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Gene expression and active virus replication in the liver after injection of duck hepatitis B virus DNA into the peripheral vein of ducklings
Journal ofHepatology 1996; 24: 328-334 Printed in Denmark All rights reserved Munkrgaard Copenhagen
Copyright 0 European Association for the Study of the Liver 1996 Journal of nepato1ogy ISSN 0168.8278
Gene expression and active virus replication in the liver after injection of duck hepatitis B virus DNA into the peripheral vein of ducklings Masami Tagawa’, Osamu Yokosuka’, Fumio Imazeki’, Masao Ohto’ and Masao 0mata2 ‘First Department of Medicine, Chiba University School of Medicine, Chiba, and 2Second Department of Internal Medicine, Tokyo University Faculty of Medicine, Tokyo, Japan
Background/Aims: Duck hepatitis B virus is a member of the hepadnavirus family, which possesses strong hepatotropism. Duck hepatitis B virus DNA serves as the replicative template for producing biologically active virus particles after transfection into cell lines established from human hepatocellular carcinoma or into duck liver by direct injection of calcium phosphate-precipitated DNA. Our aim was to develop a new method of liver-specific gene expression after intravenous DNA delivery. Methods/Results: We inoculated duck hepatitis B virus DNA with and without cationic liposomes, Lipofectin or LipofectAMINE, as DNA carriers. Two weeks after a single intravenous injection of 10 or 50 pg of plasmid DNA containing a head-totail dimer of duck hepatitis B virus DNA into 25 one-day old ducklings, duck hepatitis B virus RNA transcripts including the pregenome replicative intermediate were detected by Northern blot in the
liver of eight ducks (100%) of the Lipofectin group, five ducks (63%) of the LipofectAMINE group, and three ducks (50%) of the group which received DNA without carrier. Duck hepatitis B virus RNA transcription was almost exclusively liver specific, even though the lipsomes had no tissue specificity. Replicative forms of duck hepatitis B virus DNA were detected in the liver and DHBsAg was observed in the cytoplasm of the hepatocytes by immunostaimng. The serum of transfected ducklings contained virus particles which were infectious in other ducklings. Conclusion: The efficient and liver-specific expression of inoculated DNA was due to the amplification of nucleic acids by active virus replication process under the control of hepatocyte specific regulation.
C
tion in which pregenome RNA can be expressed, such as a head-to-tail dimer of the DHBV genome, can initiate viral infection after transfection into human hepatocellular carcinoma cell lines, HepG2 and HUH-~ (7) and the avian hepatocellular carcinoma cell line, LMH (8, 9), and the duck liver by direct injection of calcium-phosphate-precipitated DNA (3). Recently, liposomes have served as a strong tool for the transfection of nucleic acids to a wide variety of cultured cells (10, 11). Several kinds of liposome reagents are commercially available. The cationic liposome, Lipofectin, was used as the carrier for systemic gene expression after intravenous DNA delivery into adult mice (12). In that report, cytomegaloviruschloramphenicol acetyltransferase (CMV-CAT) ex-
characteristics of members of the hepadnavirus family include replication in the hepatocytes without integration of viral DNA into the genome of the host cell (l), and establishment of a chronic carrier state by horizontal or vertical transmission in the neonatal period (2). Hepadnaviral DNA becomes the replication template and initiates the replication cycle and eventual secretion of mature virus after transfection into hepatocellular carcinoma cell lines (3-8). Duck hepatitis B virus (DHBV) DNA, which possesses a configuraOMMON
Received 8 May: revised 7 August; accepted 9 August 1995
Correspondence: Masami Tagawa, M.D., First Department of Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260 Japan.
328
Key words: Duck hepatitis B virus DNA; Liposome; Systemic inoculation; Transfection.
DHBV replication
pression plasmid was expressed in a variety of tissues, especially in the lung, spleen, lymph node and bone marrow. In the present study, we used Lipofectin and LipofectAMINE for transfection of DHBV head-totail dimer DNA into the liver of l-day-old ducklings by direct injection of the liposome-DNA complex into a peripheral vein, and observed efficient viral replication in the liver at 2 weeks after injection. DHBV RNA transcription, protein translation, and virus particle formation were observed in some ducklings inoculated with DHBV DNA without using any carrier, but liposomes increased the efficiency of transfection, resulting in virus replication in the hepatocytes. DHBV expression was exclusively liver specific at 2 weeks after systemic transfection.
after peripheral
injection of DNA
groups I and II were inoculated with 10 and 50 pg of plasmid DNA, respectively. Each group included the ducklings which were inoculated with or without carrier liposomes. The amount of cationic liposome injected to one duckling was 19 nmol of Lipofectin and 60 nmol of LipofectAMINE in group I, and 95 nmol of Lipofectin and 300 nmol of LipofectAMINE in group II. The number of ducklings of each group is given in Table 1. Two or four hundred microliters of DNA-liposome complex solution per animal were injected into the leg vein. Samples were collected at 2 weeks after inoculation of DNA alone or DNA-liposome complex.
Ducklings One-day-old white Pekin ducklings (Anus domesticus) were purchased from a farm in Chiba Prefecture, Japan, whose flock was reported to be DHBV-free (2, 13). The mean body weight of a l-day-old duckling before feeding was 57 g (14). Twenty-five ducklings were inoculated with DHBV DNA, and 22 of them were used for the analysis.
Nucleic acids analysis Poly[A]‘RNA was extracted from frozen tissues by the QuickPrep mRNA purification kit or the QuickPrep Micro mRNA purification kit (Pharmacia Biotech AB, Uppsala, Sweden). DNA extraction from frozen liver and serum samples was performed as reported before (14). RNA and DNA samples were electrophoresed, transferred to Hybond-N+ (Amersham International plc, Bucks, U.K.) and hybridized with 32P-labeled 3.0 kb DHBV DNA fragment using Rapid Hybridization Buffer (Amersham International plc).
