The central role of Italy in the spatial spread of USUTU virus in Europe.

USUTU virus (USUV) is an arbovirus maintained in the environment through a bird-mosquito enzootic cycle. Previous surveillance plans highlighted the endemicity of USUV in North-eastern Italy. In this work, we sequenced 138 new USUV full genomes from mosquito pools (Culex pipiens) and wild birds collected in North-eastern Italy and we investigated the evolutionary processes (phylogenetic analysis, selection pressure and evolutionary time-scale analysis) and spatial spread of USUV strains circulating in the European context and in Italy, with a particular focus on North-eastern Italy. Our results confirmed the circulation of viruses belonging to four different lineages in Italy (EU1, EU2, EU3 and EU4), with the newly sequenced viruses from the North-eastern regions, Veneto and Friuli Venezia Giulia, belonging to the EU2 lineage and clustering into two different sub-lineages, EU2-A and EU2-B. Specific mutations characterize each European lineage and geographic location seem to have shaped their phylogenetic structure. By investigating the spatial spread in Europe, we were able to show that Italy acted mainly as donor of USUV to neighbouring countries. At a national level, we identified two geographical clusters mainly circulating in Northern and North-western Italy, spreading both northward and southward. Our analyses provide important information on the spatial and evolutionary dynamics of USUTU virus that can help to improve surveillance plans and control strategies for this virus of increasing concern for human health.

Phylogenetic characterization of Central/Southern European lineage 2 West Nile virus: analysis of human outbreaks in Italy and Greece, 2013-2014.

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.

The challenge of West Nile virus in Europe: knowledge gaps and research priorities.

West Nile virus (WNV) is continuously spreading across Europe, and other continents, i.e. North and South America and many other regions of the world. Despite the overall sporadic nature of outbreaks with cases of West Nile neuroinvasive disease (WNND) in Europe, the spillover events have increased and the virus has been introduced into new areas. The high genetic diversity of the virus, with remarkable phenotypic variation, and its endemic circulation in several countries, require an intensification of the integrated and multidisciplinary research efforts built under the 7th Framework Programme of the European Union (FP7). It is important to better clarify several aspects of WNV circulation in Europe, including its ecology, genomic diversity, pathogenicity, transmissibility, diagnosis and control options, under different environmental and socio-economic scenarios. Identifying WNV endemic as well as infection-free areas is becoming a need for the development of human vaccines and therapeutics and the application of blood and organs safety regulations. This review, produced as a joint initiative among European experts and based on analysis of 118 scientific papers published between 2004 and 2014, provides the state of knowledge on WNV and highlights the existing knowledge and research gaps that need to be addressed with high priority in Europe and neighbouring countries.

Modeling HPV early promoter regulation.

In high risk forms, human papillomaviruses (HPV) can either induce or promote cancerous lesions, especially cervical cancer which is considered the second most common cancer in the women worldwide. HPV life cycle is tightly linked to the infected cell differentiation program and its evolution is strictly joined to the switch between the early and the late viral polycistronic promoters.The aim of this study is to develop a novel mathematical model which collects and structures the available biologic knowledge on the early promoter regulation for HPV in episomal form. The model includes the main regulation by E2 viral protein as well as a novel discovered co-regulation function mediated by the viral E1 protein. Only by including both E2 and E1 regulatory effect the model is able to correctly predict the temporal behaviour of the early promoter switching off. A possible use of the model as in silico tool to evaluate new antiviral therapies is discussed.

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.

Accurate human papillomavirus genotyping by 454 pyrosequencing.

Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.

Novel West Nile virus lineage 1a full genome sequences from human cases of infection in north-eastern Italy, 2011.

During 2008-2009, several human cases of WNV disease caused by an endemic lineage 1a strain were reported in areas surrounding the Po river in north-eastern Italy. Since 2010, cases have been recorded in nearby northern areas, where, in 2011, both lineage 1a and 2 were detected. We describe here two new WNV complete genome sequences from human cases of WNV infection occurring in 2011 in the Veneto Region. Phylogenetic analysis showed that both genome sequences belonged to lineage 1a and were related to WNV strains of the Western Mediterranean subtype. The novel WNV genomes had high nucleotide and amino acid sequence divergence from each other and from the WNV strain circulating in Italy in 2008-2009. The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease that requires further investigation.

