鸭乙型肝炎病毒感染的研究

Cherry valley ducks were infected by intravenous injection of DHBV positive serum in order to study the intrahepatic distribution of DHBsAg and the relation of the degree of hepatic lesions to viremia and humoral immunologic deficiency after surgical removal of Bursa of Fabricius. The anti-DHBsAg serum prepared in our laboratory showed high specificity. There was no cross reaction with HBsAg and DHBsAg was found to be located in the cytoplasm of hepatocytes as well as bile duct epithelial cells which usually showed stronger staining quality. The histopathology of liver revealed normal/mild hepatitis in the control group, moderate/severe hepatitis in the infected group. In comparison, hepatitis in the infected group was more severe in the older ducks than the ducklings, in those viremia-positive ones than in the negative ones, and in the bursectomized than in the non-bursectomized ducks. Evidently, the hepatic lesions were mostly due to DHBV infection in this series, although some other environmental factors could not be ruled out entirely. The present investigation shows that Cherry valley ducks are one of the best spices for experimental DHBV infection, and bursectomized ducks with humoral immunodeficiency might provide a reliable and useful model for the study of pathogenesis of hepatitis B virus infection.     

The interferon system in ducks infected with viral hepatitis B

The duck peripheral blood lymphocytes and spleen cells were shown to be relatively resistant to interferon induction by viral (NDV) and nonviral (dsRNA) inducers as well as to Con A induction of gamma-interferon. The concentration of the inducers had to be increased 4-5-fold to induce interferon production. Charry valley ducks produced more alpha-interferon than Peking ducks. The level of interferon production induced by NDV and dsRNA (Ridostin) was similar in DHBV-positive Peking ducks and in the control group, but in the infected Charry valley ducks interferon production was lower than in the controls (224 to 180 U/0.1 ml in NDV-induced and from 192 to 43.3 U/0.1 ml in dsRNA-induced). Early bursectomy brought down the interferon production in non-infected ducts, but in DHBV-positive bursectomized ducks (Y) the level of interferon was higher than in the control bursectomized ducks.     

Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection

As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies. Control ducks were injected with either 0.7% NaCl or non-transfected PDEF. The ducks were then challenged at 18 days of age by intravenous inoculation with DHBV (5 x 10(8) viral genome equivalents). Liver biopsies obtained on day 4 post-challenge demonstrated that vaccination did not prevent infection of the liver as similar numbers of infected hepatocytes were detected in all vaccinated and control ducks. However, analysis of liver tissue obtained 9 or more days post-challenge revealed that 9 out of 11 of the PDEF-DHBcAg vaccinated ducks and 8 out of 11 ducks vaccinated with PDEF-DHBcAg plus anti-DHBc antibodies had rapidly resolved the DHBV infection with clearance of infected cells. In contrast, 10 out of 11 of the control unvaccinated ducks developed chronic DHBV infection. In conclusion, vaccination of ducks with a whole-cell PDEF vaccine expressing DHBcAg elicited immune responses that induced a rapid resolution of DHBV infection. The results establish that chronic infection can be prevented via the vaccine-mediated induction of a core-antigen-specific immune response.     

Cellular immune response of ducks to duck hepatitis B virus infection

Duck hepatitis B virus (DHBV) has been a useful model for hepadnavirus infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P< 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P< 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P< 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV infection.     

反义寡脱氧核苷酸在鸭体内抑制鸭乙型肝炎病毒复制与 …

目的 研究反义核酸的抗病毒作用。方法 设计合成了针对鸭乙型肝炎病毒（ＤＨＢＶ）前Ｓ（ＰｒｅＳ）基因区第９５１－９６８位核苷酸的硫代反义寡脱氧核苷酸（ＡＳ－ＯＤＮ），以２０μｇ／ｇ体重／日剂量对３只腹腔感染ＤＨＢＶ５．２毒株后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性鸭连续静脉注射１０天，同时以等体积生理盐水注射另３只感染鸭作为对照。结果 对照鸭注射生理盐水后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性未     

DNA vaccines expressing the duck hepatitis B virus surface proteins lead to reduced numbers of infected hepatocytes and protect ducks against the development of chronic infection in a virus dose-dependent manner

