reserves collected between 1984 and 1990, 54 (10"2~o) were positive for DHBV D N A (Table 1). The frequency of DHBV infection varied in different years, from 1 to 20 ~. DHBV was also detected in two out of 130 necropsy liver samples from ducks shot by hunters and in 1.8 ~ of wild mallards originating from north-east U.S.A. Altogether, these results demonstrate that DHBV is endemic in the wild duck populations. A closely related virus, the heron hepatitis B virus has been isolated recently from grey heron living in the wild (Sprengel et al., 1988). In addition, DHBV had been also detected in wild maned ducks (Chemonettajubata) in Australia (R. J. Dixon, personal communication). Thus, wild birds may represent a natural reservoir of avian hepadnaviruses. Our investigation of liver samples from naturally

0000-9832 © 1991 SGM

418

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Table 1. Detection of D H B V in American and French wild duck populations Year 1979 1981

Origin Michigan* Michigan* Illinois*

1985

North Dakota* Dombes§ Sommel] Dombe§

1986

Dombes shot¶ Dombes§

1989 1990

Dombes§ Alsaceshot¶

1984

Number of birds 2 juvenile

Number DHBVpositive

~

Number of livers examined

1

50

MAt

10 adults

1

l0

NA

89 age ND~ 26 juvenile 27 adults

0 1 0

0 1"9 0

17 age

0

0

ND

20 adults 20 adults 20 adults 174 juvenile 105 adults 38 adults 84 juvenile 45 adults 25 adutlts

2 1 3 36 1 0 6 4 1

10 5 15 20.7 0.95 0 7.15 8.9 4

HCC

DHBV status in liver

-

-

-

-

NA NA

-

-

NA

--

-

0/2 0/2 0/6 0/1

Free Free Free Free

2 NA 2 6 1 NA NA NA 1

-

-

0/1

-

Free

* Ducks from north-east U.S.A. as part of a study of wild waterfowl undertaken by the American National Wildlife Health Laboratory. t NA, Not available. :~ND, Not determined. § National wildlife reserve of Dombes. IINational wildlife reserve of Somme. ¶ Ducks shot by hunters in the Dombes lake district or in the Alsace district. infected wild mallard ducks revealed neither H C C nor viral D N A integration (Table 1). However, the limited number of samples available, of which the majority orginated from young birds, does not allow us to rule out the occurrence of H C C in wild mallards. H C C could develop in wild ducks, although this is difficult to test because surveying liver disease in waterfowl is hampered by the lack of systematic autopsy studies of birds which die either on their flyway or on reserve territory. The initial screening of ducks for D H B V D N A suggested that wild mallards exhibited higher serum levels of viral D N A than observed in Pekin ducks (data not shown). It was therefore of interest to compare further the course of viraemia in wild and domestic birds. Wild mallard eggs were collected in the reserve of Dombes, France and hatched ducklings were screened for D H B V infection. Pekin ducklings congenitally infected with D H B V were a progeny of a flock of D H B V carrier ducks maintained at our institute (Cova et al., 1985). The two groups of 1-week-old D H B V carrier mallard (14 birds) and Pekin (12 birds) ducklings were individually monitored for D H B V D N A in serum over a 1 year period. To quantify D H B V D N A , 50 ~tl of duck sera and cloned D H B V D N A ranging from 100 ng to 0.1 pg were spotted in duplicates onto nitrocellulose filters (Cova et al., 1990) and probed with cloned, genome length, D H B V D N A labelled by nick translation (Rigby et al., 1977) to a specific activity of 0-8 to 1.2 x 10 s c.p.m./~tg. The filters were hybridized overnight with radiolabelled D H B V probe in 5 0 ~ formamide at 42 °C, as previously described (Cova et al., 1985), washed under

stringent conditions, dried, and liquid scintillation counting of each individual spot was done. From the curve derived from D H B V D N A blots, the counts obtained for each spot were converted into pg D H B V D N A and then to virus genome equivalents (v.g.e.), assuming each D H B V genome to be 3-3 × 10 - 6 pg (Jilbert et al., 1988). Fig. 1 indicates that at the peak of viraemia (1 to 5 weeks post-hatch), wild mallards scored significantly higher D H B V titres (P < 0.001), reaching 2 × 1011 v.g.e./ml for some animals compared to Pekin ducks for which titres did not exceed 3 × 101° v.g.e./ml by the same age. Decreased and fluctuating D H B V D N A titres were observed thereafter. All naturally infected ducks were chronic virus carriers 1 year posthatch and no D H B V D N A integration was observed in the liver samples analysed (data not shown). To assess whether the high viraemia titres observed in wild mallards were related to the greater susceptibility of wild birds to D H B V infection or to the higher infectivity of some viral isolates, an experimental transmission of three D H B V isolates (Lambert et al., 1990) orginating from a highly viraemic wild mallard ( D H B V w ~ ) , a Chinese brown duck (DHBVc) and a domestic Pekin duck (DHBVP) was therefore undertaken. Ten-day-old Pekin and mallard ducklings (12 to 15 birds for each D H B V isolate) were inoculated intravenously with DHBV-positive serum diluted to contain similar amounts of virus corresponding to 109 v.g.e./ml. Fig. 2(a) shows that at the peak of viraemia (day 8), Pekin ducklings inoculated with DHBVwM developed significantly higher ( P < 0 - 0 0 1 ) viraemia titres than

