Publications by authors named "Arianna Calistri"

56 Publications

Persistent Herpes Simplex Virus Type 1 Infection of Enteric Neurons Triggers CD8 T Cell Response and Gastrointestinal Neuromuscular Dysfunction.

Front Cell Infect Microbiol 2021 18;11:615350. Epub 2021 May 18.

Department of Molecular Medicine, University of Padova, Padova, Italy.

Behind the central nervous system, neurotropic viruses can reach and persist even in the enteric nervous system (ENS), the neuronal network embedded in the gut wall. We recently reported that immediately following orogastric (OG) administration, Herpes simplex virus (HSV)-1 infects murine enteric neurons and recruits mononuclear cells in the myenteric plexus. In the current work, we took those findings a step forward by investigating the persistence of HSV-1 in the ENS and the local adaptive immune responses against HSV-1 that might contribute to neuronal damage in an animal model. Our study demonstrated specific viral RNA transcripts and proteins in the longitudinal muscle layer containing the myenteric plexus (LMMP) up to 10 weeks post HSV-1 infection. CD3CD8INFγ lymphocytes skewed towards HSV-1 antigens infiltrated the myenteric ganglia starting from the 6 week of infection and persist up to 10 weeks post-OG HSV-1 inoculation. CD3CD8 cells isolated from the LMMP of the infected mice recognized HSV-1 antigens expressed by infected enteric neurons. , infiltrating activated lymphocytes were involved in controlling viral replication and intestinal neuromuscular dysfunction. Indeed, by depleting the CD8 cells by administering specific monoclonal antibody we observed a partial amelioration of intestinal dysmotility in HSV-1 infected mice but increased expression of viral genes. Our findings demonstrate that HSV-1 persistently infects enteric neurons that in turn express viral antigens, leading them to recruit activated CD3CD8 lymphocytes. The T-cell responses toward HSV-1 antigens persistently expressed in enteric neurons can alter the integrity of the ENS predisposing to neuromuscular dysfunction.
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http://dx.doi.org/10.3389/fcimb.2021.615350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169984PMC
July 2021

Organoid modeling of Zika and herpes simplex virus 1 infections reveals virus-specific responses leading to microcephaly.

Cell Stem Cell 2021 Apr 7. Epub 2021 Apr 7.

Institute of Molecular Biotechnology (IMBA), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna 1030, Austria; Medical University of Vienna, Vienna 1030, Austria. Electronic address:

Viral infection in early pregnancy is a major cause of microcephaly. However, how distinct viruses impair human brain development remains poorly understood. Here we use human brain organoids to study the mechanisms underlying microcephaly caused by Zika virus (ZIKV) and herpes simplex virus (HSV-1). We find that both viruses efficiently replicate in brain organoids and attenuate their growth by causing cell death. However, transcriptional profiling reveals that ZIKV and HSV-1 elicit distinct cellular responses and that HSV-1 uniquely impairs neuroepithelial identity. Furthermore, we demonstrate that, although both viruses fail to potently induce the type I interferon system, the organoid defects caused by their infection can be rescued by distinct type I interferons. These phenotypes are not seen in 2D cultures, highlighting the superiority of brain organoids in modeling viral infections. These results uncover virus-specific mechanisms and complex cellular immune defenses associated with virus-induced microcephaly.
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http://dx.doi.org/10.1016/j.stem.2021.03.004DOI Listing
April 2021

TaSCA, an Agile Survey on Chemosensory Impairments for Self-Monitoring of COVID-19 Patients: A Pilot Study.

Front Neurol 2021 24;12:633574. Epub 2021 Feb 24.

Padova Neuroscience Center, Padua, Italy.

