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Biomedical subjects

R H Purcell

Publications and source records attributed to R H Purcell.

At least 217 records · Page 12Linked to original sources
PubMed1987

Complete nucleotide sequence of an attenuated hepatitis A virus: comparison with wild-type virus.

The complete nucleotide sequence of an attenuated hepatitis A virus, HAV HM-175/7 MK-5, was determined from cloned cDNA. This virus was derived from wild-type HAV HM-175 after 32 passages in African green monkey kidney cells. The resultant cell culture-adapted virus is attenuated for chimpanzees. This virus was passaged an additional three times in monkey kidney cells to obtain sufficient virus for molecular cloning and was designated HM-175/7 MK-5. Three overlapping cDNA clones were obtained that together spanned the entire genome. Comparison of the nucleotide sequence of cDNA from wild-type virus (propagated in marmoset liver in vivo) with attenuated virus (grown in cell culture) showed 24 nucleotide changes distributed throughout the genome. Five base deletions occurred in the 5' noncoding region, and 12 of the 16 base substitutions in the coding region resulted in amino acid changes. Amino acid changes occurred in viral capsid proteins VP1 and VP2 and several of the nonstructural proteins. Thus, a small number of nucleotide changes are responsible for adaptation to cell culture and attenuation of HAV strain HM-175.

Animals↗
PubMed1987

Titration of the infectivity of hepatitis D virus in chimpanzees.

The infectivity of hepatitis D virus (HDV) was evaluated by intravenous inoculation of chimpanzees. HDV was present in the inoculum at a titer of 10(11) chimpanzee infectious doses (CID). In contrast, the titer of hepatitis B virus (HBV) in the same inoculum was 10(6) CID. All HBV-infected chimpanzees inoculated with less than or equal to 10(-11) dilutions of the HDV-positive plasma were superinfected; an animal receiving a 10(-12) dilution did not develop markers of HDV replication in serum or liver. All HDV-infected chimpanzees had marked elevations of serum alanine aminotransferase activities. The incubation period from exposure to development of hepatitis was inversely related to the dose of HDV inoculum, although the severity and duration of hepatitis were independent of it. All animals recovered and rapidly developed antibody to hepatitis D antigen.

Alanine Transaminase↗
PubMed1987

A human monoclonal antibody that recognizes viral polypeptides and in vitro translation products of the genome of the hepatitis D virus.

Peripheral blood lymphocytes from a patient chronically infected with hepatitis D virus (HDV) were immortalized by Epstein-Barr virus transformation. Two stable monoclonal cell lines, derived from the same parent culture, were established and produced antibodies of the IgG isotype that were specific for the hepatitis delta antigen (HDAg). Both monoclonal antibodies (MAbs) recognized the major HDAg polypeptides of 24 kilodaltons and 27 kilodaltons that were previously detected by polyclonal antibodies to HDAg in both liver and serum from HDV-infected humans, chimpanzees, and woodchucks. This result indicates that the major polypeptides of HDAg share common epitopes. The MAbs also reacted with minor polypeptides of lower molecular weight, which were present in infected liver. In vitro translation products of HDV-specific RNA from infected liver were also detected by the MAbs; these polypeptides were 24 kilodaltons and 27 kilodaltons, respectively, and comigrated with liver- or serum-derived HDAg. In contrast, HDV RNA isolated from virions in serum was not translated into HDAg polypeptides in the in vitro system.

Animals↗
PubMed1987

Determining the size of non-A, non-B hepatitis virus by filtration.

The approximate size of the H strain of non-A, non-B (NANB) hepatitis virus was determined by filtration through polycarbonate membranes. The accuracy and reproducibility of such filtrations were monitored by filtering selected reference viruses. These studies indicate that strain H, representative of the principal blood-borne NANB hepatitis virus, is 30-60 nm in diameter. It is therefore highly unlikely that NANB hepatitis virus is a retrovirus, as has been suggested.

Animals↗
PubMed1987

An epidemic of hepatitis A in an institution for young children.

A common-source epidemic of hepatitis A occurred in an Athenian institution boarding 38 children (mean age 4.8 years). All children were examined, and blood was drawn from each at the onset of the study and repeatedly during the next three months. Only one child (2.6%) was initially immune to hepatitis A virus as a result of prior infection. The attack rate (62.2%) and the ratio of icteric to anicteric cases (1:1.3) were high despite the administration of immunoglobulin (IG). Assays for anti-HAV IgM and a rising titer of anti-HAV IgG identified 19 (82.6%) and 22 (95.7%) of the 23 hepatitis A infections, respectively. One case (4.3%) was detected only by the presence of hepatitis A virus antigen and hepatitis A virus RNA in a fecal specimen, but these assays were otherwise marginally useful in this study. Nevertheless, the use of all available tests for the detection of hepatitis A virus is mandatory for the most accurate estimation of an epidemic of hepatitis A. Prompt administration of immunoglobulin had no effect on the number of clinical cases that were in the late incubation period, but it may have diminished the clinical expression of the infection and thus made diagnosis of infection more difficult.

