The Effect of Gankang Suppository on Duck Hepatitis B Virus, Serum Biochemistry and Liver Histology in Ducklings

To examine the effect of Gankang Suppository on duck hepatitis B virus （DHBV）, the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups： a Gankang Suppository treatment group, an acyclovir （ACV） group and a DHBV model group （control）, with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medica- tion and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values （log） of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment （P=0.0092, P=0.0070, P=0.0080, respectively）. Compared with DHBV model control group, the ALT level was significantly decreased （P=0.0020, P=0.0019, respectively） on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment （P=0.0298）. Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal （P=0.0016）. Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group （P=0.0282, P=0.0084, P=0.0195, respectively）. It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1(DHAV-1) Isolates in Shandong Province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn't cause virulence of DHAV-1 change.

The Potential of Duck Hepatitis Virus(DHV-1) Stimulating the Body Weight Gain and the Effects of Silymarin on It in Duckling

To evaluate the effects of duck hepatitis virus-1 （DHV-1） on the body weight gain in duck and the effects of silymarin on it in vivo, 100 10-d-old ducks, both male and female, were collected to be subjected to the test. The experiments were conducted in 8 groups： in group 1-3, the animals were inoculated with 1：105 diluted duck hepatitis virus （DHV-1） infected allantoic fluid and given 0, 30, and 50 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 4-6, the animals were inoculated with 1：5 × 105 diluted DHV-1 infected allantoic fluid and given 0, 10, and 30 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 7, the animals were given 10 mg kg^-1 BW d^-1 silymafin only. Group 8 was the control one treated by injecting sterillized saline into the leg muscles. All the silymarin was given from 0 to 4 d after inoculation of the virus. By the 5th d after inoculation, the vein blood was drawn from the dorsal foot vein and the plasma samples were collected and stored at -20℃. The body weight gain （BWG） was measured from 0 to 10 d after inoculation. The plasma IGF-I, T3, and T4 concentrations were measured by radioimmunoassay （RIA）. At the virus dose of 1：5 ×105 diluted virus infected allantoic fluid, the inoculation of the virus enhanced the BWG significantly compared with that of the control （P〈 0.01）, while 10-50 mg kg^-1 BW d^-1 silymarin could counteract the effects of the virus on the BWG dose-dependently. The plasma IGF-I levels showed no correlation with the BWG, but the T3 levels showed a same tropism with the body weight gain. The present results indicated that sublethal DHV-1 enhanced the body weight gain of ducklings significantly, and the silymarin could counteract this effect in vivo.

Molecular Characterization and SYBR Green I-Based Quantitative PCR for Duck Hepatitis Virus Type 1

To determine the genomic sequence of a duck hepatitis virus type 1 （DHV-1） strain, real-time quantitative polymerase chain reaction （RTQ-PCR） assay based on SYBR Green I technology was developed to target 3D gene of DHV-1, Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5＇ untranslated region （UTR）-VP0-VP3-VPI-2A1-2A2-2B-2C-3A-3B-3C-3D-3＇ UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6℃ and the real-time PCR provides a broad dynamic range, detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.

Isolation,Identification and Pathogenic Characteristics of Duck Hepatitis Virus Isolates

[ Objective] The aim of this study was to determine and analyze the pathogen and the reasons of duck viral hepatitis which is prevalent recently and difficult to control. [Method] Viruses were isolated from livers and spleens of ducks with typical clinical symptoms in Linyi, Weifang, Binzhou and other regions of Shandong Province. The pathogenic characteristics were observed by inoculation in chicken or duck embryo, RT- PCR, serological test, and duck regression. [ Result] Four duck hepatitis virus （DHV） strains were isolated, and the 5th passage allantoic fluid contained 10^3.41 -10^5.20 ELD50/mI. The serum cross protection rate was 20% -80% between the DHV stains and DHV type I. The mortalities of 4- day-old healthy ducks challenged by these four stains were 50% -100%. All challenged ducks had typical lesions of duck viral hepatitis, and the death peak appeared after 24-48 h. [Conclusion] The virulence of different DHV isolates has regional difference.

Molecular Characterization of Duck Hepatitis B Virus Isolated from Hubei Brown Ducks

The objective of this study was to characterize the genome structure of duck hepatitis B virus （DHBV） isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction （PCR） and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 % This strain （GenBank accession number DQ276978） had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the ＂Chinese＂ DHBV strains than those of the ＂Western＂ country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.

