Michael S. Halpern

Experimental transmission of duck hepatitis B virus

Susceptibility to experimental infection with duck hepatitis B virus (DHBV) was explored, with the objective of defining procedures that were both rapid and reproducible. For the purpose of these experiments, a small flock of DHBV-free breeders was established as a source of susceptible eggs and ducklings, since ca. 10% of the ducks (all ages) from commercial flocks were DHBV infected. Intravenous inoculation of DHBV into 15-day duck embryos from the DHBV-free flock produced a persistent infection, with a high-titer viremia, in at least 80% of the injected animals. The tissue tropism of DHBV in these experimentally infected animals was similar to that associated with natural, congenital infections from viremic ducks to their progeny. Virus antigen was found not only in hepatocytes and bile duct epithelium of liver, but also in cells associated with exocrine and endocrine pancreas, and in proximal convoluted tubular epithelium of kidney. Infection of embryonic liver was rapid, as evidenced by active synthesis of DHBV-DNA by reverse-transcription of RNA by 24 hr postinjection. During this latter analysis, formation of supercoiled viral DNA appeared to precede the reverse-transcription phase of viral DNA synthesis, suggesting that this species may be important in initiation of infection.

A structural protein complex in Moloney leukemia virus.

Abstract Comparison of the electrophoretic mobilities of the proteins of Moloney leukemia virus under reducing and nonreducing conditions has shown that approximately 50% of the gp69/71 glycoprotein is linked by disulfide bonds to a nonglycosylated protein(s) having a molecular weight of about 15,000.

Major histocompatibility (B) complex control of the growth pattern of v-src DNA-induced primary tumors

Observations that the major histocompatibility (B) complex is a determinant of the growth pattern of Rous sarcoma virus (RSV)-induced tumors raised the question as to whether control is exerted at the level of a v-src-determined, i.e., transformation-specific, function. To investigate this point, the tumor size scores and tumor profile indices of v-src-induced tumors were compared in two lines of chickens congenic for B complex genotypes. The finding that the growth patterns of tumors, induced by v-src DNA inoculation at 6 weeks posthatch, differ in these two lines establishes that the B complex exerts control over tumor growth at the level of a v-src-determined function. The potential importance of this control, in terms of the naturally occurring case of an avian sarcoma virus infection, is suggested by the observation that the patterns of tumor growth in a given congenic line are similar whether the tumors are induced by v-src DNA or by RSV.

Viral antigen expression in the pancreas of DHBV-infected embryos and young ducks

The time course of appearance of viral antigen-positive pancreatic cells was examined in both congenitally duck hepatitis B virus (DHBV)-infected duck embryos and experimentally DHBV-infected posthatch ducks. In the embryos, the earliest detectable viral antigen-positive pancreatic cells were localized to islets and identifiable as endocrine on the basis of hormone expression. Non-islet-associated, viral antigen-positive cells appeared at a late stage of embryogenesis, following the onset of chymotrypsinogen production by exocrine tissue; a number of these viral antigen-positive cells were directly identifiable as exocrine on the basis of chymotrypsinogen expression. By contrast, in the pancreas of experimentally infected posthatch ducks, the appearance of viral antigen-positive exocrine cells (chymotrypsinogen-positive) predated the appearance of antigen-positive islet cells. These results are consistent with the possibility that viral antigen expression in exocrine tissue is dependent on the state of cell maturation.

Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores

Double immunofluorescence assays of fixed sections of tissues from Pekin ducks congenitally infected with duck hepatitis B virus were carried out to score cells reactive to antiserum elicited either to viral DNA synthesis complexes (immature cores) purified from liver of infected ducks or to viral surface antigen particles and virions purified from sera of viremic ducks. Subpopulations of doubly fluorescent cells were detectable in liver (hepatocytes and bile duct epithelia), in kidney (proximal tubular epithelia), and in pancreas (acinar-associated cells and endocrine alpha and beta cells); all cells reactive with one antiserum were reactive with the second antiserum. Competition assays established that the cell-associated antigens recognized by the two antisera were mutually non-crossreactive. The cell-associated antigen recognized by the antiserum elicited to the purified immature cores appears to be crossreactive with a 35,000-Da polypeptide fraction associated with immature cores.

Duck hepatitis B virus is tropic exocrine cells of the pancreas

Earlier observations had established that duck hepatitis B virus (DHBV) is tropic for pancreatic endocrine cells, including cells localized to islets and to acini. Because cells identifiable as endocrine represented only a minor fraction of the total acinar-associated, infected subpopulation, the possibility was addressed in the present study that this subpopulation also comprises exocrine cells. Fixed preparations of cells from pancreas of congenitally DHBV-infected young ducks were reacted in double immunofluorescence assay with anti-virus serum and either anti-avian pancreatic polypeptide (APP) serum, a probe for a major subclass of acinar-associated endocrine cells, or anti-chymotrypsin serum, a probe for exocrine cells. Approximately 2-5% of the cells in these preparations were viral antigen-positive, comprising a minor fraction positive for APP and a much larger fraction positive for chymotrypsinogen. The detection of the latter establishes that DHBV is tropic for exocrine cells.

