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We report here the results of a study on the prenatal diagnosis of congenital cytomegalovirus (CMV) infection. The study was carried out by both PCR and virus isolation from amniotic fluid (AF) for 82 pregnant women at risk of transmitting CMV for the detection of (i) seroconversion to CMV immunoglobulin G (IgG) positivity during the first trimester of pregnancy, (ii) symptomatic CMV infection in the mother during the first trimester of pregnancy or intrauterine growth retardation detected by ultrasound or abnormal ultrasonographic findings suggestive of fetal infections, and (iii) seropositivity for CMV-specific IgM. For 50 women, fetal blood (FB) was also obtained and tests for antigenemia and PCR were performed. The results indicate that AF is better than FB for the prenatal diagnosis of CMV infection. PCR with AF has a sensitivity (SNS) of 100%, a specificity (SPE) of 83.3%, a positive predictive value (PPV) of 40%, and a negative predictive value (NPV) of 100%; rapid virus isolation with the same material has an SNS of 50%, an SPE of 100%, a PPV of 100%, and an NPV of 94.7%. Fewer than 10% of the women positive for IgM by enzyme immunoassay (EIA) had a congenitally infected fetus or newborn infant. When EIA IgM positivity was confirmed by Western blotting (WB) and the WB profile was considered, the percent transmission detected among women with an "at-risk" profile was higher than that observed among IgM-positive women and was the same as that among women who seroconverted during the first trimester of pregnancy (transmission rates of 29 and 25%, respectively).

We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a mu-specific conjugate. The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals. The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100%. We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history. Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests. Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring.

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.

beta2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5' sequence of the transcript, which harbors two short upstream ORFs.

Objective: Human cytomegalovirus (HCMV) is often isolated from HIV-1-infected patients and the two viruses can infect the same cell type giving rise to direct bidirectional interactions. Whereas the long terminal repeat (LTR) transactivation ability of HCMV immediate early gene (IE1/IE2) is well documented, no information is available on the possible role of other HCMV proteins. In this study, the activity of ppUL44, an early DNA-binding protein, on HIV LTR transactivation was investigated., Methods: HIV LTR transactivation by ppUL44 in presence or absence of HIV-1 Tat and HCMV IE1/IE2 was determined in J-Jhan and U973 cells through transient transfection experiments with a series of different expression vectors. Some experiments were also performed on U373-MG astrocytoma cells permanently transfected with UL44 or with another HCMV gene used as a control (UL55)., Results: The basal transactivation activity of the HIV LTR was not influenced by the presence of ppUL44. On the contrary, the transactivation observed in the presence of Tat, IE1/IE2 or both factors in synergy was strongly downregulated by ppUL44 in a dose-dependent manner. Deletion constructs of ppUL44 demonstrated that the region of the molecule responsible for the inhibition of the LTR is located within the last 114 amino acids at the carboxyl-terminal region., Conclusions: The results obtained indicate that within the last 114 amino acids of ppUL44 there is a domain that has a negative effect on the ability of HIV-1 LTR to be activated by both its autologous transactivator Tat and the heterologous transactivator HCMV IE1/IE2 functioning individually or synergistically.

The diagnostic problems linked to human cytomegalovirus (HCMV) in pregnancy are many and not all have been fully defined. In long-term seropositive women there is a tacit agreement that no laboratory testing for HCMV should be carried out. In seronegative women a test for HCMV-specific IgG should be performed at least twice during the first 4 months of pregnancy, and if the seronegativity persists, further follow-up might be stopped. On the other hand, if a seropositivity appears the diagnosis of a primary HCMV infection is established and prenatal diagnosis should be offered to the mother. Finally, in the case of a pregnant woman with unknown serological status, the diagnosis of HCMV infection is a complex problem and several different questions need to be addressed. In our opinion they should be screened with a reliable IgM test (confirmed by blot if necessary) followed, in the case of positivity, by an avidity assay. Pregnant women undergoing a primary HCMV infection should be encouraged to seek prenatal diagnosis to be performed by PCR and virus isolation from amniotic fluid at the 21st to 23rd week of gestation.

