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Biomedical subjects

R H Purcell

Publications and source records attributed to R H Purcell.

At least 145 records · Page 8Linked to original sources
PubMed1993

ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates.

A recombinant baculovirus containing the complete open-reading frame (ORF)-2 region of the hepatitis E virus (HEV) genome was constructed. The major protein synthesized in insect cells infected with recombinant virus was about the size expected for the complete ORF-2 product. This protein reacted in a Western blot assay with plasma from an HEV-infected chimpanzee. Lysates of the recombinant virus-infected insect cells were used in ELISA to monitor seroconversion of eight primate species (chimpanzees, four species of Old World monkeys, and three species of New World monkeys) inoculated with HEV. Homologous detector anti-immunoglobulin was more sensitive than heterologous anti-immunoglobulin for detecting anti-HEV by ELISA. All primate species except tamarins seroconverted after inoculation with HEV, although anti-HEV titers of Old World monkey species were generally higher than those of New World monkey species. The ELISA with complete ORF-2 antigen appeared to be a sensitive and practical method for detecting anti-HEV.

Animals↗
PubMed1993

High prevalence of hepatitis C virus (HCV) RNA in dialysis patients: failure of commercially available antibody tests to identify a significant number of patients with HCV infection. Copenhagen Dialysis HCV Study Group.

Results of serologic tests were correlated with hepatitis C virus (HCV) viremia, determined by a cDNA polymerase chain reaction assay to detect HCV RNA, in 340 Danish dialysis patients; of these, 28 (8.2%) were positive for antibodies to HCV (anti-HCV) with second-generation ELI-SAs. HCV RNA was found in sera from 27 of these 28 anti-HCV-positive patients. However, 8 dialysis patients had detectable levels of HCV RNA but were anti-HCV-negative with second-generation ELISAs. Among the 35 HCV-infected dialysis patients 16 were positive, 7 indeterminate, and 12 negative with the second-generation RIBA. More than 60% of patients with evidence of ongoing liver disease had HCV infection. Thus, current commercially available antibody tests did not accurately reflect the HCV status in dialysis patients. A relatively high prevalence (> 10%) of HCV RNA, closely associated with liver disease, was found among dialysis patients in a low-prevalence area of the world.

Base Sequence↗
PubMed1993

The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks.

All mammalian hepadnaviruses possess a gene, termed X, that encodes a protein capable of transactivating virus gene expression. The X gene overlaps the polymerase and precore genes as well as two newly identified open reading frames (ORFs) termed ORF5 and ORF6. In this investigation, we examined whether ORF5, ORF6, and the X gene were important for the replication of woodchuck hepatitis virus (WHV) in susceptible woodchucks. First, we investigated whether proteins were produced from ORF5 and ORF6 by in vitro translation of appropriate viral transcripts, searched for antibodies against the putative proteins in the sera of animals infected with wild-type virus, and looked for an antisense WHV transcript, necessary for expression of a protein from ORF6, in the livers of acutely or chronically infected woodchucks. All such experiments yielded negative results. Next, we used oligonucleotide-directed mutagenesis to introduce termination codons into ORF5 and ORF6 at two locations within each ORF. Adult woodchucks in groups of three were transfected with one of the four mutant genomes. All of these woodchucks developed WHV infections that were indistinguishable from those of animals transfected with the wild-type WHV recombinant. Polymerase chain reaction amplification and direct DNA sequencing confirmed that reversion of the mutants to a wild-type genotype did not occur. Taken together, these data indicate that ORF5 and ORF6 are not essential for virus replication and are unlikely to represent authentic genes. Finally, we generated five WHV X-gene mutants that either removed the initiation codon for protein synthesis or truncated the carboxyl terminus of the protein by 3, 16, 31, or 52 amino acids. Groups of three adult woodchucks were transfected with one of the five X-gene mutants. Only the mutant that possessed an X gene lacking 3 amino acids from the carboxyl terminus was capable of replication within the 6-month time frame of the experiment. In contrast, all seven woodchucks transfected with wild-type WHV DNA developed markers consistent with viral infection. Thus, it is likely (P < 0.01) that the WHV X gene is important for virus replication in the natural host.

Amino Acid Sequence↗
PubMed1993

Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.

Animals↗
PubMed1992

Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line.

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis C virus (HCV) infection. The cell cultures were inoculated with a serum containing HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigens showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.

Animals↗
PubMed1992

Sequence analysis of the 5' noncoding region of hepatitis C virus.

