Arboviruses transmitted by mosquito vectors represent a (re-)emerging threat for human health worldwide due to the expansion of vector distribution. The invasive Asian tiger mosquito Aedes albopictus transmits more than 20 different arboviruses and is the main vector in Europe. To date, vector control represents the best approach to limit arboviral diseases transmission. Due to increased insecticide resistance, developing novel genetic-based transmission-blocking strategies is essential. For this purpose, a deep understanding of the mosquito-virus interaction, their co-evolution and the mechanisms underlying this interaction is primordial. In this context, the analysis of Ae. albopictus genome revealed that it is enriched with sequences from non-retroviral RNA viruses. These non-retroviral Endogenous Viral Elements (nrEVEs) are mostly fragmented viral sequences and are enriched in PIWI-interacting RNA (piRNA) clusters and produce piRNAs, which are suggested to limit cognate viral infections. We also identified 12 nrEVEs which encompass complete open reading frames (ORFs) with similarities to RNA-dependent RNA polymerases (RdRPs). Hereafter, I will refer to these EVEs as RdRP-like nrEVEs. In viruses RdRPs are involved in genome replication, transcription and encapsulation. RdRPs are also present in plants and fungi where they are involved in the RNA interference (RNAi) pathway. Instances of viral RdRPs horizontally transferred to eukaryotes are also known in chelicerates and bats. I hypothesize that the RdRP-like nrEVEs of Ae. albopictus also derived from horizontal gene transfer from viruses and may contribute to limit further viral infections. To confirm my hypothesis, I designed several functional experiments. First, I verified that RdRP-like nrEVEs are constitutively expressed in different mosquito developmental stages and female tissues. In addition, several RdRP-like nrEVEs showed to be regulated in response to infection with the flavivirus dengue virus and the alphavirus chikungunya virus. Based on expression results, I selected two RdRP-like nrEVEs, Rhabdo 21 and Flavi 24, for further characterization. Each of the two ORFs was fused with the ORF encoding for the green fluorescent protein (GFP) and cloned into an expression plasmid. A recombinant plasmid for the GFP was generated as a control plasmid. The three plasmids were used to separately transfect mosquito Aag2 cells. Two days post transfection, I could detect high expression of Rhabdo 21 as well as Flavi 24. Transfected cells were subsequently infected with the insect-specific virus Cell Fusion Agent Virus (CFAV; Flaviviridae). My results show that in comparison to infected non-transfected cells and infected GFP-transfected cells, the expression of Rhabdo 21 or Flavi 24 reduced CFAV replication. My results suggest Rhabdo 21 and Flavi 24 may be new antiviral effectors. These Ae. albopictus RdRP-like nrEVEs responded in vivo to viral infection and negatively affected viral replication in ex vivo experimental conditions. Aedes albopictus RdRPs-like nrEVEs can represent a possible target for the development of genetic-based control strategies.
Gli arbovirus trasmessi da zanzare (vettori) rappresentano una minaccia (ri-)emergente per la salute umana a causa dell'espansione della distribuzione dei vettori in tutto il mondo. La zanzara tigre asiatica Aedes albopictus è una specie invasiva che trasmette più di 20 diversi arbovirus ed è il principale vettore in Europa. Ad oggi, il controllo dei vettori rappresenta il miglior approccio per limitare la trasmissione delle malattie arbovirali. A causa dell'aumento della resistenza agli insetticidi, è essenziale sviluppare nuove strategie di blocco della trasmissione. A questo scopo, una profonda comprensione dell'interazione zanzara-virus, la loro co-evoluzione ed i meccanismi alla base di questa interazione è fondamentale per sviluppare nuove strategie di controllo genetico. L'analisi del genoma di Ae. albopictus ha portato alla scoperta di sequenze virali. Questi elementi virali endogeni non-retrovirali (nrEVEs) sono perlopiù sequenze virali frammentate, sono arricchiti nei cluster di PIWI-interacting RNA (piRNA) e producono piRNAs, che possono limitare infezioni di virus con sequenza corrispondente alle sequenze degli nrEVEs. Il genoma di Ae. albopictus ha anche 12 nrEVEs che comprendono open reading frame (ORF) complete codificanti per RNA polimerasi RNA dipendenti (RdRPs). In seguito, mi riferirò a questi EVE come RdRP-like nrEVEs. Nei virus le RdRP sono coinvolte nella replicazione del genoma, nella trascrizione e nell'incapsulamento. Le RdRP sono presenti anche in piante e funghi, dove sono coinvolte nell’RNA interference (RNAi). Casi di RdRP virali trasferite orizzontalmente agli eucarioti sono noti anche in chelicerati e chirotteri. Ipotizzo che anche gli RdRP-like nrEVEs di Ae. albopictus siano il risultato di un trasferimento genico orizzontale e possano contribuire a limitare successive infezioni virali. Per confermare la mia ipotesi, ho eseguito diversi