Hepatitis E virus infection in North Italy: high seroprevalence in swine herds and increased risk for swine workers.

We determined the hepatitis E virus (HEV) seroprevalence and detection rate in commercial swine herds in Italy's utmost pig-rich area, and assessed HEV seropositivity risk in humans as a function of occupational exposure to pigs, diet, foreign travel, medical history and hunting activities. During 2011-2014, 2700 sera from 300 swine herds were tested for anti-HEV IgG. HEV RNA was searched in 959 faecal pools from HEV-seropositive herds and in liver/bile/muscle samples from 179 pigs from HEV-positive herds. A cohort study of HEV seropositivity in swine workers (n = 149) was also performed using two comparison groups of people unexposed to swine: omnivores (n = 121) and vegetarians/vegans (n = 115). Herd-level seroprevalence was 75·6% and was highest in farrow-to-feeder herds (81·6%). Twenty-six out of 105 (24·8%) herds had HEV-positive faecal samples (25 HEV-3, one HEV-4). Only one bile sample tested positive. HEV seropositivity was 12·3% in swine workers, 0·9% in omnivores and 3·0% in vegetarians/vegans. Factors significantly associated with HEV seropositivity were occupational exposure to pigs, travel to Africa and increased swine workers' age. We concluded that HEV is widespread in Italian swine herds and HEV-4 circulation is alarming given its pathogenicity, with those occupationally exposed to pigs being at increased risk of HEV seropositivity.

Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories.

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

A surveillance system of Invasive Pneumococcal Disease in North-Eastern Italy.

BACKGROUND: From 2007, in the Veneto Region (Italy), a surveillance system for invasive pneumococcal diseases (IPD) was implemented to estimate the regional epidemiology of IPD and to evaluate the impact of 13-valent pneumococcal conjugate vaccine (PCV13) vaccination. METHODS: Data were collected from 2007 to 2014 and the total, annual and age-specific IPD notification rates were calculated. A Poisson regression model was used to identify the possible risk factors for developing IPD. RESULTS: A total of 713 IPD cases were notified and the overall IPD notification rate was equal to 2.0 cases per 100,000 population (95% CI: 1.7-2.1), with an increasing trend between 2007 and 2014. The pneumococcal serotypes were identified in 608 (85.3%) isolates from biological specimens, and the most distributed serotypes were those contained in PCV13. Children <5 year-old and the adults over 65 year-old showed the highest PCV13 vaccine-type IPD notification rate, equal to 2.7/100,000 and 2.8/100,000, respectively. The risk to develop IPD was greater in children aged <5 years (RR = 8.9, 95% CI: 5.1-15.9; p<0.0001) and in adults aged >65 years (RR = 4.3, 95% CI: 2.7-6.9; p<0.0001), especially in males > 65 years of age (RR = 1.7, 95% CI: 1.0-2.8; p = 0.042). The invasive pneumococcal disease was mainly caused by the PCV13 serotypes (RR = 2.9, 95%CI: 2.3-3.9; p<0.0001), principally after the PCV13 introduction (RR = 2.3, 95% CI: 1.4-3.8; p<0.001). In spite of that, a significant reduction of the overall IPD incidence is evident in the period following the PCV13 vaccine introduction (RR = 0.4, 95% CI: 0.3-0.5; p<0.0001), particularly in children aged <5 years (RR = 0.3, 95% CI: 0.2-0.7; p = 0.002), demonstrating the real efficacy of PCV13 immunization for children. CONCLUSIONS: In the Veneto Region, the surveillance system has allowed to describe the detailed epidemiological profile of invasive pneumococcal disease, pointing out that the most circulating pneumococcal serotypes were those contained in the PCV13 vaccine.

Functional Interaction Between the ESCRT-I Component TSG101 and the HSV-1 Tegument Ubiquitin Specific Protease.

Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de-ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de-conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de-ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N-terminal variant of the herpes simplex virus (HSV) type-1 large tegument protein VP1/2 (VP1/2(1-767)), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)-I, functionally interact. In particular, VP1/2(1-767) modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV-1 specific de-ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family.

Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Whole genome sequencing and phylogenetic analysis of West Nile virus lineage 1 and lineage 2 from human cases of infection, Italy, August 2013.

A human outbreak of West Nile virus (WNV) infection caused by WNV lineage 2 is ongoing in northern Italy. Analysis of six WNV genome sequences obtained from clinical specimens demonstrated similarities with strains circulating in central Europe and Greece and the presence of unique amino acid changes that identify a new viral strain. In addition, WNV lineage 1 Livenza, responsible for a large outbreak in north-eastern Italy in 2012, was fully sequenced from a blood donor during this 2013 outbreak.

New endemic West Nile virus lineage 1a in northern Italy, July 2012.

