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Biomedical subjects

R H Purcell

Publications and source records attributed to R H Purcell.

At least 73 records · Page 4Linked to original sources
PubMed1998

Experimental infection of chimpanzees with hepatitis G virus and genetic analysis of the virus.

Hepatitis G virus (HGV) was transmitted to 2 chimpanzees by inoculation with human plasma containing approximately 10(8) genome equivalents (GE) of HGV. The infection was characterized by the late appearance (weeks 10 and 11 after inoculation [pi]) of viremia that persisted throughout the 120-week follow-up. Serum HGV titer increased steadily until it plateaued at 10(6)-10(7) GE/mL. However, despite this relatively high titer, neither of the chimpanzees developed hepatitis. The sequence of the viral genome, recovered from each chimpanzee at week 77 pi, differed from that of the inoculum by 5 nt (2 aa) and 27 nt (2 aa). Two more chimpanzees were inoculated with a first-passage plasma pool. The chimpanzee inoculated with approximately 10(6.7) GE of HGV had viremia at week 1 pi. However, the viral titer increased with the same kinetics as observed in the first passage. The second chimpanzee inoculated with approximately 10(4.7) GE of HGV had late appearance (week 7 pi) of viremia.

Amino Acid Substitution↗
PubMed1998

Infection of a chimpanzee with hepatitis C virus grown in cell culture.

Culture supernatant harvested from Daudi cells, a lymphoplastoid cell line, after 58 days of infection with the H77 strain of hepatitis C virus (HCV), was inoculated into a chimpanzee. HCV RNA, as detected by RT-PCR, first appeared in the serum and liver 5 and 6 weeks, respectively, after inoculation. Peripheral blood mononuclear cells (PBMC) collected on week 7 were also positive for HCV RNA. The major sequences of hypervariable region 1 (HVR1) of the viral genome recovered from the inoculated chimpanzee were the ones which were the majority in the original H77 inoculum and not those which were in the majority in the culture supernatant. Only the sequence recovered from PBMC was the same as the major one found in the cell culture.

Animals↗
PubMed1998

Characterization of a strain of infectious hepatitis E virus isolated from sewage in an area where hepatitis E is not endemic.

Raw sewage samples from an area where hepatitis E is not endemic (Barcelona, Spain) were analyzed by reverse transcriptase-PCR followed by nested PCR. One of the 37 tested samples showed a positive result for hepatitis E virus (HEV). The detected strain was amplified by inoculation into rhesus monkeys, and the course of the infection was studied by analyzing serological and biochemical parameters and by monitoring the presence of HEV in serum and feces. Fecal suspensions from the rhesus monkeys were used as the source of viral particles for sequence analysis. Eighty percent of the genome of the isolated strain, named BCN, was sequenced and found to be phylogenetically related to Asian (Indian) strains, with a 98% nucleotide identity with an isolate from Madras, India. Since this was a single isolation we cannot conclude that HEV is regularly present in the sewage. However, the finding of viable HEV in sewage has implications for contamination of the environment and shellfish by HEV and must be considered in the diagnosis of viral hepatitis in regions of nonendemic hepatitis.

Animals↗
PubMed1998

Genetic and experimental evidence for cross-species infection by swine hepatitis E virus.

Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared >/=97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses.

Animals↗
PubMed1998

Utilization of chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus to study the molecular basis of virulence.

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5' or 3' half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3' half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.

Animals↗
PubMed1998

Comparison of tests for antibody to hepatitis E virus.

The results of serologic tests for hepatitis E virus have varied widely from laboratory to laboratory, making interpretation of seroepidemiologic studies difficult. The present study compares serologic results with different antigens and tests developed in two laboratories for their ability to diagnose hepatitis E and measure antibody prevalence in a high risk population in Saudi Arabia. The results confirm that tests based upon open reading frame (ORF) 3 of HEV are of limited value for seroepidemiologic studies, whereas ORF2-based antigens have broad utility and yield data that are reproducible in more than one laboratory.

Adolescent↗
PubMed1997

How Escherichia coli can bias the results of molecular cloning: preferential selection of defective genomes of hepatitis C virus during the cloning procedure.

Cloned PCR products containing hepatitis C virus (HCV) genomic fragments have been used for analyses of HCV genomic heterogeneity and protein expression. These studies assume that the clones derived are representative of the entire virus population and that subsets are not inadvertently selected. The aim of the present study was to express HCV structural proteins. However, we found that there was a strong cloning selection for defective genomes and that most clones generated initially were incapable of expressing the HCV proteins. The HCV structural region (C-E1-E2-p7) was directly amplified by long reverse transcription-PCR from the plasma of an HCV-infected patient or from a control plasmid containing a viable full-length cDNA of HCV derived from the same patient but cloned in a different vector. The PCR products were cloned into a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins. Twenty randomly picked clones derived from the HCV-infected patient all contained nucleotide mutations leading to absence or truncation of the expected HCV products. Of 25 clones derived from the control plasmid, only 8% were fully functional for polyprotein synthesis. The insertion of extra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number of fully functional clones derived from the patient (42%) and from the control plasmid (72-92%). Nonrandom selection of clones during the cloning procedure has enormous implications for the study of viral heterogeneity, because it can produce a false spectrum of genomic diversity. It can also be an impediment to the construction of infectious viral clones.

