Establishment of a Hepatitis B Virus Recombinant Baculovirus System and the
Evaluation of its Function in Viral Antigen Production
Yen-Hsuan Ni, Hong-Yuan Hsu, Huey-Ling Chen, Mei-Hwei Chang
Department of Pediatrics, College of Medicine, National Taiwan University
ABSTRACT
A handy in vitro viral replication system is mandatory for hepatitis B virus (HBV) study.
A recombinant baculovirus with 1.3XHBV DNA construct was previously designed to
infect HepG2 cells. We adapted this system and set up another one using 1.5XHBV
DNA construct to generate our recombinant baculovirus, and we use Huh7 cells instead
of HepG2 cells to establish this system. HBV genome was inserted into the
baculovirus by recombination and the novel HBV recombinant baculovirus was
identified by enzyme digestion. The viral stock was purified and its titre was
determined. We then use the HBV recombinant baculovirus to infect Huh7 cell culture
and demonstrate its ability to produce HBsAg. The production of HBsAg was first
detected in the media three days after infection. Its production was in proportion to the
loading amount of HBV recombinant baculovirus. A sustained HBsAg production
could be achieved by superinfection of this recombinant virus to the already infected
Huh7 cell culture. This system can be applied to the basic and clinical studies of HBV.
We then introduced the recombinant baculovirus into the portal vein of the mice to test
the in vivo model. The histologic examination of the mice showed the
infection found in human pathology was not demonstrated in the mice model. A
microarray study was done to compare the RNA expression in the mouse liver infected
with HBV-recombinant baculovirus and with baculovirus vector only for the future
study.
INTRODUCTION
The major obstacles in the study of HBV have been the inability of the virus to
infect cells in vitro, and the lack of good animal model systems due to a relatively strict
virus-host range. The chimpanzee system is very expensive and difficult to handle, and
only available in very few centers in the world. The duck system and the woodchuck
system are valuable models for studying HBV lifecycle, however, many significant
differences still existed between animal hepadanviruses and human HBV. An
easy-access HBV producing cell line is thus still of necessity for any new chemicals
which might be effective in antiviral therapy for HBV.
Within the last decade, several HBV expressing cell line have been established. These cell
lines were liver-derived human cell lines transfected with HBV DNA and the novel cell
lines containing stably integrated HBV genomes were selected out (1). These cell lines
did make contributions to the study of HBV biology and were useful for the trial of newly
developed antiviral therapy (2,3). There are drawbacks for these cell lines, because they
contained multiple copies of integrated HBV DNA and express HBV DNA with some
integrated into host genome, but this event is not required for the lifecycle of HBV.
Besides, in human hepatocytes, the integrated HBV DNA is frequently rearranged and is
often transcriptionally silent (4). The viral gene expression and replication is continuous
in these cell line and impossible to be synchronized. Thus, it is impossible to precisely
regulate the replication of HBV DNA at a specific time point. It is also impossible to
superinfect the cell line to increase the dosage of HBV.
To overcome the above problems, an HBV recombinant baculovirus system
was established (5). The previous study showed that by way of its endogenous
promoter, a 1.3x genome of HBV construct inside a baculovirus could be successfully
generated and transfected HepG2 cells. This HBV recombinant baculovirus-infected
HepG2 cell line had advantages over the previous cell lines: (i) high-level of HBV
expression. (ii) HBV replication level can be regulated over a wide range simply by
changing the baculovirus multiplicity of infection (MOI). (iii) HBV replication in this
system is detectable one day after infection and persisted for at least 11 days. (iv) The
superinfection with this baculovirus can enhance or extend the infection period of the
test our new drugs and/or antiviral therapy in the future. In our study, we use a
1.5-unit length HBV DNA for generating the HBV expression baculovirus, and
subsequently test in Huh7 cell line.
