F. De Conto

Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus

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.

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.