Phylogenetic analysis of West Nile virus isolates, Italy, 2008-2009.

To determine the lineage of West Nile virus that caused outbreaks in Italy in 2008 and 2009, several West Nile virus strains were isolated from human specimens and sequenced. On the basis of phylogenetic analyses, the strains isolated constitute a distinct group within the western Mediterranean cluster.

Cytomegalovirus gN genotypes distribution among congenitally infected newborns and their relationship with symptoms at birth and sequelae.

BACKGROUND: Cytomegalovirus (CMV) is the leading cause of congenital infection, with morbidity and mortality at birth and sequelae. Both host and viral factors may affect the outcome of infection. CMV strain virulence may depend on genetic variability in "key genes," such as UL73, which encodes the envelope glycoprotein gN. This study aimed to ascertain the role of gN variants as markers of pathogenicity and prognosis in newborns congenitally infected with CMV. METHODS: Seventy-four congenitally infected newborns were monitored for symptoms of CMV disease at birth and during long-term follow-up. The distribution of gN variants was analyzed in relation to virological parameters, clinical signs, laboratory and instrumental abnormalities at birth, and sequelae. Multivariate cluster analysis was used to test for differences in the distribution of variables. An independent validation cohort of the same size and modality of recruitment as the original population was examined by logistic regression to validate results. RESULTS: Univariate and cluster analyses suggest that newborns congenitally infected with CMV fall into 2 subpopulations on the basis of definite parameters of CMV disease. The first population with no symptoms at birth, negative instrumental findings, and a favorable long-term outcome was significantly associated with gN-1 and gN-3a genotypes. The second group with symptoms at birth, abnormal imaging results, and sequelae was associated with gN-4 genotypes (P<.05). The validation cohort further supports the results, indicating that genotypes gN-1 or gN-3a reduce the risk of sequelae 5 fold (95% confidence interval, 1.3-15.6 fold), whereas variants belonging to group gN-4 increase the risk of sequelae 8 fold (95% confidence interval, 2.6-25.8 fold). CONCLUSIONS: Results suggest that gN genotypes might be markers for virulence of CMV wild-type strains and a discriminating factor for selection of CMV-infected newborns who are at risk of developing sequelae.

Dendritic cell function in cytomegalovirus-infected patients with mononucleosis.

Dendritic cells (DCs) are important target cells for human cytomegalovirus (HCMV) infection, and the virus has been shown to hamper the differentiation and maturation pathways of these cells in vitro. In the present study, we examined the function of monocyte-derived DCs obtained from immunocompetent individuals undergoing symptomatic HCMV infection in terms of immunophenotypic characteristics, pinocytosis, lymphocyte stimulation capacity, and cyto-chemokine secretion in comparison with DCs obtained from healthy controls. Immature and lipopolysaccharide (LPS)-stimulated DCs obtained from patients actively infected with HCMV expressed significantly lower levels of major histocompatibility complex (MHC) class II molecules. The inhibition of expression of MHC class II molecules by HCMV appeared to be functionally relevant, as mature DCs obtained from patients with HCMV mononucleosis were inefficient in stimulating proliferation of allogenic lymphocytes. Finally, the pattern of cyto-chemokines secreted by DCs obtained from patients with HCMV mononucleosis was characterized by a proinflammatory profile with an increased production of interleukin (IL)-1beta, tumor necrosis factor alpha, CC chemokine ligand 2 (CCL2) and CCL3, and reduced secretion of IL-10 upon LPS stimulation. During symptomatic HCMV infection in the immunocompetent host, DCs exhibit an impaired immunophenotype and function. These effects may contribute to the viral-induced immunomodulation, which is often observed in HCMV-infected patients.

Phlebotomine sand fly-borne pathogens in the Mediterranean Basin: Human leishmaniasis and phlebovirus infections.

