The relationship between duck hepatitis B virus infection and duck liver diseases in Qitong, China

The relationship between duck hepatitis B virus (DHBV) infection and duck liver diseases was analysed by spot and gel blot hybridization in sera and liver tissues. One hundred and forty ducks were obtained from Qitong county in China. The DHBV-infected rate was 65.7% and the incidence of duck liver diseases was 70%. The state of DHBV DNA was free in the hepatocytes. The detection rate of DHBV DNA in liver was higher than in serum. The results showed that Qitong duck liver diseases are closely correlated to DHBV infection. The incidence of Qitong duck liver diseases and the DHBV infection rate were different in various species of ducks. Qitong ducks have several hepatic pathological changes as seen in human beings. Therefore, Qitong ducks are most appropriate as an experimental model for human HBV infection.     

Electron microscopic studies on duck hepatitis B virus particles in hepatocytes and sequential changes in various patterns of infection

To investigate the critical factors involved in the elimination of the duck hepatitis B virus (DHBV) in acute infection, the sequential changes in the number of DHBV particles in hepatocytes were studied electron microscopically in ducks experimentally infected by DHBV. Twenty Japanese white Peking ducks were infected with DHBV on the day of hatching, and on the 7th day and 14th day after hatching. Inoculation of DHBV on the day of hatching, and on the 7th and 14th day after hatching resulted in persistent viremia, transient viremia and no viremia, respectively in ducks as tested by spot hybridization assay. The number of DHBV particles in the liver correlated well with the amount of serum DHBV-DNA, DHBV particles decreased in hepatocytes without any interaction of inflammatory cells over the observation period, and the number of particles was not associated with the degree of hepatic inflammation. From these results, the elimination of the virus was thought to be induced by a reduction of viral replication in the hepatocytes and not by destruction of their host cells. There must be an age-dependent factor which strongly suppresses the viral replication.     

Electron microscopic studies on duck hepatitis B virus particles in hepatocytes and sequential changes in various patterns of infection

To investigate the critical factors involved in the elimination of the duck hepatitis B virus (DHBV) in acute infection, the sequential changes in the number of DHBV particles in hepatocytes were studied electron microscopically in ducks experimentally infected by DHBV. Twenty Japanese white Peking ducks were infected with DHBV on the day of hatching, and on the 7th day and 14th day after hatching. Inoculation of DHBV on the day of hatching, and on the 7th and 14th day after hatching resulted in persistent viremia, transient viremia and no viremia, respectively in ducks as tested by spot hybridization assay. The number of DHBV particles in the liver correlated well with the amount of serum DHBV-DNA, DHBV particles decreased in hepatocytes without any interaction of inflammatory cells over the observation period, and the number of particles was not associated with the degree of hepatic inflammation. From these results, the elimination of the virus was thought to be induced by a reduction of viral replication in the hepatocytes and not by destruction of their host cells. There must be an age-dependent factor which strongly suppresses the viral replication.     

Replication of duck hepatitis B virus in primary duck hepatocytes and its dependence on the state of differentiation of the host cell

Primary duck hepatocytes obtained from Pekin ducks congenitally infected with duck hepatitis B virus were used to monitor expression of viral proteins and replication of viral DNA in cell culture. Duck hepatitis B virus core antigen, duck hepatitis B virus pre-surface antigen and duck hepatitis B virus DNA were detectable for at least 12 days after cell plating. Whereas expression of duck hepatitis B pre-surface antigen was constant during this time, expression of duck hepatitis B core antigen and of viral DNA rapidly declined. This diminished production of viral components in vitro was paralleled by a change of the hepatocytes toward a fibroblast-like morphology. Supplementation of cell culture medium with 2% dimethyl sulfoxide, a solvent known to maintain the differentiated state of cultured cells, retained competence of the cultured hepatocytes to express duck hepatitis B core antigen and duck hepatitis B virus DNA at high levels. In a second set of experiments, duck hepatitis B virus negative hepatocytes were infected with duck hepatitis B virus from serum of congenitally infected ducks. Dimethyl sulfoxide remarkably improved the competence of cultured duck hepatocytes to become productively infected. This function was maintained for at least 12 days postplating.     

Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma.

The presence of duck hepatitis B virus (DHBV) DNA in liver and serum and its state (integrated vs. free) were studied in 23 ducks from Chi-tung county in China by spot hybridization and Southern blot hybridization, respectively. In 16 of 23 (70%), DHBV DNA was detected in serum and/or in liver tissue. These infected ducks showed a variety of pathological changes including advanced chronic disease in the liver. In contrast, none of the virus-negative ducks had advanced hepatic changes. One DHBV DNA-seropositive duck had a large hepatocellular carcinoma. Southern blot analysis demonstrated integrated DHBV DNA in neoplastic tissue and abundant episomal DHBV DNA in non-neoplastic tissue of the liver. In one noninfected duck with a small adenoma, no viral DNA was detected in tumor or non-neoplastic tissue. The detection of integrated DHBV DNA in hepatocellular carcinoma suggests that DHBV behaves like human and woodchuck hepatitis viruses in relation to chronic liver disease and hepatocarcinogenesis.     

Inflammation of the liver causes mutations in duck hepatitis B virus genome

To investigate whether hepatitis causes mutation in the viral genome, DNA sequences in the pre-core region of duck hepatitis B virus (DHBV) DNA were analyzed in both ducks with hepatitis and without hepatitis. Five DHBV carrier ducks were injected with DHBV particle proteins purified from duck serum with Freund’s complete adjuvant (FCA) intrahepatically from 14 day posthatch for 9 weeks (immunized group). Serum was drawn at the end of the 1st and 4th week after the 1st injection of DHBV particle protein and ducks were killed at the end of the 9th week to obtain the liver. Another five ducks without treatment were used as controls. All ducks of the immunized group showed moderate to severe hepatitis at the 9th week. All ducks in the immunized group showed one mutation except one duck that showed two mutations only at the 9th week. Mutations were observed in the 5th, 13th, 21st, 22nd, and 28th codon of the precore region. All of them were point mutation at the 3rd base in the triplets. The frequency of mutation was different in each duck from 20% to 60% but not 100%. There was no mutations in ducks in control group. These results suggest that hepatitis causes mutation in the pre-core lesion genome of duck hepatitis B virus.     

Naturally occurring infection of Pekin duck embryos by duck hepatitis B virus.

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 polymerase activity characteristic of hepatitis B-related viruses. The presence 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 present in serum. These additional liver forms probably represent viral replication intermediates. These observations suggest that the vertical route is a major pathway of DHBV transmission and that viral replication may be initiated by the 12th day of embryonic life.     

Liver orcein stain and viral DNA in duck hepatitis B virus infection in Chinese ducks and experimentally infected Japanese ducklings

Liver sections were stained with orcein, and duck hepatitis B virus was identified in sera and livers by the hybridization technique in 106 ducks (44 Chinese ducks, 15 Japanese ducks and 47 Japanese ducklings). Orcein-positive hepatocytes were found in 18 of 38 (47%) duck hepatitis B virus DNA seropositive ducks, and only in 3 of 68 (4%) seronegative ducks. The three ducks were all from a heavily infected flock in southern China. Serial analyses of viral DNA by Southern blot and spot hybridizations in experimentally infected Japanese ducklings revealed a dissociation or a time gap between the amount of viral DNA in serum and the emergence of orcein positive hepatocytes. Orcein-positive hepatocytes were generally associated with prolonged presence of viral infection for at least 4 to 6 months. These findings support the clinical hypothesis that the presence of orcein-positive hepatocytes indicates persistent rather than acute infection. Since orcein-positive hepatocytes have been seen in infection with hepatitis B, woodchuck hepatitis, ground squirrel and duck hepatitis B viruses, accumulation of orcein-positive material in liver cells may be one of the common properties these viruses share. This stain may be utilized for screening new hepatitis B virus-like viruses.     