We then planned to use the HBV-recombinant baculovirus to infect human hepatocytes in vitro and rat hepatocytes in vivo. The RNA extracted from the human and rat hepatocytes after the HBV-recombinant baculovirus infection were subjected to microarray study (the strategy is listed below). Based upon the microarray results, we are likely to find out some possible candidate genes that may regulate the antiviral responses. It is also likely for us to find out possible viral receptor(s) in the mammalian hepatocytes. As the duck model system, it was shown the overexpression of gp180 (receptor) could enhance the duck hepatitis B virus entry into the hepatocytes (6). The microarray possibly will help us to find out overexpression of candidate receptor(s) genes when the recombinant HBV-baculovirus enters the hepatocytes, either in vitro or in vivo in the future.
MATERIALS AND METHODS
Cell culture. Sf21 insect cells (kindly provided by Prof. Bor-Leung Chiang) were
maintained in complete TNM-FH medium (Grace’s insect medium supplemented with
Huh7 cells were maintained in Dulbecco's modified essential medium supplemented with
10% fetal bovine serum and 1% PS and were grown in humidified incubator at 37℃ and 5% CO2. (All medium, serum, and antibiotic are from Gibco, Life Technology).
Construction of Baculovirus Transfer Vector. A recombinant transfer vector was
constructed by excising a PvuII/PvuII fragment containing 1.5×HBV DNA (~5 kb) from pGEM3Z4.8 (kindly provided by Dr. Hui-Ling Wu) and cloned into the SmaI site of the
multiple cloning region of pBlueBac4.5 (Invitrogen, Chatsworth, CA, USA). The
insertion and orientation of the recombinant transfer vector were then confirmed by
HindIII/KpnI enzyme digestion and DNA sequencing (Figs. 1 and 2).
Generation of Recombinant Baculovirus Containing 1.5× HBV DNA. Seed 2×106
Sf21 cells in a 60 mm dish, and gently wash twice with Grace’s insect medium without
FBS to remove the serum. Six μg of the purified recombinant transfer vector, 0.5μg
of the Bac-N-BlueTM DNA (linear AcMNPV baculovirus DNA), and 20μl of Cellfectin®
reagent were then co-transfected into the Sf21 cells according to the manufacturer’s
instructions (BAC-N-BLUE transfection kit, Invitrogen). After 4-hour of incubation at
sealed with paraffin and incubated at 27℃ for 72 hours before harvesting the recombinant baculovirus.
Harvest the Recombinant Baculovirus by Plaque assay. Seed 5×106
Sf21 cells to 100
mm plate and infect the cells with 102, 103, and 104 dilutions of the transfection viral
stock in TNM-FH medium at room temperature for one hour. The medium was then
aspirated and an agarose/medium/X-gal mixture, consisting of 2.5 ml of 2.5% agarose
solution (47℃), 2.5 ml of complete TNM-FH medium (47℃), and 5 ml of complete TNM-FH medium with X-gal (concentration: 150μg/ml, room temperature) was gently poured into the plate to overlay the virus-infected Sf21 cultured cells. The plates were
sealed and incubated at 27℃ for 5 days until the distinguishable blue plaques are formed.
Select the Recombinant Baculovirus by PCR. The putative recombinant viruses were
amplified from blue plaques. 72 hrs after infection, 0.75 ml of the medium was mixed
with the same volume of 20%polyethylene/1M NaCl to precipitate the released virions
from the blue plaques. The medium were then collected and labelled as P-1 stocks and
phenol/chloroform extraction, and isopropanol precipitation. The DNA was used as
templates for polymerase chain reaction (PCR) (Primers P1: 5’-TCA CCA TAT TCT
TGG GAA CAA GA-3’ and P8: 5’-TTA GGG TTT AAA TGT ATG CCC-3’) to detect if
the virus isolates contain the 1.5× HBV DNA.
Preparation of High-Titer Viral Stocks. Seed 25 cm2 flask with 2×106 log-phase Sf21
cells and add 20 μl of the P-1 viral stock in 5 ml complete TNM-FH medium.