Pathogens transmitted to humans by phlebotomine sand flies are neglected, as they cause infectious diseases that are not on the priority list of national and international public health systems. However, the infections caused by protozoa of the Leishmania genus and viruses belonging to the Phlebovirus genus (family Phenuiviridae)-the most significant group of viruses transmitted by sand flies-have a relevant role for human pathology. These infections are emerging in the Mediterranean region and will likely spread in forthcoming decades, posing a complex threat to human health. Four species and 2 hybrid strains of Leishmania are pathogenic for humans in the Mediterranean Basin, with an estimated annual incidence of 239,500-393,600 cases of cutaneous leishmaniasis and 1,200-2,000 cases of visceral leishmaniasis. Among the phleboviruses, Toscana virus can cause neuroinvasive infections, while other phleboviruses are responsible for a typical "3-day fever"; the actual incidence of Phlebovirus infections in the Mediterranean area is unknown, although at least 250 million people are exposed. Here, we reviewed the current literature on epidemiology of sand fly-borne infections in the Mediterranean Basin, with a focus on humans. Our analysis indicates the need for increased public health activities directed to determine the disease burden of these infections as well as to improve their surveillance. Among the emerging challenges concerning sand fly-borne pathogens, the relationships between sand fly-borne protozoa and viruses should be considered in future studies, including epidemiological links between Leishmania and phleboviruses as well as the conditional capacity for these pathogens to be involved in interactions that may evolve towards increased virulence.

Serological and molecular tools to diagnose visceral leishmaniasis: 2-years' experience of a single center in Northern Italy.

The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIV-positive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an optimal screening test for Mediterranean VL.

Hydroxymethyl-glutaryl coenzyme a reductase inhibition limits cytomegalovirus infection in human endothelial cells.

BACKGROUND: Statins exert anti-inflammatory effects independently of cholesterol-lowering properties. Cytomegalovirus (CMV) infection appears to be implicated in the pathophysiology of atherosclerosis by inducing inflammatory modifications in endothelial cells, especially in immunosuppressed patients. We investigated whether the activity of statins can inhibit replication of CMV in human endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were infected with CMV and coincubated with fluvastatin at 0.1 and 0.2 micromol/L. Fluvastatin inhibited (P<0.001) CMV antigen expression, and this effect was dose related (P<0.001). Quantitative polymerase chain reaction showed that CMV DNA concentration was consistently lower in supernatants from fluvastatin-treated cells than in infected controls, and viral particle concentration was up to 30 times lower in 0.2 micromol/L fluvastatin-treated cells than in infected controls (10.5+/-0.9 versus 0.34+/-0.03 per 10(3) pfu/mL, P<0.001). Addition of mevalonate to treated cultures almost completely abolished fluvastatin inhibition of viral growth. Electrophoretic mobility shift assay showed that fluvastatin reduced nuclear factor-kappaB binding activity in CMV-infected cells. CONCLUSIONS: HMG-CoA inhibition by fluvastatin restrains CMV replication in HUVECs by inhibiting viral antigen expression, DNA synthesis, and viral particle production, conceivably by involving a reduction of nuclear factor-kappaB binding activity.

Monitoring for human cytomegalovirus infection in solid organ transplant recipients through antigenemia and glycoprotein N (gN) variants: evidence of correlation and potential prognostic value of gN genotypes.

Human cytomegalovirus (HCMV) ORF UL73 encodes the envelope glycoprotein gpUL73-gN, which shows seven genotypes (gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, gN-4c). The goal of this study was to determine retrospectively the distribution of gN variants in solid organ transplant recipients with HCMV infection and to establish an association with parameters important for monitoring post-transplantation clinical course during a follow-up of up to 2 years. Peripheral blood leukocytes from 40 solid organ transplant recipients were analysed for pp65-antigen by immunofluorescence and gN genotyped by sequencing or RFLP analysis. A correlation between gN genotypes and antigenemia peak was found, showing a highly significant difference between gN-1 and gN-4b variants (P<0.005). In particular, gN-1 seems to be associated with patients developing low level antigenemia (<50 pp65-positive cells/2 x 10(5) PBLs; PPV = 90%), whereas gN-4b predicts significantly higher values (>50 pp65-positive cells/2 x 10(5) PBLs; PPV = 80%). Furthermore, the onset of positive antigenemia is significantly earlier in patients infected with a gN-4b strain, compared with those infected by a gN-1 variant. Reported data further support a role for gN genotypes in HCMV pathogenesis. gN-1 and gN-4b show a significantly different virulence and could serve as early predictors for the progression of HCMV infection in transplant patients.

