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Biomedical subjects

V C Emery

Publications and source records attributed to V C Emery.

At least 91 records · Page 5Linked to original sources
PubMed1994

Phylogenetic relationship between human immunodeficiency virus type 1 (HIV-1) long terminal repeat natural variants present in the lymph node and peripheral blood of three HIV-1-infected individuals.

PCR has been used to amplify a 540 base pair fragment of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat region encompassing the U5/R/U3 regions from proviral HIV DNA that was present in the lymph nodes and peripheral blood mononuclear cells of three HIV-infected individuals. The resulting PCR products were cloned and the DNA sequence of multiple clones from each reaction was determined to enable the distribution and phylogenetic relationship between the variants in each body site to be assessed. The results of bootstrapped parsimony phylogenetic analyses showed that a distinct polarization was evident between peripheral blood and lymph node variants in the patient who possessed a histologically intact lymph node. In contrast, similar analyses conducted on samples from two patients with extensive lymph node disruption showed similar variants in lymph node and peripheral blood. In the patient with an intact lymph node, lymph node variants were significantly more heterogeneous than those present in blood samples taken either simultaneously or 11 months later. No differences in heterogeneity between lymph node and peripheral blood were observed in patients with disrupted lymph nodes. The significance of these results for our understanding of HIV pathogenesis is discussed.

Base Sequence↗
PubMed1993

Sequence microheterogeneity in the long control region of clinical isolates of human papillomavirus type 16.

The polymerase chain reaction (PCR) has been used to amplify the long control region (LCR) of episomal human papillomavirus type 16 from cervical scrape DNA obtained from a woman with no evidence of cervical disease and a woman with cervical intraepithelial neoplasia grade 3 (CIN 3). An 883 base pair fragment containing the entire LCR was cloned into pUC13 and the DNA sequence determined for both isolates and compared with the prototype HPV type 16 LCR DNA sequence. Nucleotide variation was apparent in the LCRs derived from both women. In the case of the sample derived from the woman with no cervical disease, there were three nucleotide deletions, one insertion, four transversions, and three transitions (overall conservation: 98.7%). In contrast, the LCR derived from the woman with CIN 3 showed significantly more nucleotide variation with two nucleotide deletions, one insertion, nine transversions, and ten nucleotide transitions (overall conservation 97.6%). Using computer analyses coupled with available data from DNA footprint studies, the effects of these sequence variations on established transcription factor binding sequences were investigated. Cloning of the LCR derived from the woman without cervical disease into a chloramphenicol acetyl transferase (CAT) promoter screening vector, followed by transfection of HeLa cells with the LCR-CAT construct, revealed that the LCR was a functional promoter but was 4.6-fold less active than an equivalent SV40 early promoter-CAT construct.

Base Sequence↗
PubMed1993

Sequence variation within the human immunodeficiency virus V3 loop at seroconversion.

Analysis of the HIV-1 V3 quasispecies present in an individual at the time of seroconversion was carried out. The polymerase chain reaction (PCR) was used to amplify proviral HIV-1 DNA extracted from peripheral blood mononuclear cells from a patient who was viraemic (p24 = 15 pg/ml) and had an equivocal HIV-1 antibody status. The PCR products were cloned and the DNA sequence determined for 15 clones. These data showed that the V3 region contained only limited sequence heterogeneity with a major variant accounting for 66% of the protein quasispecies present. The protein sequence of the principal neutralising domain on all species contained the relatively rare GPGKTL motif rather than GPGRAF. The relevance of these data for early stages of HIV infection are discussed.

Amino Acid Sequence↗
PubMed1993

Use of semi-quantitative PCR for human papillomavirus DNA type 16 to identify women with high grade cervical disease in a population presenting with a mildly dyskaryotic smear report.

