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Biomedical subjects

R H Purcell

Publications and source records attributed to R H Purcell.

At least 127 records · Page 7Linked to original sources
PubMed1994

Attenuated hepatitis A virus: genetic determinants of adaptation to growth in MRC-5 cells.

A live candidate hepatitis A virus vaccine, developed from the HM-175 strain and adapted to growth in primary African green monkey kidney (AGMK) cells, was adapted to growth in MRC-5 cells. The nucleotide sequence of the MRC-5 cell-adapted virus was determined and compared with the known sequence of the AGMK cell-adapted virus. Thirteen unique mutations, which occurred during passage in MRC-5 cells, were identified. Four of the unique mutations were located in a cluster in the 5' noncoding region (NC), and three of the remaining nine mutations encoded amino acid changes. Infectious chimeric cDNAs were constructed from infectious cDNA clones of the AGMK cell-adapted and wild-type HM-175 viruses and PCR-amplified cDNA segments of the MRC-5 cell-adapted virus. The viruses encoded by these plasmids were recovered after transfection of cultured cells with in vitro transcripts, and their growth phenotypes in fetal rhesus kidney 4 (FRhK-4) and MRC-5 cells were determined. The important growth-enhancing mutations could be divided into three sets. Two of these were located in the 5' NC region, and the third was located in the 2C nonstructural gene. The mutations in the 5' NC region that developed during passage in MRC-5 cells were indispensable for efficient growth in MRC-5 cells, but a combination of the two groups in the 5' NC region and one in the 2C gene were required to increase growth dramatically in MRC-5 cells.

Adaptation, Physiological↗
PubMed1994

Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.

Cell Line↗
PubMed1994

Expression of the c-myc protooncogene product in cells infected with the hepatitis delta virus.

The intrahepatic accumulation of the c-myc protooncogene product was observed on immunofluorescence in each of six patients with chronic hepatitis delta virus infection who exhibited the hepatitis D antigen in their livers. The c-myc product was stained in the same nuclei that contained the hepatitis D antigen. C-myc was not observed in acute hepatitis D or in cases of chronic hepatitis delta virus infection without expression of the hepatitis D antigen. The protooncogene product was detected in only 1 of 32 viral and nonviral liver disorders unrelated to hepatitis delta virus. To confirm these observations, we transfected HBsAg-positive (PCL/PRF/5) and HBsAg-negative (HepG2) transformed liver cell lines with a plasmid containing a hepatitis delta virus cDNA trimer under the control of the SV40 early enhancer/promoter sequences. Whereas baseline c-myc expression was barely detectable in mock-transfected PLC/PRF/5 or HepG2 cells, strong c-myc nuclear fluorescence was observed when these same cells were transfected with the hepatitis D antigen expression vector. Similar results were obtained after infection of HeLa cells with a recombinant vaccinia virus expressing the hepatitis D antigen. Detection of c-myc mRNA sequences by means of in situ hybridization suggested that the c-myc product accumulation was not due to increased amounts of its mRNA. The c-myc protein accumulates selectively in the livers of patients with chronic hepatitis delta virus infection and in the same nuclei that contain the hepatitis D antigen. The expression of c-myc in hepatitis D antigen-containing cells does not require the presence of hepatitis B virus infection.

Amino Acid Sequence↗
PubMed1994

Virology of the hepatitis A epidemic in Italy.

We studied the virologic aspects of a hepatitis A epidemic that occurred among hemophilia patients in Italy between 1989 and 1992. Twelve lots of factor VIII concentrate manufactured by the solvent-detergent chromatographic technique and suspected of contamination by the hepatitis A virus (HAV) were analyzed by a two-step, nested polymerase chain reaction (PCR) procedure. PCR was applied to 1-ml samples of factor VIII concentrate and 100-microliters serial serum samples available from 2 patients. Particular care was taken to rule out the possibility of false-positive results during analysis. Results demonstrated PCR amplification of the 3'-region of the VP3 gene in 5 of the 12 implicated lots of factor VIII and in the serial serum samples of both patients. PCR amplification also revealed that the gene sequences detected in patients' sera were identical to the sequences detected in the product they had received. In all, 3 VP3 sequences (found to be 96-99% identical) were amplified. Further characterization of the HAV found in the factor VIII concentrate and the patients' sera was attempted by PCR amplification of the VP1/2A region. Successful amplification of this region was achieved in the serum of only 1 patient and in the concentrate he received. This fourth amplified sequence was identical in both serum and factor VIII concentrate. Attempts to transmit hepatitis A from the contaminated lots to 3 chimpanzees resulted in no signs of infection after 10 months of observation. Based on the Italian experience, persons with severe hemophilia who receive large-pool concentrate are at potential risk for HAV infection and should be vaccinated against HAV or use an alternative to solvent-detergent-prepared concentrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma.