Plasmid DNA, preparation of DNA-carrier complexes and inoculation to ducklings The 8913 bp plasmid pGDBVdi contains a head-to-tail dimerofDHBVDNA(DHBV16)(15)attheEcoRIsite of pGEM4 (Promega Corp., Madison, WI, USA). The supercoiled form of pGDBVdi was used as the inoculum. Two kinds of cationic liposomes were purchased from GIBCO-BRL (Grand Island, NY,USA): Lipofectin reagent, containing N-[ 1-(2,3-dioleyloxy)propyl]n,n,n-trimethlammonium chloride (16), and LipofectAMINE reagent, containing 2,3-dioleyloxy-N-[2 (sperminecarboxamido) ethyl]-N,N-dimethyl1-propanaminium trifluoroacetate (17). One microgram of DNA was mixed with the cationic liposome at the ratio of 1.9 nmol Lipofectin to 6 nmol LipofectAMINE. The DNA-liposome complex formed during incubation for 15 min at room temperature. One-day-old ducklings of
Analysis of DHBsAg One hundred milligrams of frozen liver tissue were homogenized in 1 ml of 0.1 M NaCl, 5 mM MgCl,, 1% Nonidet P-40, 0.5% Sodium deoxycholate, 20 mM Tris-HCl (pH 7.5) and 8 TIU of aprotinin (Sigma Chemical Company, St Louis, MO, USA), and centrifuged at 15 000 t-pm for 5 min at 4°C (18). Ten microliters of the supernatant was mixed with 5 pl of 200 mM Tris-HCl (pH 7.5), 3% SDS, 2% 2-mercaptoethanol, 10% sucrose, 0.1% bromophenol blue, 2 mM EDTA and heated at 100°C for 5 min. The mixture was separated on 12.5% of acrylamide-SDS gel and transferred to a nitrocellulose filter, which was stained by the indirect immunoperoxidase method with rabbit anti-DHBs antibody (a generous gift from Dr. PL. Marion, Stanford University) as the first antibody. Formalin-fixed liver specimens were also
Materials and Methods
TABLE 1 Groups of ducklings inoculated with pGDBCdi DNA
Group I Group II
1oFg 50 kg
Carrier No carrier
Lipofectin
LipofectAMINE
3 3
5 51’2’
5 4”
‘)one duckling died within 24 h. ‘) one duckling died within 3 days.
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Acute toxicity of liposomes In group II, two ducklings, one inoculated with Lipofectin and the other with LipofectAMINE, died within 24 h, and another duckling inoculated with Lipofectin died within 3 days after injection of the complex (Table 1). They took less food than other treated ducklings but no signs of injury at the site of injection or macroscopic changes of abdominal organs were seen. Histological examination of liver sections did not show any special findings. None of the group I ducklings and none of those inoculated without carrier died during the follow-up period.
stained with anti-DHBs antibody by the immunoperoxidase method.
Detection of virus in the serum The presence of infectious virus particles in the serum of ducklings which were injected with pGDBVd-liposome complex, was confirmed by a second transmission of DHBV to other ducklings by inoculation of the serum to be tested. Three hundred microliters of serum of duckling 20, which was bled at 2 weeks after injection of pGDBVdi, was used as the inoculum. One milliliter of the same serum sample was subjected to a 15-30% sucrose gradient by centrifugation 25 000 t-pm for 7 h at 4°C with a Beckman SW27 rotor, and 21 fractions of 1.6 ml were collected from the bottom (19, 20). The concentration of DHBV DNA of each fraction was analyzed by slot blot, and 200 or 400 l.tl of the peak fraction of DHBV DNA (fraction 7, sucrose 26.3%) was also used as the inoculum for the second transmission experiment. Serum samples were collected from 1 to 8 weeks after inoculation and the presence of DHBV DNA was tested by Southern blotting.
DHBV RNA and DNA in the liver Northern blot analysis was done using 2 pg (group I) or 3 p.g (group II) of poly[A]‘RNA. DHBV RNA was detected in some ducklings as three major bands of 3.4,2.2 and 1.9 kb, corresponding to the C, preS, and S mRNAs in the liver of DHBV-infected ducks (Fig. 1) (20, 21). One and two ducklings inoculated without carrier in group I (No. 3) and group II (No. 14 and 16) had a positive signal of DHBV RNA. Replicative forms of DHBV DNA, relaxed circular and linear forms of DNA and shorter size of smear DNA were detected by Southern blotting in the liver of the same ducklings (data not shown). The positive rate of DHBV DNA and RNA was increased in ducklings inoculated with the DNA-Lipofectin complex to 4/5 (80%) and 5/5 (100%) in group I (Table 2). LipofectAMINE was less effective than Lipofectin in group I, but the positive rate of DHBV DNA and RNA was increased to 2/3 (66%) and 313 (100%) in group II.
Results Twenty-five duckling were inoculated with 10 or 50 yg of plasmid DNA containing dimer construct of DHBV DNA through the leg vein, and DHBV expression and replication was analyzed at 2 weeks after inoculation.
t-1 123
1oJa
Lipofectin 45678
LipofectAMlNE 9 1011
1213
kb* 7.54.4-
-3.4k”
2.474%
1.4-
50 IJS
14 15 16 kb 7.54.42.41.4-
330
1718
19
20 21 22
-2.2 -1.9
N
Fig. 1. Northern blot analysis of RNA samples extracted from the liver of ducklings 2 weeks after inoculation with DNA. Upper panels (lane 1 to 13) are 2 pg of poly[A]‘RNA of group I ducklings inoculated with 10 pg of pGDBVdi, and lower panels (lane 14 to 22) are 3 pg of poly[A]+ RNA of group II ducklings inoculated with 50 pg of pGDBVdi. 3.4, 2.2 and 1.9 kb bands correspond to the major RNA transcripts, C, preS and S mRNA detected in the DHBV replicating liver. Lane numbers correspond to the number of ducklings. N; negative control.
