Proc.-Natl Acad. Sci.
USA
Vol. 80, pp. 1703-1706, March 1983
Medical Sciences
Naturally occurring infection of Pekin duck embryos by duck
hepatitis B virus
(maternal transmission/viral replication/DNA polymerase/agarose gel electrophoresis)
ANNA P. O'CONNELL, MICHAEL K. URBAN, AND W. THOMAS LONDON
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Communicated by Baruch S. Blumberg, December 2, 1982
ABSTRACT We tested the hypothesis that duck hepatitis B
virus (DHBV) is a naturally occurring congenital infection of Pekin
duck embryos. Of 219 embryos, 5-25 days after being laid, sera
from 30 were found to be positive for endogenous DNA polymer-
ase activity characteristic of hepatitis B-related viruses. The pres-
ence of the duck virus was confirmed by hybridization with cloned
DHBV DNA. Viral DNA was also found in the livers of embryos
incubated for 12 or 18 days. Electrophoretically different forms
of DHBV DNA were identified in liver extracts that were not pres-
ent in serum. These additional liver forms probably represent viral
replication intermediates. These observations suggest that the ver-
tical route is a major pathway of DHBV transmission and that viral
replication may be initiated by the 12th day of embryonic life.
Infection with human hepatitis B virus (HBV) is a worldwide
health problem. The majority of HBV infections are acute and
are followed by complete recovery with the development of an-
tibody to the surface antigen of the virus (anti-HBs). In areas of
the world where the virus is endemic (Southeast Asia, China,
and Africa), most of the members of the population have been
infected and a significant proportion (3-20%) are chronically in-
fected. Persistent infection with HBV is associated with chronic
liver disease and primary hepatocellular carcinoma (PHC) (1-3).
Study of the role of HBV in liver carcinogenesis has been
hampered by the limited host range of HBV (man and chim-
panzees) and the lack of an in vitro system for viral propagation.
Recently, it has been shown that HBV belongs to a family of
hepatitis B viruses (4) that includes woodchuck hepatitis virus
(WHV) (5), ground squirrel hepatitis virus (6), and Pekin duck
HBV (DHBV) (7). Since persistent infection of woodchucks with
woodchuck hepatitis virus leads to the development of PHC (5)
and domestic ducks in China that are carriers of DHBV have
been reported to develop PHC (ref. 8; M. Omata, personal
communication), these animal systems may provide a means to
study the relationship between HBV infection and the patho-
genesis of PHC.
A question of particular importance in humans is whether
true vertical transmission of HBV occurs. Transmission of HBV
from infected mothers to their infants is well documented (9-
12). Most workers believe that in utero infection does not occur
and that babies become infected horizontally by contact with
maternal blood during delivery, since serological signs of in-
fection do not become evident until 6 wk of age. An alternative
possibility is that the fetus is infected in utero, but the fetal liver
is unable to replicate HBV or release viral products into the blood
until further maturation of hepatocytes occurs. The study of
duck embryos makes possible an investigation of whether ver-
tical transmission of DHBV occurs and at what stage of devel-
opment and maturation virus replication begins.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertise-
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
1703
In this paper, we report that, under natural conditions, Pekin
ducks are frequently infected with DHBV early in embryonic
life and that the virus replicates in the embryonic liver.
MATERIALS AND METHODS
Materials. Fertilized Pekin (Anas domesticus) duck eggs
were purchased from a commercial supplier. [a-32P]TTP (800
Ci/mmol; 1 Ci = 37 GBq) was purchased from New England
Nuclear. Sodium dGTP, sodium dCTP, and disodium dATP were
from P-L Biochemicals. Nuclease S1 of Aspergillus oryzae was
purchased from Miles. Escherichia coli DNA polymerase I and
endonuclease EcoRI were from Boehringer Mannheim. BamHI
(Bacillus amyloliquefaciens H.) was from New England BioLabs.
Electrophoresis grade agarose was from Seakem (Rockland, ME).
Nitrocellulose sheets (BA85) (0.45 ,Am) were from Schleicher &
Schuell. Pronase was from Calbiochem-Behring. Enzyme in-
cubations were conducted according to supplier's recommen-
dations.
