Grahame-Clarke C, Chan NN, Andrew D, Ridgway GL, Betteridge DJ, Emery V, Colhoun HM, Vallance P (2003) Human cytomegalovirus seropositivity is associated with impaired vascular function., Circulation108(6)pp. 678-683
BACKGROUND: Herpesvirus infection is a possible risk factor for atherogenesis, and diabetics may be at particular risk. Endothelial dysfunction is an early marker for atherosclerosis, and the present study tests the hypotheses that (1) prior infection with cytomegalovirus (CMV) and herpes simplex virus (HSV) is associated with endothelial dysfunction and (2) this may be more marked in diabetics. METHODS AND RESULTS: Serum samples were tested for anti-IgG antibodies to CMV and HSV from 400 subjects (mean age for diabetics and nondiabetics, 37.8+/-4.3 and 37.9+/-3.7 [SD]). We also assessed Helicobacter pylori and Chlamydia pneumoniae serology. Coronary atheroma was quantified by means of electron beam computed tomography. Subjects (n=157) underwent venous occlusion plethysmography with acetylcholine, bradykinin, glyceryl trinitrate, norepinephrine, and l-NG-monomethyl-l-arginine. Individuals who were seropositive for CMV had reduced responses to bradykinin (P=0.005) and glyceryl trinitrate (P=0.006). The reduced response to bradykinin remained significant (P=0.045) after adjusting for the response to glyceryl trinitrate and was independent of conventional risk factors. Positive serology for the other organisms did not have an independent effect on reactivity. There was a weaker association between CMV and coronary artery calcification (P=0.09). Positive serology for each of the other pathogens did not affect reactivity, but there was a relation between total pathogen burden and impaired vascular reactivity. No significant differences were found between diabetics and nondiabetics. CONCLUSIONS: This study shows that CMV-seropositive individuals have endothelial dysfunction and impaired responses to NO. This association was independent of conventional risk factors and may be associated with increased atherosclerosis burden.
Nebbia G, Grillo F, Clewley G, Burroughs AK, Emery VC (2008) Comparison of hepatitis C replication dynamics during acute infection in immunocompetent hosts and re-infection of the donor liver after transplantation, AMERICAN JOURNAL OF TRANSPLANTATION8pp. 634-634 WILEY-BLACKWELL PUBLISHING, INC
Griffiths PD, McLean A, Emery VC (2001) Encouraging prospects for immunisation against primary cytomegalovirus infection., Vaccine19(11-12)pp. 1356-1362
Congenital cytomegalovirus (CMV) infection is the leading infectious cause of mental retardation in children. Using seroprevalence data from two large antenatal populations (in excess of 14000 women) coupled with a mathematical modelling approach, we have shown that CMV has a low force of infection (ca. 0.03 per seronegative per annum) and its basic reproductive number R0 is relatively modest at 2.4. On the basis of these results, the critical vaccination proportion required for eradication of CMV is between 59-62%. In contrast to the predicted and observed effects of rubella vaccination on the incidence of congenital rubella, the increase in the average age of infection following instigation of a CMV vaccine programme will not increase the number of congenital infections. In conclusion, CMV is a prime candidate for eradication from the human population through vaccination.
Clark DA, Emery VC, Griffiths PD (2003) Cytomegalovirus, human herpesvirus-6, and human herpesvirus-7 in hematological patients, Seminars in Hematology40(2)pp. 154-162
The prototype member of the Betaherpesvirinae subfamily, cytomegalovirus (CMV), is the most important infectious pathogen in transplant recipients, including those receiving bone marrow or stem cell grafts. Overt CMV disease such as pneumonitis is notoriously difficult to treat. Antiviral prophylaxis, rapid diagnostic tests to identify CMV infection, and preemptive antiviral chemotherapy are significant improvements in the management of CMV. As the kinetics of the immune response to CMV become better defined, immunotherapeutic approaches should be introduced to complement current management strategies. Two newly identified betaherpesviruses, human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), are genetically more closely related to each other than to CMV. Both are highly prevalent in the general population and infections post-bone marrow transplantation are common. These viruses are not as pathogenic as CMV but HHV-6 at least can cause disease such as encephalitis, hepatitis, and bone marrow suppression. Both of these newer herpesviruses are potentially susceptible to existing and licensed antiherpesvirus drugs. © 2003 Elsevier Inc. All rights reserved.
Bowen EF, Cherrington JM, Lamy PD, Griffiths PD, Johnson MA, Emery VC (1999) Quantitative changes in cytomegalovirus DNAemia and genetic analysis of the UL97 and UL54 genes in AIDS patients receiving cidofovir following ganciclovir therapy., J Med Virol58(4)pp. 402-407
Five AIDS patients with cytomegalovirus (CMV) retinitis who had received ganciclovir (GCV) therapy were followed with serial blood sampling to detectchanges both in CMV load and in the genetic composition of genes UL97 and UL54 whilst receiving cidofovir (CDV) therapy. CDV neither reduced CMV load in blood nor prevented its quantitative resurgence during therapy. These effects were not explained by the initial presence or development of CDV-associated drug resistance mutations in UL54. In two patients, UL97 genotypic resistance to GCV involving either a L595S mutation or a deletion of amino acids 590-603 were present at the initiation of CDV and, in both patients, repopulation of CMV strains with wild-type UL97 sequences occurred during CDV therapy. These data are consistent with GCV-resistant strains containing UL97 mutations being less fit than their wild-type counterparts and so being able to persist only with the selective pressure of GCV.
Manuel O, Asberg A, Pang X, Rollag H, Emery VC, Preiksaitis JK, Kumar D, Pescovitz MD, Bignamini AA, Hartmann A, Jardine AG, Humar A (2009) Impact of Genetic Polymorphisms in Cytomegalovirus Glycoprotein B on Outcomes in Solid-Organ Transplant Recipients with Cytomegalovirus Disease, CLINICAL INFECTIOUS DISEASES49(8)pp. 1160-1166 UNIV CHICAGO PRESS
Shannon-Lowe CD, Emery VC (2010) The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein., Herpesviridae1(1)
BACKGROUND: The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required. METHODS: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities. RESULTS: Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM. DISCUSSION: Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients.
Emery VC, Einsele H, Atabani S, Haque T (2011) Immunotherapy and vaccination after transplant: the present, the future., Hematol Oncol Clin North Am25(1)pp. 215-229
Vaccination and adoptive immunotherapy for herpes virus infections has become an attractive option for the control of a virus family that negatively affects transplantation. In the future, enhanced ability to select antigen-specific T cells without significant in vitro manipulation should provide new opportunities for refining and enhancing adoptive immunotherapeutic approaches. This article focuses on advances in the area of vaccinology for some of these infections and in the use of adoptive immunotherapy. At present, many of these approaches in transplant recipients have focused on infections such as human cytomegalovirus, but the opportunity to use these examples as proof of concept for other infections is discussed.
Aubert G, Hassan-Walker AF, Madrigal JA, Emery VC, Morte C, Grace S, Koh MB, Potter M, Prentice HG, Dodi IA, Travers PJ (2001) Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants., J Infect Dis184(8)pp. 955-963
The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.
GRIFFITHS PD, EMERY VC, LEE C, JOHNSON MA, MCLAUGHLIN J (1994) HHV-6 IN AIDS, LANCET343(8905)pp. 1104-1105 LANCET LTD
Gillespie SH, Ullman C, Smith MD, Emery V (1994) Detection of Streptococcus pneumoniae in sputum samples by PCR., J Clin Microbiol32(5)pp. 1308-1311
A method for the detection of Streptococcus pneumoniae in sputum samples by PCR has been developed. The assay employs oligonucleotide primers specific for a portion of the autolysin gene lytA of S. pneumoniae. Other closely related streptococci, Haemophilus influenzae, and Moraxella catarrhalis do not give a positive result in the assay. The assay was capable of detecting between 10 and 100 CFU of S. pneumoniae in distilled water and 1.4 x 10(4) CFU/ml in simulated sputum samples. Sputum samples from 33 patients with acute pneumonia were collected and subjected to culture, PCR, and C-polysaccharide antigen detection by enzyme-linked immunosorbent assay (ELISA). A significant isolate of S. pneumoniae was isolated from 14 patients, of which 13 were positive by PCR and C-polysaccharide antigen ELISA. No positive results were obtained for the 19 patients in whom other pathogens or upper respiratory tract floras only were isolated. The sensitivity of the autolysin PCR is 92.8%, the specificity is 100%, the predictive value of a positive result is 100%, and the predictive value of a negative result is 95%. This suggests that autolysin PCR is suitable for the detection of S. pneumoniae in clinical samples.
Kidd IM, Clark DA, Ait-Khaled M, Griffiths PD, Emery VC (1996) Measurement of human herpesvirus 7 load in peripheral blood and saliva of healthy subjects by quantitative polymerase chain reaction., J Infect Dis174(2)pp. 396-401
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.
Pillay D, Emery VC, Mutimer D, Ogilvie MM, Carman W, Mutton K, Wreghitt T, Westmoreland D, Breuer J, Zuckerman M (2002) Guidelines for laboratory monitoring of treatment of persistent virus infections, JOURNAL OF CLINICAL VIROLOGY25(1)PII S1386-6532(02)00018-5pp. 73-92 ELSEVIER SCIENCE BV
Drew WL, Paya CV, Emery V (2001) Cytomegalovirus (CMV) resistance to antivirals, AMERICAN JOURNAL OF TRANSPLANTATION1(4)pp. 307-312 WILEY-BLACKWELL
Ansari A, Emery VC (1999) The U69 gene of human herpesvirus 6 encodes a protein kinase which can confer ganciclovir sensitivity to baculoviruses., J Virol73(4)pp. 3284-3291
The protein encoded by the U69 open reading frame (ORF) of human herpesvirus 6 (HHV-6) has been predicted to be a protein kinase. To investigate its functional properties, we have expressed the U69 ORFs from both HHV-6 variants, A and B, by using recombinant baculoviruses (BV6AU69 and BV6BU69). Nickel agarose and antibody affinity chromatography was used to purify the proteins to homogeneity and when incubated with [gamma-32P]ATP, both U69 proteins became phosphorylated on predominantly serine residues. These data strongly suggest that U69 is a protein kinase which autophosphorylates. The phosphorylation reaction was optimal at physiological pH and low NaCl concentrations. It required the presence of Mg2+ or Mn2+, and Mg2+ was able to support phosphorylation over a wider range of concentrations than Mn2+. Both ATP and GTP could donate phosphate in the protein kinase assay and the former was more efficient. U69 was capable of phosphorylating histone and casein (serine/threonine kinase substrates) but not enolase (a tyrosine kinase substrate). For the autophosphorylation reaction, the Michaelis constants for ATP of baculovirus-expressed HHV-6A and HHV-6B U69 were calculated to be 44 and 11 microM, respectively. U69 is a homologue of the UL97 gene encoded by human cytomegalovirus which has been shown to phosphorylate the antiviral drug ganciclovir (GCV). We analyzed whether the U69 ORF alone was capable of conferring GCV sensitivity on baculoviruses BV6AU69 and BV6BU69. In plaque reduction experiments, these baculoviruses displayed a GCV-sensitive phenotype compared to a control baculovirus (BVLacZ). The 50% inhibitory concentrations (IC50) of BV6AU69 and BV6BU69 were calculated to be 0.35 and 0.26 mM, respectively, whereas the control baculovirus had an IC50 of >1.4 mM. This shows that the U69 gene product is the only one required to confer GCV sensitivity on baculovirus.
Kidd IM, Clark DA, Emery VC (2000) A non-radioisotopic quantitative competitive polymerase chain reaction method: application in measurement of human herpesvirus 7 load., J Virol Methods87(1-2)pp. 177-181
Quantitative-competitive polymerase chain reaction (QCPCR) is a well-optimised and objective methodology for the determination of viral load in clinical specimens. A major advantage of QCPCR is the ability to control for the differential modulation of the PCR process in the presence of potentially inhibitory material. QCPCR protocols were developed previously for CMV, HHV-6, HHV-7 and HHV-8 and relied upon radioactively labelled primers, followed by autoradiography of the separated and digested PCR products to quantify viral load. Whilst this approach offers high accuracy and dynamic range, non-radioactive approaches would be attractive. Here, an alternative detection system is reported, based on simple ethidium bromide staining and computer analysis of the separated reaction products, which enables its adoption in the analysis of a large number of samples. In calibration experiments using cloned HHV-7 DNA, the ethidium bromide detection method showed an improved correlation with known copy number over that obtained with the isotopic method. In addition, 67 HHV-7 PCR positive blood samples, derived from immunocompromised patients, were quantified using both detection techniques. The results showed a highly significant correlation with no significant difference between the two methods. The applicability of the computerised densitometry method in the routine laboratory is discussed.
Emery VC, Sabin CA, Hassan-Walker AF, Griffiths PD (2000) Viral-load kinetics and CMV disease - Reply, LANCET356(9238)pp. 1353-1353 ELSEVIER SCIENCE INC
Emery V, Zuckerman M, Jackson G, Aitken C, Osman H, Pagliuca A, Potter M, Peggs K, Clark A (2013) Management of cytomegalovirus infection in haemopoietic stem cell transplantation, British Journal of Haematology
Yi CK, Charalambous BM, Emery VC, Baldwin SA (1992) Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus., Biochem J283 ( Pt 3)pp. 643-646
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.
MCLEAN AR, EMERY VC, WEBSTER A, GRIFFITHS PD (1991) POPULATION-DYNAMICS OF HIV WITHIN AN INDIVIDUAL AFTER TREATMENT WITH ZIDOVUDINE, AIDS5(5)pp. 485-489 RAPID SCIENCE PUBLISHERS
Dargan DJ, Douglas E, Cunningham C, Jamieson F, Stanton RJ, Baluchova K, McSharry BP, Tomasec P, Emery VC, Percivalle E, Sarasini A, Gerna G, Wilkinson GWG, Davison AJ (2010) Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture, JOURNAL OF GENERAL VIROLOGY91pp. 1535-1546 SOC GENERAL MICROBIOLOGY
Prentice HG, Potter MN, Mattes FM, Hainsworth E, Murdin-Geretti A, Nebbia G, Burroughs AK, Sweeny P, Hassan-Walker AF, Okwuadi S, Emery VC, Griffiths PD (2001) A randomized, controlled trial comparing ganciclovir or ganciclovir plus foscarnet (each at half dose) for pre-emptive therapy of cytomegalovirus infection in transplant recipients., BLOOD98(11)pp. 395A-395A AMER SOC HEMATOLOGY
Emery VC (1991) Baculovirus expression vectors for the production of viral proteins, Reviews in Medical Virology1(1)pp. 11-17
Emery VC, Hassan-Walker AF (2002) Focus on new drugs in development against human cytomegalovirus., Drugs62(13)pp. 1853-1858
The limitations of current therapies for human cytomegalovirus (HCMV) coupled with the continued impact of HCMV disease in the immunocompromised host are the driving force for the development of new drugs against HCMV. This review predominantly focuses on new non-DNA polymerase inhibitors of HCMV replication. Drugs such as tomeglovir (BAY-384766), 2-bromo-5,6-dichloro-1beta-D-ribofuranosyl benzimidazole (BDCRB) and GW-275175X act as inhibitors of the terminase complex that is involved in cleavage and packaging of the unit length DNA into the capsids. Although the viral protein kinase UL97 has been exploited as an activator of ganciclovir and its prodrug valganciclovir, a new inhibitor maribavir (benzamidavir) has been shown to be a highly potent inhibitor of this enzyme. Many of these compounds have undergone successful phase I clinical trials. There are other compounds which have been identified through drug-screening but are at the earlier stages of development.
Rankin PF, Prentice HG, Moore J, Clark DA, Potter M, Knight SN, Sabin CA, Mattes FM, Noibi SN, Emery VC, Griffiths PD (1998) Significance of quantitative-competitive PCR for human herpes virus-6, human herpes virus-7 and cytomegalovirus following bone marrow transplantation., BLOOD92(10)pp. 518A-518A W B SAUNDERS CO
Bowen EF, Emery VC, Wilson P, Johnson MA, Davey CC, Sabin CA, Farmer D, Griffiths PD (1998) Cytomegalovirus polymerase chain reaction viraemia in patients receiving ganciclovir maintenance therapy for retinitis., AIDS12(6)pp. 605-611
OBJECTIVES: To determine whether recurrence of polymerase chain reaction (PCR) viraemia during maintenance ganciclovir for cytomegalovirus (CMV) retinitis correlates with (i) CMV disease at a new anatomical site, (ii) progression of the presenting retinitis, or (iii) acquisition of genetic changes in gene UL97 associated with resistance to ganciclovir. DESIGN: A previously described cohort of 45 patients presenting with first episode retinitis was followed clinically using ophthalmoscopy and serial tests for PCR viraemia for a median of 7 months. CMV viral load and genetic markers of ganciclovir resistance were measured in PCR-positive samples. METHODS: PCR amplification of the glycoprotein B region of CMV and quantitative competitive PCR assays were employed. Genetic changes in UL97 were identified by sequencing/point mutation assay. RESULTS: PCR viraemia correlated significantly with new episodes of CMV disease (P=0.011) and a trend was seen for the association with progression of retinitis (P=0.07). Amongst the 14 patients PCR-positive during maintenance ganciclovir, 10 (71%) had genetic markers of resistance. None of these patients became PCR-negative in blood after reinduction ganciclovir therapy compared with three out of four without markers of resistance (P=0.022). CONCLUSIONS: CMV PCR viraemia correlated strongly with the development of new episodes of CMV disease. Most patients with progression of retinitis remained PCR-negative in blood, consistent with therapeutic failure due to poor intraocular penetration of ganciclovir. However, the minority who were PCR-positive in blood may have reinfected their eye, and frequently had markers of ganciclovir resistance. The implications of these findings for the management of patients with CMV disease are discussed.
Emery VC (2013) CMV infected or not CMV infected: That is the question, European Journal of Immunology43(4)pp. 886-888
Cytomegalovirus (CMV) infection is widespread in the human population. Normally, in adolescents and adults, prior exposure to CMV can readily be determined by IgG assays. However, in individuals where antibody production is impaired, such as patients with common variable immunodeficiency disease or in the very young where maternal antibodies are present, diagnosis of CMV infection is problematic using such assays. In this issue of the European Journal of Immunology, a study by Sester and colleagues [Eur J Immunol 2013. 43: 1099-1108] using CD4+ T-cell immunity as a marker of infection clearly differentiates young children with prior exposure to CMV from those who only have passive maternal antibody. This information will quickly find application in the pretransplant screening of young children for CMV infection and help with the stratification of these children to identify those who are truly CMV negative and are therefore at risk for future CMV infection and disease if receiving an organ from a CMV-positive donor as discussed in this Commentary. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Hassan-Walker AF, Emery VC, Griffiths PD (2002) Molecular diagnosis of cytomegalovirus disease, BIOMARKERS OF DISEASE: AN EVIDENCE-BASED APPROACHpp. 467-473 CAMBRIDGE UNIV PRESS
Herrero-Martínez E, Sabin CA, Evans JG, Griffioen A, Lee CA, Emery VC (2002) The prognostic value of a single hepatitis C virus RNA load measurement taken early after human immunodeficiency virus seroconversion., J Infect Dis186(4)pp. 470-476
Hepatitis C virus (HCV) RNA loads are measured sporadically in HCV-positive individuals. However, the prognostic value of these isolated measurements for predicting progression to acquired immune deficiency syndrome (AIDS) and all-cause mortality in coinfected individuals remains unclear. In this study, the prognostic value of a single HCV RNA load measurement taken early after human immunodeficiency virus (HIV) seroconversion was investigated in a cohort of 96 male patients with inherited bleeding disorders. Dates of HIV seroconversion had been estimated for all patients, and at least 4 HCV RNA load measurements per patient were done retrospectively after HIV seroconversion. HCV RNA load stabilized at 4 years after HIV seroconversion, and this point was used for analysis. There was a significant correlation between increased age and early HCV RNA load (r=0.25; P=.01). Adjusting for HIV RNA levels, CD4 cell counts, and the age effect, HCV RNA load >5.90 log(10) copies/mL was predictive of progression to AIDS and all-cause mortality over a period of at least 15 years.
Cope AV, Sweny P, Sabin C, Rees L, Griffiths PD, Emery VC (1997) Quantity of cytomegalovirus viruria is a major risk factor for cytomegalovirus disease after renal transplantation., J Med Virol52(2)pp. 200-205
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.
Sabin CA, Griffioen A, Yee TT, Emery VC, Herrero-Martinez E, Phillips AN, Lee CA (2002) Markers of HIV-1 disease progression in individuals with haemophilia coinfected with hepatitis C virus: a longitudinal study., Lancet360(9345)pp. 1546-1551
BACKGROUND: Low serum albumin concentration is associated with short-term survival in individuals with HIV-1. However, few investigators have assessed whether individuals with a low serum albumin concentration have delayed progression to AIDS, or survive in the long term. We aimed to assess the relation between markers of liver function and progression to AIDS and death in individuals with haemophilia infected with HIV-1 and hepatitis C virus. METHODS: We measured markers of liver function and took CD4 counts every 3 months in 111 patients registered at the Royal Free Hospital Haemophilia Centre, London, UK. HIV RNA concentrations were measured yearly and then every 3-6 months from 1996. We used Cox's regression models to assess the independent prognostic value of these markers for AIDS and death. FINDINGS: As a fixed covariate, albumin concentrations measured shortly after HIV-1 seroconversion were associated with risk of AIDS (relative hazard 0.91 [95% CI 0.84-1.00], p=0.04) and death (0.89 [0.82-0.96], p=0.004) over a 15-year period. These findings were independent of the CD4 count and HIV-1 RNA concentration. As a time-updated covariate, after adjustment for CD4 count and HIV-1 RNA concentrations, albumin was not associated with progression to AIDS (0.96 [0.90-1.01], p=0.13), but was strongly associated with death (0.88 [0.84-0.93], p<0.0001) in the short term. INTERPRETATION: Low concentrations of albumin in individuals infected with HIV-1 could indicate a poor outlook and should therefore prompt concern at any stage of infection.
Whalley SA, Murray JM, Brown D, Webster GJ, Emery VC, Dusheiko GM, Perelson AS (2001) Kinetics of acute hepatitis B virus infection in humans., J Exp Med193(7)pp. 847-854
Using patient data from a unique single source outbreak of hepatitis B virus (HBV) infection, we have characterized the kinetics of acute HBV infection by monitoring viral turnover in the serum during the late incubation and clinical phases of the disease in humans. HBV replicates rapidly with minimally estimated doubling times ranging between 2.2 and 5.8 d (mean 3.7 +/- 1.5 d). After a peak viral load in serum of nearly 10(10) HBV DNA copies/ml is attained, clearance of HBV DNA follows a two or three phase decay pattern with an initial rapid decline characterized by mean half-life (t(1/2)) of 3.7 +/- 1.2 d, similar to the t(1/2) observed in the noncytolytic clearance of covalently closed circular DNA for other hepadnaviruses. The final phase of virion clearance occurs at a variable rate (t(1/2) of 4.8 to 284 d) and may relate to the rate of loss of infected hepatocytes. Free virus has a mean t(1/2) of at most 1.2 +/- 0.6 d. We estimate a peak HBV production rate of at least 10(13) virions/day and a maximum production rate of an infected hepatocyte of 200-1,000 virions/day, on average. At this peak rate of virion production we estimate that every possible single and most double mutations would be created each day.
Li YT, Emery VC, Surah S, Jarmulowicz M, Sweny P, Kidd IM, Griffiths PD, Clark DA (2010) Extensive Human Cytomegalovirus (HCMV) Genomic DNA in the Renal Tubular Epithelium Early After Renal Transplantation: Relationship With HCMV DNAemia and Long-Term Graft Function, JOURNAL OF MEDICAL VIROLOGY82(1)pp. 85-93 WILEY-LISS
Gamett G, Boeckh M, Einsele H, Emery V (2009) The natural history and control of cytomegalovirus infection following allogeneic stem cell transplantation, BONE MARROW TRANSPLANTATION43pp. S237-S238 NATURE PUBLISHING GROUP
Emery VC (2001) Progress in understanding cytomegalovirus drug resistance., J Clin Virol21(3)pp. 223-228
The molecular basis for cytomegalovirus drug resistance against currently used antivirals comprises two genetic loci. In the case of ganciclovir, mutations in both the UL97 protein kinase and UL64 DNA polymerase can lead to resistance, whereas for cidofovir and foscarnet only mutations in UL54 give rise to resistance. Clinically, resistance strains of cytomegalovirus appear after prolonged periods of antiviral therapy especially when treatment has been interrupted or is at sub-optimal doses. Knowledge of the replication dynamics of cytomegalovirus in vivo can be used to predict the virologic course of patients who develop resistance virus. Using such models, a good agreement between experimentally determined viral load and resistance patterns is observed.