Impaired dendritic cell immunophenotype and function in heart transplant patients undergoing active cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) infects dendritic cells (DCs) in vitro and inhibits their maturation properties and their ability to stimulate T-cell proliferation and cytotoxicity. This study analyzed HCMV infection of DCs in vivo. METHODS: We compared blood DCs and monocyte-derived DCs from heart-transplant patients undergoing an acute HCMV infection with DCs obtained from HCMV-negative transplant patients. Diagnosis of active HCMV infection was established by antigenemia test. RESULTS: We detected viral RNA and antigens in defined DC subsets obtained from patients undergoing an active HCMV infection. In addition, we found an impaired immunophenotype in immature DCs from HCMV-positive subjects and a reduced ability of mature DCs from the same group of patients to stimulate allogenic T-cell proliferation. CONCLUSIONS: The impaired immunophenotype and function detected in DCs from transplant patients undergoing an active HCMV infection may be a mechanism used by the virus to interfere with early immune functions and thereby contributing to the HCMV-induced immunosuppression in these patients.

Relevance of cytomegalovirus infection and coronary-artery remodeling in the first year after heart transplantation: a prospective three-dimensional intravascular ultrasound study.

BACKGROUND: Transplant coronary artery disease (TxCAD) is a major cause of long-term mortality after heart transplantation. Although vascular remodeling has been implicated in the pathophysiology of TxCAD, its determinants remain unknown. METHODS: Twenty-nine consecutive heart-transplant recipients prospectively received intravascular ultrasound (IVUS) of the left-anterior descending artery 1 and 12 months after transplant, with volumetric reconstruction of the proximal 30 mm. RESULTS: Overall, patients exhibited intimal volume increase (+83%, P<0.001), wheras vessel volume remained largely unchanged (+4%, P=0.270); consequently, overall lumen volume decreased (-6%, P=0.058). Among the clinical and laboratory variables, cytomegalovirus (CMV) infection requiring treatment (occurring in 12 patients), as assessed by pp65 antigenemia, was independently associated with the impaired ability of the vessel wall to enlarge in response to intimal volume increase, ultimately resulting in lumen loss (OR [95% CI]=0.098 [0.010-0.920]; P=0.042). However, adequate vessel response to intimal hyperplasia with consequent lumen preservation was observed in the remaining 17 patients who did not present CMV infection requiring treatment. CONCLUSIONS: The present study demonstrates that either adequate or inadequate coronary remodeling may occur during the first year after transplantation. Moreover, for the first time, it strongly suggests that remodeling modalities may be negatively influenced by the occurrence of clinically relevant CMV infection. Randomized prospective trials are warranted to investigate whether aggressive treatment of CMV infection may help prevent TxCAD.

Intrauterine cytomegalovirus infection and glycoprotein N (gN) genotypes.

BACKGROUND: Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, supposed to be related with strain-specific tissue-tropism and HCMV-induced immunopathogenesis. One recently discovered polymorphic gene is ORF UL73, encoding for the envelope glycoprotein gN. Among HCMV clinical strains, it shows four distinct genomic variants denoted as gN-1, gN-2, gN-3 and gN-4. OBJECTIVES: Aims of this study were to assess the prevalence of the different gN types in the populations examined and to investigate the possible relationship between genotypes and severity of congenital CMV disease. STUDY DESIGN: The gN genotyping was carried out by sequencing analysis of the HCMV ORF UL73. Comparisons were made by chi-square test and contingency tables. RESULTS: All the four gN genotypes can cause congenital infections and the overall distribution was as follows: gN-1, 23.6%; gN-2, 1.1%; gN-3, 12.9%; gN-4, 62.4%. None of them seems to be preferentially associated with vertical transmission or with acute outcome of congenital infection. However, considering the chronic outcome and long-term sequelae, there was a statistically significant (P<0.05) difference between congenitally infected infants with or without adverse chronic outcome. CONCLUSIONS: HCMV congenital infections, which displayed a prevalence of the gN-1 variants, seem to be associated with favorable chronic outcome.

Cytomegalovirus as a hepatotropic virus.

