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Introduction
Hepatitis B virus (HBV) is the causative agent of acute
and chronic hepatitis B in humans. More than 350 million
people worldwide are chronic virus carriers and face a
significantly increased risk of developing liver cirrhosis and
primary hepatocellular carcinoma. Despite the availability of
a safe and effective vaccine against hepatitis B, chronic
infection with HBV remains a major health problem
worldwide[1_4]. The duck model of HBV (DHBV) infection represents a
suitable system for the study of the activity of anti-HBV
agents. It provides relevant tools for the detailed
molecular studies of hepadnavirus replication and its
inhibition[5_7].
To date, conventional therapy for chronic HBV infection
has involved the use of either α-interferon
or 3-thiacytidine (3TC, lamivudine). α-Interferon therapy is only partially
effective and is frequently limited by adverse effects and is
also expensive. Although 3TC efficiently inhibits HBV
replication, the emergence of drug-resistant mutants which
carry mutations affects the reverse transcriptase domain.
In order to control viral replication and delay the
emergence of virus-resistant mutants, it is critical to look for new
valid medicines and develop new antiviral
strategies[8,9]. Only
a few antiviral drugs that are currently under clinical trial
for the treatment of persistent HBV infection are Entecavir
(ETV)[10_12], Chinese medicinal herbs etc.
The present study was designed to test the efficacy of
PNA, a new potent inhibitor of the hepadnaviral polymerases.
PNA is a
2-amine-9-(2,3-dideoxy-2,3-dihydro-β-D-arabino-
furanosyl)-6-methoxy-9H-purine. It was conducted to
evaluate the antiviral activities of PNA in this alternative HBV
animal model by measuring the levels of DHBV DNA in
infected duck serum and livers.The changes of duck hepatitis
B surface antigen (DHBsAg) and liver pathological changes
were also observed.
Materials and methods
Animals One-day-old Sichuan Mallard ducklings
(Anas platyrhynchos domesticus) were purchased from Sichuan
Agriculture University's hatchery (Yaan, China) and held in
the animal house facilities of the Institute of
Veterinary Science, Sichuan Agriculture University. Congenitally
DHBV-infected ducklings detected by PCR were excluded
from the experiments.
Inoculum preparation A pooled, sera-derived DHBV
containing 9.6×1010 viral
genomes (vge)/mL that was collected from the congenitally-infected Peking ducks during a period
of 2 weeks (6_20 d old) was used as the inoculum. The viral
inoculum was prepared by diluting DHBV-infected sera with
uninfected duck sera to obtain the appropriate dose of
1.6×107 DHBV genome equivalents in 100 µL and was
inoculated via the vena cruralis.
Drug source, preparation, and uptake
PNA was synthesized and supplied in powder form by Nanjing Changao
Pharmaceutical Technology (Nanjing, China).
100 mg PNA was added to 1 mL distilled water. 100
mg/mL stock solutions of PNA were stored
at 4 °C for up to 1 d and allowed to reach room
temperature before use. A stock solution of 3TC was prepared as earlier mentioned.
PNA, 3TC, or distilled water was administered twice a day
to the ducks in each group.
Ninety ducklings were divided into 6 groups. Fifteen
ducklings from each group started daily treatment at d 7 after
inoculum preparation. Control group I received a placebo of
2 mL distilled water and control group II was administered
perorally (po) with 3TC at the dose of 50 mg/kg.
Treatment group I was administered with PNA at a dose of 40
mg/kg and treatment group II was administered with PNA
at a dose of 80 mg/kg by po. Treatment group III was
administered with PNA at a dose of 20 mg/kg and treatment
group IV was administered with PNA at a dose of 40 mg/kg
PNA intra-venously. The administration lasted 10 d. The
experimental plan is summarized in Figure 1.
Sample collection The serum samples were collected
daily at 1 d before treatment, 5 and 10 d after treatment, and
at 3 d withdrawal for monitoring viremia in each animal. The
blood samples were collected from the jugular vein, and
serum was collected following the incubation of the blood
samples at 37 °C for 90 min and then stored at -20 °C.
An autopsy was performed in 5 ducks in each group at
the same time for determining the amount of inoculum virus
bound in the liver. The liver samples from all the ducks were
divided into 2 parts. Part I was snap-frozen
in liquid nitrogen and then stored at -80 °C and part II was
fixed in 4% paraformaldehyde buffered with phosphate-buffered
solution (PBS). The experimental plan is summarized in Figure 1.
Analysis of serum samples
Isolation of DNA from sera From each serum sample,
300 µL was incubated at 65 °C for 4 h in lysis buffer (20
mmol/L Tris-HCl [pH 8.0] 10 mmol/L EDTA, 0.1% SDS, and 0.8 %
proteinase K). DNA was then extracted with
phenol-chloroform and precipitated with ethanol. The pellet was dissolved
in 50 µL of RNase- and DNase-free
ddH2O.
