Influence of aflatoxin B1 intoxication on duck livers with duck hepatitis B virus infection.

In an attempt to determine the effect of aflatoxin B1 (AFB) intoxication on livers with duck hepatitis B virus (DHBV) infection, domestic ducks were given 0.1 mg of AFB/kg body weight twice a week for a maximum period of 54 weeks employing various experimental designs. The ducks were infected with DHBV by i.v. inoculation of DHBV-positive sera within 24 h posthatch. The livers were examined histologically, immunohistochemically, and ultrastructurally, and the livers and sera were examined by molecular hybridization for DHBV DNA. AFB administration induced hepatocellular necrosis and marked biliary cell proliferation of the periportal areas, and finally liver cirrhosis. On short-term administration, the hepatocytes of DHBV-infected livers revealed a marked increase in incomplete particles of DHBV by immunostaining and electron microscopy, as compared to those without its administration. Long-term AFB administration provoked frequent nodular or cirrhotic changes. There was no significant increase in frequency of these changes in DHBV-positive ducks as compared to DHBV-negative ones. AFB administration induced hepatocellular carcinoma (HCC) in one DHBV-positive duck and in two DHBV-negative ducks. The HCC and cirrhotic livers revealed extrachromosomal but no integrated form of DHBV DNA by Southern blot hybridization analysis. Immunostaining demonstrated a heterogeneous distribution of DHBV from area to area in nodular and cirrhotic livers. Thus, AFB intoxication provoked various liver disorders independent of DHBV infection, and neither a cocarcinogenic effect of AFB and DHBV nor integration of viral DNA into the genome of neoplastic and nonneoplastic tissues was observed in the present experiments. Generally speaking, DHBV infection did not appear to accelerate hepatic disorders induced by AFB intoxication. However, AFB administration altered the DHBV in the liver in terms of its amount and distribution.

Geographical pathology of duck livers infected with duck hepatitis B virus from Chiba and Shimane in Japan and Shanghai in China.

In order to evaluate geographical differences in the liver pathology of ducks infected with duck hepatitis B virus (DHBV), ducks in Chiba and Shimane, Japan, and Shanghai, China, were investigated. The numbers (DHBV positive/negative) and the maximum age of the ducks examined were 18/10 at 19 mo, 15/1 at 3 yr 4 mo, and 72/27 at 18 mo, respectively. DHBV infection was induced experimentally in ducks from Chiba and Shimane but was present congenitally in those from Shanghai. Ducks were examined regarding liver function tests, conventional histology, immunohistology, electron microscopy, and molecular hybridization for DHBV DNA in the serum and liver. There was no significant difference between DHBV-positive and -negative ducks in bilirubin and transaminase and alkaline phosphatase activities in the sera. Histologically, while the livers of ducks from Chiba and Shimane did not show necroinflammatory (hepatitis) activity, those from Shanghai frequently did (52.5%). Necroinflammatory activity of the Shanghai ducks was present almost equally in both DHBV-positive and -negative livers. The livers of Shanghai ducks but not the other two areas often (8.3%) had ground-glass inclusions which corresponded ultrastructurally to numerous virus particles in the dilated cisternae of the proliferated endoplasmic reticulum. No advanced liver disease, such as cirrhosis or hepatocellular carcinoma, was observed. There was no significant difference in the amount of DHBV DNA in the sera or in its pattern in the liver tissue among ducks of the three areas. In addition, the livers of Chiba ducks frequently had amyloidosis, while those of Shanghai ducks were contaminated with parasites. In conclusion, DHBV infection did not appear to provoke significant hepatitis activity or advanced liver disease in the examined ducks of all three areas, and the DHBV-positive livers from Shanghai ducks showed a different morphological appearance from those of the other two areas. This variation might reflect the difference in the strain of ducks, subtypes of DHBV, environmental factors, or a combination of these influences.

Clonal origin of human hepatoma determined by integration of hepatitis B virus DNA.

The hepatitis B virus genome is integrated in cellular DNA of human hepatocellular carcinoma from hepatitis B surface antigen-positive patients. Using this phenomenon, we determined the clonal origin of hepatocellular carcinoma from the integration mode of hepatitis B virus DNA. The molecular size and the number of restriction fragments of integrated hepatitis B virus DNA in several parts of tumors in the same liver and in metastatic tumors were compared by Southern blot analysis. Of 14 cases of hepatoma, 13 cases were monoclonal; in one case, a different clone of hepatoma was found in one part of the tumor. In three of 13 cases of monoclonal hepatoma, metastatic tumors in lymph nodes and the lung were also examined and found to be the same clone as the liver tumors. These results indicate that hepatocellular carcinomas were usually generated from a single tumor cell even though tumor cells spread in the liver and invaded other organs for a long time. Development of different clones of tumor was apparently unusual but was observed in one case of hepatocellular carcinoma.