This work demonstrates that antibodies to the product of the recombinant pUS22 of human cytomegalovirus (HCMV) are present in human sera during natural infection. US22 gene product has been identified as a member of the US22 family which may be secreted from infected cells. It is an early protein of 593 amino acids, 76 Kd in molecular weight. US22 seems to be an antigen which stimulates a good IgG response. In fact specific IgGs were found in approximately 40% of the CMV positive sera irrespective of their anti-CMV IgG titer. Specific IgM antibodies to pUS22 were observed exclusively during primary infection and in the sera with a high anti-CMV IgM titer. pUS22 could be considered for inclusion in a cocktail of CMV recombinant proteins to determine seropositivity to CMV and also to diagnose an active CMV infection.

Human cytomegalovirus (HCMV) open reading frame UL32 codes for the basic phosphoprotein pp150 (ppUL32), an abundant constituent of the virion tegument. In order to study its potential role in the assembly and/or transport of progeny particles, astrocytoma cell lines (U373MG) were generated, stably expressing a 2.1 kb 5' fragment of UL32 in antisense orientation under the control of the HCMV major immediate early promoter. The steady-state level of the UL32 sense mRNA and pp150 synthesis were strongly reduced in infected antisense cell lines. Neither immediate early and early gene expression, nor viral DNA replication, was inhibited; the expression of the late gene product gB (gpUL55) was also reduced, but mainly at the level of translation. Control experiments indicated that this differential effect of UL32 antisense expression on the synthesis of viral products was specific. As a consequence of the inhibitory effect, virus yield was significantly reduced in antisense mRNA cell lines. Ultrastructural comparison of control and antisense cells revealed no difference in nucleocapsid forms in the nucleus. However, in the cytoplasm of antisense cells, DNA-containing C capsids and virions were absent and abnormal forms of non-infectious enveloped particles were observed. The data suggest the involvement of pp150 either in the transport of DNA-containing particles through the nuclear envelope or in the stabilization of capsids in the cytoplasm. Thus, UL32 antisense mRNA appears to interfere strongly with virus maturation during the late phase of the infectious cycle.

Chemokines play a major role in inflammatory responses and affect hematopoiesis both negatively and positively. We show that fresh isolates and laboratory strains (Towne and Ad-169) of human cytomegalovirus (HCMV) induce production of the CC chemokine RANTES in fibroblasts. Induction of extracellular RANTES production occurred as early as 8 h after infection, peaked around 24 h after infection, and was almost undetectable by 48 and 72 h. Upregulation occurred in the absence of viral DNA synthesis, suggesting that it was due to immediate-early-early HCMV gene expression. CMV infection stimulated RANTES transcription, since reverse transcription-PCR detected a sharp increase in RANTES RNA which persisted even when extracellular RANTES was no longer detected. Induction of RANTES in fibroblasts was not due to prior induction of tumor necrosis factor alpha or interleukin 1 beta. Down-regulation required an active viral genome. Decrease of RANTES in culture supernatants may be associated with the appearance of the HCMV CC chemokine receptor US28, since we show that this gene is transcribed as early as 8 h after infection. Modulation of CC chemokine production early during CMV infection might have a regulatory effect on viral replication, as well as affect immune surveillance.

The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalovirus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were tested against HCMV-positive PBLs from a patient with ongoing HCMV infection. Three clones were found to react specifically with the nuclei of these cells. These three recombinant Fabs were subsequently tested, individually and pooled together, against 60 PBL samples taken from immunosuppressed patients. The reactivity observed was comparable to that obtained with mouse monoclonal antibodies commercially available for this purpose. The three recombinant Fabs were shown to react specifically with the 65-kDa viral tegument phosphoprotein encoded by UL83 (pUL83), which is the most abundant viral antigen in HCMV-infected PBLs.

We tested 101 serum samples obtained from pregnant women for the presence of human cytomegalovirus (HCMV)-specific IgM with nine different commercially available kits and with two Western blotting (WB) tests previously developed in our laboratory. The conventional WB test contains viral structural proteins separated on a gel from purified HCMV particles and transferred to nitrocellulose. The new WB test contains viral structural proteins and three recombinant proteins which contain the significant immunogenic portions of pp150 (ppUL32), pp52 (ppUL44), and p130 (pUL57). The results obtained indicate that the new WB test combines high specificity (92.5%) with high sensitivity (95.0%), characteristics that, in combination, have not been obtained with any of the other tests.

