Global Stability of Generalized Within-host Chikungunya Virus Dynamics Models

This paper proposes two models of a general nonlinear within-host Chikungunya virus (CHIKV) dynamics. The production, incidence, proliferation and removal rates of all compartments are modeled by general nonlinear functions that satisfy a set of reasonable conditions. The second model takes into consideration two forms of infected host cells: (i) latently infected cells which do not produce the CHIKV, (ii) actively infected cells which generate the CHIKV particles. We show that all the solutions of the models are nonnegative and bounded. The global stability of the steady states of the models is proven by applying Lyapunov method and LaSalle’s invariance principle. We perform numerical simulations to complement the obtained theoretical results

Understanding the Wolbachia-mediated inhibition of arboviruses in mosquitoes: progress and challenges

Arthropod-borne viruses (arboviruses) pose a considerable threat to human and animal health, yet effective control measures have proven difficult to implement, and novel means of controlling their replication in arthropod vectors, such as mosquitoes, are urgently required. One of the most exciting approaches to emerge from research on arthropods is the use of the endosymbiotic intracellular bacterium Wolbachia to control arbovirus transmission from mosquito to vertebrate. These α-proteobacteria propagate through insects, in part through modulation of host reproduction, thus ensuring spread through species and maintenance in nature. Since it was discovered that Wolbachia endosymbiosis inhibits insect virus replication in Drosophila species, these bacteria have also been shown to inhibit arbovirus replication and spread in mosquitoes. Importantly, it is not clear how these antiviral effects are mediated. This review will summarize recent work and discuss determinants of antiviral effectiveness that may differ between individual Wolbachia/vector/arbovirus interactions. We will also discuss the application of this approach to field settings and the associated risks

Determination of the biological functions of undefined domains of VEEV nsP2.

Alphaviruses are positive sense, single strand, RNA viruses. These viruses occur on every populated continent. Alphaviruses are divided into two clades, the New-World and Old-World viruses. The New-World viruses include Eastern (EEEV), Western (WEEV), and Venezuelan equine encephalitis viruses (VEEV), and cause neuroinvasive disease. The Old-World viruses include Chikungunya (CHIKV) and Ross River viruses (RRV), and typically cause multijoint arthralgia. There are currently no approved antiviral therapeutics or vaccines for any alphavirus, making them a high priority for antiviral drug design and discovery. A benzamidine inhibitor (ML336) of VEEV was characterized, and determined to inhibit replication of VEEV RNA during infection of BHK-21 cells, a fibroblast model. This activity was due a loss of synthesis of new viral RNA. This compound had no effect on RNA synthesis in uninfected cells, making it a promising target for therapeutic development. The inhibitory activity of ML336 was highly specific for VEEV, having no effect on RNA synthesis of CHIKV. A potential interaction between ML336 and the VEEV nsPs was examined, but these results were inconclusive. ML336 and related compounds were used to generate resistant mutant VEEV. These isolates were sequenced and it was revealed that mutations were concentrated in a region of nsP2 of unknown function. Analysis of these mutant viruses revealed delayed growth, RNA synthesis, and translation of viral proteins in BHK cells. There was also a growth delay seen in SH-SY5Y cells, a model of neuronal infection. These findings indicate that this region of nsP2 is likely involved in RNA synthesis of VEEV, and shows promise as a target of antiviral drug development