The aim of this study was to assess whether qualitative or semi-quantitative detection of human papillomavirus type 16 (HPV 16) can help to identify women with major grade cervical intraepithelial neoplasia (CIN 2 and CIN 3) among those referred with a smear suggesting mild dyskaryosis. The study population consisted of 200 women sequentially attending the Royal Free Hospital colposcopy clinic. All women were investigated by cytology, colposcopy and, where appropriate, histopathology, and HPV 16 DNA was detected in cervical scrape samples using the polymerase chain reaction (PCR). A final clinical diagnosis of normal, wart virus infected (WVI), CIN 1, CIN 2 or CIN 3 was made in 179 women. On the basis of the qualitative PCR data, the presence of HPV 16 DNA was of borderline use in identifying women with high grade cervical disease [63/113 (normal/WVI/CIN 1) vs 46/66 (CIN 2/CIN 3); P = 0.065]. However, semi-quantitative PCR analysis showed that a high/medium HPV 16 result was significantly associated with high-grade disease [29/113 (normal/WVI/CIN 1) vs 38/66 (CIN 2/CIN 3); P = 0.0001]. Furthermore, semi-quantitative PCR and cytology were performed on the repeat smear taken immediately prior to colposcopy. The combined laboratory results show that 53/60 women with biopsy proven high-grade disease were identified, as were 26/95 women who were either normal or who had low grade cervical disease. The possibility of using such an approach for selecting women for more rapid or for routine colposcopy appointments in the two groups respectively is discussed.

Adult↗
PubMed1993

Provision of prognostic information in immunocompromised patients by routine application of the polymerase chain reaction for cytomegalovirus.

A polymerase chain reaction (PCR) assay that amplifies a 149 base pair fragment of the cytomegalovirus glycoprotein B gene was used in the routine screening of 548 urine and 248 blood specimens from immunocompromised patients. The PCR results were compared with those obtained for the same specimens tested by the methods of conventional cell culture (CCC) and detection of CMV-specific immediate-early antigen fluorescent foci (DEAFF). For both urine and blood, PCR positivity correlated with a positive result in CCC (urine 93.2%; blood 86%). As expected for a more sensitive assay, PCR also identified CMV in samples that were negative by CCC and DEAFF such that there was no concordance between tests (Kappa test P > 0.05). The sensitivity, specificity, positive predictive value, and negative predictive values of PCR positivity in blood with respect to CMV disease were 0.8, 0.86, 0.62, and 0.94, respectively, with an associated relative risk of 5.84 (95% CI; 3.2-10.8). PCR detection of CMV in urine was more sensitive than either DEAFF or CCC (0.6 vs. 0.35 and 0.5, respectively) and had a high negative predictive value (0.89) but the positive predictive value was lower than either CCC or DEAFF (0.32 vs. 0.41 and 0.37, respectively) with respect to disease. Longitudinal data on patients with disease showed that CMV in blood was detected at a median of 5 days (range; -20 to +3 days) before disease onset whereas CMV was detected by CCC at a median of 13 days (range -4 to +20 days) after disease onset. In addition, the PCR assay was integrated into the battery of tests routinely performed on transplant patients in the diagnostic laboratory at this institution.

AIDS-Related Opportunistic Infections↗
PubMed1993

Sequence variation within neutralizing epitopes of the envelope glycoprotein B of human cytomegalovirus: comparison of isolates from renal transplant recipients and AIDS patients.

The envelope glycoprotein B of human cytomegalovirus (CMV) is a major target of the neutralizing antibody response against this virus, and hence has importance as a potential subunit vaccine. PCR was utilized to amplify DNA encoding the dominant antigenic determinant on this molecule, AD-1 (codons 552 to 635), and DNA sequencing was carried out in order to compare nucleotide variation in AD-1 between clinical isolates of CMV and the laboratory strain AD169. Wild-type CMV strains isolated from AIDS patients were not only more likely to possess nucleotide substitutions (19/24 compared to 5/25, P < 0.0001) than those from renal transplant recipients, but they also exhibited a greater degree of nucleotide sequence divergence (6.94 versus 0.82 substitutions/1000 bp, P < 0.0001; 96.0 to 100% versus 99.4 to 100% similarity). Increased sequence variation in the AIDS patients did not correlate with absolute peripheral blood CD4+ T cell level (r = 0.33, P > 0.1). Only two strains from AIDS patients and one strain from the renal transplant recipients possessed nucleic acid substitutions that resulted in codon changes, indicating that AD-1 is relatively well conserved amongst clinical isolates of CMV. The demonstration of strains with codon changes within neutralizing epitopes, however, highlights the importance of taking into consideration the presence of these strains within the wild-type virus population when preparing subunit vaccines.

AIDS-Related Opportunistic Infections↗
PubMed1992

Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus.

The human erythrocyte-type glucose transporter (GLUT1) has been abundantly expressed in insect cells by using a recombinant baculovirus. At 4 days after infection with the virus, the insect cell-surface and intracellular membranes were found to contain greater than 200 pmol of D-glucose-sensitive binding sites for the transport inhibitor cytochalasin B per mg of protein. The characteristics of binding were identical with those of the erythrocyte transporter, although the two proteins differed substantially in apparent Mr, probably as a result of glycosylation differences.