Sims S, Colston J, Emery V, Klenerman P (2014) CD73 is dispensable for the regulation of inflationary CD8+ T-cells after murine cytomegalovirus infection and adenovirus immunisation, PLoS ONE9(12)
© 2014 Sims et al.The ecto-5'-nucleotidase (CD73) is expressed by T-cell subsets, myeloid derived suppressive cells and endothelial cells. It works in conjunction with CD39 to regulate the formation and degradation of adenosine in vivo. Adenosine has previously been shown to suppress the proliferation and cytokine secretion of T-cells and recent evidence suggests that inhibition of CD73 has the potential to enhance T-cell directed therapies. Here we utilised a CD73 knockout mouse model to assess the suppressive ability of CD73 on CD8+ T-cell classical memory and memory "inflation", induced by murine cytomegalovirus (MCMV) infection and adenovirus immunisation. We show that CD73 is dispensable for normal CD8+ T-cell differentiation and function in both models. Thus CD73 as a suppressor of CD8+ T-cells is unlikely to play a deterministic role in the generation and functional characteristics of antiviral memory in these settings. Copyright:
Webster A, McLaughlin JE, Johnson MA, Emery VC, Griffiths PD (1995) Use of the polymerase chain reaction to detect genomes of human immunodeficiency virus and cytomegalovirus in post-mortem tissues., J Med Virol47(1)pp. 23-28
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.
Cope AV, Sabin C, Burroughs A, Rolles K, Griffiths PD, Emery VC (1997) Interrelationships among quantity of human cytomegalovirus (HCMV) DNA in blood, donor-recipient serostatus, and administration of methylprednisolone as risk factors for HCMV disease following liver transplantation., J Infect Dis176(6)pp. 1484-1490
Longitudinal analysis of 162 liver transplant recipients identified 51 patients who were viremic. Virus load was determined in 47 of these patients using quantitative-competitive polymerase chain reaction. Peak virus load was significantly higher in 20 symptomatic patients than 27 asymptomatic patients (P < .0001). Elevated virus load, donor seropositivity, and total methylprednisolone dosage were risk factors for human cytomegalovirus (HCMV) disease (odds ratio [OR], 2.22/0.25 log10 increase in virus load, P = .001; OR, 4.11, P = .05; OR, 1.30/1-g increment in methylprednisolone, P = .01). Methylprednisolone and virus load were independent risk factors in a multivariate analysis (OR, 2.70/1-g increase, P = .003; OR, 1.61/0.25 log10 increase, P = .03, respectively). Virus loads of 10(4.75)-10(5.25) genomes/mL of blood were associated with an increased disease probability; the latter was shifted to lower virus loads with increasing quantities of methylprednisolone. These data illustrate the central role of virus load in HCMV pathogenesis.
Downey GP, Bavin PJ, Deery A, Crow J, Griffiths PD, Emery VC, Walker PG (1995) Relation between human papillomavirus type 16 and potential for progression of minor-grade cervical disease, ACOG Current Journal Review8(1)
Emery VC, Griffiths PD (1990) Molecular biology of cytomegalovirus., International journal of experimental pathology71(6)pp. 905-918
Hassan-Walker AF, Vargas Cuero AL, Mattes FM, Klenerman P, Lechner F, Burroughs AK, Griffiths PD, Phillips RE, Emery VC (2001) CD8+ cytotoxic lymphocyte responses against cytomegalovirus after liver transplantation: correlation with time from transplant to receipt of tacrolimus., J Infect Dis183(6)pp. 835-843
The effects of the immunocompromised state after liver transplantation on the frequency of cytomegalovirus-specific cytotoxic T lymphocytes (CTL) were investigated in 93 patients by using HLA class I tetrameric complexes corresponding to HLA-A*0201, HLA-B*0702, HLA-B*0801, and HLA-B*3501 refolded with peptides from the ppUL83 matrix protein. ppUL83 CTL frequencies were suppressed during the first 6 months after transplantation. Patients with >1 HLA-restricted response detected had high correlation among ppUL83 CD8(+) CTL frequencies restricted by different HLA haplotypes (Spearman's rho=.67; P<.0001). There was an inverse correlation among levels of the calcineurin inhibitor, tacrolimus, and ppUL83 CD8(+) CTL frequencies (r=-.31; P=.005), which is consistent with the presence of a large proportion (70%) of activated (CD38(+)) ppUL83 CD8(+) CTL within the population of HLA class I tetramer-positive cells.
Hassan-Walker AF, Vargas AL, Mattes FM, Klenerman P, Lechner F, Burroughs AK, Griffiths PD, Phillips RE, Emery VC (2000) Immunosuppression for liver transplantation with tacrolimus impairs specific cytomegalovirus CD8 immune responses, CLINICAL INFECTIOUS DISEASES31(1)pp. 317-317 OXFORD UNIV PRESS INC
Nokta MA, Holland F, De Gruttola V, Emery VC, Jacobson MA, Griffiths P, Pollard RB, Feinberg JE (2002) Cytomegalovirus (CMV) polymerase chain reaction profiles in individuals with advanced human immunodeficiency virus infection: Relationship to CMV disease, JOURNAL OF INFECTIOUS DISEASES185(12)pp. 1717-1722 OXFORD UNIV PRESS INC
Lao WC, Lee D, Burroughs AK, Lanzani G, Rolles K, Emery VC, Griffiths PD (1997) Use of polymerase chain reaction to provide prognostic information on human cytomegalovirus disease after liver transplantation., J Med Virol51(3)pp. 152-158
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.
Gor D, Lee D, Emery VC (1996) Detection of human cytomegalovirus polymerase chain reaction products using oligonucleotide probes directly conjugated to alkaline phosphatase., J Virol Methods61(1-2)pp. 145-150
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.
Regoes RR, Bowen EF, Cope AV, Gor D, Hassan-Walker AF, Prentice HG, Johnson MA, Sweny P, Burroughs AK, Griffiths PD, Bonhoeffer S, Emery VC (2006) Modelling cytomegalovirus replication patterns in the human host: factors important for pathogenesis, PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES273(1596)pp. 1961-1967 ROYAL SOC
Fryer J, Emery V, Griffiths P, Clark D (2003) Susceptibility of porcine cytomegalovirus to antiviral drugs, XENOTRANSPLANTATION10(5)pp. 520-520 BLACKWELL MUNKSGAARD
Stefanov R, Dimitrov BD, Emery VC (2000) Viral-load kinetics in post-transplantation cytomegalovirus infections  (multiple letters), Lancet356(9242)pp. 1685-1686
Fryer J, Tucker D, Hughes A, Emery V, Griffiths P, Clark D (2001) Porcine cytomegalovirus in pigs being bred for xenograft organs: prospects for eradication, XENOTRANSPLANTATION8pp. 15-15 WILEY-BLACKWELL
Griffiths PD, Stanton A, McCarrell E, Smith C, Osman M, Harber M, Davenport A, Jones G, Wheeler DC, O'Beirne J, Thorburn D, Patch D, Atkinson CE, Pichon S, Sweny P, Lanzman M, Woodford E, Rothwell E, Old N, Kinyanjui R, Haque T, Atabani S, Luck S, Prideaux S, Milne RS, Emery VC, Burroughs AK (2011) Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant recipients: a phase 2 randomised placebo-controlled trial., Lancet377(9773)pp. 1256-1263
BACKGROUND: Cytomegalovirus end-organ disease can be prevented by giving ganciclovir when viraemia is detected in allograft recipients. Values of viral load correlate with development of end-organ disease and are moderated by pre-existing natural immunity. Our aim was to determine whether vaccine-induced immunity could do likewise. METHODS: We undertook a phase-2 randomised placebo controlled trial in adults awaiting kidney or liver transplantation at the Royal Free Hospital, London, UK. Exclusion criteria were pregnancy, receipt of blood products (except albumin) in the previous 3 months, and simultaneous multiorgan transplantation. 70 patients seronegative and 70 seropositive for cytomegalovirus were randomly assigned from a scratch-off randomisation code in a 1:1 ratio to receive either cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant or placebo, each given at baseline, 1 month and 6 months later. If a patient was transplanted, no further vaccinations were given and serial blood samples were tested for cytomegalovirus DNA by real-time quantitative PCR (rtqPCR). Any patient with one blood sample containing more than 3000 cytomegalovirus genomes per mL received ganciclovir until two consecutive undetectable cytomegalovirus DNA measurements. Safety and immunogenicity were coprimary endpoints and were assessed by intention to treat in patients who received at least one dose of vaccine or placebo. This trial is registered with ClinicalTrials.gov, NCT00299260. FINDINGS: 67 patients received vaccine and 73 placebo, all of whom were evaluable. Glycoprotein-B antibody titres were significantly increased in both seronegative (geometric mean titre 12,537 (95% CI 6593-23,840) versus 86 (63-118) in recipients of placebo recipients; p<0.0001) and seropositive (118,395; 64,503-217,272) versus 24,682 (17,909-34,017); p<0.0001) recipients of vaccine. In those who developed viraemia after transplantation, glycoprotein-B antibody titres correlated inversely with duration of viraemia (p=0.0022). In the seronegative patients with seropositive donors, the duration of viraemia (p=0.0480) and number of days of ganciclovir treatment (p=0.0287) were reduced in vaccine recipients. INTERPRETATION: Although cytomegalovirus disease occurs in the context of suppressed cell-mediated immunity post-transplantation, humoral immunity has a role in reduction of cytomegalovirus viraemia. Vaccines containing cytomegalovirus glycoprotein B merit further assessment in transplant recip
Griffiths PD, Clark DA, Emery VC (2000) Betaherpesviruses in transplant recipients., J Antimicrob Chemother45 Suppl T3pp. 29-34
The three betaherpesviruses known to infect humans are cytomegalovirus (CMV) and human herpesviruses 6 and 7 (HHV-6 and -7). All three viruses can infect opportunistically after organ transplantation. CMV causes a variety of end-organ diseases, including pneumonitis, hepatitis and gastrointestinal ulceration. Patients who develop overt CMV disease have significantly higher CMV viral loads than infected patients without evidence of clinical disease. A high CMV viral load largely explains the previously described risk factors for the development of CMV disease, which include donor/recipient serostatus before transplant and viraemia after transplant. CMV also causes some cases of allograft rejection, which can be prevented by antiviral prophylaxis. Application of similar quantitative methods for the study of HHV-6 and -7 have shown that HHV-6 and CMV are significantly and independently associated with biopsy-proven graft rejection after liver transplantation. The full clinicopathological significance of the betaherpesviruses may, thus, be greater than is currently appreciated.
PHILLIPS AN, SABIN CA, ELFORD J, BOFILL M, EMERY V, GRIFFITHS PD, JANOSSY G, LEE CA (1994) VIRAL BURDEN IN HIV-INFECTION, NATURE367(6459)pp. 124-124 MACMILLAN MAGAZINES LTD
Human (Homo sapiens)micro-RNAs (hsa-miRNAs) regulate
virus and host-gene translation, but the biological
impact in patientswith human cytomegalovirus (hCMV)
infection is notwell defined in a clinically relevantmodel.
First, we compared hsa-miRNA expression profiles in
peripheral blood mononuclear cells from 35 transplant
recipients with and without CMV viremia by using a
microarray chip covering 847hsa-miRNAs. This approach
demonstrated a set of 142 differentially expressed hsamiRNAs.
Next, we examined the effect of each of these
miRNAs on viral growth by using human fibroblasts
(human foreskin fibroblast-1) infected with the hCMV
Towne strain, identifyinga subset of proviral andantiviral
hsa-miRNAs. miRNA-target prediction software indicated
potential binding sites within the hCMV genome
(e.g., hCMV-UL52 and -UL100 [UL¼unique long]) and
host-genes (e.g., interleukin-1 receptor, IRF1). Luciferaseexpressing
plasmid constructs and immunoblotting
confirmed several predicted miRNA targets. Finally, we
determined the expression of selected proviral and
antiviral hsa-miRNAs in 242 transplant recipients with
hCMV-viremia. We measured hsa-miRNAs before and
after antiviral therapy and correlated hsa-miRNA expression
levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase
during treatment, concurrent with viral decline. In
contrast, six of eight proviral hsa-miRNAs showed a
decrease during viral decline. Our results indicate that a
complex and multitargeted hsa-miRNA response occurs
during CMV replication in immunosuppressed patients.
This study provides mechanistic insight and potential
novel biomarkers for CMV replication.
Mattes FM, McLaughlin JE, Emery VC, Clark DA, Griffiths PD (2000) Histopathological detection of owl's eye inclusions is still specific for cytomegalovirus in the era of human herpesviruses 6 and 7., J Clin Pathol53(8)pp. 612-614
BACKGROUND: Cytomegalovirus (CMV) is the prototype member of the beta-herpesvirinae, which can cause multiple organ dysfunction in the immunocompromised host. Human herpesvirus 6 (HHV-6) and HHV-7 are newer members of the beta-herpesvirinae that can cause febrile illness in young children and are also possible pathogens in the immunocompromised patient. AIM: CMV is detected in histopathological sections by visualisation of owl's eye inclusion bodies. The aim of this study was to quantify the relation between CMV, HHV-6, and HHV-7 viral loads and the presence of owl's eye inclusions in histological sections. METHODS: Histopathological examination of postmortem material and recording of owl's eye inclusion bodies were performed. CMV, HHV-6, and HHV-7 were detected by qualitative and quantitative polymerase chain reaction (PCR) from the same postmortem samples. Statistical analysis of the histopathological and PCR results was performed. RESULTS: There was a significant association between the detection of owl's eye inclusion bodies and positive CMV PCR (p < 0.001); the median CMV viral load was significantly higher in samples that were positive for owl's eye inclusions (p < 0.001). No association was found between the presence of owl's eye inclusions and HHV-6 or HHV-7 positivity. CONCLUSION: Histological detection of owl's eye inclusion bodies is an insensitive but highly specific method for detecting CMV organ involvement. Owl's eye inclusion bodies are not associated with HHV-6 or HHV-7 infection.
Griffiths DJ, Cooke SP, Herve C, Rigby SP, Mallon E, Hajeer A, Lock M, Emery V, Taylor P, Pantelidis P, Bunker CB, du Bois R, Weiss RA, Venables PJW (1999) Detection of human retrovirus 5 in patients with arthritis and systemic lupus erythematosus, ARTHRITIS AND RHEUMATISM42(3)pp. 448-454 LIPPINCOTT WILLIAMS & WILKINS
Devereux H, Telfer P, Brown D, Morris A, Dusheiko G, Emery V, Lee C (1996) Longitudinal genotype analysis and quantification of hepatitis C virus in haemophilic patients receiving interferon-alpha therapy., J Viral Hepat3(1)pp. 43-48
Haemophilic patients have a high prevalence of hepatitis C virus (HCV) infection because of the use of unsterilized clotting factor concentrates. Six major genotypes of HCV have been distinguished so far, with epidemiological evidence suggesting that genotypes 1-3 are common in the indigenous UK and US populations. The aim of this study was to analyse the changes in viral load and composition of the HCV quasispecies in haemophilic patients receiving therapy with interferon-alpha (IFN-alpha) using the four major methods currently available for HCV genotyping. The most consistent genotype results were obtained using restriction fragment-length polymorphism (RFLP) analysis when compared with the DNA sequence analysis, and showed that the dominant genotype can change in patients with mixed genotype infections treated with IFN-alpha. This study indicates the difficulties in studying this group of patients with mixed HCV genotype infections, and that frequent sampling is necessary, together with viral load measurement to monitor response to IFN-alpha therapy.
Sabin CA, Devereux HL, Clewley G, Emery VC, Phillips AN, Loveday C, Lee CA, Griffiths PD (2000) Cytomegalovirus seropositivity and human immunodeficiency virus type 1 RNA levels in individuals with hemophilia., J Infect Dis181(5)pp. 1800-1803
The effect of cytomegalovirus (CMV) seropositivity on the course of human immunodeficiency virus (HIV) type 1 RNA levels and HIV disease progression was assessed in a cohort of 109 hemophilic men infected with HIV-1 for a median of 12.7 years. There was no evidence of higher HIV RNA levels in the first year after HIV seroconversion (P=. 88) or faster rates of increase over infection (P=.20) in the 59 CMV-seropositive individuals than in the CMV-seronegative individuals. In univariate analyses, CMV seropositivity was associated with significantly faster progression to AIDS and death (relative hazards of 1.58 and 2.22, respectively). These effects were unchanged after adjusting for the RNA level, but they were reduced after adjusting for the CD4 cell count, age at seroconversion, and calendar year of follow-up. Thus, the effect of CMV seropositivity on clinical progression remains significant in this cohort but does not appear to be mediated through an increase in HIV RNA levels.
Andrews PA, Emery VC, Newstead C (2011) Summary of the British Transplantation Society Guidelines for the Prevention and Management of CMV Disease After Solid Organ Transplantation., Transplantation92(11)pp. 1181-1187
The third edition of the British Transplantation Society Guidelines for the Prevention and Management of CMV Disease after Solid Organ Transplantation was published in March 2011. This article summarizes the important changes and advances in management in this rapidly evolving field. The pros and cons of universal, or targeted anti-cytomegalovirus (CMV) prophylaxis, and pre-emptive anti-CMV therapy are discussed, especially with respect to advances in CMV polymerase chain reaction monitoring. The evidence for oral anti-CMV prophylaxis using valganciclovir is presented, together with a summary of the treatment of CMV disease and emerging fields such as CMV vaccination, CMV genotyping, and drug resistance.
Nebbia G, Northfield J, Barnes E, Emery V, Brown D, Willberg C, Dusheiko G, Klenerman P, Fabris P (2006) Role of circulating IL-10 in acute viral hepatitis, JOURNAL OF HEPATOLOGY44pp. S149-S150 ELSEVIER SCIENCE BV
Darlington J, Super M, Patel K, Grundy JE, Griffiths PD, Emery VC (1991) 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., J Gen Virol72 ( Pt 8)pp. 1985-1989
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.
Deayton JR, Prof Sabin CA, Johnson MA, Emery VC, Wilson P, Griffiths PD (2004) Importance of cytomegalovirus viraemia in risk of disease progression and death in HIV-infected patients receiving highly active antiretroviral therapy., Lancet363(9427)pp. 2116-2121
BACKGROUND: Before highly active antiretroviral therapy (HAART) became available, cytomegalovirus was a major cause of opportunistic infection in HIV-infected patients and was associated with accelerated progression to AIDS and death. We have investigated whether cytomegalovirus viraemia remains a significant risk factor for progression of HIV disease and death in the era of HAART. METHODS: 374 patients whose CD4-cell count had ever been below 100 per microL were enrolled in a prospective study. Serial blood samples were tested for cytomegalovirus by PCR. Rates of new cytomegalovirus disease, new AIDS-defining disorders, and death were calculated over a median follow-up of 37 months after stratification according to baseline and most recent cytomegalovirus PCR status at any point during follow-up. FINDINGS: Of 2969 PCR assays, 375 (12.6%) were positive for cytomegalovirus DNA. 259 (69.3%) patients were persistently negative for cytomegalovirus by PCR; 15 were persistently positive; and 100 were intermittently positive and negative. In multivariate models, cytomegalovirus PCR-positive status as a time-updated covariate was significantly associated with increased relative rates of progression to a new AIDS-defining disorder (2.22 [95% CI 1.27-3.88] p=0.005) and death (4.14 [1.97-8.70] p=0.0002). INTERPRETATION: Detection of cytomegalovirus in blood by PCR continues to identify patients with a poor prognosis, even in the era of HAART. Randomised controlled clinical trials of drugs active against cytomegalovirus are needed to investigate whether this virus is a marker or a determinant of HIV disease progression.
Nashan B, Gaston R, Emery V, Säemann MD, Mueller NJ, Couzi L, Dantal J, Shihab F, Mulgaonkar S, Seun Kim Y, Brennan DC (2012) Review of cytomegalovirus infection findings with mammalian target of rapamycin inhibitor-based immunosuppressive therapy in de novo renal transplant recipients, Transplantation93(11)pp. 1075-1085
Cytomegalovirus (CMV) infection and disease are major complications in the renal transplant recipient. The occurrence of CMV is associated with acute rejection, allograft dysfunction, significant end-organ disease, and mortality. Several clinical studies have indicated that the use of certain immunosuppressive drugs can delay the reconstitution of CMV-specific cell-mediated immune responses, thereby leading to uncontrolled CMV replication. Accumulating evidence indicates, however, that the use of the mammalian target of rapamycin (mTOR) inhibitors, sirolimus, and everolimus, may decrease the incidence and severity of CMV infection in renal transplant recipients. The purpose of this article is to review CMV infection data from randomized clinical trials that investigated the use of sirolimus-and everolimus-based treatment regimens in de novo renal transplantation. The mTOR inhibitor clinical trials included were primarily identified using biomedical literature database searches, with additional studies added at the authors' discretion. This review will summarize these studies to discuss whether mTOR inhibitor-based immunosuppressive therapy can reduce the magnitude of CMV-related complications in the de novo renal transplantation setting. © 2012 Lippincott Williams & Wilkins.
Atkinson C, Emery VC (2011) Cytomegalovirus quantification: where to next in optimising patient management?, J Clin Virol51(4)pp. 223-228
BACKGROUND: Over the years quantification of cytomegalovirus (HCMV) load in blood has become a mainstay of clinical management helping direct deployment of antiviral therapy, assess response to therapy and highlight cases of drug resistance. AIMS: The review focuses on a brief historical perspective of HCMV quantification and the ways in which viral load is being used to improve patient management. METHODS: A review of the published literature and also personal experience at the Royal Free Hospital. RESULTS: Quantification of HCMV is essential for efficient patient management. The ability to use real time quantitative PCR to drive pre-emptive therapy has improved patient management after transplantation although the threshold viral loads for deployment differ between laboratories. The field would benefit from access to a universal standard for quantification. CONCLUSIONS: We see that HCMV quantification will continue to be central to delivering individualised patient management and facilitating multicentre trials of new antiviral agents and vaccines in a variety of clinical settings.
Emery VC (1996) Zidovudine resistance and HIV-1 load in multiple autopsy tissues of AIDS patients, Pediatric AIDS and HIV Infection7(5)
Objective. This study aims to investigate the relationship between the quantitative prevalence of ZDV resistance in peripheral blood and lymphoid organs to that present in multiple other organs of the same individual. Methods Proviral HIV-1 load was measured by quantitative-competitive PCR in multiple organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine.The percentage of wildtype and mutant sequences at the positions 41,67,70,215 and 219" of the reverse transcriptase was assessed using a point mutation assay. Results HIV-1 proviral load in 90 samples from multiple organs obtained for 12 patients dying with AIDS was significant associated with increasing resistance to ZDV (p = 0.008). Proviral loads were significantly higher in lymph node and spleen than all other organs analysed (p <0.01 ).The distribution of wildtype and mutant sequences at codons 41,67,70. 215 and 219 was not uniform and could differ markedly between lymphoreticular system and other organs such as brain. Conclusion These results demonstrate that treatment of HI V-1 infection with odovudine does not exert uniform selective pressures in multiple organs.These findings have implications for the interpretation of resistance data and design of treatment strategies for HfV. arguing in particular, that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in sites other than the peripheral circulation.
Emery VC (1999) Viral dynamics during active cytomegalovirus infection and pathology., Intervirology42(5-6)pp. 405-411
The central role that cytomegalovirus (CMV) load plays in its pathogenesis is being unravelled. In AIDS patients with active CMV replication, many months prior to the development of CMV disease, elevated CMV load in the blood and urine are significantly associated with an increased risk of disease progression. In addition, elevated load in blood is associated with an increased risk of death. Intervention with ganciclovir acts to rapidly inhibit CMV replication in vivo and has allowed estimates of the clearance/replication rate of CMV to be performed. These data indicate that CMV replicates dynamically in the human host with a doubling time of approximately 1 day. This knowledge has been used to determine the relative contribution of initial viral load and rate of change of viral load as predictors of CMV disease in organ transplant recipients. The data show that both these parameters have prognostic value in multivariate models and should allow the development of novel patient management strategies.
Nejati A, Shoja Z, Shahmahmoodi S, Tafakhori A, Mollaei-Kandelous Y, Rezaei F, Hamid KM, Mirshafiey A, Doosti R, Sahraian MA, Mahmoudi M, Shokri F, Emery V, Marashi SM (2015) EBV and vitamin D status in relapsing-remitting multiple sclerosis patients with a unique cytokine signature, MEDICAL MICROBIOLOGY AND IMMUNOLOGY205(2)pp. 143-154 SPRINGER
Griffiths PD, Emery VC (2014) Taming the transplantation troll by targeting terminase, New England Journal of Medicine370(19)pp. 1844-1846
Manicklal S, Emery VC, Lazzarotto T, Boppana SB, Gupta RK (2013) The "Silent" global burden of congenital cytomegalovirus, Clinical Microbiology Reviews26(1)pp. 86-102
Human cytomegalovirus (CMV) is a leading cause of congenital infections worldwide. In the developed world, following the virtual elimination of circulating rubella, it is the commonest nongenetic cause of childhood hearing loss and an important cause of neurodevelopmental delay. The seroprevalence of CMV in adults and the incidence of congenital CMV infection are highest in developing countries (1 to 5% of births) and are most likely driven by nonprimary maternal infections. However, reliable estimates of prevalence and outcome from developing countries are not available. This is largely due to the dogma that maternal preexisting seroimmunity virtually eliminates the risk for sequelae. However, recent data demonstrating similar rates of sequelae, especially hearing loss, following primary and nonprimary maternal infection have underscored the importance of congenital CMV infection in resource-poor settings. Although a significant proportion of congenital CMV infections are attributable to maternal primary infection in well-resourced settings, the absence of specific interventions for seronegative mothers and uncertainty about fetal prognosis have discouraged routine maternal antibody screening. Despite these challenges, encouraging results from prototype vaccines have been reported, and the first randomized phase III trials of prenatal interventions and prolonged postnatal antiviral therapy are under way. Successful implementation of strategies to prevent or reduce the burden of congenital CMV infection will require heightened global awareness among clinicians and the general population. In this review, we highlight the global epidemiology of congenital CMV and the implications of growing knowledge in areas of prevention, diagnosis, prognosis, and management for both low (50 to 70%)- and high (>70%)-seroprevalence settings. © 2013, American Society for Microbiology. All Rights Reserved.