We have determined the nucleotide sequence of the 5' noncoding (NC) region of the hepatitis C virus (HCV) genome in 44 isolates from around the world. We have identified several HCV isolates with significantly greater sequence heterogeneity than reported previously within the 5' NC region. The most distantly related isolates were only 90.1% identical. Nucleotide insertions were seen in three isolates. Analysis of the nucleotide sequence from 44 HCV isolates in this study combined with that of 37 isolates reported in the literature reveals that the 5' NC region of HCV consists of highly conserved domains interspersed with variable domains. The consensus sequence was identical to the prototype HCV sequence. Nucleotide variations were found in 45 (16%) of the 282 nucleotide positions analyzed and were primarily located in three domains of significant heterogeneity (positions -239 to -222, -167 to -118, and -100 to -72). Conversely, there were three highly conserved domains consisting of 18, 22, and 63 completely invariant nucleotides (positions -263 to -246, -199 to -178, and -65 to -3, respectively). Two nucleotide domains within the 5' NC region, conserved among all HCV isolates studied to date, shared statistically significant similarity with pestivirus 5' NC sequences, providing further evidence for a close evolutionary relationship between these two groups of viruses. Additional analysis revealed the presence of short open reading frames in all HCV isolates. Our sequence analysis of the 5' NC region of the HCV genome provides additional information about conserved elements within this region and suggests a possible functional role for the region in viral replication or gene expression. These data also have implications for selection of optimal primer sequences for the detection of HCV RNA by the PCR assay.

Base Sequence↗
PubMed1992

Hepatitis B and hepatitis C in emergency department patients.

BACKGROUND: Infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and the human immunodeficiency virus type 1 (HIV-1) are common in inner-city populations, but their frequency and interrelations are not well established. METHODS: During a six-week period, excess serum samples were collected, along with information on risk factors, from all adult patients presenting to an inner-city emergency department. The samples were assayed for hepatitis B surface antigen (HBsAg) and antibodies to HCV and HIV-1. RESULTS: Of the 2523 patients tested, 612 (24 percent) were infected with at least one of the three viruses. Five percent were seropositive for HBV, 18 percent for HCV, and 6 percent for HIV-1. HCV was found in 145 of the 175 intravenous drug users (83 percent), 36 of the 171 transfusion recipients (21 percent), and 5 of the 24 homosexual men (21 percent). Among black men 35 to 44 years of age, the seroprevalence of HCV was 51 percent. HBsAg was present in 9 percent of those whose only identifiable risk was possible heterosexual exposure. At least one viral marker was found in about 30 percent of the patients who were actively bleeding or in whom procedures were performed. Testing for HIV-1 alone would have failed to identify 87 percent of the patients infected with HBV and 80 percent of those infected with HCV. CONCLUSIONS: In a population of patients in an inner-city emergency room, HBV, HCV, and HIV-1 are all highly prevalent. However, routine screening for HIV-1 alone would identify only a small fraction of the patients who pose risks of severe viral infections, including HBV and HCV, to providers.

Adolescent↗
PubMed1992

Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.

Alanine Transaminase↗
PubMed1992

Characterization of a prototype strain of hepatitis E virus.

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia.

Amino Acid Sequence↗
PubMed1992

Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay.

We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.

Base Sequence↗
PubMed1992

Molecular basis of virulence and growth of hepatitis A virus in cell culture.

The ability of engineering variants of hepatitis A virus (strain HM175) to replicate in cell culture or to cause disease in marmosets was evaluated. Virus variants were encoded by chimeric genomes constructed from infectious cDNA clones of two viruses (wild type and cell-culture-adapted) which differed in their ability to grow in vitro and to cause acute hepatitis in marmosets. Transfection and infectivity assays indicated that virus growth in vitro could be enhanced by subcloning the cell substrate prior to infection or by introducing multiple combinations of two or more mutations into the wild type genome. Various chimeric viruses induced liver enzyme elevations in marmosets, indicating that attenuation of virulence also required multiple mutations.

Animals↗
PubMed1992

Clinical and laboratory observations following oral or intramuscular administration of a live attenuated hepatitis A vaccine candidate.

