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R H Purcell

Publications and source records attributed to R H Purcell.

At least 91 records · Page 5Linked to original sources
PubMed1997

Sequence analysis of the hepatitis C virus genome recovered from serum, liver, and peripheral blood mononuclear cells of infected chimpanzees.

Of 13 different strains of hepatitis C virus (HCV) in the inoculum used, only 1 persisted in human lymphocyte cell lines infected in vitro (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). To determine whether that particular strain (designated H1-2) has a tropism for lymphocytes in vivo, we sequenced hypervariable region 1 (HVR1) of the genome of HCV recovered from the sera, livers, and peripheral blood mononuclear cells (PBMC) of chimpanzees infected with plasma H77, the same inoculum used for the in vitro studies. In the PBMC collected from two chimpanzees during the early phase of infection, H1-2 was detected as the only or predominant HVR1 sequence. H1-2 was also detected in PBMC obtained during persistent infection from a chimpanzee that had been treated with immunosuppressants. From the livers of these chimpanzees, two to six different strains were recovered but H1-2 was not detected. Thus, H1-2 appeared to have an affinity for lymphocytes not only in vitro but also in vivo. In samples collected from a chimpanzee after 6 years of infection, however, such tissue compartmentalization of the HCV genome was not observed; a single strain became predominant in the serum, liver, and PBMC. An HCV strain capable of replicating in both the liver and PBMC probably emerged during in vivo replication and persisted.

Animals↗
PubMed1997

African strains of hepatitis E virus that are distinct from Asian strains.

Partial genomic sequences of four hepatitis E virus (HEV) strains from Africa (Morocco and Tunisia) and one from Central Asia (Tashkent, Uzbekistan) were obtained. The reverse transcriptase-polymerase chain reaction was used to amplify 5' and hypervariable regions of open reading frame 1 (ORF1) and a region overlapping all 3 ORFs. Sequence analysis of these regions revealed the African strains to be quite distinct from all known Asian strains but more similar to them than to the Mexican strain. Sequence analysis of the Tashkent strain revealed almost complete identity with another central Asian strain from Osh, Kirgizia. These results thus further confirm the geographical origin of HEV strain divergence.

Animals↗
PubMed1996

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Amino Acid Sequence↗
PubMed1996

A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures.

To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77. The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1. The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient. The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1.

Amino Acid Sequence↗
PubMed1996

DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines.

Vaccination of two chimpanzees against hepatitis B virus (HBV) by intramuscular injection of plasmid DNA encoding the major and middle HBV envelope proteins induced group-, subtype- and preS2-specific antibodies. These were initially of IgM isotype, and then they were of IgG (predominantly IgGl) isotype. The chimpanzee injected with 2 mg of DNA attained >100 milli-international units/ml of anti-HBs antibody after one injection and 14,000 milli-international units/ml after four injections. A smaller dose (400 microg) induced lower and transient titers, but a strong anamnestic response occurred 1 year later. Comparison with responses in 23 chimpanzees receiving various antigen-based HBV vaccines suggests that the DNA approach is promising for prophylactic immunization against HBV.

Animals↗
PubMed1996

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Animals↗
PubMed1996

Varicella in Chimpanzees.

Two chimpanzees were inoculated subcutaneously with the wild-type Oka strain of varicellazoster virus (VZV). Viral DNA was detected in peripheral blood mononuclear cells of both animals using the polymerase chain reaction (PCR) shortly after inoculation. Ten days after inoculation both animals developed an erythematous, papular rash near the site of inoculation that extended into the adjacent dermatome. Viral DNA was found by PCR in a skin biopsy from one of the animals at the time of the rash. While only two animals were studied, the development of a mild form of varicella in chimpanzees indicates that these animals might be useful for molecular studies of viral genes involved in virulence or attenuation of VZV.

Animals↗
PubMed1996

Hepatitis C virus: detection of intracellular virus particles by electron microscopy.

We previously demonstrated that a human T-cell line, HPBMa10-2 derived from HPBALL, was capable of supporting a productive infection of hepatitis C virus (HCV). We subsequently found Daudi cells, a human B-cell line, to be susceptible to HCV infection. Employing these cell lines infected with HCV as well as liver obtained during the acute phase of hepatitis C from a chimpanzee, we observed intracellular HCV particles by electron microscopy (EM). We detected viruslike particles with a diameter of approximately 50 nm in the cytoplasmic vesicles. In Daudi cells harvested 15 days after virus inoculation, the appearance of cytoplasmic tubular structures which we originally described for chimpanzee hepatocytes in association with HCV infection was noted. Immunoperoxidase EM using antibodies against HCV core and envelope demonstrated that the particles contained the viral antigens.