Methods. Cells and nuclei. Embryos from eggs incubated 5
days were explanted by trimming around the sinus terminalis
and blood was isolated from these embryos by cannulation of the
vitelline vessels. Blood was obtained from older embryos by car-
diac puncture. Twelve- and 18-day-incubated duck embryos were
dissected free of extra-embryonic membranes and rinsed in
chicken Ringer's solution to remove adhering yolk; the livers
were removed and frozen at -70°C. Nuclei were isolated from
the frozen livers as described (13). The supernatant (25 ml)
from the first homogenization and centrifugation in 10 mM K/Tris
maleate, pH 7.4/5 mM CaC12/25 mM glycine (the postnuclear
supernatant) and the nuclei were saved for DNA extraction and
assay of DHBV-specific DNA.
Extraction of DNA. The nuclei and postnuclear supernatant
were digested with Pronase at 100 ,ug/ml in 0.15 M NaCl/0. 1
M EDTA/0.2% NaDodSO4 for 1 hr at 37°C. Nucleic acids were
deproteinized by two extractions with Tris-buffered phenol/
chloroform, pH 8.0, followed by one extraction with chloro-
form. The aqueous phase was adjusted to 0.2 M NaOAc and the
nucleic acids were precipitated with 2.5 vol of absolute ethanol.
The pellet was suspended in 0. 15 M NaCl/0. 5% NaDodSO4 and
digested with Pronase at 100 ,ug/ml as above. The DNA was
extracted as above and precipitated with absolute ethanol. In
addition, some samples were digested with RNase A at 100 ,g/
ml in 0.15 M NaCl/0.015 M Na citrate (NaCl/Cit) for 1 hr at
370C.
Assay of serumfor endogenous DNA polymerase activity. Fifty
to 100 ,ul of duck embryo serum was layered onto 5 ml of a 10-
20% (wt/vol) sucrose gradient in 0.15 M NaCl/0.02 M Tris HC1,
pH 7.4, and centrifuged for 3 hr at 48,000 rpm in a Spinco SW
Abbreviations: HBV, hepatitis B virus; PHC, primary hepatocellular
carcinoma; DHBV, duck HBV; NaCl/Cit, 0.15 M NaCl/0.015 M Na
citrate; kb, kilobase pair(s).
1704 Medical Sciences: O'Connell et al Proc. Natl. Acad. Sci. USA 80 (1983)
50.1 rotor at 20'C. The supernatant was discarded and the pellet 1 6 7 A kb
was assayed for incorporation of [a-32P]dTTP into viral DNA by qww.l* * _

an endogenous DNA polymerase activity (7).

Assay of duck DNA for DHBV sequences. For the hybrid-
ization spot test, 10 Al of serum or 5-25 ,ul of DNA in distilled
water was spotted directly on nitrocellulose filter paper and air
dried (14). The nitrocellulose was submerged in 0.1 M NaOH/1.5
M NaCl for 3 min and then in 0.1 M Tris HCl, pH 7.4/1.5 M
NaCl for 3 min and baked at 80'C at reduced pressure for 2 hr.
The filter was treated by the method of Wahl et al. (15) and hy-
bridized with [a-32P]dTTP-labeled DHBV DNA isolated from
an EcoRI digest of the viral genome cloned in the bacteriophage
p
S -4.3

A vector Charon 16A. The viral clone was a gift of William S.

Mason et al. (16). The filter was incubated as described (15),
washed three times at room temperature with 0.1% NaDodSO4
in double-strength NaCl/Cit (a total of 20 min) and three times
at 560C with 0. 1% NaDodSO4 in 10% NaCl/Cit (20 min per wash),
dried, and autoradiographed.