esperimenti funzionali. In primo luogo, ho verificato che gli RdRP-like nrEVEs sono costitutivamente espressi in diversi stadi di sviluppo della zanzara e nei tessuti femminili. Inoltre, ho dimostrato che gli RdRP-like nrEVEs sono regolati in risposta all'infezione con il virus dengue (Flaviviridae) ed il virus chikungunya (Togaviridae). Sulla base dei risultati di espressione, ho selezionato due RdRP-like nrEVEs, Rhabdo 21 e Flavi 24, per un'ulteriore caratterizzazione. Ciascuna delle due ORF è stata fusa con l'ORF che codifica per la proteina fluorescente verde (GFP) e clonata in un plasmide di espressione. Un plasmide ricombinante per la GFP è stato generato come plasmide di controllo. I tre plasmidi sono stati usati per trasfettare separatamente le cellule Aag2 di zanzara. Due giorni dopo la trasfezione, ho potuto rilevare un'alta espressione di Rhabdo 21 così come di Flavi 24. Le cellule trasfettate sono state successivamente infettate con il virus specifico per gli insetti Cell Fusion Agent Virus (CFAV; Flaviviridae). I miei risultati mostrano che in confronto ai controlli (cellule infettate non trasfettate; cellule infettate e trasfettate con plasmide GFP), l'espressione di Rhabdo 21 o Flavi 24 ha ridotto la replicazione del CFAV. I miei risultati suggeriscono che Rhabdo 21 e Flavi 24 sono nuovi effettori antivirali. Questi RdRP-like nrEVEs hanno risposto in vivo all'infezione virale e hanno influenzato negativamente la replicazione virale in condizioni sperimentali condotte ex vivo. Gli RdRPs-like nrEVEs di Ae. albopictus possono rappresentare un possibile bersaglio per lo sviluppo di strategie di controllo basate sulla modificazione genetica del vettore.
Caratterizzazione funzionale di sequenze di origine virale integrate nel genoma del vettore Aedes albopictus
SOGLIANI, DAVIDE
2020/2021
Abstract
Arboviruses transmitted by mosquito vectors represent a (re-)emerging threat for human health worldwide due to the expansion of vector distribution. The invasive Asian tiger mosquito Aedes albopictus transmits more than 20 different arboviruses and is the main vector in Europe. To date, vector control represents the best approach to limit arboviral diseases transmission. Due to increased insecticide resistance, developing novel genetic-based transmission-blocking strategies is essential. For this purpose, a deep understanding of the mosquito-virus interaction, their co-evolution and the mechanisms underlying this interaction is primordial. In this context, the analysis of Ae. albopictus genome revealed that it is enriched with sequences from non-retroviral RNA viruses. These non-retroviral Endogenous Viral Elements (nrEVEs) are mostly fragmented viral sequences and are enriched in PIWI-interacting RNA (piRNA) clusters and produce piRNAs, which are suggested to limit cognate viral infections. We also identified 12 nrEVEs which encompass complete open reading frames (ORFs) with similarities to RNA-dependent RNA polymerases (RdRPs). Hereafter, I will refer to these EVEs as RdRP-like nrEVEs. In viruses RdRPs are involved in genome replication, transcription and encapsulation. RdRPs are also present in plants and fungi where they are involved in the RNA interference (RNAi) pathway. Instances of viral RdRPs horizontally transferred to eukaryotes are also known in chelicerates and bats. I hypothesize that the RdRP-like nrEVEs of Ae. albopictus also derived from horizontal gene transfer from viruses and may contribute to limit further viral infections. To confirm my hypothesis, I designed several functional experiments. First, I verified that RdRP-like nrEVEs are constitutively expressed in different mosquito developmental stages and female tissues. In addition, several RdRP-like nrEVEs showed to be regulated in response to infection with the flavivirus dengue virus and the alphavirus chikungunya virus. Based on expression results, I selected two RdRP-like nrEVEs, Rhabdo 21 and Flavi 24, for further characterization. Each of the two ORFs was fused with the ORF encoding for the green fluorescent protein (GFP) and cloned into an expression plasmid. A recombinant plasmid for the GFP was generated as a control plasmid. The three plasmids were used to separately transfect mosquito Aag2 cells. Two days post transfection, I could detect high expression of Rhabdo 21 as well as Flavi 24. Transfected cells were subsequently infected with the insect-specific virus Cell Fusion Agent Virus (CFAV; Flaviviridae). My results show that in comparison to infected non-transfected cells and infected GFP-transfected cells, the expression of Rhabdo 21 or Flavi 24 reduced CFAV replication. My results suggest Rhabdo 21 and Flavi 24 may be new antiviral effectors. These Ae. albopictus RdRP-like nrEVEs responded in vivo to viral infection and negatively affected viral replication in ex vivo experimental conditions. Aedes albopictus RdRPs-like nrEVEs can represent a possible target for the development of genetic-based control strategies.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13054