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V C Emery

Publications and source records attributed to V C Emery.

At least 73 records · Page 4Linked to original sources
PubMed1997

Diagnosis of primary human herpesvirus 6 and 7 infections in febrile infants by polymerase chain reaction.

Primary human herpesvirus 6 (HHV-6) and 7 (HHV-7) infections were identified in febrile children by qualitative and quantitative polymerase chain reaction (PCR) assays. Diagnosis was based on the differential detection of viral DNA in peripheral blood mononuclear cells (PBMC), but not in saliva. Six of 41 febrile infants, but none of seven non-febrile controls, were identified with primary infections (three HHV-6, three HHV-7). These children had significantly higher viral loads in PBMC (HHV-6, median 24213 genomes/10(6) PBMC; HHV-7, median 6,040,000 genomes/10(6) PBMC) than DNA-aemic, saliva PCR positive children (HHV-6, median 1606 genomes/10(6) PBMC, p < 0.01; HHV-7, median 7089 genomes/ 10(6) PBMC, p < 0.05). Viral DNA was detected in serum by PCR in only 50% of primary infections. All three children with primary HHV-7 infection had febrile convulsions. Thus PCR, including quantitative assays, may identify primary HHV-6 and HHV-7 infections when an appropriate combination of clinical specimens is used.

DNA, Viral↗
PubMed1997

Use of polymerase chain reaction to provide prognostic information on human cytomegalovirus disease after liver transplantation.

Sixty-four consecutive liver transplant patients receiving 76 organs have been monitored for human cytomegalovirus (HCMV) in blood and urine posttransplantation using a polymerase chain reaction (PCR) assay that amplifies a 149 base pair fragment of the glycoprotein B gene. Six hundred and twenty-six blood and 310 urine samples were analysed during surveillance. Thirty-two patients had CMV infection (50%), 12 of whim progressed to HCMV disease. Detection of HCMV in either blood or urine was significantly associated with the presence or development of HCMV disease (blood, P < 0.00001; urine, P = 0.0033). All cases of HCMV disease were detected as PCR-positive in blood, although due to sampling only 50% of these patients were PCR-positive prior to disease onset. HCMV infection and disease were more likely in patients who suffered rejection (P < 0.001). In addition, the median amounts of augmented prednisolone were higher in patients with HCMV infection and disease. In all cases, augmented prednisolone preceded HCMV infection/disease. There was no statistical association between CMV infection and death. Overall, the results show that routine use of PCR for HCMV in surveillance samples of blood and urine of liver transplant recipients can provide diagnostic and prognostic information. However, its ability to provide prognostic information is directly related to the availability of appropriate surveillance samples, emphasising the importance of the routine acquisition of such samples in patient management to allow preemptive anti-HCMV therapy.

Acyclovir↗
PubMed1997

Quantity of cytomegalovirus viruria is a major risk factor for cytomegalovirus disease after renal transplantation.

Studies have shown that risk factors for human cytomegalovirus (HCMV) disease after renal transplant include primary infection (virus of donor origin infecting a non-immune individual), re-infection (virus of donor origin infecting a immune individual), and the detection of viraemia (as a marker of virus dissemination). We now report that viral load in the urine is also a significant factor in HCMV disease and is one of the main mechanisms underlying the risk associated with viraemia and donor serostatus. Longitudinal analysis of a group of 196 renal recipient identified 35 recipients who were PCR positive for HCMV in urine. Elevated viral loads were present in symptomatic patients, viraemic patients, and patients experiencing primary HCMV infection. Disease was associated with the peak quantity of virus present in the urine during the post-transplant period (P = 0.0001), with viraemia (P = 0.0003), and with transplantation of a seropositive donor (P = 0.03). Univariate logistic regression analysis showed that increases of 0.25 log10 in viral load were associated with a 179% increased risk of disease (odds ratio = 2.79; 95% C.I. 1.22-6.39; P = 0.02). This effect persisted in a multivariate logistic analysis when viraemia was incorporated (odds ratio = 2.77; 95% C.I. 1.07-7.18; P = 0.04). In contrast, the significant association between viraemia and disease observed in univariate analysis (odds ratio = 23.75; 95% C.I. 3.69-152.90; P = 0.0009) became marginally non-significant in multivariate analysis once viral load had been controlled for (odds ratio = 34.54; 95% C.I. 0.75-1599.00; P = 0.07). The computed probability of disease showed that a rapid transition occurred at viral loads between 10(5.7) and 10(6.5) genomes/ml urine in non-viraemic patients compared to viral loads between 10(5.0) and 10(5.7) genomes/ml urine in patients with concurrent viraemia. The implications of these findings for understanding HCMV pathogenesis, improving patient management, and optimising trials of antiviral treatment are discussed.

