M.C. Medici

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification

Virus detection and/or identification traditionally rely on methods based on cell culture, electron microscopy and antigen or nucleic acid detection. These techniques are good, but often expensive and/or time-consuming; furthermore, they not always lead to virus identification at the species and/or type level. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was tested as an innovative tool to identify human polioviruses and to identify specific viral protein biomarkers in infected cells. The results revealed MALDI-TOF MS to be an effective and inexpensive tool for the identification of the three poliovirus serotypes. The method was firstly applied to Sabin reference strains, and then to isolates from different clinical samples, highlighting its value as a time-saving, sensitive and specific technique when compared to the gold standard neutralization assay and casting new light on its possible application to virus detection and/or identification.

Human Cytomegalovirus Proteins PP65 and IEP72 Are Targeted to Distinct Compartments in Nuclei and Nuclear Matrices of Infected Human Embryo Fibroblasts

The cellular distribution of the human cytomegalovirus (HCMV)‐specific UL83 phosphoprotein (pp65) and UL123 immediate‐early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so‐called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non‐extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV‐specific products into the pre‐existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.

Epidemiological and molecular features of norovirus infections in Italian children affected with acute gastroenteritis.

During a 5-year (2007-2011) surveillance period a total of 435 (15·34%) of 2834 stool specimens from children aged <14 years with acute gastroenteritis tested positive for norovirus and 217 strains were characterized upon partial sequence analysis of the polymerase gene as either genogroup (G)I or GII. Of the noroviruses, 99·2% were GII with the GII.P4 genotype being predominant (80%). GII.P4 variants (Yerseke 2006a, Den Haag 2006b, Apeldoorn 2008, New Orleans 2009) emerged sequentially during the study period. Sequence analysis of the capsid gene of 57 noroviruses revealed that 7·8% were recombinant (ORF1/ORF2) viruses including GII.P7_GII.6, GII.P16_GII.3, GII.P16_GII.13, GII.Pe_GII.2, and GII.Pe_GII.4, never identified before in Italy. GII.P1_GII.1, GII.P2_GII.1, GII.P3_GII.3 and GII.P6_GII.6 strains were also detected. Starting in 2011 a novel GII.4 norovirus with 3-4% nucleotide difference in the polymerase and capsid genes from variant GII.4 New Orleans 2009 was monitored in the local population. Since the epidemiology of norovirus changes rapidly, continuous surveillance is necessary to promptly identify the onset of novel types/variants.

Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages.

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle.