Kirk E. Fry

Molecular Cloning and Disease Association of Hepatitis G Virus: A Transfusion-Transmissible Agent

An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

Molecular cloning and sequencing of the mexico isolate of hepatitis E virus (HEV)

Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.

Synthesis of Hybrid Bacterial Plasmids Containing Highly Repeated Satellite DNA

Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes.

Characterization of a Helicobacter pylori vaccine candidate by proteome techniques

In a previous two-dimensional (2D) gel electrophoretic study of protein antigens of the gastric pathogen, Helicobacter pylori recognized by human sera, one of the highly and consistently reactive antigens, a protein with Mr of approximately 30,000 (Spot 15) seemed to be of special interest because of low yields on N-terminal protein sequencing. This suggested possible N-terminal modification, as the N-terminal sequence analysis of this 30,000 protein (Spot 15) did not provide a definitive match within the H. pylori genomic database. This protein was isolated by 2D polyacrylamide gel electrophoresis, evaluated by liquid chromatography-mass spectrometry, and found to consist of two related species of approximately 28,100 and 26,500. In parallel, the proteins within this spot were digested in situ with the endoprotease Lys-C. Analysis of the Lys-C digest by matrix-assisted laser desorption time-of-flight mass spectrometry, peptide mapping, and sequence analysis was conducted. Comparison of the mass and sequence of the Lys-C peptides with those derived from a H. pylori genomic library identified an open reading frame of approximately 300 base pairs as the source of the Spot 15 protein. This corresponded to HP0175 in the recently reported H. pylori genome sequence, an open reading frame with some homology to Campylobacter jejeuni cell binding protein 2. Mass spectral and sequence analysis indicated that Spot 15 was a processed product generated by proteolytic cleavage at both the carboxy and amino termini of the 34 open reading frame precursor.

Hepatitis E virus (HEV): Strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site

Hepatitis is transmitted by a number of infectious agents. The epidemiological characterization of waterborne or enterically transmitted non-A, non-B hepatitis (ET-NANBH) is unique when compared with other known hepatitides. We have reported on the molecular cloning of a cDNA clone derived from the etiologic agent associated with ET-NANBH, the hepatitis E virus (HEV). The complete sequence of these first molecular clones, isolated from an HEV-infected human after passage inMacaca fascicularis (cynomolgus macaques), illustrates a distant relationship to other known positive-strand RNA viruses of plants and animals. The translated major open reading frame (ORF-1) from these clones indicates that this portion of the genome encodes a polyprotein with consensus sequences found in RNA-dependent RNA polymerase and ATP/GTP binding domains. The latter activity has been associated with putative helicases of positive-strand RNA viruses. These viral-encoded enzymatic activities identify this region and ORF-1 as containing at least two different nonstructural genes involved in HEV replication. Molecular clones obtained from two other geographically distinct HEV isolates demonstrated sequence heterogeneity in this nonstructural gene region. Further study will be required to elucidate the pathogenic significance (if any) of this observed divergence in the nonstructural region.

Hepatitis E virus (HEV): The novel agent responsible for enterically transmitted non-A, non-B hepatitis

SummaryA normally endemic form of viral hepatitis is the cause of major epidemic outbreaks in developing countries. This disease has a global distribution and has been referred to as water-borne, epidemic or enterically transmitted non-A, non-B hepatitis (ET-NANBH). Although the fecal-oral route of transmission predominates, person-toperson routes of exposure were also suggested in some epidemiologic studies. The disease has been documented as having an extremely high mortality in pregnant women (~20%). Sporadic cases of ET-NANBH, as well as imported travel exposures, have been reported in developed countries. Molecular cloning was hampered by the lack of a tissue culture system for virus propagation, however, an available animal model and a newly developed non-specific amplification procedure were used to clone and identify an exogenous cDNA (ET1.1) from a Burma-isolate infected animal. Molecular clones were also identified by immunoscreening of a cDNA library made from a fecal specimen collected from a Mexican outbreak of ET-NANBH. The isolation and sequencing of a set of overlapping cDNA clones had led to the recognition that this form of hepatitis is caused by a virus unlike any of the other viral hepatitis agents. The molecular characterization of HEV will lead to important pathobiologic insights and hasten the development of potentially useful diagnostic and therapeutic products for ET-NANBH.