Adolescent↗
PubMed1996

Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein.

Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli. The carboxyl terminal fusion (HBc3-144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc1-183) could not be purified or characterised immunologically, although it formed core like particles. HBc3-144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7-17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc3-144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.

Animals↗
PubMed1996

Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.

Amino Acid Sequence↗
PubMed1996

Detection of human cytomegalovirus polymerase chain reaction products using oligonucleotide probes directly conjugated to alkaline phosphatase.

A 24 base pair oligonucleotide probe directly conjugated to alkaline phosphatase has been used to detect immobilised amplicons derived from a cytomegalovirus specific polymerase chain reaction (PCR). The sensitivity of detection using a highly amplified alkaline phosphatase detection system was four genome equivalents and was comparable to the limit of detection using agarose gel methods. The mean optical density at 492 nm of samples not known to contain cytomegalovirus DNA was 0.085 +/- 0.006 and was well separated from the optical density generated from four genome equivalents (absorption at 492 nm: 0.132). The assay was used to identify the presence of cytomegalovirus in blood DNA extracts from immunocompromised patients in whom conventional ethidium bromide stained agarose gel electrophoresis revealed the presence of multiple amplicons. Samples yielding an uninterpretable result at both neat and diluted 1 in 20 in the PCR gave rise to the highest proportion of positive results (68%) whilst samples that produced uninterpretable results neat but were negative at 1 in 20 and vice versa gave positive rates of 33.6 and 21.7%, respectively. The use of this assay for identifying cytomegalovirus specific PCR products in problematic samples is discussed.

Alkaline Phosphatase↗
PubMed1996

Measurement of human herpesvirus 7 load in peripheral blood and saliva of healthy subjects by quantitative polymerase chain reaction.

Qualitative and competitive-quantitative nested polymerase chain reaction (PCR) assays were developed for human herpesvirus 7 (HHV-7). These assays amplify a DNA sequence encoding part of the HHV-7 homologue of the human herpesvirus 6 (HHV-6) U42 gene. The PCR assays were used to analyze peripheral blood DNA (pbDNA) and saliva from 24 healthy volunteers. The prevalence of HHV-7 in saliva was 96%, with a median virus load of 1.1 x 10(6) copies/mL. Longitudinal analysis revealed sustained virus load, suggesting continued active viral replication. Analysis of 1 microgram of pbDNA showed the prevalence of HHV-7 to be 83%, with a median virus load of 40 copies (267 copies/10(6) cells). Analysis of sequential pbDNA samples showed individuals to have stable levels of HHV-7 virus load. These data demonstrate persistence of HHV-7 at two distinct sites and provide baseline data allowing comparisons with HHV-7 load in immunocompromised patients.

Base Sequence↗
PubMed1996

Quantification of human herpesvirus 6 in immunocompetent persons and post-mortem tissues from AIDS patients by PCR.

A quantitative competitive PCR assay for human herpesvirus 6 (HHV-6) was developed. Firstly, viral burden was determined in the blood of 25 healthy persons. Using 1 microgram of DNA, the prevalence of HHV-6 was 36% (9/25). Eight persons had viral loads of < or = 32 HHV-6 genomes/microgram DNA. The viral burden in the ninth individual was 1.2 x 10(6) HHV-6 genome copies/microgram DNA, which remained constant over a period of 10 months. This demonstrates the persistence of a high HHV-6 load in the absence of apparent disease. Secondly, HHV-6 burden was determined in 100 post-mortem tissues from seven AIDS patients and three controls. For all tissues combined, there was a statistically significant higher median viral load in AIDS patients (56 copies/microgram DNA, range 0-43321) compared to controls (10 copies/microgram DNA, range 0-423) (P = 0.04). The precision and reproducibility of this assay will allow hypotheses concerning the pathogenic potential of HHV-6 to be tested quantitatively.

AIDS-Related Opportunistic Infections↗
PubMed1996

A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood.

The presence of cytomegalovirus (CMV) in the blood has important consequences for patient management, and an external quality control study of its detection by the PCR was conducted by the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation. Forty-eight coded peripheral blood samples from bone marrow transplant recipients were processed in parallel in three European centers by using the routine in-house PCR assay. Protocols varied in choice of primers, specificity and amplificability controls, and sample processing. Results for 38 of 47 samples agreed, 35 being negative and 3 positive. Of the 12 samples reported as positive by a least one center, only 3 were found to be positive by all three centers, 1 was found to be positive by two centers, and the remaining 8 were found to be positive by one center only. The nine discrepant samples appeared to contain around 1,000-fold less viral DNA than the three concordant positive samples. CMV detection was affected both by the number of leukocytes from which DNA was extracted and by the number of cell equivalents added per PCR. External quality control schemes for CMV PCR are clearly necessary in order to compare data from different centers, and recommendation for standardizing the PCR detection of CMV in blood leukocytes are made.

