ORIGINE E DISPERSIONE DI WNV-2 IN ITALIA DAL 2011 AL 2018: RICOSTRUZIONE FILOGENETICA E FILOGEOGRAFICA

La presente tesi di dottorato, sviluppata in collaborazione con il Policlinico San Matteo di Pavia e l\u2019Istituto Zooprofilattico della Lombardia e dell\u2019Emilia Romagna, \ue8 incentrata sulla epidemiologia del virus di West Nile (WNV), un patogeno emergente che rappresenta il flavivirus pi\uf9 diffuso a livello globale, essendo presente in tutti i continenti, con la sola esclusione dell\u2019Antartide. WNV rappresenta oggi un importante problema di salute pubblica, potendo causare patologie neurologiche potenzialmente letali soprattutto nelle fasce pi\uf9 fragili della popolazione, come gli anziani e i malati cronici. Ad oggi sono descritti 9 lineages virali, di cui l\u20191 e il 2 rappresentano i principali patogeni per l\u2019uomo. WNV-1 \ue8 diffuso in tutto il Mondo eccetto l\u2019Antartide; WNV-2, isolato inizialmente in Africa Sub-Sahariana e in Madagascar, dal 2011 \ue8 comparso in Italia e attualmente sembrerebbe essere l\u2019unico lineage circolante nel nostro territorio. In accordo con i dati epidemiologici riportati dall\u2019ECDC, 1832 casi umani di malattia neuro-invasiva sono stati registrati nel periodo 2011-2017 nei Paesi membri dell\u2019Unione Europea e nelle Nazioni limitrofe. Nel 2018, la trasmissione del virus in Italia e in diversi Paesi europei \ue8 iniziata precocemente e ha causato in una sola stagione un numero di casi nell\u2019uomo pari a quelli che si sono verificati nei 7 anni precedenti. Solo nel nostro Paese, da Giugno 2018 sono stati segnalati 577 casi umani confermati di infezione, di cui 230 si sono manifestati nella forma neuro invasiva e 42 sono deceduti. Inoltre, \ue8 stata evidenziata una elevata circolazione nelle zanzare e negli uccelli, che rappresentano rispettivamente il vettore e il serbatoio del virus. Non essendo disponibile attualmente alcun vaccino per l\u2019uso nell\u2019uomo, di fondamentale importanza risultano le attivit\ue0 di sorveglianza di questa importante arbovirosi, istituita e regolamentata dal \u201cPiano Nazionale integrato di sorveglianza e risposta ai virus West Nile e Usutu - 2019\u201d. E\u2019 in questo contesto che si inserisce il presente lavoro, finalizzato alla ricostruzione della storia epidemiologica del virus attraverso l\u2019utilizzo di approcci bioinformatici e filogenetici avanzati. Nel corso di questi tre anni di attivit\ue0 di ricerca sono stati collezionati i ceppi di West Nile circolanti in Italia nelle stagioni estivo-autunnali 2013- 2018; dopo una messa a punto dei protocolli di amplificazione dell\u2019intero genoma virale, questi ceppi sono stati caratterizzati tramite tecnologie di Next Generation Sequencing (NGS). Le sequenze ottenute sono state analizzate alla ricerca di mutanti importanti da un punto di vista epidemiologico o patogenetico, ed \ue8 stato condotto uno studio di evoluzione molecolare del genoma virale e delle forze che ne guidano l\u2019evoluzione, in relazione allo spazio e al tempo. Attraverso l\u2019applicazione di modelli filogenetici il nostro gruppo di ricerca ha cercato di ricostruire la storia di WNV-2 dal momento del suo primo isolamento ad oggi, utilizzando i genomi home-made e quelli depositati in banche dati pubbliche alla data di inizio dello studio, corredati di informazioni relative all\u2019anno e al luogo di isolamento. L\u2019utilizzo di un approccio filogeografico ci ha consentito, unicamente sulla base della conoscenza del luogo di isolamento delle sequenze incluse nell'analisi, di ricostruire la localit\ue0 pi\uf9 probabile dei nodi interni del nostro albero, e di stimare la diffusione geografica del virus, in scala di tempo reale. Allo scopo di studiare le dinamiche di propagazione virale sono stati anche utilizzati modelli filo-epidemiologici che permettono di stimare importanti parametri, quali il numero riproduttivo effettivo (Re) e i suoi cambiamenti nel tempo. Interessanti risultati, pubblicati recentemente dal nostro gruppo, sono stati ottenuti sulla circolazione di WNV-2 nel nostro Paese. La ricostruzione filogenetica ha confermato l\u2019ingresso del virus in Italia nel 2008 e ha evidenziato la contemporanea circolazione di due varianti, una delle quali sembrerebbe essersi estinta nel 2013/2014. Inoltre, l\u2019osservazione della esistenza di differenti gruppi significativi all\u2019interno della variante ancora circolante nel 2018, ha suggerito la presenza di ceppi endemici e supporta l\u2019ipotesi che il virus endemico sia mantenuto tra una stagione estiva e l\u2019altra attraverso diversi possibili meccanismi di \u201coverwintering\u201d. I metodi utilizzati di filodinamica e filogeografia sono risultati particolarmente adatti alla ricostruzione della storia di infezioni emergenti, come l\u2019arbovirosi da West Nile, potendo fornire informazioni utili alla sorveglianza. Pertanto, ci proponiamo in futuro, al fine di comprendere al meglio l\u2019origine e le modalit\ue0 di diffusione del virus, di migliorare la qualit\ue0 dei dati