Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1.

Liver cytosol antibody type 1 (LC1) is regarded as a serologic marker of type 2 autoimmune hepatitis, in addition to liver kidney microsomal antibody type 1. Among 38 patients with type 2 autoimmune hepatitis, 23 were positive for LC1 antibodies. The antigen recognized by LC1 has been identified as a liver-specific 58-kd metabolic enzyme named formiminotransferase cyclodeaminase (FTCD). All 23 LC1-positive sera immunoprecipitated rat FTCD, and 22 gave an identity reaction with rat FTCD by immunodiffusion. No reaction was observed with sera from 10 patients with type 1 autoimmune hepatitis, 10 with primary biliary cirrhosis, 10 with chronic hepatitis C, and 10 healthy controls. By Western immunoblotting all 23 LC1-positive sera and all the controls tested negative, suggesting that all the antigenic epitopes were destroyed by denaturation. FTCD is a bifunctional protein composed of distinct globular FT and CD domains connected by a short linker. To identify epitopes that trigger the LC1 autoimmune response, we tested LC1 antibodies against FTCD constructs encoding the N-terminal FT domain (amino acids 1-339), or the C-terminal CD domain (amino acids 332-541). Of 20 sera positive against full-length FTCD, 8 (40%) recognized the FT domain and the CD domain, 7 (35%) recognized only the FT domain, and 5 (25%) did not recognize either construct. No sera reacted with only the CD domain. These data indicate that multiple regions of FTCD trigger the LC1 autoimmune response, and that LC1 reactivity is mainly directed to conformation-sensitive epitopes located in the FT region of FTCD.

[Amino acid sequence analysis of peptides that can react to the nuclear localization signals of human cytomegalovirus PPUL44].

OBJECTIVE: To find the peptides that have strong binding ability to the nuclear localization signals (NLS) of human Cytomegalovirus (HCMV) PPUL44 protein, and to analyze their amino acid sequences. METHODS: Peptide clones that have binding ability to the NLSs were selected from a random peptide display library by using synthesized HCMV PPUL44 NLSA and NLSB respectively. The DNA sequences of these clones were detected by using "ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit". The amino acid sequences of the clones were analyzed for their homology, and the homologous sequences were compared with known sequences in protein bank. RESULTS: The peptides that can bind to the HCMV PPUL44 NLSA(named as bNLSApep) have higher homologous amino acid sequence to importin alpha subunit, the bNLSApep48 sequence AVVTPVLTEILK is more similar to the sequence of the importin alpha subunit Arm 7 from amino acid 18 to 29, ANIFPVLTEILQ; The bNLSBpep 39 is similar to the sequence of all 8 Arm regions of importin alpha subunit from amino acid 10 to 21. CONCLUSION: It is possible that the HCMV PPUL44 NLSA is a specific recognition site by importin alpha subunit, it can bind to the Arm 7 region of importin alpha subunit; HCMV PPUL44 NLSB is a non-specific recognition site for importin alpha subunit. This study provided an experimental data for the proposal that the Arm repeat region of importin alpha subunit is the binding region of NLS.

Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer.

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1, 608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

BACKGROUND: Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. METHODS: The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. RESULTS: Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. CONCLUSIONS: AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

Expression and characterization of a novel structural protein of human cytomegalovirus, pUL25.

Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.

Procedure for the quantitation of Gadd45 expression levels in clonal hematopoietic progenitor cells by competitive polymerase chain reaction.

OBJECTIVE: The growth arrest and DNA damage-inducible (gadd) genes represent a family of stress-inducible genes that are coordinately regulated at transcriptional level. Gadd45, in particular, has been linked to a p53-dependent inducible network required for regulated transition from G1 to S phase of cell cycle following genotoxic insult and growth arrest treatments and has seemingly a pivotal role in DNA repair. DESIGN AND METHODS: Here we show that competitive polymerase chain reaction (PCR) is an adequate method to quantitate gadd45 expression levels in hematopoietic progenitor cell line 32D, whose constitutive gene expression is very low. RESULTS: The sensitivity and reproducibility of our strategy support its usefulness for clinical purposes, to assess the DNA repair capacity of highly purified early myeloid progenitors, whose failure may be responsible for either short-term chemotherapy side effects (bone marrow hypoplasia and peripheral blood cytopenia) or long-term consequences of antiblastic drugs (leukemia and myelodysplasia).

Development of a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot for detection of CMV-specific IgM.

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.

The major open reading frame of the beta2.7 transcript of human cytomegalovirus: in vitro expression of a protein posttranscriptionally regulated by the 5' region.

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.

Human cytomegalovirus product UL44 downregulates the transactivation of HIV-1 long terminal repeat.

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.

A novel Western blot test containing both viral and recombinant proteins for anticytomegalovirus immunoglobulin M detection.

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.

Human cytomegalovirus late-phase maturation is blocked by stably expressed UL32 antisense mRNA in astrocytoma cells.

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.

Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris.

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.

Stably expressed antisense RNA to cytomegalovirus UL83 inhibits viral replication.

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 infection: a complex diagnostic problem in which molecular biology has induced a rapid evolution.

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.

Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay.

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.

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.

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.

Construction of a polyepitope fusion antigen of human cytomegalovirus ppUL32 and detection of specific antibodies by ELISA.

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.

Construction of polyepitope fusion antigens of human cytomegalovirus ppUL32: reactivity with human antibodies.

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.

Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies.

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.

Lack of correlation between virus detection and serologic tests for diagnosis of active cytomegalovirus infection in patients with AIDS.

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.