Therapeutic effect of Styela plicata on duck hepatitis B virus in vivo

Objective:To evaluate the antiviral activity of the alcohol extract of Styela plicata on DHBV (duck hepatitis B virus) in vivo. Methods: Guangzhou-Sheldrake ducklings congenitally infected with DHBV were assigned to receive the alcohol extract of Styela plicata or lamivudine for 30 consecutive days. The DHBV DNA of sera was detected by RT-PCR. and the histological analysis of duckling liver was evaluated. Results:Thirty days after therapy,histological analysis of duckling liver showed that the ducklings receiving the alcohol extract of Styela plicata or lamivudine exhibited catabatic status in the degree of liver cell degeneration and inflammation compared with the ducklings receiving normal diet. DHBV DNA of sera from alcohol extract of Styela plicata-treated ducklings and lamivudine-treated ducklings all produced significantly lower levels compared with ducklings receiving normal diet (P<0. 01 ). Although these treatment groups all exhibited a rebound phenomenon 10 d after withdrawal of medication, they still exhibited a significant lower level of serum DHBV DNA compared with the control group responded to normal diet (P<0. 05, P<0. 01). Conclusion:Styela plicata may be an effective antiviral medicine in treating chronic hepatitis B. The data of this experiment will be valuable in studying the therapeutic role and the potential therapeutic mechanism of Styela plicata.

DUCK HEPATITIS B VIRUS DNA WITHIN HEPATIC MULTICENTRIC CANCER AND/OR METASTATIC CANCER

Duck hepatitis B vims (DHBV) DNA was detected in different tumorous nodules of ducks with hepatic multicentric cancer or intrahepatic metastasis by Southern blot technique. Among 7 ducks with hepatocellular carcinoma of multiple tumor nodules, the hybridization pattern of Integrated DHBV DNA In different tumorous nodules was identical in 3 cases and different in 2 cases. One case showed a similar hybridization pattern in two tumorous nodules and other one was negative tor DHBV DNA. Integrated DHBV DNA was also identified in a metastatic lung cancer of ducks with hepatocellular carcinoma. The hybridization pattern of metastasis of lungs was as the some as that in primary hepatocellular carcinoma. The same discrete hybridization bands In the different tumorous nodules indicate that these nodules might arise from one transformed cell. The different hybridization patterns In various tumorous nodules show that these tumorous nodules might arise from various transformed cells. The results suggest that the hybridization pattern of different nodules of hepatocellular carcinoma with viral DNA probe could make a cell clone origin marker of tumor nodule to differentiate hepatic multlcentric cancer from Intrahepatic metastatic cancer.

DUCK HEPATITIS B VIRUS MODEL FOR SCREENING OF ANTIVIRAL AGENTS FROM MEDICINAL HERBS

The effects of the extracts of 20 Chinese medicinal herbs and an antiviral drug foscarnet on duck hepatitis B virus (DHBV) endogenous DNA polymerase (DNAp) activity were compared. The extracts of P. urinaria showed a dose-dependent inhibition on DHBV DNAp. And those of other herbs showed little inhibition effect. Primary duck hepatocyte (PDH) cultures were used for evaluating effects of the extract of P. urinaria, foscarnet and acyclovir (ACV) on DHBV, and all the drugs or the extracts showed inhibition of DHBV DNA replication. Furthermore, in vivo trials were carried out. Peking ducks infected with LJ-76 strain of DHBV were treated with the extract of P. urinaria or ACV and compared with placebo treated control ducks. The treatment results in the loss or reduction of circulating viral DHBV DNA and DHBsAg.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Is hepatitis B-virucidal validation of biocides possible with the use of surrogates?

The hepatitis B virus(HBV)is considered to be a major public health problem worldwide,and a significant number of reports on nosocomial outbreaks of HBV infections have been reported.Prevention of indirect HBV transmission by contaminated objects is only possible through the use of infection-control principles,including the use of chemical biocides,which are proven to render the virus non-infectious.The virucidal activity of biocides against HBV cannot be predicted;therefore,validation of the virucidal action of disinfectants against HBV is essential.However,feasible HBV infectivity assays have not yet been established.Thus,surrogate models have been proposed for testing the efficacy of biocides against HBV.Most of these assays do not correlate with HBV infectivity.Currently,the most promising and feasible assay is the use of the taxonomically related duck hepatitis B virus(DHBV),which belongs to the same Hepadnaviridae virus family.This paper reviews the application of DHBV,which can be propagated in vitro in primary duck embryonic hepatocytes,for the testing of biocides and describes why this model can be used as reliable method to evaluate disinfectants for efficacy against HBV.The susceptibility levels of important biocides,which are often used as ingredients for commercially available disinfectants,are also described.

Viral and cellular determinants involved in hepadnaviral entry

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus （HBV） constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses （e.g. HBV） and avihepadnaviruses （e.g. duck hepatitis B virus [DHBV]）. Two important discoveries have been made recently regarding HBV： （1） primary hepatoo/tes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and （2） a human hepatoma cell line （HepaRG） was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D （CPD） has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes （PDH）, investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to： （1） functionally dissect the hepadnaviral entry pathways, （2） perform reverse genetics （e.g. test the fitness of escape mutants）, （3） titrate and map neutralizing antibodies, （4） improve current vaccines to combat acute and chronic infections of hepatitis B, and （5） develop entry inhibitors for future clinical applications.

