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The present work investigated antigenic variations of the most immunogenic polypeptides of Cytomegalovirus (CMV), namely two nonstructural polypeptides of MW 72 Kd and 52 Kd, and 5 structural proteins of MW 150, 65, 58, 38 and 28 Kd, in several low-passage CMV isolates from urine of infected children. The polypeptides showing the greatest variations were the non structural p72 and p52, and the structural proteins p65 and p38. Furthermore the reactivity of the children's sera was studied with regard to the antigenic proteins of the homologous virus strain. We failed to recognize any reactivity pattern typical of an acute CMV infection and the results obtained indicate that CMV-infected children may have a very low and variable IgG and IgM response, p150 and p65 being the two most often recognized antigens.

Rabbit antisera raised against the product of ORF UL 80 of human cytomegalovirus (CMV) genome (HindIII L fragment of AD169 strain) as well as IgM from acutely infected patients recognize an antigen of Mr 38 kDa. In the viral particle this antigen is bound via S-S bridge to a lower Mr compound to form a final complex of 62 kDa that is also recognized by rabbit antisera as well as patients' IgM. P38 is present both in the nucleus and in the cytoplasm of CMV-infected cells starting from 24 h p.i. and increasingly thereafter. Immunoelectron microscopy revealed that this antigen is mainly associated with the internal portion of viral capsids both in the nucleus and in the cytoplasm.

Although human cytomegalovirus (HCMV) has a genome of 150 x 10(6) Da, and a protein-coding content of over 200 open reading frames, few viral proteins seem able to elicit a strong antibody response in the natural host during viral infection. The immunodominant polypeptides include a component of 72 kDa among immediate early proteins, a polypeptide of 52 kDa among delayed early proteins and a glycoprotein complex of 58 and 93-130 kDa and two phosphoproteins of mol. wt 150 and 65 kDa among the structural proteins. Following a general overview of the humoral immune response, this brief survey mainly deals with the antibody response to these proteins. As significant epitopes of the major HCMV immunogenic polypeptides have been expressed in procaryotic cells over the last few years, an overview of the state of the art in this particular field will also be given.

One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulins G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.

The tegument of human cytomegalovirus (HCMV) contains a phosphorylated protein of 65 kilodaltons, termed pp65, which was reported to carry significant epitopes for the stimulation of the humoral immune response during natural infection. A monoclonal antibody directed against this protein was used to screen a lambda gt11 cDNA library for recombinant polypeptides. Two DNA fragments from purified lambda clones and one fragment from genomic DNA were used for cloning in a bacterial high-level expression vector. The resulting fusion proteins were tested for their reactivity with a panel of monoclonal antibodies directed against pp65 and with polyspecific anti-HCMV rabbit antisera. The binding site for all the monoclonal antibodies tested was found to be contained in one of the recombinant proteins with a viral portion of 26 amino acids. Immunoblot analyses with HCMV-positive human sera revealed that pp65 alone is not a reliable antigen for serodiagnosis but may be very useful in combination with other HCMV proteins.

We compared cytomegalovirus (CMV) isolation in tissue culture and viral DNA detection by DNA:DNA hybridization in 60 clinical urine samples concentrated by different procedures. Our results showed that urine concentration by filtration is the easiest and quickest procedure allowing further detection of CMV. With optimized working conditions, the CMV detection in urine can be done in 5 hr without requiring cell cultures or costly instruments.

A series of indole-3-carboxaldehyde thiosemicarbazones with substituent variations at the 1 and 2 positions were prepared and evaluated for in vitro antiviral activity against vaccinia virus strain IHD and parainfluenza virus type 3, HA-I/CR-8 strain. The inhibitory activity of most compounds was restricted to the vaccinia virus, only compounds (III e) and (III f) being effective against both viruses. Structure-activity relationship are discussed.