During the COVID-19 pandemic, smell and taste disorders emerged as key non-respiratory symptoms. Due to widespread presence of the disease and to difficult objective testing of positive persons, the use of short surveys became mandatory. Most of the existing resources are focused on smell, very few on taste or trigeminal chemosensation called chemesthesis. However, it is possible that the three submodalities are affected differently by COVID-19. We prepared a short survey (TaSCA) that can be administered at the telephone or through online resources to explore chemosensation. It is composed of 11 items on olfaction, taste, and chemesthesis, in order to discriminate the three modalities. We avoided abstract terms, and the use of semiquantitative scales because older patients may be less engaged. Statistical handling included descriptive statistics, Pearson's chi-squared test and cluster analysis. The survey was completed by 83 persons (60 females and 23 males), which reported diagnosis of COVID-19 by clinical ( = 7) or molecular ( = 18) means, the others being non-COVID subjects. Cluster analysis depicted the existence of two groups, one containing mostly asymptomatic and one mostly symptomatic subjects. All swab-positive persons fell within this second group. Only one item, related to trigeminal temperature perception, did not discriminate between the two groups. These preliminary results indicate that TaSCA may be used to easily track chemosensory symptoms related to COVID-19 in an agile way, giving a picture of three different chemosensory modalities.
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http://dx.doi.org/10.3389/fneur.2021.633574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943440PMC
February 2021

Why Cells and Viruses Cannot Survive without an ESCRT.

Cells 2021 Feb 24;10(3). Epub 2021 Feb 24.

Department of Molecular Medicine, University of Padua, 35121 Padua, Italy.

Intracellular organelles enwrapped in membranes along with a complex network of vesicles trafficking in, out and inside the cellular environment are one of the main features of eukaryotic cells. Given their central role in cell life, compartmentalization and mechanisms allowing their maintenance despite continuous crosstalk among different organelles have been deeply investigated over the past years. Here, we review the multiple functions exerted by the endosomal sorting complex required for transport (ESCRT) machinery in driving membrane remodeling and fission, as well as in repairing physiological and pathological membrane damages. In this way, ESCRT machinery enables different fundamental cellular processes, such as cell cytokinesis, biogenesis of organelles and vesicles, maintenance of nuclear-cytoplasmic compartmentalization, endolysosomal activity. Furthermore, we discuss some examples of how viruses, as obligate intracellular parasites, have evolved to hijack the ESCRT machinery or part of it to execute/optimize their replication cycle/infection. A special emphasis is given to the herpes simplex virus type 1 (HSV-1) interaction with the ESCRT proteins, considering the peculiarities of this interplay and the need for HSV-1 to cross both the nuclear-cytoplasmic and the cytoplasmic-extracellular environment compartmentalization to egress from infected cells.
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http://dx.doi.org/10.3390/cells10030483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995964PMC
February 2021

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line.

Viruses 2020 09 18;12(9). Epub 2020 Sep 18.

Department of Molecular Medicine, University of Padua, 35121 Padua, Italy.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.
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http://dx.doi.org/10.3390/v12091044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551853PMC
September 2020

Alix-Mediated Rescue of Feline Immunodeficiency Virus Budding Differs from That Observed with Human Immunodeficiency Virus.

J Virol 2020 05 18;94(11). Epub 2020 May 18.

Department of Molecular Medicine, University of Padua, Padua, Italy

The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the CS double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding. FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.
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http://dx.doi.org/10.1128/JVI.02019-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269445PMC
May 2020

Coronaviruses: a paradigm of new emerging zoonotic diseases.

Pathog Dis 2019 12;77(9)

Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121, Padova, Italy.

A novel type of coronavirus (2019-nCoV) infecting humans appeared in Wuhan, China, at the end of December 2019. Since the identification of the outbreak the infection quickly spread involving in one month more than 31,000 confirmed cases with 638 death. Molecular analysis suggest that 2019-nCoV could be originated from bats after passaging in intermediate hosts, highlighting the high zoonotic potential of coronaviruses.
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http://dx.doi.org/10.1093/femspd/ftaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108526PMC
December 2019

A clinical trial investigating biodistribution and shedding of an oncolytic virus.

EBioMedicine 2019 09 13;47:4-5. Epub 2019 Aug 13.

Department of Molecular Medicine, University of Padua, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2019.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796555PMC
September 2019

Antiviral treatment and virological monitoring of oseltamivir-resistant influenza virus A(H1N1)pdm09 in a patient with chronic B lymphocytic leukemia.