Although hepadnaviruses are implicated in the etiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection of mutant woodchuck hepatitis virus (WHV) genomes cloned from virions in serum that are strikingly similar to the rearranged hepadnaviral genomes found previously as integrated sequences in cellular DNA. Of 102 cloned genomes studied, 2 had large inverted duplications, 1 a 219-nucleotide direct duplication, and 1 a 219-nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at preferred topoisomerase I cleavage sites. Since these sites are located in the single-stranded regions of the genome, cleavage by topoisomerase I would produce linear molecules that would be expected to be highly recombinogenic since this enzyme, possessing nicking and ligating activities, would remain covalently attached. Sucrose density gradient centrifugation coupled with polymerase chain reaction studies confirmed that the mutant WHV DNA forms resided in virions and did not represent free viral DNA released from infected cells or were unlikely to be an artifact of the cloning process. Thus, the finding in virions of mutant WHV DNA similar to WHV DNA integrated into cellular DNA suggests that the processes of mutation and integration are linked in some instances. Furthermore, the mutant genomes that are preferentially integrated into cellular DNA may have an etiologic role in hepatocarcinogenesis.

Animals↗
PubMed1993

At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.

In a previous study we sequenced the 5' noncoding (NC) region of 44 isolates of hepatitis C virus (HCV) and identified heterogeneous domains that provided evidence for additional genetic groups of HCV not previously recognized. In this study we have determined the complete nucleotide sequence of the putative envelope 1 (E1) gene in 51 HCV isolates from around the world and found that they could be grouped into at least 12 distinct genotypes. The E1 gene sequence of 8 of these genotypes has not been reported previously. Although the genetic relatedness of HCV isolates determined by the previous analysis of the 5' NC region predicted the relationships observed in the E1 gene, analysis of the 5' NC sequence alone did not accurately predict all HCV genotypes. The nucleotide and amino acid sequence identities of the E1 gene among HCV isolates of the same genotype were in the range of 88.0-99.1% and 89.1-98.4%, respectively, whereas those of HCV isolates of different genotypes were in the range of 53.5-78.6% and 49.0-82.8%, respectively. The latter differences are similar to those found when comparing the envelope gene sequences of the various serotypes of the related flaviviruses as well as other RNA viruses. We found that some genotypes of HCV were widely distributed around the world, whereas others were identified only in discreet geographical regions. Four genotypes were identified exclusively in Africa and comprised the majority of HCV isolates on that continent. The E1 gene was exactly 576 nucleotides in length in all 51 HCV isolates with no in-frame stop codons. Analysis of the predicted E1 protein identified several conserved domains that may be important for maintaining its biological function: (i) eight invariant cysteine residues, (ii) three potential N-linked glycosylation sites, (iii) a domain of nine amino acids (GHRMAWDMM), and (iv) an amino acid doublet (GV) near the putative cleavage site at the C terminus of the protein. In conclusion, the discovery of at least 12 genotypes of HCV has important implications for HCV diagnosis and vaccine development.

Amino Acid Sequence↗
PubMed1993

Correlation between the infectivity of hepatitis C virus in vivo and its infectivity in vitro.

A murine retrovirus-infected human T-cell line, HPB-Ma, supported replication of hepatitis C virus (HCV) at least as well as the previously reported Molt4-Ma cells. Cloning of HPB-Ma cells revealed a clonal variation of cellular susceptibility to HCV infection. Using one of the sensitive clones, we tested HCV inocula from different sources for their infectivity titer in cell culture. The in vitro titers obtained correlated with the reported infectivity titers of the inocula in chimpanzees. Thus, the system appears to be useful for estimating the in vivo infectivity of HCV.

Adsorption↗
PubMed1993

Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen.

The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 10(3.5) chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B approximately 1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant of HBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.

Amino Acid Sequence↗
PubMed1993

Hepatitis C virus RNA in southern African blacks with hepatocellular carcinoma.