Akter P, Cunningham C, McSharry BP, Dolan A, Addison C, Dargan DJ, Hassan-Walker AF, Emery VC, Griffiths PD, Wilkinson GWG, Davison AJ (2003) Two novel spliced genes in human cytomegalovirus, JOURNAL OF GENERAL VIROLOGY84pp. 1117-1122 SOC GENERAL MICROBIOLOGY
Emery VC (1992) Baculovirus expression vectors : choice of expression vector., Methods Mol Biol8pp. 287-307
Baculoviruses have been used for many years as effective pest-control agents; however, their interest to molecular virologists stems from their exploitation as helper-independent viral expression vectors for the production of proteins in a eukaryotic environment. The pioneering work that led to the development of the system came from the laboratory of Max Summers at Texas A & M, USA. Since its inception, the system has been increasingly utilized to express a broad range of eukaryotic proteins (reviewed in ref. 1) and has been extensively modified (2), in particular by David Bishop and Bob Possee at the Institute of Virology and Environmental Microbiology, Oxford, UK. This work has led to the construction of single and multiple baculovirus expression vectors. Extensive reviews on the subject of baculoviruses are available (1,3-5), including a manual of methods specific for their use as expression systems (6). Chapters 26 - 28 aim to provide a practical account of baculoviruses as expression vectors, along with the experimental methodologies required to produce a baculovirus transfer vector containing the gene to be expressed, the generation and selection of a recombinant expressing this gene product, and the methods available for processing downstream the baculovirus-produced protein.
Manuel O, Pang X, Preiksaitis JK, Emery VC, Kumar D, Asberg A, Hartmann A, Pescovitz MD, Bignamini AA, Rollag H, Jardine AG, Humar A (2008) A prospective study of viral genetic polymorphisms in CMV glycoprotein B and their association with clinical and virologic outcomes in patients with CMV disease, AMERICAN JOURNAL OF TRANSPLANTATION8pp. 184-184 WILEY-BLACKWELL
Dolan A, Cunningham C, Hector RD, Hassan-Walker AF, Lee L, Addison C, Dargan DJ, McGeoch DJ, Gatherer D, Emery VC, Griffiths PD, Sinzger C, McSharry BP, Wilkinson GWG, Davison AJ (2004) Genetic content of wild-type human cytomegalovirus, JOURNAL OF GENERAL VIROLOGY85pp. 1301-1312 SOC GENERAL MICROBIOLOGY
Whalley SA, Brown D, Webster GJM, Jacobs R, Teo CG, Emery VC, Dusheiko GM (2002) Evolution of hepatitis B virus during primary infection, GUT50pp. A118-A118 BRITISH MED JOURNAL PUBL GROUP
Van Damme E, Sauviller S, Lau B, Kesteleyn B, Griffiths P, Burroughs A, Emery V, Sinclair J, Van Loock M (2015) Glucocorticosteroids trigger reactivation of human cytomegalovirus from latently infected myeloid cells and increase the risk for HCMV infection in D+R+ liver transplant patients, Journal of General Virology96(1)pp. 131-143
© 2015 The Authors.Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediateearly promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R- group.
Emery VC (2001) Cytomegalovirus and the aging population., Drugs Aging18(12)pp. 927-933
Human cytomegalovirus (HCMV) has the largest genome of any virus known to infect man. The virus has evolved many strategies to manipulate the host immune systems and so can remain latent and evade important immune responses. The human host mounts a substantial immune response against the virus, with up to 1% of the virus-specific CD8+ T cells being directed against specific epitopes. Acquisition of HCMV occurs progressively from an early age, and in developed countries the overall seroprevalence is approximately 60%. In contrast, specific communities such as gay men, lower socioeconomic groups and people residing in developing countries have seroprevalence rates that can exceed 90%. It is a widely held belief that successful control of viral infections decreases with increasing age because of a reduction in the capacity of the immune system. Studies in aging populations have shown a specific expansion of the CD8+, CD28- and CD57+ subset of cells in patients who are HCMV-seropositive. Prior infection with HCMV has also been associated with a significantly increased number of CD4+ and CD8+ lymphocytes, as well as cells expressing CD56 and HLA-DR. Thus, HCMV infection can cause substantial perturbations in T cell subsets and these effects persist in the aging population. In the context of solid organ transplantation, older age of both recipients and donors may serve to increase the frequency of donor-positive recipient-positive (D+R+) transplants, which have only a moderate risk of HCMV disease. In the context of HIV infection, age has been a dominant risk factor for progression to AIDS and death. At present, it does not appear that this can be explained by lack of immune control of HCMV in the aging population, although studies have identified prior HCMV infection as a risk factor for AIDS and death independent of age. We await further investigations to determine whether the immune control of HCMV in the elderly patient is as effective as in the younger adult, and whether this is linked to pathological consequences.
Hassan-Walker AF, Cope AV, Griffiths PD, Emery VC (1998) Transcription of the human cytomegalovirus natural killer decoy gene, UL18, in vitro and in vivo., J Gen Virol79 ( Pt 9)pp. 2113-2116
Multiplex RT-PCR analysis of human cytomegalovirus (HCMV) replication in human fibroblasts showed transcription of the natural killer (NK) cell decoy gene, UL18, from 72 h onwards. Transcription of glycoprotein B (gpUL55; a late gene) occurred from early time-points and peaked at 24 h post-infection. UL18 mRNA was also detected in the peripheral blood mononuclear cells of organ transplant recipients with HCMV viraemia, especially those with HCMV DNA virus loads greater than 10(5) genomes/ml whole blood. Thus, UL18 is produced via a low abundance transcript late during the infectious cycle at a time coincidental with the increased risk of NK cell lysis as a consequence of class I HLA down-regulation.
Razonable RR, Emery VC (2004) Management of CMV infection and disease in transplant patients, Herpes11(3)pp. 77-86
The International Herpes Management Forum (IHMF®) has published guidelines for the diagnosis and management of cytomegalovirus (CMV) infection and disease in solid organ (SOT) and haematopoietic stem cell transplant (HSCT) recipients. These recommendations have been updated to include, among others: (1) use of whole blood for the polymerase chain reaction (PCR) diagnosis of CMV infection; (2) CMV load measurements for prognostication and for monitoring response to anti-CMV therapy; (3) valganciclovir prophylaxis in CMV donor-positive/recipient-negative (D+/R-) SOT patients for prevention of CMV disease; (4) oral ganciclovir prophylaxis, in preference to aciclovir, to reduce incidence of CMV disease in SOT patients; (5) pre-emptive therapy with oral ganciclovir to reduce incidence of CMV disease and viraemia in liver transplant patients; (6) valaciclovir prophylaxis, in preference to high-dose oral aciclovir, to prevent CMV infection in allogeneic HSCT patients; and (7) foscarnet as an alternative to intravenous ganciclovir for pre-emptive treatment of CMV infection in allogeneic HSCT patients. New developments in the field requiring further research were highlighted, including: optimal frequency of CMV monitoring in CMV D+/R- SOT patients; optimal duration of prophylaxis for the prevention of late CMV disease; need for an acceptable viral threshold for initiation of pre-emptive therapy; and assessment of the clinical efficacy of valganciclovir for the treatment of CMV disease and as pre-emptive therapy in SOT and HSCT patients. This article presents supporting evidence for these recommendations and statements.
Clark DA, Ait-Khaled M, Wheeler AC, Kidd IM, McLaughlin JE, Johnson MA, Griffiths PD, Emery VC (1996) Quantification of human herpesvirus 6 in immunocompetent persons and post-mortem tissues from AIDS patients by PCR., J Gen Virol77 ( Pt 9)pp. 2271-2275
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.
Griffiths PD, Feinberg JE, Fry J, Sabin C, Dix L, Gor D, Ansari A, Emery VC (1998) The effect of valaciclovir on cytomegalovirus viremia and viruria detected by polymerase chain reaction in patients with advanced human immunodeficiency virus disease, JOURNAL OF INFECTIOUS DISEASES177(1)pp. 57-64 UNIV CHICAGO PRESS
Mattes FM, Hainsworth EG, Geretti AM, Nebbia G, Prentice G, Potter M, Burroughs AK, Sweny P, Hassan-Walker AF, Okwuadi S, Sabin C, Amooty G, Brown VS, Grace SC, Emery VC, Griffiths PD (2004) A randomized, controlled trial comparing ganciclovir to ganciclovir plus foscarnet (each at half dose) for preemptive therapy of cytomegalovirus infection in transplant recipients., J Infect Dis189(8)pp. 1355-1361
Forty-eight patients who provided 2 consecutive blood samples that tested positive for cytomegalovirus DNA by polymerase chain reaction (PCR) were randomized to receive either full-dose ganciclovir (5 mg/kg intravenously [iv] twice daily) or half-dose ganciclovir (5 mg/kg iv once daily) plus half-dose foscarnet (90 mg/kg iv once daily) for 14 days. In the ganciclovir arm, 17 (71%) of 24 patients reached the primary end point of being CMV negative by PCR within 14 days of initiation of therapy, compared with 12 (50%) of 24 patients in the ganciclovir-plus-foscarnet arm (P = .12). Toxicity was greater in the combination-therapy arm. In patients who failed to reach the primary end point, baseline virus load was 0.77 log10 higher, the replication rate before therapy was faster (1.5 vs. 2.7 days), and the viral decay rate was slower (2.9 vs. 1.1 days) after therapy. Bivariable logistic regression models identified baseline virus load, bone-marrow transplantation, and doubling time and half-life of decay as the major factors affecting response to therapy within 14 days. This study did not support a synergistic effect of ganciclovir plus foscarnet in vivo.
Griffiths PD, Cope AV, Hassan-Walker AF, Emery VC (1999) Diagnostic approaches to cytomegalovirus infection in bone marrow and organ transplantation., Transpl Infect Dis1(3)pp. 179-186
Cytomegalovirus (CMV) continues to be a clinical problem, impairing the overall success rate of transplantation, either through direct involvement of a variety of end-organs or by inducing indirect effects such as graft rejection. We review here how the virus manages to evade host immune responses and replicate extensively in allograft recipients. Recent studies show that the quantity of CMV (viral load) is related directly to the development of CMV disease. We review how clinically significant levels of CMV viral load can be defined and summarize the results of studies showing that a high CMV viral load is the major determinant of CMV disease, explaining the previously reported risk factors of pre-transplant serostatus and the post-transplant detection of CMV viremia.Note
Griffiths PD, Feinberg JE, Fry J, Sabin C, Dix L, Gor D, Ansari A, Emery VC (1998) The effect of valaciclovir on cytomegalovirus viremia and viruria detected by polymerase chain reaction in patients with advanced human immunodeficiency virus disease. AIDS Clinical Trials Group Protocol 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group., J Infect Dis177(1)pp. 57-64
Samples of blood and urine were collected at baseline, week 4, and week 8 and then every 8 weeks from 310 patients entering a controlled trial of prophylaxis with valaciclovir versus acyclovir. Samples were tested under code by polymerase chain reaction (PCR) in one laboratory. The median number of samples collected from each patient was 5 for blood (range, 0-15) and 5 for urine (range, 0-15). Both baseline PCR viremia and PCR viruria were significantly associated with future cytomegalovirus (CMV) disease (P = .002 and P = .02, respectively). The greatest effect of valaciclovir on CMV disease was seen in patients who were PCR-positive in blood at baseline (P = .002), although a significant effect was also seen in those who were PCR-negative in urine (P = .02). Thus, PCR viremia provides prognostic information about CMV disease in AIDS patients, and valaciclovir showed activity as both a preemptive and prophylactic agent.
Slyker JA, Lohman-Payne BL, Rowland-Jones SL, Otieno P, Maleche-Obimbo E, Richardson B, Farquhar C, Mbori-Ngacha D, Emery VC, John-Stewart GC (2009) The detection of cytomegalovirus DNA in maternal plasma is associated with mortality in HIV-1-infected women and their infants, AIDS23(1)pp. 117-124 LIPPINCOTT WILLIAMS & WILKINS
Bowen EF, Wilson P, Atkins M, Madge S, Griffiths PD, Johnson MA, Emery VC (1995) Natural history of untreated cytomegalovirus retinitis., Lancet346(8991-8992)pp. 1671-1673
Cytomegalovirus infection is common in patients with AIDS, and often causes retinitis. Treatment is rarely curative, but the progression of retinitis is delayed. The untreated course of cytomegalovirus retinitis in AIDS is unknown. We report a 35-year-old man with retinitis who refused treatment. Retinitis resulted in blindness within 6 months. Measurement of cytomegalovirus genomes showed an increasing viral load in blood and urine.
Atkinson C, Emery VC, Griffiths PD (2014) Development of a novel single tube nested PCR for enhanced detection of cytomegalovirus DNA from dried blood spots, Journal of Virological Methods196pp. 40-44
Newborn screening for congenital cytomegalovirus (CCMV) using dried blood spots (DBS) has been proposed because many developed countries have DBS screening programmes in place for other diseases. The aim of this study was to develop a rapid, single tube nested polymerase chain reaction (PCR) method for enhanced detection of CMV from DBS compared to existing (single target) real time PCRs. The new method was compared with existing real time PCRs for sensitivity and specificity. Overall sensitivity of the single target PCR assays in both asymptomatic and symptomatic infants with laboratory confirmed congenital CMV was 69% (CMV PCR or culture positive before day 21 of life). In contrast, the single tube nested assay had an increased sensitivity of 81% with100% specificity. Overall the assay detected CMV from a DBS equivalent to an original blood sample which contained 500. IU/ml. In conclusion this single tube nested methodology allows simultaneous amplification and detection of CMV DNA in 1.5. h removing the associated contamination risk of a two step nested PCR. Owing to its increased sensitivity, it has the potential to be used as a screening assay and ultimately allow early identification and intervention for children with congenital CMV. © 2013 Elsevier B.V.
Mattes FM, Vargas A, Kopycinski J, Hainsworth EG, Sweny P, Nebbia G, Bazeos A, Lowdell M, Klenerman P, Phillips RE, Griffiths PD, Emery VC (2008) Functional impairment of cytomegalovirus specific CD8 T cells predicts high-level replication after renal transplantation., Am J Transplant8(5)pp. 990-999
Human cytomegalovirus (HCMV) remains an important cause of morbidity after allotransplantation, causing a range of direct effects including hepatitis, pneumonitis, enteritis and retinitis. A dominant risk factor for HCMV disease is high level viral replication in blood but it remains unexplained why only a subset of patients develop such diseases. In this detailed study of 25 renal transplant recipients, we show that functional impairment of HCMV specific CD8 T cells in the production of interferon gamma was associated with a 14-fold increased risk of progression to high level replication. The CD8 T-cell impairment persisted during the period of high level replication and was more prominent in patients above 40 years of age (odds ratio = 1.37, p = 0.01) and was also evident in dialysis patients. Threshold levels of functional impairment were associated with an increased risk of future HCMV replication and there was a direct relationship between the functional capacity of HCMV ppUL83 CD8 T cells and HCMV load (R(2)= 0.83). These results help to explain why a subset of seropositive individuals develop HCMV replication and are at risk of end-organ disease and may facilitate the early identification of individuals who would benefit from targeted anti-HCMV therapy after renal transplantation.
Ullman CG, Haris PI, Kell B, Cason J, Jewers RJ, Best JM, Emery VC, Perkins SJ (1994) Hypothetical structure of the membrane-associated E5 oncoprotein of human papillomavirus type 16., Biochem Soc Trans22(4)
Griffiths PD, Ait-Khaled M, Bearcroft CP, Clark DA, Quaglia A, Davies SE, Burroughs AK, Rolles K, Kidd IM, Knight SN, Noibi SM, Cope AV, Phillips AN, Emery VC (1999) Human herpesviruses 6 and 7 as potential pathogens after liver transplant: prospective comparison with the effect of cytomegalovirus., J Med Virol59(4)pp. 496-501
Because cytomegalovirus (CMV) is an important opportunistic infection after liver transplant, we conducted a prospective study to see if the same applied to human herpesviruses (HHV)-6 and -7. We used polymerase chain reaction (PCR) methods optimised to detect active, not latent, infection and studied patients not receiving antiviral prophylaxis for CMV. Post-transplant, 536 blood samples were tested by PCR (median 7; range 4-50). Active infection with CMV was detected in 28/60 (47%), HHV-6 in 19/60 (32%), and HHV-7 in 29/60 (48%) of patients. The PCR-positive samples were tested by quantitative-competitive PCR to measure the virus load of each betaherpesvirus. The median peak virus load for CMV was significantly greater than that for HHV-6 or HHV-7. Detailed clinicopathological analyses for the whole population showed that CMV and HHV-6 were each significantly associated with biopsy-proven graft rejection. Individual case histories suggested that HHV-6 and HHV-7 may be the cause of some episodes of hepatitis and pyrexia. It is concluded that HHV-6 is a previously unrecognized contributor to the morbidity of liver transplantation, that HHV-7 may also be important and that both viruses should be included in the differential diagnosis of graft dysfunction.
Ullman CG, Harris PI, Smith KF, Sim RB, Emery VC, Perkins SJ (1996) beta-Sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy (vol 371, pg 199, 1995), FEBS LETTERS379(2)pp. 201-201 ELSEVIER SCIENCE BV
Ljungman P, Wang FZ, Clark DA, Emery VC, Remberger M, Ringden O, Linde A (2000) High levels of human herpesvirus 6 DNA in peripheral blood leucocytes are correlated to platelet engraftment and disease in allogeneic stem cell transplant patients, BRITISH JOURNAL OF HAEMATOLOGY111(3)pp. 774-781 WILEY-BLACKWELL
Fox JC, Kidd IM, Griffiths PD, Sweny P, Emery VC (1995) Longitudinal analysis of cytomegalovirus load in renal transplant recipients using a quantitative polymerase chain reaction: correlation with disease., J Gen Virol76 ( Pt 2)pp. 309-319
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.
Emery VC, Sabin C, Feinberg JE, Grywacz M, Knight S, Griffiths PD (1999) Quantitative effects of valacyclovir on the replication of cytomegalovirus (CMV) in persons with advanced human immunodeficiency virus disease: baseline CMV load dictates time to disease and survival. The AIDS Clinical Trials Group 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group., J Infect Dis180(3)pp. 695-701
Virus load is a major risk factor for disease in many human viral infections, especially human immunodeficiency virus (HIV) disease. The effect of cytomegalovirus (CMV) load on disease progression and the influence of antiviral chemotherapy on surrogate markers of replication was investigated in 310 patients with advanced HIV disease in a randomized controlled trial that compared the effects of valacyclovir with those of acyclovir. Sequential blood and urine samples were analyzed by polymerase chain reaction (PCR), for human CMV (HCMV) DNA. In multivariate analyses, elevated virus load in both blood and urine at baseline was associated with increased risk of HCMV disease (relative hazard, 1.49 and 1.44 per log increase, respectively). Elevated virus load in blood at baseline was also associated with a significantly shorter survival time (log rank, P=. 0001). In time-updated analyses, valacyclovir significantly suppressed the virus load in subjects who were PCR positive at baseline (in blood or urine), when compared with the combined acyclovir arms.
ULLMAN CG, HARIS PI, SMITH KF, SIM RB, EMERY VC, PERKINS SJ (1995) BETA-SHEET SECONDARY STRUCTURE OF AN LDL RECEPTOR DOMAIN FROM COMPLEMENT FACTOR-I BY CONSENSUS STRUCTURE PREDICTIONS AND SPECTROSCOPY, FEBS LETTERS371(2)pp. 199-203 ELSEVIER SCIENCE BV
Emery VC (2000) Tuning to the right frequency: cytotoxic T lymphocytes and cytomegalovirus., Transplantation69(11)pp. 2241-2242
Fabio G, Knight SN, Kidd IM, Noibi SM, Johnson MA, Emery VC, Griffiths PD, Clark DA (1997) Prospective study of human herpesvirus 6, human herpesvirus 7, and cytomegalovirus infections in human immunodeficiency virus-positive patients., J Clin Microbiol35(10)pp. 2657-2659
Blood samples from human immunodeficiency virus (HIV)-positive patients were monitored for cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and HHV-7 by PCR. We detected CMV in 17% of the patients, HHV-6 in 6%, and HHV-7 in 3%. The viral loads of CMV were significantly higher than those of HHV-6 (P = 0.007) or HHV-7 (P = 0.01). Detection of CMV and HHV-6 was associated with low and high CD4 counts, respectively.
Emery VC, Rose J, Manuel O, Humar A, Asberg A, Neumann A (2012) A Full Dynamic Model To Explain Declines in Cytomegalovirus (CMV) Load after Ganciclovir Therapy in Patients with CMV Disease, AMERICAN JOURNAL OF TRANSPLANTATION12pp. 69-69 WILEY-BLACKWELL
Slyker JA, Lohman-Payne BL, John-Stewart GC, Maleche-Obimbo E, Emery S, Richardson B, Dong T, Iversen AKN, Mbori-Ngacha D, Overbaugh J, Emery VC, Rowland-Jones SL (2009) Acute cytomegalovirus infection in Kenyan HIV-infected infants, AIDS23(16)pp. 2173-2181 LIPPINCOTT WILLIAMS & WILKINS
Ullman CG, Chamberlain D, Ansari A, Emery VC, Haris PI, Sim RB, Perkins SJ (1998) Human complement factor I: its expression by insect cells and its biochemical and structural characterisation., Mol Immunol35(9)pp. 503-512
Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.
Clark DA, Kidd IM, Collingham KE, Tarlow M, Ayeni T, Riordan A, Griffiths PD, Emery VC, Pillay D (1997) Diagnosis of primary human herpesvirus 6 and 7 infections in febrile infants by polymerase chain reaction., Arch Dis Child77(1)pp. 42-45
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.
Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker AF, Griffiths PD (2000) Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation., Lancet355(9220)pp. 2032-2036
BACKGROUND: Cytomegalovirus (CMV) continues to be a major problem post-transplantation; early markers for predicting patients at risk of CMV disease are needed. Peak CMV load in the blood correlates with CMV disease but frequently occurs too late to provide prognostic information. METHODS: 359 transplant recipients (162 liver, 87 renal, and 110 bone marrow) were prospectively monitored for CMV DNA in the blood with qualitative and quantitative PCR. 3873 samples were analysed. The CMV load in the first PCR-positive sample and the rate of increase in CMV load in blood during the initial phase of replication were assessed as risk factors for CMV disease using logistic regression. FINDINGS: 127 of the 359 patients had CMV DNA in the blood and 49 developed CMV disease. Initial viral load correlated significantly with peak CMV load (R2=0.47, p=<0.001) and with CMV disease (odds ratio 1.82 [95% CI 1.11-2.98; p=0.02; 1.34 [1.07-1.68], p=0.01, and 1.52 [1.13-2.05], p=0.006, per 0.25 log10 increase in viral load for liver, renal, and bone-marrow patients, respectively). The rate of increase in CMV load between the last PCR-negative and first PCR-positive sample was significantly faster in patients with CMV disease (0.33 log10 versus 0.19 log10 genomes/mL daily, p<0.001). In multivariate-regression analyses, both initial CMV load and rate of viral load increase were independent risk factors for CMV disease (1.28 [1.06-1.52], p=0.01, per 0.25 log10 increase in CMV load and 1.52 [1.06-2.17], p=0.02, per 0.1 log10 increase in CMV load/mL daily, respectively). INTERPRETATION: CMV load in the initial phase of active infection and the rate of increase in viral load both correlate with CMV disease in transplant recipients; in combination, they have the potential to identify patients at imminent risk of CMV disease.
Kidd IM, Clark DA, Sabin CA, Andrew D, Hassan-Walker AF, Sweny P, Griffiths PD, Emery VC (2000) Prospective study of human betaherpesviruses after renal transplantation: association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection., Transplantation69(11)pp. 2400-2404
BACKGROUND: Human herpesvirus 6 (HHV-6) and HHV-7 are two lymphotropic herpesviruses, which, like cytomegalovirus (CMV), have the potential to be pathogenic in immunocompromised individuals. We have conducted a prospective investigation to compare the natural history of HHV-6 and HHV-7 infection with that of CMV after renal transplantation. METHODS: Polymerase chain reaction was used to identify infections and quantify the viral load of CMV, HHV-6, and HHV-7 in peripheral blood samples from 52 renal transplant recipients. Betaherpesvirus infections were related to defined clinical criteria obtained by detailed examination of the clinical records of each patient for the immediate 120-day posttransplant period. RESULTS: CMV was the most commonly detected virus after transplant (58% of patients), followed by HHV-7 (46%) and HHV-6 (23%). Examining the time to first polymerase chain reaction positivity, HHV-7 infection was detected earlier than CMV (P=0.05). The median maximum CMV viral load was significantly higher than those for HHV-6 (P=0.01) and HHV-7 (P<0.0001) and a trend for HHV-7 viral load to be greater than HHV-6 (P=0.08). Clinicopathological analyses revealed that, in those patients with rejection, HHV-7 was associated with more episodes of rejection (P=0.02). In addition, there was a significant increase in CMV disease occurring in patients with CMV and HHV-7 co-infection compared to those with CMV infection only (P=0.04). CONCLUSIONS: HHV-7 should be further investigated as a possible co-factor in the development of CMV disease in renal transplant patients and may potentially exacerbate graft rejection. No clear pathological role was observed for HHV-6.