epidemiologici disponibili, attraverso l\u2019implementazione di metodi molecolari, per una pi\uf9 precoce individuazione della circolazione virale, e lo sviluppo di una rete di competenze e di collaborazioni con figure professionali diverse per fare chiarezza sul complesso ciclo epidemiologico del virus.The present thesis, developed in collaboration with the Policlinico San Matteo in Pavia and the Zooprophylactic Institute of Lombardia and Emilia Romagna, is focused on the epidemiology of West Nile virus (WNV), an emerging pathogen globally widespread, affecting all continents, with the only exception of Antarctica. Today WNV represents an important public health problem, being able to cause lethal neurological pathologies, especially in the most fragile sections of the population, such as the elderly and chronic patients. To date, 9 viral lineages have been described, whose lineages 1 and 2 represent the main pathogens for human beings. WNV-1 can be found all over the World except for Antarctica; WNV-2, initially isolated in Sub-Saharan Africa and Madagascar, first appeared in Italy in 2011 and it currently seems to be the only lineage circulating in our territory. In accordance with the epidemiological data recorded by the ECDC, 1832 human cases of this disease were recorded in the period 2011-2017 in several countries of European Union, closer to each other. In 2018, the transmission of the virus in Italy and in other European countries started earlier and in just one season the number of cases in human beings equaled those that occurred in the previous 7 years. In our country alone, since June 2018, 577 infection cases in humans had been reported, with 230 neuro-invasive events and a total of 42 deaths. Furthermore, there was evidence of high viral circulation in mosquitoes and birds that represent respectively the vector and the virus reservoir. Since no vaccine is currently available for use in humans, surveillance activities, established and regulated by the "Integrated National Surveillance and Response Plan to West Nile and Usutu Virus - 2019", are of main importance. It is in this context that this work is inserted, aiming at reconstructing the epidemiological history of the virus through the use of advanced bioinformatic and phylogenetic approaches. During these three years of research, the West Nile strains circulating in Italy in the summer-autumn seasons between 2013 and 2018 were collected; after the optimization of the amplification protocols of the entire viral genome, these strains were characterized by Next Generation Sequencing (NGS) technologies. The sequences obtained were analyzed in order to search important mutants from an epidemiological or pathogenetic point of view; moreover a study of molecular evolution of the viral genome and the forces that guide its evolution, has been done in relation to space and time. Through the application of phylogenetic models our research group tried to reconstruct the history of WNV-2 from the time of its first isolation to the present, using the home-made genomes and those deposited in public databases at the beginning of the study, accompanied by information relating to the year and place of isolation. The use of a phylogeographic approach allowed us, on the basis of the knowledge of the place of isolation of the sequences included in the analysis, to reconstruct the most probable location of the internal nodes of our tree, and to estimate the geographical spread of the virus, in real time scale. In order to study the dynamics of viral propagation, phylo-epidemiological models have also been used which has allowed us to estimate important parameters such as the actual reproductive number (Re) and its changes over time. Interesting results, recently published by our group, have been obtained on the circulation of WNV-2 in our country. The phylogenetic reconstruction confirmed the entry of the virus in Italy in 2008 and highlighted the simultaneous circulation of two variants, one of which apparently became extinct in 2013\u20132014 Furthermore, the observation of the existence of different significant groups within the variant still circulating in 2018, suggested the presence of endemic strains and supports the hypothesis that the endemic virus is maintained alive between one summer season and another through different possible overwintering mechanisms. The methods used in philodynamics and phylogeography are particularly suitable for the reconstruction of the history of emerging infections, such as arbovirosis like West Nile virus, providing useful information for surveillance. Therefore, in the future, in order to better understand the origin and the spread of the virus, we intend to improve the quality of epidemiological data available through the implementation of molecular methods, for an earlier identification of the viral circulation, and the development of a network of skills and collaborations with different professional figures. In this way we will able to clarify the complex epidemiological cycle of the virus