We used a sensitive and specific cDNA polymerase chain reaction assay to detect hepatitis C virus (HCV) RNA in serum samples from 128 unselected southern African Blacks with hepatocellular carcinoma (HCC) and found HCV RNA in 26 (20.3%) of these patients. Antibodies to HCV (anti-HCV) were detected in 59 patients (46.1%) with a first-generation ELISA test, and only 19 of these patients were HCV RNA-positive. Anti-HCV was detected in 25 patients (19.5%) with a second-generation ELISA test, and 17 of these patients were HCV RNA-positive. Among the second-generation ELISA- and HCV RNA-positive patients, 14 were anti-HCV positive, 2 were indeterminate, and 1 was anti-HCV negative in a second-generation recombinant immunoblot assay, whereas all patients who were second-generation ELISA positive, but HCV RNA negative, were anti-HCV negative in this immunoblot assay. A total of 5 patients were negative in both ELISA tests but were HCV RNA positive. Seventy-one patients (55.5%) had evidence of a current HBV infection, 50 (39.1%) had evidence of a previous infection, and 7 (5.5%) had no evidence of a current or previous HBV infection. The prevalence of current HBV infection was significantly lower in patients who were positive for HCV RNA than in those who were negative (P = 0.001). This difference was not dependent on sex, age, or geographical location of the patients. The mean age of HCC patients positive for HCV RNA (52.3 years) was significantly higher (P < 0.001) than that of negative patients (40.3 years), and the difference was not dependent on HBV status or geographical location. Patients positive for HCV RNA were more likely to be urban than were negative patients. Thus, a significant number of southern African Blacks with HCC had a current HCV infection but not a current HBV infection, further suggesting that infection with HCV plays a role, albeit minor, in the development of HCC in this population.

Adult↗
PubMed1993

Partial characterization of hepatitis A viruses from three intermediate passage levels of a series resulting in adaptation to growth in cell culture and attenuation of virulence.

Viruses from passages 9/10, 21, and 32 of a serially passaged human isolate of hepatitis A virus, strain HM-175, were partially sequenced and compared for their abilities to grow in cell cultures and to cause disease in primates. Viruses from all passages grew more efficiently in cell culture than did the wild-type virulent virus from which they were derived, and all displayed some degree of attenuation of virulence for primates. Within the 5' noncoding region and the 2B2C region of the HAV genome, passage 9/10 virus differed in sequence from wild-type at a single and novel position in the 2C gene, while the sequence of the passage 32 virus was almost identical to that of a fully attenuated passage 35 virus. Passage 21 viruses were found to consist of a mixture of viruses which included all but two of the 13 mutations present in the sequenced regions of the virus from passage 32.

Animals↗
PubMed1993

Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable.

Partial genomic sequences representing 420 nucleotides of a nonstructional region, 480 nucleotides of the putative RNA polymerase region, and 540 nucleotides of the structural region of epidemic-associated Chinese strains of hepatitis E virus (HEV) were obtained by direct sequencing of PCR-amplified DNA. Comparison with previously published HEV sequences showed a clear relatedness of all Chinese strains to each other and to a Pakistani strain (Sar-55). All eight Chinese strains examined had very similar sequences (98.5-99.8% homology) in the regions examined and were much closer to the Pakistani strain (Sar-55) (97.9-98.4% homology) than to the Burmese strain (92.5-93.3% homology). Sequence comparisons of the three genomic regions in the Chinese strains indicated that the RNA polymerase region was much more conserved than the other nonstructural region or the structural region. HEV isolates from three remote geographic regions of China had sequences closely related to each other.

Amino Acid Sequence↗
PubMed1993

The complete nucleotide sequence of a pre-core mutant of hepatitis B virus implicated in fulminant hepatitis and its biological characterization in chimpanzees.

Hepatitis B virus (HBV) with an in-frame stop codon within the pre-core region of the virus genome caused fulminant hepatitis in two individuals. Serum from a chronically infected patient who was the source of the virus was inoculated into three chimpanzees at dilutions of 10(-1), 10(-3), and 10(-7). All three chimpanzees developed acute hepatitis B with relatively high peak values of liver enzymes in their serum. The complete nucleotide sequence of virus DNA recovered from the chimpanzee serum by enzymatic amplification was identical with that from the human serum. By comparing the sequence of this strain (HT) with that of 32 published HBV genomes, changes in nucleotides and predicted amino acids that were rarely or never found in other HBV isolates were identified. Thirteen such nucleotides were found within the cis-acting regulatory elements, of which 6 were within the enhancer II-core promoter region. Twenty-four rare or unique changes in amino acids were found in open reading frames, of which 15 occurred in the region that spanned the 3' half of the X gene, through the pre-core/core gene, to the 5' end of the polymerase gene. Thus, an HBV pre-core stop mutant implicated in fulminant hepatitis is highly infectious, induces severe hepatitis in chimpanzees, and possesses significant genetic variation from reported HBV isolates.

Animals↗
PubMed1993

2B and 2C mutations are essential but mutations throughout the genome of HAV contribute to adaptation to cell culture.