Child↗
PubMed1987

Detection of hepatitis B virus DNA in asymptomatic hepatitis B surface antigen carriers: relation to sexual transmission.

The authors tested, by molecular hybridization, for hepatitis B virus DNA in serum specimens of 182 asymptomatic hepatitis B surface antigen (HBsAg) Greek carriers who were heterosexual partners of patients with acute hepatitis B (group A: 96 cases) or healthy subjects who were susceptible to hepatitis B (group B: 86 cases). The mean age (34.1 +/- 10.4 vs. 33.9 +/- 8.4 years) and the mean duration of sexual contact (6.9 +/- 8.9 vs. 7.2 +/- 6.3 years) were similar in the two groups of carriers. Hepatitis B virus DNA was detected significantly more frequently in group A than in group B (59.4% vs. 11.6%, p less than 0.001). In particular, in group A, hepatitis B virus DNA was detected in 96.9% of hepatitis B e antigen (HBeAg)-positive and 41% of antibody to HBeAg (anti-HBe)-positive carriers. In contrast, in group B, hepatitis B virus DNA was identified in only 10.8% of anti-HBe-positive carriers (p less than 0.001). These differences were especially significant in the young and middle-aged carriers (16-49 years old) and during the first four years of sexual contact. These data suggest that 1) there is a positive correlation between the presence of hepatitis B virus DNA in serum and the epidemiologic evidence of sexual transmission of hepatitis B virus, 2) hepatitis B virus DNA is a better indicator of infectivity than HBeAg/anti-HBe, and 3) the detection of hepatitis B virus DNA in serum probably identified carriers with high infectivity and potentially higher risk of transmitting hepatitis B virus to their sexual partners.

Adolescent↗
PubMed1987

Detection of hepatitis A virus by extraction of viral RNA and molecular hybridization.

Hepatitis A virus (HAV) RNA was extracted from cell culture, serum, liver, and feces and then detected by molecular hybridization with cloned HAV cDNA. Hybridization was approximately 10-fold more sensitive than immune electron microscopy or radioimmunoassay was and less sensitive than was assays of HAV infectivity in primates or in cell culture. As little as 10(3) 50% infective doses of HAV, or approximately 0.1 pg of viral RNA, was detected by this method. Analysis of fecal specimens from an experimentally infected marmoset and an epidemic of hepatitis A showed that HAV excretion could often be detected later in the illness by hybridization than by radioimmunoassay. This technique should be widely applicable for detection and analysis of HAV RNA.

Animals↗
PubMed1987

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.

Base Sequence↗
PubMed1987

Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture.

A full-length cDNA copy of an attenuated, cell culture-adapted hepatitis A virus (HAV HM-175/7 MK-5) genome was constructed in the PstI site of plasmid vector pBR322. Transfection of monkey kidney cells with this plasmid failed to induce the production of hepatitis A virus (HAV). The HAV cDNA was excised from pBR322 and inserted, without the oligo(dG) X oligo(dC) tails, into an RNA transcription vector to yield plasmid pHAV/7. Transfection of monkey kidney cells with pHAV/7 DNA induced HAV infection. Transfection with RNA transcripts produced in vitro from pHAV/7 yielded about 10-fold more HAV than did transfection with pHAV/7 DNA. Marmosets inoculated with transfection-derived virus developed anti-HAV antibodies and had liver enzyme patterns that closely resembled the liver enzyme patterns seen in animals inoculated with virus from a comparable level of cell culture passage. Infectious RNA transcripts from HAV cDNA should be useful for studying the molecular basis of cell culture adaptation and attenuation as well as for studying specific viral functions.

Animals↗
PubMed1987

Relation between non-A, non-B and delta hepatitis: studies with monoclonal antibodies.