Phylogenetic analysis of human immunodeficiency virus type 2 group B

Context: Human immunodeficiency virus type 2 (HIV-2) infections are mainly restricted to West Africa; however, in the recent years, the prevalence of HIV-2 is a growing concern in some European countries and the Southwestern region of India. Despite the presence of different HIV-2 groups, only A and B Groups have established human-to-human transmission chains. Aims: This work aimed to evaluate the phylogeographic inference of HIV-2 Group B worldwide to estimate their data of origin and the population dynamics. Materials and Methods: The evolutionary rates, the demographic history for HIV-2 Group B dataset, and the phylogeographic analysis were estimated using a Bayesian approach. The viral gene flow analysis was used to count viral gene out/in flow among different locations. Results: The root of the Bayesian maximum clade credibility tree of HIV-2 Group B dated back to 1957. The demographic history of HIV-2 Group B showed that the epidemic remained constant up to 1970 when started an exponential growth. From 1985 to early 2000s, the epidemic reached a plateau, and then it was characterized by two bottlenecks and a new plateau at the end of 2000s. Phylogeographic reconstruction showed that the most probable location for the root of the tree was Ghana. Regarding the viral gene flow of HIV-2 Group B, the only observed viral gene flow was from Africa to France, Belgium, and Luxembourg. Conclusions: The study gives insights into the origin, history, and phylogeography of HIV-2 Group B epidemic. The growing number of infections of HIV-2 worldwide indicates the need for strengthening surveillance

Mir-21-Sox2 Axis Delineates Glioblastoma Subtypes with Prognostic Impact.

UNLABELLED: Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21-Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21-Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21-Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes. SIGNIFICANCE STATEMENT: Molecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21-Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development

Bayesian reconstruction of the evolutionary history and cross-species transition of variola virus and orthopoxviruses

Variola virus (VARV), the causative agent of smallpox, is an exclusively human virus belonging to the genus Orthopoxvirus, which includes many other viral species covering a wide range of mammal hosts, such as vaccinia, cowpox, camelpox, taterapox, ectromelia and monkeypox virus. The tempo and mode of evolution of Orthopoxviruses were reconstructed using a Bayesian phylodynamic framework by analysing 80 hemagglutinin sequences retrieved from public databases. Bayesian phylogeography was used to estimate their putative ancestral hosts. In order to estimate the substitution rate, the tree including all of the available Orthopoxviruses was calibrated using historical references dating the South American variola minor clade (alastrim) to between the XVI and XIX century. The mean substitution rate determined by the analysis was 6.5\u2009 7\u200910-6substitutions/site/year. Based on this evolutionary estimate, the time of the most recent common ancestor of the genus Orthopoxvirus was placed at about 10,000 years before the present. Cowpox virus was the species closest to the root of the phylogenetic tree. The root of VARV circulating in the XX century was estimated to be about 700 years ago, corresponding to about 1300 AD. The divergence between West African and South American VARV went back about 500 years ago (falling approximately in the XVI century). A rodent species is the most probable ancestral host from which the ancestors of all the known Orthopoxviruses were transmitted to the other mammal host species, and each of these species represented a dead-end for each new poxvirus species, without any further inter-specific spread