Hepatitis is a frequent complication of cytomegalovirus infection in the immunocompetent as well as the immunocompromised host. Current knowledge of liver involvement in different groups of subjects undergoing cytomegalovirus infection, such as congenitally infected newborns, immunocompetent adults, transplant recipients and AIDS patients, will be summarized in this chapter. Furthermore, a recently identified population at risk for cytomegalovirus infection, hepatopathic patients, will be described.

Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-alpha secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4(+) and CD8(+) T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.

Cytomegalovirus (CMV)-encoded UL144 (truncated tumor necrosis factor receptor) and outcome of congenital CMV infection.

BACKGROUND: Cytomegalovirus (CMV) is the most common congenital infection in humans. The effect of viral strains on the outcome of congenital CMV is debated. We evaluated whether UL144 polymorphisms in amniotic fluid from CMV-infected Italian women were associated with terminations of pregnancy, subsequent disease in their offspring, or viral load. METHODS: The study was nested within a prenatal CMV program. We sequenced the UL144 gene from 66 amniotic-fluid samples, without knowledge of pregnancy outcome. We performed data analyses on 56 samples for which all information was available. RESULTS: Genotype C was associated with termination of pregnancy (P=.03). Genotype B was associated with fewer terminations of pregnancy (P=.003). A possible association was found between genotype C and symptomatic disease in newborns (odds ratio, 8.81 [95% confidence interval, 0.48-164.02]; P=.05). There was no association between specific genotype and the viral load in amniotic fluid. Symptomatic newborns who had the most common UL144 genotype (B) were more likely to have higher viral loads than were asymptomatic infants (P=.003). CONCLUSIONS: UL144 polymorphisms may be associated with the outcome of congenital CMV infection. Larger studies should be conducted to confirm this association, before genotype analysis can be used, along with other factors, in considering terminations of pregnancy.

Vascular anastomoses in dichorionic diamniotic-fused placentas.

A case of fetal twin-to-twin cytomegalovirus infection through a dichorionic diamniotic (DiDi)-fused placenta prompted our search for possible vascular anastomoses in this type of placenta. This case and three additional DiDi-fused placentas were studied with gross (macro) sections and a three-dimensional (3D) stereomicroscopic technique. Two twins were dizygotic (they differed in gender and blood groups) and the other two were probably monozygotic. Macrosections and 3D-image analysis demonstrated side-to-side connections between small subchorionic vessels. These findings demonstrate that vascular anastomoses are present in DiDi-fused placentas.

Autoantibody appearance in cytomegalovirus-infected liver transplant recipients: correlation with antigenemia.

The presence of anti-endothelial cells (AECA), smooth muscle (SMA), antinuclear (ANA) and antimitochondrial (AMA) autoantibodies, and liver/kidney microsomal antibody type 1 (LKM1) was investigated retrospectively in sera of liver transplant patients and correlated with cytomegalovirus (CMV) infection as determined by the antigenemia test and with the appearance of acute or chronic allograft rejection. Indirect immunofluorescence analysis was carried out in sequential sera from 40 liver transplant patients. Ten out of 23 antigenemia-positive and none of the antigenemia-negative patients developed serum autoantibodies (P = 0.002, Fisher's exact test). Anti-endothelial cell autoantibodies were found in nine cases and SMA in four patients. Antinuclear antibodies were detected in one otherwise autoantibody-negative patient. All but one case of autoantibody positivity were observed in the high antigenemia group (P < 0.0001, Fisher's exact test). In all but one case, autoantibodies were detected in blood during the antigenemia phase and in most cases in coincidence with or after the antigenemia peak. Even though a statistically significant correlation was not found between autoantibody production and the development of acute or chronic allograft rejection, proportionally more acute rejection cases were observed in the autoantibody-positive than in the autoantibody-negative group. It has been speculated that CMV-induced endothelial damage may be a potent antigenic stimulus, which leads to the production of anti-endothelial cells autoantibodies. Anti-endothelial cell autoantibodies may represent not only a marker of cell injury but also contribute to the progression of the inflammatory response leading to the exposure of tissue-privileged self antigens and the induction of other autoantibodies such as SMA.