M. P. Landini

Human cytomegalovirus morphogenesis: an ultrastructural study of the late cytoplasmic phases

SummaryThe 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.

New developments in the diagnosis of maternal and congenital CMV infection

Recent advances in the screening of pregnant women with Cytomegalovirus (CMV) IgM, CMV IgG and CMV IgG avidity serologic tests, has led to a more accurate diagnosis of CMV infection. When serologic screening is performed early in gestation, it is possible to identify those women at risk of intrauterine transmission of the virus, i.e., those women with a primary CMV infection, who should be enrolled in prenatal diagnosis. The use of quantitative PCR on amniotic fluid from pregnant women at 21–22 weeks of gestation in prenatal diagnosis is an effective diagnostic tool to distinguish between CMV infection and CMV disease in the fetus and newborn. Quantitative PCR on peripheral blood leukocytes from CMV infected newborns can be used to monitor viral load, especially during treatment with ganciclovir. These advances in serology and quantitative virology should lead to more accurate diagnosis of maternal and congenital CMV infection.

Congenital cytomegalovirus infection: patterns of fetal brain damage

Cytomegalovirus (CMV) is the most prevalent infectious agent causing neurological dysfunction in the developing brain. This study analysed the different patterns of tissue damage, particularly in the brain, of fetuses with documented CMV infection. We studied 45 fetuses at 20-21 weeks of gestation with congenital CMV infection documented by invasive positive prenatal diagnosis. At the time of amniocentesis, abnormal ultrasound findings had been recorded for 13 of the 45 fetuses (29%). Histological and immunohistochemical characterization was performed on the placenta, brain, heart, lung, liver, kidney, and pancreas. The different degrees of brain damage were correlated with tissue viral load, inflammatory response, placental functionality, and extramedullary haematopoiesis. Even though a high CMV load was detected in all amniotic fluids, brain infection occurred in only 62% of the fetuses and with different degrees of severity. Tissues with a low viral load showed a globally weak inflammatory response, and fetuses had only mild brain damage, whereas tissues with a high CMV load showed prominent infiltration of the activated cytotoxic CD8(+) T-lymphocytes responsible for immune-mediated damage. Furthermore, severe placental infection was associated with diffuse villitis and necrosis, consistent with functional impairment and possible consequent hypoxic cerebral damage. Brain injury induced by CMV congenital infection may be the result of uncontrolled viral replication, immune-mediated damage by cytotoxic CD8(+) T-lymphocytes, and, in the presence of placental insufficiency, fetal hypoxia.

Serum Antibodies to Individual Cytomegalovirus Structural Polypeptides in Renal Transplant Recipients during Viral Infection

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 hosts 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.

Laboratory diagnosis of late-onset sepsis in newborns by multiplex real-time PCR.

Bloodstream infections (BSIs) are an important cause of neonatal morbidity and mortality, and often result in prolonged hospitalization of infants who are admitted to neonatal intensive care units (VerboonMaciolek et al., 2006). Late-onset neonatal sepsis (occurring in newborns aged older than 3 days) occurs in approximately 0.1 % of all newborns and in up to ~25 % of very low birth weight infants (birth weight ,1500 g) (Kaufman & Fairchild, 2004). Early diagnosis of sepsis and prompt treatment are critical in preventing severe and life-threatening complications in these patients (Harbarth et al., 2003; Kollef, 2003; Lodise et al., 2003). The clinical recognition of sepsis in neonates is difficult, however, because the signs and symptoms are often non-specific (Gerdes, 1991; Verboon-Maciolek et al., 2006) and blood cultures (BCs) are rarely positive.

Relationship between IgM Antibody to Human Cytomegalovirus, Virus Load, Donor and Recipient Serostatus, and Administration of Methylprednisolone as Risk Factors for Cytomegalovirus Disease after Liver Transplantation

A retrospective study was performed on a selected cohort of 40 liver transplant recipients derived from the previous prospective follow-up of 162 liver transplant patients. The criterion for selection of this cohort was the presence of human cytomegalovirus (HCMV) DNAemia after transplantation, as determined by qualitative polymerase chain reaction (PCR). These 40 patients were followed longitudinally by quantitative PCR and by the new recombinant antigen-based AxSYM immunoassay for IgM to HCMV. The detection of IgM to CMV after transplantation was significantly associated with the development of HCMV disease in patients who had evidence of active HCMV replication in the blood by PCR (P=.01). On the basis of multivariate logistic regression analyses, the maximum titer of IgM detected after transplantation was a risk factor that was independent of augmented methylprednisolone and donor seropositivity. However, in multivariate analyses, elevated virus load continued to be the predominant risk factor for progression to HCMV disease.

Humoral immune response to human cytomegalovirus proteins: A brief review

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.

Structural Components of Human Cytomegalovirus: In situ Localization of the Major Glycoprotein

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.

TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2.

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 transactivaton 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-κB response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated or 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.

IgM antibody detection of ppUL80A and ppUL32 by immunoblotting: an early parameter for recurrent cytomegalovirus infection in renal transplant recipients.

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.