Marta Trevisan

Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016.

We describe the dynamics of Zika virus (ZIKV) infection in a man in his early 40s who developed fever and rash after returning from Haiti to Italy, in January 2016. Follow-up laboratory testing demonstrated detectable ZIKV RNA in plasma up to day 9 after symptom onset and in urine and saliva up to days 15 and 47, respectively. Notably, persistent shedding of ZIKV RNA was demonstrated in semen, still detectable at 181 days after onset.

Human papillomavirus genotyping by 454 next generation sequencing technology.

BACKGROUND An accurate tool for human papillomavirus (HPV) typing is important both for management of patients with HPV infection and for surveillance studies. OBJECTIVES Design and evaluation of an HPV typing method based on 454 next generation sequencing (NGS) technology. STUDY DESIGN Development of an HPV typing method based on 454 NGS of HPV L1 amplicons generated with MY09/11-based primers. Evaluation of the NGS method in control samples and in a panel of cervical cytological samples. Comparison of the NGS typing method with cycle sequencing and with the reverse hybridization-based INNO-LiPA HPV Genotyping Extra assay (LiPA). RESULTS In control samples carrying mixtures of HPV16 and HPV18 DNA, the NGS method could reliably detect genotype sequences occurring at a frequency of 1% in multiple infections with a sensitivity of 100 genome equivalents/μL. In cervical cytology samples, comparison with cycle sequencing demonstrated accuracy of HPV typing by NGS. The NGS method had however lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/11-based primers. At variance, LiPA could not detect HPV types which were present in low proportion in multiple infections (<10% of HPV reads obtained by NGS). In addition, NGS allowed identifying the presence of different variants of the same HPV type in a specimen. CONCLUSIONS NGS is a promising method for HPV typing because of its high sensitivity in multiple infection and its potential ability to detect a broad spectrum of HPV types, subtypes, and variants.

Expression of aromatase and estrogen receptors in human adrenocortical tumors

We recently demonstrated that adrenocortical carcinoma cells express aromatase and estrogen receptors (ERs) and that 17β-estradiol enhances adrenocortical cell proliferation. To provide a clue to the role of estrogens in adrenal tumorigenesis, we investigated the expression profile of genes involved in sex steroid hormone production and activity in a large series of normal and neoplastic human adrenocortical tissues. Quantitative reverse transcriptase–polymerase chain reaction, Western blotting, and immunohistochemistry showed that ERα and ERβ, androgen receptor (AR), and aromatase were expressed in the adrenal cortex and in adrenocortical tumors. ERβ was the predominant ER subtype and was mainly expressed in the zona glomerulosa and fasciculata. Western blot analysis revealed the presence of a truncated form of AR in adrenocortical tissues. With respect to the normal adrenal cortex and adrenocortical adenomas, carcinomas were characterized by significantly lower ERβ levels, ERα upregulation, and aromatase overexpression. ER expression correlated with expression of nuclear hormone receptors, suggesting they could be involved in ER modulation. In agreement with our in vitro findings, the results of this study suggest that estrogens, locally produced by aromatase, could enhance adrenocortical cell proliferation though autocrine/paracrine mechanisms. This study opens new perspectives on the potential use of antiestrogens and aromatase inhibitors as therapeutic agents against ACC.

WU and KI Polyomaviruses in the Brains of HIV-Positive Patients With and Without Progressive Multifocal Leukoencephalopathy

The polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified mainly in respiratory samples from children and immunosuppressed patients. Investigation of 54 autopsy brain tissue samples from 22 subjects demonstrated that WUPyV DNA and KIPyV DNA could be detected in 1 of 4 human immunodeficiency virus (HIV)-positive individuals with progressive multifocal leukoencephalopathy (PML) and in 3 of 10 HIV-positive individuals without PML, but not in 8 HIV-negative individuals. Viruses were localized in all regions of the central nervous system that were analyzed, that is, the cerebral hemispheres, cerebellum, pons, and medulla oblongata. No specific histopathological findings were found to be associated with the presence of WUPyV and KIPyV.

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.

Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer

Valentina Militello, Marta Trevisan, Laura Squarzon, Maria Angela Biasolo, Massimo Rugge, Carmelo Militello, Giorgio Palù* and Luisa Barzon Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova, Italy Department of Medical Diagnostic Sciences and Special Therapies, University of Padova, Padova, Italy Department of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy

Primary bilateral adrenal B-cell lymphoma associated with EBV and JCV infection

Primary lymphoma of the adrenal gland is a rare and highly aggressive disease, with only a few reports in the literature. The pathogenesis is unknown, but detection of Epstein Barr virus (EBV) genome sequences and gene expression in some cases of primary adrenal lymphomas suggested the virus might be a causative agent of the malignancy. While investigating the presence of genome sequences of oncogenic viruses in a large series of adrenal tumors, both EBV and JC polyomavirus (JCV) DNA sequences were detected in a diffuse large primary bilateral B-cell non-Hodgkin lymphoma of the adrenal gland, which was diagnosed only at postmortem examination in a 77 year-old woman with incidentally discovered adrenal masses and primary adrenal insufficiency. The presence of both EBV and JCV genome sequences suggests the relevance of EBV and JCV coinfection in the pathogenesis of this rare form of B-cell lymphoma.

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.

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study.

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.

Investigation on human adrenocortical cell response to adenovirus and adenoviral vector infection.

After systemic administration, adenoviral vectors (AdVs) are sequestered in the liver and adrenal glands. Adenoviral vector transduction has been shown to cause cytopathic effects on human hepatocytes and to induce an inflammatory response, whereas the effect of AdVs on human adrenocortical cells has never been investigated. In this study, human adrenocortical carcinoma cell lines and primary cell cultures were used to assess the effects of wild‐type adenovirus (Ad5WT) and E1/E3‐deleted AdVs on cell proliferation and steroidogenesis. Ad5WT could efficiently replicate in adrenocortical cells, leading to S phase induction, followed by cell death, whereas high titer AdVs transduction had only mild effects on adrenocortical cell proliferation, with accumulation of cells in G2/M. Both AdVs and Ad5WT induced expression of inflammatory cytokines, such as interleukin‐6 and tumor necrosis factor‐α, but, most importantly, they led to a marked and dose‐dependent increase of cortisol and other steroid hormone production and consistently modulated expression of key steroidogenic enzymes and regulators of steroidogenesis. This effect, which was already apparent at 6 h post‐infection, probably represented a response to adenoviral entry and/or early phases of infection. The result of this study contribute to the understanding of host response to adenoviral vector administration. J. Cell. Physiol. 220: 45–57, 2009.