Atkins M, Strappe P, Kaye S, Loveday C, McLaughlin JE, Johnson MA, Tedder RS, Griffiths PD, Emery VC (1998) Quantitative differences in the distribution of zidovudine resistance mutations in multiple post-mortem tissues from AIDS patients., J Med Virol55(2)pp. 138-146
Replication of HIV introduces errors into the genome which are responsible for conferring a growth advantage over wildtype virus when drugs such as zidovudine (ZDV) exert a selective pressure. The molecular basis for HIV-1 resistance to ZDV has been mapped to codons 41, 67, 70, 215 and 219 of the reverse transcriptase gene both in vitro and in clinical samples of blood. This study has investigated the relationship between the quantitative prevalence of ZDV resistance in multiple organs of the same individual. Proviral HIV-1 load was measured by quantitative-competitive PCR in 90 samples from organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine. The distribution of wildtype and mutant sequences at the positions 41, 67, 70, 215 and 219 of the reverse transcriptase was assessed using a point mutation assay. The results showed that the highest proviral loads were predominately found in lymph node, spleen and lung and there was a significant association between viral load and resistance to ZDV (P=0.008). Inter-organ distribution of wildtype and mutant sequences at codons 41, 67, 70, 215 and 219 was frequently not uniform and in some patients differed markedly between the lymphoreticular system and other organs. These results demonstrate that treatment of HIV-1 infection with zidovudine does not exert uniform selective pressures in multiple organs. These findings have implications for the interpretation of resistance data and design of treatment strategies for HIV, arguing in particular that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in multiple sites within the same individual.
Emery V (2007) Facing the facts: The indirect effects of cytomegalovirus, TRANSPLANTATION84(6)pp. S7-S10 LIPPINCOTT WILLIAMS & WILKINS
Ullman CG, Haris PI, Smith KF, Sim RB, Emery VC, Perkins SJ (1995) ²-Sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy, FEBS Letters371(2)pp. 199-203
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 ²-strands was predicted. Sequence threading against known protein folds indicated consistency with small ²-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 ²-sheet together with turns and loops. LDLr is proposed to have a ²-sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop. © 1995.
Hassan-Walker AF, Kidd IM, Sabin C, Sweny P, Griffiths PD, Emery VC (1999) Quantity of human cytomegalovirus (CMV) DNAemia as a risk factor for CMV disease in renal allograft recipients: relationship with donor/recipient CMV serostatus, receipt of augmented methylprednisolone and antithymocyte globulin (ATG)., J Med Virol58(2)pp. 182-187
A prospective longitudinal study of 87 renal allograft recipients identified 31 patients with cytomegalovirus (CMV) viraemia. Previous studies have identified CMV viraemia, donor positivity, and CMV load in urine as independent risk factors for disease following renal transpl antation. We used quantitative-competitive polymerase chain reaction (QC-PCR) to quantify the CMV DNA load in blood from these patients, and report that it is a significant and independent risk factor for CMV disease. Patients with symptomatic CMV infection had significantly higher maximum CMV loads than those with no disease (P = .0003). We also found that peak loads were significantly higher in individuals experiencing primary CMV infection (P < .01), and CMV re-infection (P < .05) compared with recipients reactivating endogenous CMV. Univariate analysis revealed that CMV DNA load in blood, donor seropositivity, and receipt of antithymocyte globulin (ATG) were all significantly associated with disease (P = .005, .04, and .05, respectively). However, the association of donor/recipient serostatus, and receipt of ATG became nonsignificant in multivariate analyses whereas the significance of the quantity of CMV DNAemia was maintained, illustrating that CMV load plays a central role in the pathogenesis of CMV disease.
Bavin PJ, Giles JA, Hudson E, Williams D, Crow J, Griffiths PD, Emery VC, Walker PG (1992) Comparison of cervical cytology and the polymerase chain reaction for HPV 16 to identify women with cervical disease in a general practice population., J Med Virol37(1)pp. 8-12
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.
Zanghellini F, Boppana SB, Emery VC, Griffiths PD, Pass RF (1999) Asymptomatic primary cytomegalovirus infection: Virologic and immunologic features, JOURNAL OF INFECTIOUS DISEASES180(3)pp. 702-707 OXFORD UNIV PRESS INC
Nebbia G, Mattes FM, Sabin CA, Samonakis D, Rolando N, Burroughs AK, Emery VC (2007) Differential effects of prednisolone and azathioprine on the development of human cytomegalovirus replication post liver transplantation., Transplantation84(5)pp. 605-610
BACKGROUND: We sought to investigate the impact of different immunosuppressive regimens on human cytomegalovirus (HCMV) incidence and replication dynamics in a cohort of 256 patients after liver transplantation. METHODS: A time-updated approach was used to determine the risk of developing HCMV replication (>200 genomes/mL blood) within the first 100 days after liver transplantation according to the immunosuppressive regimen being received at specific time points. RESULTS: In patients receiving tacrolimus, the addition of prednisolone was associated with a significant increased risk of HCMV replication both at baseline (relative rate of infection [RRI]=4.34; P=0.0001) and in a time-updated analysis (RRI=4.68; P=0.0001). However, the addition of azathioprine substantially reduced the risk of HCMV replication to that observed with tacrolimus alone. As expected donor/recipient HCMV serostatus was also a risk factor for viraemia. Multivariable models showed that the tacrolimus plus prednisolone regimen and donor/recipient serostatus were independent risk factors for HCMV replication. Viral replication dynamics showed that the duration of HCMV viraemia, the peak viral load, and the growth rate of HCMV were greatest in patients receiving tacrolimus plus prednisolone although these differences did not reach statistical significance. CONCLUSIONS: The combination of prednisolone plus tacrolimus as baseline immunosuppression after liver transplantation is associated with a high risk of HCMV replication. This effect can be negated by the addition of azathioprine.
Ait-Khaled M, Emery VC (1993) Sequence variation within the human immunodeficiency virus V3 loop at seroconversion., J Med Virol41(4)pp. 270-274
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.
Papagno L, Spina CA, Marchant A, Salio M, Rufer N, Little S, Dong T, Chesney G, Waters A, Easterbrook P, Dunbar PR, Shepherd D, Cerundolo V, Emery V, Griffiths P, Conlon C, McMichael AJ, Richman DD, Rowland-Jones SL, Appay V (2004) Immune activation and CD8(+) T-cell differentiation towards senescence in HIV-1 infection, PLOS BIOLOGY2(2)ARTN e20pp. 173-185 PUBLIC LIBRARY SCIENCE
Whalley SA, Murray JM, Brown D, Webster GJM, Emery VC, Dusheiko GH, Perelson AS (2000) The kinetics of acute hepatitis B virus infection in humans., HEPATOLOGY32(4)pp. 446A-446A W B SAUNDERS CO
Kopycinski J, Osman M, Griffiths PD, Emery VC (2010) Sequence flexibility of the immunodominant HLA A*0201 restricted ppUL83 CD8 T-cell epitope of human cytomegalovirus., J Med Virol82(1)pp. 94-103
The cytomegalovirus ppUL83 protein contains an immunodominant A*0201 restricted epitope between residues 495 and 503. We investigated the tolerance of this epitope to sequence variation in the context of peptide binding to HLA A*0201 and the ability to induce an Interferon gamma (IFNgamma) response through engagement with the T-cell receptor (TCR). The majority of mutations investigated resulted in a decrease in the production of IFNgamma indicating that if such variants occurred in vivo they would not be recognized by CD8 T-cell clones specific for the wild-type epitope. The mechanistic basis for the majority of the mutant peptides was their failure to bind and stabilize class I HLA cell surface expression. However, one peptide with a mutation at the P5 position (methionine to cysteine) resulted in a significant enhanced binding to HLA A*0201 and also an increase in cell surface expression over the wild-type peptide but was unable to engage with the CD8 TCR and trigger IFNgamma production. This peptide acted as a competitive inhibitor of the wild-type peptide but could not fully inhibit IFNgamma production by the latter. We subsequently investigated whether mutations of the HLA A*0201 epitope were evident in immunocompromized patients experiencing either rapid exponential or persistent cytomegalovirus replication.
Mattes FM, Hainsworth EG, Hassan-Walker AF, Burroughs AK, Sweny P, Griffiths PD, Emery VC (2004) Kinetics of CMV load reductions following pre-emptive therapy with valganciclovir or intravenous ganciclovir in adult solid organ transplant patients, AMERICAN JOURNAL OF TRANSPLANTATION4pp. 535-535 WILEY-BLACKWELL
Emery VC, Manuel O, Asberg A, Pang X, Kumar D, Hartmann A, Preiksaitis JK, Pescovitz MD, Rollag H, Jardine AG, Gahlemann CG, Humar A (2012) Differential decay kinetics of human cytomegalovirus glycoprotein B genotypes following antiviral chemotherapy, JOURNAL OF CLINICAL VIROLOGY54(1)pp. 56-60 ELSEVIER SCIENCE BV
Samonakis DN, Triantos CK, Thalheimer U, Quaglia A, Leandro G, Teixeira R, Papatheodoridis GV, Sabin CA, Rolando N, Davies S, Dhillon AP, Griffiths P, Emery V, Patch DW, Davidson BR, Rolles K, Burroughs AK (2005) Immunosuppression and donor age with respect to severity of HCV recurrence after liver transplantation., Liver Transpl11(4)pp. 386-395
In HCV cirrhotic patients after liver transplantation, survival and recurrence of HCV appears to be worsening in recent years. Donor age has been suggested as a cause. However, it is not clear if early and/or late mortality is affected and whether donor age is a key factor, as opposed to changes in immunosuppression. The aim of this study was to assess impact of donor age and other factors with respect to the severity of HCV recurrence posttransplant. A consecutive series of 193 HCV cirrhotic patients were transplanted with cadaveric donors, median age 41.5 years (13-73) and median follow-up of 38 months (1-155). Donor age and other factors were examined in a univariate/multivariate model for early/late survival, as well as fibrosis (grade 4 or more, Ishak score) with regular biopsies, 370 in total, from 1 year onwards. Results of the study indicated that donor age influenced only short-term (3 months) survival, with no significant effect on survival after 3 months. Known HCC independently adversely affected survival, as did the absence of maintenance azathioprine. Severe fibrosis (stage > or = 4) in 51 patients was related to neither donor age nor year of transplantation, but it was independently associated with combined biochemical/histological hepatitis flare (OR 2.9, 95% CI 1.76-4.9) whereas maintenance steroids were protective (OR 0.4, 95% CI 0.23-0.83). In conclusion, in this cohort donor age did not influence late mortality in HCV transplanted cirrhotic patients or development of severe fibrosis, which was related to absence of maintenance steroids and a hepatitis flare. Maintenance azathioprine gave survival advantage.
Emery VC, Einsele H, Atabani S, Haque T (2010) Immunotherapy and vaccination after transplant: the present, the future., Infect Dis Clin North Am24(2)pp. 515-529
Vaccination and adoptive immunotherapy for herpes virus infections has become an attractive option for the control of a virus family that negatively affects transplantation. In the future, enhanced ability to select antigen-specific T cells without significant in vitro manipulation should provide new opportunities for refining and enhancing adoptive immunotherapeutic approaches. This article focuses on advances in the area of vaccinology for some of these infections and in the use of adoptive immunotherapy. At present, many of these approaches in transplant recipients have focused on infections such as human cytomegalovirus, but the opportunity to use these examples as proof of concept for other infections is discussed.
Soderberg-Naucler C, Emery VC (2001) Viral infections and their impact on chronic renal allograft dysfunction, TRANSPLANTATION71(11)pp. SS24-SS30 LIPPINCOTT WILLIAMS & WILKINS
O'Neil C, Lee D, Clewley G, Johnson MA, Emery VC (1997) Prevalence of anti-vif antibodies in HIV-1 infected individuals assessed using recombinant baculovirus expressed vif protein., J Med Virol51(3)pp. 139-144
A 630 base pair fragment of the HIV-1 genome encompassing the entire vif open reading frame has been produced by the polymerase chain reaction and cloned into the baculovirus transfer vector pAcYM1. Extracts from insect cells infected with a recombinant baculovirus expressing the HIV-1 vif gene product were used in a radioimmunoassay to analyse 238 sera from HIV infected individuals for the presence of anti-vif antibodies. The overall prevalence of anti-vif antibodies in this group of patients was 25.3%. Stratification of the group according to CD4 levels showed that anti-vif antibodies were more prevalent in patients with CD4 counts below the median of the group (155 x 10(6) cells/L; P = 0.005). A significant increase in anti-vif antibodies was observed in patients with CD4 levels less than 280 x 10(6) cells/L (P < 0.01) and in patients with symptomatic HIV infection (P = 0.0003). However, there was no significant difference in the prevalence of anti-vif antibodies in patients stratified according to p24 antigen status. The implications of these findings in the context of HIV replication are discussed.
Whalley SA, Brown D, Webster GJM, Mattes F, Emery V, Dusheiko GM (1999) Viral dynamics in vivo of acute hepatitis B infection, GUT44pp. A52-A52 BRITISH MED JOURNAL PUBL GROUP
Marashi SM, Raeiszadeh M, Enright V, Tahami F, Workman S, Chee R, Webster AD, Milne RS, Emery VC (2012) Influence of cytomegalovirus infection on immune cell phenotypes in patients with common variable immunodeficiency., J Allergy Clin Immunol129(5)pp. 1349-1356.e3
BACKGROUND: A subset of patients with common variable immunodeficiency (CVID) have debilitating inflammatory complications strongly associated with cytomegalovirus (CMV) infection and a hyperproliferative CMV-specific T-cell response. OBJECTIVES: We studied the T-cell response to CMV and the global effect of this virus on immune effector cell populations in patients with CVID. METHODS: Antibody staining, peptide stimulation, and proliferation assays were used to profile CMV-specific T-cell function. RESULTS: CMV infection drives the CD4/CD8 ratio inversion that is characteristic of CVID. The late effector CD8(+) T-cell subset is expanded in CMV-infected patients with CVID. This expansion is largely attributable to CMV-specific cells and correlates with inflammatory disease; within the CMV-specific population, the frequency of late effector cells correlates inversely with the frequency of cells expressing programmed death 1. Supernatants from proliferating CMV-specific CD8(+) cells from patients with inflammatory disease can confer proliferative potential on cells from patients with noninflammatory CVID and healthy subjects. Blocking experiments showed that this proliferation is mediated in part by IFN-³ and TNF-±. CONCLUSIONS: These data strengthen the association of CMV with inflammatory pathology in patients with CVID, explain some of the well-known T-cell abnormalities associated with this condition, and provide a plausible mechanism for the documented therapeutic activity of anti-TNF-± and antiviral chemotherapy in managing CVID-associated inflammatory disease.
Slyker JA, Rowland-Jones SL, Dong T, Reilly M, Richardson B, Emery VC, Atzberger A, Mbori-Ngacha D, Lohman-Payne BL, John-Stewart GC (2012) Acute Cytomegalovirus Infection Is Associated with Increased Frequencies of Activated and Apoptosis-Vulnerable T Cells in HIV-1-Infected Infants, JOURNAL OF VIROLOGY86(20)pp. 11373-11379 AMER SOC MICROBIOLOGY
Egli A, Lisboa L, O'Shea D, Emery V, Asberg A, Hartmann A, Kumar D, Humar A (2013) A Complex Host MicroRNA Response to Cytomegalovirus Infection: A Bench to Bedside Analysis, AMERICAN JOURNAL OF TRANSPLANTATION13pp. 57-57 WILEY-BLACKWELL
Raeiszadeh M, Kopycinski J, Paston SJ, Diss T, Lowdell M, Hardy GA, Hislop AD, Workman S, Dodi A, Emery V, Webster AD (2006) The T cell response to persistent herpes virus infections in common variable immunodeficiency., Clin Exp Immunol146(2)pp. 234-242
We show that at least half of patients with common variable immunodeficiency (CVID) have circulating CD8(+) T cells specific for epitopes derived from cytomegalovirus (CMV) and/or the Epstein-Barr virus (EBV). Compared to healthy age-matched subjects, more CD8(+) T cells in CVID patients were committed to CMV. Despite previous reports of defects in antigen presentation and cellular immunity in CVID, specific CD4(+) and CD8(+) T cells produced interferon (IFN)-gamma after stimulation with CMV peptides, and peripheral blood mononuclear cells secreted perforin in response to these antigens. In CVID patients we found an association between a high percentage of circulating CD8(+) CD57(+) T cells containing perforin, CMV infection and a low CD4/CD8 ratio, suggesting that CMV may have a major role in the T cell abnormalities described previously in this disease. We also show preliminary evidence that CMV contributes to the previously unexplained severe enteropathy that occurs in about 5% of patients.
Atabani SF, Atkinson CE, Aldridge RW, Harber M, Emery VC, Griffiths PD (2011) Pre-Emptive Therapy for the Prevention of CMV Infection Post Solid Organ Transplantation: The London Experience, AMERICAN JOURNAL OF TRANSPLANTATION11pp. 456-456 WILEY-BLACKWELL
Fox JC, Griffiths PD, Emery VC (1992) Quantification of human cytomegalovirus DNA using the polymerase chain reaction., J Gen Virol73 ( Pt 9)pp. 2405-2408
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.
Phillips AN, McLean A, Johnson MA, Tyrer M, Emery V, Griffiths P, Bofill M, Janossy G, Loveday C (1997) HIV-1 dynamics after transient antiretroviral therapy: implications for pathogenesis and clinical management., J Med Virol53(3)pp. 261-265
Simple models of CD4 lymphocyte interactions with human immunodeficiency virus (HIV) lead to the hypothesis that progression of HIV infection involves an increase in viral replicative capacity, due either to changes in the virus or in the host environment, or both. In order to consider how changes in plasma virus load after transient, potent antiretroviral therapy can be used to test the above hypothesis--a simple mathematical model that encompasses the processes of (1) arrival of new CD4 lymphocytes, (2) death/removal of these cells by HIV-independent mechanisms, (3) infection of susceptible CD4 lymphocytes by HIV, and (4) death/removal of infected cells was investigated. This showed that the in vivo rate of increase in plasma virus load immediately after transient therapy provides a measure of the viral replicative capacity. Thus, the hypothesis that progression of HIV infection involves an increase in viral replicative capacity can be tested by measuring this viral growth rate in patients with high CD4 counts and in patients with low CD4 counts. Studies should thus investigate dynamics of changes in virus levels after stopping antiretroviral therapy and, in particular, measure rates of increase in virus in patients at high and low CD4 counts. In practice, such data may assist in therapeutic management of patients with HIV infection.
Atabani SF, Emery VC, Smith C, Harber M, Thorburn D, Griffiths PD (2012) Response to letter regarding "cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy"., Am J Transplant12(10)pp. 2859-2860
Nebbia G, Mattes FM, Cholongitas E, Garcia-Diaz A, Samonakis DN, Burroughs AK, Emery VC (2007) Exploring the bidirectional interactions between human cytomegalovirus and hepatitis C virus replication after liver transplantation., Liver Transpl13(1)pp. 130-135
Recurrence of Hepatitis C (HCV) post-liver transplantation (LT) is universal and its course is more aggressive than in immunocompetent individuals. Human cytomegalovirus (CMV) infection is a common post-LT infection and has immunomodulatory effects that could adversely affect the outcome of HCV. To date, the effect of HCV replication on the dynamics of CMV have not been investigated. From 2000 to 2004, a cohort of 69 HCV-infected liver transplant recipients and 188 HCV-negative liver transplant recipients (NON-HCV cohort) were monitored for CMV infection twice weekly by CMV polymerase chain reaction (PCR) with preemptive therapy initiated after 2 consecutive positive results. None of the patients received CMV prophylaxis. A subset of 18 HCV-infected patients had their HCV viral load monitored regularly post-LT by quantitative PCR. CMV DNAemia (>200 genomes/mL blood) did not influence the level of HCV replication within 150 days posttransplantation or the stage of liver fibrosis in liver biopsies at 1 yr post-LT. There were no differences in the incidence of CMV DNAemia or replication dynamics in the HCV cohort compared to the NON-HCV cohort. In conclusion, short term CMV viremia does not enhance the replication of HCV after LT, while HCV replication does not alter the replication dynamics of CMV.
Webster A, Emery VC (1996) Developments in anti-herpesvirus therapy, Reviews in Contemporary Pharmacotherapy7(2)pp. 75-89
Developments in the area of anti-herpesvirus research are proceeding along several fronts. Aciclovir is currently the drug of choice for the treatment of herpes simplex virus infections, but has poor oral bioavailability, and lower activity against other members of the herpesvirus group. Two prodrugs with improved oral bioavailability are currently being developed, with the aim of improving efficacy against the less sensitive herpesviruses, and with less frequent dosing. Valaciclovir is a prodrug of aciclovir, and famciclovir is a prodrug of penciclovir, a new nucleotide analogue with similar activity to that of aciclovir. The possible benefits of a variety of different anti-herpesvirus prophylactic regimens are also being assessed in various immunocompromised populations. New nucleoside analogues are also under development, and have reached clinical trial. Sorivudine has high specific activity against varicella zoster virus, and is being investigated for the treatment of shingles. HPMPC has broad spectrum anti-herpesvirus activity, but preliminary data on its safety profile do not appear to support systemic use. Other viral molecular processes are being investigated as targets for antiviral therapy, and many potential compounds are at the early stages of development. Resistance to antiviral agents is an area of concern, and has helped fuel the search for agents with alternative modes of action. However, the correlation between emergence of resistant strains and clinical outcome needs further study.
TUDORWILLIAMS G, EMERY VC (1992) DEVELOPMENT OF INVITRO RESISTANCE TO ZIDOVUDINE IS LIKELY TO BE CLINICALLY SIGNIFICANT, REVIEWS IN MEDICAL VIROLOGY2(3)pp. 123-129 JOHN WILEY & SONS LTD
Atabani SF, Smith C, Atkinson C, Aldridge RW, Rodriguez-Perálvarez M, Rolando N, Harber M, Jones G, O'Riordan A, Burroughs AK, Thorburn D, O'Beirne J, Milne RS, Emery VC, Griffiths PD (2012) Cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy., Am J Transplant12(9)pp. 2457-2464
After allotransplantation, cytomegalovirus (CMV) may be transmitted from the donor organ, giving rise to primary infection in a CMV negative recipient or reinfection in one who is CMV positive. In addition, latent CMV may reactivate in a CMV positive recipient. In this study, serial blood samples from 689 kidney or liver transplant recipients were tested for CMV DNA by quantitative PCR. CMV was managed using preemptive antiviral therapy and no patient received antiviral prophylaxis. Dynamic and quantitative measures of viremia and treatment were assessed. Median peak viral load, duration of viremia and duration of treatment were highest during primary infection, followed by reinfection then reactivation. In patients who experienced a second episode of viremia, the viral replication rate was significantly slower than in the first episode. Our data provide a clear demonstration of the immune control of CMV in immunosuppressed patients and emphasize the effectiveness of the preemptive approach for prevention of CMV syndrome and end organ disease. Overall, our findings provide quantitative biomarkers which can be used in pharmacodynamic assessments of the ability of novel CMV vaccines or antiviral drugs to reduce or even interrupt such transmission.
Deayton JR, Sabin CA, Johnson MA, Griffiths PD, Emery VC (2000) Efficacy of the regenerating immune system in the inhibition of CMV replication after highly active antiretroviral therapy (HAART)), AIDS14pp. S5-S5 LIPPINCOTT WILLIAMS & WILKINS
Buyck HC, Griffiths PD, Emery VC (2010) Human cytomegalovirus (HCMV) replication kinetics in stem cell transplant recipients following anti-HCMV therapy., J Clin Virol49(1)pp. 32-36
BACKGROUND: Cytomegalovirus (HCMV) remains an important infection following stem cell transplantation (SCT) and is managed via pre-emptive therapy. In some patients HCMV loads continue to increase after therapy and they experience multiple episodes of replication. OBJECTIVES: To identify the risk factors associated with failure to immediately control HCMV replication after antiviral therapy and for recurrence of replication. STUDY DESIGN: Replication kinetics of human cytomegalovirus (HCMV) were studied a cohort of 153 T-cell depleted allogeneic SCT patients. RESULTS: In 57 patients (31%) who experienced HCMV DNAemia, the mean growth rate of HCMV was 0.35 day(-1) equivalent to a doubling time of 2.2 days. In patients requiring anti-HCMV treatment with either ganciclovir or ganciclovir/foscarnet (n=49), HCMV load increased to a peak value of >2 days after initiation of therapy in 21 patients and only the growth rate prior to therapy was a risk factor (Odds ratio=1.4 per 0.1 day(-1) increase; p=0.004). In patients where antiviral intervention occurred after peak virus load the decline rate of HCMV load was accelerated 4-fold if the patient was subsequently initiated on anti-HCMV therapy (p=0.02). A subset of patients (38%) experienced a recurrence of their DNAemia at a mean of 20 days after the cessation of their first replication episode and this was only associated with receiving stem cells from a seronegative donor (Odds ratio=6.59; p<0.001). CONCLUSIONS: The kinetics of response to therapy is closely associated with HCMV replication kinetics prior to therapy while recurrence of replication is associated with HCMV serostatus of the donor.
Roy DM, Grundy JE, Emery VC (1993) Sequence variation within neutralizing epitopes of the envelope glycoprotein B of human cytomegalovirus: comparison of isolates from renal transplant recipients and AIDS patients., J Gen Virol74 ( Pt 11)pp. 2499-2505
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.