Uudsed vaated alfaviiruste poolt kodeeritud nsP2 valgu funktsioonide kohta

Väitekirja elektrooniline versioon ei sisalda publikatsiooneAlfaviirused (sugukond Togaviridae) on sfäärilise ümbrisega virionide ja positiivse polaarsusega RNA genoomiga viirused, mis enamasti levivad lülijalgsete vektorite vahendusel. Paljud alfaviirused on olulised inimeste patogeenid, sealhulgas ulatuslikke puhanguid põhjustav Chikungunya viirus (CHIKV), Austraalias leviv Ross River viirus (RRV), Ameerikas leviv ida hobuste entsefaliidi viirus (EEEV) ja ka Eestis leiduv Sindbis viirus (SINV). Alfaviiruste põhjustatud haigused sõltuvad viiruse liigist ja selle päritolust,kus üldistatult põhjustavad Vana Maailma alfaviirused palaviku, löövet ja artriiti, samal ajal kui Uue Maailma alfaviirused on sageli kõrge patogeensusega põhjustades neuroloogilisi haiguseid, sealhulgas entsefaliiti. Alfaviiruste RNA genoom kodeerib nelja viiruse RNA replikaasi allühikut, mis toodetakse liitvalgust eellase (polüproteiini) kujul. Liitvalk lõigatakse neljaks valmis replikaasi valguks viiruse nsP2 proteaasi abil. Viimastel aastakümnetel läbiviidud uurimistööd on võimaldanud välja selgitada viiruse replikaasi kompleksi kui ka selle komponentide peamised funktsioonid ning meie arusaamine viiruse valkude struktuuridest on oluliselt täienenud. Ka need uurimised on näidanud, et alfaviiruse infektsiooni üks esimesi ja kõige olulisemaid sündmusi on replikaasi valkude eellase lõikamine valmis valkudeks. Eelvalgu lõikamised toimuvad kindlas järjekorras ja onkindlalt ajastatud, mis määrab ära selle, kas vabanevad valgud moodustavad toimiva RNA replikaasi või mitte. Seega kujutab eelvalgu lõikamise protsessi läbi viiv nsP2 endast RNA replikatsiooni käivitamise ja efektiivsuse regulaatorit. Samas on tegemist multifunktsionaalse valguga, mis on lisaks RNA sünteesi reguleerimisele oluline nii raku viirusvastaste kaitsereaktsioonide aktiveerimisel kui ka nende mahasurumiseks. Sellest tulenevat mõjutavad muudatused nsP2 valgus, sealhulgas punktmutatsioonid valgu funktsionaalselt olulistes regioonides, oluliselt viiruse bioloogilisi omadusi. Seejuures ei ole mitmete nsP2 aktiivsuste taga seisvad molekulaarsed mehhanismid lõpuni selged. Küll aga on selgunud, et nsP2 funktsioone mõjutavad interaktsioonid alfaviiruse ülejäänute replikaasi valkude nsP1, nsP3 ja nsP4-ga. Käesoleva uurimistöö raames selgitati välja ja analüüsiti mitmeid nsP2 valgu ja RNA replikaasi seni tundmatuid funktsioone. Töö peamised tulemused saab kokku võtta järgnevalt: 1. alfaviiruste genoomide alusel konstrueeritud trans-replikatsiooni süsteemid võimaldavad uurida nsP2 valgu funktsioone, mis on seotud viiruse RNA replikaasi kompleksi moodustamisega, aga ka selle moodustamise blokeerimise ja/või selle aktiivsuse mahasurumisega. Viimati nimetatud aktiivsused on olulised viiruste superinfektsiooni blokeerimisel ja tõenäoliselt ka viiruse replikatsiooni ajalisel reguleerimisel. CHIKV ja SINV trans-replikatsiooni süsteeme kasutades tehti kindlaks, et superinfektsiooni blokeerimise puhul on võtmesündmuseks ühe viiruse replikaasi eellase lõikamine teise viiruse nsP2 poolt ja et seda mõjutavad mutatsioonid nii sihtmärk-liitvalgus kui ka nsP2 valgus; 2. leiti, et individuaalse nsP2 süntees sääse (vektorputuka) rakkudes surub maha alfaviiruse replikatsiooni aktiivsust. Seda nähtust ei põhjusta ainult nsP2 proteaasne aktiivsus – olulised on ka nsP2 proteaassest aktiivsusest sõltumatud mehhanismid. See, milline on konkreetsete mehhanismide osakaal, sõltub nii viiruse RNA replikaasi kui ka selle moodustamist inhibeeriva nsP2 valgu päritolust. Homoloogse viiruse replikatsiooni mahasurumisel domineerib nsP2 proteaassest aktiivsustest sõltuv mehhanism, kus nsP2 poolt