Evolutionary Dynamics of the Lineage 2 West Nile Virus That Caused the Largest European Epidemic: Italy 2011-2018

Lineage 2 West Nile virus (WNV) caused a vast epidemic in Europe in 2018, with the highest incidence being recorded in Italy. To reconstruct the evolutionary dynamics and epidemiological history of the virus in Italy, 53 envelope gene and 26 complete genome sequences obtained from human and animal samples were characterised by means of next-generation sequencing. Phylogenetic analysis revealed two Italian strains originating between 2010 and 2012: clade A, which apparently became extinct in 2013-2014, and clade B, which was responsible for the 2018 epidemic. The mean genetic distances in clade B increased over time and with the distance between sampling locations. Bayesian birth-death and coalescent skyline plots of the clade B showed that the effective number of infections and the effective reproduction number (Re) increased between 2015 and 2018. Our data suggest that WNV-2 entered Italy in 2011 as a result of one or a few penetration events. Clade B differentiated mainly as a result of genetic drift and purifying selection, leading to the appearance of multiple locally circulating sub-clades for different times. Phylodynamic analysis showed a current expansion of the infection among reservoir birds and/or vectors

Distribution of Marburg virus in Africa: An evolutionary approach

The aim of this study was to investigate the origin and geographical dispersion of Marburg virus, the first member of the Filoviridae family to be discovered. Seventy-three complete genome sequences of Marburg virus isolated from animals and humans were retrieved from public databases and analysed using a Bayesian phylogeographical framework. The phylogenetic tree of the Marburg virus data set showed two significant evolutionary lineages: Ravn virus (RAVV) and Marburg virus (MARV). MARV divided into two main clades; clade A included isolates from Uganda (five from the European epidemic in 1967), Kenya (1980) and Angola (from the epidemic of 2004-2005); clade B included most of the isolates obtained during the 1999-2000 epidemic in the Democratic Republic of the Congo (DRC) and a group of Ugandan isolates obtained in 2007-2009. The estimated mean evolutionary rate of the whole genome was 3.3 7 10-4 substitutions/site/year (credibility interval 2.0-4.8). The MARV strain had a mean root time of the most recent common ancestor of 177.9 years ago (YA) (95% highest posterior density 87-284), thus indicating that it probably originated in the mid-XIX century, whereas the RAVV strain had a later origin dating back to a mean 33.8 YA. The most probable location of the MARV ancestor was Uganda (state posterior probability, spp = 0.41), whereas that of the RAVV ancestor was Kenya (spp = 0.71). There were significant migration rates from Uganda to the DRC (Bayes Factor, BF = 42.0) and in the opposite direction (BF = 5.7). Our data suggest that Uganda may have been the cradle of Marburg virus in Africa

Sofosbuvir selects for drug-resistant amino acid variants in the zika virus RNA-dependent RNA-polymerase complex in vitro

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure signifi-cantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half-maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofos-buvir as a possible lead for anti-ZIKV drug development

Ricostruzione filogenetica dell’origine e dispersione geografica di WNV2 in Italia