Incubate at 27℃ for 7 days until all the cells were lysed. Centrifuge at 1000g for 20 minutes to remove the cell debris and store at 4℃ as P-2 stock. Virus titres were determined by the plaque assay using 106, 107, and 108 dilutions of P-2 stock.
Infection of Huh7 cells with Recombinant Baculovirus Containing 1.5X HBV DNA.
Seed 6-well culture plates with 105 Huh7 cells per well and the cells were grown for 16
to 24 hours before infection. On the day of infection, P-2 virus stock was diluted with
DMEM/FBS/PS according to their titers to achieve the desired multiplicity of infection
(moi) and adjust the final infection volume to1 ml. After 1 hour of infection at 37℃, the inoculum was aspirated and the cells were gently washed with HBSS (Gibco, Life
DMEM/FBS/PS was re-fed and maintained at 37℃ incubator.
Analysis of Secreted Hepatitis B Surface Antigen (HBsAg). Detection of HBsAg was
performed by enzyme linked immunoassay kit (EIA, Abbott). The media from Huh7
cells were collected at several time points after infection and stored at -80℃ until analysis.
Animals and cannula insertion into portal vein. F-344 rats (160-200 gm) were fed a laboratory diet with water and food ad libitum until use and were kept under constant environmental conditions with a 12-hour light-dark cycle. The animals received humane care in compliance with the guidelines of the National Science Council (NSC 1997). Animals were maintained in separate cages. The cannula was an 18 cm length of polyethylene tubing (PE-50, ID 0.58 mm, OD 0.96 mm; Becton Dickinson, Sparks, MD). One end was sheathed with a 2.5 cm length of PE-160 tubing (ID 1.14 mm, OD 1.59 mm; Becton Dickinson, Sparks, MD). The two tubings were fused at one end with heat. The other end of the PE-50 cannula was beveled. A 3 mm section of a 20 GA BD Vialon catheter (ID 1.1 mm; Becton Dickinson, Sandy, Utah) was placed 7 mm from the tip of the bevel as a collar. The finished cannula was sterilized with ethylene oxide. A novel spring-guide-wire introducer needle was created by the modification of the spring-wire guide of the pediatric central venous catheterization set (0.46 mm x 45 cm, Arrow International, Inc). The 25 GA introducer needle was cut 6 cm from its point, and the cut edge was smoothed. This needle was adapted to the straight-tip end of the spring-wire
guide.
Rats were anesthetized intramuscularly with 75 mg/kg ketamine hydrochloride and 5 mg/kg xylazine. A midline laparotomy was performed, and the portal vein was exposed. The spring-guide-wire introducer needle was introduced through the cannula. Using this introducer needle, we punctured the portal vein about 1 mm down-stream from the stitch, advanced the needle 5 mm into the vein, and slid the cannula through the guide-wire into the portal vein up to the Vialon collar. The cannula was secured with a triple knot over the Violon collar with another triple knot just behind the collar. The guide-wire introducer needle was then withdrawn. After free blood returned into the cannula, the cannula was cleared with 0.1 ml heparinized saline (50 U/ml) and stopped with a blunt-ended 23 GA needle. Ampicillin (40 mg) and gentamicin (4 mg) were administered intraperitoneally. The abdominal wall incision was closed with 4-0 chromic surgical guts. The cannula was led through the subcutaneous tunnel to the interscapular incision (Fig. 3). The distal end of the cannula was anchored to the interscapula by suturing the cannula around the PE-160 tubing sheath to the skin. The patency of the cannula was reconfirmed and checked twice a week.
Histologic examination and RNA extraction from the animal livers. The rats were infused the recombinant virus through the portal cannula. In the meantime, the other rats infused with the wild type baculovirus served as the control group. Both the
experimental group and the control group were sacrificed 72 hours after the infusion of virus. When sacrificed, (1) blood samples were collected through superior vena cava and sent for HBsAg assay; (2) a piece of liver tissue was sent for histologic examination;
the specimens were serially sectioned in 10-µm slicesand stained with H&E to define the analyzed regions; (3) another piece of liver was homogenized with Trizol (Qiagen, Chatsworth, CA, USA) to extract RNA.