DHBV replication
after peripheral
injection
of DNA
TABLE 2 Analysis of DHBV expression at 2 weeks after inoculation Carrier
Liver
Serum
DHBV DNA
DHBV RNA
DHBsAg
DHBV DNA
Group I
No carrier Lipofectin LipofectAMINE
l/3 (33%) 4/5 (80%) 2/5 (40%)
l/3 (33%) 5/5 (100%) 2/5 (40%)
113(33%) 3/5 (60%) 2/5 (40%)
l/3 (33%) 3/5 (60%) l/5 (20%)
Group II
No carrier Lipofectin
213 (67%) 3/3 (100%)
2/3 (67%) 313 (100%)
113(33%) 2/3 (67%)
l/3 (33%) 213 (67%)
LipofectAMINE
213 (67%)
3/3 (100%)
2/3 (67%)
2/3 (67%)
The presence of DHBV DNA integrated into the genome of host cell was not confirmed because free viral DNA gave a strong signal. DHBsAg in the liver Immunostaining of a Western blot of proteins from liver extracts of a positive control duck with rabbit anti-DHBs antibody detected bands of 37 and 28 kD (Fig. 2), indicating that this antibody recognized preS1 and preS2 proteins but did not recognize the major S protein (18). Bands with the same mobility were detected in DHBV DNA-transfected ducklings (Fig. 2, lanes 1 to 3). Immunohistochemical analysis with the same anti-DHBs antibody revealed that cytoplasm of scattered hepatocytes were stained. In one duckling (No. 9), which was positive for DHBV
DNA and RNA in the liver without evidence of viremia (DHBV DNA was not detected in the serum), DHBsAg was expressed mainly in hepatocytes in the periportal area (Fig. 3). DHBsAg was probably translated from the transfected DNA template but not from the secondary infected virus genome, indicating that pGDBVdi-liposome complex reached the liver by the blood flow and transfected the hepatocytes in the periportal area. DHBV
DNA in the serum
The relaxed circular DNA that is the form of DHBV DNA in the virion became detectable in the sera at 2 weeks after inoculation (Fig 4, duckling No. 20)(Table 2) All ducklings with positive DHBsAg in the liver by Western blot analysis, except No. 9, were positive for the relaxed circular form of DHBV DNA in the serum. To analyze whether DHBV DNA detected in the serum of transfected ducklings represented the infec-
N123P kD 69-
46-
-p37 30-
-p28 21.5-
Fig. 2. Western blot analysis of the liver reacted with rabbit anti-DHBs serum. Lanes I, 2 and 3 are samples from ducklings No. 7, 14 and 9. Two bands of p37 and ~28 were detected in positive control and all samples, which correspond to pre-SI and pre-S2 antigen, respectively.
Fig. 3. Detection of DHBsAg in the liver of duckling No. 9 2 weeks after inoculation with IO pg of pGDBVdi-LipofectAMINE complex into the leg vein. Cytoplasm of hepatocytes in the periportal area and some hepatocytes in the lobules were stained with rabbit anti-DHBs antibody by the indirect peroxidase method.
331
M. Tagawa et al.
Lipofectin 20
20.1
20.2
20.3 Li
1248
LipofectAMlNE
P
KSLuH
Li
P
K
SLuH
1
c-
preS S’
Fig. 4. Secondary transmission of DHBVfrom the serum of duckling No. 20 to other ducklings. One-day-old duckling No. 20. 1 was intravenously injected with 300 ~1 of the serum of No. 20 bled at 2 weeks after inoculation. Ducklings No. 20.2 and 20.3 were injected with 200 and 400 ~1 of a peak fraction of DHBV DNA collected after ultracentrifigation of the serum of duckling No. 20 through 15% to 30% sucrose gradient. DNA samples extracted from 50 ~1 of the serum from 1 to 8 weeks after injection were analyzed by Southern blot hybridization. P; 40 pg of DHBV DNA fragment 3.0 kb long.
Fig. 5. Liver-specijc transcription of DHBV RNA after inoculation of plasmid DNA-liposome complex. Twenty micrograms of pGDBVdi-Lipofectin complex and 10 pg of pGDBVdi-LipofectAMINE complex were inoculated into a leg vein of l-day-old ducklings, and samples of liver (L), pancreas (P), kidney (K). spleen (S), lung (Lu) and heart (H) were obtained at 2 weeks after inoculation. Two micrograms of poly[A]+RNA extracted from those organs were analyzed by Northern blot. Exposure time of hybridized$lter was 16 h.
tious virus particles, we did a secondary transmission of DHBV using the serum of duckling No. 20 (Fig. 4). Three 1-day-old ducklings were intravenously injected with: i) 300 ~1 of the serum (duckling No. 20.1), ii) 200, and iii) 400 ~1 of the fraction of sucrose gradient (ducklings No. 20.2 and 20.3). DHBV DNA was detected in the serum of these ducklings from 1 or 2 weeks until 8 weeks after injection, indicating that virus particles in the serum of duckling No. 20 were infectious.
ions. Infectious virus particles were also detected in the serum, indicating that the proteins expressed in the hepatocytes were completely processed to produce biologically active proteins involved in the initial steps of virus replication, even with the peripherally injected DNA template. Because DHBV DNA was amplified by multiple replication cycles and viral RNA was transcribed from both the inoculurn DNA and also from the newly synthesized DNA template, DHBV DNA and RNA were easily detected by hybridization techniques. It was noteworthy that virus production was detected after a peripheral injection of naked DNA. A small population of inoculum DNA might escape attack by DNase during circulation in the blood, and successfully reach the hepatocytes. The positive rate of DHBV RNA, l/3 in group I and 2/3 in group II, indicates that it was not a rare event for the 9-kb supercoiled DNA, and nonspecific absorption of DNA was expected to occur in a concentration dependent manner. In an in vivo mouse transfection model, Zhu et al. reported that 20-g mice were each inoculated with 100 pg of CMV-CAT plasmid-liposome complex into the tail vein and the CAT gene was expressed in the lung, spleen, lymph node, bone marrow and other tissues (12). Transfected DNA was barely detectable at
Liver specific
expression
of DHBV
Tissue specificity of DHBV expression was analyzed by Northern blotting using 2 l,tg of poly[A]+RNA extracted from the liver, pancreas, kidney, spleen, lung and heart (Fig. 5). DHBV RNA was mainly transcribed in the liver, even though a weak signal was detected in the spleen and lung of ducklings transfected with Lipofectin and LipofectAMINE on autoradiography with a long-exposure film.