J Infect Chemother 2019 Jul 20;25(7):543-546. Epub 2019 Apr 20.

Department of Molecular Medicine, University of Padova, Padova, Italy; Microbiology and Virology Unit, Azienda Ospedaliera di Padova, Padova, Italy. Electronic address:

We report the virological monitoring and the antiviral therapy adopted for the treatment of a patient affected by chronic B lymphocytic leukemia, who experienced a severe pneumonia with long-term shedding of influenza virus A(H1N1)pdm09, characterized by an early development of oseltamivir resistance.
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http://dx.doi.org/10.1016/j.jiac.2018.11.008DOI Listing
July 2019

Lentiviral Vectors as Tools for the Study and Treatment of Glioblastoma.

Cancers (Basel) 2019 Mar 24;11(3). Epub 2019 Mar 24.

Department of Molecular Medicine, University of Padova, 35121 Padova, Italy.

Glioblastoma (GBM) has the worst prognosis among brain tumors, hence basic biology, preclinical, and clinical studies are necessary to design effective strategies to defeat this disease. Gene transfer vectors derived from the most-studied lentivirus-the Human Immunodeficiency Virus type 1-have wide application in dissecting GBM specific features to identify potential therapeutic targets. Last-generation lentiviruses (LV), highly improved in safety profile and gene transfer capacity, are also largely employed as delivery systems of therapeutic molecules to be employed in gene therapy (GT) approaches. LV were initially used in GT protocols aimed at the expression of suicide factors to induce GBM cell death. Subsequently, LV were adopted to either express small noncoding RNAs to affect different aspects of GBM biology or to overcome the resistance to both chemo- and radiotherapy that easily develop in this tumor after initial therapy. Newer frontiers include adoption of LV for engineering T cells to express chimeric antigen receptors recognizing specific GBM antigens, or for transducing specific cell types that, due to their biological properties, can function as carriers of therapeutic molecules to the cancer mass. Finally, LV allow the setting up of improved animal models crucial for the validation of GBM specific therapies.
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http://dx.doi.org/10.3390/cancers11030417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468594PMC
March 2019

Ebola Virus Entry: From Molecular Characterization to Drug Discovery.

Viruses 2019 03 19;11(3). Epub 2019 Mar 19.

Department of Molecular Medicine, University of Padova, IT-35121 Padova, Italy.

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the family. The recent large outbreak of EVD in Western Africa (2013⁻2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle , allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.
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http://dx.doi.org/10.3390/v11030274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466262PMC
March 2019

Perspectives on immunotherapy via oncolytic viruses.

Infect Agent Cancer 2019 11;14. Epub 2019 Feb 11.

Department of Molecular Medicine, University of Padua, Via A. Gabelli, 63, 35121 Padua, Italy.

Background: With few exceptions, current chemotherapy and radiotherapy protocols only obtain a slightly prolonged survival with severe adverse effects in patients with advanced solid tumors. In particular, most solid malignancies not amenable to radical surgery still carry a dismal prognosis, which unfortunately is also the case for relapsing disease after surgery. Even though targeted therapies obtained good results, clinical experience showed that tumors eventually develop resistance. On the other hand, earlier attempts of cancer immunotherapy failed to show consistent efficacy. More recently, a deeper knowledge of immunosuppression in the tumor microenvironment (TME) allowed the development of effective drugs: in particular, monoclonal antibodies targeting the so-called immune checkpoint molecules yielded striking and lasting effects in some tumors. Unfortunately, these monoclonal antibodies are not effective in a majority of patients and are ineffective in several solid malignancies. Furthermore, due to their mechanism of action, checkpoint inhibitors often elicit autoimmune-like disease.

Main Body: The use of viruses as oncolytic agents (OVs) was considered in the past, while only recently OVs revealed a connection with immunotherapy. However, their antitumoral potential has remained largely unexplored, due to safety concerns and some limitations in the techniques to manipulate viruses. OV research was recently revived by a better knowledge of viral/cancer biology and advances in the methodologies to delete virulence/immune-escape related genes from even complex viral genomes or "to arm" OVs with appropriate transgenes. Recently, the first oncolytic virus, the HSV-1 based Talimogene Laherparepvec (T-VEC), was approved for the treatment of non-resectable melanoma in USA and Europe.