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.

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.

Urinary JCV-DNA testing during natalizumab treatment may increase accuracy of PML risk stratification.

The risk of progressive multifocal leukoencephalopathy (PML) in patients treated with natalizumab for multiple sclerosis (MS) is a serious concern. The presence of anti-JC virus antibodies is a risk factor for PML development, but 2.5 % of the patients result falsely-negative, while the prognostic relevance of testing JCV-DNA in biological fluids of treated patients is debated. Aim of this work was to evaluate the utility of testing JCV-DNA, together with anti-JCV antibodies, in biological samples of treated patients as a tool for PML risk stratification. 126 subjects from 5 MS Centers in Italy were included in the study. We performed a cross-sectional study in 63 patients testing JCV-DNA in blood, peripheral blood cells and urine. We longitudinally assessed the presence of JCV-DNA in a cohort of 33 subjects, one of which developed PML. We could test retrospectively serum samples from another PML case occurred during natalizumab therapy. Anti-JCV antibodies and urinary JCV-DNA were both tested in 73 patients. No changes in JCV-DNA status occurred during natalizumab treatment. The subject who developed PML in the longitudinal cohort had detectable JCV-DNA in urine at all time-points while serum or blood from both PML patients were always negative before the onset of disease and, in one case, after. Four subjects with JCV-DNA in urine and undetectable anti-JCV antibodies were retested for anti-JCV antibodies and three out of four resulted positive. In conclusion, testing JCV-DNA in urine is complementary to testing anti-JCV antibodies in identifying patients at risk of PML.

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.

Genome sequence analysis of the first human West Nile virus isolated in Italy in 2009.

In 2009, six new human cases of West Nile neuroinvasive disease (WNND) were identified in Veneto region, following the six cases already reported in 2008. A human West Nile virus (WNV) isolate was obtained for the first time from an asymptomatic blood donor. Whole genome sequence of the human WNV isolate showed close phylogenetic relatedness to the Italy-1998-WNV strain and to other WNV strains recently isolated in Europe, with the new acquisition of the NS3-Thr249Pro mutation, a trait associated with avian virulence, increased virus transmission, and the occurrence of outbreaks in humans.

West Nile virus transmission with human cases in Italy, August - September 2009.

In 2009, to date 16 human cases of West Nile neuroinvasive disease (WNND) have been reported in Italy, in three regions: Veneto, Emilia-Romagna and Lombardia. The number of cases is higher compared with last year when nine cases were identified (eight cases of WNND and one case of West Nile fever) and the geographical distribution indicates spread from east to west.

West Nile virus infection in Veneto region, Italy, 2008-2009.

We report here an update on human cases of West Nile virus (WNV) infection in Veneto region, northeastern Italy. In addition to two cases of WNV neuroinvasive disease notified through a surveillance programme started in September 2008, further four cases were retrospectively identified (in May 2009) by investigating patients with aseptic meningoencephalitis of unknown aetiology occurring in Veneto region in June-September 2008. All six patients had symptom onset in August-September 2008 and were resident in a wetland area close to the Po river delta in Rovigo province. Further five cases of asymptomatic WNV infection, including four residents of the same area in Rovigo, were identified in a seroprevalence study in farm workers from Veneto region. To date, no human cases have been notified in 2009.

Investigation of BRAF and CTNNB1 activating mutations in adrenocortical tumors.