Effects of acyclovir and vidarabin 5′-monophosphate on anti-duck hepatitis B virus in an in vitro culture system

Various anti-viral agents, e.g., interferon, have recently been used for the treatment of viral hepatitis. In the present study, duck hepatitis B virus (DHBV) was cultured in vitro and the anti-DHBV effects of acyclovir (ACV) and vidarabin 5′-monophosphate (VMP) were studied. The portal perfusion method was applied to the livers of 7-day-old white ducks weighing 100g, bred in Japan, and hepatocytes were infected with DHBV in vitro. Duck hepatocytes infected with DHBV were cultured in medium containing ACV or VMP, and the anti-DHBV effects of these drugs were assessed by determining DHBV-DNA and duck hepatitis B surface antigen in the medium. Both ACV and VMP had anti-DHBV effects when used immediately after infection; however, both drugs were ineffective in hepatocytes obtained from a DHBV carrier duck. In conclusion, the anti-DHBV effects of these drugs were very limited. However, this culture system appears to be useful for studies of hepatitis virus and anti-viral drugs.     

拉米夫定联合泛昔洛韦抗鸭乙型肝炎病毒的实验研究

观察核苷酸类似物拉米夫定联合泛昔洛韦体内抗鸭乙型肝炎病毒（DHBV）的作用。方法 采用重庆麻鸭乙型肝炎动物模型,用拉米夫定联合泛昔洛韦口服治疗4周,停药观察1周,检测用药前后血清中的DHBV DNA、DHBsAg及血清转氨酶（ALT、AST）、肝组织HE染色病理。并以单用拉米夫定、泛昔洛韦、阿昔洛韦作对照。结果 拉米夫定联合泛昔洛韦用药后能使血清中DHBV DNA含量总体水平显著降低（P＜0．01）,停药1周后DHBV DNA较用药4周时DHBV DNA含量回升现象不明显。用药前后清血DHBsAg的吸光度值（490nm)的变化与DNA含量改变相似;此外,肝脏病理检查及治疗4周、停药1周后血清转氨酶检测未发现联合用药对鸭肝组织有明显的毒性损害。结论 拉米夫定联合泛昔洛韦连续用药4周在鸭体内有抗鸭乙型肝炎病毒的作用,且停药后DHBV DNA无明显“反跳”,二者用药有协同作用。     

Ampligen及其与Ganciclovir和Coumermycin Al联合应用对血清中鸭乙型肝炎病毒抑制效果

Ａｍｐｌｉｇｅｎ是一种免疫调节剂或干扰素诱导剂。单独和联合Ｇａｎｃｉｃｌｏｖｉｒ和／或ＣｏｕｍｅｒｍｙｃｉｎＡｌ治疗了鸭乙型肝炎病毒（ＤＨＢＶ）阳性鸭模型。结果表明,上述三种治疗方。案对鸭血清中ＤＨＢＶ均有抑制作用,但治疗结束ＤＨＢＶ回升到治疗前水平。任何一种方案对血清循环ＤＨＢＶ表面抗原（ＤＨＢｓＡｇ）均未产生明显抑制效果。本研究结果为人乙型肝炎治疗时合理使用抗病毒药物和免疫调节、诱导剂提供了重要参考。     

Alterations in intrahepatic expression of duck hepatitis B viral markers with ganciclovir chemotherapy

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the guanosine analogue, ganciclovir, and the effect on serum and intrahepatic expression of DHBV DNA and viral proteins was examined. After 21 days of ganciclovir treatment, a substantial reduction in viraemia occurred; in contrast, the level of circulating DHBV surface antigen was unchanged. Ganciclovir therapy also substantially reduced the level of DHBV DNA replicative intermediates and the expression of viral core and surface antigen in hepatocytes. However, despite the antiviral treatment some liver cells, including the bile duct epithelial cells and putative oval cells, maintained their intense staining for the viral proteins. Furthermore, DHBV-infected cells in extrahepatic sites such as the pancreas, kidney and spleen were also unaffected by ganciclovir treatment. These results suggest that monotherapy with nucleoside analogues is unlikely to eliminate chronic hepadnaviral infection, and antiviral programs should be designed to target all cell populations infected by the virus.     