For hybridization of DHBV to embryonic duck DNA sepa- FIG. 1. Agarose gel electrophoresis of the product of the endoge-
rated by agarose gel electrophoresis, the DNA was transferred nous DNA polymerase reaction. Five serum samples from 18-day em-
to a nitrocellulose filter by the following modification of the pro- bryonic ducks and serum from A, an adult DHBV-positive duck, were
cedure of Southern (17). Before denaturation, the gel was soaked centrifuged through a 10-20% sucrose gradient in 0.15 M NaCl/0.02
in 0.05 M NaOAc (pH 4.2) at room temperature for 1 hr and at M Tris HCl, pH 7.4, for 3 hr at 48,000 rpm in a Spinco SW 50.1 rotor
53°C for 1 hr, and 1 M NH4OAc (pH 8. 0) was used as the transfer at 2000. The pellets were incubated for 2 hr at 370C in the presence of
buffer instead of NaCl/Cit. The filter was then baked dry and [a32P]TTP and unlabeled dATP, dGTP, and dCTP as described by Ma-
son et al. (7). Gel electrophoresis was run for 16 hr. Serum samples 1
hybridized as described above for the spot test. One Southern and 7 are positive for DHBV DNA polymerase by this assay.
transfer filter was hybridized with 32P-labeled plus-strand DHBV
DNA, cloned in M13 phage, a gift of William S. Mason et al. markers is increased when the concentration of agarose in the
(14). gel is decreased from 1.5% (Fig. 3) to 0.7% (Fig. 4B) [4.2 kilo-
Agarose gel electrophoresis. One-tenth volume of 64% (wt/vol) base pairs (kb) -* 3 kb] (7) and it is converted to band 2 after
sucrose/1% Sarkosyl/0. 1% bromophenol blue was added to the digestion with restriction endonucleases that cut within DHBV
samples from the endogenous DNA polymerase assay and to the DNA only once (BamHI, EcoRI; Fig. 3) (14). Band 2 has an elec-
DNA extracted from duck embryos. This mixture was electro- trophoretic mobility identical to that of cloned linear duplex
phoresed at 0.8 V/cm on a horizontal slab gel of 1.5% (wt/vol) DHBV DNA and probably results from nicking the relaxed-cir-
or 0.7% agarose in 0.04 M Tris acetate, pH 7.2/20 mM NaOAc/1 cular partially double-stranded DHBV DNA within the single-
mM EDTA. stranded region (14). This is further substantiated by finding
RESULTS discrete DHBV components of =2.5 kb after digestion with EcoRI
and =2.2 kb after digestion with BamHI, suggesting cleavage
Sera from duck embryos were first tested for virus-associated of the linear DHBV DNA (band 2) a short distance from the sin-
DNA polymerase activity. About 14% of sera from duck em- gle-stranded nicking site (14). The second fragment generated
bryos at each incubation stage studied incorporated labeled nu- from these digestions would have migrated out of the gel during
cleotides into a DNA species having a size similar to that of the electrophoresis. The faster migrating species have not been de-
duck HBV-like virus (Fig. 1 and Table 1). tected in the sera of DHBV-infected 18-day duck embryos (Fig.
To confirm that the endogenous polymerase activity repre- 4A). The bands labeled SS are resistant to EcoRI and BamHI
sented DHBV, a spot hybridization assay was carried out with digestion but sensitive to nuclease S1 digestion, suggesting that
cloned DHBV DNA. Fig. 2 shows the spot hybridization pat- they contain single-stranded DNA. Incubation of the liver DNA
terns of serum samples from forty-six 12-day embryos, five of in 10% NaCl/Cit at 1000C for 5 min, which dissociated the strands
which were positive for DHBV DNA and from seven 18-day em-
bryos, three of which were positive for DHBV. Sera that tested Table 1. Prevalence of DHBV in sera of Pekin duck embryos
positive for DHBV by the DNA polymerase assay also tested DNA polymerase
positive for DHBV DNA by hybridization. However, 6 of the positive embryos
189 embryonic duck sera in which polymerase activity was not Days of Embryos,
detected were positive for DHBV DNA by hybridization (sam- incubation no. No. %total
ple 6: compare Fig. 1 with Figs. 2B and 4B). 5 14 5 35.7
If congenital infection is a major mode of transmission for 10 20 2 10.0
DHBV, then replicating viral DNA should be present in the tis- 12 55 6 10.9
sues (liver) of embryonic ducks. The endogenous polymerase- 15 25 2 8.0
labeled DHBV DNA from the sera of infected ducks migrates 17 23 2 8.7
as two components when electrophoresed in 1.5% agarose gels 18 10 2 20.0
(Fig. 1). However, when DNA isolated from 18-day embryonic 21 9 1 11.2
duck liver was electrophoresed on 1.5% agarose gels, at least 23 26 5 19.2
four components hybridized to DHBV DNA (Fig. 3). The two 24 18 2 11.1
slower migrating bands are similar in electrophoretic mobility 25 19 3 15.7
to the DNA of the DHBV virus in serum (Fig. 1). Band 1 is Total 219 30 13.7
probably the relaxed circular conformation of DHBV DNA since Virus was detected by the endogenous DNA polymerase assay, using
its electrophoretic mobility relative to that of linear duplex gel electrophoresis to identify labeled DHBV DNA molecules.