Short communication

250 ~

60

,

r

J

~

~

~

~

~

~

i

~

419

i

(a) 200

x

eh

1°°II111111 0

22

27

34

41

48

I ,55

62

69

1 76

120

160

180 ,200

0

~ 4

Time (days) Fig. 1. Comparative viraemia follow-up in mallard and Pekin ducks naturally infected with DHBV. Two groups of 1-week-old DHBV carrier mallard and Pekin ducklings were individually monitored for DHBV DNA in serum over a 1 year period. To quantify viral DNA 50 lal of duck serum was spotted onto nitrocellulose filters and probed with radiolabelled DHBV DNA probe followed by liquid scintillation counting of each individual spot as described in the text. Each column corresponds to the mean value + S.E.M. of serum virus genome equivalents/ml from ( i ) mallard (14 birds) and (IS])Pekin (12 birds) ducks.

6

7

~ 8

i 11

~ 18

i 25

~ 3

t 39

~ 46

~ 53

J 67

~ J 106 120 140

6O

(b)

~' x _~ d

4O

2O

those w h i c h r e c e i v e d D H B V P . I n m a l l a r d d u c k l i n g s D H B V w M also p e a k e d ( d a y 4) to significantly h i g h e r viral D N A titres t h a n D H B V P (day 8) (Fig. 2b). T h e v i r a e m i a in m a l l a r d d u c k l i n g s was r e m a r k a b l y s i m i l a r for D H B V c a n d D H B V w M , r e a c h i n g its highest titres 4 d a y s p o s t - i n f e c t i o n (Fig. 2b). T a b l e 2 shows t h a t the histological e x a m i n a t i o n o f liver s a m p l e s from highly v i r a e m i c m a l l a r d d u c k s n a t u r a l l y i n f e c t e d with D H B V (group 1) did n o t reveal significant liver lesions. This is c o n s i s t e n t with findings in h u m a n s a n d w o o d c h u c k s w h i c h i n d i c a t e t h a t the severity o f liver disease is not r e l a t e d to the level o f virus r e p l i c a t i o n , but to the host i m m u n e r e s p o n s e ( A l b e r t i et al., 1983; F r o m m e l et al., 1984). T h e a b s e n c e o f liver disease in d u c k l i n g s c o n g e n i t a l l y i n f e c t e d w i t h D H B V is s i m i l a r to c h r o n i c a s y m p t o m a t i c p e r i n a t a l H B V infection o f b a b i e s who usually do not d e v e l o p liver p a t h o l o g y . By contrast, the significant p o r t a l i n f l a m m a t o r y r e a c t i o n a n d necrosis w h i c h we o b s e r v e d in P e k i n as well as in

I1

7

18

25

32

39

46

53

67

Experimental infection

Degre•,•e

Group Ducks

DHBVP

DHBVwM

DHBVc

Controls

1 Mallard

2 Pekin

3 Mallard

4 Pekin

5 Mallard

6 Pekin

7 Mallard

8 Pekin

9 Mallard

10 Pekin

11/13 2/13 0/13

6/9 3/9 0/9

2/5 2/5 1/5

4/6 0/6 2/6

2/6 2/6 2/6

4/6 1/6 1/6

4/5 1/5 0/5

4/6 0/6 2/6

6/6 0/6 0/6

5/6 1/6 0/6

of hepatitis* 0 + to + + +++

120

140

mallard ducklings experimentally infected with DHBVP, D H B V c or D H B V w M at 10 d a y s ( T a b l e 2, groups 3 to 8) seem to correlate w i t h the d e v e l o p m e n t o f the i m m u n e response o f birds w h i c h begins to function 3 to 5 d a y s p o s t - h a t c h (Solomon, 1966; F u k u d a et al., 1987), T h e m a i n finding o f this study was the c h a r a c t e r i z a tion o f the D H B V isolate f r o m n a t u r a l l y i n f e c t e d wild m a l l a r d s , w h i c h i n d u c e d unusually high v i r a e m i a titres