L\u2019infezione dal virus West Nile (WNV) rappresenta, a tutt\u2019oggi, un problema rilevante di Sanit\ue0 Pubblica. In particolare, il lineage 2 negli ultimi anni ha causato estese epidemie in Europa centrale diffondendosi in Italia ad aree geografiche fino ad ora indenni. Le tecniche di analisi filogenetica avanzate (filodinamica e filogeografia) applicate a sequenze/genomi virali, consentono la ricostruzione spazio-temporale dell\u2019origine e della diffusione dei virus, dando un notevole contributo alla sorveglianza delle infezioni emergenti. Dalla analisi filogenetica e filogeografica condotta su 91 genomi completi di WNV-2 isolati in varie regioni del Mondo, di cui 5 isolati in Italia nel 2015 e caratterizzati nel nostro laboratorio, \ue8 emerso che il virus \ue8 probabilmente originato in Sud Africa verso la fine del 1700. Dalla stessa analisi \ue8 emerso che WNV-2 \ue8 stato introdotto in Ungheria nel 2002 e da questa si \ue8 diffuso da un lato in Grecia, dall\u2019altro in Austria, e da qui in Italia, il cui ingresso risale all\u2019anno 2007 dalle regioni del Nord-Est. L\u2019analisi filogeografica del clade italiano suggerisce la circolazione in Italia di due differenti ceppi. Uno sarebbe originato direttamente nelle regioni del Nord-Est e si sarebbe diffuso intorno al 2012 in direzione Ovest. L\u2019altro ceppo si sarebbe, invece, differenziato nelle regioni Nord Occidentali nel 2012 e si starebbe differenziando in parte anch\u2019esso ad Ovest, verso il Piemonte ed in parte verso Est, ancora nelle direzioni delle regioni Centro settentrionali, in cui, quindi, troviamo ambedue i ceppi. Le ragioni di questi flussi virali sono ancora da chiarire; tuttavia \ue8 possibile ipotizzare che dopo un singolo ingresso del virus avvenuto dall\u2019Europa Centro-orientale attraverso le regioni del Nord Est, il virus si sia amplificato e differenziato in Italia e si stia diffondendo attraverso serbatoi e vettori autoctoni seguendo il tragitto del Po. Questo escluderebbe quindi la necessit\ue0 di molteplici reintroduzioni virali, sottolineando l\u2019importanza della trasmissione verticale del virus tra i vettori. Questi dati mostrano una complessa epidemiologia di WNV in Italia e sottolineano l\u2019importanza di una attenta sorveglianza della sua circolazione, che \ue8 resa possibile dalla integrazione fra diverse competenze in ambito entomologico, veterinario e umano

Analisi filogenetica spazio-temporale di virus della Border disease nel camoscio

Il virus della Border disease (BDV) appartiene al genere Pestivirus ed \ue8 ampiamente diffuso nell\u2019ovino, che rappresenta l\u2019ospite principale. Infezioni sono state segnalate anche in altri ruminanti domestici e selvatici, fra cui il camoscio. Gli stipiti di BDV segregano in almeno sette gruppi filogenetici diffusi con diversa frequenza a livello mondiale. Dal 2001 numerosi focolai associati a BDV-4 hanno determinato un forte impatto demografico nel camoscio dei Pirenei (Rupicapra pyrenaica pyrenaica). Al fine di ricostruire origine e diffusione spazio-temporale di BDV nel camoscio, \ue8 stata condotta un\u2019analisi filogenetica di 95 sequenze (5\u2019UTR) di stipiti virali isolati dal camoscio pirenaico, camoscio alpino (R. rupicapra rupicapra) e da ungulati domestici, includendo sia sequenze nuove che disponibili in banca dati. Gli alberi filogenetici sono stati costruiti mediante approccio bayesiano con tecniche Markov Chain Monte Carlo, utilizzando il programma BEAST v1.7.4. L\u2019unica sequenza disponibile di BDV nel camoscio alpino (Alpi francesi) si \ue8 confermata appartenere a BDV-6 ovino, mentre le sequenze del camoscio pirenaico si raggruppano in un unico cluster altamente significativo, che origina da BDV-4 ovino. L\u2019introduzione di BDV-4 nella popolazione di camoscio pirenaico \ue8 risultata risalire indicativamente al 1989 nei Pirenei Orientali. L\u2019analisi filogeografica ha permesso di ricostruire gli eventi di infezione fra le diverse aree interessate dai focolai di BDV del camoscio pirenaico, evidenziando due flussi di diffusione principali, rispettivamente verso i Pirenei Centrali e Occidentali. In conclusione, i dati ad oggi disponibili evidenziano due quadri epidemiologici differenti nel camoscio alpino rispetto a quello pirenaico. Il camoscio alpino presenta una frequenza di infezione sporadica associata a stipiti ovini ed in assenza di manifestazioni conclamate, contrariamente all\u2019ampia diffusione ed elevata mortalit\ue0 osservata nei Pirenei. BDV-4 nel camoscio pirenaico \ue8 risultato di recente introduzione, precedendo infatti di un decennio il primo focolaio del 2001. In seguito flussi di dispersione di BDV-4 hanno permesso la diffusione ed il mantenimento dell\u2019infezione, che attualmente risulta endemica in alcune aree dei Pirenei Occidentali