Nucleotide sequence of the large terminal repeat of two different strains of gibbon ape leukemia virus

Gibbon ape leukemia virus, SEATO strain (GaLV-SEATO), a virus that induces myeloid leukemia in gibbon apes, and GaLV, San Francisco strain (GaLV-SF), a virus associated etiologically with lymphocytic leukemia in gibbon apes, have been molecularly cloned. The complete nucleotide sequence of the large terminal repeats (LTRs) of both viruses are reported and compared to the previously published nucleotide sequence of the LTR of another member of the same virus group, the simian sarcoma virus (SSV). Substantial homology is evident among all three LTR sequences. The most striking feature of the GaLV-SEATO LTR is the presence of a 45-bp tandem direct repeat in the U3 region, an area likely to contain transcriptional enhancers. Both GaLV-SEATO and GaLV-SF contain a deletion in U3 when compared to SSV. Each of the three LTRs differ from the other two by short deletions in R-U5 and short additions in U3, as well as by numerous point mutations. The possibility that the structural changes observed in the LTR contribute to the differences in the pathogenic effects of these viruses is discussed.

Detection and resolution of closely related satellite DNA sequences by molecular cloning

Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm3 satellite DNA of Drosophila melanogaster into two distinct components: polydA-A-G-A-GT-T-C-T-C and polydA-A-G-A-G-A-GT-T-C-T-C-T-C. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.

Patterns of thymocyte differentiation markers on virus and radiation induced lymphomas of C57BL/Ka mice

To better understand the biology of tumorigenesis in virus and radiation lymphomas of C57Bl/Ka mice, we have examined the cell surface phenotypes of a large series of primary tumors induced by both agents. Data derived using flow cytometry and recently available monoclonal antibodies to thymocyte differentiation antigens supports three major conclusions. First, tumor cell populations are unimodal for staining with most antibodies and are probably of clonal origin. Second, many, but not all, tumor cells show surface phenotypes similar to those of previously defined subpopulations of normal thymocytes. Third, at the cell surface level, no major differences between virus- and radiation-induced lymphomas can be discerned. Our data thus further define the relationship between thymomas induced by these two agents.

Strategies for Stable Human Monoclonal Antibody Production

The development of hybridoma technology by Kohler and Milstein (1975) opened a new era not only in immunology, but in all fields of biological science. Hybridoma cell lines formed by the fusion of mutant mouse myeloma cells with spleen cells from an immunized mouse assure the permanent availability of monoclonal antibody of defined specificity. The clinical use of these xenoantibodies in human patients, however, will be limited by the fact that they themselves will be immunogenic upon repeated administration. Accordingly, for therapeutic applications in man, the availability of human monoclonal antibodies would be advantageous. The advance of human hybridoma technology has, however, been slowed by the unavailability of suitable fusion partners. Early attempts to generate immortalized human immunoglobulinproducing cells involved the fusion of human lymphoid cells with mouse myeloma cells to create chimeric hybridomas (Levy and Dilley, 1978; Schwaber, 1975; Schwaber and Cohen, 1973). Although exceptions have been reported (Schlom et al., 1980; Lane et al., 1982), such mouseiahuman hybridomas have tended to be unstable and cease immunoglobulin production due to the selective loss of human chromosomes (Weiss and Green, 1967; Nabholz et al., 1969), or to disturbances of gene expression (Raison et al., 1982). A second approach has involved the transformation of antigen-primed human B lymphocytes with Epstein-Barr virus (EBV) (Zurawski et al., 1978; Steinitz et al., 1979; Kozbor et al., 1979; Hirano et al., 1980; Tsuchiya et al., 1980; Yoshie and Ono, 1980). This method has also had some success, but in most instances, such cultures have tended to be unstable and produce low yields of antibody (Zurawski et al., 1978; Tsuchiya et al., 1980).