Conclusion: OVs have the potential to become powerful agents of cancer immune and gene therapy. Indeed, in addition to their selective killing activity, they can act as versatile gene expression platforms for the delivery of therapeutic genes. This is particularly true for viruses with a large DNA genome, that can be manipulated to address the multiple immunosuppressive features of the TME. This review will focus on the open issues, on the most promising lines of research in the OV field and, more in general, on how OVs could be improved to achieve real clinical breakthroughs in cancers that are usually difficult to treat by immunotherapy.
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http://dx.doi.org/10.1186/s13027-018-0218-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371415PMC
February 2019

Herpes Simplex Virus Type 1 Engages Toll Like Receptor 2 to Recruit Macrophages During Infection of Enteric Neurons.

Front Microbiol 2018 11;9:2148. Epub 2018 Sep 11.

Department of Molecular Medicine, University of Padua, Padua, Italy.

virus type 1 (HSV-1) is a widespread neurotropic pathogen responsible for a range of clinical manifestations. Inflammatory cell infiltrate is a common feature of HSV-1 infections and has been implicated in neurodegeneration. Therefore, viral recognition by innate immune receptors (i.e., TLR2) and the subsequent inflammatory response are now deemed key players in HSV-1 pathogenesis. In this study we infected with HSV-1 the enteric nervous system (ENS) of wild-type (WT) and TLR2 knock-out (TLR2) mice to investigate whether and how TLR2 participates in HSV-1 induced neuromuscular dysfunction. Our findings demonstrated viral specific transcripts suggestive of abortive replication in the ENS of both WT and TLR2 mice. Moreover, HSV-1 triggered TLR2-MyD88 depend signaling in myenteric neurons and induced structural and functional alterations of the ENS. Gastrointestinal dysmotility was, however, less pronounced in TLR2 as compared with WT mice. Interesting, HSV-1 caused up-regulation of monocyte chemoattractant protein-1 (CCL2) and recruitment of CD11b macrophages in the myenteric ganglia of WT but not TLR2 mice. At the opposite, the myenteric plexuses of TLR2 mice were surrounded by a dense infiltration of HSV-1 reactive CD3CD8INFγ lymphocytes. Indeed, depletion CD3CD8 cells by means of administration of anti-CD8 monoclonal antibody reduced neuromuscular dysfunction in TLR2 mice infected with HSV-1. During HSV-1 infection, the engagement of TLR2 mediates production of CCL2 in infected neurons and coordinates macrophage recruitment. Bearing in mind these observations, blockage of TLR2 signaling could provide novel therapeutic strategies to support protective and specific T-cell responses and to improve neuromuscular dysfunction in pathogen-mediated alterations of the ENS.
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http://dx.doi.org/10.3389/fmicb.2018.02148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141724PMC
September 2018

A first molecular characterization of Listeria monocytogenes isolates circulating in humans from 2009 to 2014 in the Italian Veneto region.

New Microbiol 2018 07 20;41(3):232-234. Epub 2018 Jul 20.

Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121 - Padova, Italy.

Listeriosis is a disease usually associated with the consumption of low-processed ready-to-eat food products contaminated by Listeria monocytogenes. In Italy, listeriosis has an incidence of 0.19-0.27 cases per 100000 persons. Since detailed information concerning the molecular characterization of listeriosis in the Italian Veneto region is currently lacking, we analyzed 36 L. monocytogenes clinical isolates collected between 2009 and 2014. Results show that the serotype 1/2a was the most represented among the tested samples. No antimicrobial resistance was detected in selected isolates representing the main pulsotypes.
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July 2018

Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment.

Front Cell Infect Microbiol 2018 15;8:74. Epub 2018 Mar 15.

Department of Molecular Medicine, University of Padova, Padova, Italy.

Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection and allow recognition of an original pathophysiologic mechanism underlying gastrointestinal diseases as well as identification of novel therapeutic targets.
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http://dx.doi.org/10.3389/fcimb.2018.00074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862801PMC
March 2019

Amiodarone affects Ebola virus binding and entry into target cells.

New Microbiol 2018 Apr 2;41(2):162-164. Epub 2018 Mar 2.

Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Several research studies have aimed to identify effective antiviral agents. Amiodarone, a drug used for the treatment of arrhythmias, has been shown to inhibit filovirus infection in vitro by acting at the early step of the viral replication cycle. Here we demonstrate that amiodarone reduces virus binding to target cells and slows down the progression of the viral particles along the endocytic pathway. Overall our data support the notion that amiodarone interferes with Ebola virus infection by affecting cellular pathways/ targets involved in the viral entry process.
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April 2018

Antiviral activity of cationic amphiphilic drugs.

Expert Rev Anti Infect Ther 2017 05 20;15(5):483-492. Epub 2017 Mar 20.

a Department of Molecular Medicine , University of Padova , Padova , Italy.

Introduction: Emerging and reemerging viral infections represent a major concern for human and veterinary public health and there is an urgent need for the development of broad-spectrum antivirals. Areas covered: A recent strategy in antiviral research is based on the identification of molecules targeting host functions required for infection of multiple viruses. A number of FDA-approved drugs used to treat several human diseases are cationic amphiphilic drugs (CADs) that have the ability to accumulate inside cells affecting several structures/functions hijacked by viruses during infection. In this review we summarized the CADs' chemical properties and effects on the cells and reported the main FDA-approved CADs that have been identified so far as potential antivirals in drug repurposing studies. Expert commentary: Although there have been concerns regarding the efficacy and the possible side effects of the off-label use of CADs as antivirals, they seem to represent a promising starting point for the development of broad-spectrum antiviral strategies. Further knowledge about their mechanism of action is required to improve their antiviral activity and to reduce the risk of side effects.
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http://dx.doi.org/10.1080/14787210.2017.1305888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103695PMC
May 2017

Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

Mol Ther Nucleic Acids 2016 Apr 19;5:e312. Epub 2016 Apr 19.

Department of Molecular Medicine, University of Padova, Padova, Italy.

Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.
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http://dx.doi.org/10.1038/mtna.2016.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014525PMC
April 2016

Dissecting the Role of K61/K59 Residue in VPS4 Functions.

Protein Pept Lett 2016 ;23(6):518-24

Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, Padova, Italy.

ESCRTs (Endosomal Sorting Complexes Required for Transport) are required for the formation of the intraluminal vesicles in the multivesicular bodies and are involved in other topologically similar processes such as cytokinesis, nuclear envelope sealing and viral egress. The final complex ESCRT-III is disassembled by the recruitment and activation of the AAA-ATPase VPS4 to the endosomal membranes. This recruitment is due to the binding of VPS4 N-terminal MIT with MIM1 and MIM2 domains present in the CHMPs proteins. By analyzing different cellular membrane remodeling events in which VPS4 is involved, here we provide evidence that the K61 residue, mapping within the MIT domain of VPS4B (K59 in VPS4A), is involved in VPS4 functioning. Posttranslational modifications of this residue might modulate MIT-MIM2 binding affinity and, as a consequence, VPS4 functions.
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http://dx.doi.org/10.2174/0929866523666160331143850DOI Listing
March 2017

Small RNAs targeting the 5' end of the viral polymerase gene segments specifically interfere with influenza type A virus replication.