Diagnosis of primary human cytomegalovirus (HCMV) infection is accomplished exclusively by serologic testing. Among the possible methods, the determination of immunoglobulin G (IgG) avidity is one of the least explored. In this work, we used a commercially available kit to test anti-HCMV IgG avidity in 336 serum samples from pregnant women and transplant recipients undergoing virologically proven HCMV primary or nonprimary infections and from latently infected blood donors. Our results demonstrate that the anti-HCMV IgG avidity test differentiates primary from nonprimary HCMV infections in both pregnant women and solid organ transplant recipients. In fact, 88.6% of primary infections and no secondary infections showed low-avidity IgG to HCMV. In particular, low IgG avidity is a marker of primary infection for 18 to 20 weeks after onset of symptoms in both immunocompromised and immunocompetent subjects.

Objective: The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat., Methods: HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors., Results: We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted., Conclusions: The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.

We devised a novel Western blot (WB) test for anti-human cytomegalovirus (HCMV) immunoglobulin M (IgM) detection which contains viral structural polypeptides, significant portions of recombinant p150 (ppUL32), and a significant portion of the most immunogenic nonstructural protein p52 (ppUL44). This new test was evaluated in latently infected blood donors, pregnant women, and transplant recipients with ongoing HCMV infection and shown to be more sensitive and specific than traditional WB and conventional enzyme immunoassay for the detection of HCMV-specific IgM.

We have previously shown that single or multiple epitopes of the major human cytomegalovirus (HCMV) antigens, produced as fusion proteins in prokaryotes can be valuable diagnostic material in the serology of HCMV infection. In this work we moved to a eukaryotic system, to produce one of the most immunogenic HCMV antigens, ppUL44 (also called pp52 due to its apparent molecular size on acrylamide gels), as a non-fusion protein, in an attempt to eliminate some non-specific reactivity of human sera with bacterial carrier proteins. We expressed the DNA encoding ppUL44 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. Good levels of intracellular, soluble pp52 were produced. We observed an indistinguishable pattern of the yeast pp52 from the viral native protein in immunoblotting and a good reactivity with human sera.

Astrocytoma cells were transfected with an expression vector for the structural phosphoprotein ppUL83 (pp65) of human cytomegalovirus (HCMV). Once made, pp65 showed the same characteristics as naturally produced protein and was stable for several days in astrocytoma cells, as it is during HCMV replication in fibroblasts. In permanently transfected cells, pp65 was homogeneously dispersed in the cell nucleus and showed a preferential methanol-stable association with condensed chromatin throughout cell division. Furthermore, pp65 could be detected bound to metaphase-arrested chromosomes in pp65-expressing astrocytoma cells, and in fibroblasts and astrocytoma cells during productive virus infection.

Western blotting (immunoblotting) with proteins separated from purified human cytomegalovirus (HCMV) particles (viral WB) has repeatedly been shown to be a reliable and sensitive method for detecting HCMV-specific immunoglobulin M (IgM). The aim of the present work was to determine whether IgM detected by viral WB correlates with virological diagnosis better than conventional enzyme immunoassay (conv-EIA). The presence of an active HCMV infection was documented on the basis of isolation of virus from urine and/or saliva and on the basis of antigenemia and/or PCR with polymorphonuclear leukocytes for immunocompetent and immunocompromised subjects, respectively. The agreement observed between IgM detected by viral WB and the results obtained by virological detection of HCMV was significantly higher (88.7%) than the agreement of IgM detected by conv-EIA and virological results (67.5%).

A quantitative assay is necessary to differentiate between low and high Cytomegalovirus load in the blood, a high load frequently being correlated with clinical disease. A new method for the quantitative determination of viral DNA (HCMV-hybrid-capture) was compared with antigenemia and PCR. Hybrid-capture proved to be a simple and rapid method for the quantitative determination of viral load in the blood. It is less sensitive than PCR and antigenemia, but seems to correlate with clinical symptoms as well as the antigenemia test and better than non quantitative PCR.

In this work we used PCR to amplify the DNA regions coding for two polypeptides from pUL57 of human cytomegalovirus (amino acids 540 to 601 and 1144 to 1233) and showed that both portions reacted very efficiently with immunoglobulin M in sera of acutely infected subjects. However, pUL57 is not an essential antigen for the replacement of or supplement to a cocktail of recombinant protein antigens containing portions of ppUL32, -44, -83, and -80a in immunoglobulin M serology.