teostatud replikaasi eelvalgu lõikamine 2/3 saidist välistab funktsionaalse RNA replikaasi moodustumise. Mõne alfaviiruse (SINV) puhul on nsP2 proteaassest aktiivsusest sõltuv mehhanism ka peamiseks mehhanismiks millega surutakse alla heteroloogsete alfaviiruste replikaaside moodustamist; 3. nii SINV kui ka CHIKV nsP2 valk takistab heteroloogsete alfaviiruste RNA replikatsiooni ka proteaassest aktiivsusest sõltumatute mehhanismide abil, kus CHIKV nsP2 puhul on need peamisteks meetoditeks heteroloogsete alfaviiruste replikatsiooni mahasurumisel. Selle inhibeerimise täpne põhjus on hetkel teadmata, küll aga näitasime, et erinevad mutatsioonid nsP2 valgus mõjutavad taolise inhibeerimise efektiivsust. Need andmed viitavad sellele, et proteaassest aktiivsusest sõltumatu replikatsiooni inhibeerimine saavutatakse tõenäoliselt mitmete paralleelsete mehhanismide abil; 4. väga patogeensele ja seetõttu vähe uuritud EEEV-le konstrueeritud trans-replikastiooni süsteem osutus kõrgelt efektiivseks nii inimese kui ka sääse rakkudes. Samuti osutus võimalikuks EEEV replikaasi eelvalku (P1234) kodeeriva järjestuse jagamine kaheks (P123 + nsP4) ja kolmeks (nsP1+P23+nsP4) komponendiks. See võimaldas uurida kas ja kuidas replikaasi komponentide vahekord mõjutab EEEV RNA replikaasi aktiivsust. Läbiviidud katsed näitasid, et erinevalt seni uuritud alfaviirustest sõltub EEEV RNA replikaasi aktiivsus teda moodustavate komponentide optimaalsest vahekorrast ja et RNA replikaasi katalüütilise allühiku (nsP4) ülehulk vähendab EEEV RNA replikaasi aktiivsust. Selline aktiivsuse langus tuleneb sellest, et väheneb nii rakkude hulk milles RNA replikatsioon aktiveeritakse, kuid ka RNA replikatsiooni aktiivsus nendes rakkudes. Hetkel pole veel selge kas tegemist on EEEV RNA replikaasi unikaalse omadusega või leidub ka teisi sarnaste omadustega alfaviiruste replikaase; 5. uuriti RRV loodusliku isolaadi RRV 2528 omadusi. Leiti, et see viiruse isolaat põhjustab väga tugevat tüüp-I interferoonide vastust. Viiruse genoomi analüüs tõi välja, et RRV 2528 erineb teistest vähemal määral interferoonide tootmist indutseerivatest RRV isolaatidest mitmete mutatsioonide poolest, millest paljud paiknevad just nsP2 valgus Näitasime, et need mutatsioonid mõjutavad RRV nakkuse käigus toimuvat interferoonide tootmist, kuid ei mõjuta viiruse replikaasi eelvalgu protsessimist valmis valkudeks, viiruse võimet suruda maha raku valgusünteesi ega viiruse struktuurvalkude sünteesi. Seega ei tulene suurenenud tüüp-I interferoonide vastus defektist RRV 2528 RNA replikatsioonikomplekside moodustamisel või võimetusest maha suruda raku üldist geeniekspressiooni.Over the last decades, the intensive studies of factors/activities responsible for multiple aspects of alphavirus infection have been performed. In particular, our understanding about structures and functions of viral RNA replicase and its components has significantly increased. Novel findings emphasize that one of the first and most essential event in alphavirus infection is processing of the ns polyprotein carried out by its nsP2 region and an individual nsP2; this process not only ensures the release of functional replicase subunits but also determines would these proteins form the active RCs or not. Thus, it is increasingly evident that nsP2 is one of the “main driving forces” of successful RNA replication. Furthermore, due to its versatile functions and various activities nsP2 is involved in other aspects of infection. It is one of the key determinants associated with activation as well as counteracting of antiviral response in infected cells. Therefore, different modifications of the protein, including point mutations, often have drastic impact on alphavirus infection. However, much