Discussion DHBV DNA inoculated into the peripheral vein of lday-old ducklings resulted in successful expression in the liver of viral antigen, and the forms of DHBV DNA and species of DHBV RNA which strikingly resembled those of duck liver infected by DHBV vir332
DHBV replication after peripheral injection of DNA
21 days or later by the hybridization technique, even though it was detected by PCR. In our system, less inoculum DNA, 10 pg of plasmid DNA per 57-g duckling, was amplified using the virus replication cycle, and long-term expression might be expected once chronic infection was established. We previously reported that 100% of l-day-old ducklings became chronic carriers of DHBV by the inoculation of DHBV positive serum (2). In the infection experiments, the incubation period correlated well to the inoculum size, and viremia was detected from 6 days and 10 days after inoculation of serum containing lo9 and lo7 genome equivalents, respectively (14, 22). In the present study, viremia was not detected at 1 week (data not shown), but was detected at 2 weeks after transfection (Fig. 4). This phenomenon was roughly equivalent to infection with lo7 genomes of DHBV. As 10 pg of pGDBVdi corresponded to 1012 genome equivalents, 0.01% of the inoculum DNA might act as the template in the hepatocytes to initiate infection. More analysis is necessary to conclude whether liposomes shorten the incubation period as the result of the more efficient transfection of the replicative template into the hepatocytes. DHBV RNA transcripts were found exclusively in the liver, and DHBsAg staining indicated that hepatocytes were the place where virus replication occurred in the liver. Even though hepadnaviruses are hepatotropic viruses, it was reported that DHBV could replicate in the pancreas, kidney, spleen and lymphocytes (14, 23, 24). Copy numbers of DHBV in the liver, pancreas, kidney and spleen of 2-week-old ducklings which were experimentally infected with DHBV positive serum at 1 day after hatching, were calculated from the density of dot blot test of DHBV DNA to 1284,9,5 and 24 copies per cell, respectively (14). In the present study, the concentration of DHBV RNA in the different organs was used as the indicator of virus replication instead of DHBV DNA, because inoculum plasmid DNA might give a high background if the concentration of DHBV DNA or copy number of DHBV was used for the comparison. A faint signal of DHBV RNA was barely detected in the spleen and lung, and the density of that signal was much less than in the liver, with the ratio of DHBV RNA concentration estimated at 1: 1000-10 000 Transfection of DHBV DNA could initiate hepatocyte-specific virus replication without a need for the DHBV receptor. At 2 weeks after transfection, the organ distribution of transfected and replicating DHBV was similar to experimental infection. By this time, transient expression of foreign genes was sus-
petted to be reduced and the products of DHBV replication cycles accumulated to the detectable level. Hepatotropism of DHBV or other members of the hepadnavirus family might be explained or partially explained by the transcriptional regulation of DHBV genome by the hepatocyte specific factors, or by hepatocyte specific nuclear factors), as described in several reports (25-29). In other words, even though DHBV DNA was introduced without using DHBV receptor, transcriptional regulatory elements of the DHBV genome were not fully functional in other types of cells. We could not clarify the reason for the deaths of three animals. Other ducklings in groups I and II appeared normal from the time of injection and grew as well as the non-treated ducklings. Zhu et al. reported no treatment-related toxicity of the liposome to mice by histological examinations of a wide variety of tissues (12) A low dose of liposome is thought to be less toxic to the ducklings. This transfection system facilitates the functional analysis of the DHBV genome, because deleted or mutated DHBV DNA could be introduced into hepatocytes by simply injecting a DNA-liposome complex into a peripheral vein, and DHBV would replicate more efficiently than in the culture cells. Hepatitis B virus or other members of hepadnavirus DNA could be introduced to other experimental animals using this systemic injection system to analyze the regulatory elements of gene expression and viral replication and immunological reaction to the viral proteins.
Acknowledgement We are grateful to Patricia L. Marion (Stanford University, USA) for critical reading of the manuscript.
References 1. Summers J, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 1982; 29: 403-15. 2. Omata M, Yokosuka 0, Imazeki F, Matsuyama Y, Uchiumi K, Ito Y, Mori J, Okuda K. Transmission of duck hepatitis B virus from Chinese carrier ducks to Japanese ducklings: a study of viral DNA in serum and tissue. Hepatology 1984; 4: 603-7. 3. Sprengel R, Kuhn C, Manso C, Will H. Cloned duck hepatitis B virus DNA is infectious in Pekin ducks. J Virol 1984; 52: 932-7. 4. Sureau C, Romet-Lemonne J-L, Mullins JI, Essex M. Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 1986; 47: 37-47. 5. Sells MA, Chen M-L, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Nat1 Acad Sci USA 1987; 84: 1005-9.
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M. Tagawa et al. 6. Yaginuma K, Shirakata Y, Kobayashi M, Koike K. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci USA 1987; 84: 2678-82. 7. Galle PR, Schlicht HJ, Fischer M, Schaller H. Production of infectious duck hepatitis B virus in a human hepatoma cell line. J Vim1 1988; 62: 1736-40. 8. Condreay LD, Aldrich CE, Coates L, Mason WS, Wu ‘IT. Efficient duck hepatitis B virus production by an avian liver tumor cell line. J Virol 1990; 64: 3249-58. 9. Kawaguchi T, Nomura K, Hirayama Y, Kitagawa T. Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res 1987; 47: 446w. 10. Faller DV, Baltimore D. Liposome encapsulation of retrovirus allows efficient superinfection of resistant cell lines. J Virol 1984; 49; 269-72. 11. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenze M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Nat1 Acad Sci USA 1987; 84: 7413-17. 12. Zhu N, Liggitt D, Liu Y, Debs R. Systemic gene expression after intravenous DNA delivery into adult mice. Science 1993; 261: 209-11. 13. Omata M, Uchiumi K, Ito Y, Yokosuka 0, Mori J, Terao K, Wei-Fa Y, O’Connel AP, London WT, Okuda K. Duck hepatitis B virus and liver diseases. Gastroenterology 1983; 85: 260-7. 14. Tagawa M, Omata M, Yokosuka 0, Uchiumi K, Imazeki F, Okuda K. Early events in duck hepatitis B virus infection: sequential appearance of viral deoxyribonucleic acid in the liver, pancreas, kidney, and spleen. Gastroenterology 1985; 89: 1224-9. 15. Mandart E, Kay A, Lopez-Cabrera M, Letovsky J, Hu K-Q, Siddiqui A. Multiple liver-specific factors bind to the hepatitis B virus core/pregenomic promoter: trans-activation and repression by CCAAT/enhancer binding protein. Proc Nat1 Acad Sci USA 1990; 87: 5069-73. 16. Innes CL, Smith PB, Langenbach R, Tindall KR, Boone LR. Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors. J Virol 1990; 64; 95761. 17. Hawley-Nelson P, Ciccarone V, Gebeyehu G, Jessee J. Lipofectamine reagent: a new, higher efficiency polycationic liposome transfection reagent. Focus 1993; 15; 73-9.