The human cytomegalovirus (HCMV) open reading frame UL83 encodes a phosphoprotein of 64 to 68kDa (pp65) which is a major constituent of this virion and dense bodies. To determine the importance of the HCMV gene in the virus cycle, we studied HCMV replication in astrocytoma cells stably transfected with a retroviral vector carrying an antisense UL83 cDNA. Reverse transcription-PCR detected antisense RNA in the cytoplasm. The steady-state level of a 4-kb RNA containing coding sequences for pp65 was significantly reduced after infection of antisense cells. Concomitant with this, levels of expression of pp65 and pp71 (UL82) were severely reduced. Extracellular HCMV production was almost completely blocked, irrespective of the multiplicity of infection or the time after infection studied. The block occurred at an early phase, since immediate-early protein synthesis occurred normally, while several late proteins (e.g., pp150 [ppUL32] and assembly protein [UL80]) were absent or strongly inhibited. Normal replication of herpes simplex virus and of a pp65 deletion mutant of HCMV (RVAd65), lacking target sequences of antisense RNA, demonstrated the specificity of the block for wild-type HCMV in the antisense-stabilized cells and indicated that the block was not due to indirect interference with cellular genes. Our results appear to contradict those of Schmolke et al (S. Schmolke, H.F. Kern, P. Drescher, G. Jahn, and B. Plachter, J. Virol. 69:5959-5968, 1995), which show that UL83 is a nonessential gene for HCMV replication in vitro. This contradiction is discussed in light of the fact that the 4-kb mRNA, which codes for pp65 and was targeted in UL83-antisense cell lines, may be a bicistronic mRNA which also codes for pp71 (UL82). Thus, interference of expression from the genes encoding pp65 and pp71 by blocking of this putative bicistronic message leads to severe impairment of viral replication.

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another.

The value of IgM detection for the early diagnosis of an active cytomegalovirus (CMV) infection in renal transplant recipients was evaluated prospectively. Sequential serum samples obtained from 22 allograft recipients with active CMV infection were tested for the presence of CMV-specific immunoglobulin M antibodies (IgM) by an enzyme-linked immunosorbent assay (ELISA) and a microparticle enzyme immunoassay (MEIA) and were compared with the Western-immunoblotting technique (IB). The time course of CMV IgM antibody detection was evaluated in relation to the shell vial assay (SVA), CMV disease, and immunosuppressive regimen. By IB, IgM antibodies against the capsid protein ppUL80a and the basic matrix phosphoprotein ppUL32 were detected in all 22 recipients with active CMV infection. Using the MEIA and the ELISA, the presence of CMV IgM antibodies was detected in 17 (77%) and ten (46%) of these 22 recipients, respectively. The SVA was the earliest parameter for detection of primary CMV infection in seven of nine (78%) recipients, in contrast to two of 13 (15%) patients with recurrent CMV infection (P < .05). The detection of IgM antibodies by IB was the earliest parameter for detection of recurrent CMV infection in seven out of 13 (54%) recipients in contrast to one out of nine (11%) patients with primary CMV infection (P < .05). During a primary CMV infection, the development of an abundant IgM antibody response was associated with recovery from CMV disease and the end of the viremic phase.

Herpesviruses are highly disseminated in nature, and nearly 100 Herpesviruses have been at least partially characterized. Seven Herpesviruses have been isolated so far from humans and one has recently been detected by sequence analysis. Consequently, the diagnosis of human Herpesvirus infection is based on eight different viruses. The diagnosis of a Herpesvirus infection can be achieved through the direct demonstration of the presence of the virus or its components (mainly nucleic acids and antigens) in pathological materials or indirectly through serology. In recent years much progress has been made in both directions and thanks to the detailed study of the viral genomes and their antigenic gene products and by the determination of the immune reactivity against individual antigenic polypeptides in different clinical settings. Progress in the direct detection of the viruses in pathological materials mainly regards the production of specific probes and their use with or without DNA amplification for the detection of viral genomes. Serological advances are mainly linked to the production, characterization and use of specific viral antigens by DNA recombinant procedures and by the production and use of synthetic peptides acting as good antigenic epitopes.

Human cytomegalovirus (HCMV) is associated with several diseases in immunocompromised individuals. CMV infection can be diagnosed directly by demonstration of the virus or virus components in pathological materials or indirectly through serology. Molecular biology has allowed detailed studies of the viral genome and its antigenic gene products and has led to major advances in CMV diagnosis providing new tools for both the analysis of the CMV-specific immune response and the detection of virus-specific antigens or genetic material. In an attempt to provide a guide for the correlation between laboratory findings and clinical interpretation, we discuss in this work the clinical settings in which the presence of CMV needs to be diagnosed, and how a CMV diagnosis should be asked for or interpreted by the clinician in view of the variety of new or improved laboratory tests now available.

Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.

Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with an apparent molecular weight of 38 kD, ppUL99 (MW of 28 kD), the basic phosphoprotein ppUL32 (MW of 150 kD), the lower matrix protein ppUL83 (65 kD), and the DNA binding protein ppUL44 (MW of 52 kD). Some fragments overlapped and responses to these recombinant proteins were compared. Antibody responses appeared to be predominately directed at fusion proteins of ppUL32, the basic phosphoprotein and at ppUL44, the major DNA binding protein. Some differences were found in the responses to these partly overlapping fusion proteins. In addition, an IgM response mainly present in primary infections to fusion proteins XP1 and G2, again representing ppUL32 and ppUL44, was found.

Background: Unstable angina is most frequently caused by coronary thrombosis, with or without plaque fissure, but the mechanisms underlying these events are still speculative. Since cytomegalovirus (CMV) antigens and DNA encoding CMV major immediate-early (MIE) gene have been detected in atherosclerotic arterial walls, the active replication of CMV may be responsible for plaque instability. Therefore the expression of CMV MIE gene mRNA, an early marker of viral replication, was assessed in coronary atherectomy specimens from patients with stable or unstable angina., Methods and Results: Twenty patients with unstable angina (12 men and 8 women; mean age, 62 years; range, 44 to 89 years) and 20 patients with stable angina (16 men and 4 women; mean age, 62 years; range, 43 to 81 years) who underwent successful directional coronary atherectomy were enrolled in the study. The efficiency of mRNA extraction, transcription, and amplification from each coronary atherectomy specimen was assessed by performance of reverse transcription and thermal cycling amplification of a 548-bp human beta-actin cDNA segment. After Southern blotting and hybridization with a specific probe, all specimens but one showed a positive hybridization signal. The negative sample was excluded from the study. Reverse transcription and thermal cycling amplification of a 145-bp CMV cDNA segment of the MIE gene were then carried out. After Southern blotting and hybridization with a specific probe, none of the specimens showed a positive hybridization signal. Plasmid pACYC 184 containing the Xba I-inserted MIE gene cDNA was used as a positive control: as few as 10 molecules of the plasmid per reaction were detectable after amplification., Conclusions: Our results do not support the hypothesis that, in patients with unstable angina, replication of CMV in coronary atherosclerotic plaques is a major cause of plaque instability. These findings suggest that the research for the causes of unstable angina should be directed toward processes other than CMV replication.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.

Diagnosis of human cytomegalovirus (CMV) infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology. Serological diagnosis gives only indirect evidence of the presence of the virus, and is problematic because of the immunological disorders occurring in most patients at risk of developing a CMV infection. Furthermore, antigenic reagents used in commercially available kits are not standardized and discordant results are often obtained. However, serology is cheaper than the other diagnostic tests, requires a short execution time, is safe and can be completely automated. Therefore, it is worthwhile exploring the possible application fields in which the use of serology is justified nowadays. This is what this review will attempt to do.

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides can be valuable diagnostic material in the serology of HCMV infection (5, 6, 13). In this work we fused sequences expressing two different epitopes (aa 1005-1048 and aa 595-614) contained in the basic phosphoprotein of 150 KD coded by UL32 (1, 2), (ppUL32), which has repeatedly been shown to be the strongest immunogen present in the viral particle. The fusion protein was tested by ELISA with HCMV-positive human sera in comparison with other fusion proteins of ppUL32. We found that the double epitope fusion protein was recognised by IgM present in a larger number of sera and with more intense reactions than all the other ppUL32 fusion proteins. The double epitope reacted positively with 81.3% and, when denatured, with 94.7% of IgM-positive sera respectively. IgG reactivity was also high, reaching a percentage of 90.7.

Human cytomegalovirus (CMV) is a major cause of severe disease in HIV-infected persons and some findings suggest that it may accelerate HIV disease. In this study, a total of 621 blood samples from patients with LAS-ARC and AIDS were analysed in parallel for CMV and HIV-I antigenaemias. Results indicate that the presence of CMV antigenaemia and the presence of HIV-I p24 in the blood are highly correlated statistically and encourage other studies on the role of CMV in the evolution of AIDS. In a smaller group of cases, CMV was also isolated from saliva and/or urine. The correlation with HIV replication was positive (although much lower) with CMV detected in saliva and completely negative with CMV isolated from urine.