Deayton JR, Sabin CA, Britt WB, Jones IM, Wilson P, Johnson MA, Griffiths PD, Emery VC (2002) Rapid reconstitution of humoral immunity against cytomegalovirus but not HIV following highly active antiretroviral therapy., AIDS16(16)pp. 2129-2135
OBJECTIVE: To determine the kinetics of reduction in human cytomegalovirus (HCMV) load and specific anti-glycoprotein B (gB) immune responses in patients with concurrent HCMV DNAaemia following the initiation of highly active antiretroviral therapy (HAART). DESIGN: Sequential analysis of eleven patients with HCMV DNAaemia who received HAART and eleven control patients with HCMV DNAaemia. METHODS: HCMV load was measured by quantitative competitive polymerase chain reaction and anti-gB, anti-HIV Env and Gag responses by an end-point dilution immunofluorescence assay using recombinant antigens expressed in insect cells. Estimates of the efficacy of the reconstituting immune system at controlling HCMV replication were based on previous dynamic models. RESULTS: In patients initiating HAART, HCMV DNA levels in blood declined rapidly, with a median half-life of 5.2 days, consistent with an efficacy of the reconstituting immune system at inhibiting HCMV replication of 52.8-85% (median, 61%). Commensurate with this decrease, a significant increase in anti-gB titres was observed in the post-HAART period (corresponding to an average fourfold increase in titre by 1 month rising to an eightfold increase at month 3; = 0.01). No changes in titre were observed in the control group or for anti-HIV Gag antibody levels, while anti-HIV Env antibody levels decreased after HAART. CONCLUSIONS: In patients with HCMV DNAaemia, reconstitution of humoral immunity to HCMV gB occurs rapidly following the initiation of HAART. These changes contrast with the patterns observed for anti-HIV humoral immune responses.
Emery V, Zuckerman M, Jackson G, Aitken C, Osman H, Pagliuca A, Potter M, Peggs K, Clark A (2013) Management of cytomegalovirus infection in haemopoietic stem cell transplantation, British Journal of Haematology162(1)pp. 25-39
Emery VC, Clark DA (2007) HHV-6A, 6B, and 7: Persistence in the population, epidemiology and transmission, pp. 875-882
© Cambridge University Press 2007 and 2009.In common with all human herpesviruses, HHV-6 and HHV-7 establish lifelong infection following initial exposure and seroconversion. True latency as exemplified by HSV-1 and VZV, in which the genome is maintained in a transcriptionally restricted state, has not been conclusively shown for HHV-6 or HHV-7. However, the betaherpesviruses may persist in individuals via low grade replication which is continuously suppressed by a functional immune response. In this chapter we will summarize the current understanding of the epidemiology and persistence of HHV-6 and HHV-7 in the human host and its relevance to transmission. In addition, we will highlight a novel form of persistence for HHV-6 which involves integration into host chromosomal DNA. In the case of both HHV-6 and HHV-7, PCR analysis of peripheral blood mononuclear cells (PBMC) shows that a sensitive nested assay and an adequate quantity of input DNA (at least equivalent to approximately 150000 mononuclear cells or 1?g DNA) can detect viral DNA in healthy immunocompetent individuals suggestive of low levels of latent/persisting virus in peripheral blood (Jarrett et al., 1990; Clark et al., 1996; Kidd et al., 1996). In contrast, viral loads are maintained at high levels in saliva of seropositive individuals, particularly in the case of HHV-7 (Kidd et al., 1996; Fujiwara et al., 2000; see Fig. 49.1).
Marashi SM, Raeiszadeh M, Workman S, Rahbar A, Soderberg-Naucler C, Klenerman P, Chee R, Webster AD, Milne RS, Emery VC (2011) Inflammation in common variable immunodeficiency is associated with a distinct CD8(+) response to cytomegalovirus., J Allergy Clin Immunol127(6)pp. 1385-93.e4
BACKGROUND: Common variable immunodeficiency is the most common primary immunodeficiency. A subset of patients has debilitating inflammatory complications. OBJECTIVES: We investigated the role of cytomegalovirus (CMV), and the T-cell response targeted at this virus, in this inflammatory disease. METHODS: Phenotypic and functional assays were used to profile CMV-specific T cells in patients with common variable immunodeficiency with and without inflammatory complications. Highly sensitive immunohistochemistry was used to detect CMV antigens at sites of inflammation. RESULTS: Cytomegalovirus was significantly associated with inflammatory disease, which occurred in 31 of 43 (72%) virus-exposed patients and 8 of 31 (26%) naive patients (P = .0001). CMV pp65-NLVPMVATV epitope-specific CD8(+) T-cell frequencies were significantly elevated in inflammatory patients, but these cells did not show evidence of exhaustion, with low levels of programmed death-1 and high T-cell receptor avidity. Rather, they showed features consistent with high in vivo functionality and proliferative activity including reduced levels of the anti-inflammatory marker CD73 (1.67% of NLV(+) cells were CD73(+) vs 42.01% in noninflammatory patients; P = .004) and increased Ki-67 expression (37% vs 2% in noninflammatory patients; P < .0001). In vitro, the CMV-specific T cells showed high antigen-specific proliferative potential compared with cells from noninflammatory patients. By using sensitive immunohistochemistry, we detected for the first time viral antigen at the sites of inflammation, indicative of active viral replication. CONCLUSION: Our data strongly support a direct role for CMV and a hyperreactive CMV-specific immune response in the debilitating chronic inflammatory complications of common variable immunodeficiency.
Mattes FM, Hainsworth EG, Hassan-Walker AF, Burroughs AK, Sweny P, Griffiths PD, Emery VC (2005) Kinetics of cytomegalovirus load decrease in solid-organ transplant recipients after preemptive therapy with valganciclovir., J Infect Dis191(1)pp. 89-92
The availability of valganciclovir (VGCV) has significantly simplified the treatment of human cytomegalovirus (HCMV) infection after solid-organ transplantation. We show that there was no difference in the kinetics of the decrease in HCMV load after preemptive therapy with VGCV in 22 solid-organ transplant recipients (T1/2=2.16 days), compared with that in 23 patients treated with intravenous ganciclovir (GCV) (T(1/2) = 1.73 days; P=.63). Preemptive therapy with VGCV provides control of HCMV replication that is comparable to that achieved with preemptive intravenous therapy with GCV.
Devereux HL, Emery VC, Johnson MA, Loveday C (2001) Replicative fitness in vivo of HIV-1 variants with multiple drug resistance-associated mutations., J Med Virol65(2)pp. 218-224
The relative fitness of HIV-1 viral variants containing a broad range of drug resistance-associated mutations has been little studied in vivo. Understanding the relative fitness associated with viruses containing mutations may aid future therapeutic management. The aim of this study was to investigate the relative fitness of mutant viruses by assessing a cohort of patients who had developed resistance to many drugs and subsequently stopped all therapy. Eleven patients were assessed for drug resistance associated mutations in the protease (PR) and reverse transcriptase (RT) genes before and, at multiple time points, after stopping therapy. Relative fitness was calculated as a function of the rate of disappearance of mutant viruses when therapy was stopped. The least fit viruses were associated with the RT mutation M184I/V (11.6% less fit) and the PR mutations D30N (12.4% less fit) and M46I/L (21% less fit). Mutations at these codons were associated with significant reductions in fitness levels compared to wild-type viruses. Mutations at codons 10, 20, 36, and 63 in the PR gene remained fairly constant when therapy was stopped and may not significantly reduce viral fitness. The rapid re-population of wild-type viruses may allow the recycling of antiretroviral drugs prescribed previously.
Gor D, Sabin C, Prentice HG, Vyas N, Man S, Griffiths PD, Emery VC (1998) Longitudinal fluctuations in cytomegalovirus load in bone marrow transplant patients: relationship between peak virus load, donor/recipient serostatus, acute GVHD and CMV disease., Bone Marrow Transplant21(6)pp. 597-605
Quantitative competitive PCR was used to monitor the quantity of cytomegalovirus (HCMV) in 1647 blood samples from 110 BMT recipients. DNAemia was detected in 49/110 (45%) of the patients, of whom 15/49 experienced HCMV disease. Peak virus load during surveillance was elevated in symptomatic (median 4.5 log10 genomes/ml) vs asymptomatic patients (median 3.6 log10 genomes/ml, P=0.002) and was also significantly elevated in HCMV seropositive recipients of seronegative marrow, (R+D-, median 5.0 log10), compared to those in the R-D- and R+D+ groups (P < 0.01 and <0.005). Odds ratios for disease per 0.25 log10 increase in viral load, recipient seropositivity and aGVHD were 1.43 (P=0.004), 6.60 (P=0.05) and 3.17 (P=0.08), respectively. In multivariate logistic regression analysis only elevated viral load remained a significant risk factor for HCMV disease. The computed disease probability viral load curve showed a rapid increase in disease risk at viral loads between 3.8 and 5.5 log10 genomes/ml in blood, and odds ratios for disease were determined for different threshold viral loads. These data demonstrate the central role of viral load in the pathogenesis of HCMV in BMT recipients and provide an additional marker for targeting and monitoring therapy.
Emery VC, Mattes FM (2012) Pre-emptive therapy for the management of cytomegalovirus disease after transplantation, pp. 193-202
Over the last decade a growing appreciation of the replication kinetics of cytomegalovirus (CMV) in the immunocompromised host has enabled key parameters to be identified as major risk factors for CMV disease including rate of replication, peak and cumulative viral load in blood. The advent of rapid, accurate methods for the quantification of CMV including the antigenemia assay and real time PCR based approaches has allowed viral replication directed therapeutic interventions known as preemptive therapy to be deployed. In this chapter we review how pre-emptive therapy (antiviral therapy initiated on the basis of signs of active CMV replication such as antigenemia positivity or DNAemia) aimed at stopping CMV replication at the earliest stages of the replication history is an effective approach to minimise the incidence of CMV disease after solid organ and stem cell transplantation. We will also highlight some of the remaining challenges associated with pre-emptive therapy including the the influence of different clinical samples (whole blood and plasma), viral load thresholds for the initiation of therapy, the incidence of recurrent DNAemia and the impact of pre-emptive therapy on long term graft function. © 2012 by Nova Science Publishers, Inc. All rights reserved.
Bowen EF, Johnson MA, Griffiths PD, Emery VC (1997) Development of a point mutation assay for the detection of human cytomegalovirus UL97 mutations associated with ganciclovir resistance., J Virol Methods68(2)pp. 225-234
A point mutation assay was developed to detect the quantitative prevalence of mutations at codons 460 (M to I; M to V), 520 (H to Q), 594 (A to V) and 595 (L to F; L to S) within the UL97 gene of human cytomegalovirus which segregate with ganciclovir resistance. Synthetic mixtures of wild-type and mutant plasmids containing the UL97 gene were amplified by nested polymerase chain reaction and the 700 base pair amplicon subsequently subjected to the point mutation assay. In plasmid reconstruction experiments, there was a high correlation between experimentally derived percentage mutant with the theoretical values. The assay was then used to assess the changes in the genetic composition of the UL97 gene in three patients on prolonged ganciclovir therapy. All three patients developed genotypic resistance against ganciclovir involving mutation at codon L595S, L595F and double mutation at codons L595F and M460I. In one patient, alteration of therapy to foscarnet did not affect the composition of UL97 and virus remained genotypically resistant to ganciclovir. In contrast, in two patients whose therapy was altered to cidofovir (HPMPC), repopulation with cytomegalovirus strains carrying the wild-type (ganciclovir-sensitive) codon at positions 595 and 460 occurred. The potential use of this assay for the rapid detection of cytomegalovirus resistance in patients on long-term ganciclovir therapy is discussed.
Emery VC, Milne RSB (2011) Recent developments in antiviral drugs for cytomegalovirus, FUTURE VIROLOGY6(5)pp. 633-651 FUTURE MEDICINE LTD
Deayton J, Mocroft A, Wilson P, Emery VC, Johnson MA, Griffiths PD (1999) Loss of cytomegalovirus (CMV) viraemia following highly active antiretroviral therapy in the absence of specific anti-CMV therapy., AIDS13(10)pp. 1203-1206
OBJECTIVE: To determine the effect of highly active antiretroviral therapy (HAART) on cytomegalovirus (CMV) viraemia and retinitis in patients at high risk of disease. DESIGN: Sixteen patients with CMV viraemia, but no evidence of end organ disease at the time of first receipt of HAART including a protease inhibitor, were studied. No patient had ever received specific anti-CMV therapy. METHODS: CMV load in blood was measured using quantitative competitive PCR at baseline and for a median follow-up of 21 months. Regular ophthalmological screening for retinitis was conducted throughout the study period. RESULTS: All 16 patients became CMV negative by PCR following the commencement of HAART. CMV loads prior to treatment ranged from 2.0 x 10(3) to 4.1 x 10(6) copies/ml (median, 7.6 x 10(4) copies/ml). The median time to becoming PCR negative was 13.5 weeks (range, 5-40 weeks). Fourteen patients remained CMV negative throughout follow-up. CMV viraemia recurred in two patients; these individuals were indistinguishable with respect to either baseline parameters or response to antiretroviral therapy. None of the 16 patients developed CMV retinitis. CONCLUSIONS: HAART including a protease inhibitor can result in the complete suppression of CMV viraemia, an effect not previously observed in HIV-infected patients in the absence of specific anti-CMV therapy. This response correlated with protection against CMV retinitis in a group of patients at high risk of development of disease. These results help to explain why the natural history of CMV disease has altered since the introduction of such therapeutic regimens.
Danta M, Semmo N, Fabris P, Brown D, Pybus OG, Sabin CA, Bhagani S, Emery VC, Dusheiko GM, Klenerman P (2008) Impact of HIV on host-virus interactions during early hepatitis C virus infection., J Infect Dis197(11)pp. 1558-1566
BACKGROUND: Human immunodeficiency virus (HIV) may influence the outcome and natural history of hepatitis C virus (HCV) infection through an impact on acute HCV-specific T cell responses. METHODS: Fifty-five HIV-positive males with acute HCV infection were identified; monoinfected individuals (n = 8) were used for peripheral blood mononuclear cell comparison. In 14 coinfected and 8 HCV-monoinfected patients, HCV-specific T cell responses against a range of HCV antigens were assessed using interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) and proliferation assays. E1/E2 region genetic diversity and the selection pressure on the virus were measured in 8 coinfected patients by use of cloned sequences over time. RESULTS: HCV persisted in 52 (95%) coinfected individuals. HCV/HIV coinfection significantly reduced IFN-gamma ELISpot responses versus those in HCV-monoinfected individuals, especially against nonstructural proteins (1/10 vs. 5/8; P = .008). In coinfected patients, increased HCV genetic diversity was observed between the first and subsequent time points, with no evidence for positive selection in the E1/E2 region sequenced. CONCLUSION: HIV coinfection is associated with increased rates of HCV persistence and a lack of critical CD4 T cell responses, with no evidence of immune selection pressure during early HCV infection. Loss of key cellular immune responses against HCV during acute disease may contribute to the failure of early host control of HCV in HCV/HIV-coinfected patients.
Emery VC (1998) Relative importance of cytomegalovirus load as a risk factor for cytomegalovirus disease in the immunocompromised host, CMV-RELATED IMMUNOPATHOLOGY21pp. 288-301 KARGER
MOCROFT AJ, JOHNSON MA, SABIN CA, LIPMAN M, ELFORD J, EMERY V, MORCINEK J, YOULE M, JANOSSY G, LEE CA, PHILLIPS AN (1995) STAGING SYSTEM FOR CLINICAL AIDS PATIENTS, LANCET346(8966)pp. 12-17 LANCET LTD
Kidd IM, Fox JC, Pillay D, Charman H, Griffiths PD, Emery VC (1993) Provision of prognostic information in immunocompromised patients by routine application of the polymerase chain reaction for cytomegalovirus., Transplantation56(4)pp. 867-871
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.
Teixeira R, Pastacaldi S, Davies S, Dhillon AP, Emery VC, Rolles K, Davidson B, Patch D, Burroughs AK (2000) The influence of cytomegalovirus viraemia on the outcome of recurrent hepatitis C after liver transplantation., Transplantation70(10)pp. 1454-1458
BACKGROUND: Several interrelated host and hepatitis C virus (HCV) associated factors have been proposed to explain the variable outcomes in HCV recurrence. Recent evidence suggests that cytomegalovirus (CMV) infection not only is co-factor in progression of HCV recurrence but may precipitate allograft rejection. We investigated whether short-term CMV viremia influences HCV recurrence, the number and grade of acute rejection episodes, and the histological course of HCV recurrence during the first year after orthotopic liver transplantation (OLT) for HCV-related cirrhosis. METHODS: A cohort of 39 patients transplanted for cirrhosis HCV-related was analyzed. Patients were evaluated twice weekly for CMV infection by a blood polymerase chain reaction (PCR) assay. Triple therapy with cyclosporine or tacrolimus, azathioprine and prednisolone was the initial immunosuppressive regimen. Preemptive treatment with ganciclovir was started when two consecutive PCRs for CMV were positive. Liver biopsies were performed on day 7 after OLT or when indicated. A 3-day IV 1 g methilprednisolone was given to patients with moderate or severe rejection. Ishak's score was used to grade inflammation and to stage fibrosis. RESULTS: Neither CMV viremia nor CMV disease after OLT for HCV-related cirrhosis adversely influenced the incidence and grade of acute rejection episodes nor the histological outcome of post transplant HCV recurrence, during the first year after liver transplantation. CONCLUSION: CMV viremia as detected by PCR does not affect the progression of HCV recurrence in liver grafts.
Emery VC (2002) Expert comments: Diagnostic value of enzyme linked immuno-sorbent assay, Journal of Postgraduate Medicine48(3)
Strappe P, Atkins M, Kaye S, Loveday C, Johnson M, Tedder R, Griffiths PD, Emery VC (1995) Distribution quantity of zidovudine-resistant genotypes in multiple organs of HIV-infected patients dying with AIDS, JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES10pp. 79-79 LIPPINCOTT WILLIAMS & WILKINS
Emery VC (1996) Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal VirusReproduced from Proc. Natl Acad. Sci 38, 747-752 (1952) with kind permission of Proceedings of the National Academy of Sciences, USA., Rev Med Virol6(2)pp. 61-64
Fraile-Ramos A, Pelchen-Matthews A, Risco C, Rejas MT, Emery VC, Hassan-Walker AF, Esteban M, Marsh M (2007) The ESCRT machinery is not required for human cytomegalovirus envelopment, Cellular Microbiology9(12)pp. 2955-2967
The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment. © 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd.
Background.?Cytomegalovirus (CMV) is associated with morbidity and mortality in human immunodeficiency virus (HIV)?exposed infants. We assessed the effect of and relative contribution of breastfeeding to CMV acquisition among infants delivered by HIV-infected mothers.
Methods.?Between 1993 and 1998 pregnant, HIV-infected women in Nairobi, Kenya, were randomly assigned to breastfeed or formula-feed their infants in an HIV transmission study. Women were allocated equally between treatment arms, and the study was not blinded. The primary endpoint of this nested study was time to infant CMV infection.
Results.?CMV infection was assessed in 138 breastfed and 134 formula-fed infants. Baseline characteristics were similar between arms. Breastfed infants acquired CMV earlier than formula-fed infants (median age of acquisition, 4.26 vs 9.87 months; P < .001) and had a higher 1-year probability of CMV infection (0.89 vs 0.69; P < .001). Breastfeeding was associated with a 1.6-fold increased risk of infant CMV acquisition independent of infant HIV status (multivariable hazard ratio, 1.61; 95% confidence interval, 1.20?2.16; P = .002). Approximately one third of CMV infections occurred during the peripartum period, with 40% acquired through breastfeeding and the remainder acquired through modes other than breast milk.
Fryer JF, Griffiths PD, Emery VC, Clark DA (2004) Susceptibility of porcine cytomegalovirus to antiviral drugs., J Antimicrob Chemother53(6)pp. 975-980
OBJECTIVES: Re-activation of porcine cytomegalovirus (PCMV) in the xenograft has been reported in pig-to-baboon models of xenotransplantation and is associated with invasive disease and consumptive coagulopathy. If xenotransplantation of porcine organs into human recipients is to proceed, donor organs will have to be free from a wide range of infectious agents including PCMV. However, it is prudent to characterize the antiviral susceptibility of this virus. We therefore investigated the effect of selected antiviral agents, currently licensed for the treatment of human herpesvirus infections, on PCMV replication. METHODS: Antiviral susceptibility was determined using real-time PCR and indirect immunofluorescence measurements in a porcine fallopian tube cell line infected with PCMV. RESULTS: PCMV replication was significantly inhibited by ganciclovir and cidofovir (both EC(50) < 1 mg/L) and to a lesser extent by foscarnet (EC(50) within range 25-50 mg/L) and aciclovir (EC(50) > 25 mg/L). CONCLUSIONS: These results show that, if it proves necessary, ganciclovir and cidofovir should be considered as first-line drugs to treat PCMV infections in xenograft recipients.
Deayton JR, Shannon-Lowe C, Wilson P, Johnson MA, Griffiths PD, Emery VC (2000) Recurrence of CMV viraemia and development of anti-CMV drug resistance in patients receiving highly active antiretroviral therapy after CMV retinitis, AIDS14pp. S5-S6 LIPPINCOTT WILLIAMS & WILKINS
Emery VC (1992) Baculovirus expression vectors., Methods Mol Biol8pp. 309-318
This chapter aims to provide the reader with the experimental protocols required to produce a baculovirus containing the gene of interest, including transfection of Spodoptera frugiperda cells and the screening of progeny virus by plaque assay. In addition, an outline of the biochemical methods routinely used to characterize the expressed protein product within the baculovirus system will be given. Throughout the experimental procedures, areas that researchers generally find technically difficult will be highlighted in the hope that this may ameliorate some of the problems encountered in using the system ab initio.
Slyker J, Farquhar C, Atkinson C, Ásbjörnsdóttir K, Roxby A, Drake A, Kiarie J, Wald A, Boeckh M, Richardson B, Odem-Davis K, John-Stewart G, Emery V (2014) Compartmentalized cytomegalovirus replication and transmission in the setting of maternal HIV-1 infection, Clinical Infectious Diseases58(4)pp. 564-572
Background. Cytomegalovirus (CMV) infection is associated with adverse outcomes in human immunodeficiency virus (HIV)-exposed infants. Determinants of vertical CMV transmission in the setting of maternal HIV-1 infection are not well-defined.Methods. CMV and HIV-1 levels were measured in plasma, cervical secretions, and breast milk of 147 HIV-1-infected women to define correlates of maternal CMV replication and infant CMV acquisition.Results. Although few women had detectable CMV in plasma (4.8%), the majority had detectable CMV DNA in cervical secretions (66%) and breast milk (99%). There was a strong association between cervical CMV detection during pregnancy and later breast milk levels (= 0.47; P =. 005). Plasma HIV-1 level and CD4 counts were associated with CMV in the cervix and breast milk. However HIV-1 levels within the cervix and breast milk were not associated with CMV within these compartments. Maternal breast milk CMV levels (hazard ratio [HR], 1.4; P =. 003) and maternal CD4 < 450 cells/mm3 (HR, 1.8; P =. 008) were independently associated with infant CMV acquisition; each log10 increase in breast milk CMV was associated with a 40% increase in infant infection. The breast milk CMV level required to attain a 50% probability of CMV transmission increased with higher maternal CD4 counts, increasing from 3.55 log10 CMV DNA copies/mL at a CD4 count of 350 cells/mm3 to 5.50 log10 CMV DNA copies/mL at a CD4 count of 1000 cells/mm3.Conclusions. Breast milk CMV levels and maternal CD4 count are major determinants of CMV transmission in the setting of maternal HIV-1. Maternal immune reconstitution or lowering breast milk CMV levels may reduce vertical CMV transmission. © 2013 The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.
Bowen EF, Wilson P, Atkins M, Madge S, Griffiths PD, Johnson MA, Emery VC (1995) Natural history of untreated cytomegalovirus retinitis, LANCET346(8991-2)pp. 1671-1673 LANCET LTD
Lock MJ, Thorley N, Teo J, Emery VC (2002) Azidodeoxythymidine and didehydrodeoxythymidine as inhibitors and substrates of the human herpesvirus 8 thymidine kinase., J Antimicrob Chemother49(2)pp. 359-366
Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), encodes many core genes that have been maintained during evolution of the Herpesviridae. Among these is a thymidine kinase (TK) homologue (ORF21), which has 12% homology to the related TK encoded by herpes simplex virus. We show that the HHV-8 TK is a functional deoxythymidine (dT) kinase, with Michaelis constants (K(m)) for dT and ATP of 18.5 and 6.6 microM, respectively. Using homology modelling coupled with site-directed mutagenesis, we identify Gly265, Asp362 and Phe372 as key amino acid residues involved in the catalytic process. The HHV-8 TK is competitively inhibited by azidodeoxythymidine (zidovudine) and didehydrodeoxythymidine (stavudine) and can also accept these anti-retroviral compounds as substrates. These data have implications for our understanding of changes in AIDS-KS incidence following the clinical licensing of these compounds and in the development of new therapies for KS.
Ait-Khaled M, Emery VC (1994) 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., J Gen Virol75 ( Pt 7)pp. 1615-1621
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.