J Biotechnol 2015 Sep 16;210:85-90. Epub 2015 Jun 16.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. Electronic address:

Human and avian influenza A viruses, associated with seasonal epidemics and occasionally with pandemics, have a high impact on public health. The development of new antivirals to counteract the emergence of drug resistant influenza virus variants is a main concern. The aim of this study was to develop systems for the efficient and stable expression of small therapeutic RNAs into influenza virus infected cells in order to get further insights on the efficacy of nucleic acid-based antiviral strategies. To this end, lentiviral vectors expressing either microRNAs or antisense-RNAs targeting the 5' end of the PA, PB1 and PB2 influenza virus genomic sequences were generated. Derivative recombinant lentiviral particles were employed to transduce the influenza virus highly susceptible human alveolar basal epithelial A549 cells. The expression of both RNA molecules led to a reduction up to 3 logs of the viral titer when transduced A549 cells were challenged with different human and avian subtypes of influenza type A virus. Importantly, no inhibition of influenza type B virus was observed. Overall our data support the development of nucleic acid-based antiviral strategies to control human and avian influenza A virus infection.
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http://dx.doi.org/10.1016/j.jbiotec.2015.06.391DOI Listing
September 2015

Amiodarone and metabolite MDEA inhibit Ebola virus infection by interfering with the viral entry process.

Pathog Dis 2015 Jul 30;73(5). Epub 2015 Apr 30.

Department of Microbiology, The Public Health Agency of Sweden, Solna 171 82, Sweden Department for Laboratory Medicine, Karolinska Institute, Huddinge/Stockholm 141 83, Sweden National Veterinary Institute, Uppsala 751 89, Sweden.

Ebola virus disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work, we investigated how amiodarone interferes with Ebola virus infection. Wild-type Sudan ebolavirus and recombinant vesicular stomatitis virus, pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate that the antiviral activity is correlated with drug ability to accumulate into and interfere with the endocytic pathway. Considering that it is well tolerated, especially in the acute setting, amiodarone appears to deserve consideration for clinical use in EVD.
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http://dx.doi.org/10.1093/femspd/ftv032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108539PMC
July 2015

The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex: Implications for the antiviral activity of a G-quadruplex ligand.

Antiviral Res 2015 Jun 3;118:123-31. Epub 2015 Apr 3.

Department of Molecular Medicine, University of Padua, via Gabelli, 63, 35121 Padua, Italy. Electronic address:

Guanine-rich nucleic acids can fold into G-quadruplexes, secondary structures implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. The remarkably high guanine content of the Herpes Simplex Virus-1 (HSV-1) genome prompted us to investigate both the presence of G-quadruplex forming sequences in the viral genome and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with a novel mechanism of action. Using biophysical, molecular biology and antiviral assays, we showed that the HSV-1 genome displays multiple clusters of repeated sequences that form very stable G-quadruplexes. These sequences are mainly located in the inverted repeats of the HSV-1 genome. Treatment of HSV-1 infected cells with the G-quadruplex ligand BRACO-19 induced inhibition of virus production. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. This work, presents the first evidence of extended G-quadruplex sites in key regions of the HSV-1 genome, indicates the possibility to block viral DNA replication by G-quadruplex-ligand and therefore provides a proof of concept for the use of G-quadruplex ligands as new anti-herpetic therapeutic options.
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http://dx.doi.org/10.1016/j.antiviral.2015.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113899PMC
June 2015

Editorial commentary: Unbiased next-generation sequencing and new pathogen discovery: undeniable advantages and still-existing drawbacks.

Clin Infect Dis 2015 Mar 7;60(6):889-91. Epub 2015 Jan 7.

Department of Molecular Medicine, University of Padova, Italy.

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http://dx.doi.org/10.1093/cid/ciu913DOI Listing
March 2015

The ubiquitin-conjugating system: multiple roles in viral replication and infection.

Cells 2014 May 6;3(2):386-417. Epub 2014 May 6.

Department of Molecular Medicine, University of Padova, via Gabelli 63, Padova 35121, Italy.

Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells.
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http://dx.doi.org/10.3390/cells3020386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092849PMC
May 2014

New generation sequencing in pathogen discovery and microbial surveillance.

Expert Rev Anti Infect Ther 2013 Sep;11(9):877-9

Department of Molecular Medicine, University of Padova, Via Aristide Gabelli 63, 35121 Padova, Italy.