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides, can be valuable diagnostic material in the serology of HCMV infection (M. P. Landini, M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti, A. Necker, T. Lazzarotto, and B. Plachter, J. Clin. Microbiol. 28:1375-1379, 1990; M. P. Landini, T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa, J. Clin. Microbiol. 27:2324-2327, 1989; A. Ripalti, M. P. Landini, E. S. Mocarski, and M. La Placa, J. Gen. Virol. 70:1247-1251, 1989). In this work we addressed the question of whether the expression of more than one linear epitope on a single fusion protein could increase the reactivity of genetically engineered antigenic material with human antibody. To answer this question we fused sequences expressing two different epitopes contained in the basic phosphoprotein of 150 kDa encoded by UL32 (M. S. Chee, A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, T. Cerny, T. Hornsel, C. A. Hutchinson, T. Kouzarides, J. A. Martignetti, and B. G. Barrell, Curr. Top. Microbiol. Immunol. 154:125-169, 1990; G. Jahn, T. Kouzarides, M. Mach, B.-C. Scholl, B. Plachter, B. Traupe, E. Preddie, S. C. Satchwell, B. Fleckenstein, and B. G. Barrell, J. Virol. 61:1358-1367, 1987), ppUL32, which was repeatedly shown to be the strongest immunogen present in the viral particle. We also made fusions with sequences expressing a single epitope repeated once, twice, or three times. The different fusion proteins were tested with HCMV-positive human sera. We found that fusion proteins expressing different epitopes together were recognized by a larger number of serum specimens and with more intense reactions in Western blot (immunoblot) experiments. We also found evidence that expression on the same polypeptide of the two distinct epitopes produced a stronger antigen than the mere addition of two fusion proteins which each carried one copy of one of these epitopes. Furthermore, we found that while the same epitope expressed two or three times on the same fusion protein was not better recognized by immunoglobulin G than the single epitope, immunoglobulin M reactivities to the double and triple epitopes were enhanced.

We isolated and characterized from a lambda gt11 expression library clones expressing portions of human cytomegalovirus (HCMV)-p52. This nonstructural viral protein is encoded by UL44 and is known to be one of the best IgM reactive antigens. The reactivity of these clones was studied with human antibody and the gene fragment coding for the most immune-reactive portion of p52 (aa 202-434) was cloned in a prokaryotic expression vector, pROS, which overexpresses the antigen as a fusion protein to a truncated molecule of beta-galactosidase.

Human cytomegalovirus (HCMV) infection of a CD34+ hematopoietic progenitor cell line (TF1) was studied before and after TPA differentiation. TF1 cells were found to be infected but the virus does not replicate, while differentiated TF1 cells can be infected and allow HCMV complete replication. In the same system we studied the interaction between HCMV and HIV and found that while contact between HIV gp 120 and the HCMV-infected cell has an inhibitory effect, exogenous Tat protein stimulates HCMV replication. The interaction between HCMV and HIV in hematopoietic progenitor cells is complex and depends on several factors that can have opposite effects.

The IgG subclass pattern against linear antibody binding sites on glycoproteins of human cytomegalovirus (HCMV) was investigated in HCMV-positive healthy blood donors, human immunodeficiency virus-infected persons, sera from mothers with primary HCMV infection during pregnancy and their children, and sequential sera from transplant recipients. As antigens, three immunodominant domains capable of inducing neutralizing antibodies during natural infection were selected on glycoproteins gp58/116 (gB) and gp86 (gH). Bacterial fusion proteins representing these regions were used as antigens in a subclass-specific ELISA. Reactivity against the antibody binding site on gp86 was detected in both the IgG1 and IgG3 subclasses. In contrast, exclusively IgG1 antibodies were found against both linear domains on glycoprotein complex gp58/116 and also against full-length gp58/116 expressed in insect cells. The data demonstrate a differential regulation of the antibody response to envelope components of HCMV.