BACKGROUND: Activating mutations of the BRAF oncogene play a central role in the development of various cancer types, but their role in human adrenocortical tumors is unknown. At variance, activating mutations of another oncogene, CTNNB1, which encodes beta-catenin, have been shown to be common events in both benign and malignant adrenocortical tumors. AIM: To investigate the prevalence of BRAF and CTNNB1 activating mutations in sporadic adrenocortical tumors. MATERIALS AND METHODS: Tissue samples from 15 adrenocortical carcinomas and 41 adrenocortical adenomas were investigated for the presence of BRAF and CTNNB1 activating mutations by PCR amplification and direct sequencing. RESULTS: An advanced invasive non-functioning adrenocortical carcinoma carried a somatic heterozygous BRAF V600E mutation, while 4 functioning and 4 non-functioning adenomas and 3 functioning carcinomas carried different CTNNB1 activating mutations. CONCLUSIONS: Activating BRAF somatic mutations may be occasionally found in advanced adrenocortical carcinomas, while CTNNB1 activating mutations are early and common events in adrenal tumorigenesis.

Detection of polyomaviruses and herpesviruses in human adrenal tumors.

The presence of polyomaviruses and herpesviruses in adrenal tumors and their role in adrenal tumorigenesis has never been investigated, even though the adrenal gland seems to be a preferential site of infection by these viruses and adrenal steroid hormones have been shown to activate their replication. We examined in a large series of normal adrenal gland tissues (n=20) and adrenal tumors (n=107) the presence of herpesviruses and polyomaviruses sequences and gene expression, which were detected in a high proportion of both normal and neoplastic adrenal samples (overall, viruses were found in 15% normal adrenals, 27.8% benign adrenal tumors and 35.3% malignant tumors). The polyomaviruses SV40 and BK virus were more frequently found in malignant adrenal tumors, whereas herpesviruses, especially Epstein-Barr virus and human cytomegalovirus, were more frequently detected in functioning benign adrenocortical tumors, often as coinfection. Moreover, tumors from patients with severe hypercortisolism frequently showed herpesvirus coinfections at high viral genome copy number. Our study suggests that the adrenal gland could be a reservoir of infection for these viruses and that hormone overproduction by the adrenal gland could represent a trigger for virus reactivation. On the other hand, these viruses could also contribute to adrenal cell proliferation and tumorigenesis.

The role of 21-hydroxylase in the pathogenesis of adrenal masses: review of the literature and focus on our own experience.

An exaggerated response of 17- hydroxyprogesterone (17-OHP) to exogenous ACTH stimulation has been found in 30 to 70% of patients with incidentally discovered adrenal tumors, supporting the concept that congenital 21- hydroxylase deficiency may be a predisposing factor for adrenocortical tumorigenesis. Decreased expression of 21-hydroxylase gene has been observed in sporadic non-functioning adrenocortical adenomas and adrenocortical carcinomas, in agreement with the reduced steroidogenic activity found in these types of tumors. Screening studies for the presence of mutations in CYP21A2 gene, encoding 21-hydroxylase, in patients with sporadic adrenocortical tumors yielded discordant results. Overall, a higher frequency of germline 21-hydroxylase mutation carriers has been found among patients with adrenal tumors, including incidentalomas, than in the general population. However, the presence of mutations did not correlate with endocrine test results and tumor mass features, suggesting that 21-hydroxylase deficiency does not represent a relevant mechanism in adrenal tumorigenesis. Mechanisms leading to reduced 21-hydroxylase expression and activity are still unknown.

Cytomegalovirus and BK-Virus co-infection of a clinically non-functioning adrenal adenoma: innocent bystanders or new pathogenetic agents?

We report a case of a 64-year-old woman who underwent left adrenalectomy with removal of a 8,5 cm clinically non-functioning adrenocortical adenoma and a 4-cm myelolipoma. Molecular testing for viral infection demonstrated the presence of cytomegalovirus (CMV) DNA sequences in the adrenal adenoma, but not in the myelolipoma (confirmed by immunohistochemistry). Moreover, the adrenal adenoma was also positive for parvovirus B19, and both adrenal tumor samples were positive for polyomavirus BK (BKV) and adenovirus DNA sequences. This is the first report of co-infection of an adrenocortical adenoma by CMV and BKV. The role of these viruses in adrenal tumorigenesis was postulated.