of the precise mechanisms of P2 action remain enigmatic. What is clear is that nsP2 does not act alone, its activities are modulated by other components of viral replicase. The current study allowed us to identify and confirm new functions and properties of nsP2 and alphavirus RNA replicase. The general conclusions of this study can be presented as follows: 1. Alphavirus trans-replicase systems can be applied as a tool for studies of functions of nsP2 associated with inhibition of RC formation/activity. These functions are essential for SIE and are likely related to these used to regulate RNA replication in the alphavirus infected cells. Using trans-replicase systems of CHIKV and SINV, it was found that the key event in SIE is targeting of replicase precursor (P1234) by an individual nsP2 protein and that this ability of nsP2 can be altered by mutations present in its functionally important regions. 2. It was found that synthesis of an individual (free) nsP2 in mosquito cells has an inhibitory effect on the alphavirus RC formation/functionality. This is not, however, a result of a single mechanism but results from combination of nsP2 protease-activity dependent mechanism and protease-activity independent mechanisms. The level and dominant mode of inhibition of alphavirus RNA replication depends form the virus, source of free nsP2 and substitutions present in this protein. The protease-activity mediated mechanism is important for suppression of replication of matching virus and relies mostly on ability of nsP2 to cleave 2/3 site in ns polyprotein. For some viruses such as SINV it is also dominant mechanism used to suppress formation of RNA replicases of heterologous alphaviruses. 3. nsP2 of SINV and CHIKV can inhibit formation/activity of RNA replicase of heterologous alphavirus using protease-activity independent mechanisms; for nsP2 of CHIKV this is the dominant mechanism to suppress activity of RNA replicases of heterologous alphaviruses. The precise details of protease-activity independent mode of action of nsP2 remain unknown; however, it is clear that this property can be enhanced by introduction of certain mutations into nsP2. It is likely that the protease-activity independent inhibitory effect originates not from a single mechanism but from several mechanisms. 4. Trans-replicase of highly pathogenic EEEV was found to be highly active in human and mosquito cells. Splitting of construct of EEEV P1234 expression into two (P123 and nsP4) or three (nsP1, P23 and nsP4) expression construct allowed analysis of requirements of active RC formation. It was found that activity of EEEV RNA replicase depends from correct ratio of P123 (or nsP1+P23) component to nsP4 component and that in contrast to previously studied alphaviruses an excess of nsP4 reduced activity of EEEV RNA replicase. The reduction was due to the decrease of a number of cells were RNA replication was initiated as well as to the reduced RNA replicase activity in such cells. It remains unclear, is this property unique for EEEV RNA replicase. 5. Natural isolate RRV 2528 was found to be a prominent inducer of type-I IFN expression. This property was associated with specific amino acid substitutions in the nsP2 encoded by this isolate. None of these substitutions or their combination affected ability of RRV to induce shutdown of cellular protein synthesis or level of viral structural proteins expression. Similarly, no effect on the processing of P1234 was detected. Combined, these findings indicate that the excessive type-I IFN induction was not due to the lack of the ability to induce general shutdown of cellular gene expression or due to the defects in RC formation.https://www.ester.ee/record=b553517

