Feathers as bioindicators of PCB exposure in clapper rails.

In this study we used feathers to biomonitor exposure to the polychlorinated biphenyl (PCB) Aroclor 1268 congener mixture in clapper rails (Rallus longirostris). This species has been used as an indicator species of environmental damage for the LCP superfund site located in Brunswick, GA, USA which is contaminated with Aroclor 1268, a congener mixture that has been used in limited amounts elsewhere and therefore can be used as a contaminant marker. The Aroclor 1268 congener mixture, including congener profiles, were quantified in feathers using gas chromatography (GC). Concurrently, each sample was quantified for the total Aroclor 1268 congener mixture using an enzyme-linked immunosorbant assay (ELISA) and compared to the GC results to determine if ELISA was an efficient method for quantifying or qualifying PCBs in feathers. ELISA consistently quantified PCB loads over an order of magnitude lower than the GC. Based on sample replication, extraction recovery, and sample spike, it appears that GC is the more reliable method of detection and that ELISA methods may be more suitable for qualitative exposure assessment for this particular Aroclor. Moreover, since all clapper rails from the LCP site had the Aroclor 1268 congener mixture in their feathers, this experiment showed that birds were returning to the site to breed despite the adverse effects experienced by this population from the contamination revealed in previous studies. This study also supports the utility of feathers as a non-lethal mechanism by which to biomonitor PCBs in the environment.

Infection and uptake of duck hepatitis B virus by duck hepatocytes maintained in the presence of dimethyl sulfoxide.

Primary duck hepatocytes maintained in serum-free culture medium containing dimethyl sulfoxide support efficient replication of duck hepatitis B virus following infection in vitro. Cells remain susceptible to infection for at least 2 weeks after plating, allowing spread of virus via repeat cycles of infection. Up to 100% of cultured hepatocytes can be infected by prolonged exposure to high titer virus inoculum at 37 degrees. We have identified a fraction of infecting virus which binds tightly to cells at 4 degrees, presumably due to association with high affinity receptors on the hepatocyte membrane. The rate at which tightly bound virus is internalized at 37 degrees appears slow, with uptake occurring over a period of at least 16 hr.

Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles.

DNA from the pre-S region of the duck hepatitis B virus (DHBV) genome was inserted into an open reading frame vector designed to give high-level expression in Escherichia coli. The resulting fusion protein contained the first 8 amino acids of beta-galactosidase, 86 amino acids of the DHBV pre-S region, and 219 amino acids of chloramphenicol acetyltransferase at the C terminus (beta-gal:pre-S:CAT). Rabbit antiserum against purified beta-gal:pre-S:CAT was used to identify pre-S-containing polypeptides in DHBV particles by Western blotting. A dominant species of 36 kilodaltons (kDa) was identified. Antiserum against the major 17-kDa DHBsAg polypeptide also reacted with the 36-kDa protein. This suggests that the DHBV envelope gene polypeptides share the same carboxyl terminus, but differ in the sites from which translation is initiated. N-linked carbohydrate was not detected on either the 17- or 36-kDa envelope proteins. Anti-beta-gal:pre-S:CAT abolished infectivity of the virus in an in vitro assay. Thus, the pre-S region is exposed on the surfaces of infectious virions and may be directly involved in binding of virus to host-cell receptors.

In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus.

Duck hepatitis B virus (DHBV) obtained from the serum of congenitally infected ducks was used to infect primary duck hepatocyte cultures 1 to 4 days after plating. Virus replication was demonstrated by the appearance, beginning at 2 days after infection, of intracellular covalently closed-circular and single-stranded DHBV DNA replicative intermediates which were not present in the inoculating virus preparation. With increasing time after infection there was further amplification of intracellular relaxed circular, covalently closed-circular, and single-stranded DHBV DNA. Cultures of primary duck hepatocytes are competent for infection with DHBV only during the first 4 days of culture. Synthesis of DHBV core antigen and DHBV surface antigen was detected by immunofluorescence in 10% of the hepatocytes in culture. De novo synthesis and release of infectious virus was also demonstrated. Therefore, all stages of viral replication were carried out by these experimentally infected primary hepatocyte cultures. This system makes it possible to study DHBV replication in vitro.

Mapping of the cohesive overlap of duck hepatitis B virus DNA and of the site of initiation of reverse transcription.

The hepatitis B-like viruses have a approximately 3.2 kilobase, partially double-stranded DNA genome that is held in a circular conformation by a cohesive overlap between the 5' ends of the two strands. In addition, a protein is covalently bound to the 5' end of the minus strand of virion DNA. The sequence of the cohesive overlap region and its location relative to open reading frames and to the initiation site for minus-strand DNA synthesis, which occurs by reverse transcription of viral RNA, were investigated in duck hepatitis B virus. The 5' ends of virion DNA were mapped by restriction endonuclease analysis of labeled virion DNA, S1 nuclease digestion, and primer extension, using avian myeloblastosis virus DNA polymerase. The cohesive overlap region was shown to be 69 +/- 4 base pairs in length. It contained a 10-base pair inverted repeat in approximately the middle and a 12-base pair direct repeat near each end. The apparent initiation site of reverse transcription was determined by partial sequence analysis of dideoxynucleotide-truncated minus-strand DNA intermediates and comparison of their lengths with the length of a known DNA sequence. It mapped within two to four nucleotides of the 5' end of the minus strand of virion DNA. The results are consistent with the interpretation that the 5' end of the minus strand of virion DNA is the origin of reverse transcription and that the protein covalently bound to virion DNA is the primer of reverse transcription.

Woodchuck hepatitis virus in natural woodchuck populations.

Woodchuck hepatitis virus (WHV) was discovered in serum samples from captive woodchucks (Marmota monax) at the Penrose Research Laboratory in December, 1977. WHV belongs to the same class of viruses as hepatitis B virus (HBV), the cause of serum hepatitis in man. Both appear to be associated with chronic hepatitis and hepatocellular carcinoma in their respective hosts. Woodchucks were trapped and blood samples collected to determine the prevalence of WHV in natural woodchuck populations. Sera from 217 woodchucks trapped from southeastern Pennsylvania, central New Jersey, and north central Maryland during the spring and summer of 1978 and 1979 were tested for evidence of WHV infection. In 1978, 7 of 51 (13.7%) woodchucks were positive for WHV antigens and in 1979, 28 of 166 (16.9%) tested positive. In addition, 49 of 166 (29.5%) woodchucks trapped in 1979 had antibodies to WHV antigens. The data indicate a high prevalence of WHV in woodchucks from the areas surveyed.

Biotin-deficient growth of Bacillus polymyxa.

When Bacillus polymyxa, a wild-type biotin auxotroph, is grown in biotin-deficient medium, a retardation of cell division and consequential cell elongation are the initial detectable consequences of limited biotin. Subsequent events in biotin-deficient cells include, in chronological order: inhibition of net ribonucleic acid (RNA) synthesis and a simultaneous arithmetical accumulation of protein; loss of net RNA, deoxyribonucleic acid, and protein synthesis; morphological aberration, death, and lysis. Incorporation studies employing (32)P-phosphate and (14)CO(2) demonstrate an initial selective inhibition of net ribosomal RNA synthesis over that of ribosomal protein or total protein. Biotin could not be replaced by various extracts from which biotin had been removed, nor could osmotic stabilizers be found which could prevent lysis of the culture.