Whalley SA, Brown D, Webster GJ, Jacobs R, Reignat S, Bertoletti A, Teo CG, Emery V, Dusheiko GM (2004) Evolution of hepatitis B virus during primary infection in humans: transient generation of cytotoxic T-cell mutants., Gastroenterology127(4)pp. 1131-1138
BACKGROUND & AIMS: Acute hepatitis B is a highly dynamic human viral infection during which the hepatitis B virus can generate many genetic variants. METHODS: We analyzed the evolution of the hepatitis B virus genome in sequential serum samples from a unique cohort of patients with acute infection acquired from a single source. RESULTS: We showed that most mutations were nonsynonymous, that genetic diversity was greatest at the peak of viremia, and that patients who resolved their infection ("resolvers") showed a significantly higher level of diversity in the core, surface, and polymerase genes compared with those who progressed to chronic infection. Overall, the core gene showed the greatest genetic diversity. In resolvers who possessed an HLA-A*0201 haplotype, the emergence of mutants in the immunodominant HLA-A*0201-restricted core 18-27 epitope was observed. Functional studies showed that these mutants were less able to stimulate interferon-gamma release from core 18-27 specific CD8 + T-cell lines. However, they appeared only as a transient low-abundance species and were rapidly displaced by wild-type sequences before resolution of infection, and their overall significance is uncertain. CONCLUSIONS: Overall, genetic evolution of the hepatitis B virus differs at early time points between patients who experience acute resolving hepatitis B and those who progress to chronicity. These observations suggest that the rapid development of broadly reactive host immune responses leads to clearance of hepatitis B virus, even in the presence of possible CD8+ T-cell immune escape variants.
Atabani SF, Atkinson C, Aldridge RW, Smith C, Harber M, Thornbun D, Rolando N, Emery VC, Griffiths PD (2011) A QUANTITATIVE ANALYSIS OF CMV INFECTION IN SOLID-ORGAN TRANSPLANT RECIPIENTS MANAGED EXCLUSIVELY ON PRE-EMPTIVE THERAPY, TRANSPLANT INTERNATIONAL24pp. 175-175 WILEY-BLACKWELL
Emery VC, Cope AV, Bowen EF, Gor D, Griffiths PD (1999) The dynamics of human cytomegalovirus replication in vivo., J Exp Med190(2)pp. 177-182
Cytomegalovirus (CMV) is generally described as a slowly replicating virus. During studies of immunocompromised patients, we observed rapid changes in the quantity of CMV DNA present in serial blood samples by quantitative-competitive polymerase chain reaction commensurate with a doubling time of <2 d. To further investigate the dynamics of replication in vivo, patients in three distinct situations were studied in detail: (a) those receiving intravenous ganciclovir; (b) those in whom ganciclovir-resistant strains appeared during long-term therapy; and (c) those in whom ganciclovir-resistant strains disappeared with alternative drug therapy. In all cases, it was possible to provide accurate estimates of the doubling time of CMV and its half-life of disappearance after antiviral chemotherapy. The results from all three approaches demonstrated that the doubling time/half-life of CMV in blood is approximately 1 d when frequent samples are collected. These results show that CMV DNA replication in vivo is a highly dynamic process. We conclude that the reputation of CMV as a slowly replicating virus based on the time taken to produce cytopathic effects in vitro is unwarranted. These findings have implications for the potency, dose, and duration of antiviral chemotherapy needed for the effective treatment of this important human pathogen.
Atkins MC, Carlin EM, Emery VC, Griffiths PD, Boag F (1996) Fluctuations of HIV load in semen of HIV positive patients with newly acquired sexually transmitted diseases., BMJ313(7053)pp. 341-342
Hassan-Walker AF, Mattes FM, Griffiths PD, Emery VC (2001) Quantity of cytomegalovirus DNA in different leukocyte populations during active infection in vivo and the presence of gB and UL18 transcripts., J Med Virol64(3)pp. 283-289
The quantity of human cytomegalovirus (HCMV) DNA in the blood of immunocompromised individuals correlates with the development of HCMV disease. We wished to determine which leukocytes harboured DNA and whether this represented active viral replication. Magnetic bead separation techniques were used to obtain pure polymorphonuclear leukocyte (PMNL), monocyte, B and T cell fractions, and RT-PCR and quantitative-competitive PCR (QC-PCR) to detect HCMV glycoprotein B (gB; UL55) transcripts and quantify HCMV DNA levels, respectively, in each cell fraction. QC-PCR revealed that PMNLs contribute the greatest to the overall viral load in blood (median viral load: PMNLs, 10(5.37) genomes/ml of blood; monocytes, 10(4.40) genomes/ml; B cells, 10(3.70) genomes/ml; and T cells, 10(4.08) genomes/ml). However, monocytes have a viral burden of 0.65 genomes/monocyte which is greater than that within the other leukocyte populations (0.11 genomes/PMNL, 0.23 genomes/B cell, and 0.20 genomes/T cell). Glycoprotein B transcripts were detected in all four cell populations: 3/10 PMNL fractions, 6/13 monocyte fractions, 5/13 B cell fractions, and 4/13 T cell fractions. The data show that productive infection of these leukocyte subpopulations, including PMNLs, can occur in vivo. Furthermore, transcripts of gpUL18, the putative natural killer (NK) cell decoy, were detected in 2/6 monocyte fractions with active replication, and 1/4 T cell fractions but not in the other leukocyte fractions. The transient nature of UL18 gene expression, and the low abundance of the transcript relative to gB were confirmed.
Atabani SF, Smith C, Atkinson C, Aldridge RW, Rodriguez-Perálvarez M, Rolando N, Harber M, Jones G, O'Riordan A, Burroughs AK, Thorburn D, O'Beirne J, Milne RSB, Emery VC, Griffiths PD (2012) Cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy, American Journal of Transplantation12(9)pp. 2457-2464
After allotransplantation, cytomegalovirus (CMV) may be transmitted from the donor organ, giving rise to primary infection in a CMV negative recipient or reinfection in one who is CMV positive. In addition, latent CMV may reactivate in a CMV positive recipient. In this study, serial blood samples from 689 kidney or liver transplant recipients were tested for CMV DNA by quantitative PCR. CMV was managed using preemptive antiviral therapy and no patient received antiviral prophylaxis. Dynamic and quantitative measures of viremia and treatment were assessed. Median peak viral load, duration of viremia and duration of treatment were highest during primary infection, followed by reinfection then reactivation. In patients who experienced a second episode of viremia, the viral replication rate was significantly slower than in the first episode. Our data provide a clear demonstration of the immune control of CMV in immunosuppressed patients and emphasize the effectiveness of the preemptive approach for prevention of CMV syndrome and end organ disease. Overall, our findings provide quantitative biomarkers which can be used in pharmacodynamic assessments of the ability of novel CMV vaccines or antiviral drugs to reduce or even interrupt such transmission. The authors show that preemptive antiviral therapy is an effective approach for control and prevention of cytomegalovirus replication after renal and liver transplantation, even in high-risk patients. © Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.
Emery VC (2001) Human herpesviruses 6 and 7 in solid organ transplant recipients., Clin Infect Dis32(9)pp. 1357-1360
The impact of cytomegalovirus, a member of the beta-herpesvirus subgroup of the Herpesviridae, on patients who have undergone transplantation cannot be overstated. However, in the last 15 years, 2 additional members of the human beta-herpesvirus family have been discovered: human herpesviruses 6 and 7 (HHV-6 and HHV-7). The impact of HHV-6 and HHV-7 is assessed, as is the well-being of transplant recipients. Also discussed is whether the data on the pathological consequences of infection warrant routine screening for these viruses in solid organ transplant recipients.
Tarar MR, Emery VC, Harrison TJ (1996) Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein., FEMS Immunol Med Microbiol16(3-4)pp. 183-192
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.
Downey GP, Bavin PJ, Deery ARS, Griffiths PD, Emery VC, Walker PG (1994) The relationship between HPV 16 semi-quantitation and the potential for progression of minor grade cervical disease, British Journal of Obstetrics and Gynaecology101(3)
Kidd IM, Emery VC (1993) The use of baculoviruses as expression vectors., Appl Biochem Biotechnol42(2-3)pp. 137-159
The use of recombinant baculoviruses as high level expression systems is becoming more and more popular. This review aims to provide a summary of the impact of this expression system in biochemistry and biotechnology, highlighting important advances that have been made utilizing the system. The potential of newly developed multiple baculovirus expression systems to enable the reconstruction of complex biological molecules and processes is also reviewed.
Brennan DC, Aguado JM, Potena L, Jardine AG, Legendre C, Säemann MD, Mueller NJ, Merville P, Emery V, Nashan B (2013) Effect of maintenance immunosuppressive drugs on virus pathobiology: Evidence and potential mechanisms, Reviews in Medical Virology23(2)pp. 97-125
Recent evidence suggesting a potential anti-CMV effect of mTORis is of great interest to the transplant community. However, the concept of an immunosuppressant with antiviral properties is not new, with many accounts of the antiviral properties of several agents over the years. Despite these reports, to date, there has been little effort to collate the evidence into a fuller picture. This manuscript was developed to gather the evidence of antiviral activity of the agents that comprise a typical immunosuppressive regimen against viruses that commonly reactivate following transplant (HHV1 and 2, VZV, EBV, CMV and HHV6, 7, and 8, HCV, HBV, BKV, HIV, HPV, and parvovirus). Appropriate immunosuppressive regimens posttransplant that avoid acute rejection while reducing risk of viral reactivation are also reviewed. The existing literature was disparate in nature, although indicating a possible stimulatory effect of tacrolimus on BKV, potentiation of viral reactivation by steroids, and a potential advantage of mammalian target of rapamycin (mTOR) inhibition in several viral infections, including BKV, HPV, and several herpesviruses. © 2012 John Wiley & Sons, Ltd.
Emery VC, Griffiths PD (1990) Molecular biology of cytomegalovirus., Int J Exp Pathol71(6)pp. 905-918
Fraile-Ramos A, Pelchen-Matthews A, Risco C, Rejas MT, Emery VC, Hassan-Walker AF, Esteban M, Marsh M (2007) The ESCRT machinery is not required for human cytomegalovirus envelopment, CELLULAR MICROBIOLOGY9(12)pp. 2955-2967 WILEY-BLACKWELL
Aldridge RW, Mattes FM, Rolando N, Rolles K, Smith C, Shirling G, Atkinson C, Burroughs AK, Milne RSB, Emery VC, Griffiths PD (2015) Effects of donor/recipient human leukocyte antigen mismatch on human cytomegalovirus replication following liver transplantation, Transplant Infectious Disease17(1)pp. 25-32
© 2015 The Authors. Transplant Infectious Disease Published by John Wiley & Sons Ltd.Background: Natural immunity against cytomegalovirus (CMV) can control virus replication after solid organ transplantation; however, it is not known which components of the adaptive immune system mediate this protection. We investigated whether this protection requires human leukocyte antigen (HLA) matching between donor and recipient by exploiting the fact that, unlike transplantation of other solid organs, liver transplantation does not require HLA matching, but some donor and recipient pairs may nevertheless be matched by chance. Methods: To further investigate this immune control, we determined whether chance HLA matching between donor (D) and recipient (R) in liver transplants affected a range of viral replication parameters. Results: In total, 274 liver transplant recipients were stratified according to matches at the HLA A, HLA B, and HLA DR loci. The incidence of CMV viremia, kinetics of replication, and peak viral load were similar between the HLA matched and mismatched patients in the D+/R+ and D-/R+ transplant groups. D+/R- transplants with 1 or 2 mismatches at the HLA DR locus had a higher incidence of CMV viremia >3000 genomes/mL blood compared to patients matched at this locus (78% vs. 17%; P = 0.01). Evidence was seen that matching at the HLA A locus had a small effect on peak viral loads in D+/R- patients, with median peak loads of 3540 and 14,706 genomes/mL in the 0 and combined (1 and 2) mismatch groups, respectively (P = 0.03). Conclusion: Overall, our data indicate that, in the setting of liver transplantation, prevention of CMV infection and control of CMV replication by adaptive immunity is minimally influenced by HLA matching of the donor and recipient. Our data raise questions about immune control of CMV in the liver and also about the cells in which the virus is amplified to give rise to CMV viremia.
Emery VC (2015) Restimulating interest in cytomegalovirus as a cofactor for HIV infection, Journal of Infectious Diseases211(2)pp. 169-171
JEFFRIES D, BARTON S, JOHNSON R, EMERY V (1994) ISSUES IN HERPES MANAGEMENT, ANTIVIRAL CHEMISTRY & CHEMOTHERAPY5pp. 23-29 BLACKWELL SCIENCE LTD
Emery VC (2011) Could a vaccine against immune-evading cytomegalovirus become a reality?, EXPERT REVIEW OF VACCINES10(8)pp. 1109-1111 EXPERT REVIEWS
Le[nikowski ZJ, Paradowska E, Przepiórkiewicz M, Olejniczak A, Emery VC (2002) [Drugs against human cytomegalovirus]., Pol Merkur Lekarski13(75)pp. 242-249
Human cytomegalovirus (HCMV) infects about 60% of adults in developed world and more than 90% of developing countries population. In the immunocompetent host, initial infection and reactivation of latent infection are usually asymptomatic. However, in hosts with impaired cellular immune functions, such as transplant recipients, patients infected with human immunodeficiency virus (HIV) or undergoing anticancer chemo- and/or radiotherapy, the full pathogenic potential of the virus may be realized. HCMV is also among the most common causes of viral intrauterine infection affecting from 0.4 to 2.3% of live-born infants. Though in pregnant, immunocompetent women infections with HCMV are usually asymptomatic, severe infections may occur among congenitally infected fetuses and infants due to immaturity of their immune system. Approximately 40% of mothers with primary HCMV infections during gestation transmit virus to their infants. Although only 10% of infected infants are symptomatic at birth, 20 to 30% of these die. In addition, 5 to 15% of asymptomatic neonates are at risk of developing congenital anomalies later. In this outline we present anti-CMV drugs currently in clinical use and give examples of new molecules under laboratory and clinical development.
WEBSTER A, LEE CA, COOK DG, GRUNDY JE, EMERY VC, KERNOFF PBA, GRIFFITHS PD (1989) CYTOMEGALO-VIRUS INFECTION AND PROGRESSION TO AIDS, LANCET2(8664)pp. 681-681 LANCET LTD
Paradowska E, Przepiorkiewicz M, Nowakowska D, Studzinska M, Wilczynski J, Emery VC, Lesnikowski ZJ (2006) Detection of cytomegalovirus in human placental cells by polymerase chain reaction, APMIS114(11)pp. 764-771 WILEY-BLACKWELL PUBLISHING, INC
FOX JC, EMERY VC (1992) QUANTIFICATION OF VIRUSES IN CLINICAL-SAMPLES, REVIEWS IN MEDICAL VIROLOGY2(4)pp. 195-203 JOHN WILEY & SONS LTD
Grundy JE, Ehrnst A, Einsele H, Emery VC, Hebart H, Prentice HG, Ljungman P (1996) A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood., J Clin Microbiol34(5)pp. 1166-1170
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.
Temperton NJ, Quenelle DC, Lawson KM, Zuckerman JN, Kern ER, Griffiths PD, Emery VC (2003) Enhancement of humoral immune responses to a human cytomegalovirus DNA vaccine: adjuvant effects of aluminum phosphate and CpG oligodeoxynucleotides., J Med Virol70(1)pp. 86-90
A human cytomegalovirus (HCMV) glycoprotein B (gpUL55) DNA vaccine has been evaluated in BALB/c mice. Intramuscular immunization of these mice with pRc/CMV2-gB resulted in the generation of high levels of gpUL55-specific antibody (geometric mean titer [GMT] 1:8900) and neutralizing antibody (GMT 1:74) after 2 booster doses given 5 and 10 weeks after primary inoculation. Emulsifying the construct with the aluminum phosphate gel adjuvant Adju-Phos before immunization enhanced gpUL55-specific antibody responses (GMT 1:17800, P = 0.04). Co-immunization with CpG oligodeoxynucleotides was shown to enhance levels of neutralizing antibodies generated by immunization of mice with a pRc/CMV2-gB/Adju-Phos emulsion (P = 0.04). The results provide a rationale for evaluating combinations of other HCMV proteins for incorporation into a multi-target DNA vaccine, and for the optimization of adjuvant usage, to elicit enhanced levels of neutralizing antibodies. 2003.
Walton JA, Lydyard PM, Nathwani A, Emery V, Akbar A, Glennie MJ, Porakishvili N (2010) Patients with B cell chronic lymphocytic leukaemia have an expanded population of CD4+perforin expressing T cells enriched for human cytomegalovirus specificity and an effector-memory phenotype, BRITISH JOURNAL OF HAEMATOLOGY148(2)pp. 274-284 WILEY-BLACKWELL PUBLISHING, INC
Emery VC (2013) Human herpesvirus vaccines and future directions., Am J Transplant13 Suppl 3pp. 79-86
Over the last few years there has been an impressive increase in the virological and immunological tools available to detect both human herpesvirus (HHV) and immune control of replication post-solid organ transplantation. This has allowed a greater appreciation of pathogenesis, studies to be designed to evaluate potential vaccines, new approaches adopted for antiviral deployment and the success of interventions to be judged. This chapter aims to summarize the state-of-the-art in vaccine development and look forward to the role that vaccines, immune monitoring, viral kinetics and new antiherpesvirus agents may play in the future management of HHV infections after transplantation.
Emery VC (1998) Cytomegalovirus drug resistance., Antivir Ther3(4)pp. 239-242
Clinical resistance of cytomegalovirus (CMV) against the currently licensed antiviral drugs is becoming an increasingly recognized problem. This review focuses on the molecular basis of resistance and describes mutations in the UL54 DNA polymerase leading to resistance against cidofovir, foscarnet and ganciclovir. The review highlights two important developments in our appreciation of resistance. Firstly, the use of more rapid molecular based assays to detect genotypic resistance and secondly, the relationship between resistance profiles in multiple organ systems of the same host. Finally, the changing face of CMV disease in the era of highly active antiviral chemotherapy is considered with respect to its impact on the frequency of CMV resistance in the clinic.
Fishman JA, Emery V, Freeman R, Pascual M, Rostaing L, Schlitt HJ, Sgarabotto D, Torre-Cisneros J, Uknis ME (2007) Cytomegalovirus in transplantation - challenging the status quo, CLINICAL TRANSPLANTATION21(2)pp. 149-158 WILEY-BLACKWELL PUBLISHING, INC
Webster A, Lee CA, Cook DG, Grundy JE, Emery VC, Kernoff PB, Griffiths PD (1989) Cytomegalovirus infection and progression towards AIDS in haemophiliacs with human immunodeficiency virus infection., Lancet2(8654)pp. 63-66
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.
ORCHARD K, PERKINS N, CHAPMAN C, HARRIS J, EMERY V, GOODWIN G, LATCHMAN D, COLLINS M (1990) A T-CELL PROTEIN WHICH RECOGNIZES A PALINDROMIC DNA-SEQUENCE IN THE NEGATIVE REGULATORY ELEMENT OF THE HIV-1 LONG TERMINAL REPEAT WITH HOMOLOGY TO STEROID THYROID-HORMONE RECEPTOR-BINDING SITES, BIOCHEMICAL SOCIETY TRANSACTIONS18(4)pp. 555-556 PORTLAND PRESS
Sabin CA, Phillips AN, Lee CA, Janossy G, Emery V, Griffiths PD (1995) The effect of CMV infection on progression of human immunodeficiency virus disease is a cohort of haemophilic men followed for up to 13 years from seroconversion., Epidemiol Infect114(2)pp. 361-372
The effect of prior infection with cytomegalovirus (CMV) on progression of HIV disease in a cohort of 111 men with haemophilia was studied after 13 years follow-up. The relative hazards associated with CMV positivity on progression to AIDS, death and a CD4 count of 0.05 x 10(9)/l were 2.28, 2.42 and 2.34, respectively. CMV seropositive patients were significantly older than the seronegative and this was controlled for by using a Cox proportional hazards model. The relative hazards for the three endpoints decreased to 1.89, 1.82 and 1.93 respectively and were marginally non-significant (P = 0.05, 0.08 and 0.08 for the three endpoints respectively). We conclude that this cohort continues to show evidence of a 'co-factor' effect associated with prior infection with CMV which is confounded by age but not completely explained by age differences. The potential biological significance of these results is discussed in the context of recent controlled clinical trials which show a survival benefit from long-term high-dose acyclovir, a drug with activity in vivo against CMV and other herpesviruses.
Kidd IM, Clark DA, Bremner JA, Pillay D, Griffiths PD, Emery VC (1998) A multiplex PCR assay for the simultaneous detection of human herpesvirus 6 and human herpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products., J Virol Methods70(1)pp. 29-36
A multiplex polymerase chain reaction (PCR) method was developed for the simultaneous detection of human herpesviruses 6 and 7 (HHV-6; HHV-7) in clinical samples, using primers which amplify a section of the HHV-6 U67 gene and the HHV-7 homologue of the HHV-6 U42 gene. Comparison of the multiplex assay with the respective single PCR assays, using cloned HHV-6 and HHV-7 sequences as targets for amplification, showed equivalent sensitivity and specificity for the assays. To demonstrate the use of multiplex PCR for the analysis of clinical samples, serum and saliva from infants were analysed using this technique. The results showed that a clear distinction can be made between the amplicons of HHV-6 and HHV-7, without loss of sensitivity or specificity. There was complete concordance between the respective single PCR assays, and the multiplex PCR. HHV-6 amplicons derived from the multiplex PCR analysis were typed by differential AvaII restriction endonuclease digestion, in which HHV-6 variant A amplicons are cleaved but those of variant B remain undigested. These results were compared to HHV-6 variant typing by an established method, the results of which showed complete concordance between assays. It is proposed that this multiplex assay, where HHV-6 positive samples may be typed directly from the reaction products, is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of HHV-6 and HHV-7.
Mocroft AJ, Johnson MA, Sabin CA, Lipman M, Elford J, Emery V, Morcinek J, Youle M, Janossy G, Lee CA (1995) Staging system for clinical AIDS patients. Royal Free/Chelsea and Westminster Hospitals Collaborative Group., Lancet346(8966)pp. 12-17
Although there are wide differences in prognosis between patients with AIDS they are often thought of as a single homogeneous group. We think a simple staging system that accounts for important prognostic factors including type and number of AIDS diseases and the CD4 lymphocyte count is required. We followed 363 AIDS patients at the Royal Free Hospital and reported the occurrence of 680 AIDS-defining diseases (ADDs). We measured CD4 counts at approximately monthly intervals. Severity of AIDS diseases was defined a priori on the basis of survival in the AIDS in Europe study of 6578 AIDS patients: mild-oesophageal candidiasis, Kaposis sarcoma (cutaneous), Pneumocystis carinii pneumonia, extrapulmonary tuberculosis; severe-all other ADDs except lymphoma; very severe-lymphoma. The risk of death increased by 15% (p = 0.08) for each mild condition experienced, by 89% (p < 0.0001) for each new severe condition and by 535% (p < 0.0001) when a lymphoma developed. Estimates from the Cox model were used to derive a score reflecting the risk of death. Patient experience was divided into three categories. Patients in AIDS Grade I had an average death rate of one per 10.1 years, compared with one per 2.8 years in AIDS Grade II and one per 1.1 years in AIDS Grade III. Similar rates were seen in an independent validation study on 1230 AIDS patients at different hospital. Our grading system should be useful for patient management, clinical trial design, surveillance, and resource management.
Emery VC, Asher K, Sanjuan CDEJ (2012) Importance of the cytomegalovirus seropositive recipient as a contributor to disease burden after solid organ transplantation., J Clin Virol54(2)pp. 125-129
BACKGROUND: The incidence of cytomegalovirus (CMV) syndrome/disease after adult solid organ transplantation in the era effective antiviral therapy has not been fully assessed. OBJECTIVE: To determines the incidence of CMV syndrome/disease after solid organ transplantation in the UK. STUDY DESIGN: A retrospective analysis of 1807 solid organ transplants from 12 UK solid organ transplant centres representing 32.7% of all transplant activity occurring in the UK between 1/04/2004 and 31/03/2006. Patients were categorised into those experiencing an episode of symptomatic CMV infection after transplant or those who remained free of symptoms. All patients were followed up for 2 years for the occurrence of CMV syndrome/disease. RESULTS: The majority of the transplant centres used valganciclovir prophylaxis in the high risk D+R- patients (91.6%) whereas management of the lower risk D+R+ and D-R+ patients was more variable with deployment of both prophylactic and pre-emptive strategies in <50% of centres. CMV syndrome/disease occurred in 20.5% of the D+R- patients representing 55 cases whereas the incidence was only 8.1% and 9% in the D+R+ and D-R+ group, respectively (p<0.001 compared to the D+R- group), but representing a further 58 cases of CMV syndrome/disease. CMV viraemia in the D+R- group was associated with a high probability (65%) of CMV syndrome/disease in renal transplant recipients whereas this was less apparent in the intermediate risk groups. CONCLUSIONS: CMV syndrome/disease remains an important healthcare burden after solid organ transplantation with the intermediate risk groups contributing similar numbers of cases as the high risk group.
Fox JC, Ait-Khaled M, Webster A, Emery VC (1991) Eliminating PCR contamination: is UV irradiation the answer?, J Virol Methods33(3)pp. 375-382
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.