Identification of three essential regions of hepatitis B virus X protein for trans-activation function.

The X protein of hepatitis B virus (HBV) consists of 154 amino acids and trans-activates various cellular and viral promoters and enhancers. To investigate the essential amino acid sequences of X protein for trans-activation function, various mutations were introduced into the X open reading frame and analysed for trans-activation activity by chloramphenicol acetyltransferase assay. The amino acid sequences 46-52 (especially Pro-46, His-49 and His-52), 61-69 (especially Cys-61, Gly-67 to Pro-68 and Cys-69) and 132-139 (especially Phe-132, Cys-137 and His-139) of HBV X protein were found to be essential for the trans-activation function. These three sequences are included in the conserved amino acid sequences among hepadna virus X proteins. The first one could form a domain-like structure characteristic of histidine/aspartic acid requirement. The second and the third are homologous to the Kunitz domain of Kunitz-type serine protease inhibitors. The amino acids 5-27 region was found to make no positive contribution to the trans-activation function like the last 12 amino acids in the carboxy-terminal region [Takada, S. & Koike, K. (1990). Proc. Natl. Acad. Sci. USA, 87, 5628-5632]. From these findings, the trans-activation function of X protein appears to be dependent on at least two types of domain-like structures.

Occurrence and ultrastructural localization of duck hepatitis B virus in the liver of ducks after experimental infection.

A sequential study was performed to investigate the occurrence and localization of duck hepatitis B virus in the liver of domestic ducks utilizing the indirect immunoperoxidase method and electron microscopy. Seventeen ducklings were injected intravenously with duck hepatitis B virus-positive serum within 24 hr after hatching and were subsequently sacrificed on the 2nd, 3rd, 4th, 5th, 27th and 44th day after injection. Nine ducklings were not injected and were used as a negative control. Duck hepatitis B virus DNA by spot hybridization using a [3P]-labeled probe occurred in trace amounts on the 2nd day and in large amounts on the 4th day after inoculation. Immunoreactivity for DHBV was seen in the hepatocytes, sporadically on the 2nd day and diffusely on the 4th day, and also in the biliary epithelial cells on the 27th day. Both kinds of cells revealed staining in the cytoplasm but not in the nucleus. Virus particles were recognized by electron microscopy in the hepatocytes beginning on the 4th day. The hepatocytes had many incomplete virus particles, 40 to 61 nm in diameter, and a few complete virus particles, 40 nm in diameter, in the cisternae of the rough and smooth endoplasmic reticula. Such particles and the endoplasmic reticulum showed reaction products for duck hepatitis B virus by immunoelectron microscopy. There were clusters of core particles, 27 nm in diameter, in the hyaloplasm around peroxisomes where an assembly of cores appeared to occur. No conspicuous virus particles were recognized in the biliary epithelial cells. The similarities and differences in virus localization between duck hepatitis B virus and hepatitis B virus are discussed.

Increase of eosinophilic cell population in bone marrow of rats by immunization with Ascaris suum antigen.

Immunization of rats with the antigen, Ascaris suum extract, increased the number of peripheral eosinophils. Analysis by Western blot and reverse transcription-polymerase chain reaction revealed that the levels of major basic protein and its mRNA in the bone marrow were also increased, suggesting that eosinophilic cell population in the bone marrow is increased by the immunization. These findings indicate that immunization with this antigen stimulates differentiation of progenitor cells to eosinophils in the bone marrow, and induces blood eosinophilia.

Oncogenic potential of hepatitis B virus.

The function of the X gene was clarified by examination of the transient production of hepatitis B virus (HBV) particles by transfected recombinant HBV DNA (pHBV-3 DNA) and its frameshift mutant (delta X) of the X open reading frame into hepatocellular carcinoma HuH-7 and HepG2 cells. No reduction of viral mRNAs was observed in the HuH-7 cells by the delta X mutant, whereas mRNAs underwent marked reduction in HepG2 cells. No reduction in core particle production was observed in HuH-7 cells, but in HepG2 cells reduction was considerable. To clarify the significance of the delta X mutation in the trans-acting function of the X gene in hepatoma cells, the chloramphenicol acetyl/transferase (CAT) assay was conducted. Transfection of plasmid pHBV-3 into HepG2 cells increased CAT activity of pSV2-CAT, while the delta X mutation clearly showed no stimulation of activity. On the other hand, in the HuH-7 cells, pHBV-3 exhibited no such stimulation. The trans-acting function of the X gene product in two different hepatoma cells was clearly shown to differ. Furthermore, transfection of X gene expression plasmid pKSV-HBx into mouse NIH3T3 cells increased the CAT activity of pSV2-CAT. Trans-activation was still detectable even following deletion of enhancer sequences in the pSV2CAT. The oncogenic potential of HBV is discussed with special attention to the X gene product, which may be able to activate a cellular transcription factor at the viral and cellular promotor sequences in the cells.