Sequence comparison of woodchuck hepatitis virus replicative forms shows conservation of the genome.

The complete nucleotide sequence of virion DNA from two isolates of woodchuck hepatitis virus (WHV 7 and WHV 59) was determined along with the sequence of supercoiled DNA from one of those isolates (WHV 7). The sequences of the two WHV isolates were compared with the previously published sequences of two other isolates (WHV 1 and WHV 8). The range of nucleotide sequence variation of the four isolates (1 to 3.5%) was similar to those of HBVs of the same subtype (1.5 to 2%), but less than those of HBVs of different subtypes (8 to 10%). When amino acid sequences from the four WHV isolates were aligned, a consensus sequence could not be obtained at 13 sites. At 11 of 13 sites (7 in polymerase, 2 in presurface, 1 in surface, and 1 in the X region) the viruses could be grouped into pairs, so that WHV 1 and WHV 59 shared one amino acid and/or WHV 7 and WHV 8 shared a different amino acid. WHV 7 was passaged twice in woodchucks and supercoiled DNA was isolated. The nucleotide sequence of WHV 7 supercoiled DNA (derived from liver) was compared to that of WHV 7 virion DNA (derived from serum). Sequence comparison of virion and free supercoiled DNA from WHV 7 showed no nucleotide changes, except for a single nucleotide deletion thought to represent an error during molecular cloning or a defective viral genome. Thus, the nucleotide sequence of two different replicative forms of WHV DNA, separated by two in vivo passages, were virtually identical.

Type D hepatitis: the clinical significance of hepatitis D virus RNA in serum as detected by a hybridization-based assay.

Hepatitis D virus is a defective human pathogen that requires hepatitis B virus for its replication. A hybridization-based assay for the 1.75 kb RNA genome of hepatitis D virus was developed using as probe a radiolabeled transcript of a cloned cDNA fragment (pKD3 hepatitis D virus DNA). Sera from 120 chronic carriers of HBsAg with confirmed hepatitis D virus infection were analyzed for the presence of hepatitis D virus RNA. Serum hepatitis D virus RNA was detected in 43 of 74 (58%) patients with chronic liver disease; some patients were positive for hepatitis D virus RNA in multiple samples over a period of several years. Serum hepatitis D virus RNA was present in 17 of 28 (61%) patients during the acute phase of clinical hepatitis and was not detected after recovery from acute disease or in 18 asymptomatic chronic HBsAg carriers with antibody to hepatitis D virus. The presence of hepatitis D virus RNA correlated with other known markers of active hepatitis D virus replication; all chronic active liver disease patients with serum hepatitis D virus RNA were positive for antihepatitis D antigen IgM, and 34 of 37 (92%) had hepatitis D antigen in their liver biopsy specimens. The assay for hepatitis D virus RNA provides a direct and noninvasive method for the detection of hepatitis D virus in serum and will be useful in the study of the natural history of type D hepatitis, the identification of chronic hepatitis D virus carriers likely to transmit hepatitis D virus and the selection and monitoring of patients for potential antiviral therapy.

Structure, sequence and expression of the hepatitis delta (delta) viral genome.

Biochemical and electron microscopic data indicate that the human hepatitis delta viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis delta viral infections.

Cloned fragment of the hepatitis delta virus RNA genome: sequence and diagnostic application.

Hepatitis delta virus (HDV) is a replication-defective etiological agent of hepatitis that requires hepatitis B virus (HBV) as a helper. A complementary DNA (cDNA) fragment of the RNA genome of HDV was cloned into the plasmid vector pBR322, and the primary nucleotide sequence and predicted protein products of the cDNA fragment were determined. This cloned cDNA fragment has been used as a sensitive radioactive probe for the detection of HDV RNA in the serum of patients with either acute or chronic HDV infections.

Expression of hepatitis B virus surface and e antigen genes cloned in bovine papillomavirus vectors.

Sequences of the hepatitis B virus (HBV) genome coding for the surface antigen (HBsAg), but lacking the regulatory (preS) sequences, were cloned into a bovine papillomavirus (BPV) vector consisting of the transforming 69% of the BPV genome (BPV 69T), and transformed into mouse c127 cells. Clones carrying HBV and BPV sequences in the same transcriptional orientation did not produce immunologically active HBsAg, while those with the opposite orientation produced and secreted HBsAg. RNA species of an HBsAg producer were 3400, 9600, 11000 and 18000 nucleotides long and hybridized with both HBV and BPV probes. Mouse cells (c127) transformed with the whole BPV genome (BPV-1) carrying both the HBsAg-coding sequences and the regulatory preS sequences of HBV DNA were stable and produced and secreted HBsAg 22 nm particles. These yielded 11500 and 12500 nucleotide RNA transcripts which also hybridized with both BPV and HBV DNA probes. BPV-1 carrying whole HBV DNA monomer or dimer genomes yielded transformants which initially produced HBsAg as well as HBV e antigen (HBeAg), but these were not stable. The hybrid genomes, with the exception of those carrying the HBV dimers, existed as multicopy plasmids (50-100 copies per cell) and often acquired new sequences.

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles.

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.

Sequence organization of the origins of DNA replication in lambdoid coliphages.

We have determined the sequences of the ori region DNA of several phage lambda mutants and hybrids, which shed light on the mechanism of DNA replication in the lambdoid phages. These include the heterologous substitution hybrids lambda rep82:lambda and lambda rep80:lambda, a pseudorevertant of the ori-r93 mutant lambda r93hot5, and the insertion mutant lambda pk35. The ori regions of the three lambdoid phages, lambda, phi 80 and 82, all have repeated sequences, termed iterons, and A . T-rich zones. We note that a similar arrangement of DNA is also found in several other prokaryotic origins of replication. lambda and phi 80 have four iterons, and 82 has five. The origin of lambda r93hot5 is unusual in that contains only three iterons, yet the phage grows normally. Analysis of this mutant indicates that the spacing of iterons is crucial to ori function, whereas their number is not. This argues against the cloverleaf model for lambda ori structure (Hobom et al., 1979). In lambda pk35 the drug resistance element Tn903 is inserted into the "inceptor" (ice) site, proposed to be crucial for lambda replication initiation (Hobom et al., 1979); yet this phage grows normally.