The hepatitis 6 virus can be found in the serum and liver of some hepatitis B virus patients. We now report that the RNA genome of serum-derived 6 virus is single-stranded and circular. Livers of infected chimpanzees or woodchucks contained as many as 300,000 copies of genomic strand RNA per average cell, and at least some of this RNA had a circular conformation. Also present in the livers were RNA species complementary to the virion RNA. The genomic RNA was 5-22 times more abundant than this antigenomic strand. Some of the antigenomic RNA was complexed with genomic RNA, as evidenced by the fact that at least 34% of the antigenomic RNA was resistant to digestion with either RNase A in 0.3 M NaCl or S1 nuclease. Some of the antigenomic RNA was in a circular conformation. These and other fmdings showed that the structure and replication of hepatitis 6 virus are in many ways similar to those of the previously described plant viroids, virusoids, and satellite RNAs.The human hepatitis 8 virus, as described by Rizzetto et al.(1), is considered to be a defective virus, in that its natural transmission has been detected only in the presence of hepatitis B virus. This dependence has been demonstrated by experimental transmission of the agent to chimpanzees chronically infected with hepatitis B (2, 3). It has also been extended by transmission studies to woodchucks in which the woodchuck hepatitis B virus provided the transmission function (4). Antibodies to the woodchuck hepatitis virus envelope have been used to show that the concomitant hepadnavirus infection provides the packaging for the 8 viral genome. The characteristic antigen of the 8 particle, the 8 antigen, is packaged, with the virion RNA, inside a 36-nmdiameter lipoprotein envelope in which is incorporated hepatitis B surface antigen (2, 5).Bonino et al. have previously reported that the RNA of the 8 particle is a 1.7-kilobase (kb) single-stranded species (5).Denniston et al. used this RNA to make a cDNA clone in pBR322, designated pKD3, with a 166-base 8-specific insert (6). As presented here, we have derived an independent cDNA library, screened it with pKD3, made strand-specific probes, and used these probes to study both the virion and intracellular RNA from livers of infected chimpanzees and woodchucks. Our findings on the structure and replication of the 8 genome support the previous conjecture (7) that the agent bears similarities to the viroids and virusoids of plant cells (8).MATERIALS AND METHODS Viral RNA. For most experiments, RNA was directly extracted from serum after dilution (1:20) into a buffer containing 10 mM Tris, 10 mM EDTA, 0.1% sodium dodecyl sulfate, and Pronase at 1 mg/ml. After digestion for 1 hr at 370C, the sample was extracted once with phenol and twice with ether and then collected by precipitation with ethanol, using dextran as carrier. In some cases the virions were first purified by centrifugation through a sucrose cushion (5) or by isopycnic centrifugation in cesium chloride (5). RNA was then extracted as described.Li...

cDNA prepared from the single-stranded circular RNA genome of hepatitis delta virus was cloned in lambda gt11 by using RNA from the liver of an infected woodchuck. From the sequence of overlapping clones, we assembled the full sequence of 1,679 nucleotides. The sequence indicated an exceptional ability for intramolecular base pairing, yielding a rod structure with at least 70% of the bases paired and a predicted free energy of -805 kcal (-3,368 kJ)/mol. Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody. These clones were sequenced so as to establish the reading frame of the delta antigen on the antigenomic strand. The fusion protein produced by one clone was purified by immunoaffinity chromatography and then was used to raise rabbit antibodies specific for the delta antigen.

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