A monoclonal antibody (MAb), N2, neutralized human cytomegalovirus (HCMV) infectivity in the absence of complement and recognized a normal cell protein. By immunofluorescence, MAb N2 detected antigens in uninfected human cells, but not in cells of non-human origin. Antigens were present at the membrane and were dispersed diffusely within the cytoplasm. MAb N2 immunoprecipitated a glycoprotein with an Mr of 94K from uninfected and infected cells. In infected cells only, it also recognized a protein of Mr 34K which was not linked to the 94K Mr glycoprotein by disulphide bonds. N2 neutralized both laboratory and field isolates of HCMV. A study of the distribution of the N2 neutralization epitope recognized among fresh isolates of HCMV showed that only 67% of the isolates could be neutralized by this antibody. There was no correlation between the number of in vitro passages and the level of neutralization. The 34K Mr polypeptide was present in cells infected by all isolates. It thus appears that, during virus assembly, HCMV acquires a normal cell protein that bears a neutralization epitope.

Before replication of the human cytomegalovirus (HCMV) genome takes place, two major antigenic complexes are induced in the infected cell by an early expression of parental viral genome: one (Early antigen or EA) is localised in the nucleus, the other (Early membrane antigen or EMA) on the plasma-membrane. Sequential samples of serum from renal transplant recipients, pregnant women, blood donors and patients with known HCMV disease were examined by means of indirect immunofluorescence tests for the presence of IgG, IgM and IgA antibodies against EA and EMA. Serum antibody reacting with EMA belongs almost exclusively to the IgM class while that reacting with EA may be of the IgG, IgM or IgA class. IgM antibody to EMA seems to be preferentially associated with primary active HCMV infection.

ADP ribosylation has been studied during human Cytomegalovirus (HCMV) replication in human embryo fibroblasts. The intranuclear level of this enzyme is higher during the whole viral replication cycle with respect to mock infected cells. The increase begins during the first hour ater the addition of the viral stock to the cells. Results are discussed in view of the possibility that this enhanced activity might be correlated with HCMV-induced alteration of the cell chromatin leading to an increase in host cell macromolecular synthesis.

The distribution of a lipophilic cation, tetraphenylphosphonium (TPP+), has been used to monitor changes in mitochondrial and plasma membrane potentials within human embryo fibroblasts infected with human cytomegalovirus. An increase in TPP+ accumulation was observed throughout the whole viral replication cycle, beginning 6 h after infection. This effect requires an active viral DNA and seems to be dependent on the early transcription of the viral genome. In the early phase of viral replication, the increase in TPP+ accumulation is insensitive to mitochondrial inhibitors and sensitive to ouabain, and could be due to a hyperpolarization of the plasma membrane. Later during the infectious cycle, enhanced accumulation of the lipophilic cation is sensitive to mitochondrial inhibitors.

The Authors report the results of an ultrastructural study of the developmental cycle of Chlamydia trachomatis in cell cultures in vitro in the presence of inhibitors of te synthesis of DNA (mitomycin-C), RNA (actinomycin-D and rifampin) and proteins (oxytetracycline and chloramphenicol). The chlamidiae were grown to IUDR-pre-treated McCoy cell cultures and the drugs were added to the medium either immediately after the infection (in the presence of "elementary bodies" only) or after the appearance of the first "initial bodies" (14 hours after infection). The appearance of "initial bodies" or their multiplication was completely prevented in the infected cell cultures in the presence of mitomycin-C, actinomycin-D, rifampin and oxytetracycline added immediately after the infection or 14 hours after the infection, respectively. In the presence of chloramphenicol the appearance of "initial bodies" or their multiplication was possible, but their differentiation into "elementary bodies" was completely inhibited.

Indirect immunofluorescence and enzyme-linked immunosorbent assay to detect serum antibodies against Legionella pneumophila serotype 1 were compared. A very good agreement was found with the great majority of serum samples (85.07%). For detecting high antibody levels and rise in antibody titres, both methods seem to be suitable, however for the detection of relatively low antibody titres, very important in epidemiological studies, enzyme immunoassay could be more accurate.