PCR was employed to exclude congenitally
DHBV-infected ducklings. The serum samples were tested
by PCR for the presence of DHBV DNA. The PCR mixture also
contained 5 µL of 10×reaction buffer (TaKaRa, Dalian, China),
4 µL of 25 mmol/L MgCl2, 1 µL of 10×dNTP, 1 µL
Taq polymerase (TaKaRa, Dalian,China),
1 µL of both forward and reverse primers, and 50 µL
ddH2O. The PCR primers were designed according to the sequence in GenBank
(Accession No gi325448) and were as follows:
5'-CCATTG-AAGCAA-TCACTAGAC-3' and
5'-ATCTATGGTGGCTGC-TCGAAC-3'. The PCR protocol was 5 min at 94 °C, followed
by 30 cycles of 35 s at 94 °C, 35 s at 55 °C, and 45 s
at 72 °C. The PCR product was detected by 1.5% agarose gel
electro-phoresis.
Oligonucleotide primers and probe for real-time
PCR Real-time PCR was performed using the BioRad Lightcycler
(BioRad, Idaho, USA) on extracted DHBV DNA samples and
quantitated using fluorescence probe. Each 25 µL reaction
mixture contained 50 ng DNA, 2 µL probes
(Genecore,Shanghai,China), 2.5 µL
MgCl2 (at a final concentration of
2.5 mmol/L), 0.25 µL TaKaRa Ex Taq HS (5 U/µL) (TaKaRa,
Dalian, China), and 0.5 µL of 10 µmol/L primers. The probe
sequence (5'-CGGGCTCCCCTCTCCCACG-3') was labeled with 6-carboxy-fluorescein dye at the 5'-end and with the
quencher dye 6-carboxy-tetramethyl-rhodamine at the 3'-end.
The sense primer sequence was 5'-GAGCCCCTTCACCCC-AAC-3' and antisense primer sequence was
5'-ATCTATGGT-GGCTGCTCGAACT-3'.The primers and probe were
synthesized by Shanghai Genecore (TaqMan minor groove binder,
Shanghai, China). The PCR protocol required initial
incubation for 5 min at 95 °C and then 40 cycles of 5 s at 95 °C, 10
s at 55 °C, and 15 s at 72 °C. Each sample was detected in
triplicate.
Preparation of standards The 998 bp PCR product of
the S gene was inserted into the vector of a
pMD18-T(TaKaRa, Dalian, China). It resulted in plasmid and named
pDHBV (kept by the Key Laboratory of Animal Disease and
Human Health of Sichuan Province, China). Plasmid DNA
was then isolated with the DNA purification system (Beijing
SaiBeiSeng Genetech, Beijing, China) and quantified by
spectrophotometry (BioRad SmartSpec3000, BioRad, USA) . Concentrations were converted to copies/µL
by use of the Avogadro constant. Serial dilutions of plasmids were
used for the construction of the standard curve and for the
validation of the real-time PCR
assay[13_16]. Plasmids were detected in triplicate. The correlation between the
quantification of DHBV and pDHBV plasmids was evaluated by the
regression analysis.
Analysis of the virus in liver tissues
Aliquots of 0.1 g (fidelity 0.0001) liver tissue of each sample were extracted
using a DNeasy tissue kit (Tiangen, Beijing, China). DNA
was eluted in 50 µL TE buffer (50 mmol/L EDTA, 20 mmol/L
Tris-HCl [pH 8.0]). Viral load
was quantified by real-time PCR
as earlier mentioned.
The fixed samples were sectioned and examined for
DHBsAg by immunoperoxidase staining as a marker of
infected cells[17,18]. The expression of DHBsAg was detected
by a monclonal anti-DHB antibody (kindly provided by the
Institute for Viral Hepatitis, Chongqing Medical University,
Chongqing, China). The specimens collected from control
group I, positive for DHBV, served as positive controls. The
negative control was established by omitting the primary
antibody incubation. The assessment criterion for IHC was
that cells bearing a brown-yellow stain in the nuclei were
considered positive. Immunoreactivity was quantified by
drawing around the whole section using Image Pro Plus
software (Media Cybernetics, Silver Spring, MD, USA) and
determining the mean intensity. Tissues were also sectioned
and stained with hematoxylin-eosin (HE) for the
histological analysis[19,20].
Statistics The data were expressed as mean±SD and
analyzed by t-test. P<0.05 was considered statistically
significant.
Results
Detection of DHBV-related avihepadnaviruses in serum
samples In total, 4 of 100 ducklings detected were found to
be congenitally-infected birds by PCR and were excluded
from experiments (Figure 2).
Standard curve The concentration of plasmid DNA was
221.72 µg/L. Concentrations were converted to
1.14×1010 copies/µL by
use of the Avogadro constant. The amplification lines of reaction templates were
1.14×1010copies/L diluting as 10×grad
(10-1, 10-2, 10-3,
10-4, 10-5, 10-6). The standard
curve was drawn by 40 cycles of amplification of the plasmid
samples and we obtained a linear quantification with a slope
of -3.825 and an R2 of >0.996 (Figure 3). According to the
standard curve, the sample copies were equal to lg
(48.632-Y)/3.825, where Y is the Ct (cycle threshold). The
sensitivity limit was 5 copies/µL (100% positive results).