Animals↗
PubMed1997

A novel virus in swine is closely related to the human hepatitis E virus.

A novel virus, designated swine hepatitis E virus (swine HEV), was identified in pigs. Swine HEV crossreacts with antibody to the human HEV capsid antigen. Swine HEV is a ubiquitous agent and the majority of swine >/=3 months of age in herds from the midwestern United States were seropositive. Young pigs naturally infected by swine HEV were clinically normal but had microscopic evidence of hepatitis, and developed viremia prior to seroconversion. The entire ORFs 2 and 3 were amplified by reverse transcription-PCR from sera of naturally infected pigs. The putative capsid gene (ORF2) of swine HEV shared about 79-80% sequence identity at the nucleotide level and 90-92% identity at the amino acid level with human HEV strains. The small ORF3 of swine HEV had 83-85% nucleotide sequence identity and 77-82% amino acid identity with human HEV strains. Phylogenetic analyses showed that swine HEV is closely related to, but distinct from, human HEV strains. The discovery of swine HEV not only has implications for HEV vaccine development, diagnosis, and biology, but also raises a potential public health concern for zoonosis or xenozoonosis following xenotransplantation with pig organs.

Amino Acid Sequence↗
PubMed1997

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5' and 3' termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase-PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6-28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Animals↗
PubMed1997

Replication and immunogenicity of Ad7-, Ad4-, and Ad5-hepatitis B virus surface antigen recombinants, with or without a portion of E3 region, in chimpanzees.

Human adenovirus vectors containing intact or largely deleted E3 region were used to construct adenovirus-hepatitis B recombinant viruses (Ad-HepB) and shown to produce substantial amount of recombinant protein, hepatitis B surface antigen (HBsAg), in tissue culture. Previously we showed that these viruses were able to elicit good anti-HBs antibodies in a dog model. In the present study, the Ad-HepB viruses were evaluated for replication and immunogenicity in chimpanzees which sustain permissive infection by human adenoviruses. Recombinants containing entire E3 region showed better replication pattern than their E3 deleted counterparts as evidenced by longer duration and high titers of virus shedding. The effect of E3 region was also seen in the antibody titers against HBsAg in that the E3 containing viruses showed better response than the E3 deleted viruses. The importance of E3 region for the development of adenovirus vectored vaccines is further discussed.

Adenovirus E3 Proteins↗
PubMed1997

Recombinant vaccine against hepatitis E: dose response and protection against heterologous challenge.

Thirty-two rhesus monkeys were used to evaluate the dose response of a recombinant HEV vaccine, and the efficacy of the vaccine based on the ORF2 protein of the Pakistani strain for pre- and post-exposure vaccination against intravenous challenge with homologous or heterologous virus was examined. Post-exposure vaccination did not protect animals against hepatitis. Although primates vaccinated twice with 50-microgram, 10-microgram, 2-microgram, or 0.4-microgram doses of the recombinant 55 kDa ORF-2 protein were infected, they were protected from hepatitis when they were challenged with very high doses of the homologous strain of HEV. Primates vaccinated twice with a 50 micrograms dose of the recombinant protein were protected from hepatitis after heterologous challenge with the Mexican strain, the strain of HEV most genetically distant from the Pakistani strain.

Animals↗
PubMed1997

In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

3C Viral Proteases↗
PubMed1997

Hepatitis A among homosexual men and injection drug users: more evidence for vaccination.

As agencies develop guidelines for administering the newly developed hepatitis A virus (HAV) vaccine, information is needed regarding the occurrence of HAV infection in groups putatively at risk for the infection. We tested serum samples from 300 injection drug users (IDUs), 300 homosexual males, and 300 blood donors for the presence of total antibody to HAV (anti-HAV). Anti-HAV was detected in 66% of IDUs, 32% of homosexual males, and 14% of blood donors. Anti-HAV was not significantly associated (P > .10) with high-risk drug-using behaviors but was more prevalent among IDUs with annual incomes of <$5,000 (P = .018). The occurrence of anti-HAV increased among homosexual males as the number of sexual partners increased (P < .001) but was similar to the age-adjusted prevalence (30.6%) estimated for the general United States population. IDUs are at increased risk for HAV infection; however, our data suggest that factors related to low socioeconomic status contribute more to the occurrence of HAV infection among IDUs than does injection drug use. IDUs and persons at risk for injection drug use should receive HAV vaccine.