The uptake rate of some sugars was demonstrated in human embryo fibroblasts following infection by human cytomegalovirus (HCMV) and a significant increase in glucose uptake was demonstrated. Quantitative and sequential analysis of glucose uptake during the HCMV replication cycle showed that the enhanced uptake began during the first 20 h after infection and occurred even when a high glucose level was constantly present in the medium. The increase in sugar uptake requires an active viral genome, de novo protein synthesis and seems to be dependent only on the early transcription of the viral genome, as it did occur when replication of the viral genome was limited (non-permissive cells or cells infected in the presence of DNA synthesis inhibitors).

Using specific monoclonal antibodies, we have localized two structural proteins (p65-69 and p28) of human cytomegalovirus in infected cells and in virions and/or virus-related particles by immunoelectron microscopy using protein A-gold. Protein p65-69 is present in some roundish structures in the nuclei, often in contact with the viroplasm, and in the cytoplasm, exclusively within the dense body matrix. Protein p28 is present only in the outline of cytoplasmic capsids, and reaches the highest density in the large aggregates of virions and dense bodies which are particularly numerous during the late phases of the viral replication cycle.

180 serum specimens from pregnant women and renal transplant recipients, were studied for the presence of antibodies to human cytomegalovirus (HCMV) by complement fixation (CF), enzyme-linked-immunosorbent assay (EIA) for IgG and IgM, and indirect immunofluorescence (IIF). IIF was carried out separately against immediate-early, early and late HCMV-induced antigens. A positive CF test was always associated with the presence of antibodies against late virus antigens. EIA procedures were able to detect HCMV antibodies also when CF was negative and when IIF was positive only against immediate-early and/or early HCM-induced antigens.

The late cytoplasmic phases of human Cytomegalovirus (HCMV) morphogenesis in cultured fibroblasts have been studied by transmission electron microscopy focusing attention on the relationship between the viral particles and host cell organelles. The results obtained largely reflect changes in cells subjected to sublethal injurious stimuli induced by many viruses as well as different noxious agents. A great increase in the number of Golgi apparatuses and lysosomes was observed, both of them interacting with the viral progeny. HCMV seems to acquire its final envelope from Golgi-derived structures and, less frequently, from the plasma membrane.

An ultrastructural comparative analysis is reported on the replication cycle of six strains of Chlamydia trachomatis, recently isolated, and Chlamydia psittaci strain 6BC, grown in cell cultures. Important morphological distinctions of the bacterial walls are documented. In Chlamydia trachomatis the cell wall seems more rigid. This causes, during the morphogenesis of the elementary body, its separation from the cell membrane with formation of an electron transparent space, which is not demonstrable in Chlamydia psittaci. As a result of this process, the appearance of "ghosts" and "mini reticular bodies" is documented. Budding and multiple fission are suggested as possible mechanisms of replication of Chlamydia trachomatis, in coexistence or/as alternative to binary fission.

Purified human cytomegalovirus nucleocapsids are able to transform supercoiled plasmid DNA into nicked circular DNA. The activity exhibits a ionic strength optimum of 0.15 M; has a broad pH and temperature dependence and is enhanced after pre-incubation of the nucleocapsids with NP40.

Several sera with different IgG and IgM titres measured by enzyme immunoassay were studied in serial dilutions for their reactivity with human Cytomegalovirus structural polypeptides separated in SDS-PAGE and electrotransferred to nitrocellulose. The different immunogenicity of the viral proteins is documented and a hypothetical evolution of the humoral immune response to individual polypeptides is presented.

This study aimed at broadening the understanding of the immunogenic potential of cytomegalovirus (CMV) structural polypeptides during natural infection and to ascertain their possible use in serological diagnosis. Immunoblotting was used to analyse the appearance and development of serum IgG and IgM against human CMV structural polypeptides in sequential sera from renal transplant recipients during the first 1-3 months of primary CMV infection. The results showed that the first IgG to appear is specific for a polypeptide of 66 kDa and these antibodies appear either alone or together with others against another polypeptide of 82 kDa. IgG to the 150 kDa protein appears at least one week later. The first IgM to appear reacts preferentially with a 38 and 66 kDa polypeptide. The early detection of antibody against the major viral antigenic proteins in the diagnosis of CMV primary infection is discussed.