We tested the efficacy of DNA vaccines expressing the duck hepatitis B virus (DHBV) pre-surface (pre-S/S) and surface (S) proteins in modifying the outcome of infection in 14-day-old ducks. In two experiments, Pekin Aylesbury ducks were vaccinated on days 4 and 14 of age with plasmid DNA vaccines expressing either the DHBV pre-S/S or S proteins, or the control plasmid vector, pcDNA1.1Amp. All ducks were then challenged intravenously on day 14 of age with 5 x 10(7) or 5 x 10(8) DHBV genomes. Levels of initial DHBV infection were assessed using liver biopsy tissue collected at day 4 post-challenge (p.c.) followed and immunostained for DHBV surface antigen to determine the percentage of infected hepatocytes. All vector vaccinated ducks challenged with 5 x 10(7) and 5 x 10(8) DHBV genomes had an average of 3.21% and 20.1% of DHBV-positive hepatocytes respectively at day 4 p.c. and 16 out of 16 ducks developed chronic DHBV infection. In contrast, pre-S/S and S vaccinated ducks challenged with 5 x 10(7) DHBV genomes had reduced levels of initial infection with an average of 1.38% and 1.93% of DHBV-positive hepatocytes at day 4 p.c. respectively and 10 of 18 ducks were protected against chronic infection. The pre-S/S and the S DNA vaccinated ducks challenged with 5 x 10(8) DHBV genomes had an average of 31.5% and 9.2% of DHBV-positive hepatocytes on day 4 p.c. respectively and only 4 of the 18 vaccinated ducks were protected against chronic infection. There was no statistically significant difference in the efficacy of the DHBV pre-S/S or S DNA vaccines. In conclusion, vaccination of young ducks with DNA vaccines expressing the DHBV pre-S/S and S proteins induced rapid immune responses that reduced the extent of initial DHBV infection in the liver and prevented the development of chronic infection in a virus dose-dependent manner.     

Antiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.     

Natural infection of duck embryos with duck hepatitis B virus: time and tissue sequence of infection

There have been no studies addressing the detailed sequence of embryonic infection with duck hepatitis B virus (DHBV). Therefore, duck embryos from flocks infected with DHBV were examined to study the sequence of infection by DHBV in various embryonic tissues. Embryos from flocks infected with DHBV were harvested in duplicates from 7 to 25 days of incubation. Whole embryos (to 12 days) or dissected embryonic tissues were fixed, paraffin embedded, and stained for DHBV surface antigen (DHBsAg) using a peroxidase-antiperoxidase technique. Isolated hepatic cells were infected in 7-day-old embryos, and these increased in number until 11 days, when most cells were positive for DHBsAg. Endocrine pancreatic cells were positive from day 10, but only an occasional exocrine pancreatic cell was infected after day 20. Renal tubule cells were positive for DHBV by day 11, increasing in number until about day 18, after which a decline in numbers of infected cells occurred. Renal glomeruli became positive for DHBsAg from day 24. When present in the developing embryo, thymus, bursa of Fabricius, spleen, bone marrow, lung, and duodenum remained negative for DHBsAg. It was concluded that the timing of infection of specific tissues was not necessarily related to cellular maturity but may reflect a need for specific metabolic functions that permit viral replication.     

鸭乙型肝炎病毒在鸭胆管上皮细胞内增殖的超微结构研究

Liver specimens from Shanghai Ma ducks infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. The results showed that DHBV and DHBV antigen were found not only in the hepatocytes but also in the biliary epithelial cells of infected ducks. Electron microscopy also revealed that incomplete spherical particles, 40-50 nm in diameter, were present in the dilated cisternae of rough endoplasmic reticulum (RER), and complete spherical virions, 55-65 nm in diameter, existed in the cytoplasmic vesicles and cytoplasm in small amounts. The results of the present study seem to confirm the possibility of infection and replication of DHBV not only in the liver cells but also in the biliary epithelial cells of ducks.     

The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis

Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~ 80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes.     

In vivo antiviral effects of mismatched double-stranded RNA on duck hepatitis B virus

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)_n′r(C₁₂-U)_n′ to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2′-5′ Oligo-adenylate synthetase (2′-5′ AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 ± 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 ± 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2′–5′AS activity contribute to the inhibition of DHBV replication by m-dsRNA. © 1994 Wiley-Liss, Inc.     

Evaluation of anti-hepadnavirus activity of Phyllanthus amarus and Phyllanthus maderaspatensis in duck hepatitis b virus carrier Pekin ducks

Extracts of the two traditional Indian herbs, Phyl lanthus amarus (P. amarus) and Phyllanthus maderaspatensis (P. maderaspatensis), described by others as useful in the treatment of chronic hepatitis B virus infection were studied for antiviral properties on duck hepatitis B virus infection. One hundred and fourteen ducks infected posthatch with the duck hepatitis B virus (DHBV) were divided into groups at three months of age and treated intraperitoneally with the aqueous, butanol, and alcoholic extracts of these two plants at doses of 25, 50, or 200 mg/kg body weight. Saline-treated animals served as controls. In the ducks negative for DHBV in serum after treatment, we observed replicative intermediates in the liver. There was no definite antiviral property observed in the treated ducks.     