Human Cytomegalovirus (CMV) infection can result in a dramatic disease in immunosuppressed patients. For effective treatment sensitive and specific procedures are required for prompt and early diagnosis of active CMV infection. In the present study five different serological assays were used for detection of CMV-specific immunoglobulin M antibodies (IgM) in 78 sera, obtained from patients with a clinical suspicion of CMV infection. In addition, sequential sera, obtained from renal transplant recipients who experienced a primary CMV infection, were analysed by the five IgM detection assays. The assays used were two enzyme-linked immunosorbent assays (ELISA): the Vironostika CMV ELISA from Organon Teknika (ELISA-1) and the Monosan CMV ELISA from Sanbio (ELISA-2), the CMV microparticle enzyme immunoassay (MEIA) from Abbott and two Western-(immuno)blotting techniques using purified CMV structural viral proteins of the AD169 strain (IB-AD169) and the major non structural DNA binding protein of 52 kiloDalton (IB-Rp52) as antigenic material. The results of the assays were compared to each other with respect to sensitivity, specificity and overall agreement, as well as incidence of false positive and false negative results. Although the MEIA gave the highest sensitivity, the ELISA-1 combined a high sensitivity with a high specificity and therefore appears the most suitable method for determination of active CMV infection.

In this work, we show that antibodies to baculovirus recombinant DNA polymerase of human cytomegalovirus are present in human sera during natural infection. Specific antibody was found to be of the immunoglobulin G class, produced at low titers only for a short period of time at the beginning of acute infection and not detectable in sera from patients with a convalescent or a latent phase of infection. These results were compared with those obtained with procaryotically expressed p52, the polymerase accessory protein.

Two prokaryotically expressed fusion proteins encompassing amino acids 484-650 (AD-1) and 27-100 (AD-2) of glycoprotein gp58/116 of human cytomegalovirus (HCMV) were purified from E. coli lysates and used in ELISA to determine antibody levels in human sera. The specificity of the test was established by comparison of 116 randomly selected sera with commercially available HCMV-ELISA tests. The recombinant polypeptides were then used for the analysis of antibody titers in 112 human sera and were compared to the capacity to neutralize HCMV. A strong correlation between the neutralization titer and antibody levels against AD-1 and a weaker correlation for AD-2 was observed. Of 29 sera with a high neutralization titer (> 1:128), 96% and 62% were positive for AD-1 and AD-2, respectively, while 44% and 19% were positive in sera with low neutralization titer (< 1:8). Serum pools prepared from human sera selected on the basis of recognition of the recombinant antigens had a 10-fold higher neutralization capacity than pools prepared from sera with a high titer in commercially available HCMV tests. A synchronous increase in neutralization capacity and titer against recombinant antigens was observed in transplant patients.

The IgG and IgM immunoblotting patterns for the major HCMV antigens p65 and p55/52 were studied in 5 renal transplant recipients during primary HCMV infections. Sequential sera from the 5 patients were tested in parallel with standardized antigens derived from the reference strain AD169 and from the patient'own isolates. The immunoblotting patterns differed from patient to patient as well as between strain AD169 and the patient's homologous isolates. The differences consisted in a better response to the antigens derived from the reference strain AD169, suggesting a delay of the IgM/IgG switch and of the affinity maturation of IgG antibodies to the homologous p65 and p55/52 antigens. The results suggest caution in the interpretation of immunoblotting data derived from laboratory strains such as AD169.

Complement (C')-dependent neutralising cytomegalovirus (CMV) antibody titres were determined in 4150 serum samples obtained from infants and children, as well as women of childbearing age. In a smaller group of samples, C'-independent neutralising antibody titres were also measured together with their reactivity to each individual CMV structural polypeptide in addition to total CMV antibody concentration. Results showed that primary CMV infection takes place mainly in early infancy and that 30-40% women of childbearing age do not have any or have very low titres of CMV neutralising antibody. Since poor correlation was found between the results of neutralisation tests and those of enzyme immunoassay, routine testing of neutralising antibody is warranted in those subjects, such as pregnant women, at risk of CMV infection. Results of immunoblotting suggest a correlation between high titres of neutralising antibody and antibody reactivity with three proteins of MW 86, 65, 60 kDa.

The aim of this study was to evaluate the usefulness of serologic analysis for the diagnosis of active cytomegalovirus infection in patients with AIDS. Active cytomegalovirus infection was diagnosed by virus isolation from urine and saliva and detection of antigenemia. Serologic analysis was done by several conventional and innovative procedures. The results indicate no correlation between any of the most popular serologic procedures and virus detection by culture in urine or saliva or antigenemia.

Four serological methods were compared and evaluated for use in detecting cytomegalovirus antibody in blood and organ donors. Western blotting (immunoblotting), latex agglutination, enzyme-linked immunosorbent assay, and a recent available microparticle enzyme immunosorbent assay were used. The microparticle enzyme immunoassay appears to compare favorably with each of the other three assays tested for screening blood and organ donors for a previous cytomegalovirus infection.