Animals↗
PubMed1992

Comparison of cervical cytology and the polymerase chain reaction for HPV 16 to identify women with cervical disease in a general practice population.

A comparison of the ability of cervical cytology and the polymerase chain reaction (PCR) for human papilloma virus type 16 (HPV 16) to identify women with cervical disease has been performed in a general practice population of 249 women, none of whom were believed to have current cervical disease prior to examination. Within this population, 29 women were found by colposcopy and subsequent histopathology to have evidence of cervical disease [5 with cervical intraepithelial neoplasia (CIN) 3; 8 with CIN 2; and 16 with CIN 1]. The prevalence of HPV 16 in this population was 18.9% (CIN 3, 80%; CIN 2, 50%, CIN 1, 12.5%, normal, 16.8%). Women with severe disease (CIN 2 and CIN 3) had a significantly higher incidence of HPV 16 DNA than those with mild cervical disease (CIN 1) or no cervical abnormality (P = 0.001). There was no significant difference in the ability of either PCR for HPV 16 or cytology to identify women with cervical disease. The combination of screening by cytology and the presence of HPV 16 DNA resulted in the identification of a higher proportion of the women with disease, but this observation did not reach statistical significance. Although the failure to detect disease by the two screening methods was similar, HPV 16 DNA positivity was associated with a higher false-positive rate for disease detection than cytology (P less than 0.03). The PCR assay for detecting HPV 16 in this investigation was shown to have a false-positive rate of 2.4% and a false-negative rate of 10.4%. The prospect of screening women for cervical disease using PCR for HPV 16 is discussed.

Adolescent↗
PubMed1992

Detection of herpesvirus DNA in the large intestine of patients with ulcerative colitis and Crohn's disease using the nested polymerase chain reaction.

The prevalence of herpesvirus DNA was examined in inflammatory bowel disease tissue. DNA was extracted from resection and biopsy specimens of the large intestine from patients with ulcerative colitis (n = 21), patients with Crohn's disease (n = 29), and patients with noninflammatory bowel disease (controls) (n = 21). The nested polymerase chain reaction was used to detect viral DNA using primer pairs specific for either cytomegalovirus (CMV), herpes simplex virus 1 (HSV1), human herpesvirus 6 (HHV6), varicella zoster virus (VZV), or Epstein Barr virus (EBV). HSV1 and VZV DNA were not detected in any of tissue samples. There was a high prevalence of CMV (81%), HHV6 (76%), and EBV (76%) DNA in ulcerative colitis tissue compared to Crohn's disease tissues (CMV 66%, HHV6 45%, EBV 55%). Control tissue had a relatively low frequency of CMV (29%) and EBV (19%) DNA but a prevalence of HHV6 DNA similar to that of ulcerative colitis (86%). However, the simultaneous presence of HHV6 and CMV and/or EBV DNA in ulcerative colitis tissue (76%) was much greater than in either Crohn's disease tissues (38%) or control tissue (29%) (P < 0.05). There was a low prevalence of CMV, HHV6, and EBV DNA in peripheral blood mononuclear cells from all patient groups. CMV and EBV are capable of reactivating HHV6: the high prevalence of coexistent HHV6 infection with either or both of these two viruses in ulcerative colitis tissue suggests that they may play a synergistic role in the pathogenesis of this disease.

Adolescent↗
PubMed1992

Quantification of human cytomegalovirus DNA using the polymerase chain reaction.

The important goal of developing quantitative assays for viral nucleic acids in clinical samples has been achieved for human cytomegalovirus (HCMV) by using a modified polymerase chain reaction (PCR). A control PCR target sequence was constructed by PCR mutagenesis to allow the post-amplification quantification of HCMV DNA. The control region was identical to a naturally occurring sequence within the glycoprotein B (gB) coding part of the virus genome, except that a unique restriction site, introduced by the aforementioned mutagenesis step, allowed post-amplification differentiation of control/non-control target amplified product. This technique was initially validated using known amounts of cloned control/non-control target DNA, and was found to be sufficiently sensitive to allow the quantification of a range of 10 to 10(6) genome equivalents of virus. The method was applied to urine samples of congenitally infected infants for which infectious virus titres were available. The results obtained demonstrated that the number of infectious virions determined by conventional cell culture represented a small proportion of the HCMV genome present in the samples, as assessed by the quantitative PCR methodology.