The role of RNA structure in Chikungunya virus early replication events

Chikungunya virus (CHIKV) is a pathogenic, single-stranded, positive-sense RNA virus transmitted to humans by Aedes spp. mosquitoes. After decades of low-level endemic circulation, CHIKV has re-emerged to establish local transmission on five continents, infecting upwards of 3,000,000 people. There are no currently available vaccines or direct-acting anti-viral therapeutic agents. A greater understanding of the CHIKV replication cycle is essential, as much of what is known about the replication cycle is assumed from studies of related but divergent viruses, which have provided conflicting reports. Preliminary work carried out by A. Tuplin (University of Leeds) suggested a highly ordered structured region at the 5′ end of the CHIKV genome, spanning ~300 nt including the 5′ untranslated region (UTR) and the 5′ coding sequence of nsp1. The aim of this project was to determine the phenotypic importance of secondary structure in this region for the CHIKV lifecycle in human and mosquito cells at multiple stages of viral replication and to investigate the sequence and structure requirements for functional interactions. This study represents the first investigation of functional elements within the 5′ UTR and adjacent nsP1-coding region in CHIKV. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, the wild-type secondary structure of the 5′ 300 nt of the CHIKV genome was found to be essential for genome replication in human- and Ae. albopictus-derived cells. Six RNA stem-loops were determined to individually enhance CHIKV genome replication - including novel structures analysed for the first time in this study. Comparative analysis in human and mosquito-derived cell lines revealed that the novel stem-loop SL47 in the 5′ UTR functions in a host-independent manner while stem-loops in nsp1 function in a host-dependent manner. Stem-loops were demonstrated to function within the positive-strand genomic RNA, via predominantly structure-dependent mechanisms. Furthermore, single-host passaging studies suggested strong selection pressure to regenerate secondary structures and highlighted potential differences in translational recoding between host species. Finally, the potential for tertiary structure formation was explored. In addition to furthering knowledge of fundamental aspects of the molecular virology of this important human pathogen, this study will inform rational design of a genetically stable attenuated vaccine

Alphavirus-host interactions : how to unravel the proviral activity of G3BP

The genus Alphavirus belongs to the virus family Togaviridiae, a group of arthropod-borne enveloped RNA viruses with single-stranded, positive-sense genome. Members of this genus can be found worldwide and are geographically distinguished into Old World and New World alphaviruses. Old World alphaviruses include the re-emerging human pathogen chikungunya virus (CHIKV) and the model virus Semliki Forest virus (SFV). Upon cellular infection, the released viral genome is directly translated to produce the non-structural polyprotein, which is subsequently processed into four non-structural proteins. The non-structural protein 3 (nsP3) of Old World alphaviruses contains two FGDF motifs, which facilitate binding to the NTF2-like domain of the host protein G3BP and its recruitment to viral replication complexes. G3BP1 and G3BP2 (hereafter jointly referred to as G3BP) are homologous proteins, best characterized for their ability to assemble stress granules in response to a variety of cellular stressors, such as viral infection. In paper I we investigated the structural and functional significance of the two FGDF motifs for the Old World alphaviruses SFV and CHIKV. A 3-dimensional structure of the NTF2-like domain of G3BP1 in complex with a SFV nsP3-derived peptide showed that the two FGDF motifs crosslink dimers of G3BP1 into a nsP3:G3BP1 oligomer. Mutational analysis of the FGDF motifs furthermore revealed that both motifs are required for efficient growth of SFV and suggest a critical role for the formation of nsP3:G3BP oligomeric structures for SFV. CHIKV is non-viable if the nsP3:G3BP1 interaction is abrogated through mutation of both motifs. The presence of a single functional FGDF motif is sufficient to rescue CHIKV replication, albeit to a lesser extent than in the presence of both motifs. Together, the results of this paper highlight similarities, but also discrepancies between SFV and CHIKV for the two G3BP-binding motifs. In paper II we studied potential proviral roles of G3BP for SFV and CHIKV. To this end, we used a panel of human osteosarcoma (U2OS) cell lines lacking endogenous G3BP proteins and stably expressing G3BP1 mutants and truncation variants. SFV replication is attenuated in the absence of G3BP and efficiently rescued by the presence of only the NTF2-like domain of G3BP1, which is accompanied by clustering of replication complexes. On the contrary, CHIKV strictly depends on the presence of the NTF2-like and the RGG domains of G3BP1. By immunoprecipitation we show that the RGG domain of G3BP1 facilitates binding of nsP3:G3BP1 complexes to 40S ribosomal, which correlates with enhanced localized translational activity in close proximity to viral replication complexes. The results suggest that G3BP exerts several proviral activities by mediating clustering of viral replication complexes and the recruitment of the translation initiation machinery. The results of paper III demonstrate that Old Wold alphaviruses differ remarkably in their dependence on G3BP. We describe a role for the P4 residue of the cleavage site between nsP1 and nsP2 (1/2 site) of the alphavirus non-structural polyprotein in conferring the extent of G3BP sensitivity. An Arg residue at the P4 position of the 1/2 site, as for CHIKV, is associated with fast a cleavage rate at this site and a high sensitivity towards G3BP deletion. An His residue at this position, as for SFV4, confers a slower cleavage rate at this site, accompanied with partial resistance towards G3BP deletion. Arg-to-His substitution of the P4 residue of CHIKV allows partially rescues replication even in the absence of G3BP. However, our data suggest that G3BP proteins do not influence the processing of the non-structural polyprotein. Instead we propose a critical role for G3BP proteins during the initiation of viral RNA replication. In summary, the work presented in this thesis improves our understanding of alphavirus-host interactions and highlights previously unanticipated proviral roles of G3BP for Old World alphavirus infections. The results mark G3BP as a potential target for the development of antivirals and provide a platform for future investigations