RESULT
Generation of the HBV Expressing Baculovirus
The 1.5X HBV DNA was excised from pGem3Z4.8, cloned into the baculovirus
transfer vector pBlueBac4.5, and confirmed by restriction mapping (Fig. 1 and 2) and
DNA sequencing. The recombinant baculovirus transfer vector and Bac-N-BlueTM DNA,
a modified linear baculovirus DNA, were then co-transfected into Sf21 cells. The
recombinant baculovirus would produce β-galactosidase and yield blue plaques (Fig. 4).
To confirm the successful construction of the recombinant baculovirus, a PCR with
primers P1/P8 was designed to detect the presence of HBV DNA (Fig. 5) (8). The
viral isolates were used for all subsequent viral amplification and titered by plaque
assay.
Secretion of Hepatitis B Surface Antigen by infected Huh7 cells
products after infecting Huh7 cells. Huh7 cells were infected at different MOI and 1
ml of the medium was collected daily for analysis of HBsAg by EIA. At MOI=50,
HBsAg was first detected 3 days after infection, and the titer of HBsAg increase daily
during the 8-day period (Fig. 6). With different MOIs (= 0, 10, and 20), the production
of HBsAg was basically dose-dependent (Fig. 7).
Superinfection of Infected Huh7 Cells with HBV Baculovirus
To determine whether the HBV expression in Huh7 cells could be bolstered by the
recombinant baculovirus, we superinfected the already infected Huh7 cells 4 days after
the initial infection at MOI=200. The media were collected daily for detection of
HBsAg. The level of HBsAg titer increased after the second infection (Fig. 8).
Histologic finding of the HBV baculovirus-infected rat liver
The rat livers were process for H&E staining 72 hours after HBV baculovirus
infusion through portal vein. We did not detect any ground glass pattern of human
HBV infection. However, the necrosis of hepatocytes, the acidophilic bodies, and
DISCUSSION
Baculovirus is a natural hepatotropic agent (9). It tends to predominantly infect the
hepatocytes and won’t replicate in mammalian cells (10,11). This nature renders this
HBV recombinant baculovirus become a powerful in vitro HBV replication system. In
this project, we aimed to set up an easy and quick system of HBV replication. Such a
system will be very useful when we apply it to the study of the efficacy of antiviral
therapies, the molecular and cellular profiles of viral-hepatocyte interaction, and even the
hepatocytes responses to multiple viral infections.
We have now established the HBV recombinant baculovirus system. A 1.5X HBV
genome was inserted into a baculovirus to construct this recombinant baculovirus. This
system was well demonstrated to be successful in terms of the production of HBsAg, the
dose-dependent manner of the HBsAg production, and the sustained production of
HBsAg by a superinfection of the recombinant virus. We also know the temporal profile
of HBsAg production in this system.
The next step is to prove the existence of all the replication intermediates of HBV in
system. In the future, this system can be applied to: (i) the evaluation of efficacy of
antiviral therapies. (ii) The model for acute response to HBV infection in hepatocytes;
we may check the molecular profiles of hepatocytes before and after infected by HBV
recombinant baculovirus. (iii) The model of co-infection of HBV plus other viruses or
superinfection of other viruses upon an HBV-infected hepatocyte cell lines.
By this study, we did demonstrate a successful establishment of HBV baculovirus
system in vitro. The portal vein infusion of this recombinant virus into the rat liver is
not as successful as the in vitro model. The failure to demonstrate ground glass pattern
of human HBV infection in the rat liver may be due to: (i) different species with
different manifestation of HBV infection (ii) failure of HBV baculovirus to enter the
hepatocytes through the portal vein. We have to do other studies, like HBcAg
immunohistochemical staining, HBV RNA expression, to prove the in vivo expression
of HBV baculovirus. If all the above studies proved that this is not the right way for
HBV baculovirus to express, it is likely that we should seek for some other animal
ACKNOWLEDGEMENTS
The authors thank Prof Chu-Fang Lo for her help in baculovirus system set-up. The
authors also thank Ms. Y Lin for her technical assistance and help to prepare the
manuscript. This study is supported by a grant from National Science Council,
REFERENCES
1. Shih C, Li LS, Roychoudhury S, Ho MH. In vitro propagation of human hepatitis B virus in a rat hepatoma cell line. Proc Natl Acad Sci USA 1989;86:6323-7.
2. Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in HepG2 cells
transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci USA
1987;84:1005-9.
3. Tsurimoto T, Fujiyama A, Matsubara K. Stable expression and replication of hepatitis
B virus genome in an integrated state in a human hepatoma cell line transfected with
the cloned viral DNA. Proc Natl Acad Sci USA 1987;84:444-8.
4. Nagaya T, Nakamura T, Tokino T et al. The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev 1987;1:773-82.
5. Delaney WE, Isom HC. Hepatitis B virus replication in human HepG2 cells mediated
by hepatitis B virus recombinant baculovirus. Hepatology 1998;28:1134-46.
6. Breiner KM, Schaller H. Cellular receptor traffic is essential for productive for duck
hepatitis B virus infection. J Virol 2000; 74:2203-9.
7. Remie R. Experimental surgery. In: Krinke GJ, eds. The laboratory rat. London:
8. Lindh M, Anderson AS, Gusdal A. Genotypes, nt 1858 variants, and geographic origin
of hepatitis B virus - large scale analysis using a new genotyping method. J Infect Dis
1997;175:1285-1293.
9. Boyce FM, Bucher NL. Baculovirus-mediated gene transfer into mammalian cells.
Proc Natl Acad Sci U S A. 1996; 93:2348-52.
10. Sandig V, Hofmann C, Steinert S, Jennings G, Schlag P, Strauss M. Gene transfer
into hepatocytes and human liver tissue by baculovirus vectors. Hum Gene Ther 1996
7:1937-45.
11. Hoffmann C, Sandig V, Jennings G, Rudolph M, Schlag P, Strauss M. Efficient
gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S
LEGENDS OF THE FIGURES
Figure 1. HBV recombinant transfer vector, a PvuII/ PvuII fragments containing the
entire 1.5X-genome length HBV construct was excised from pGem3Z4.8 and cloned
into the baculovirus transfer vector pBlueBac4.5.
Figure 2. HBV recombinant transfer vector was confirmed by restriction mapping.
The PvuII/ PvuII fragment containing the entire 1.5-genome length HBV construct is
approximately 5 kb and pBlueBac4.5 is 4.9 kb. The linear size of the recombinant
vector is approximately 10 kb. If this vector is cut with HindIII and KpnI
simultaneously, the resulting 4.9 kb plus 5 kb will be difficult to separate.
Figure 3. Long-term portal cannulation in rats. The portal cannula was (A) inserted into
the main portal vein, remaining secured onto the wall of portal vein by one stitch with a
triple knot over the Vialon collar, and (B) was anchored in the interscapular area of the rat.
Figure 4. Successful recombinant baculovirus yield blue plaques
Figure 5. PCR result of recombinant baculovirus isolates. pGem3Z4.8 is used as the
template of positive control.
baculovirus.
Figure 7. Analysis of HBsAg in the medium of Huh7 cells infected with different MOIs
of HBV recombinant baculovirus.
Figure 8. The effect of superinfection of HBV recombinant baculovirus. The already
infected Huh7 cells were superinfected with HBV recombinant baculovirus 4 days after
the initial infection at MOI=200. The booster effect of HBsAg production could be
seen.
Figure 9. The histological examination of the rat liver sacrificed 72 hours after HBV
baculovirus infusion through the portal vein. (a) necrosis of hepatocytes (arrow) (b) an
acidophilic body (arrow) showed an acute hepatitis reaction (c) perihapatocyte hyaline
membrane changes, perhaps also due to the acute hepatitis reaction (d) the control rat
