Graphical abstract

Acute respiratory tract infections (ARTIs) are among the leading causes of morbidity and mortality in children. The viral etiology of ARTIs was investigated over 3 years (October 2012-September 2015 in 2575 children in Parma, Italy, using indirect immunofluorescent staining of respiratory samples for viral antigens, cell culture, and molecular assays. Respiratory viruses were detected in 1299 cases (50.44%); 1037 (79.83%) were single infections and 262 (20.17%) mixed infections. The highest infection incidence was in children aged N6 months to ≤3 years (57.36%). Human respiratory syncytial virus (27.12%) and human adenovirus (23.58%) were the most common viruses identified. The virus detection rate decreased significantly between the first and third epidemic season (53.9% vs. 43.05%, P b 0.0001). The simultaneous use of different diagnostic tools allowed us to identify a putative viral etiology in half the children examined and to provide an estimate of the epidemiology and seasonality of respiratory viruses associated with ARTIs.

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.

SARS-CoV-2 emerged in China in December 2019 and has now been declared a pandemic by the World Health Organization. This paper described the case of a 7-week-old suckling baby from Italy who was SARS-CoV-2-positive only by the cell culture method, with no clinical suspicion of and/or risk factors for SARS-CoV-2 infection. The baby was referred to hospital, with signs and symptoms of upper respiratory tract infection, before the virus had spread to the province. Nasal and pharyngeal swabs and a nasopharyngeal aspirate were used for conventional and molecular diagnostic assays not including the SARS-CoV-2 virus. Bacteria referred to the resident population were revealed in nasal and pharyngeal swabs. No viruses were detected using both immunofluorescence assay and nucleic acid amplification assays in the nasopharyngeal aspirate. The baby was discharged in good condition after 3 days of hospitalisation. Later, a cytopathic effect on the cell monolayers currently used for respiratory viruses was observed and the viral particles were identified as Coronaviridae by transmission electron microscopy. SARS-CoV-2 was identified by RT-PCR performed both on cell culture and on the stored aliquot of the original sample. The virus isolate was named SARS-Cov-2/human/Parma/1/2020. Cell culture still remains the only reference diagnostic method also for emerging viruses, allowing it to reveal cytopathogenic viruses and demonstrate their infectivity.

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.

The nucleolus is a nuclear domain involved in the biogenesis of ribosomes, as well as in many other important cellular regulatory activities, such as cell cycle control and mRNA processing. Many viruses, including herpesviruses, are known to exploit the nucleolar compartment during their replication cycle. In a previous study, we demonstrated the preferential targeting and accumulation of the human cytomegalovirus (HCMV) UL83 phosphoprotein (pp65) to the nucleolar compartment and, in particular, to the nucleolar matrix of lytically infected fibroblasts; such targeting was already evident at very early times after infection. Here we have investigated the possible effects of rRNA synthesis inhibition upon the development of HCMV lytic infection, by using either actinomycin D or cisplatin at low concentrations, that are known to selectively inhibit RNA polymerase I activity, whilst leaving RNA polymerase II function unaffected. Following the inhibition of rRNA synthesis by either of the agents used, we observed a significant redistribution of nucleolar proteins within the nucleoplasm and a simultaneous depletion of viral pp65 from the nucleolus; this effect was highly evident in both unextracted cells and in nuclear matrices in situ. Of particular interest, even a brief suppression of rRNA synthesis resulted in a very strong inhibition of the progression of HCMV infection, as was concluded from the absence of accumulation of HCMV major immediate-early proteins within the nucleus of infected cells. These data suggest that a functional relationship might exist between rRNA synthesis, pp65 localization to the nucleolar matrix and the normal development of HCMV lytic infection.

No abstract

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.