Chimeric viruses constructed from various portions of two infectious cDNA clones representing the genomes of the wild-type and cell culture-adapted mutants of the HM-175 strain of hepatitis A virus were compared for their ability to replicate in cultures of fetal rhesus kidney cells. Mutations located in either the 5' or 3' third of the genome could markedly enhance growth in vitro but only when they were combined with mutations in the P2 region within either the 2B or the 2C gene. Therefore, mutations in 2B and 2C are essential for cell culture adaptation but mutations elsewhere in the genome also contribute significantly to the enhanced growth rate.

Adaptation, Biological↗
PubMed1993

Importance of the polymerase chain reaction in the study of hepatitis C virus infection.

Recently, the principal etiological agent of parenterally transmitted non-A, non-B hepatitis was molecularly cloned from the plasma of an experimentally infected chimpanzee and has been named hepatitis C virus. Determination of the complete nucleotide sequence of the hepatitis C virus genome was a crucial step in preparing the way for future study of this medically important human pathogen. Due to the very low concentration of virus in serum, amplification of viral RNA sequences by reverse transcription and polymerase chain reaction is the only practical method currently available for demonstrating viremia in patients with hepatitis C virus infection. This review examines the pivotal role of the polymerase chain reaction in understanding the biology of hepatitis C virus.

Acute Disease↗
PubMed1993

Heterogeneity of the woodchuck hepatitis virus genome in a chronically infected woodchuck.

The nucleotide sequence of an isolate of woodchuck hepatitis virus (WHV) from the serum of a woodchuck trapped in New York state (WHVNY) was compared with the sequences of previously published isolates. The nucleotide sequence of WHVNY was closest to that of an isolate originating from New Jersey: the two genomes shared a 15 nucleotide in-frame deletion in the region where the presurface and polymerase genes overlap (nucleotides 3260-3274) and differed by 54 point mutations (1.6% of genome). Amino acid differences ranged from 0.4% in the surface gene to 5.7% in the X gene. Three isolates from woodchucks that originated in Pennsylvania and Maryland did not contain the deletion and differed from WHVNY by 102 to 106 point mutations (3.0% to 3.2% of nucleotides). Amino acid changes ranged from 0.5% in the core gene to 5.7% in the X-gene. Thus, WHVNY differed little from previous isolates. Next, the genomes from 102 independent clones of WHVNY were compared to ascertain the extent of sequence variation among WHV genomes in a chronically infected animal. A total of 98 clones had genomes of unit length while 2 clones had genomes shorter than unit length and 2 clones had genomes longer than unit length. The clones not of unit length possessed deletions or inverted duplications of sequence. The rate of mutation in the viral genes was 2.65 mutations per 10,000 nucleotides in the precore domain, 1.27 per 10,000 in the X-gene, 0.98 per 10,000 in the presurface gene, and 3.77 per 10,000 at the 5' end of the core gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Control of hepatitis A by inactivated vaccines: unanswered questions and unresolved issues.

The recent development of safe and effective inactivated hepatitis A virus vaccines provides for the first time the opportunity to control hepatitis A. However, control will depend upon the intelligent use of the vaccines. How and when hepatitis A vaccines should be used will be determined from the results of studies of the levels and duration of protective antibody, determination of optimum immunization schedules, estimations of the continuing need for normal immune globulin and discussions of how hepatitis A vaccine can be incorporated into national and international vaccination programs. Cost and feasibility will determine whether live attenuated hepatitis A vaccines will become useful adjuncts or replacements for inactivated hepatitis A vaccines.

Hepatitis A↗
PubMed1993

Variation in course of hepatitis E in experimentally infected cynomolgus monkeys.

Five cynomolgus monkeys (Macaca fascicularis) developed hepatitis after inoculation with a prototype strain of hepatitis E virus (HEV) from Pakistan. Although all 5 monkeys displayed liver enzyme elevations, viremia, virus secretion in feces, and seroconversion, two different patterns of these parameters were observed. For 4 monkeys, increased alanine aminotransferase (ALT) activity was first observed on days 21-26, viremia occurred before and during enzyme elevation, and the animals seroconverted coincidentally with the end of viremia or shortly thereafter. One of these monkeys had a more severe hepatitis, with peak ALT values more than twice the peak levels of the other monkeys. The fifth monkey developed biphasic hepatitis with peaks of ALT activity on days 26 and 54. In this case, viremia and seroconversion were correlated only with the second peak of enzyme elevation and liver histopathology only with the first peak. Viral shedding in this fifth animal lasted two times longer than in other animals.

Alanine Transaminase↗