Hepatiit C viiruse ja Chikungunya viiruse vastased lähenemised

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Tänapäeval on võimalik ennast erinevate viiruste vastu vaktsineerida ning ka viirushaiguste ravi on muutunud oluliselt tõhusamaks. Samas leidub endiselt meditsiiniliselt olulisi viiruseid, mille vastu puudub vaktsiin ja/või mille poolt põhjustatud haigustele pole siiani adekvaatset ravi. Viirus-vastaste ühendite ja vaktsiinide väljatöötamist raskendavad nii viiruste suur mitmekesisus kui ka nende keeruline elutsükkel. Üheks selliseks viiruseks on C hepatiidi viirus (HCV), mis on kroonilise maksahaiguse levinuimaks tekkepõhjuseks. Hinnanguliselt on selle viirusega krooniliselt nakatunud ~3% inimkonnast. Kuigi HCV infektsiooni ravis on toimunud suur läbimurre, on viiruse geneetilise mitmekesisuse, ravimresistentsete vormide tekkimise ning patsientide ravile mitteallumise tõttu endiselt väga oluline uute HCV vastaste ravimite väljatöötamine. Antud uurimustöö üheks eesmärgiks oli analüüsida erinevaid tehnoloogilisi lahendusi HCV vastaste ühendite loomiseks. Ühe lähenemisena valiti FQSAR arvutiprogrammi põhiselt välja madal-molekulaarsed ühendid, mis seondudes HCV NS3/4A proteaasiga inhibeerivad HCV replikatsiooni, ja iseloomustati nende mõju viiruse infektsioonile. Kõik analüüsitud seitse ühendit omasid HCV-vastast efekti, kuid ainult üks ühend (23332) oli kasutatavas kontsentratsioonis mitte-toksiline. Teine lähenemisviis seisnes looduslikult esineva modifikatsiooni (8-oxo-dG) mõju analüüsimises oligonukleotiidsete (ON) inhibiitorite efektiivsusele. Kombineerides erinevaid modifikatsioone leiti ON ühend, mis inhibeeris HCV replikatsiooni nanomolaarsetel kontsentratsioonidel. Lisaks HCV uurimisele on võimalik käsitletud lähenemise kasutada ka teiste viiruste vastu suunatud ühendite väljatöötamisel. Chikungunya viirus (CHIKV, perekond Alfaviirus) on troopilistes piirkondades leviv arboviirus, mis on viimasel aastakümnel korduvalt väljunud oma tavalisest levialast ja põhjustanud epideemiaid erinevates maailmajagudes. Antud töö kolmandaks eesmärgiks oli analüüsida uudsete CHIKV-vastaste vaktsiinikandidaatide geneetilist stabiilsust ning uurida nendes sisalduvate viirust nõrgestavate mutatsioonide mõju CHIKV elutsüklile. Leiti, et viirustel CHIKVΔ5nsP3 ja CHIKVΔ6K on nõrgestatud fenotüüp ka pärast mitmekordset passeerimist koekultuuri rakkudes. Mitmetest analüüsitud CHIKV-vastastest vaktsiini kandidaatidest osutus kõige efektiivsemaks CHIKVΔ5nsP3. See nõrgestatud viirus sisaldab suurt deletsiooni nsP3 valgu C-terminaalses regioonis. Katsetest selgus, et nimetatud regioon interakteerub sama valgu keskmise domeeni ning nsP2 valgu C-terminaalse osaga ja need kontaktid on olulised