T cell tolerance in the chicken. II. Lack of evidence for suppressor cells in tolerant agammaglobulinemic and normal chickens.

Tolerance to human γ-globulin (HGG) at the T cell level was readily induced in both normal and bursectomized FP and EL 6 strain chickens by intravenous injection of 5 to 50 mg of soluble HGG. T cells from tolerant chickens, subsequently immunized with HGG in complete Freund's adjuvant (CFA), failed to cooperate in the adoptive immune response to 2, 4, 6-trinitrophenylated (TNP) HGG with TNP-immune spleen cells. However, suppressive activity could not be demonstrated when cells from tolerant chickens were combined with cells from HGG and CFA-sensitized chickens in the recipients, even at a 5 : 1 ratio. HGG-specific helper activity could be induced, both in bursectomized FP and in intact EL6 spleen cells, but neither tolerant bursectomized nor tolerant intact chicken spleen cells showed suppressor activity in this assay. Such tolerant cells also failed to inhibit formation of helper cell activity upon transfer to intact or bursectomized recipients subsequently exposed to HGG and CFA. Cells mediating delayed hypersensitivity (T_DH cells) were also tolerized by intravenous injection of soluble HGG. Injection of cyclophosphamide (CY), 100 mg/kg intraperitoneally, two days prior to the HGG did not interfere with this tolerance induction. CY also failed to augment the DH reactivity of immunized bursectomized animals, although it did increase the reactivity of intact animals. Thus, a CY-sensitive suppressor cell did not appear to be responsible for induction of the tolerant state. Moreover, spleen or thymus cells from tolerant bursectomized or intact animals, transferred into normal chickens, were unable to prevent sensitization of the T_DH cells in recipients. This was true in both bursectomized and intact recipients and was also not affected by treatment of recipients with CY, used to facilitate entry of tolerant donor cells into recipients' lymphoid tissue. Tolerance was also readily induced in thymectomized chickens. It was considered unlikely that the generation of suppressor cells was responsible for induction of tolerance to HGG at the T cell level in bursectomized or intact chickens. Since antibody formation could be excluded as a factor in the bursectomized birds, these results were in favor of a T cell deletion model of tolerance. Various ways in which deletion of T_DH cells might be achieved are discussed.     

Thymus dependency of antigen-binding cells in the bone marrow and spleen of chicks.

The bone marrow and spleen of normal unimmunized chicks contain lymphoid cells which form rosettes with quail red blood cells (QRBC), but not with sheep red blood cells. The number of these antigen binding cells (QRBC-ABC) were restored to their original level 2-3 weeks after whole body X-irradiation following a temporary but drastic decrease in the number of QRBC-ABC. This recovery is not observed in chicks which have been thymectomized or thymectomized and bursectomized. However, QRBC-ABC numbers were restored in bursectomized chicks comparable with those levels in control chicks. Anti-thymic serum effectively blocked rosette formation and it seems that QRBC-ABC are a thymus-dependent population. Possibly, the thymus exerts an influence on the genesis of QRBC-ABC through a humoral mechanism.     

Intracellular factors, but not virus receptor levels, influence the age-related outcome of DHBV infection of ducks.

Previous serological studies of experimental infection with duck hepatitis B virus (DHBV) have shown that the outcome of infection depends largely on the age of the duck at the time of inoculation. To examine the hypothesis that decreased susceptibility with increased age might be due to the loss of the virus receptor on hepatocyte membranes in adult ducks, we performed receptor binding studies using intact serum-derived DHBV virions and purified liver plasma membranes from both young ducklings and adult ducks. These studies showed that (1) DHBV was able to bind specifically to duck liver plasma membranes but not to internal membranes; (2) this binding could be inhibited by a monoclonal antibody to DHBV preS, a corresponding region in hepatitis B virus that binds to human hepatocytes; and (3) there was no significant difference in the receptor binding ability between plasma membranes from ducklings and from adult ducks. Since hepatocytes in the neonatal ducks are actively dividing, in contrast to the situation in adult ducks, we examined the effect of partial hepatectomy on DHBV-carrier ducks. A sharp increase was noted in the level of DHBV in the serum after partial hepatectomy suggesting that DHBV replication was enhanced in dividing hepatocytes. Thus the age-related difference in susceptibility of ducks to DHBV infection is not due to loss of the receptor but may be related to an intracellular event associated with cell division.     