Ullman CG, Emery VC (1996) Transforming Proteins of Human Papillomaviruses., Rev Med Virol6(1)pp. 39-55
Nebbia G, Mattes FM, Smith C, Hainsworth E, Kopycinski J, Burroughs A, Griffiths PD, Klenerman P, Emery VC (2008) Polyfunctional cytomegalovirus-specific CD4+ and pp65 CD8+ T cells protect against high-level replication after liver transplantation., Am J Transplant8(12)pp. 2590-2599
To determine whether polyfunctional CD4+ T-cell responses coupled with CD8+ T-cell responses against human cytomegalovirus (HCMV) are key to the control of HCMV replication we prospectively analyzed 29 liver transplant recipients for CD4+ T-cell responses against soluble HCMV antigen, pp65 and IE1 proteins, CD8+ T-cell responses against pp65 and IE1 proteins and a range of T helper (Th) 1 and Th2 cytokines. Eleven patients (38%) developed HCMV DNAemia at a median of 21 days post-liver transplantation (range 17-31 days). There was a significantly lower frequency and absolute number of total HCMV CD4+ T cells producing IFNgamma, IFNgamma+IL2 and IL2 and pp65-CD8+ T cells producing IFNgamma in patients with DNAemia. The quantities of Th1 and Th2 cytokines present during the first 20 days posttransplant were not predictive of DNAemia. Cut-off levels during the first 20 days posttransplant of 0.1% of lysate stimulated CD4+ T cells producing IL2, and pp65-CD8+ T cells producing IFNgamma above 0.4% had positive and negative predictive values for DNAemia of 54% and 100% and 50% and 92%, respectively. Measuring polyfunctional CD4+ T cells against HCMV early posttransplant may allow targeted intervention to minimize the occurrence and acute and long-term consequences of HCMV replication.
Emery VC, Griffiths PD (2000) Prediction of cytomegalovirus load and resistance patterns after antiviral chemotherapy., Proc Natl Acad Sci U S A97(14)pp. 8039-8044
Cytomegalovirus (CMV) replicates rapidly in its human host with a doubling time of approximately 1 day. Using simple mathematical models we show that the efficacy of the anti-CMV drug ganciclovir (GCV) against wild-type strains is 91.5% [95% confidence interval (CI) 89-94%] when given i.v. (5 mg/kg twice a day) but only 46.5% (95% CI 45-47.5%) when given orally (1 g three times a day) whereas the corresponding figures for a typical GCV-resistant virus are 62% (95% CI 57-66%) and 35% (95% CI 33-37%), respectively. During prolonged periods of GCV therapy we show that the apparent sudden appearance of GCV resistance is explained by the combination of two exponentially increasing populations (wild type and mutant) at doses of GCV that do not completely inhibit CMV replication. Cell culture methods to assess CMV drug resistance in vivo will underestimate its prevalence because of the fitness loss of resistant virus in the absence of therapy. The parameters determined from these models then were used to predict the likely viral load and resistance patterns in patients on prolonged therapy with GCV. The modeled and experimental data showed excellent agreement over extended time periods (up to 270 days of therapy) and provide a framework to predict the virologic course of patients at therapeutic initiation.
Griffiths SJ, Riddell NE, Masters J, Libri V, Henson SM, Wertheimer A, Wallace D, Sims S, Rivino L, Larbi A, Kemeny DM, Nikolich-Zugich J, Kern F, Klenerman P, Emery VC, Akbar AN (2013) Age-associated increase of low-avidity cytomegalovirus-specific CD8 + T cells that re-express CD45RA, Journal of Immunology190(11)pp. 5363-5372
The mechanisms regulating memory CD8+ T cell function and homeostasis during aging are unclear. CD8+ effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8+ T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8+ T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8+ T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-± that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA2 CD45RO+ CMV-specific CD8+ T cells induced CD45RA expression while Ag activated cells remained CD45RO+. This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8+CD45RA+ T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging. Copyright © 2013 by The American Association of Immunologists, Inc.
Whalley S, Brown D, Webster G, Jacobs R, Teo CG, Emery V, Dusheiko G (2002) Evolution of hepatitis B virus during primary infection: Relevance to virulence and outcome, JOURNAL OF HEPATOLOGY36pp. 95-95 ELSEVIER SCIENCE BV
WAKEFIELD AJ, FOX JD, SAWYERR AM, TAYLOR JE, SWEENIE CH, SMITH M, EMERY VC, HUDSON M, TEDDER RS, POUNDER RE (1992) DETECTION OF HERPESVIRUS DNA IN THE LARGE-INTESTINE OF PATIENTS WITH ULCERATIVE-COLITIS AND CROHNS-DISEASE USING THE NESTED POLYMERASE CHAIN-REACTION, JOURNAL OF MEDICAL VIROLOGY38(3)pp. 183-190 WILEY-LISS
Downey GP, Emery VC, Walker PG (1999) A longitudinal study of human papillomavirus 16 positivity in the development of lower genital intraepithelial neoplasia in immunosuppressed women, Journal of Lower Genital Tract Disease3(3)pp. 163-170
Objectives. We studied a group of female renal transplant patients and patients receiving dialysis to determine the natural history of human papillomavirus (HPV) type 16 DNA infection in the two groups and its effect on lower genital tract intraepithelial neoplasia development and progression. Materials and Methods. Female renal transplant and dialysis patients were recruited from the renal unit of the hospital. Follow-up was every 6 months, and all women were seen at least twice. Assessment was obtained by cytology, colposcopy, directed biopsy, and HPV 16 DNA semiquantitative analysis by polymerase chain reaction. The oligonucleotide primers were designed to amplify a 233-base pair region of the HPV 16 E6 and E7 genes (nucleotides 491-714), with confirmation of the amplified product performed by Southern blotting and probing with an oligonucleotide probe specific to an internal portion of the ampli-con. Results. The 28 transplant patients and 14 dialysis patients were followed up for an overall mean of 14.6 months (range, 5-24 months; median, 18 months). Only one dialysis patient was positive for the HPV 16 genome, and this was in low copy numbers. The HPV status changed in some patients throughout the study period; however, no one who had medium-high or high copy numbers at enrollment became HPV 16 DNA-negative. From study entry, HPV 16 DNA positivity was associated with progression of preexisting intraepithelial disease and new disease development, although the association was not statistically significant by either log-rank or Cox's proportional hazard model (p = .76 and .77, respectively). Neither coexisting cervical intraepithelial neoplasia (CIN) nor type of renal disease nor the therapeutic regimen used at study entry appeared to influence disease development and progression (p = .42 and .7, respectively). The only factor that influenced development and progression of CIN from study entry was the length of time from transplantation (p = .01). Time from transplantation did appear to influence HPV 16 DNA positivity (p = .04), but the combined influence did not hold up to scrutiny in a multivariate analysis using Cox's proportional model (p = .34), suggesting that HPV 16 positivity was a surrogate marker for time from transplantation. Conclusions. HPV 16 DNA positivity alone is an inadequate explanation for the development and progression of lower genital tract intraepithelial disease in women with renal transplant. The length of time from transplanta
Bowen EF, Wilson P, Cope A, Sabin C, Griffiths P, Davey C, Johnson M, Emery V (1996) Cytomegalovirus retinitis in AIDS patients: influence of cytomegaloviral load on response to ganciclovir, time to recurrence and survival., AIDS10(13)pp. 1515-1520
OBJECTIVES: Despite life-long maintenance therapy, cytomegalovirus (CMV) retinitis frequently progresses in patients with AIDS. Virological markers that could clarify pathogenesis and identify risk factors for progression are required. DESIGN AND METHODS: We prospectively recruited 45 patients with CMV retinitis. Blood and urine samples were collected before and after induction therapy, and on a monthly basis thereafter during routine medical and ophthalmological assessment, and at any time retinitis progressed. CMV load was measured by quantitative-competitive polymerase chain reaction (PCR). RESULTS: The median time to first progression of retinitis was 78 days and to death was 8.7 months. Eighty-five per cent of patients who were PCR-positive at diagnosis of retinitis became PCR-negative after 21 days of ganciclovir induction therapy. Six patients who remained PCR-positive after 21 days of treatment had a significantly higher CMV load at presentation (P = 0.005), and a shorter time to first progression of retinitis of 40 days. High CMV loads in blood at presentation were associated with a shorter time to progression (P = 0.16; relative hazard, 1.57) and a significantly shorter time to death (P = 0.004; relative hazard, 1.76). This significant relationship with survival remained after adjustment for potential confounding variables (CD4 count, age, method of drug administration). CONCLUSIONS: We conclude that CMV load in the blood of AIDS patients is an important factor in the pathogenesis of retinitis, and quantification of CMV could be used to both select patients for controlled clinical trials and to optimize individual anti-CMV induction therapy.
Carpenter B, Haque T, Dimopoulou M, Atkinson C, Roughton M, Grace S, Denovan S, Fielding A, Kottaridis PD, Griffiths P, Mackinnon S, Emery V, Chakraverty R (2010) Incidence and dynamics of Epstein-Barr virus reactivation after alemtuzumab-based conditioning for allogeneic hematopoietic stem-cell transplantation., Transplantation90(5)pp. 564-570
BACKGROUND: Reactivation of Epstein-Barr virus (EBV) infection and posttransplant lymphoproliferative disorder (PTLD) pose a significant risk after T-cell-depleted (TCD) allogeneic hematopoietic stem-cell transplantation (HSCT). The pattern of EBV reactivation in patients receiving allogeneic HSCT, incorporating in vivo or in vitro alemtuzumab as the method of TCD, is not known. METHODS: Monitoring for EBV DNA was performed by quantitative polymerase chain reaction on whole blood in 111 consecutive adults undergoing HSCT using alemtuzumab-based TCD. Patients with more than 40,000 copies/mL were screened for PTLD, followed by the withdrawal of immunosuppression and a single infusion of rituximab. RESULTS: The 2-year cumulative incidence of EBV DNAemia was 40.3%. In vivo alemtuzumab was associated with earlier EBV reactivation than in vitro alemtuzumab (100-day incidence 22.7% vs. 2.8%, P=0.006). Eighteen patients (16%) had EBV DNAemia of more than 40,000 copies/mL. In evaluable patients, the initial rate of increase in EBV DNA levels was significantly faster in those who went on to treatment with rituximab than in patients who were left untreated (mean doubling time 3.5 days vs. 4.2 days, P=0.003). Rituximab treatment induced rapid declines in EBV DNA with an average half-life of 1.2+/-0.7 days. Only one patient (0.9%) had histologic confirmation of PTLD and subsequently attained a complete remission with rituximab that persists at 18 months. CONCLUSIONS: Alemtuzumab-based TCD is associated with a high frequency of EBV reactivation but a low (<1%) risk of PTLD using a strategy of preemptive rituximab therapy.
Emery VC, Cope AV, Sabin CA, Burroughs AK, Rolles K, Lazzarotto T, Landini MP, Brojanac S, Wise J, Maine GT (2000) Relationship between IgM antibody to human cytomegalovirus, virus load, donor and recipient serostatus, and administration of methylprednisolone as risk factors for cytomegalovirus disease after liver transplantation., J Infect Dis182(6)pp. 1610-1615
A retrospective study was performed on a selected cohort of 40 liver transplant recipients derived from the previous prospective follow-up of 162 liver transplant patients. The criterion for selection of this cohort was the presence of human cytomegalovirus (HCMV) DNAemia after transplantation, as determined by qualitative polymerase chain reaction (PCR). These 40 patients were followed longitudinally by quantitative PCR and by the new recombinant antigen-based AxSYM immunoassay for IgM to HCMV. The detection of IgM to CMV after transplantation was significantly associated with the development of HCMV disease in patients who had evidence of active HCMV replication in the blood by PCR (P=.01). On the basis of multivariate logistic regression analyses, the maximum titer of IgM detected after transplantation was a risk factor that was independent of augmented methylprednisolone and donor seropositivity. However, in multivariate analyses, elevated virus load continued to be the predominant risk factor for progression to HCMV disease.
Emery VC (1992) Baculovirus expression vectors., Methods Mol Biol8pp. 319-326
The baculovirus system has the potential for the large-scale production of protein products by two methods. First, as a result of recent advances (1,5), large-scale cell culture is now possible and, second, the recombinant baculovirus can be used to infect susceptible insect larvae (3). Whether using small-scale cell culture or the aforementioned methods for scale-up, the requirement for downstream processing of the protein product is manifest. Purification of the product is greatly facilitated when expression is high (ca. 30% of total cell protein); however, when expression is relatively low or membrane proteins are required, the researcher faces the same problems encountered in ascertaining the optimum conditions for any de novo protein purification. Obviously, the degree of purity required for a given product is indicative of its final use; consequently, a number of antigens for use in diagnostic procedures have been relatively crude preparations (4,5). This chapter highlights the methods that have been used to purify baculovirusexpressed protein products, and is aimed at providing general guidelines to the purification of certain types of protein product rather than a definitive guide to protein purification.
Gor D, Lee D, Emery VC (1996) Detection of human cytomegalovirus polymerase chain reaction products using oligonucleotide probes directly conjugated to alkaline phosphatase (vol 61, pg 145, 1996), JOURNAL OF VIROLOGICAL METHODS62(2)pp. 185-185 ELSEVIER SCIENCE BV
Walter S, Atkinson C, Sharland M, Rice P, Raglan E, Emery VC, Griffiths PD (2008) Congenital cytornegalovirus: association between dried blood spot viral load and hearing loss, ARCHIVES OF DISEASE IN CHILDHOOD-FETAL AND NEONATAL EDITION93(4)pp. F280-F285 B M J PUBLISHING GROUP
Ullman CG, Haris PI, Galloway DA, Emery VC, Perkins SJ (1996) 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., Biochem J319 ( Pt 1)pp. 229-239
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.
Downey GP, Bavin PJ, Deery AR, Crow J, Griffiths PD, Emery VC, Walker PG (1994) Relation between human papillomavirus type 16 and potential for progression of minor-grade cervical disease., Lancet344(8920)pp. 432-435
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.
Clark DA, Fryer JF, Tucker AW, McArdle PD, Hughes AE, Emery VC, Griffiths PD (2003) Porcine cytomegalovirus in pigs being bred for xenograft organs: progress towards control., Xenotransplantation10(2)pp. 142-148
In human medicine, human cytomegalovirus (HCMV) is readily transmitted by organ transplant causing end-organ disease and triggering graft rejection in recipients. Because of a chronic shortage of human organs, pigs transgenic for human complement control proteins are being considered as potential donors. Such xenotransplantation raises concerns about the potential zoonotic transmission of viruses including porcine cytomegalovirus (PCMV), an endemic infection of pigs. Similar to HCMV and PCMV transmission is thought to occur in utero and perinatally. We used quantitative polymerase chain reaction to examine the prevalence, organ distribution and viral load of PCMV in human decay accelerating factor (CD55) transgenic pigs. In animals reared under conventional farm conditions, virus was identified in a wide range of organs including potential xenografts (liver, kidney and heart). The spleen was PCMV DNA positive in all infected pigs. Examination of foetal spleens failed to identify evidence of transplacental infection and prospective monitoring of two litters showed that infection occurred in the postnatal period. This transmission was prevented by hysterotomy derivation and barrier rearing. Our findings demonstrate that PCMV could be eradicated from pig herds being bred for xenotransplantation and argue that the spleen from donor animals should be examined as part of quality control procedures if clinical trials proceed.
Emery VC, Atkins MC, Bowen EF, Clark DA, Johnson MA, Kidd IM, McLaughlin JE, Phillips AN, Strappe PM, Griffiths PD (1999) Interactions between beta-herpesviruses and human immunodeficiency virus in vivo: evidence for increased human immunodeficiency viral load in the presence of human herpesvirus 6., J Med Virol57(3)pp. 278-282
In vitro, beta-herpesviruses can stimulate or inhibit HIV replication under particular circumstances. In order to investigate the effects of beta-herpesvirus infection on HIV replication and vice versa at an organ level, we determined the quantitative relationships between cytomegalovirus (CMV), human herpesviruses (HHV) 6 and 7, and HIV-1 proviral DNA using quantitative competitive PCR methods in 141 organs collected at autopsy from 11 AIDS patients. The presence of HHV-6 DNA in an organ was significantly associated with elevated HIV-1 proviral DNA (difference in HIV median loads, 1.3 log10 genomes; P = 0.004). Consistent with this, there was a trend for the presence of HIV-1 proviral DNA to be associated with an elevated HHV-6 load (0.44 log10 difference; P = 0.07). In contrast, there were no significant differences between viral loads in the combinations of either CMV or HHV-7 with HIV-1 proviral DNA load. Pairwise combinations of the beta-herpesviruses revealed that the quantity of HHV-7 was increased in the presence of HHV-6 (difference in median loads, 1.3 log10; P = 0.001) and the quantity of HHV-6 was increased in the presence of HHV-7 (difference in median loads, 0.7 log10; P=0.002). These results demonstrate that the presence of HHV-6 in an organ is significantly associated with an elevated HIV-1 proviral load and have implications for understanding HIV pathogenesis in the human host and the role that beta-herpesviruses, especially HHV-6, might play as cofactors in the HIV disease process.
Sabin CA, Emery V, Devereux HL, Griffioen A, Bishop J, Dusheiko G, Yee TT, Herrero-Martinez E, Lee CA (2002) Long-term patterns of hepatitis C virus RNA concentrations in a cohort of HIV seronegative men with bleeding disorders., J Med Virol68(1)pp. 68-75
Little is known about the natural history of hepatitis C virus (HCV) RNA concentrations over the course of infection. The aim of this study was to describe the natural history of HCV RNA concentrations in 85 HIV negative men with bleeding disorders infected with HCV for up to 30 years. HCV RNA concentrations were measured in yearly serum samples using a branched DNA assay. HCV RNA concentrations increased over time in this cohort. Two years after exposure to HCV, 53% of patients had undetectable concentrations and no patients had levels >7 log(10)(genome Eq/ml); by 20 years, these proportions had changed to 23% and 32% respectively. The RNA concentration correlated strongly with alanine aminotransferase (ALT; correlations of 0.41-0.71 depending on stage of infection) and aspartate aminotransferase (AST; 0.20-0.51) levels. Patients with haemophilia A had significantly higher HCV concentrations than those with other disorders. An effect of HCV genotype on HCV RNA concentrations became nonsignificant after excluding patients who were persistently HCV PCR negative and who could not be genotyped. The correlation of HCV RNA concentrations with other markers of liver function, such as ALT, means that studies with clinical outcomes are required to assess whether HCV RNA concentrations provide additional prognostic information to that provided by these other markers.
Bowen EF, Sabin CA, Wilson P, Griffiths PD, Davey CC, Johnson MA, Emery VC (1997) Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIV-positive patients at high risk of CMV disease., AIDS11(7)pp. 889-893
BACKGROUND: Cytomegalovirus (CMV) disease is a major cause of morbidity in patients with HIV infection. Despite treatment, CMV retinitis causes substantial visual loss, especially in patients with CD4 cell counts below 50 x 10(6)/l. Although routine ophthalmological screening of these patients has been recommended, no controlled trials have evaluated how frequently it should be performed. The aim of this study was to assess whether CMV polymerase chain reaction (PCR) results could direct ophthalmological screening to patients at high risk of CMV retinitis. METHODS: In a prospective study of HIV-positive patients with CD4 cell counts below 50 x 10(6)/l, CMV viraemia was detected by qualitative PCR of whole blood. Patients who were CMV PCR-viraemic were allocated to monthly virological and ophthalmological follow-up; patients who were PCR-negative received 3-monthly virological and ophthalmological follow-up. CMV viral load was determined in all CMV-positive samples using a quantitative competitive PCR. RESULTS: Nineteen out of 97 patients developed CMV disease over the first 12 months of the study. Sixteen (59%) out of 27 patients who were CMV-positive developed disease compared with three (4%) out of 70 of patients who were PCR-negative (P = 0.0001). A positive CMV PCR result was significantly associated with the development of disease (P = 0.0001), with a relative hazard of 20.15 [95% confidence interval (CI), 5.80-69.98]. Median CMV viral load was significantly higher in those individuals who went on to develop CMV disease (P = 0.02). In PCR-positive patients, each 0.25 log10 increase in viral load increased the risk of disease (relative hazard, 1.37; 95% CI, 1.15-1.63; P = 0.0004). CONCLUSIONS: CMV PCR predicts the development of CMV disease and can be used to target ophthalmological resources to those patients at highest risk of retinitis. Asymptomatic patients who are PCR-positive represent a high-risk group in whom controlled trials of pre-emptive therapy could be conducted.
Phillips AN, Mocroft A, Emery V, Janossy G, Johnson M (1995) Mathematical model of the effects of therapy and resistance to therapy in primary HIV infection, JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES AND HUMAN RETROVIROLOGY10pp. 83-83 LIPPINCOTT-RAVEN PUBL
Bowen EF, Griffiths PD, Davey CC, Emery VC, Johnson MA (1996) Lessons from the natural history of cytomegalovirus., AIDS10 Suppl 1pp. S37-S41
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.
Caplin B, Sweny P, Burroughs A, Emery V, Griffiths P (2005) Antiviral treatment after solid organ transplantation, LANCET366(9488)pp. 806-807 ELSEVIER SCIENCE INC
Emery VC (2001) Prophylaxis for CMV should not now replace pre-emptive therapy in solid organ transplantation., Rev Med Virol11(2)pp. 83-86
Pre-emptive therapy (PET) initiated on the basis of HCMV positivity in the blood using sensitive methods such as PCR, nucleic acid sequence based amplification or antigenaemia offers several advantages for the management of HCMV infection. These include the ability to target antiviral drug therapy to those most at risk of future disease, minimising drug exposure and maximising cost-benefit. In addition, allowing limited replication to occur also provides immune stimulation which will be important for future control of HCMV replication. In contrast, prophylaxis is a high-cost strategy which exposes all patients to potentially toxic drugs, does not facilitate immune priming and leads to the development of late HCMV infection and disease in high-risk patients.
ORCHARD K, PERKINS N, CHAPMAN C, HARRIS J, EMERY V, GOODWIN G, LATCHMAN D, COLLINS M (1990) A NOVEL T-CELL PROTEIN WHICH RECOGNIZES A PALINDROMIC SEQUENCE IN THE NEGATIVE REGULATORY ELEMENT OF THE HUMAN-IMMUNODEFICIENCY-VIRUS LONG TERMINAL REPEAT, JOURNAL OF VIROLOGY64(7)pp. 3234-3239 AMER SOC MICROBIOLOGY
Emery VC (1995) Nucleoside analogues as antiviral agents: Learning from experience?, International Antiviral News3(3)pp. 42-44
Lock MJ, Griffiths PD, Emery VC (1997) Development of a quantitative competitive polymerase chain reaction for human herpesvirus 8., J Virol Methods64(1)pp. 19-26
A quantitative competitive polymerase chain reaction (PCR) assay for human herpesvirus 8 (HHV8) was developed. The assay uses a competitive internal control sequence which differs from the wild type sequence by the presence of an EcoRI restriction endonuclease site. The quantitative method was validated by co-amplifying known amounts of control sequence DNA and wild type DNA, and shown to accurately quantify HHV8 within the range of 10(1)-10(6) genome copies (R = 0.998). The assay was used to quantify HHV8 in sequential blood samples of a renal transplant patient diagnosed with Kaposi's sarcoma (KS). A peak viral load of 28320 genomes/ml blood was present at diagnosis of KS, which reduced to 13680 genomes after 2 days and was undetectable 4 months later. Control of HHV8 replication in this patient parallelled the cessation of immunosuppressive therapy.
Whalley SA, Webster GJM, Brown D, Teo CG, Bertoletti A, Emery VC, Dusheiko GM (2002) Transient generation of core CD8+ cytotoxic T-cell escape mutants during primary HBV infection, GUT50pp. A112-A112 B M J PUBLISHING GROUP
Clark DA, Emery VC, Griffiths PD (2003) Cytomegalovirus, human herpesvirus-6, and human herpesvirus-7 in hematological patients., Semin Hematol40(2)pp. 154-162
The prototype member of the Betaherpesvirinae subfamily, cytomegalovirus (CMV), is the most important infectious pathogen in transplant recipients, including those receiving bone marrow or stem cell grafts. Overt CMV disease such as pneumonitis is notoriously difficult to treat. Antiviral prophylaxis, rapid diagnostic tests to identify CMV infection, and preemptive antiviral chemotherapy are significant improvements in the management of CMV. As the kinetics of the immune response to CMV become better defined, immunotherapeutic approaches should be introduced to complement current management strategies. Two newly identified betaherpesviruses, human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), are genetically more closely related to each other than to CMV. Both are highly prevalent in the general population and infections post-bone marrow transplantation are common. These viruses are not as pathogenic as CMV but HHV-6 at least can cause disease such as encephalitis, hepatitis, and bone marrow suppression. Both of these newer herpesviruses are potentially susceptible to existing and licensed antiherpesvirus drugs.