Eight commercially available enzyme-linked immunoadsorbent assays (ELISA) for the detection of cytomegalovirus (CMV)-specific IgM were used in parallel to determine the presence of CMV-IgM in 123 serum samples from pregnant women. The results obtained with the eight kits were compared. Based on concordance of six or more of the eight kits, we assessed sensitivity, specificity, and overall agreement, as well as incidence of false-positive and -negative results for each kit. The results obtained by ELISA were then compared with those obtained by immunofluorescence (IF) and immunoblotting (IB). Our study did not single out one outstanding ELISA kit among the eight evaluated, nor did it suggest that IF or IB are better than ELISA. Furthermore our results indicate that IB might be useful in several cases as, beside its good sensitivity, most IB-false-positive sera are easily recognized as reacting exclusively with pp150, the unique reactivity to pp150 not being among the IB profiles of IB-true-positive sera. Nevertheless 14.6% of sera remained CMV-IgM-indeterminate.

One of the characteristic features of cytomegalovirus (CMV) replication is the formation of cytoplasmic dense bodies. Recent findings revealed similar structures also in the nuclei of CMV-infected cells. By transmission electron microscopy, immuno electronmicroscopy, and cytochemistry, we have studied the morphogenetic steps and macromolecular composition of both structures. Our results show that both structures contain DNA, RNA and viral antigenic proteins. Nuclear dense bodies are probably an expression of a stimulated cellular metabolism, while cytoplasmic dense bodies may represent the site where surplus cellular and viral molecules are stored before being eliminated.

Immunoblotting (IB) was used to detect the presence of serum antibody against each individual CMV structural polypeptide and the major nonstructural antigen during CMV hepatitis in 12 orthotopic liver transplant recipients to evaluate the diagnostic utility of this technique. The basic phosphoprotein p150 primarily, and to a much lesser extent p65 (lower matrix protein) and p52 (nonstructural DNA-binding protein) were the most recognized antigens by both IgG and IgM class antibodies. Collectively, IgM antibodies were detected to p150 in 9 of 12 (75%) patients and were detected before (2 cases), at the same time (1 case), or after (6 cases) detection of virus in cell cultures. IB was equal to indirect immunofluorescence (IIF) and enzyme immune assay (EIA) for the detection of IgG and superior to EIA for IgM class antibodies to CMV in OLT patients. Our results indicate that the p150 phosphoroprotein is an essential component of serologic assays, especially for the detection of IgM antibodies to CMV.

Immunological abnormalities of an autoimmune nature often develop during acute primary human cytomegalovirus (HCMV) infection. IgM antibodies reacting with the membrane of uninfected human embryonic fibroblasts can be detected in most patients undergoing a primary HCMV infection. In this work, we have found that there is a common antigenic epitope shared by a cell membrane component of Mr 60K (mp60), which is recognized by IgM in sera from patients with primary HCMV infection, and a linear determinant in the C-terminal half of the HCMV assembly protein of Mr 38K (vp38), which is known to be one of the most IgM-reactive antigens of HCMV. While vp38 seems to contain other specific IgM-reactive regions, IgM reactivity to mp60 is due exclusively to this shared epitope. Furthermore, mp60 is found abundantly on the surface of human red blood cells, a possible explanation for the pathogenesis of the haemolytic anaemia that may appear during primary HCMV infection.

In the present study, several peptides of the major structural antigen (pp150) of human cytomegalovirus (CMV) have been chemically synthesized and tested by a modified slot blotting procedure for their ability to bind CMV-specific immunoglobulin G (IgG) and IgM present in human sera. The sequences of the peptides were deduced on the basis of either (i) their presence in a fusion protein already known to be frequently recognized by human antibody or (ii) their high content of hydrophilic amino acids as deduced from the published nucleotide sequence. An important IgM-binding epitope was found to be located in the last 38 amino acids at the carboxy terminus of the molecule. This region reacts with anti-CMV IgM present in the great majority (83.3%) of IgM-positive human sera, and adsorption experiments have shown that IgM titers to the entire pp150 decrease 25 to 50% in most sera previously absorbed with this region. The overall results obtained endorse the continued synthesis of other sequences in order to define a group of peptides sensitive and specific enough to replace the virus and infected cells as an antigenic substrate in the serological evaluation of anti-CMV antibody.