Effect of treatment with PNA on DHBV replication
level in duck serum and liver tissues The DHBV
replication level in the duck serum and liver was markedly
decreased in the PNA- and 3TC-treatment groups. PNA
treatment significantly downregulated the DHBV replication
levels in the serum and liver tissue (Figure 4). Compared with
control group II, there was no significant difference in
inhibiting efficacy in treatment groups I and III
(P>0.05), but the inhibiting efficacy of treatment groups II and IV were better
than that of control group II (P<0.05). Interestingly,
significant differences were seen at 3 d withdrawal. The DHBV
replication levels slightly increased at 3 d withdrawal, but
rebounded slightly in the PNA-treatment groups than in
control group II (P<0.05). The DHBV replication levels in the
treated groups were lower than that of control group I (Figure
4 and Table 1). The DHBV replication levels in sera were in a
positive relationship with that in the livers, but the DHBV
replication levels in the livers were lower than that in the
sera.
Histopathological features Positive DHBsAg
immu-nostaining showed a yellow-brown color localized in the
liver cells. The intensity of the DHBsAg stain varied with
the samples (Figure 5). The proportion of positive liver
cells ranged from 5% to 85%. The majority of liver cells
showed negative DHBsAg staining and was observed as blue
with a hematoxylin counterstain. The negative control was
not immunostained.
Necrosis, steatosis, and often swelling of the hepatic
cytoplasm were found in control group, as well as focal
necrosis and vacuolar degeneration. In contrast,
pathological changes in the treatment groups were obviously
improved. A significant improvement of the
hepatocellular architecture and a considerable reduction in necrosis
and vacuolation were found. Pathological changes were
associated with DHBsAg and DHBV replication levels
(Figure 5, 6 and Table 1).
Discussion
In total, 4 of 100 ducklings detected were found as
congenitally-infected birds, but 10%_50% of congenital
infection was reported. An epidemiological survey of the
prevalence of DHBV in different duck varieties and areas should
be conducted. Analysis of data from the reports can supply
some useful information for DHBV or HBV morphogenesis
and will help to define their epidemiology more
precisely[21].
The development of new antiviral strategies has created
a need for methods that are able to monitor the low viremic
levels reached during therapy, both for the early detection
of drug-resistant mutants and for the evaluation of the
treatment outcome. This study was carried out with a simplified
system for real-time PCR by TaqMan MGB technology. The
method for the detection of DHBV was accurate and had a
high degree of sensitivity. This result was consistent with
previous reports using similar
approaches[16,22]. It showed that this system allowed reliable quantification of the viral
genome in biological samples, and therefore, may be a
valuable tool for real-time quantification when the detection of
multiple targets in a single well is not required. In conclusion,
we developed a sensitive, specific, and reproducible test for
the quantification of DHBV DNA. This assay potentially
allowed for the analysis of sera DHBV DNA without the need
for concentration or dilution of samples, independent of
treatment administration. The same approach could also be used
to distinguish and quantify viral variants emerging during
therapy.
Our results showed that PNA exhibited a potent
inhibitory effect on DHBV replication in
vivo. The originality of our study relied on the association of compounds that study
the efficacy of antiviral treatment in a complete set of
experimental systems with a real-time PCR, IHC, pathology and
in vivo studies in infected ducklings on treatment pathway-
and dose-related outcomes[11,12]. The DHBV replication
levels appeared to be directly related to the amount of
DHBsAg-positive liver cells.
The results of our study showed that PNA was effective
in suppressing DHBV replication of DHBV in models po
and iv. Administered perorally or
intravenously at concentrations as
little as 20 or 40 mg/kg can reduce serum
viral DNA levels. Perhaps the
favorable pharmacokinetic properties of
PNA and the relatively long intracellular half-life of
PNA, a strategy of dosing regimens,
was undertaken to
enable a long-term study to be
more easily conducted.
In the immunohistochemical study, the reported
intensity and rate of DHBsAg positivity could be influenced by
the experimental procedures. IHC can assess the relative
content of a special protein in degeneration cells and
reliably evaluate the level of DHBV DNA expression.
This study highlights a number of significant gaps in our
understanding of hepadnavirus persistence during treatment.
What is the mechanism of continued virus production
during treatment in control group II? Are some
producer cell types less sensitive
to PNA or is there an ongoing breakthrough of the suppression of
replication? In the stable state of reduced
production of viruses during PNA treatment, how
are the clearance mechanisms recalibrated to balance
production and result in steady serum levels of DHBV DNA and
DHBsAg?
In summary, PNA, a potent inhibitor of the hepadnaviral
polymerases, prevents the development of infection when
administered in the early stages of DHBV infection. It is a
potent and rapid-acting suppressor of DHBV replication. The
mechanism which allows DNA to rebound withdrawal and
the spontaneous viral genome variability leading to the
emergence of drug-resistant mutants requires further study. A
long-term study and the toxicity of PNA also require further
study. It is likely that dosage form is important for PNA.
This study has opened the way for further studies, including
an optimal dosing regimen and effective treatment
approaches.
Acknowledgements
We thank Yu-fei GUO and Xu CHAO for excellent
technical assistance.
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