The annual congress of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) is recognized as the largest European congress for the presentation and discussion of the key priorities and more recent scientific developments in the fields of clinical microbiology and infection. This year, it attracted almost 10,000 participants from all over the world. Keynote lectures, symposia, meet-the-expert sessions, educational workshops, poster and oral sessions covered the diagnosis, treatment, epidemiology and prevention of infectious diseases, as well as related basic microbiology. Moreover, interactive sessions addressing specific subjects underlined the important educational aspect of the ECCMID's congress. The scientific program, abstracts, oral presentations are available at their website [101] . This meeting report is focused on one of the several challenging and one of the most transversal topics of the meeting: the application of the next-generation sequencing (NGS) to the microbial world.
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http://dx.doi.org/10.1586/14787210.2013.827883DOI Listing
September 2013

Tsg101 interacts with herpes simplex virus 1 VP1/2 and is a substrate of VP1/2 ubiquitin-specific protease domain activity.

J Virol 2013 Jan 17;87(1):692-6. Epub 2012 Oct 17.

Department of Molecular Medicine, University of Padova, Padova, Italy.

Ubiquitination/deubiquitination of key factors represent crucial steps in the biogenesis of multivesicular body (MVB) and sorting of transmembrane proteins. We and others previously demonstrated that MVB is involved in herpes simplex virus 1 (HSV-1) envelopment and budding. Here, we report that the HSV-1 large tegument protein, VP1/2, interacts with and regulates the ubiquitination of Tsg101, a cellular protein essential in MVB formation, thus identifying the first cellular substrate of a herpesviral deubiquitinating enzyme.
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http://dx.doi.org/10.1128/JVI.01969-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536428PMC
January 2013

A novel de novo missense mutation in TP63 underlying germline mosaicism in AEC syndrome: implications for recurrence risk and prenatal diagnosis.

Am J Med Genet A 2012 Aug 27;158A(8):1957-61. Epub 2012 Jun 27.

The Veneto Eye Bank Foundation, Venice, Italy.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in TP63, encoding a transcriptional factor of the p53 family. Mutations in TP63, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of mutations in individuals with AEC syndrome. Of these, ≥70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation appears de novo, it is not possible to differentiate between a sporadic mutation, or germline mosaicism in the parents. In this latter case, there is a risk of having additional affected offspring. We describe two sisters with AEC syndrome, whose parents were unaffected. Both patients carried the heterozygous c.1568T>C substitution in exon 13 of TP63, resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from parental blood cells, seminal fluid (from the father) and maternal cells (buccal, vaginal, and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells (very low grade somatic mosaicism) or, more likely, maternal gonadal mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and pre-implantation/prenatal genetic diagnosis should be offered to all couples, even when the mutation is apparently de novo.
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http://dx.doi.org/10.1002/ajmg.a.35414DOI Listing
August 2012

Feline tetherin is characterized by a short N-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein.

J Virol 2012 Jun 18;86(12):6688-700. Epub 2012 Apr 18.

Department of Molecular Medicine, University of Padua, Padua, Italy.

Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.
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http://dx.doi.org/10.1128/JVI.07037-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393548PMC
June 2012

Report of the 2011 annual meeting of the Italian Society for Virology.

J Cell Physiol 2012 Jul;227(7):2965-8

Division of Microbiology and Virology, Department of Molecular Medicine, University of Padova, Padova, Italy.

The 10th annual meeting of the Italian Society for Virology (SIV) comprised seven plenary sessions focused on: General virology and viral genetics; Virus-Host interaction and pathogenesis; Viral oncology; Emerging viruses and zoonotic, foodborne and environmental pathways of transmission; Viral immunology and vaccines; Medical virology and antiviral therapy; Viral biotechnologies and gene therapy. The meeting had an attendance of 143 virologists, about 60% were senior, and the other were young scientists. The submitted abstracts amounted to 88 and the abstracts selected for oral presentation were 41. Complete abstracts of oral and poster presentations are available at the web site www.siv-virologia.it. A summary of the plenary lectures and oral selected presentations is reported.
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http://dx.doi.org/10.1002/jcp.24055DOI Listing
July 2012
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