Clinical observations made after immunising volunteers with a live attenuated hepatitis A vaccine are described. The candidate vaccine was prepared with the HM175 strain of hepatitis A virus and shown to be safe, immunogenic and efficacious in experimental animals. When the candidate vaccine was tested by oral administration in humans at increasing doses--10(4), 10(5), 10(6) and 10(7) median tissue culture infective doses (TCID50)--an antibody response was not observed at any dose. Volunteers who received similar doses by the intramuscular route developed antibody to hepatitis A three weeks after immunization with 10(6) or 10(7) TCID50. The antibody response was sustained for the 12 weeks of the observation period. All volunteers remained healthy with normal results from liver tests throughout the monitoring period. Further clinical observations of this product are in progress.

Administration, Oral↗
PubMed1992

Inactivated hepatitis A vaccine: active and passive immunoprophylaxis in chimpanzees.

Studies of active and passive immunoprophylaxis were carried out in chimpanzees to determine whether a candidate hepatitis A virus (HAV) vaccine could stimulate antibody to HAV (anti-HAV) that was qualitatively similar to anti-HAV stimulated by natural infection. Normal immune globulin (Ig) was prepared from plasma obtained from human volunteers before and after vaccination with the HAV vaccine, and these preparations or commercially prepared Ig were administered to chimpanzees. Protective efficacy was compared to that obtained after vaccination of chimpanzees. As expected, pre-vaccination Ig did not protect chimpanzees against challenge with virulent hepatitis A. In contrast, chimpanzees were protected against hepatitis A by Ig prepared from volunteers who had received hepatitis A vaccine. The protection was qualitatively similar to that afforded by commercial normal Ig containing convalescent anti-HAV. The minimum protective dose of passively acquired anti-HAV was approximately the minimum dose detectable by serological means. This information will be useful in calculating minimum acceptable titres of anti-HAV in normal Ig. Whereas administration of Ig protected chimpanzees against hepatitis A pathology, it did not protect them from infection with HAV. Thus, these chimpanzees were protected by classical passive-active immunoprophylaxis. In contrast, chimpanzees actively immunized with HAV vaccine were apparently protected against both hepatitis A pathology and HAV infection. The mechanism of this complete protection is unknown but may simply represent the higher titre of anti-HAV in the vaccinated chimpanzees, compared to the passively protected animals.

Animals↗
PubMed1992

The natural history of infection with hepatitis C virus (HCV) in chimpanzees: comparison of serologic responses measured with first- and second-generation assays and relationship to HCV viremia.

The sensitivity of first- and second-generation tests for antibody to hepatitis C virus (HCV) and the relationship among the patterns of antibody response and HCV viremia were examined in serial serum samples from 6 chimpanzees experimentally infected with HCV and followed less than or equal to 3 years. HCV infection was transient in 4 chimpanzees and became chronic in 2. All chimpanzees developed antibodies to HCV detectable by second-generation assays, while only 5 of the 6 became positive by first-generation assay. Second-generation were consistently more sensitive than first-generation assays for the early diagnosis of primary HCV infection. The pattern observed with second-generation assays was not influenced by the outcome of HCV infection, since antibodies remained persistently detectable throughout follow-up regardless of whether viremia was transient or persistent. In contrast, the first-generation antibody response was variable: It usually disappeared after loss of viremia, whereas its presence paralleled HCV viremia in chimpanzees with chronic infection.

Animals↗
PubMed1992

cDNA clone of hepatitis A virus encoding a virulent virus: induction of viral hepatitis by direct nucleic acid transfection of marmosets.

Direct inoculation of marmoset livers with an in vitro transcription mixture containing cDNA and full-length genomic RNA transcripts of hepatitis A virus resulted in acute viral hepatitis. Elevations in serum levels of liver enzymes were correlated with appearance of antibody to hepatitis A virus. Genomes of infectious hepatitis A virus isolated from the feces of transfected marmosets contained the same mutation as the cDNA template used for transfection. Liver biopsies confirmed that the virus encoded by the cDNA clone induced histopathological changes equivalent to those caused by virulent wild-type virus.

Animals↗
PubMed1992

Mutations in both the 2B and 2C genes of hepatitis A virus are involved in adaptation to growth in cell culture.

Oligonucleotide-directed mutagenesis of an infectious cDNA clone of wild-type hepatitis A virus was performed to determine which mutations acquired in the nonstructural 2B and 2C genes during adaptation to growth in cell culture were effective in enhancing virus growth in vitro. Results of transfection assays demonstrated that one mutation in the 2B gene and two mutations in the 2C gene were responsible for an increased efficiency in growth, but growth enhancement required the participation of at least two of the three mutations.

Animals↗
PubMed1992

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.

Animals↗