Animals↗
PubMed1996

A simian strain of hepatitis A virus, AGM-27, functions as an attenuated vaccine for chimpanzees.

The AGM-27 strain OF hepatitis A virus (HAV) was originally isolated from an African green monkey with hepatitis and appears to represent a true simian strain. The virus caused acute hepatitis after intravenous inoculation into African green monkeys, rhesus monkeys, and marmosets. Cynomolgus monkeys inoculated with the virus did not develop hepatitis, probably because of prior exposure to HAV. Chimpanzees inoculated with a high dose of the virus did not develop signs of hepatitis, although the virus replicated and the animals seroconverted. Marmosets and chimpanzees convalescent from infection with the AGM-27 strain of HAV were rechallenged with the virulent HM-175 strain of human HAV. They were partially or totally protected from disease.

Animals↗
PubMed1996

Contamination of commercially available fetal bovine sera with bovine viral diarrhea virus genomes: implications for the study of hepatitis C virus in cell cultures.

The establishment of cell cultures for hepatitis C virus (HCV) is important for its study as a human pathogen. However, in reported cell lines, HCV demonstrates low levels of replication detected primarily by reverse transcription-polymerase chain reaction (RT-PCR) assays. In attempts to culture HCV, an additional complication was observed. From mock-infected cultures, cDNA of appropriate size was obtained by RT-PCR with primers deduced from conserved domains of the 5' noncoding region of HCV. However, sequence analysis revealed that the cDNA was amplified from bovine viral diarrhea virus (BVDV). All of 7 bovine sera tested were contaminated with BVDV. In conclusion, most commercially available bovine sera are contaminated with BVDV and, although there is no evidence that the virus is infectious, bovine sera should be screened for this virus by RT-PCR when used in conjunction with HCV or for the development or production of vaccine.

Base Sequence↗
PubMed1996

Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes.

In this study we amplified virtually the entire genomes of hepatitis A virus (a member of the Picornaviridae family), hepatitis B virus (a member of the Hepadnaviridae family), and hepatitis C virus (a member of the Flaviviridae family) by using the recently described technique of long PCR. In order to do this, we first demonstrated, using the lambda phage, that long PCR can be made highly sensitive and the sensitivity can be further enhanced by nested long PCR. We also showed, using tobacco mosaic virus as a model, that a reverse transcriptase reaction can be linked to a long PCR, enabling the nearly full-length amplification of the genomes of RNA viruses. We then applied these techniques to serial dilutions of titrated stocks of well-characterized strains of hepatitis A, B, and C viruses. We amplified the nearly full-length sequence of each of these viruses from a small number of viral genomes, demonstrating the sensitivity of the process.

Bacteriophage lambda↗
PubMed1996

A hydrophobic heptad repeat of the core protein of woodchuck hepatitis virus is required for capsid assembly.

The capsid particle of hepadnaviruses is assembled from its dimer precursors. However, the mechanism of the protein-protein interaction is still poorly understood. A small region in the capsid protein of woodchuck hepatitis virus (WHV) contains four hydrophobic residues, including leucine 101, leucine 108, valine 115, and phenylalanine 122, that are conserved and spaced every seventh residue in the primary sequence to form a hydrophobic heptad repeat (hhr). A hydrophobic force often plays an important role in the interaction of proteins. Therefore, to investigate the role of this region in capsid assembly, we individually changed the codons specifying these four hydrophobic amino acids to codons specifying alanine or proline. In addition, we examined the in vivo infectivity of a WHV genome bearing a naturally occurring single amino acid change (histidine 104-->proline) in the hhr region. The phenotype of each altered genome was determined in both eukaryotic and prokaryotic systems by a capsid protein assay and electron microscopic examination. We show that replacement of any one of the four hydrophobic residues with alanine did not prevent capsid assembly. However, assembled capsid particles were not detected if combinations of any two of the four residues were substituted with alanines or if the spacing of these four hydrophobic residues was changed. An individual introduction of a proline (which dramatically changes the secondary structure of proteins) into different positions of this small region also abolished capsid assembly in vitro or viral replication in vivo. These results suggested that the hhr region of the core protein of WHV was critical for capsid assembly.

Amino Acid Sequence↗
PubMed1996

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine.

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5' noncoding (NC) region. These 5' NC mutations and a separate group of 5' NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5' NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.

Animals↗
PubMed1995

Polyprotein processing in echovirus 22: a first assessment.

The major steps of the polyprotein processing of Echovirus 22 (EV22), a highly unusual member of the picornavirus family, have been characterized for the first time by employing in vitro assay systems. Cell-free expression of a P1-2ABC precursor as well as VP1-2A yielded autoproteolytically inactive proteins, suggesting that the 2A region of the EV22 polyprotein does not contain a proteolytic activity. The intra- and intermolecular cleavage specificity of proteinase 3C, the major proteolytic enzyme in picornaviruses, was studied by expressing the enzyme of EV22 in a bacterial system as well as in the framework of precursor molecules generated by in vitro transcription/translation in a cell-free system. A VP1-2A percursor could very efficiently be cleaved in trans by the recombinant 3C, whereas the junction between P2 and P3 remained uncleaved. Expression of the complete P3-region in the cell-free system led to the autocatalytic release of large amounts of p22, a protein of the predicted molecular weight of the EV22 proteinase 3C that was recognized by an antibody raised against the recombinant enzyme.

3C Viral Proteases↗
PubMed1995

Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes.

Worldwide, HCV is a major etiologic agent of chronic hepatitis that may lead to the development of liver cirrhosis and hepatocellular carcinoma. Thus, significant morbidity and mortality is caused by HCV infection and effective control measures against the spread of this virus are needed. Originally, the extent of genetic heterogeneity of HCV was not fully appreciated. However, the breadth of the genetic heterogeneity of HCV is great, and this may have important implications in diagnosis, pathogenesis, treatment, and vaccine development. In an infected individual the HCV genome population circulates as a quasispecies distribution of closely related yet heterogeneous RNA sequences centered around one dominant sequence. The dominant sequence, as well as the consensus sequence, changes sequentially during the course of the infection. A hypervariable region (HVR1) within one of the envelope proteins of HCV (E2) evolves very rapidly. Patients infected with HCV mount a humoral immune response to epitopes of HVR1. However, sequential changes in the consensus sequence of HVR1 during infection result in the generation of variants that are not recognized by preexisting antibodies. This might represent a mechanism by which HCV evades host immune surveillance and establishes and maintains persistent infection. It will be important to determine whether HVR1 of HCV, as was found for the V3 loop of HIV, contains epitopes that elicit neutralizing antibodies against HCV. Furthermore, it will be important to determine whether the quasispecies nature of HCV helps the virus evade the cytotoxic T-cell response of the host. Analysis of complete or partial HCV genomic sequences revealed that HCV exists as multiple, distinct genotypes. A total of nine major genetic groups and at least 30 subgroups have been recognized. To evaluate the current classification of HCV genotypes, we performed phylogenetic analyses of complete and partial nucleotide sequences from isolates that represent all published variants of HCV. Analysis of complete HCV sequences, which represent three major genetic groups, supports the currently used genotype classification scheme. However, analysis of the partial genomic regions (ie, C, E1, and NS5b) of HCV isolates that represent all recognized variants of HCV demonstrates that the genetic relatedness among some of the genotypes was not equivalent in the different gene regions. Furthermore, the distinction among isolates, subtypes, and types of HCV was not always clear. This finding might reflect the shortcomings of analyzing only limited gene regions or may reflect the wide spectrum of genetic variation of HCV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Long-term efficacy of active postexposure immunization of infants for prevention of hepatitis B virus infection. United States-People's Republic of China Study Group on Hepatitis B.

Perinatal transmission of hepatitis B virus (HBV) contributes to the high prevalence of chronic infection in China and many other countries. In a placebo-controlled trial among 166 infants, the 12-month efficacy of active postexposure prophylaxis to prevent chronic perinatal HBV infection varied by vaccine (range, 45%-89%). In a 5-year follow-up study, 2 additional infants became chronically infected with HBV, and the efficacy of active prophylaxis was estimated to be 38% and 72% for the two vaccines at 5 years. In addition, 80% of immunized infants continued to have protective levels of antibody at the end of 5 years. However, among 27 infants who received passive-active immunoprophylaxis with high-dose hepatitis B immune globulin, only 60% (11/19) had protective antibody levels. These data indicate that active postexposure immunization initiated soon after birth continues to provide protection during early childhood when there is a high risk of chronic HBV infection.

Chronic Disease↗