Table 2. Histological study of naturally and experimentally DHB V-infected Mallard and Pekin ducks

Natural infection

106

Time (days) Fig. 2. Experimental infection of Pekin and mallard duckings with three DHBV isolates. Three DHBV isolates originating from a highly viraemic wild mallard (DHBVwM), a Chinese brown duck (DHBVc) and a domestic Pekin duck (DHBVP) were experimentally transmitted to domestic Pekin and mallard ducklings (12 to 15 birds for each DHBV isolate) by inoculation with DHBV-positive sera containing similar amounts of virus as described in the text. Mean values +__S.E.M. of serum v.g.e./ml for (a) Pekin and (b) mallard ducks infected with DHBVP (E3), DHBVwM ( I ) and DHBVc ([~) are shown.

* Degree of hepatitis according to Popper et al. (1980) : 0, normal liver; + to + +, minor to moderate portal and parenchymal lesions; + + +, portal infiltration and parenchymal lesions associated with focal necrosis of hepatitis. Livers scored as 0 were either normal or characterized by fatty changes.

420

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in its natural host. Our results of experimental transmission clearly demonstrated that such differences in viraemia were not related to the breed of duck but indeed to virus isolate, since DHBVwM induced significantly higher viraemia in both mallard and Pekin ducks as compared to DHBVP. Until recently, our understanding of the genetic basis for biological characteristics such as host range or virulence of hepadnaviruses has been limited. HBV variants have been discovered with an inactive pre-core gene, due to stop codon or frameshift mutations, which lead to clinically significant changes in virus pathogenicity (Carman et aL, 1989, Brunetto et al., 1989; Tong et al., 1990). This finding stimulated interest in other naturally occurring hepadnavirus variants (Alberti, 1990). In this regard the isolation of DHBVwM which induces unusually high viraemia in both mallard and Pekin ducks appears of great interest. The sequence determination of molecularly cloned DHBVwM in association with in vitro transfection of hepatoma cell lines (Galle et al., 1988; Fourel et al., 1989), will allow the functional analysis of viral genes responsible for the high viraemia induced by this isolate. Whether this reflects increased viral replication or more efficient virus packaging and export is not clear at present. We are grateful to Chantal Jacquet for her excellent technical assistance with the ducks, J.-P. Allard for the statistical analysis, A. L. Parodi for valuable histological advice and J.-P. Lamelin for critically reading the manuscript. We also thank for their kind cooperation Mr X. Raymond and Mr P. Cordonnier for providing access to the birds from the Dombes wildlife reserve, Dr W. R. Hansen (National Wildlife Health Laboratory of Madison, Wisconsin, U.S.A.) for providing sera samples from American wild waterfowl and Dr Riviere for providing samples from ducks shot in Alsace district. V. L. is the recipient of a fellowship from the Association pour la Recherche sur le Cancer (ARC). This study was supported by grant 6735 from ARC.

References ALBERTI, A. (1990), Do single nucleotide mutations result in clinically significant changes in hepatitis B virus pathogenicity? Journal of Hepatology 10, 268-270. ALBERTI, A., TREMOLADA, F., FATTOVICH G., BORTOLOTTI, F. & REALDI, G. (1983). Virus replication and liver disease in chronic hepatitis B virus infection. Digestive Diseases and Sciences 28, 962-966 BRUNETTO, M. R., STEMLER,M., SCHODEL, F., WILL, H., OTTOBRELLI, A., RIZETTO, M. & BONINO, F. (1989). Identification of HBV variants which cannot produce precore derived HBeAg and may be responsible for severe hepatitis. Italian Journal of Gastroenterology 21, 151 154. CARMAN, W. E., JACYNA, M. R., HADZIYANNIS, S., KARAVIANNIS, P., MCGARVEY, M. J., MAKRIS, A. & THOMAS, H. C. (1989). Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 8663, 588-591. COVA, L., HANTZ, O., ARLIAUD-GASSIN, M., CHEVALIER, A., BERTHILLON, P., BOULAY,J., JACQUET,C., CHOMEL, B., VITVITSKI,L. & TREPO, C. (1985). Comparative study of DHBV levels and endogenous DNA polymerase activity in naturally infected ducklings in France. Journal of Virological Methods 10, 251 260. COVA, L., LAMBERT, V., CHEVALIER, A., HANTZ, O., FOUREL, I., JACQUET, C., PICHOUD, C., BOULAY,J., CHOMEL, B., VITVITSKI,L. & TREPO, C. (1986). Evidence for the presence of duck hepatitis B virus in wild migrating ducks. Journal of General Virology 67, 537-547.