For the serological diagnosis of Herpesvirus infection, increasing use is made of the determination of the antibodies against virus-specific early antigens. The presence of serum antibody to early antigens is a widely accepted marker of acute Epstein-Barr virus infection or Varicella-Zoster virus infection. Controversial data are present in the literature about the significance of Cytomegalovirus early antigens antibody response. This is discussed in view of the possibility that controversial results could be due to different methods used to produce early antigens by different authors.

To define better the human immune response to individual structural proteins of human cytomegalovirus (HCMV), 55 human sera with different IgG and IgM titres were studied for their reactivity with HCMV structural polypeptides separated by SDS-PAGE and electrotransferred to nitrocellulose paper. The results obtained showed that antibody titres detected by immunoassay correlate with the intensity and the number of polypeptides reacting by immunoblotting (IB). The IB profiles of HCMV polypeptides reacting with different sera having the same antibody titres show considerable variation. Sera with high levels of IgG antibody and that are IgM-positive frequently react with 155, 149, 82.5, 74.5, 67, 57, 55, 38.5, and 28 kD polypeptides; all these sera react with 155, 67, 57, 55, 38.5 kD polypeptides. Sera with high levels of IgG antibody but that are IgM negative frequently react with all these polypeptides, with the exception of 149 and 74.5. Only 155 and 28 kD polypeptides were recognized by all sera of this group. The sera with moderate levels of IgG antibody preferentially recognize 155, 110, 82.5, 62, 55, 38.5 and 28 kD polypeptides. The sera with low levels of antibody reacted especially with 155 and 62 kD polypeptides. IgM antibody seems to recognize preferentially 155, 110, 67, 57, 55, 38.5 kD polypeptides.

A series of 1-benzoyl- and 1-benzyl-2-chloro-3-formylindole thiosemicarbazones was synthesized and investigated for antiviral activity against vaccinia virus IHD strain and parainfluenza virus type 3, HA-I/CR-8 strain. The results confirmed the previously observed high activity against vaccinia virus of m-substituted benzoyl derivatives. On the contrary, the analogous benzyl derivatives showed marginal activity; the compound (V c), however, was the most active against the parainfluenza virus.

DNase activity can be detected in lysates of extensively purified human cytomegalovirus (HCMV) particles. The presence of activity only in purified virions and not in dense bodies and its neutralization by HCMV-positive human sera strongly suggest that the enzyme is virus-specific. In situ analysis of the enzymatic activity of individual HCMV polypeptides, separated in a DNA-containing polyacrylamide gel, has shown that the DNase activity present in purified virions is mainly associated with a polypeptide of 46,000 dalton MW (p46). A monospecific anti-p46 serum, raised in mice, neutralizes about 80% of the DNase activity present in extracts of purified virions and recognizes an enzymatically inactive, 76,000 dalton MW polypeptide both in non-infected and HCMV-infected cells.

Sequential specimens from nine allograft recipients were examined by using a variety of methods to detect primary cytomegalovirus (CMV) infection as rapidly as possible posttransplantation. Sera were examined for immunoglobulin G (IgG) and IgM antibodies by immunoblotting, enzyme immunoassay, and immunofluorescence and also by complement fixation, latex agglutination, and an immunofluorescence test for antibody to CMV early antigen. Urine and occasionally blood, tissue, and other specimens were centrifuged onto cell cultures to enhance CMV infectivity. Eight of the nine patients showed laboratory evidence of primary CMV infection, and CMV was isolated from seven of the eight: in no case was virus isolated before seroconversion had become evident. However, serological tests differed in their abilities to detect antibody response to CMV infection in different patients; while immunoblotting, latex agglutination, and enzyme immunoassay for IgG antibodies generally detected seroconversion before complement fixation, this was not invariably the case. At present, optimal laboratory detection of CMV infections in these patients can be achieved only by a combination of serological methods and virus isolation.