 Medical Sciences: O'Connell et al.
A 6
0
2-0
4%
B 1-
6_ 7*
FIG. 2. Spot hybridization analysis of sera from 12-day (A) and 18-
day (B) embryonic ducks. Ten-microliter samples of serum from forty-
six 12-day embryos and seven 18-day embryos were spotted directly on
nitrocellulose filter paper. Hybridization was with [a32P]TTP-labeled
DHBV DNA isolated from an EcoRI digest of the viral genome cloned
in the bacteriophage A vector Charon 16A. As a control (C), 3 nmol of
unlabeled probe was spotted on each filter. Numbered spots indicate
positive serum samples. Only intense spots were accepted as positive;
gray areas were considered background.
in duplexed molecules, resulted in complete conversion of all
the components except band 3 to a band having an electropho-
retic mobility identical to that of band SS (Fig. 3). Band 3 is
probably supercoiled closed-circular double-stranded DNA, since
it is resistant to digestion with nuclease S1 under conditions in
which band SS is digested, is converted to complete linear dou-
ble-stranded DHBV DNA by digestion with EcoRI, and has an
electrophoretic mobility similar to that of 2-kb linear duplex DNA.
DNA isolated from both liver nuclei and cytosol (postnuclear
a b C d e f
-4.3
2- _~~~~~~~~.DH BV
S I -2.1
FIG. 3. Southern blot analysis of embryonic duck DNAs separated
on a 1.5% agarose gel for DHBV sequences. DNA was extracted sep-
arately from nuclei and cytosol of an 18-day embryonic liver (sample
7 in Fig. 1). Lanes: a, nuclear DNA; b, cytosolic DNA~c-e, nuclease S1,
EcoRI, and BamHI digests of nuclear DNA; f, nuclear DNA denatured
by incubation with 10% NaCl/Cit at 10000 for 5 min. All samples were
incubated at 3700 for 1 hr with 0.2% NaDodSO4/Pronase 100 pug/ml
prior to electrophoresis. DHBV represents the electrophoretic mobility
of the unlabeled cloned DHBV probe. Markers (numbers on the right,
kb) are sizes of HindIll fragments of A DNA; 1, 2, 3, SS, band identi-
fication on left.
Proc. Natl. Acad. Sci. USA 80 (1983) 1705
a b a b kb a b
-4.3
wr
^ ^ 1~~,2-
as~3 _M
.U
3-^
OF-
BS
C
FIG. 4. Southern blot analysis of embryonic duck DNAs separated
on a 0.7% agarose gel for DHBV sequences. (A) Lanes: a, 1 ng of un-
labeled cloned DHBV probe; b, DNA extracted from the serum of an 18-
day duck embryo positive for DHBV. (B and C) Eighteen-day (sample
6 in Fig. 1) and 12-day total embryonic liver. Lanes: a, undigested sam-
ples; b, nuclease Sl-digested samples. All samples were incubated at
3700 for 1 hr with 0.2% NaDodSO4/Pronase 100 jtg/ml. 1, 2,3, SS, band
identification.
supernatant) of 18-day embryonic ducks include bands 1, 2, 3,
and SS but, compared with liver nuclear DNA, cytosolic DNA
contains relatively less band 3 and more band SS and compo-
nents that have a faster electrophoretic mobility than band SS
(Fig. 3). Similar DHBV-hybridizing electrophoretic forms were
detected in the liver DNA of 12-day embryos (Fig. 4C).
We investigated whether DHBV DNA was integrated into
specific sites in the host genome by digesting cellular DNA of
infected duck embryos with a restriction endonuclease (Sac I)
that does not cut within DHBV DNA (unpublished data). This
should have revealed distinct species larger than 3 kb and hy-
bridizable to cloned DHBV DNA if the virus had integrated
into the genome of the germ line or had integrated during early
developmental stages. (Integration at random sites at later stages
of development would not be detected by this procedure.) In
our analysis of EcoRI, BamHI (both cut once within DHBV),
and Sac I digests of the genomic DNA from several 12- and 18-
day-incubated duck embryos, we have not detected DHBV-hy-
bridizable species that correspond to integrated viral DNA at
specific loci, under hybridization conditions sensitive enough
to detect one viral sequence per cell.
In some cases, serial dilutions of total liver DNA were spot-
ted on nitrocellulose filters with known concentrations of cloned
DHBV DNA. To estimate the number of viral genomes per cell,
the intensity of the spots after hybridization was compared with
the dilution of liver cell DNA. Using these criteria, we esti-
mated that each hepatocyte contained 100-1,000 DHBV ge-
nomes.
DISCUSSION
Viruses of the HBV family have a double-stranded circular DNA
genome that is partially single stranded and an endogenous DNA
polymerase capable of repairing the single-stranded gap in an
in vitro reaction (18). If this reaction is carried out with DHBV
in the presence of [a-32P]dTTP, a 3-kb circular DNA species is
labeled (7). Using this assay, we have detected DHBV in duck
embryos as early as the 5th day of incubation. Furthermore, we
have confirmed that the endogenous polymerase activity is DHBV
 ~ -1
1706 Medical Sciences: O'Connell et al.
specific by showing that the DNA in the serum and liver of poly-
merase-positive embryos contains sequences that hybridize to
cloned DHBV DNA (16).
We estimated by spot hybridization, using a known amount
of cloned viral DNA as a control and DNA isolated from total
embryonic liver homogenates, that 12-day embryonic livers
contain 100-1,000 viral genomes per cell. DHBV DNA is found
in the livers of adult ducks at 1,000-2,000 copies per cell (7).
Thus together, these results show that ducks are infected during
embryonic life. Since about 15% of the sera from duck embryos
(this report) and about 10% of the adult ducks in two commercial
flocks were positive for DHBV (7), vertical transmission is prob-
ably a major route of infection of DHBV.
Mason et al. (14) have described several electrophoretic forms
of DHBV viral DNA in infected livers of Pekin ducks. These
forms include DNAs similar to those associated with virions found
in sera (relaxed-circular partially double-stranded DNA and lin-
ear double-stranded DNA) and DNAs not normally found as-
sociated with mature virions (single-stranded DNA and cova-
lently closed circular double-stranded DNA). Summers and
Mason and co-workers (14, 19, 20) have suggested that these
additional electrophoretic forms may represent viral replication
intermediates. The single-stranded DNA consists of minus-strand
DNA that is reverse transcribed from a plus-strand RNA tem-
plate (20). The plus-strand RNA may be transcribed from the
covalently closed circular viral DNAs found in the nuclei of in--
fected cells (19). These electrophoretic forms of DHBV DNA
can also be identified in 12-day and 18-day embryonic livers.
We also find that, in embryos, liver nuclei are enriched in the
closed circular form and the cytosol is enriched in the single-
stranded form. Furthermore, band SS of duck embryo liver DNA
hybridized to a probe (M13 clone of DHBV DNA, a gift of W.
Mason) complementary to the minus strand of DHBV DNA (Fig.
a b c Robinson, W. S. (1980) Proc. Nati Acad. Sci. USA 77, 2941-2945.
w J. Am. Med. Assoc. 220, 1092-1095.
*1-2
..I
FIG. 5. Southern blot analysis of embryonic duck DNAs separated
on a 1.5% agarose gel for DHBV sequences. DNA was extracted sep-
arately from the nuclei (lane b) and cytosol (lane c) of an 18-day em-
bryonic liver. Lane a: unlabeled DHBV probe cloned in Charon 16A.
The labeled probe was strand-specific M13 clone 8 phage complemen-
tary to the minus strand of DHBV. 1, 2, 3, SS, band identification.
Proc. Nad Acad. Sci. USA 80 (1983)
5). Hence, our findings suggest that DHBV DNA is replicating
in the embryonic liver by the 12th day of incubation.