Bone Marrow Transplantation↗
PubMed1996

Lessons from the natural history of cytomegalovirus.

BACKGROUND: More than 90% of patients with HIV have been infected at some time with cytomegalovirus (CMV) and up to 40% of those with advanced HIV will develop CMV disease. The incidence of CMV disease is increasing but the prognosis for the patient remains poor. MONITORING FOR CMV: It is therefore important to monitor patients with low CD4+ counts in order to identify those most at risk of developing CMV disease and to treat them before the disease becomes established. Polymerase chain reaction (PCR) is probably the most effective and sensitive method of detecting CMV and a positive result is predictive for development of CMV disease; more than 80% of patients with CMV retinitis are CMV PCR-positive at the time of diagnosis. PCR can also detect the presence of CMV up to 14 months before the development of retinitis. TREATMENT OF CMV RETINITIS: In patients with detectable CMV, but no evidence of active infection, pre-emptive treatment with ganciclovir or valaciclovir has been shown to reduce the risk of developing retinitis in these high-risk patients. Such oral therapy, which is generally better tolerated than intravenous therapy and results in a better quality of life for the patient, is likely to be more effective at this stage whilst viral loads are low. CONCLUSIONS: CMV PCR can be used to prospectively monitor patients in order to identify those most at risk of developing CMV retinitis. If CMV infection is diagnosed early, while viral loads are still low, pre-emptive oral therapy can be instituted which will reduce the chances of developing retinitis in those patients most at risk.

AIDS-Related Opportunistic Infections↗
PubMed1995

Beta-sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy.

Low density lipoprotein receptor domains (LDLrs) represent a large cell surface receptor superfamily of consensus length 39 residues. Alignment of 194 sequences indicated highly conserved Cys and Asp/Glu residues, and a consensus secondary structure with three beta-strands was predicted. Sequence threading against known protein folds indicated consistency with small beta-sheet proteins. Complement factor I contains two LDLrs, and the second of these was successfully expressed using a bacterial pGEX system. FT-IR spectroscopy on this indicated a small amount of beta-sheet together with turns and loops. LDLr is proposed to have a beta-sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop.

Amino Acid Sequence↗
PubMed1995

Use of the polymerase chain reaction to detect genomes of human immunodeficiency virus and cytomegalovirus in post-mortem tissues.

The polymerase chain reaction (PCR) was used to amplify a 149 base-pair region of the cytomegalovirus (CMV) genome and a 551 base-pair region of the HIV-1 proviral long terminal repeat (LTR) present in DNA extracted from post-mortem tissue. Multiple tissues (n = 116) obtained from 16 patients which were subjected to PCR were also subjected to cell culture and histopathological analyses. One hundred and seven samples (92%) contained CMV DNA and 66/116 (57%) contained HIV proviral DNA at a level of > or = 10 genomes. Both viruses were detected in 60/116 (51.7%) of samples, with co-infection most frequent in the lung (69%). Cell culture for CMV detected 9.3% of the PCR-positive samples, whilst histology identified CMV inclusions in 15.9% of samples, all of which were CMV PCR-positive. CMV was most frequently detected in adrenal and lung tissues by histology. These results show that co-infection with CMV and HIV is a common occurrence in organs from AIDS patients and provide further evidence for a role of cytomegalovirus in the pathogenesis of AIDS.

AIDS-Related Opportunistic Infections↗
PubMed1995

Distinct HIV-1 long terminal repeat quasispecies present in nervous tissues compared to that in lung, blood and lymphoid tissues of an AIDS patient.