Anti-HBV effect of liposome-encapsulated matrine in vitro ana in vivo

AIM: To study the anti-HBV effect of liposome-encapsulated matrine (Lip-M) in vitro and in vivo. METHODS: 2.2.15 cell line was cultured in vitro observe the effect of Lip-M and matrine on the secretion of HBsAg and HBeAg. The toxicity of Lip-M and matrine to 2.2.15 cell line was also studied by MTT method. In in vivo study, drug treatment experiment was carried out on the 13th day after ducks were infected with duck hepatitis B virus (DHBV). The ducks were randomly divided into 4 groups with 5-6 ducks in each group. Lip-M and matrine were given to DHBV-infected ducks respectively by gastric perfusion. Four groups were observed: group of Lip-M (20 mg/kg), group of Lip-M (10 mg/kg), group of matrine (20 mg/kg) and group of blank model. The drug was given once daily for 20 d continuously, and normal saline was used as control. The blood was drawn from the posterior tibial vein of all ducks before treatment (T0), after the medication for 5 (T5), 10 (T10), 15 (T15), 20 (T20) d and withdrawl of the drug for 3 d (P3). The serum samples were separated and stored at -70 ℃, DHBV-DNA was detected by the dot-blot hybridization. RESULTS: After addition of Lip-M and matrine to 2.2.15 cell line for eleven d, the median toxic concentration (TC50) of Lip-M and matrine was 7.29 mg/mL and 1.33 mg/mL respectively. The median concentration (IC50) of Lip-M to inhibit HBsAg and HBeAg expression was 0.078 mg/mL and 3.35 mg/mL respectively. The treatment index (TI) value of Lip-M for HBsAg and HBeAg was 93.46 and 2.17 respectively, better than that of matrine. The DHBV-infected duck model treatment test showed that the duck serum DHBV-DNA levels were markedly reduced in the group of Lip-M (20 mg/kg) after treated by gastric perfusion for 10, 15 and 20 d (0.43±0.22 vs 0.95±0.18, t = 4.70, P= 0.001<0.01.0.40±0.12 vs 0.95±0.18, t = 6.34, P= 0.000<0.01. 0.22±0.10 vs 0.95±0.18, t = 8.30, P= 0.000<0.01), compared to the group of matrine (20 mg/kg) (0.43±0.22 vs 0.79±0.19, t = 3.17, P= 0.01<0.05. 0.40±0.12 vs 0.73±0.24, t = 3.21, P= 0.009<0.05. 0.22±0.10 vs0.55±0.32, t = 2.27, P= 0.046<0.05.), and the control (0.43±0.22 vs50.98±0.29, t = 3.68, P = 0.005<0.01. 0.40±0.12 vs 0.97±0.30, t = 4.26, P= 0.002<0.01. 0.22±0.10 vs 0.95±0.27, t = 5.76, P= 0.000<0.01). After the treatment for 20 d and withdrawl of the drug for 3 d, duck serum DHBV-DNA level in the group of Lip-M (10 mg/kg) markedly reduced (0.56±0.26 vs0.95±0.38, t = 5.26, P= 0.003<0.05. 0.55±0.25 vs 0.95±0.38, t = 5.52, P= 0.003<0.05), and the difference was significant as compared with the control (0.56±0.26 vs 0.95±0.27, t = 2.37, P = 0.042<0.05. 0.55±0.25 vs 0.89±0.18, t = 2.55, P= 0.031<0.05), but not significant as compared with the group of matrine (20 mg/kg). After withdrawl of the drug for 3 d, the levels of DHBV-DNA did not relapse in both groups of Lip-M. CONCLUSION: Lip-M can evidently inhibit the replication of hepatitis B virus In vitro and in viva, its anti-HBV effect is better than that of matrine.

Anti-hepatitis B Virus Activity of 8-epi-Kingiside in Jasminum officinale var. grandiflorum

Objective To evaluate the effect of 8-epi-kingiside (8-Epik) derived from the buds of Jasminum officinale var. grandiflorum (JOG) on hepatitis B virus (HBV) replication in HepG2 2.2.15 cell line in vitro and duck hepatitis B virus (DHBV) replication in ducklings in vivo. Methods The concentration of extracellular hepatitis B e antigen and hepatitis B surface antigen (HBsAg) in cell culture medium was determined by ELISA, respectively. The anti-HBV effects of 8-Epik were also demonstrated in the model of DHBV. 8-Epik was ip given (20, 40, and 80 mg/kg, twice daily) to the DHBV-infected ducklings for 10 d. The isotonic saline liquid diet was ip given as negative control and Lamivudine (50 mg/kg, twice daily) was given as positive control. DHBV DNA was measured at days 0 (T0), 5 (T5), 10 (T10), and day 3 after cessation of treatment (P3) by dot blotting. Results 8-Epik effectively blocked HBsAg secretion in HepG2 2.2.15 cells in a dose-dependent manner [IC 50 = (19.4 ± 1.04) μg/mL]. 8-Epik (40 or 80 mg/kg, ip, twice daily) also reduced viremia in DHBV-infected ducks. Conclusion Therefore, 8-Epik is warranted as a potential therapeutic agent for HBV infection.