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Milani, Adelaide (2016) Genomic and bioinformatic approach to avian influenza virus evolution. [Tesi di dottorato]

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Abstract (inglese)

Viral zoonotic agents have a significant impact both on human and veterinary public health. Ecosystems changes, increasing urbanization and easy connection have influenced the balance between pathogen and related host species. In recent years most threatening viruses, originated from animal hosts causing emerging diseases; most of them are RNA viruses that thanks to a large population sizes, high mutation rate and short generation time allow rapid evolution, genetic variability and the selection of new variants. A constant and adequate surveillance program and the sharing of different professional expertise are necessaries to follow viral evolution and to formulate efficient public health policy (Howard and Fletcher, 2012).
Influenza A virus is considered one of the most challenging RNA viruses for its zoonotic potential role in the animal-human interface, for global health and economic impact; almost every year influenza epidemics cause morbidity and mortality in the human and is also associated with influenza virus pandemics.
Both wild and domestic birds are considered the primary natural reservoir of influenza A virus and in particular wild birds are thought to be the source of influenza A viruses in all other animals (http://www.cdc.gov/flu/about/viruses/transmission.htm). Different techniques are available to genetically characterize and study viruses in order to understand their behavior, the evolutionary dynamics, the host-virus interactions and their origin; the aim is to develop a valid support with appropriate treatments during the phases of surveillance and diagnosis of possible epidemics.
During my PhD it was used an integrated approach, both genomic and structural, to study the evolution of avian influenza A virus in particular focusing on the hemagglutinin, the major surface glycoprotein, belonging to the H5, H7 and H9 (the major "avian" subtypes responsible for human infection).
Next-generation sequencing (NGS) was used to investigate and characterize the complexity of the viral population to detect low-frequency mutations and to follow the evolution of the genetically related variants present in a viral population. To compare and inspect genetic data, phylogenetic approach has shown to be a useful tools in the analysis of viral evolution. It has been used to explain the molecular epidemiology, transmission and viral evolution. In order to obtain a more complete view of the ‘functional evolution’, phylogenetic analyses based on sequence comparison and resulting in trees, was integrated taking into account information from structural comparison. Three-dimensional structural approach have shown to be a useful tool to display similarities and to inspect motifs that cannot be discovered analyzing primary sequences alone. Indeed, in the primary sequences the introduction of a mutation does not take into account the effect on the protein folding or on the surface properties, while in the three-dimensional structures, since each mutation is able to influence the structural characteristics and interactions, is directly detectable. This approach has also brought a further contribution to the phylogenetic analysis. In particular the study has focused on the evolutionary dynamics and the adaptive strategies of avian influenza H7N1 and H7N3 subtypes that circulated in Northern Italy for similar periods of time under similar epidemiological conditions. Within and between host population dynamics of Avian HPAI H7N7 viruses, that affected Italy during 2013, were investigated using next generation technology. NGS analysis was used to characterize viral population complexity into two groups of animals challenged with the same virus H5N1 HPAIvirus but vaccinated with vaccine conferring different protection levels. An extensive comparison of structural domains and sub-regions was performed on the hemagglutinin of different subtypes of influenza A virus, with particular interest to different clades of HPAI H5N1 circulating in Egypt (where bird flu is endemic in poultry ), to investigate any domain-specific changes.
Influenza A viruses belonging to H9 subtype were inspected from a phylogenetic and a structural point of view to infer type-specific characteristic and confirm if surface properties could be associated to 'functional evolution' of viral surface determinants as seen in H5N1 subtype. This work suggests that integrating genomic, phylogenetic, and structural comparison can help in understanding the 'functional evolution' of avian influenza A virus.

Abstract (italiano)