Effect of nucleoside analogue therapy on duck hepatitis B viral replication in hepatocytes and bile duct epithelial cells in vivo

BACKGROUND: Recent studies have implicated bile duct epithelial cells (BDEC) as a reservoir of hepatitis B virus (HBV) infection that may be particularly important in the development of post-liver transplant recurrence of hepatitis B. The aim of this study was to compare the effects of antiviral therapy on duck HBV (DHBV) expression in hepatocytes and BDEC and to determine if this was affected by biliary hyperplasia. METHODS: Ducklings congenitally infected with DHBV received penciclovir (10 mg/kg per day) treatment from 9 days of age. In order to mimic the biliary hyperplasia that often accompanies severe post-liver transplant HBV recurrence, half the animals underwent bile duct ligation. Duck HBV-DNA in serum was measured at day 1, and serum and liver DHBV-DNA were determined when the animals were killed on day 17. Intrahepatic expression of viral preS1 antigen and DHBV-DNA was measured by immunohistochemistry and in situ hybridization, respectively. RESULTS: Viraemia became undetectable in the penciclovir-treated animals at day 17, following 8 days of therapy. Examination of liver tissue revealed that all hepatocytes and the majority of BDEC contained DHBV preS1 antigen and DHBV-DNA. Penciclovir greatly reduced the intrahepatic viral burden, but there was no antiviral effect on viral markers within BDEC. Despite the increased number of BDEC after bile duct ligation, the same proportion of BDEC was seen to be infected, and this was unaffected by antiviral therapy. CONCLUSIONS: In the duck model with and without biliary hyperplasia, penciclovir controls DHBV replication and reduces viral burden in hepatocytes, but not in BDEC. The BDEC appear to be an important reservoir of virus that is relatively unaffected by antiviral treatment, and may play an important role in disease persistence and relapse following cessation of therapy.     

Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus.

Liver, pancreas, and kidney from Pekin ducks infected with duck hepatitis B virus (DHBV) were assayed for the presence of both viral antigen and replication-specific forms of viral nucleic acid. In young congenitally infected ducks, antigen was detectable in hepatocytes and bile duct epithelia, in kidney glomeruli and tubular epithelia, and in cells localized to pancreatic acini. In older experimentally infected ducks, antigen was detectable in hepatocytes, in glomeruli and tubular epithelia, and in cells localized to presumptive pancreatic alpha-islets. All but the glomeruli-associated viral antigen appeared to be localized to the cytoplasm of antigen-positive cells. Much of the glomeruli-associated antigen appeared to be extracellular and was detected in glomeruli that were positive for the accumulation of immunoglobulin, observations suggestive of the deposition of viral antigen-antibody complexes. As analyzed with bulk tissue, replication-specific forms of viral nucleic acid were detectable in liver and pancreas from the young congenitally infected ducks and in liver and kidney from the older experimentally infected ducks.     