Base Sequence↗
PubMed1991

Eliminating PCR contamination: is UV irradiation the answer?

The sensitivity of the polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for the detection of human cytomegalovirus (CMV) and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. The sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, the ultimate sensitivity of a PCR system for the amplification of an early gene promotor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worthwhile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system.

Base Sequence↗
PubMed1991

Population dynamics of HIV within an individual after treatment with zidovudine.

A new mechanism is proposed for the apparent breakthrough of HIV that occurs approximately 6 months after the commencement of therapy with zidovudine (AZT). Using a simple mathematical model of the interacting population dynamics of HIV and its major host cell in the circulation (the CD4+ lymphocyte), predicted patterns of HIV plasma viraemia in the weeks following treatment with zidovudine are generated. These are in close agreement with observed patterns despite the fact that the model contains no mechanisms for the development of drug-resistant strains of virus. It is suggested that the patterns of viral abundance observed during the first 6 months after treatment may be the result of non-linearities in the interactions between HIV and CD4+ cells, and that it is only after the first post-treatment burst of viral production that drug resistance plays an important role.

AIDS-Related Complex↗
PubMed1991

Use of the polymerase chain reaction to analyse sequence variation within a major neutralizing epitope of glycoprotein B (gp58) in clinical isolates of human cytomegalovirus.

The heterogeneity of low passage human cytomegalovirus (HCMV) strains was determined by HindIII typing of 28 clinical isolates from transplant patients. These data have shown that, in general, each patient's strain has a unique restriction profile, usually comprising combinations of HindIII sites present in one or more of the tissue culture-adapted strains AD169, Towne and Davis. To map sequence changes in a more refined manner we performed detailed analyses of 33 low passage clinical isolates, including those aforementioned, analysing a sequence within glycoprotein B containing a major neutralizing epitope. A 149 bp sequence containing the epitope (amino acids 608 to 625) was amplified using the polymerase chain reaction, the products were cloned and their DNA sequence was determined. Comparison of the DNA and deduced amino acid sequences with those of HCMV strain AD169 revealed that there was a high degree of conservation of the epitope between the 33 clinical isolates. However 10 of the isolates possessed silent mutations and three isolates contained mutations producing amino acid changes within the neutralizing epitope. The possible functional significance of these changes is discussed.

Amino Acid Sequence↗
PubMed1989

Cytomegalovirus infection and progression towards AIDS in haemophiliacs with human immunodeficiency virus infection.

To examine whether cytomegalovirus (CMV) infection could accelerate progression of human immunodeficiency virus (HIV) infection to AIDS, serological studies were done on 108 HIV-infected haemophiliacs. In the 1.3-9 years from time of first recognised HIV seroconversion, the age-adjusted risk of CDC group IV disease in CMV-seropositive patients was 2.5 times that in CMV-seronegative patients. CMV-seropositive patients were also more likely to have detectable p24 antigenaemia. Survival analysis showed that CMV-seropositive patients were at greater risk of HIV disease than CMV-seronegative patients from about 2 years after HIV seroconversion. Thus CMV infection is associated with a more rapid progression to HIV disease.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus.

The M RNA species of a candidate vaccine strain of Rift Valley fever virus (RVFV ZH-548M12), derived by consecutive high level mutagenesis using 5-fluorouracil (H. Caplen, C. J. Peters, and D. H. L. Bishop, J. Gen. Virol., 66, 2271-2277, 1985), has been cloned and the cDNA sequenced. The data have been compared to those obtained for the parent virus strain RVFV ZH-548 as well as the previously published data for RVFV ZH-501 (M. S. Collett, A. F. Purchio, K. Keegan, S. Frazier, W. Hays, D. K. Anderson, M. D. Parker, C. Schmaljohn, J. Schmidt, and J. M. Dalrymple, Virology, 144, 228-245, 1985). Some eight nucleotide and three amino acid differences were identified between the M RNAs of ZH-501 and ZH-548. Between the M RNAs of ZH-548 and that of the M12 mutant there were 12 nucleotide and 7 amino acid changes. Unique to the mutant virus is a new AUG codon upstream of that which initiates the open reading frame of the RVFV M gene product (the viral glycoprotein precursor). The significance of this and other differences in the mutant RNA with regard to the derivation and potential attenuation of the candidate vaccine is discussed.

Amino Acid Sequence↗