To further investigate the specificity of the monoclonal antibodies (48-1 and S-1) associated with NANB hepatitis, extensive IF studies were carried out on liver biopsy specimens from chimpanzees with experimental hepatitis A, B, NANB, or D, or from normal chimpanzees. Both 48-1 and S-1 antibodies reacted in the same manner with liver biopsy specimens from 47 of 50 (94%) chimpanzees with acute or chronic NANB hepatitis and 15 of 18 (83%) chimpanzees with type D hepatitis. Examinations of serial liver biopsy specimens revealed that the duration of expression of the antigen reacting with the antibodies in hepatocytes of chimpanzees infected with NANBV appeared to be longer than that of chimpanzees infected with HDV. By thin section EM, the presence of the microtubular aggregates, identical to those previously described for chimpanzees with NANB hepatitis and shown by immuno EM to react with the antibodies, was noted in hepatocytes during the acute phase of hepatitis D. The antibodies did not react with liver biopsy specimens from chimpanzees acutely or chronically infected with HBV, or HAV, or from normal chimpanzees. The present results confirm our previous observations with the 48-1 and S-1 antibodies. Furthermore, the finding that these two antibodies were also associated with hepatitis D would support the possibility that NANB agents and the HDV may have a similar nature or may elicit a similar host response.

Animals↗
PubMed1986

Fulminant hepatitis in asymptomatic hepatitis B surface antigen carriers in Greece.

Eleven male fulminant hepatitis (FH) patients (mean age: 47.7 +/- 16 years) positive for hepatitis B surface antigen (HBsAg) but negative for IgM antibody to hepatitis B core antigen (IgM anti-HBc) were admitted consecutively to the Athens Hospital for Infectious Diseases between May 1981 and November 1983. Because of the absence of IgM anti-HBc, determined by an enzyme immunoassay, these patients were considered to be HBsAg carriers with a superimposed acute hepatitis. Three of the 11 patients received immunosuppressive chemotherapy during the six months before the onset of the acute hepatitis. None of the patients was homosexual or a drug addict. Infection with hepatitis A virus (HAV), hepatitis B virus (HBV), or hepatitis delta virus (HDV) was detected with serologic markers and/or molecular hybridization techniques. Fulminant hepatitis was attributed to spontaneous reactivation of chronic hepatitis B in four patients, chemotherapy-induced reactivation of chronic hepatitis B in three patients, HDV superinfection in one patient and possible superinfection by non-A, non-B agent(s), HDV, or HDV-like agents in three patients. Reactivation of chronic hepatitis B was an important cause of apparent acute hepatitis in heterosexual male HBsAg carriers from an area with a high prevalence of HBV infection.

Acute Disease↗
PubMed1986

Use of monoclonal antibodies to determine if HBsAg of mixed subtype is one particle or two.

Of the ten universally recognized subtypes of hepatitis B surface antigens, two contain both of what are normally mutually exclusive subtype determinants. These mixed subtypes presumably result from a concurrent infection by different viral subtypes and result in the presence of the various subtypes either on separate particles or intermixed on one particle. This study demonstrates that both of these alternatives can occur. Additionally, a mixed subtype present on one particle could result from either intermixing of separate protein subtypes into the same viral envelope (phenotypic mixing) or genetic recombination between viruses of different subtypes resulting in the production of one protein with a mixed subtype (genotypic mixing). The latter possibility would make the definition of the antigenic determinants and production of a vaccine for hepatitis B more difficult since it implies that the virus can alter its surface characteristics. Evidence is provided against genotypic mixing, demonstrating that phenotypic mixing does occur but does not 'breed true' upon viral transmission.

Antibodies, Monoclonal↗
PubMed1986

Search for hepatitis B virus DNA in sera from patients with acute type B or non-A, non-B hepatitis.

One hundred and three single sera from adults hospitalized with acute type B (78) or non-A, non-B (25) hepatitis were tested for the presence of hepatitis B virus DNA (HBV DNA). All sera from patients with type B hepatitis were IgM anti-HBc-positive. These patients were classified as benign (47) or fulminant (31) hepatitis. The 25 acute non-A, non-B patients were also classified as benign (21) or fulminant (4) hepatitis and were negative for serologic markers of past HBV infection. Serum HBV DNA was detected with similar frequency in benign (38.5%) and fulminant (FH, 34.6%) HBsAg-positive cases. HBV DNA was not detected in either the 26 acute HBsAg-negative hepatitis B cases who were positive for anti-HBc and anti-HBs or the 25 acute non-A, non-B hepatitis cases. The absence of HBV DNA in 43.8% of benign hepatitis B patients who were positive for HBsAg and HBeAg could possibly be attributed to either low level replication of HBV that was not detectable by the [32P]HBV DNA probe or to a period of delayed clearance of free HBeAg following cessation of HBV replication. Emergence of anti-HBs in the presence of HBsAg did not always correspond to clearance of HBV in fulminant type B cases. However, in acute type B hepatitis, irrespectively of severity, disappearance of HBsAg and appearance of anti-HBs was accompanied by reduction of HBV replication to undetectable levels.

Adult↗