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18. Yokosuka 0, Omata M, Ito Y. Expression of pre-Sl, preS2, and C proteins in duck hepatitis B virus infection. Virology 1988; 167: 82-6. 19. Robinson WS, Greenmen RL. DNA polymerase in the core of the human hepatitis B virus candidate. J Virol 1974; 13: 1231-6. 20. Tagawa M, Robinson WS, Marion PL. Duck hepatitis B virus replicates in the yolk sac of developing embryos. J Virol 1987; 61: 2273-9. 21. Buscher M, Reiser W, Will H, Schaller H. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription. Cell 1985; 40: 717-24. 22. Tagawa M, Omata M, Okuda K. Appearance of viral RNA transcripts in the early stage of duck hepatitis B virus infection. Virology 1986; 152: 477-82. 23. Mason WS, Taylor JM, Seal G, Summers J. An HBV-like virus of domestic ducks. In: Alter H, Maynard J, Szmuness W, eds. Proceedings of the 1981 Symposium on Viral Hepatitis. Philadelphia: Franklin Institute Press, 1982; 107-16. 24. Halpem MS, England JM, Deery DT, Petcu DJ, Mason WS, Molnar-Kimber KL. Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus. Proc Nat1 Acad Sci USA 1983; 80; 4865-9. 25. Lilienbaum A, Crescenzo-Chaigne B, Sal1 AA, Pillot J, Elfassi E. Binding of nuclear factors to functional domains of the duck hepatitis B virus enhancer. J Virol 1993; 67: 6192200. 26. Liu C, Mason WS, Burch JE. Identification of factor-binding sites in the duck hepatitis B virus enhancer and in vivo effects of enhancer mutations. J Virol 1994; 68; 228696. 27. Karpen S, Banerjee R, Zelent A, Price P, Acs G. Identification of protein-binding sites in the hepatitis B virus enhancer and core promotor domains. Mol Cell Biol 1988; 8: 515965. 28. Yee J-K. A liver-specific enhancer in the core promoter region of human hepatitis B virus. Science 1989; 246: 658-61. 29. Lopez-Cabrera M, Letovsky J, Hu K-Q, Siddigui A. Multiple liver-specific factors bind to the hepatitis B virus core/pregenomic promoter: transactivation and repression by CCAATI enhancer binding protein. Proc Nat1 Acad Sci USA 1990; 87: 5069-73.
Copyright 0 European Association for the Study of the Liver 1996 Journal of nepato1ogy ISSN 0168.8278
Gene expression and active virus replication in the liver after injection of duck hepatitis B virus DNA into the peripheral vein of ducklings Masami Tagawa’, Osamu Yokosuka’, Fumio Imazeki’, Masao Ohto’ and Masao 0mata2 ‘First Department of Medicine, Chiba University School of Medicine, Chiba, and 2Second Department of Internal Medicine, Tokyo University Faculty of Medicine, Tokyo, Japan
Background/Aims: Duck hepatitis B virus is a member of the hepadnavirus family, which possesses strong hepatotropism. Duck hepatitis B virus DNA serves as the replicative template for producing biologically active virus particles after transfection into cell lines established from human hepatocellular carcinoma or into duck liver by direct injection of calcium phosphate-precipitated DNA. Our aim was to develop a new method of liver-specific gene expression after intravenous DNA delivery. Methods/Results: We inoculated duck hepatitis B virus DNA with and without cationic liposomes, Lipofectin or LipofectAMINE, as DNA carriers. Two weeks after a single intravenous injection of 10 or 50 pg of plasmid DNA containing a head-totail dimer of duck hepatitis B virus DNA into 25 one-day old ducklings, duck hepatitis B virus RNA transcripts including the pregenome replicative intermediate were detected by Northern blot in the
liver of eight ducks (100%) of the Lipofectin group, five ducks (63%) of the LipofectAMINE group, and three ducks (50%) of the group which received DNA without carrier. Duck hepatitis B virus RNA transcription was almost exclusively liver specific, even though the lipsomes had no tissue specificity. Replicative forms of duck hepatitis B virus DNA were detected in the liver and DHBsAg was observed in the cytoplasm of the hepatocytes by immunostaimng. The serum of transfected ducklings contained virus particles which were infectious in other ducklings. Conclusion: The efficient and liver-specific expression of inoculated DNA was due to the amplification of nucleic acids by active virus replication process under the control of hepatocyte specific regulation.
C
tion in which pregenome RNA can be expressed, such as a head-to-tail dimer of the DHBV genome, can initiate viral infection after transfection into human hepatocellular carcinoma cell lines, HepG2 and HUH-~ (7) and the avian hepatocellular carcinoma cell line, LMH (8, 9), and the duck liver by direct injection of calcium-phosphate-precipitated DNA (3). Recently, liposomes have served as a strong tool for the transfection of nucleic acids to a wide variety of cultured cells (10, 11). Several kinds of liposome reagents are commercially available. The cationic liposome, Lipofectin, was used as the carrier for systemic gene expression after intravenous DNA delivery into adult mice (12). In that report, cytomegaloviruschloramphenicol acetyltransferase (CMV-CAT) ex-
characteristics of members of the hepadnavirus family include replication in the hepatocytes without integration of viral DNA into the genome of the host cell (l), and establishment of a chronic carrier state by horizontal or vertical transmission in the neonatal period (2). Hepadnaviral DNA becomes the replication template and initiates the replication cycle and eventual secretion of mature virus after transfection into hepatocellular carcinoma cell lines (3-8). Duck hepatitis B virus (DHBV) DNA, which possesses a configuraOMMON
Received 8 May: revised 7 August; accepted 9 August 1995
Correspondence: Masami Tagawa, M.D., First Department of Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260 Japan.