Viral antigen in endocrine cells of the pancreatic islets and adrenal cortex of Pekin ducks infected with duck hepatitis B virus

Endocrine cells in the pancreas and adrenal glands of duck hepatitis B virus-infected ducks were examined for the presence of viral antigen. Analysis of pancreas tissue was based on double immunofluorescence assays in which anti-duck hepatitis B virus serum was used to detect viral antigen, and anti-glucagon and anti-insulin serum were used, respectively, to identify endocrine alpha and beta cells. Assays of pancreas from infected ducks ranging in age from 3 to 20 weeks indicated that subpopulations of both alpha and beta cells expressed viral antigen. A higher percentage of viral antigen-positive cells was observed in alpha-islets than in beta-islets. As assayed by immunofluorescence and immunoperoxidase staining with anti-viral serum, small clusters of viral antigen-positive cells were detected in the adrenal glands of young infected ducks. These cells were identified as cortical cells on the basis of histologic criteria.

Wing web or intravenous inoculation of chickens with v-src DNA induces visceral sarcomas

We have previously found that sarcomas localized to visceral organs frequently arise following wing web inoculation of Rous sarcoma virus. This observation prompted an investigation of whether visceral sarcomas are also inducible by wing web inoculation of a subgenomic viral DNA fragment (v-src DNA) that includes v-src but lacks genes encoding viral structural protein. For this analysis, line SC chickens were inoculated with v-src DNA at 1-2 days posthatch and monitored for 9 weeks. Ninety percent of the chickens developed wing web (primary) tumors at the site of inoculation and, of these, about 30% exhibited visceral tumors. All tumors were histologically identifiable as sarcomas, and both the primary and visceral sarcoma cells were specifically reactive with two monoclonal antibodies elicited to different peptide fragments of pp60v-src. In a separate set of experiments, visceral sarcomas were also observed in about half of the line SC chickens that had been inoculated intravenously with v-src DNA. These results indicate that exogenous progeny virus production is not required for v-src-induced, visceral sarcoma formation. In addition, they demonstrate that intravenous inoculation of v-src DNA is a means to achieve rapid and widespread, disseminated sarcoma growth.

The defective maturation of viral progeny with a temperature-sensitive mutant of avian sarcoma virus.

Abstract LA 334 (previously ts 75) has been identified as a mutant exhibiting two genetic lesions affecting late functions of the avian sarcoma virus genome. One causes the transformed phenotype to be temperature sensitive, and the other induces a rapidly reversible inhibition of progeny virus production at the nonpermissive temperature. The nature of the latter defect has been investigated in this study with the following findings: (1) no new protein synthesis was needed to initiate synthesis of infectious virus after a shift from the nonpermissive to the permissive temperature; (2) significant amounts of physical particles (20 to 70% of that produced at the permissive temperature) were produced at the nonpermissive temperature; and (3) intracellular accumulation of structures resembling budding virus, approximately 80% of which were aberrant, were observed in mutant-infected cells at the nonpermissive temperature. The possibility that a defect in the synthesis of the viral envelope glycoprotein, gp85, would account for these findings prompted an investigation of the expression of gp85 in LA 334-infected cells. Intracellular synthesis of gp85 in normal amounts was shown by immunoprecipitation; however, only inefficient interference against superinfecting virus could be observed with mutant-infected cells at the nonpermissive temperature. Immunoferritin electron microscopic observations also indicated the presence of envelope glycoprotein in association with budding virions of normal morphology, but in contrast, little or no glycoprotein was associated with the budding structures of aberrant morphology. It is concluded that the primary virus defect is not related to expression of gp85; rather, on the basis of the abnormal core assembly, it is proposed that a defect exists in one of the core structural proteins. This hypothesis is discussed in the light of known properties of the mutant and of certain recent observations on the composition of the noninfectious virus produced by infected cells at the nonpermissive temperature.

Induction of a diffuse mesothelioma in chickens by intraperitoneal inoculation of v-src DNA.

The presence of peritoneum-based, cytokeratin-positive tumor cells, as detected under conditions of intravenous inoculation of chickens with a v-src-positive DNA fragment, raised the possibility that such DNA may be inductive for diffuse mesothelioma. To investigate this possibility, chickens were inoculated intraperitoneally with v-src DNA, and the resultant peritoneum-based tumor tissue was subjected to histopathological analysis. On the basis of (i) morphology, (ii) immunohistochemistry/histochemistry, and (iii) ultrastructure, we concluded that this tissue represents a diffuse mesothelioma.