We report here the first blood donation positive for West Nile virus (WNV) by nucleic acid amplification testing collected in north-eastern Italy in July 2012.Partial sequencing of the WNV RNA demonstrated identity with a WNV lineage 1a genome identified in the same area in 2011 and divergence from the strain responsible for the outbreak in northern Italy in 2008­09. These data indicate that WNV activity in northern Italy is occurring earlier than expected and that different WNV strains are circulating.

Clinical and virological findings in the ongoing outbreak of West Nile virus Livenza strain in northern Italy, July to September 2012.

In July-September 2012, one month earlier than in previous years, 13 confirmed human cases of West Nile virus infection were diagnosed in northern Italy, including five with neuroinvasive disease, three with West Nile fever, and five West Nile virus (WNV)-positive blood donors. In nine cases, the presence of the WNV lineage 1a Livenza strain, characterised in 2011, was ascertained. Symptomatic patients had prolonged viruria with high viral load.

HIV coreceptor tropism in paired plasma, peripheral blood mononuclear cell, and cerebrospinal fluid isolates from antiretroviral-naïve subjects.

A survey of HIV coreceptor usage in cerebrospinal fluid (CSF) samples, peripheral blood mononuclear cells (PBMCs), and plasma samples from naïve seropositive patients was conducted. One hundred patients were enrolled in this study. Of the 100 patients, 36 had a primary or recent infection (P-RI), 31 had an early chronic infection (>350 CD4 cells) (ECI), and 33 had a late chronic infection (LCI). All 3 compartments were sampled in a subset of 33 participants, while the remaining 67 patients provided plasma samples and PBMCs only. Seventy-seven patients harbored the R5 virus in plasma samples and had a significantly higher median and percentage of CD4(+) T cells than patients with X4 virus (437 and 281 cells/µl, respectively; P = 0.0086; 20.6% and 18.6%, respectively). The X4 strain was detected more frequently in patients with LCI than in patients with P-RI or ECI (39.3%, 19.4%, and 9.6%, respectively; P = 0.0063). PBMC and plasma tropism was concordant in 90 patients, and 73 had the R5 strain. Among patients with discordant results, 4 had the R5 virus in their plasma and the X4 virus in PBMCs; 6 showed the opposite profile. Plasma, PBMC, and CSF tropism determinations were concordant in 26/33 patients (21 patients had R5, and 5 had X4). The tropism was discordant in 5/33 patients, with the X4 virus in plasma and R5 in CSF; the HIV tropism in PBMCs was X4 in 3 patients. The remaining 2/33 patients had the R5 virus in plasma and PBMCs and the X4 virus in CSF; one of these patients had a P-RI. The discordant tropism in CSF and blood may have implications for chemokine (C-C motif) receptor 5 (CCR5) antagonist use in patients with limited response to antiretroviral therapy (ART) or in responding patients evaluated for simplification of treatment.

Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital.

BACKGROUND: Outbreaks of vancomycin-resistant Enterococcus faecium (VRE) strains is an emerging problem worldwide. Even if still relatively uncommon in European hospitals, infections caused by VRE have also been increasing recently in this continent. METHODS: In this study, we characterized 50 consecutive VRE and 23 vancomycin-sensitive E. faecium (VSE) isolates collected in an Italian hospital. The presence of the esp gene and that of genes encoding resistance to glycopeptides was investigated by polymerase chain reaction (PCR). All of the isolates were typed by multi-locus sequence typing (MLST), and a selection of them also by pulsed-field gel electrophoresis (PFGE). RESULTS: We found that all of the VRE and 18 (78%) of the VSE strains belonged to the single clonal complex-17 (CC17). The most represented sequence type (ST) was ST78 (34% of the isolates). When further analyzed by PFGE, ST78 isolates were subdivided into five pulsotypes, four of them closely related. The strong association between the esp gene and CC17 was confirmed. Interestingly, such an association was higher among vancomycin-resistant isolates. Most of the esp-positive isolates (34/46, 74%) encoded Esp4, a rare variant of this protein characterized by the absence of A repeats. CONCLUSIONS: Our findings underscore the role of the CC17 lineage in the nosocomial spread of VRE and VSE, and its rapid local evolution, underscoring the need for programs designed to provide early detection in order to prevent its spreading among the nosocomial population.

Measles in Italy, July 2009 to September 2010.