Replicative efficiency and pathogenicity of hepatitis B virus e-minus precore variant

Abstract To study the replicative efficiency and pathogenicity of hepatitis B virus precore variant (A1896), anti-hepatitis B virus e antigen (HBe) titre was studied in naturally occurring wild-type virus infection, A1896 variant infection and dual infection. Higher titre of anti-HBe was found in patients with no virus replication and in patients coinfected with the wild-type virus and A1896 variant, which suggest that anti-HBe may either act as an inhibitor of virus replication or as selective pressure for the A1896 variant. Three site-directed mutants were constructed in the duck hepatitis B virus (DHBV) precore region. A frame shift in the encapsidation signal region abolished replication of DHBV; mutation in the initiation codon of the precore and mutation to generate a termination codon at the distal region of the precore resulted in decreased replication in the duck model. More significant pathological changes were found in the liver tissues of ducks infected with the mutant which mimicked the HBV A1896 variant.     

Experimental duck hepatitis B virus infection: pathology and evolution of hepatic and extrahepatic infection

Seventy, 1-day-old ducklings inoculated intraperitoneally with duck hepatitis B virus and 30 controls have been studied over a 2-year period. Infection with duck hepatitis B virus occurred in all inoculated ducks, although this was not associated with clinical morbidity. Duck hepatitis B virus DNA was first detected in liver on Day 3, in pancreatic acinar cells on Day 4, serum on Day 6, splenic red and white pulp on Day 7 and in the renal glomurulus on Day 14, using a combination of dot, Southern blot and in situ hybridization techniques. Peak levels of circulating virus, as determined by DNA polymerase levels, occurred 1 to 4 weeks postinoculation. Mild degrees of portal inflammation were seen in sections of liver tissue in both infected and control ducks. However, moderately severe inflammatory changes were present in 8 of 22 infected birds compared with 0 of 18 controls (p less than 0.025). Appearance of this inflammatory infiltrate 6 weeks postinoculation coincided with a decrease in levels of duck hepatitis B virus DNA in hepatocytes and within the pancreatic acinar cells. At the same time, duck hepatitis B virus DNA became increasingly localized to the splenic germinal centers, and viral DNA was first detected in pancreatic islet cells. No histological changes accompanied the extra-hepatic tissue infection. The sequence and significance of duck hepatitis B virus infection in liver and extra-hepatic tissues is discussed in relation to the pathogenesis of hepatitis B virus infection in man.     

广西麻鸭乙型肝炎感染病毒动物模型的实验研究

目的探讨广西麻鸭作为乙型肝炎病毒感染动物模型的可能性。方法应用广西1 d龄雏麻鸭经腹腔感染鸭乙型肝炎病毒(DHBV),13 d后采用实时荧光定量PCR法检测麻鸭血清DHBV含量,筛选出DHBV强阳性鸭。结果共检测148份麻鸭血清标本,其中DHBV阳性标本136份,阳性率为91.9%。结论广西麻鸭可作为乙型肝炎病毒感染动物模型。实时荧光定量PCR法检测DHBV敏感性较高,重复性好,可用于DHBV检测。     

Duck hepatitis B virus infection of non-hepatocytes

One hundred and seventeen ducklings, 42 inoculated with duck hepatitis B virus (DHBV) 2 days after hatching and 55 connatally infected, were studied over a 6-month period in parallel with 20 ducklings without DHBV infection. Using immunohistochemical, in situ and blot hybridization analyses, the natural course of hepatic and extrahepatic infection was examined. DHBV infection started in the liver 2-4 days post-inoculation. There, DHBV was found not only in hepatocytes, but also in bile duct epithelial cells. Further, DHBV infection occurred in exocrine and endocrine pancreas (beginning 6-10 days and 20 days post-inoculation, respectively) and in germinal centers of the spleen (beginning 8 weeks post-inoculation). Occasionally viral DNA was also found in kidney glomeruli. Using strand-specific RNA probes, viral DNA in pancreas and spleen was clearly demonstrated to be replicating intermediates. Hepatic and extrahepatic infection with DHBV was not associated with histologic inflammation or pathologic changes in these tissues or the liver. These data indicate that DHBV can infect cells other than hepatocytes. The biological significance of non-hepatocyte infection for the life-cycle of the virus and its potential significance for viral persistence remain to be determined.