328
Key words: Duck hepatitis B virus DNA; Liposome; Systemic inoculation; Transfection.
DHBV replication
pression plasmid was expressed in a variety of tissues, especially in the lung, spleen, lymph node and bone marrow. In the present study, we used Lipofectin and LipofectAMINE for transfection of DHBV head-totail dimer DNA into the liver of l-day-old ducklings by direct injection of the liposome-DNA complex into a peripheral vein, and observed efficient viral replication in the liver at 2 weeks after injection. DHBV RNA transcription, protein translation, and virus particle formation were observed in some ducklings inoculated with DHBV DNA without using any carrier, but liposomes increased the efficiency of transfection, resulting in virus replication in the hepatocytes. DHBV expression was exclusively liver specific at 2 weeks after systemic transfection.
after peripheral
injection of DNA
groups I and II were inoculated with 10 and 50 pg of plasmid DNA, respectively. Each group included the ducklings which were inoculated with or without carrier liposomes. The amount of cationic liposome injected to one duckling was 19 nmol of Lipofectin and 60 nmol of LipofectAMINE in group I, and 95 nmol of Lipofectin and 300 nmol of LipofectAMINE in group II. The number of ducklings of each group is given in Table 1. Two or four hundred microliters of DNA-liposome complex solution per animal were injected into the leg vein. Samples were collected at 2 weeks after inoculation of DNA alone or DNA-liposome complex.
Ducklings One-day-old white Pekin ducklings (Anus domesticus) were purchased from a farm in Chiba Prefecture, Japan, whose flock was reported to be DHBV-free (2, 13). The mean body weight of a l-day-old duckling before feeding was 57 g (14). Twenty-five ducklings were inoculated with DHBV DNA, and 22 of them were used for the analysis.
Nucleic acids analysis Poly[A]‘RNA was extracted from frozen tissues by the QuickPrep mRNA purification kit or the QuickPrep Micro mRNA purification kit (Pharmacia Biotech AB, Uppsala, Sweden). DNA extraction from frozen liver and serum samples was performed as reported before (14). RNA and DNA samples were electrophoresed, transferred to Hybond-N+ (Amersham International plc, Bucks, U.K.) and hybridized with 32P-labeled 3.0 kb DHBV DNA fragment using Rapid Hybridization Buffer (Amersham International plc).
Plasmid DNA, preparation of DNA-carrier complexes and inoculation to ducklings The 8913 bp plasmid pGDBVdi contains a head-to-tail dimerofDHBVDNA(DHBV16)(15)attheEcoRIsite of pGEM4 (Promega Corp., Madison, WI, USA). The supercoiled form of pGDBVdi was used as the inoculum. Two kinds of cationic liposomes were purchased from GIBCO-BRL (Grand Island, NY,USA): Lipofectin reagent, containing N-[ 1-(2,3-dioleyloxy)propyl]n,n,n-trimethlammonium chloride (16), and LipofectAMINE reagent, containing 2,3-dioleyloxy-N-[2 (sperminecarboxamido) ethyl]-N,N-dimethyl1-propanaminium trifluoroacetate (17). One microgram of DNA was mixed with the cationic liposome at the ratio of 1.9 nmol Lipofectin to 6 nmol LipofectAMINE. The DNA-liposome complex formed during incubation for 15 min at room temperature. One-day-old ducklings of
Analysis of DHBsAg One hundred milligrams of frozen liver tissue were homogenized in 1 ml of 0.1 M NaCl, 5 mM MgCl,, 1% Nonidet P-40, 0.5% Sodium deoxycholate, 20 mM Tris-HCl (pH 7.5) and 8 TIU of aprotinin (Sigma Chemical Company, St Louis, MO, USA), and centrifuged at 15 000 t-pm for 5 min at 4°C (18). Ten microliters of the supernatant was mixed with 5 pl of 200 mM Tris-HCl (pH 7.5), 3% SDS, 2% 2-mercaptoethanol, 10% sucrose, 0.1% bromophenol blue, 2 mM EDTA and heated at 100°C for 5 min. The mixture was separated on 12.5% of acrylamide-SDS gel and transferred to a nitrocellulose filter, which was stained by the indirect immunoperoxidase method with rabbit anti-DHBs antibody (a generous gift from Dr. PL. Marion, Stanford University) as the first antibody. Formalin-fixed liver specimens were also
Materials and Methods
TABLE 1 Groups of ducklings inoculated with pGDBCdi DNA
Group I Group II
1oFg 50 kg
Carrier No carrier
Lipofectin
LipofectAMINE
3 3
5 51’2’
5 4”
‘)one duckling died within 24 h. ‘) one duckling died within 3 days.
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Acute toxicity of liposomes In group II, two ducklings, one inoculated with Lipofectin and the other with LipofectAMINE, died within 24 h, and another duckling inoculated with Lipofectin died within 3 days after injection of the complex (Table 1). They took less food than other treated ducklings but no signs of injury at the site of injection or macroscopic changes of abdominal organs were seen. Histological examination of liver sections did not show any special findings. None of the group I ducklings and none of those inoculated without carrier died during the follow-up period.
stained with anti-DHBs antibody by the immunoperoxidase method.
Detection of virus in the serum The presence of infectious virus particles in the serum of ducklings which were injected with pGDBVd-liposome complex, was confirmed by a second transmission of DHBV to other ducklings by inoculation of the serum to be tested. Three hundred microliters of serum of duckling 20, which was bled at 2 weeks after injection of pGDBVdi, was used as the inoculum. One milliliter of the same serum sample was subjected to a 15-30% sucrose gradient by centrifugation 25 000 t-pm for 7 h at 4°C with a Beckman SW27 rotor, and 21 fractions of 1.6 ml were collected from the bottom (19, 20). The concentration of DHBV DNA of each fraction was analyzed by slot blot, and 200 or 400 l.tl of the peak fraction of DHBV DNA (fraction 7, sucrose 26.3%) was also used as the inoculum for the second transmission experiment. Serum samples were collected from 1 to 8 weeks after inoculation and the presence of DHBV DNA was tested by Southern blotting.