Effect of early bursectomy on germinal centre and immunoglobulin production in chickens.

Chickens were bursectomized in ovo on days 18, 19 or 20 of incubation or within 6 h of hatch and immunized at day 28 after hatch by an intravenous injection of sheep red blood cells (SRBC). The immune response in the blood was measured by haemagglutination tests on the serum and by immunocyto-adherence tests for enumeration of rosette-forming cells with SRBC. Total serum 19S and 7S immunoglobulins were determined by radial immunodiffusion and the number of germinal centres per mm2 of fixed and stained spleen tissue sections was determined. In ovo bursectomy produced undetectably low serum levels of haemagglutinins, a reduction in SRBC rosette-forming cells with normal or increased 19S and reduced 7S immunoglobulins together with a complete disappearance (absence) of germinal centres in the spleen. The role of 19S antibody in the generation of germinal centres is discussed. The finding that birds which are equipped with serum 19S but no 7S Ig lack splenic germinal centres casts doubt on the hypothesis that the switch 19S leads to 7S Ig is necessarily an intrabursal event.     

Fish oil feeding enhances lymphocyte proliferation but impairs virus-specific T lymphocyte cytotoxicity in mice following challenge with influenza virus

The effect of a fish oil diet on virus-specific cytotoxicity and lymphocyte proliferation was investigated. Mice were fed fish oil (17 g fish oil and 3 g sunflower/100 g) or beef tallow (17 g tallow and 3 g sunflower/100 g) diets for 14 days before intranasal challenge with influenza virus. At day 5 after infection, lung virus-specific T lymphocyte, but not macrophage or natural killer (NK) cell, cytotoxicity was significantly lower in mice fed fish oil, while bronchial lymph node cell proliferation to virus was significantly higher. In mice fed fish oil, spleen cell proliferation to virus was also significantly higher following immunization. The results showed that, despite improved lymphocyte proliferation, fish oil impairs primary virus-specific T lymphocyte cytotoxicity. This impairment may explain the delayed virus clearance that we have previously reported in infected mice fed the fish oil diet.     

Immune capacity of the chicken bursectomized at 60 hr of incubation: cytoplasmic immunoglobulins and histological findings

Chickens were surgically bursectomized at 60 hr of incubation, before the bursal anlage appears. Completeness of the bursectomy was confirmed at autopsy at 10 weeks of age. These embryonically bursectomized (Bx)3 chickens are known to produce immunoglobulins of IgM, IgG, and IgA classes but so far no specific antibodies have been observed even after heavy immunization. The Bx chickens had mature plasma cells in an almost normal frequency when studied at 10 weeks of age. The amount of germinal center formation in the spleen and cecal tonsils was markedly decreased when compared to the control (Co) chickens. Also, the frequency of cytoplasmic IgA-positive (c-IgA+) cells was severely decreased in the Bx animals, whereas the occurrence of c-IgG+ and c-IgM+ cells was not affected to the same extent. These findings support the hypothesis that heavy-chain class switch may occur without the bursal influence, and that the bursa of Fabricius is essential only for expansion or creation of the antibody repertoire.     

Immunity in Pekin ducks experimentally and naturally infected with duck hepatitis B virus

The immune response to duck hepatitis B virus (DHBV) had not been elucidated. An assay was therefore established to detect the presence of antibody to DHB surface antigen (anti-DHBs) in serum of experimentally inoculated and naturally infected ducks. Anti-DHBs in serum was detected by indirect RIA from the percentage inhibition of binding of rabbit anti-DHBs to purified DHBsAg. Specificity was confirmed by positive and negative controls, infected and noninfected sera, and a mouse monoclonal antibody to DHB core antigen (anti-DHBc). Serum and liver samples were tested for DHBV DNA by dot-blot hybridization assay. Adult ducks repeatedly inoculated with DHBV remained non-viraemic but developed anti-DHBs. This antibody activity neutralized the infectivity of DHBV, which was experimentally inoculated into 1-day-old ducklings. In naturally infected flocks anti-DHBs was detected in a proportion of noninfected adult ducks as well as 1-day-old hatchlings. Anti-DHBs activity in hatchlings neutralized the infectivity of experimentally inoculated DHBV. Pekin ducks can therefore mount a neutralizing antibody response to DHBV, and immunity may be transferred in ovo from dam to off-spring.