viiruse replikatsioonil. Need avastused võimaldavad edaspidi välja selgitada CHIKVΔ5nsP3 mitte-patogeense fenotüübi põhjused. CHIKV Δ5nsP3 vaktsiini tüvi on kasutusele võetud edasiseks arendamiseks farmatseutilise firma poolt.Viruses have been and will be an important part of every ecosystem. In the past, viral outbreaks have left painful marks on mankind. Using vaccines and antivirals has greatly reduced the number of infections and virus-caused pathology. Despite extensive research, some viruses and viral diseases are still lacking any good vaccine or treatment. Viral features like high mutation rate, complexity of viral lifecycle and genome diversity are only some of the obstacles needed to overcome for antivirals and vaccines to be safe and efficient. Hepatitis C virus (HCV) is associated with different liver pathologies and it is estimated that approximately 3% of the world population is chronically infected with HCV. It is lacking efficient vaccine and the options for combating HCV infection, HCV-induced pathology, spread and persistence are limited to the use of antiviral drugs. One part of this dissertation is focused on the development of anti-HCV inhibitors using two different technological approaches. Firstly, a new FQSAR method based approach allowed rapid prediction of hit compounds targeting the NS3/4A protease of HCV. Seven compounds analysed in this project displayed some anti-HCV properties but only the effect caused by the non-cytotoxic compound 23332 can be considered to be direct. Secondly, a novel technology – incorporation of naturally occurring minimally modified nucleobases into ASOs – was evaluated using ASOs binding to the HCV non-structural region. This approach led to the development of ASO compounds with high anti-HCV activity. The technology based on the use of novel modified ONs is promising as well for the development antivirals for other viruses and diseases. Chikungunya virus (CHIKV) re-emerged in the past decade and is currently spreading around the world, affecting millions of people. The second part of this study is focused on the analysis of a laboratory-developed attenuated CHIKV vaccine strain. CHIKVΔ5nsP3 and CHIKVΔ6K viruses were found to have a stably attenuated phenotype and the introduced molecular changes were maintained during serial passages. From all studied vaccine candidates the CHIKVΔ5nsP3 was the most potent. Further studies revealed that the region removed from CHIKVΔ5nsP3 vaccine candidate, is apparently involved in interactions with another domain of nsP3 as well as with the C-terminal region of nsP2. These findings provide a platform for further analysis of biological reasons for the attenuation of CHIKVΔ5nsP3 vaccine candidate