DHBV RNA and DNA in the liver Northern blot analysis was done using 2 pg (group I) or 3 p.g (group II) of poly[A]‘RNA. DHBV RNA was detected in some ducklings as three major bands of 3.4,2.2 and 1.9 kb, corresponding to the C, preS, and S mRNAs in the liver of DHBV-infected ducks (Fig. 1) (20, 21). One and two ducklings inoculated without carrier in group I (No. 3) and group II (No. 14 and 16) had a positive signal of DHBV RNA. Replicative forms of DHBV DNA, relaxed circular and linear forms of DNA and shorter size of smear DNA were detected by Southern blotting in the liver of the same ducklings (data not shown). The positive rate of DHBV DNA and RNA was increased in ducklings inoculated with the DNA-Lipofectin complex to 4/5 (80%) and 5/5 (100%) in group I (Table 2). LipofectAMINE was less effective than Lipofectin in group I, but the positive rate of DHBV DNA and RNA was increased to 2/3 (66%) and 313 (100%) in group II.
Results Twenty-five duckling were inoculated with 10 or 50 yg of plasmid DNA containing dimer construct of DHBV DNA through the leg vein, and DHBV expression and replication was analyzed at 2 weeks after inoculation.
t-1 123
1oJa
Lipofectin 45678
LipofectAMlNE 9 1011
1213
kb* 7.54.4-
-3.4k”
2.474%
1.4-
50 IJS
14 15 16 kb 7.54.42.41.4-
330
1718
19
20 21 22
-2.2 -1.9
N
Fig. 1. Northern blot analysis of RNA samples extracted from the liver of ducklings 2 weeks after inoculation with DNA. Upper panels (lane 1 to 13) are 2 pg of poly[A]‘RNA of group I ducklings inoculated with 10 pg of pGDBVdi, and lower panels (lane 14 to 22) are 3 pg of poly[A]+ RNA of group II ducklings inoculated with 50 pg of pGDBVdi. 3.4, 2.2 and 1.9 kb bands correspond to the major RNA transcripts, C, preS and S mRNA detected in the DHBV replicating liver. Lane numbers correspond to the number of ducklings. N; negative control.
DHBV replication
after peripheral
injection
of DNA
TABLE 2 Analysis of DHBV expression at 2 weeks after inoculation Carrier
Liver
Serum
DHBV DNA
DHBV RNA
DHBsAg
DHBV DNA
Group I
No carrier Lipofectin LipofectAMINE
l/3 (33%) 4/5 (80%) 2/5 (40%)
l/3 (33%) 5/5 (100%) 2/5 (40%)
113(33%) 3/5 (60%) 2/5 (40%)
l/3 (33%) 3/5 (60%) l/5 (20%)
Group II
No carrier Lipofectin
213 (67%) 3/3 (100%)
2/3 (67%) 313 (100%)
113(33%) 2/3 (67%)
l/3 (33%) 213 (67%)
LipofectAMINE
213 (67%)
3/3 (100%)
2/3 (67%)
2/3 (67%)
The presence of DHBV DNA integrated into the genome of host cell was not confirmed because free viral DNA gave a strong signal. DHBsAg in the liver Immunostaining of a Western blot of proteins from liver extracts of a positive control duck with rabbit anti-DHBs antibody detected bands of 37 and 28 kD (Fig. 2), indicating that this antibody recognized preS1 and preS2 proteins but did not recognize the major S protein (18). Bands with the same mobility were detected in DHBV DNA-transfected ducklings (Fig. 2, lanes 1 to 3). Immunohistochemical analysis with the same anti-DHBs antibody revealed that cytoplasm of scattered hepatocytes were stained. In one duckling (No. 9), which was positive for DHBV
DNA and RNA in the liver without evidence of viremia (DHBV DNA was not detected in the serum), DHBsAg was expressed mainly in hepatocytes in the periportal area (Fig. 3). DHBsAg was probably translated from the transfected DNA template but not from the secondary infected virus genome, indicating that pGDBVdi-liposome complex reached the liver by the blood flow and transfected the hepatocytes in the periportal area. DHBV
DNA in the serum
The relaxed circular DNA that is the form of DHBV DNA in the virion became detectable in the sera at 2 weeks after inoculation (Fig 4, duckling No. 20)(Table 2) All ducklings with positive DHBsAg in the liver by Western blot analysis, except No. 9, were positive for the relaxed circular form of DHBV DNA in the serum. To analyze whether DHBV DNA detected in the serum of transfected ducklings represented the infec-
N123P kD 69-
46-
-p37 30-
-p28 21.5-
Fig. 2. Western blot analysis of the liver reacted with rabbit anti-DHBs serum. Lanes I, 2 and 3 are samples from ducklings No. 7, 14 and 9. Two bands of p37 and ~28 were detected in positive control and all samples, which correspond to pre-SI and pre-S2 antigen, respectively.
Fig. 3. Detection of DHBsAg in the liver of duckling No. 9 2 weeks after inoculation with IO pg of pGDBVdi-LipofectAMINE complex into the leg vein. Cytoplasm of hepatocytes in the periportal area and some hepatocytes in the lobules were stained with rabbit anti-DHBs antibody by the indirect peroxidase method.
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M. Tagawa et al.
Lipofectin 20
20.1
20.2
20.3 Li
1248
LipofectAMlNE
P
KSLuH
Li
P
K
SLuH
1
c-
preS S’
Fig. 4. Secondary transmission of DHBVfrom the serum of duckling No. 20 to other ducklings. One-day-old duckling No. 20. 1 was intravenously injected with 300 ~1 of the serum of No. 20 bled at 2 weeks after inoculation. Ducklings No. 20.2 and 20.3 were injected with 200 and 400 ~1 of a peak fraction of DHBV DNA collected after ultracentrifigation of the serum of duckling No. 20 through 15% to 30% sucrose gradient. DNA samples extracted from 50 ~1 of the serum from 1 to 8 weeks after injection were analyzed by Southern blot hybridization. P; 40 pg of DHBV DNA fragment 3.0 kb long.
