Hepatitis C virus (HCV) genotypes have became important epidemiological markers in the management of HCV-infected subjects and infection treatment. The dynamics of HCV genotypes are changing in Europe. During a five-year (2009-2013) hospital-based surveillance in the area of Parma, Northern Italy, serum/plasma samples from 1,265 HCV RNA-positive subjects were genotyped. Subtypes 1b, 3a, and 1a were predominant (32.6 %, 19.1 %, and 17.8 %, respectively), with a correlation between viral load and gender. Subtypes 1a and 3a were more frequent in adults and males with a significant difference with the over-50 age group and females (P > 0.0001). Subtype 1b, as well as 2a/2c and G2 not-subtypeable (15.7 % and 7.2 %, respectively), were more common in females and in the over-50 age group compared to males (P < 0.0001, P < 0.0001, and P < 0.05, respectively) and the under-50 age group (P < 0.0001). While subtype 1b showed a nearly constant trend, subtype 1a peaked in 2012, when a consistent decrease in G2 was observed. The increasing detection of G4, mainly in adults, and subtypes 1a and 3a suggests their epidemiological relevance in the population. The detection of more than one HCV genotype in the same sample (0.2 %) and different genotypes in distant samples (5.1 %) from the same subject reinforces the opinion that re-infection and super-infection with different genotypes are not negligible events, especially in HIV-infected subjects. The dynamics of HCV genotypes could have significant implications for infection control.

ABSTRACTA multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively).

This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase.

Differential localization of human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) has been observed within nuclear compartments of lytically infected human embryo fibroblasts. Nucleoli were the first and principal target of pp65 in non-extracted nuclei within 15 minutes after infection, while the nuclear lamina was also involved in nuclear matrix preparations. As to IEp72, it was typically distributed in bright foci in both nuclei and nuclear matrices at very early stages of infection, when a transient colocalization with PML bodies was also clearly observed. Some of the IEp72-PML interacting complexes lay within nucleoli.

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. © 2003 Wiley‐Liss, Inc.

Prosomes are the core of 26S proteasomes, although they were originally observed as 20S particles associated with cytoplasmic mRNPs. Here we show for the first time that prosomes are also genuine constituents of the nuclear matrix, chromatin and the nuclear RNP networks. Using mouse myoblasts we tested three monoclonal antibodies recognising the prosomal subunits p23K, p27K and p30K, and found that the corresponding prosome subclasses are characterised by a variable distribution pattern within the nuclei. Their presence on the nuclear matrix, and most abundantly in the perinucleolar area, is of particular importance. When myoblasts fuse into myotubes, the distribution pattern of certain types of prosomes on the nuclear matrix changes drastically. Surprisingly, DNA strongly interferes with the detection of prosomal antigens by immunofluorescence methods, whereas RNA, histones and other proteins soluble in 2 M NaCl have no such effect. This 'masking' of prosomes can be completely overcome by extensive or even mild digestion with DNase I or restriction enzymes. Many nuclear prosomes can be solubilized by combined treatment with 0.5% Triton X-100 and 2 M NaCl, and others can be released by digestion of DNA and/or RNA, and about 10-20% of nuclear prosomes remain tightly bound to the protein-based nuclear matrix.

Several studies indicate that viruses can induce different cytoskeletal modifications. The present investigation examines the possible involvement of human embryo fibroblast cytoskeleton in the replication of human cytomegalovirus (HCMV). Significant cytoskeletal modifications occur in infected cells; specifically, microfilament depolymerization is observed very early during the HCMV replicative cycle, whilst microtubules and intermediate filaments do not undergo any change for longer times after infection. Our data focus, in particular, on microfilament depolymerization, which starts within the first hour of the replicative cycle, and on the significance of this event, as a CMV-induced mechanism to modify the post-transcriptional regulation of cellular gene expression for its own benefit. Among the possible mechanisms exploited by HCMV to induce microfilament modifications, one might involve the cellular ADP-ribosylation activity, which is increased by HCMV very early in the infectious cycle. Experiments carried out on HCMV-infected cells, in the presence of ADP-ribosylation inhibitors, seem to confirm this hypothesis.