In humans, infection of the fetus is not thought to occur. This
hypothesis has not been directly tested, however. In Taiwan,
administration of hepatitis B-immune globulin to babies born
to HBV carrier mothers reduced the incidence of chronic in-
fection from 90% to 20%, but it did not reduce the overall rate
of infection. The passively immunized babies who did not be-
come chronically infected developed very high titers of anti-
body to the surface antigen (anti-HBs), much higher than could
be accounted for by the exogenous hyperimmune globulin, and
the antibody titers were still high when the babies were ex-
amined 1 year later (21). These observations are compatible with
the view that the babies became infected in utero and hyper-
immune globulin altered the response to infection. Based on our
findings in duck embryos, further investigation of human,
woodchuck, and ground squirrel embryos for evidence of in-
fection with HBV-like viruses is warranted.
We are grateful to J. Summers and W. Mason for helpful discussions.
This work was supported by Grants CA-06551, RR-05539, and CA-06927
from the National Institutes of Health and by an appropriation from the
Commonwealth of Pennsylvania.
1. Szmuness, W., Harley, E. J., Ikram, H. & Stevens, C. (1978) in
Viral Hepatitis, eds. Vyas, G. N., Cohen, S. N. & Schmid, R.
(Franklin Institute Press, Philadelphia), pp. 297-320.
2. Redeker, A. G. (1975) Am. J. Med. Sci. 270, 9-16.
3. London, W. T. & Blumberg, B. S. (1981) in Cancer: Achieve-
ments, Challenges and Prospects for the 1980s, eds. Burchenal,
J. H. & Oettgen, H. F. (Grune & Stratton, New York), Vol. 1,
pp. 161-183.
4. Summers, J. (1981) Hepatology 1, 179-183.
5. Summers, J., Smolec, J. M. & Snyder, R. A. (1978) Proc. Natl.
Acad. Sci. USA 75, 4533-4537.
6. Marion, P. L., Oshiro, L. S., Regnery, D. C., Scullard, G. H. &
7. Mason, W. S., Seal, G. & Summers, J. (1980) J. Virol. 36, 829-
836.
8. Zhou, Y. Z. (1980) Shanghai Med. J. 3, 641-644.
9. Boxall, E. & Flewett, T. H. (1975) Lancet ii, 1211.
10. Okada, K. I., Kamiyama, I., Inomata, M., Imai, M., Miyakawa,
Y. & Mayumi, M. (1976) N. Engl. J. Med. 294, 746-749.
11. Schweitzer, I. L., Wing, A., McPeak, C. & Spears, R. L. (1972)
12. Stevens, C. E., Beasley, R. P., Tsui, J. & Lew, W. (1975) N. Engl.
J. Med. 292, 771-774.
13. Urban, M. K., Franklin, S. G. & Zweidler, A. (1979) Biochem-
istry 18, 3952-3960.
14. Mason, W. S., Aldrich, C., Summers, J. & Taylor, J. M. (1982)
Proc. Natl. Acad. Sci. USA 79, 3997-4001.
15. Wahl, G. M., Stem, M. & Stark, G. R. (1979) Proc. Natl. Acad.
Sci. USA 76, 3683-3687.
16. Mason, W. S., Taylor, J. M., Seal, G. & Summers, J. (1982) in
Proceedings of the 1981 Symposium on Viral Hepatitis, eds.
Szmuness, W., Alter, H. & Maynard, J. (Franklin Institute Press,
Philadelphia), pp. 107-116.
17. Southern, E. M. (1975)J. Mol. Biol. 98, 503-517.
18. Summers, J., O'Connell, A. & Millman, I. (1975) Proc. NatW Acad.
Sci. USA 72, 4597-4601.
19. Summers, J. & Mason, W. S. (1982) Hepatology 2, 61S-66S.
20. Summers, J. & Mason, W. S. (1982) Cell 29, 403-415.
21. Stevens, C. E., Beasley, R. P., Lin, C. C., Hwang, L. Y., Sun,
J. S., Hsieh, F. J., Wang, K. Y. & Szmuness, W. (1982) in Pro-
ceedings of the 1981 Symposium on Viral Hepatitis, eds. Szmu-
ness, W., Alter, H. & Maynard, J. (Franklin Institute Press, Phil-
adelphia), pp. 527-535.