Outbreaks of measles continue to occur in Italy, as in other European countries. We present here details of cases reported through the Italian enhanced measles surveillance system from July 2009 to September 2010. In total, 2,151 cases were reported, 42% (n=895) of which were laboratory confirmed. The median age of cases was 18 years and 1,709 of 1,856 cases (92%) were unvaccinated. Many cases with complications were reported (n=305), including three with encephalitis. A total of 652 of 1,822 cases (36%) were hospitalised. Molecular characterisation revealed circulation of a limited number of measles virus genotypes (D4, D8 and B3), which is consistent with the current epidemiology of the disease in Italy. A national measles elimination plan was approved in 2003 with the aim of interrupting endemic measles transmission by 2007. Since elimination was not achieved, the target date was recently moved to 2015. The emphasis of the new elimination plan, approved in March 2011, is on strengthening surveillance, implementing evidence based-interventions to increase measles-mumps-rubella vaccine uptake in children, adolescents and young adults, and implementing communication activities related to the vaccine. The strategies proposed by the plan should be implemented fully and appropriately by all regions in order to meet the elimination goal by 2015.

West Nile virus: the Italian national transplant network reaction to an alert in the north-eastern region, Italy 2011.

We report four cases of West Nile virus (WNV) transmission following a single multiorgan donation in north-eastern Italy. The transmissions were promptly detected by local transplant centres. The donor had been tested for WNV by nucleic acid amplification test (NAT) prior to transplantation and was negative. There were no detected errors in the nationally implemented WNV safety protocols.

Human cases of West Nile Virus infection in north-eastern Italy, 15 June to 15 November 2010.

In 2010, for the third consecutive year, human cases of West Nile virus (WNV) infection, including three confirmed cases of neuroinvasive disease and three confirmed cases of West Nile fever, were identified in north-eastern Italy. While in 2008 and 2009 all human cases of WNV disease were recorded in the south of the Veneto region, cases of WNV disease in 2010 additionally occurred in two relatively small northern areas of Veneto, located outside those with WNV circulation in the previous years. WNV IgG antibody prevalence in blood donors resident in Veneto was estimated as ranging from 3.2 per 1,000 in areas not affected by cases of WNV disease to 33.3 per 1,000 in a highly affected area of the Rovigo province. No further autochthonous human cases of WNV disease were notified in Italy in 2010. The recurrence of human cases of WNV infection for the third consecutive year strongly suggests WNV has become endemic in north-eastern Italy.

Prevalence of IgM and IgG antibodies to West Nile virus among blood donors in an affected area of north-eastern Italy, summer 2009.

Following reports of West Nile neuroinvasive disease in the north-eastern area of Italy in 2009, all blood donations dating from the period between 1 August and 31 October 2009 in the Rovigo province of the Veneto region were routinely checked to exclude those with a positive nucleic acid test for West Nile virus (WNV). Only one of 5,726 blood donations was positive (17.5 per 100,000 donations; 95% confidence interval (CI): 0.4­97.3). In addition, a selection of 2,507 blood donations collected during the period from 20 July to 15 November 2009 were screened by ELISA for IgG and IgM antibodies against WNV. A positive result was received for 94 of them. The positive sera were further evaluated using immunofluorescence and plaque reduction neutralisation test (PRNT), in which only 17 sera were confirmed positive. This corresponds to a prevalence of 6.8 per 1,000 sera (95% CI: 4.0­10.9). In a case-control study that matched each of the 17 PRNT-positive sera with four negative sera with the same date of donation and same donation centre, we did not find a significant association with age and sex of the donor; donors who worked mainly outdoors were significantly more at risk to have a positive PRNT for WNV.

Tracking the time course of reproduction number and lockdown's effect on human behaviour during SARS-CoV-2 epidemic: nonparametric estimation.

Understanding the SARS-CoV-2 dynamics has been subject of intense research in the last months. In particular, accurate modeling of lockdown effects on human behaviour and epidemic evolution is a key issue in order e.g. to inform health-care decisions on emergency management. In this regard, the compartmental and spatial models so far proposed use parametric descriptions of the contact rate, often assuming a time-invariant effect of the lockdown. In this paper we show that these assumptions may lead to erroneous evaluations on the ongoing pandemic. Thus, we develop a new class of nonparametric compartmental models able to describe how the impact of the lockdown varies in time. Our estimation strategy does not require significant Bayes prior information and exploits regularization theory. Hospitalized data are mapped into an infinite-dimensional space, hence obtaining a function which takes into account also how social distancing measures and people's growing awareness of infection's risk evolves as time progresses. This also permits to reconstruct a continuous-time profile of SARS-CoV-2 reproduction number with a resolution never reached before in the literature. When applied to data collected in Lombardy, the most affected Italian region, our model illustrates how people behaviour changed during the restrictions and its importance to contain the epidemic. Results also indicate that, at the end of the lockdown, around [Formula: see text] of people in Lombardy and [Formula: see text] in Italy was affected by SARS-CoV-2, with the fatality rate being 1.14%. Then, we discuss how the situation evolved after the end of the lockdown showing that the reproduction number dangerously increased in the summer, due to holiday relax, reaching values larger than one on August 1, 2020. Finally, we also document how Italy faced the second wave of infection in the last part of 2020. Since several countries still observe a growing epidemic and others could be subject to other waves, the proposed reproduction number tracking methodology can be of great help to health care authorities to prevent SARS-CoV-2 diffusion or to assess the impact of lockdown restrictions on human behaviour to contain the spread.