In a longitudinal study we examined by immunoblotting (IB) the development and the evolution of the humoral immune response against individual cytomegalovirus (CMV) structural polypeptides in a total of 80 serum samples from 13 renal transplant recipients showing serological evidence of CMV infection and five renal transplant recipients with an anti-CMV antibody level unchanged over the observation period. The results showed that the IB reactivity at the time of transplantation may be a good index of the host's humoral immune status against CMV; by using this procedure it is possible to identify a seroconversion by the detection of antibodies reacting with some intermediate molecular weight proteins in sera examined at high dilution. Furthermore, IB is a very sensitive procedure also for IgM detection as it anticipates the positivity of the enzyme immune assay for IgM.

The study of the prevalence of drug resistances and of the frequency of R factors among resistant strains, in E. coli isolated respectively from the respiratory, intestinal and urinary tracts of patients, during 1976, has shown that the frequency of drug resistances is highest among E. coli strains isolated from the respiratory tract and lowest among the E. coli strains isolated from urines. The frequency of R factors, among resistant strains, follows an exactly opposite distribution. This behaviour could be related to the gradient of antimicrobial drug concentrations achieved in the various compartments during ordinary antimicrobial therapy.

The localization of the major cytomegalovirus glycoprotein, both in the viral particle and in the cell membrane, was studied by means of indirect immunofluorescence and immunoelectron microscopy using a guinea pig anti-glycoprotein hyperimmune serum recently obtained by Gonczol et al. [1986]. By indirect immunofluorescence a slight and uneven positivity was observed on the plasma membrane of unfixed cells starting from 72 h postinfection (p.i.) until the end of our observation time (7 days p.i.). At immunoelectron microscopy the plasma membrane proved positive only where the virus and dense bodies budded through the membrane to form their own envelope. Extracellular viral particles (both viruses and dense bodies) appeared very strongly labeled on the external surface.

Due to the emergence of drug-resistant mutants during the treatment of viral infections, an easy and rapid laboratory technique to assay the antiviral sensitivity of virus isolates could be extremely useful. In this brief report we describe the use of enzyme-linked immunoassay (EIA) to detect the susceptibility of herpes simplex virus (HSV) type 1 to three well known antiviral agents: Cytosine-arabinoside (Ara-C), 5'-iododeoxyuridine (IUDR), and acyclovir (ACG). The results indicate that EIA can easily give an accurate indication of antiviral susceptibility 24 hr after the isolation of the virus and provides important information from the therapeutical point of view.

A total of 56 sera from asymptomatic HIV-seropositive subjects and patients with symptomatic HIV infection were studied by immunoblotting for IgM, IgG and IgG subclass reactivity against cytomegalovirus structural polypeptides. The results showed a high percentage of IgM-positive sera in the asymptomatic patients, and IgM reactivity was particularly high against a polypeptide of intermediate molecular weight (p66). IgG reactivity was very high against the majority of viral structural polypeptides with the possible exceptions of p82 and p61, in all patients. Antibody to these polypeptides decreased with the increase in HIV-specific symptoms. No significant difference in the reactivity of IgG subclass to viral polypeptides was found among the different groups of subjects, except for a generalized increase in IgG subclass reactivity from asymptomatic to HIV-specific symptomatic patients.

The study of human cytomegalovirus (HCMV) in cultures of human embryo lung fibroblasts, pre-treated with actinomycin D, has shown that under these conditions the virus infection does not proceed beyond the 'early' events of the virus replication cycle. In the same experimental conditions the growth of poliovirus type I, vaccinia virus and herpes simplex type I virus, was completely unaffected. These results suggest that the complete HCMV replication cycle requires some cellular function(s) between early transcription of the input virus genome and virus DNA synthesis.

Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera. The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169. A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11. Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment. The gene was transcribed into a late 1.3-kilobase RNA. The nucleotide sequence of the coding region was determined. Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase. In Western blots these proteins were recognized by human sera. Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots. In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.

We have produced a monoclonal antibody against human cytomegalovirus (HCMV) which recognizes a structural protein of 28 000 mol. wt. which is present in both the cytoplasm of infected cells during the late phase of the viral replication cycle and in the extracellular viral particles. This antigen was detected in all HCMV strains assayed and reacted with human sera having anti-HCMV antibodies.

.5 mM of novobiocin completely suppresses the replication of human cytomegalovirus (HCMV) in vitro. Based on the evidence that murine cytomegalovirus (MCMV) titres were reduced in vitro by 60% with the same concentration of the drug, the in vivo antiviral activity of novobiocin has been tested in the murine model. The results showed that if treatment started before the infection virus was not isolated from parenchymal organs during treatment. Virus could be isolated 24 h after stopping treatment, but not from kidney or spleen.

The antibody response to cytomegalovirus structural polypeptides in sera from three groups of acutely infected subjects was analyzed. Differences in number and types of polypeptides were noted. Immunoblotting could be used to distinguish sera from patients with acute cytomegalovirus hepatitis from convalescent sera by the detection of antibody to viral proteins of 82, 66, 62, and 55 kilodalton molecular weight at a high serum dilution.

Human cytomegalovirus (CMV) has at least 15 structural polypeptides which are immunogenic during natural infection. In this report we used immunoblotting to study the reactivity of each polypeptide with the four IgG subclasses present in CMV antibody-positive human serum obtained from both convalescent patients and from patients with serological evidence of active CMV infection. The results showed that in the serum of patients reactivity to several CMV polypeptides is mostly due to all four IgG subclasses, with IgG3 being the most prevalent followed by IgG1. Intermediate molecular weight polypeptides (namely p66, p61, p55, p49, p38) appear to be the major antigens responsible for the preferential induction of the IgG3 response. During convalescence the reactivity of all four IgG subclasses is evident only against a structural polypeptide of 150 kD molecular weight (MW), while intermediate MW polypeptides show variable IgG subclass reactivity. The presence of IgG3 to one or more polypeptides of intermediate MW might be of diagnostic interest.

We used Western Blotting analysis to determine the immune profile to Campylobacter pylori polypeptides in: A) sera from patients with idiopathic dyspepsia and bacteriological evidence of C. pylori gastric colonization, B) sera from patients with the same symptoms but no bacteriological evidence of C. pylori infection and C) healthy subjects. To avoid interference of aspecific reactions due to antigenic cross reactivity with other thermophilic Campylobacter species, antisera were raised in rabbits against C. pylori as well as against C. coli and C. jejuni. Some bands (with an approximate molecular weight of 118, 85, 40, 34, 28, 18 and 12 Kd) which can be considered specific for C. pylori were identified and the IgG reaction to some of them (40, 34, 28 Kd) was shown to be significantly higher in patients with bacteriological evidence of C. pylori infection than in the other two groups. IgM reactivity to two bacterial proteins of molecular weight 118 and 40 Kd was particularly evident in the second group of patients suggesting a possible diagnostic tool to identify C. pylori infection at a very early stage.

IgM detection by enzyme-linked immunosorbent assay (ELISA) is the most used method of establishing a cytomegalovirus (CMV) infection during pregnancy. In this paper, we discuss the results obtained assaying 1,000 sera from pregnant women by two ELISA kits: a traditional indirect ELISA and a more recent IgM-capture ELISA. All the sera that gave a positive ELISA value with one or both kits were further tested by immunoblotting (IB) to establish which CMV polypeptides were detected by IgM antibodies. From the results obtained, IgM-capture ELISA seems less sensitive than indirect ELISA, but correlates better with serological evidence of active CMV infections as judged by typical IB profiles.