OBJECTIVE: To investigate the phylogenetic relationship of HIV-1 proviral long terminal repeat (LTR) variants present in postmortem samples of lymph node, spleen, lung, dorsal root ganglion and spinal cord as well as in the peripheral blood of an HIV-1-infected patient dying with AIDS. DESIGN AND METHODS: Postmortem tissues were studied by a combination of histology, cell culture and molecular analyses. The patient had a stable CD4 count of 10 x 10(6)/I during the 12 months preceding death. A 540 base-pair fragment of the LTR including U3/R/U5 was amplified using polymerase chain reaction on proviral DNA from the five postmortem tissues and peripheral blood mononuclear cells obtained 2 months prior to death. The population of viral variants was determined by sequencing at least five plasmid clones of the amplicons. The relationship between the variants present in different body sites was investigated using molecular phylogeny methods. RESULTS: HIV-1 was present in all organs analysed and correlated with the presence of abnormal histology. Genetic variation leading to divergence from the consensus sequence was more frequently present in characterized transcription factor binding sites within the LTR (P < 0.0001) although the HIV-1 LTR quasispecies in the different body sites showed similar, relatively low levels of divergence (intra-organ median heterogeneity ranging from 0.0094 to 0.017). Phylogenetic analysis showed that the spinal cord and dorsal root ganglion harboured an LTR population genetically distinct from that present in other organs and more closely related to a previously characterized neurotropic strain of HIV (strain JRcsf). CONCLUSION: The independent clustering of HIV-1 LTR variants present in spinal cord and dorsal root ganglion shows that HIV-1 LTR evolution can occur in a compartmentalized fashion. The data show that the LTR is an important region to analyse in sequence variation studies of HIV since it may play a role in nervous tissue adaptation of HIV-1 and neuropathogenicity. Outgrowth of HIV-1 LTR variants that are most fit for the utilization of tissue-specific transcription factors can occur in the nervous tissue.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Longitudinal analysis of cytomegalovirus load in renal transplant recipients using a quantitative polymerase chain reaction: correlation with disease.

Serial surveillance samples of urine collected from 103 renal transplant recipients were analysed by polymerase chain reaction (PCR) for the presence of human cytomegalovirus (HCMV) DNA. The PCR results were consistently negative in 70 patients, none of whom developed HCMV disease, and PCR positive in 33 patients of whom 10 developed HCMV disease (P < 0.001). In 12 patients, PCR results were positive in three or more consecutive samples indicating extensive HCMV replication. HCMV load in 104 samples from these patients was analysed using a quantitative co-amplification PCR system. The maximal viral burden in the symptomatic patients ranged from 10(5.9) to 10(7.12) genomes/ml urine (median 10(6.5)) and in the asymptomatic patients from 10(4) to 10(5.7) genomes/ml urine (median 10(5.2)). The 10(1.3) difference between these median values was significant (P < 0.01). Individual kinetic profiles of viral burden showed that high levels of HCMV correlated with clinically apparent disease. In the majority of the asymptomatic individuals HCMV load remained between 10(4) and 10(5.1) genomes/ml urine; however, in two patients fluctuations in viral load were observed involving higher viral levels (up to 10(5.7) genomes/ml urine) suggesting that immune responses able to modulate viral replication could be studied in individual patients. Analysis of the temporal appearance and quantity of HCMV in the urine with alterations in white cell numbers showed that leukopenia occurred following the appearance of HCMV in the urine of symptomatic patients but preceded HCMV in the urine of asymptomatic patients (P = 0.01). Overall, these results show that longitudinal analysis using fully quantitative PCR methods for HCMV can provide insight into the natural history of HCMV disease in renal transplant recipients.

Adolescent↗
PubMed1994

Relation between human papillomavirus type 16 and potential for progression of minor-grade cervical disease.

We have previously reported that among 200 women referred for colposcopy with smears suggesting mild dyskaryosis, medium or high copy numbers of human papillomavirus type 16 (HPV16) DNA identified patients with current high-grade cervical disease. We have followed up 95 women from that group who had histologically proven mild-grade cervical disease (cervical intraepithelial neoplasia grade 1, n = 37) or wart virus infection (n = 12) or who had no evidence of cervical abnormality at study entry (n = 43). Kaplan-Meier survival analysis of the 70 months' follow-up was used to identify baseline features that might affect the risk of progression. 3 women were lost to follow-up; data were available for the remaining 92. Among the whole group the probability of remaining free of high-grade cervical disease was 0.71. Women with a histological diagnosis of minor-grade disease were more likely to progress to high-grade disease than those with no evidence of abnormality (proportion disease-free 0.52 vs 0.90, p = 0.004). Stratification of the group according to median age (28 years) revealed a weak association between age and disease progression (p = 0.04). There was no difference in disease-free probability between HPV16-positive and HPV16-negative women (0.75 vs 0.65, p = 0.19). Nor was there a significant difference in disease-free probability when the group was stratified by HPV16 viral burden. These data show that a histological diagnosis of minor-grade cervical disease is a better long-term predictor of disease progression than is HPV16 positivity, irrespective of copy number. These findings do not support the simple view that HPV16 alone is the cause of high-grade cervical disease, including cancer.

Adolescent↗
PubMed1994

HHV-6 in AIDS.

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AIDS-Related Opportunistic Infections↗