COVID-19 cumulative incidence, intensive care, and mortality in Italian regions compared to selected European countries.

BACKGROUND: The high contagiousness and rapid spreading of the coronavirus disease 2019 (COVID-19) has caused a high number of critical to severe life-threatening cases, which required urgent hospital admission and treatment in intensive care units (ICUs). The pandemic has been a tough test for all European national health systems and their capability to provide an adequate reaction. METHODS: The present work aims to reveal correlations between parameters such as COVID-19 incidence, ICU bed occupancy, ICU excess area, and mortality in Italian regions. Public data for the period of March 1 to July 16, 2020, were analyzed using several mathematical and statistical methods. RESULTS: The analysis defined two separate groups of Italian regions. The examined variables considered within these groups were interlinked and dependent on each other. The regions of the two groups shared the same kind of fitted model (linear) explaining mortality as a function of cumulative incidence, but with higher value of the constant in one group, so characterized by a high intrinsic "strength" of the pandemic, certainly playing a major role in the generation of a large number of severe and life-threatening cases. These results are confirmed at European level. Other factors may condition mortality and be linked to incidence, such as ICU saturation and excess. CONCLUSIONS: These quantitative results could be a very helpful tool to set up preventive measures and optimize biomedical interventions before the pandemic, in its recurrent waves, could overcome the reaction capacity of any public health system.

Switching of inferred tropism caused by HIV during interruption of antiretroviral therapy.

After interruption of highly active antiretroviral therapy, 15 out of 53 patients with the X4 HIV strain had a significantly larger decrease in CD4(+) T cell count (P = 0.001) and shorter length of treatment interruption (P = 0.02) than patients with the R5 strain. At treatment resumption, HIV inferred tropism switched from the X4 strain to the R5 variant in 9 patients (60%). These patients had a prolonged length of treatment interruption compared to that of those who still carried the X4 strain.

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus.

Recombinant Semliki Forest virus (SFV) is an attractive viral vector system owing to its ability to allow high efficiency of viral protein expression. To produce recombinant pseudotyped human immunodeficiency virus type 1 (HIV-1) virions, we designed a chimeric SFV/HIV vector system that contains both the HIV-1 cis- and trans-acting elements under the transcriptional control of the SFV replicase and investigated the ability of the hybrid SFV/HIV system to produce lentiviral particles capable of transducing target cells. Co-transfection of target cells with the two helper SFV packaging system RNAs along with each SFV/Gag-Pol, SFV/VSV(G) as well as SFV/HIV-1 vector unit replicon led to the generation of efficient transducing competent recombinant SFV/HIV particles. In contrast, co-transduction of target cells with the SFV/HIV chimeric virions produced recombinant particles with low transducing ability. Our data suggest that both the genomic and the subgenomic RNAs containing the HIV-1 vector unit were negatively selected for incorporation into recombinant particles, despite the fact that the SFV-driven HIV-1 vector replicon was the only one containing a lentiviral packaging sequence. The results of this study provide insights relevant to the design of chimeric lentiviral vectors.

Strategies for inhibiting function of HIV-1 accessory proteins: a necessary route to AIDS therapy?

The Human Immunodeficiency Virus (HIV) genome encodes three major structural proteins common to all retroviruses (Gag, Pol and Env), two regulatory proteins (Tat and Rev) that are essential for viral replication, and four accessory proteins (Nef, Vif, Vpu, Vpr). While accessory proteins were initially reported to be unnecessary for viral growth, their importance as virulence factors is now being more and more appreciated: they can dramatically alter the course and severity of viral infection, replication and disease progression. None of the HIV accessory proteins display enzymatic activity: they rather act altering cellular pathways via multiple protein-protein interactions with a number of host cell factors. All currently approved anti-HIV drugs target pol and env encoded proteins. Therefore, widening the molecular targets of HIV therapy by additionally targeting accessory proteins may expand treatment options, resulting in high impact effective new therapy. In this review we present the state of the art of compounds that target HIV accessory proteins. Most of the research has focused on the inhibition of specific accessory proteins/host cell partner interactions. Promising compounds have been found within different classes of molecules: small natural and synthetic molecules, peptides and proteins, oligonucleotides, in particular those used as RNA interference (RNAi) tools. With the assortment of compounds available, especially against Nef and Vif functions, the demonstration of the clinical efficacy of the new anti-HIV-1 drugs targeting accessory proteins is next challenge.