COVA, L., WILD, C. P., MEHROTRA, R., TURUSOV, V., SHIRAI, T., LAMBERT, V., JACQUET, C., TOMATIS,L., TREPO, C. & MONTESANO, R. (1990). Contribution of aflatoxin B1 and hepatitis B virus infection in the induction of liver tumors in ducks. Cancer Research 50, 2156-2163. FOUREL, I., GRIPON, P., HANTZ, O., COVA, L., LAMBERT,V., JACX)UET, C., WATANABE, K., FOX, J., GUILLOUZO, C. & TREPO, C. (1989). Prolonged duck hepatitis B virus replication in duck hepatocytes cocultivated with rat epithelial cells: a useful system for antiviral testing. Hepatology 10, 186-191. FROMMEL, D., CREVAT, D., VITVITSKI, t., PICHOUD, C., HANTZ, O., CHEVALIER, M., GRIMAUD, J. A., LINDBERG, J. & TREPO, C. (1984). Immunopathologic aspects of woodchuck hepatitis. American Journal of Pathology 115, 125-134. FUKUDA, R., FUKUMOTO,S. & SHIMADA,Y. (1987). A sequential study of viral DNA in serum in experimental transmission of duck hepatitis B virus. Journal of Medical Virology 21, 311-320. GALLE, P. R., SCHLICHT, H. J., FISCHER, M. & SCHALLER, H. (1988). Production of infectious duck hepatitis B virus in a human hepatoma cell line. Journal of Virology 62, 1736-1740. JILBERT, A., FREIMAN, J., BURRELL, C. J., HOLMES, M., GOWANS, E. J., ROWLAND, R., HALL, P. & COSSART, Y. E. (1988). Virus-liver cell interaction in duck hepatitis B virus infection. Gastroenterology 95, 1375 1382. LAMBERT, V., FERNHOLZ, D., SPRENGEL, R., FOUREL, I., DELEAGE, G., WILDNER, G., PEYRET, C., TREPO, C., COVA, L, & WILL, H. (1990). Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein. Journalof Virology64, 12901297. MARION, P. L., KNIGHT, S. S., HO, B. K., Guo, Y. Y., ROBINSON, W. S. & POPPER, H. (1984). Liver disease associated with duck hepatitis B virus infection of domestic ducks. Proceedings of the National Academy of Sciences, U.S.A. 81, 898-902. MARION, P. L., VANDAVELAAR,M. J., KNIGHT, S. S., SALAZAR,F. H., GARCIA, G., POPPER, H. & ROBINSON, W. S. (1986). Hepatocellular carcinoma in ground squirrels persistently infected with ground squirrel hepatitis virus. Proceedings of the National Academy of Sciences, U.S.A. 83, 4543-4546. POPPER, H., SHIH, J. W. K., GERIN, J. L., WONG, D. C., HOYER, B. H., LONDON, W. T., SLY, D. & PURCELL, R. H. (1981). Woodchuck hepatitis and hepatocellular carcinoma: correlation of histologic with virologic observations. Hepatology l, 91-98. POPPER, H., ROTH, L., PURCELL, R. H., TENNANT, B. C. & GERIN, J. L. (1987). Hepatocarcinogenicity of the woodchuck hepatitis B virus. Proceedings of the National Academy of Sciences, USA 84, 866-870. RIGBY, P. W. J., DIECKMANN, M., R/lODES, C. & BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. Journal of Molecular Biology 113, 237-251. SCHODEL, F., SPRENGEL, R., WEIMER, T., FERNHOLZ, D., SCHNEIDER, R. & WILL, H. (1989). Animal hepatitis B viruses. In Advances in Viral Oncology, pp. 73-102. Edited by G. Klein. New York: Raven Press. SOLOMON, J. B. (1966). Induction of antibody formation to goat erythrocytes in the developing chick embryo and effects of maternal antibody. Immunology ll, 89-96. SPRENGEL, R., KALETA, E. F. & WILL, H. 0988). Isolation and characterization of a hepatitis B virus endemic in herons. Journal of Virology 62, 3832 3839. TONG, S., LI, J. S., VITVITSKI,L. & TREPO, C. (1990). Active hepatitis virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region. Virology 176, 596-603. YOKOSUKA, O., OMATA, M., ZHOU, Y. Z., IMAZEKI, F. & OKUDA, K. (1985). Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma. Proceedings of the National Academy of Sciences, U.S.A. 82, 51805184. ZHOU, Y. Z. (1980). A virus possibly associated with hepatitis and hepatoma in ducks. Shanghai Medical Journal 3, 641-644.

(Received 31 July 1990; Accepted 22 October 1990)