I virus zoonotici, cioè in grado di infettare l’uomo e alcune specie animali, hanno un impatto significativo e costituiscono una costante, potenziale minaccia sia per la salute pubblica umana che per quella animale. Ecosistemi dagli equilibri modificati, una crescente urbanizzazione e connessioni facilitate hanno influenzato sempre piu' il rapporto tra patogeni e specie ospiti affini. Negli ultimi anni la fonte della maggior parte dei virus potenzialmente pericolosi e in grado di causare malattie emergenti sembra derivi da ospiti di origine animale; si tratta prevalentemente di virus a RNA che, grazie alla possibilità di moltiplicarsi in breve tempo all'interno di una popolazione ampia ed all'alto tasso di mutazione, permettono una rapida evoluzione, un'elevata variabilità genetica e la selezione di nuove varianti. Un adeguato e costante programma di sorveglianza, la condivisione di conoscenze e una collaborazione tra diverse competenze professionali sono fondamentali e necessarie per seguire l'evoluzione virale e per formulare politiche di sanità pubblica efficienti (Howard e Fletcher, 2012).
L' Influenza virus di tipo A è considerato uno dei virus a RNA più importanti, tanto per il suo potenziale ruolo zoonotico nell'interfaccia animale-umano, quanto per la salute globale e l'impatto economico. Quasi ogni anno epidemie di influenza provocano morbilità e mortalità nell'uomo e talvolta gli stessi virus possono essere associati a pandemie.
Il serbatoio naturale dei virus influenzali di tipo A è rappresentato dagli uccelli, sia selvatici che domestici (influenza aviaria) (http://www.cdc.gov/flu/about/viruses/transmission.htm); in particolare gli uccelli selvatici sembrano costituire la fonte dell'influenza A virus tutte le altre specie animali. Diverse tecniche sono disponibili per studiare i virus e caratterizzarli geneticamente al fine di capirne il loro comportamento, le dinamiche evolutive, il loro rapporto con l'ospite e la loro origine e per sviluppare profilassi e terapie adeguate creando un valido supporto durante la fasi di sorveglianza e diagnosi di un'eventuale epidemia .
Nell'ambito del mio dottorato è stato utilizzato un approccio integrato, sia genomico che strutturale, per studiare l'evoluzione dell'influenza aviaria; particolare interesse è stato rivolto allo studio dell'emoagglutinina virale, la principale glicoproteina di superficie, appartenente ai sottotipi H5, H7 e H9 (i principali sottotipi “aviari” responsabili di infezione nell’uomo).
Le analisi mediante Next Generation Sequencing (NGS) hanno favorito lo studio e la caratterizzazione della complessità nella popolazione virale, consentendo di monitorare finemente l'evoluzione delle varianti geneticamente correlate presenti all'interno della popolazione virale tramite l'identificazione delle mutazioni a bassa frequenza. Per confrontare ed analizzare i dati genetici, l'approccio filogenetico si è rivelato un utile strumento per l'analisi dell'evoluzione virale; è stato usato per spiegare l'epidemiologia molecolare, la trasmissione e l'evoluzione virale. Al fine di ottenere una visione più completa in termini di 'evoluzione funzionale', l'analisi filogenetica è stata integrata con le informazioni provenienti dal confronto strutturale. L'approccio strutturale, considerando lo spazio tridimensionale dell’emoagglutinina, ha dimostrato di poter essere uno strumento utile per evidenziare eventuali somiglianze e per ispezionare e valutare quei motivi il cui ruolo non può essere correttamente interpretato utilizzando le sole sequenze primarie. Infatti, nelle sequenze primarie il peso delle mutazioni non tiene conto dell'effetto sul fold o sulle proprietà di superficie, mentre nelle strutture tridimensionali, quanto ciascuna mutazione sia in grado di influenzare le caratteristiche strutturali e le interazioni, è direttamente rilevabile. Questo approccio ha inoltre portato un ulteriore contributo all'analisi filogenetica. In particolare lo studio si è concentrato sull'analisi delle dinamiche evolutive e delle strategie adattative dei sottotipi H7N1 ed H7N3 dell'influenza aviaria circolanti nel Nord Italia per periodi di tempo analoghi e in condizioni epidemiologiche simili. Inoltre è stato utilizzato il deep sequencing per studiare le dinamiche evolutive e di trasmissione intra- e inter-ospiti del virus aviario sottotipo H7N7 che colpì alcuni allevamenti italiani nel 2013. L'analisi NGS è stata utilizzata per caratterizzare la complessità della popolazione virale in due gruppi di animali sperimentalmente infetti con lo stesso virus ad alta patogenicità (HPAI) H5N1 ed immunizzati con distinti vaccini. E' stato inoltre eseguito un ampio confronto strutturale su domini e sub-regioni dell'emoagglutinina di diversi sottotipi del virus dell'influenza, con particolare interesse per i diversi clades di HPAI H5N1 circolanti in Egitto (ove l’influenza aviaria è endemica nei volatili), per indagare eventuali variazioni dominio-specifiche. I virus influenzali del sottotipo H9 sono stati analizzati da un punto di vista sia filogenetico che strutturale, per rilevare caratteristiche tipo specifiche e verificare se la variazione delle proprietà di superficie possa essere un marcatore di 'evoluzione funzionale' dei determinanti di superficie virali, come dimostrato nel sottotipo H5N1. Questo lavoro suggerisce che il confronto e l'integrazione tra analisi genomica, filogenetica e strutturale può aiutare a capire l' 'evoluzione funzionale' del virus dell'influenza aviaria di tipo A.

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Tipo di EPrint:Tesi di dottorato
Relatore:Filippini, Francesco
Correlatore:Cattoli, Giovanni
Dottorato (corsi e scuole):Ciclo 27 > scuole 27 > BIOSCIENZE E BIOTECNOLOGIE > BIOTECNOLOGIE
Data di deposito della tesi:28 Gennaio 2016
Anno di Pubblicazione:28 Gennaio 2016
Parole chiave (italiano / inglese):influenza A virus NGS phylogeny molecular modeling
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:9271
Depositato il:20 Ott 2016 10:09
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