Parker A, Bowles K, Bradley JA, Emery V, Featherstone C, Gupte G, Marcus R, Parameshwar J, Ramsay A, Newstead C (2010) Management of post-transplant lymphoproliferative disorder in adult solid organ transplant recipients - BCSH and BTS Guidelines, BRITISH JOURNAL OF HAEMATOLOGY149(5)pp. 693-705 WILEY-BLACKWELL
Emery VC, Webster A, Griffiths PD (1993) Molecular and cell biology of opportunistic infections in AIDS. Herpesviruses., Mol Cell Biol Hum Dis Ser2pp. 257-277
Davison AJ, Akter P, Cunningham C, Dolan A, Addison C, Dargan DJ, Hassan-Walker AF, Emery VC, Griffiths PD, Wilkinson GWG (2003) Homology between the human cytomegalovirus PL11 gene family and human adenovirus E3 genes, JOURNAL OF GENERAL VIROLOGY84pp. 657-663 SOC GENERAL MICROBIOLOGY
Emery VC, Hassan-Walker AF, Burroughs AK, Griffiths PD (2002) Human cytomegalovirus (HCMV) replication dynamics in HCMV-naive and -experienced immunocompromised hosts., J Infect Dis185(12)pp. 1723-1728
Human cytomegalovirus (HCMV) can infect both HCMV-naive and -experienced transplant patients. In this study, the growth rate of HCMV in HCMV-naive hosts (1.82 units/day; 95% confidence interval [CI], 1.44-2.56 units/day) was shown to be significantly faster than the growth rate of virus in HCMV-experienced hosts undergoing recurrent infection (0.61 units/day; 95% CI, 0.55-0.7 units/day; P<.0001). The basic reproductive number (R(0)) for HCMV-naive liver transplant patients was 15.1 (95% CI, 8.9-44) but was only 2.4 (95% CI, 2.35-2.8) for HCMV-experienced transplant recipients, corresponding to an anti-HCMV immune efficacy of approximately 84%, despite immunosuppressive therapy. The R(0) values suggest that an anti-HCMV drug or vaccine with an efficacy of >93% (95% CI, 89%-98%) is required to eliminate viral growth during infection of HCMV-naive liver transplant recipients, whereas lower efficacy levels are sufficient to reduce the R(0) value to <1 in hosts with prior HCMV immunity.
Bradley AJ, Kovacs IJ, Gatherer D, Dargan DJ, Alkharsah KR, Chan PKS, Carman WF, Dedicoat M, Emery VC, Geddes CC, Gerna G, Ben-Ismaeil B, Kaye S, McGregor A, Moss PA, Pusztai R, Rawlinson WD, Scott GM, Wilkinson GWG, Schulz TF, Davison AJ (2008) Genotypic analysis of two hypervariable human cytomegalovirus genes, JOURNAL OF MEDICAL VIROLOGY80(9)pp. 1615-1623 WILEY-LISS
Devereux HL, Brown D, Dusheiko GM, Emery VC, Lee CA (1997) Long-term evolution of the 5'UTR and a region of NS4 containing a CTL epitope of hepatitis C virus in two haemophilic patients., J Gen Virol78 ( Pt 3)pp. 583-590
Haemophilic patients exposed to unsterilized clotting factor concentrates prior to 1985 have become infected with hepatitis C virus (HCV). We have studied the sequence evolution of the 5'UTR and a region of NS4 over 12 years in one human immunodeficiency virus (HIV) positive haemophilic patient and 14 years for one HIV negative haemophilic patient. One sample each year from the date of HCV infection to 1994 was analysed for genotype, virus load and nucleotide sequence of the two genetic loci. Both patients were infected with HCV genotype 1 throughout the study period. The virus load profiles were similar except that the profile for the HIV infected patient was displaced 4 years earlier relative to the other patient. Mean divergence of the quasispecies at both the 5'UTR and NS4 loci was higher in the HIV coinfected patient. Phylogenetic analysis indicated that evolution of the 5'UTR was host independent, whereas the NS4 region containing a CD8 restricted CTL epitope evolved in a host specific fashion.
Deane M, Gor D, Macmahon ME, Emery V, Griffith PD, Cummins M, Prentice HG (1997) Quantification of CMV viraemia in a case of transfusion-related graft-versus-host disease associated with purine analogue treatment., Br J Haematol99(1)pp. 162-164
We report a patient who developed transfusion-associated graft-versus-host-disease (GvHD) and concurrent cytomegalovirus (CMV) infection, both complications thought to be related to severe T lymphocyte depletion induced by treatment with a purine analogue drug, fludarabine. CMV viraemia was detected by qualitative PCR and the viral load in positive samples was measured using a fully quantitative PCR assay. This quantitative assay enabled the evaluation of the efficacy of antiviral interventions based on the qualitative PCR result. The case illustrates the risks associated with the use of purine analogue drugs, as well as the value of quantitative CMV PCR assays for monitoring CMV infection in immunocompromised patients.
Bavin PJ, Walker PG, Emery VC (1993) Sequence microheterogeneity in the long control region of clinical isolates of human papillomavirus type 16., J Med Virol39(4)pp. 267-272
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.
Emery VC (2000) Viral-load kinetics in post-transplantation cytomegalovirus infections - Reply, LANCET356(9242)pp. 1686-1686 ELSEVIER SCIENCE INC
Roxby AC, Atkinson C, Ásbjörnsdóttir K, Farquhar C, Kiarie JN, Drake AL, Wald A, Boeckh M, Richardson B, Emery V, John-Stewart G, Slyker JA (2014) Maternal valacyclovir and infant cytomegalovirus acquisition: A randomized controlled trial among HIV-infected women, PLoS ONE9(2)
Background: Studies in HIV-1-infected infants and HIV-1-exposed, uninfected infants link early cytomegalovirus (CMV) acquisition with growth delay and cognitive impairment. We investigated maternal valacyclovir to delay infant acquisition of CMV. Methods: Pregnant women with HIV-1, HSV-2 and CD4 count >250 cells/¼l were randomized at 34 weeks gestation to 500 mg twice-daily valacyclovir or placebo for 12 months. Maternal CMV DNA was measured in plasma at 34 weeks gestation, in cervical secretions at 34 and 38 weeks gestation, and in breast milk at 7 postpartum timepoints; infant CMV DNA was measured in dried blood spots at 8 timepoints including birth. Results: Among 148 women, 141 infants were compared in intent-to-treat analyses. Maternal and infant characteristics were similar between study arms. Infant CMV acquisition did not differ between study arms, with 46/70 infants (66%) in placebo arm and 47/71 infants (66%) in the valacyclovir arm acquiring CMV; median time to CMV detection did not differ. CMV DNA was detected in 92% of 542 breast milk specimens with no difference in CMV level between study arms. Change in cervical shedding of CMV DNA between baseline and 38 weeks was 0.40-log greater in the placebo arm than the valacyclovir arm (p = 0.05). Conclusions: In this cohort of HIV-1-seropositive mothers, two-thirds of infants acquired CMV by one year. Maternal valacyclovir had no effect on timing of infant CMV acquisition or breast milk CMV viral loads, although it modestly reduced cervical CMV shedding. Maternal prophylaxis to reduce infant CMV acquisition warrants further evaluation in trials with antiviral agents. Trials Registration: ClinicalTrials.gov NCT00530777 © 2014 Roxby et al.
Parker A, Bowles K, Bradley JA, Emery V, Featherstone C, Gupte G, Marcus R, Parameshwar J, Ramsay A, Newstead C (2010) Diagnosis of post-transplant lymphoproliferative disorder in solid organ transplant recipients - BCSH and BTS Guidelines, BRITISH JOURNAL OF HAEMATOLOGY149(5)pp. 675-692 WILEY-BLACKWELL
Kuo YH, Kuo YL, Emery VC, Sabin CA, Hassan-Walker AF, Griffiths PD (2000) Viral-load kinetics and CMV disease  (multiple letters), Lancet356(9238)pp. 1352-1353
Emery VC (2001) Investigation of CMV disease in immunocompromised patients., J Clin Pathol54(2)pp. 84-88
Cytomegalovirus (CMV) is a recognised cause of morbidity and mortality in immunocompromised individuals. This review will concentrate on recent advances in the understanding of the complex interplay between the host and parasite and the pathological consequences of perturbation of the host immune system. The classic view of CMV as a slowly replicating virus is challenged by recent in vivo findings suggesting that active replication occurs dynamically in the human host, with a doubling time of approximately one day. In addition, CMV load plays a major role in viral pathogenesis, such that increased CMV replication is a significant risk factor for disease in all immunocompromised groups studied to date. These studies focus attention on understanding the virological and immunological determinants of enhanced viral replication and its pathological consequences.
Ait-Khaled M, McLaughlin JE, Johnson MA, Emery VC (1995) Distinct HIV-1 long terminal repeat quasispecies present in nervous tissues compared to that in lung, blood and lymphoid tissues of an AIDS patient., AIDS9(7)pp. 675-683
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.
Fryer JF, Griffiths PD, Fishman JA, Emery VC, Clark DA (2001) Quantitation of porcine cytomegalovirus in pig tissues by PCR., J Clin Microbiol39(3)pp. 1155-1156
A quantitative-competitive PCR for the quantification of porcine cytomegalovirus (PCMV) was developed. The virus was detected in a variety of pig organs (including potential xenotransplant donations), with viral loads ranging from <10 to 97 genome copies/microg of DNA. This assay will have significant utility for studying the activation and replication of PCMV and in swine models for allo- and xenotransplantation.
Deayton JR, Wilson P, Sabin CA, Davey CC, Johnson MA, Emery VC, Griffiths PD (2000) Changes in the natural history of cytomegalovirus retinitis following the introduction of highly active antiretroviral therapy., AIDS14(9)pp. 1163-1170
OBJECTIVE: To determine the effect of highly active antiretroviral therapy (HAART) on the natural history of cytomegalovirus (CMV) retinitis. DESIGN AND PARTICIPANTS: Retrospective analysis of 103 consecutive patients diagnosed with CMV retinitis between 1990 and 1998. SETTING: Specialist HIV medicine department of a London hospital. MAIN OUTCOME MEASURES: Incidence of CMV retinitis, time to death following diagnosis, episodes of progression, incidence of inflammatory complications. The date of first use of HAART was January 1995. Data were censored on 30 June 1998. RESULTS: The incidence of CMV retinitis has declined dramatically following the introduction of HAART. Survival following CMV retinitis increased from a median of 0.65 years prior to 1995 to a median of 1.07 years after this date (P = 0.004). In multivariate analyses HAART was independently associated with improved survival (P = 0.02) and the association with year of diagnosis was no longer significant, suggesting that this effect is predominantly due to HAART. None of the patients receiving HAART experienced progression after 6 months of treatment. Complications of retinitis such as retinal detachment, uveitis and optic atrophy occurred in 39% of patients. The rare inflammatory complications of vitritis and cystoid macular oedema occurred only in recipients of HAART. CONCLUSIONS: The introduction of HAART has had a major impact on the natural history of CMV retinitis with improved survival time and decreased risk of progression following diagnosis. However, immune reconstitution may be associated with inflammatory complications which can result in significant visual loss in the absence of active CMV disease.
Buyck HC, Prentice HG, Griffiths PD, Emery VC (2010) The risk of early and late CMV DNAemia associated with Campath use in stem cell transplant recipients., Bone Marrow Transplant45(7)pp. 1212-1219
The risks associated with in vivo and ex vivo use of Campath-1H and -1G in a cohort of 206 stem cell transplant recipients for human CMV (HCMV) DNAemia have been quantified. DNAemia showed a biphasic incidence pattern with an inflexion at day 60. The first phase had a linear risk rate for HCMV DNAemia of 0.3% per day, whereas the second phase had a substantially lower risk rate of 0.058% per day. In multivariable analyses, risk factors for early DNAemia were HCMV serostatus, radiotherapy-based conditioning and CD34 stem cell dose, with the use of in vivo Campath-1H having the most significant risk (hazards ratio=3.68; 95% CI=2.02-6.72; P<0.001). Ex vivo use of Campath was not associated with an increased risk for HCMV DNAemia. Patients receiving either in vivo Campath-1H or -1G experienced HCMV DNAemia earlier (27 and 33 days, respectively) compared with patients receiving no Campath (time to DNAemia, 51 days; P=0.0006). Multivariable analysis of risk factors for HCMV DNAemia occurring beyond 100 days after transplant were older age, acute GVHD>grade II and a lower CD34 stem cell dose, whereas Campath-1H use was not associated with late HCMV DNAemia.
Bavin PJ, Giles JA, Deery A, Crow J, Griffiths PD, Emery VC, Walker PG (1993) 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., Br J Cancer67(3)pp. 602-605
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.
Emery VC (1998) PCR in diagnostic virology: Methodological concepts, Herpes5(2)pp. 46-50
The polymerase chain reaction (PCR) is an in vitro, qualitative assay system that amplifies nucleic acid and can be used to detect herpesvirus infection. Its suitability as an effective diagnostic, prognostic and treatment-monitoring tool would be greatly improved, however, if it were quantitative. In classical cell cultures, end-point dilution techniques were used for quantification but these are time-consuming and expensive when applied to PCR. Internal standards that could mimic the target sequence amplification but be differentiated from target amplicons were therefore required. Initial studies used primers against cellular genes, but quantitative-competitive assays that use target sequence mimetics may be more appropriate. Such assays are based on the principle that the internal control sequences experience the same amplification process as the target sequences, thereby reflecting the starting ratio of target and control. This technique is now being used to assess herpesvirus infections in immunocompromised hosts. Where a small number of mutations are involved, PCR can also be applied to identify drug-resistant herpesvirus mutants. Rapid PCR can be achieved either using restriction endonuclease followed by acquisition/removal of the restriction enzyme site or point mutation assays. The first application of PCR in the diagnosis of herpesvirus infection was the amplification and subsequent qualitative detection of viral DNA in infected hosts. Now, technology to allow quantification of PCR is being developed. This review follows the refinement of PCR for use in herpesvirus research, and presents data from studies in which PCR has been used. A glossary of common terms used in PCR is given. It is likely that PCR will have wide applications in the diagnosis, prognosis and monitoring of patients with herpesvirus infection in future.
Caplin B, Sweny P, Burroughs A, Emery V, Griffiths P, Hodson EM, Jones CA, Webster AC, Strippoli GFM, Craig JC (2005) Antiviral treatment after solid organ transplantation  (multiple letters), Lancet366(9488)pp. 806-807
Herrero-Martínez E, Sabin CA, Lee CA, Jones IM, Pillay D, Emery VC (2004) The effect of highly active antiretroviral therapy for HIV on the anti-HCV specific humoral immune response., J Med Virol72(2)pp. 187-193
The effect of highly active antiretroviral therapy (HAART) on HCV replication is controversial, with some studies reporting no effect and others increases, reductions and even clearances of HCV RNA after treatment. In this study, the effect of HAART was investigated on the titre of anti-HCV specific antibodies and on the relationship between these antibodies and HCV RNA level in a cohort of 24 patients with inherited bleeding disorders. A significant inverse correlation between antibodies to both total HCV proteins and HCV RNA (R = -0.42, P = 0.05) and between antibodies to HCV envelope glycoproteins and HCV RNA (R = -0.54, P = 0.01) was observed pre-HAART. The relationship disappeared or was obscured after therapy (R = 0.24, P = 0.30 and R = 0.16, P = 0.50, respectively). Thus, we show that HAART affects the HCV specific humoral immune responses without affecting the HCV RNA level.
Grzywacz M, Deayton JR, Bowen EF, Wilson P, Emery VC, Johnson MA, Griffiths PD (1999) Response of asymptomatic cytomegalovirus viraemia to oral ganciclovir 3 g/day or 6 g/day in HIV-infected patients., J Med Virol59(3)pp. 323-328
Reactivation of cytomegalovirus (CMV) following immunosuppression may result in the development of CMV disease and is associated with an increased risk of death. CMV viraemia detected by the polymerase chain reaction (PCR) precedes CMV disease in HIV-infected patients and identifies individuals at high risk of disease. Pre-emptive ganciclovir (GCV) therapy in patients who have evidence of CMV viraemia is effective in preventing disease. An open study was conducted to assess the response of CMV viraemia to oral GCV at a dose of 3 or 6 g/day for 28 days. HIV RNA was measured to determine if CMV inhibition affected HIV viral load. Fourteen patients were studied, three of whom entered both phases of the study. None of the patients had evidence of CMV disease at the time of entry into the trial; two patients developed CMV retinitis after completion of the trial. Oral GCV at both 3 and 6 g/day caused a decrease in CMV viral load in individual patients. However, a rebound in CMV viral load occurred in patients receiving the 3-g/day dose. None of the patients receiving oral GCV 3 g/day became PCR negative after 21 days compared with six of eight patients receiving 6 g/day. Five of eight patients (63%) receiving GCV 6 g/day were concurrently taking protease inhibitors compared with two of nine (22%) receiving 3 g/day. Ten patients remained PCR negative throughout follow up. No change was found in HIV viral load during receipt of GCV at either dose. Thus, oral GCV is effective in reducing CMV viral load, but a dose of 3 g/day is insufficiently potent for pre-emptive therapy.
Buyck HC, Paston SJ, Lowdell MW, MacKinnon S, Emery VC (2004) Functional helper T cell response to CMV predicts probability of CMV vireamia following allogeneic stem cell transplantation., BLOOD104(11)pp. 611A-611A AMER SOC HEMATOLOGY
Emery VC (2012) Cytomegalovirus: recent progress in understanding pathogenesis and control., QJM105(5)pp. 401-405
Cytomegalovirus continues to be an important pathogen in a variety of patient groups especially the neonate and the transplant recipient, and has implicated in a range of pathologies including inflammatory disease and in contributing to early death in ageing populations. This review will focus on advances in understanding the virus-host interaction and options for the new therapeutic control measures.
Hassan-Walker AF, Okwuadi S, Lee L, Griffiths PD, Emery VC (2004) Sequence variability of the alpha-chemokine UL146 from clinical strains of human cytomegalovirus., J Med Virol74(4)pp. 573-579
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects a variety of cell types in vivo. A region (referred to as UL/b') present in the Toledo strain of HCMV and low passage clinical isolates contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL146, encodes an alpha-chemokine. PCR amplification and sequencing of this gene from serial samples obtained from transplant recipients and samples from infants with suspected congenital HCMV infection, revealed that UL146 is a hypervariable gene in vivo. However, genetic changes were highly conserved in individuals and in renal transplant recipients multiple genotypes of UL146 were present. The majority of strains characterized maintained the conserved ELRCXC motif present in the Toledo strain of HCMV. These results provide further evidence that AD169 does not represent the authentic virus in vivo and although Towne and Toledo are more representative, major genetic differences still exist. Mixed populations of HCMV strains occur in vivo so cloning of these strains is essential if an authentic genotype is to be defined.
Brennan DC, Aguado JM, Potena L, Jardine AG, Legendre C, Säemann MD, Mueller NJ, Merville P, Emery V, Nashan B (2012) Effect of maintenance immunosuppressive drugs on virus pathobiology: Evidence and potential mechanisms, Reviews in Medical Virology
Recent evidence suggesting a potential anti-CMV effect of mTORis is of great interest to the transplant community. However, the concept of an immunosuppressant with antiviral properties is not new, with many accounts of the antiviral properties of several agents over the years. Despite these reports, to date, there has been little effort to collate the evidence into a fuller picture. This manuscript was developed to gather the evidence of antiviral activity of the agents that comprise a typical immunosuppressive regimen against viruses that commonly reactivate following transplant (HHV1 and 2, VZV, EBV, CMV and HHV6, 7, and 8, HCV, HBV, BKV, HIV, HPV, and parvovirus). Appropriate immunosuppressive regimens posttransplant that avoid acute rejection while reducing risk of viral reactivation are also reviewed. The existing literature was disparate in nature, although indicating a possible stimulatory effect of tacrolimus on BKV, potentiation of viral reactivation by steroids, and a potential advantage of mammalian target of rapamycin (mTOR) inhibition in several viral infections, including BKV, HPV, and several herpesviruses. © 2012 John Wiley & Sons, Ltd.
Congenital cytomegalovirus (CMV) remains a leading cause of disability in children. Understanding the pathogenesis of infection from the mother via the placenta to the neonate is crucial if we are to produce new interventions and provide supportive mechanisms to improve the outcome of congenitally infected children. In recent years, some major goals have been achieved, including the diagnosis of primary maternal CMV infection in pregnant women by using the anti-CMV IgG avidity test and the diagnosis and prognosis of foetal CMV infection by using polymerase chain reaction real-time tests to detect and quantify the virus in amniotic fluid. This review summarises recent advances in our understanding and highlights where challenges remain, especially in vaccine development and anti-viral therapy of the pregnant woman and the neonate. Currently, no therapeutic options during pregnancy are available except those undergoing clinical trials, whereas valganciclovir treatment is recommended for congenitally infected neonates with moderately to severely symptomatic disease.
Slyker J, Richardson B, Chung M, Atkinson C, Ásbjörnsdóttir K, Lehman D, Boeckh M, Emery V, Kiarie J, Grace J (2016) Maternal highly active antiretroviral therapy reduces vertical CMV transmission but not maternal breast milk CMV levels,AIDS Research and Human Retroviruses
Mary Ann Liebert
Objective: To evaluate the impact of highly active antiretroviral therapy (HAART) on CMV transmission and breast milk level in the context of maternal HIV. Design: Specimens from a randomized trial conducted in Nairobi, Kenya between 2003?2005 were used to compare CMV transmission and breast milk levels between mother-infant pairs randomized to HAART versus short-course antenatal zidovudine plus single-dose nevirapine (ZDV/sdNVP) for prevention of mother-to-child HIV transmission (PMTCT). Methods: Fifty-one antiretroviral-naïve women d32 weeks gestation, and CD4 between 200?500 cells/mm3 were randomized at 34 weeks to begin either antenatal ZDV/sdNVP, or HAART through 6 months postpartum. Mean breast milk CMV levels and transmission were compared between arms. Results: Age, sociodemographics, CD4%, and HIV plasma RNA viral load were similar between arms at baseline. CMV viral loads were measured from 243 infant plasma and 185 breast milk specimens during the first year postpartum. The probability of infant CMV infection at 12 months was 19% lower in the HAART arm compared to ZDV/sdNVP (75% vs. 94%, p = .04). All women had CMV detected in breast milk, with 72%, 98%, and 97% testing positive during the first, second, and third weeks postpartum, respectively. There was a trend for early higher mean breast milk CMV level in the HAART arm at 1 week (p = .08), and there was significantly slower decline in breast milk CMV levels (area under the curve, p = .01). Conclusions: HAART started during the third trimester may decrease infant CMV infections, by mechanisms independent of breast milk CMV levels. Clinical trials registration: NCT00167674.
Background Cytomegalovirus (CMV) is the most prevalent congenital infection in developed countries. A significant number of infected infants develop long-term neurodevelopmental and hearing impairment irrespective of whether disease is detectable at birth. Studies of viral load and replication dynamics have informed the treatment of CMV in adult populations but no similar data exist in neonates. Objectives To study CMV virus kinetics in different body fluids of babies treated for congenital infection. Study design CMV virus load was sequentially analyzed in blood, urine and saliva in 17 babies treated for symptomatic congenital CMV infection. Results Virus was detectable in the urine and saliva of all babies at baseline but in only 15/17 in blood. At the end of 6 weeks of antiviral treatment CMV remained detectable in 9/14 blood samples, 9/12 urine samples and 4/7 salivary swabs. Median half-life (T1/2) of virus decline in blood was 2.4 days (IQR 1.9?3.3) and basic reproductive number (Ro) was 2.3. Although T1/2 values were similar in urine and saliva to those observed in blood, virus dynamics differed both during and after treatment. Conclusions T1/2 and Ro in blood in this group of neonates were similar to values derived from studies of immunocompromised adults. The persistent viremia observed in treated neonates cannot therefore be adequately explained by the virus dynamics early in treatment. The different dynamics exhibited in blood and urine suggests that studying changes in distinct body compartments may assist in further understanding long-term manifestations of disease.
Cytomegalovirus (CMV) remains a cause of disease in individuals with weakened immune systems such as patients undergoing organ transplantation. The mainstay of treatment for CMV is ganciclovir: a CMV-specific drug that looks like a building block of the viral DNA and which requires activation by a protein contained within CMV. Understanding how patients respond to treatment can lead to a better understanding of the basic biology of CMV in the human and also help to optimize treatment regimens. In this study we observed that following treatment of CMV disease in organ transplant patients, CMV viral loads decay according to four distinct patterns. We have developed a novel mathematical model that is able to describe each of these decay patterns. The model could potentially be used to assess how different patients respond to treatment in clinical settings and also to design better drug regimens as per patient. These results have striking implications in terms of how long patients should remain on treatment and provide new insight into CMV infection in the human host.
Turbé Valérian, Gray Eleanor R, Lawson Victoria E, Nastouli Eleni, Brookes Jennifer C, Weiss Robin A, Pillay Deenan, Emery Vincent, Verrips C. Theo, Yatsuda Hiromi, Athey Dale, McKendry Rachel A (2017) Towards an ultra-rapid
smartphone- connected test for
infectious diseases,Scientific Reports711971
The development is reported of an ultra-rapid, point-of-care diagnostic device which harnesses surface acoustic wave (SAW) biochips, to detect HIV in a finger prick of blood within 10 seconds (sample-in-result-out). The disposable quartz biochip, based on microelectronic components found in every consumer smartphone, is extremely fast because no complex labelling, amplification or wash steps are needed. A pocket-sized control box reads out the SAW signal and displays results electronically. High analytical sensitivity and specificity are found with model and real patient blood samples. The findings presented here open up the potential of consumer electronics to cut lengthy test waiting times, giving patients on the spot access to potentially life-saving treatment and supporting more timely public health interventions to prevent disease transmission.
This editorial contextualizes the results of Vincenti et al's (page 2945) cytomegalovirus DNA vaccine study in solid organ transplant patients, helping to explain why the vaccine did not reach its primary endpoints and what the data teach for future vaccine studies to control cytomegalovirus posttransplantation.