Fig. 5. Liver-specijc transcription of DHBV RNA after inoculation of plasmid DNA-liposome complex. Twenty micrograms of pGDBVdi-Lipofectin complex and 10 pg of pGDBVdi-LipofectAMINE complex were inoculated into a leg vein of l-day-old ducklings, and samples of liver (L), pancreas (P), kidney (K). spleen (S), lung (Lu) and heart (H) were obtained at 2 weeks after inoculation. Two micrograms of poly[A]+RNA extracted from those organs were analyzed by Northern blot. Exposure time of hybridized$lter was 16 h.
tious virus particles, we did a secondary transmission of DHBV using the serum of duckling No. 20 (Fig. 4). Three 1-day-old ducklings were intravenously injected with: i) 300 ~1 of the serum (duckling No. 20.1), ii) 200, and iii) 400 ~1 of the fraction of sucrose gradient (ducklings No. 20.2 and 20.3). DHBV DNA was detected in the serum of these ducklings from 1 or 2 weeks until 8 weeks after injection, indicating that virus particles in the serum of duckling No. 20 were infectious.
ions. Infectious virus particles were also detected in the serum, indicating that the proteins expressed in the hepatocytes were completely processed to produce biologically active proteins involved in the initial steps of virus replication, even with the peripherally injected DNA template. Because DHBV DNA was amplified by multiple replication cycles and viral RNA was transcribed from both the inoculurn DNA and also from the newly synthesized DNA template, DHBV DNA and RNA were easily detected by hybridization techniques. It was noteworthy that virus production was detected after a peripheral injection of naked DNA. A small population of inoculum DNA might escape attack by DNase during circulation in the blood, and successfully reach the hepatocytes. The positive rate of DHBV RNA, l/3 in group I and 2/3 in group II, indicates that it was not a rare event for the 9-kb supercoiled DNA, and nonspecific absorption of DNA was expected to occur in a concentration dependent manner. In an in vivo mouse transfection model, Zhu et al. reported that 20-g mice were each inoculated with 100 pg of CMV-CAT plasmid-liposome complex into the tail vein and the CAT gene was expressed in the lung, spleen, lymph node, bone marrow and other tissues (12). Transfected DNA was barely detectable at
Liver specific
expression
of DHBV
Tissue specificity of DHBV expression was analyzed by Northern blotting using 2 l,tg of poly[A]+RNA extracted from the liver, pancreas, kidney, spleen, lung and heart (Fig. 5). DHBV RNA was mainly transcribed in the liver, even though a weak signal was detected in the spleen and lung of ducklings transfected with Lipofectin and LipofectAMINE on autoradiography with a long-exposure film.
Discussion DHBV DNA inoculated into the peripheral vein of lday-old ducklings resulted in successful expression in the liver of viral antigen, and the forms of DHBV DNA and species of DHBV RNA which strikingly resembled those of duck liver infected by DHBV vir332
DHBV replication after peripheral injection of DNA
21 days or later by the hybridization technique, even though it was detected by PCR. In our system, less inoculum DNA, 10 pg of plasmid DNA per 57-g duckling, was amplified using the virus replication cycle, and long-term expression might be expected once chronic infection was established. We previously reported that 100% of l-day-old ducklings became chronic carriers of DHBV by the inoculation of DHBV positive serum (2). In the infection experiments, the incubation period correlated well to the inoculum size, and viremia was detected from 6 days and 10 days after inoculation of serum containing lo9 and lo7 genome equivalents, respectively (14, 22). In the present study, viremia was not detected at 1 week (data not shown), but was detected at 2 weeks after transfection (Fig. 4). This phenomenon was roughly equivalent to infection with lo7 genomes of DHBV. As 10 pg of pGDBVdi corresponded to 1012 genome equivalents, 0.01% of the inoculum DNA might act as the template in the hepatocytes to initiate infection. More analysis is necessary to conclude whether liposomes shorten the incubation period as the result of the more efficient transfection of the replicative template into the hepatocytes. DHBV RNA transcripts were found exclusively in the liver, and DHBsAg staining indicated that hepatocytes were the place where virus replication occurred in the liver. Even though hepadnaviruses are hepatotropic viruses, it was reported that DHBV could replicate in the pancreas, kidney, spleen and lymphocytes (14, 23, 24). Copy numbers of DHBV in the liver, pancreas, kidney and spleen of 2-week-old ducklings which were experimentally infected with DHBV positive serum at 1 day after hatching, were calculated from the density of dot blot test of DHBV DNA to 1284,9,5 and 24 copies per cell, respectively (14). In the present study, the concentration of DHBV RNA in the different organs was used as the indicator of virus replication instead of DHBV DNA, because inoculum plasmid DNA might give a high background if the concentration of DHBV DNA or copy number of DHBV was used for the comparison. A faint signal of DHBV RNA was barely detected in the spleen and lung, and the density of that signal was much less than in the liver, with the ratio of DHBV RNA concentration estimated at 1: 1000-10 000 Transfection of DHBV DNA could initiate hepatocyte-specific virus replication without a need for the DHBV receptor. At 2 weeks after transfection, the organ distribution of transfected and replicating DHBV was similar to experimental infection. By this time, transient expression of foreign genes was sus-
petted to be reduced and the products of DHBV replication cycles accumulated to the detectable level. Hepatotropism of DHBV or other members of the hepadnavirus family might be explained or partially explained by the transcriptional regulation of DHBV genome by the hepatocyte specific factors, or by hepatocyte specific nuclear factors), as described in several reports (25-29). In other words, even though DHBV DNA was introduced without using DHBV receptor, transcriptional regulatory elements of the DHBV genome were not fully functional in other types of cells. We could not clarify the reason for the deaths of three animals. Other ducklings in groups I and II appeared normal from the time of injection and grew as well as the non-treated ducklings. Zhu et al. reported no treatment-related toxicity of the liposome to mice by histological examinations of a wide variety of tissues (12) A low dose of liposome is thought to be less toxic to the ducklings. This transfection system facilitates the functional analysis of the DHBV genome, because deleted or mutated DHBV DNA could be introduced into hepatocytes by simply injecting a DNA-liposome complex into a peripheral vein, and DHBV would replicate more efficiently than in the culture cells. Hepatitis B virus or other members of hepadnavirus DNA could be introduced to other experimental animals using this systemic injection system to analyze the regulatory elements of gene expression and viral replication and immunological reaction to the viral proteins.
Acknowledgement We are grateful to Patricia L. Marion (Stanford University, USA) for critical reading of the manuscript.
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