Characterisation of nanobodies directed against emerging viruses

The emergence of new viral pathogens, such as SARS-CoV-2, or re-emergence of known pathogens, like CHIKV, point out the need for further understanding of the biology behind viruses, as well as the urgent need for the development of therapeutic and diagnostic tools. Nanobodies, small antigen-binding fragments derived from camelid heavy-chain antibodies, have gained attention for their use in viral research due to their wide range of applications: from the study of protein-protein interactions, uncovering of new viral targets, to the generation of new diagnostic tools or therapeutics. In paper I, we isolated a nanobody, Ty1, targeting the receptor binding domain (RBD) of SARS-CoV-2. We showed the ability of Ty1 to neutralise SARS-CoV-2 pseudotyped lentivirus potently (IC50 of 0.77 μg mL-1). The highly neutralising ability of Ty1 was likely due to its ability to bind the RBD in the ‘up’ and ‘down’ conformations, causing direct blocking to the cellular receptor and steric hindrance, respectively. Moreover, staining of SARS-CoV-2-infected cells with Ty1 confirmed its high specificity. In paper II we made use of a novel and rapid strategy to create nanobody multimers. We first functionalised the nanobodies using sortase A ligation to attach click chemistry functional groups. Then, the functionalised nanobodies were used to create C-to-C terminal bi- and tetravalent nanobody constructs by Cu-free strain-promoted azide-alkyne click chemistry (SPAAC). The bivalent and tetrameric nanobody constructs showed an increased potency with respect to the monomeric Ty1 of 150-fold and 4000-fold, respectively. This was true both for SARS-CoV-2 spike pseudotyped lentivirus and infectious SARS-CoV-2. In paper III, we generated nanobodies targeting the spike complex of CHIKV. We used a combinatory immunisation strategy with a cDNA prime followed by a protein boost. The CHIKV spike complex is formed by homotrimers of heterodimers of the E1 and E2 proteins. While E2 binds to the cellular receptor, E1 is responsible for the fusion of viral and cellular membranes, both essential steps of viral entry. We made use of a bivariate mining approach coupled to NGS and calculation of enrichment (fold difference in frequency between basal and enriched libraries) for the quick selection of nanobodies targeting either protein. We identified 12 nanobodies that detected cells infected with the 3 CHIKV lineages (ECSA, Asian and WA). Surprisingly, neutralisation of the ECSA and Asian genotypes was below 50% for all tested nanobodies, while 2 nanobodies, Dy010 and Dy059 could neutralise the WA lineage above 50% with PRNT50 values of 563 and 722 nM, respectively. Fusion to an Fc fragment produced an increase in potency of 130- and 63-fold for Dy010 and Dy059. Moreover, 4 of the nanobodies, Dy009, Dy025, Dy027 and Dy201 cross-reacted with other alphaviruses including ONNV, RRV and SFV, while one nanobody, Dy007, showed great specificity for CHIKV. These nanobodies expand the toolbox for research of this important human pathogen and could form a basis for the development of therapeutic or diagnostic tools

PROGRAMMED TRANSLATIONAL RECODING SIGNALS AS A THERAPEUTIC TARGET AGAINST ALPHAVIRUSES

While infection from communicable diseases has posed a longstanding threat to human health throughout history, the modern realities of population expansion, global travel, and climate change have facilitated the rapid emergence and worldwide distribution of RNA viruses at an unprecedented scale. Of particular concern are the alphaviruses, mosquito borne viruses from the Togaviridae family. These viruses were previously relegated to rare outbreaks in isolated forested regions but have dramatically spread across the globe in the past decade. One of these viruses, Venezuelan equine encephalitis virus (VEEV), is a noted bioterror threat due to its ability for aerosol transmission and successful weaponization during the Cold War. While no FDA approved drugs exist against alphaviruses, their reliance on programmed translational recoding mechanisms to regulate gene expression presents a potential vulnerability for therapeutic exploitation. Two instances of translational recoding have been identified but poorly characterized in the alphavirus genome. The first is a termination codon readthrough (TCR) event required for expression of the alphavirus replicase. The second is a programmed -1 ribosomal frameshift (-1 PRF) that produces a C-terminally extended variant of viroporin 6K. In this work, the cis-acting RNA elements that mitigate alphavirus recoding were functionally and structurally characterized. The predicted TCR and -1 PRF sequences were cloned into dual luciferase reporter vectors and their ability to promote efficient recoding was verified in several mammalian cell lines. Chemical probing assays elucidated the presence of highly structured stemloop elements downstream of the alphavirus recoding sites, which function as a kinetic trap for elongating ribosomes. Notably, mutations that abrogate efficient -1 PRF not only attenuated pathogenesis of VEEV in mice, but also provided protective immunity to subsequent wild-type challenge. These findings suggest a novel approach to the development of a safe and effective live attenuated vaccine strategy against VEEV, closely related alphaviruses, and potentially all viruses that rely on translational recoding mechanisms for optimal gene expression