Baltimore↗
PubMed1997

Detection of antibodies to the nonstructural 3C proteinase of hepatitis A virus.

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and nonstructural proteins of the virus. However, vaccination with an inactivated vaccine produces antibodies exclusively to the structural proteins. Current diagnostic assays, such as the Abbott HAVAB test, used to determine exposure to HAV detect antibodies only to the structural proteins and as a result are not able to distinguish between a natural infection and vaccination with an inactivated virus. Therefore, an ELISA was developed that is specific for antibodies to the nonstructural protein 3C of HAV and thus serves to document the occurrence of viral replication. Antibodies to the proteinase were not detected by this assay in serum from HAVAB-seropositive primates that were immunized with inactivated HAV. However, antibodies to the proteinase were detected in the serum of all primates experimentally infected with virulent HAV and in the serum of naturally infected humans.

3C Viral Proteases↗
PubMed1997

Infectivity and pathogenicity in chimpanzees of a surface gene mutant of hepatitis B virus that emerged in a vaccinated infant.

Hepatitis B virus (HBV) variants with amino acid mutations in the a epitope of the major surface protein have been identified, and questions have been raised regarding their biologic properties. Dilutions of serum that contained the first such described HBV mutant, with an Arg-for-Gly substitution at codon 145 of the S gene, were inoculated into 6 seronegative chimpanzees. Five of the animals developed serologic and/or biochemical evidence of hepatitis B. A polymerase chain reaction-based assay that discriminated between the wild type and mutant viral genomes revealed that a pure population of the mutant genome was present in the 10(-6) and 10(-7) dilutions of the index serum, resulting in infection of the chimpanzees receiving these dilutions only with the mutant virus. A clone of the mutant virus replicated normally following transfection in vitro. Thus, this HBV surface gene mutant is viable, infectious, and pathogenic in chimpanzees.

Animals↗
PubMed1997

Evaluation of commercially available and in-house reverse transcription-PCR assays for detection of hepatitis G virus or GB virus C.

Serum samples from 96 Spanish hemodialysis patients, as well as serial dilutions of RNA extracted from a reference strain of hepatitis G virus (HGV), were tested for HGV or GB virus C (GBV-C) RNA. Two different reverse transcription (RT)-PCR-based methods of detection were compared for the ability to detect RNA extracted from the samples: an RT-nested PCR assay with primers derived from the 5' noncoding region (5'NC) or nonstructural region 3 (NS3) sequences and a commercially available RT-PCR assay with primers derived from the 5'NC or NS5A sequences. When RT-nested PCR was performed on 10-fold serial dilutions of RNA from the HGV reference strain, the last positive dilution was 10(-7) to 10(-8). With the commercial RT-PCR assay, the last positive dilution was 10(-6) to 10(-7). When equal amounts of RNA extracted from serum samples from 96 hemodialysis patients were tested for HGV or GBV-C RNA, 25 patients (26%) were positive by the RT-nested PCR. However, only 21 (84%) of these 25 positive patients were positive for HGV or GBV-C by the commercial RT-PCR assay. Analysis of the 5'NC and NS3 sequences amplified by RT-nested PCR demonstrated that all but two positive patients had unique HGV or GBV-C sequences. In summary, RT-nested PCR and a commercially available RT-PCR assay for HGV or GBV-C gave concordant results for 96% of the patients tested.

Base Sequence↗
PubMed1997

Seroreactivity to hepatitis E virus in areas where the disease is not endemic.

If the occurrence of hepatitis E virus antibody (anti-HEV) in regions where the disease is not endemic represents infection, rates may be greater in high-risk populations and behavioral correlates may reflect recognized transmission modes. Serum samples from 300 homosexual males, 300 injection drug users (IDUs), and 300 blood donors from Baltimore, Md., were tested for anti-HEV by enzyme immunoassay. Anti-HEV was found in an unexpectedly high percentage of homosexual men (15.9%) and IDUs (23.0%). However, anti-HEV was present in a similar proportion of blood donors (21.3%) (P > 0.05), while hepatitis A, B, and C virus antibodies were more prevalent in the high-risk groups (P < 0.001). Among homosexual men, anti-HEV was not significantly correlated with a history of hepatitis, high-risk sexual practices, or sexually transmitted infections, in contrast to hepatitis A and B antibodies. Among IDUs, anti-HEV was not significantly associated with a history of hepatitis or high-risk drug-using practices, as was found with hepatitis C antibodies. In a setting without endemic hepatitis E disease, there was no evidence that anti-HEV reflected subclinical infection. Until the basis for HEV seroreactivity in such areas is elucidated, anti-HEV results should be interpreted with caution.

Hepatitis E↗