Rift Valley fever phlebovirus (RVFV) is an ongoing threat to both humans and animals across the continent of Africa. RVFV is a member of the Phlebovirus genus and Phenuviridae family, within the Bunyavirales order. Members of the Phlebovirus genus are characterised by a negative sense tripartite RNA genome. The large (L) segment encodes the RNA-dependent RNA polymerase (L), the medium (M) segment encodes the two glycoproteins Gn and Gc, and the small segment (S) encodes the nucleocapsid (N) protein and the non-structural protein NSs. The N protein performs a number of important functions, including encapsidation of the viral genome allowing viral RNA replication and transcription. Research into N protein-protein interactions has been limited. The work presented in this thesis characterises previously unidentified functional residues of RVFV N protein and describes new insights into virus-host protein-protein interactions. Two previously uncharacterized N protein residues, F11 and F149, when substituted for alanine, performed all its known functions; Encapsidation of the viral genome, N-N multimerisation and L protein interaction. However, utilising a minigenome assay still showed these mutants lack replication capacity. This indicates that currently unknown interactions with these residues are disrupted. Furthermore, a proteomics study on N protein immunoprecipitated from lung epithelial A549 cell infections was performed to identify RVFV N interaction partners, revealing 23 potential candidates. A subsequent siRNA knockdown of candidates identified β-catenin, Polyadenylate binding protein 1 and 4, Annexin 1 and 2, and Scaffold attachment factor B as important for functional viral replication. Previous research indicated β-catenin, the effector molecule of the WNT pathway, was involved with RVFV replication. Utilising a TOPFlash reporter assay, it was determined that the WNT pathway, of which β-catenin is the effector molecule, was inhibited by RVFV infection. The generation of a CRISPR-Cas9 β-catenin knockout cell line provided a useful tool for further study into N protein-protein and RVFV-β-catenin interactions. The knockout of β-catenin significantly reduced RVFV replication, similarly to siRNA-mediated knock down. Additionally, it was observed through the use of confocal microscopy that upon infection with RVFV, β-catenin relocalised from the plasma membrane to a diffuse pattern across the cytoplasm. Furthermore, during the course of this study, it was investigated whether RVFV N protein can affect mosquito antiviral pathway(s), similarly to yellow fever virus (genus flavivirus) capsid protein. Using alphavirus Semliki Forest virus (SFV) as a model, allowing work to be carried out in a CL-2 lab setting, it was found that N protein does not possess such properties. However, Zika virus (genus flavivirus) capsid protein (ZIKV C) showed significant proviral properties, however, this effect did not occur via disruption of the siRNA pathway, the most efficient mosquito antiviral mechanism, as evidenced by ZIKV C having no effect within our siRNA assay. To summarise, the data in this thesis reveals new interactions between RVFV nucleocapsid protein and mammalian host proteins that are important for RVFV replication. It provides a basis for future research on RVFV (or phleboviruses, in general) nucleocapsid research. The disruption of RVFV N-host protein interactions or direct disruption of N function could lead to new therapeutic strategies against this important emerging virus.

Approximately half the world's human population is at risk of infection from mosquito-borne arboviruses. Currently, interactions between the mosquito antiviral response and infecting arboviruses remains poorly understood; deciphering these will be crucial to the development of novel methods to limit replication and transmission that could help control future outbreaks. Previous mammalian studies have shown that the Homo sapiens Small Ubiquitin-related Modifier (SUMO) pathway plays a fundamental role in multiple aspects of cell biology, including the regulation of host cell immunity. However, this pathway and its impact on arbovirus replication remain uncharacterised in mosquito hosts such as Aedes aegypti (Ae. aegypti; Aa). Comparison between the Ae. aegypti and H. sapiens (Hs) SUMOylation pathways demonstrated a high degree of amino acid sequence and structural similarity. The most notable predicted difference is the lack of ability of AaSUMO to form poly-SUMO chains, which have important functions in H. sapiens. Biochemical analysis of the AaSUMOylation pathway identified a conserved function, and confirmed that AaSUMO could not efficiently form poly-SUMO chains, unlike HsSUMO3 (its closest H. sapiens homologue) due to the absence of an internal SUMO conjugation motif. Catalytically inactive mutants revealed the necessity of AaPIAS (Protein inhibitor of activated STAT) to induce the formation of poly-SUMO chains. Confocal microscopy confirmed that AaSUMO protein is expressed in haemocytes, the salivary glands, ovaries, and midgut, all of which are sites of arboviral replication. Q-PCR investigations have also revealed the AaSUMOylation pathway to be ubiquitously expressed. In vitro depletion of the AaSUMOylation pathway led to significantly enhanced levels of Zika, Semliki Forest, and Bunyamwera virus replication, identifying a vital role for AaSUMOylation in the suppression of these arboviruses. Subsequent studies in H. sapiens cells have also identified a significant role for HsPIAS1 in suppressing the replication of Zika, Semliki Forest, and Bunyamwera viruses. Furthermore, depletion of HsSUMO1 significantly enhanced the replication of Bunyamwera virus, indicating that SUMOylation suppresses arbovirus in a virus-dependent manner. Collectively, these data have identified a novel role for the SUMOylation pathway in suppressing arbovirus replication in both the vertebrate and invertebrate species in which arboviruses replicate.

The paramyxovirus family can cause a broad spectrum of diseases from mild febrile illnesses to more severe diseases that may require hospitalisation and can in the most serious cases have fatal outcomes. Understanding the virus infection dynamics is fundamental to the development of novel targets for therapeutic and vaccine development. The advancement of High-throughput sequencing (HTS) has revolutionised biomedical research providing unparalleled opportunities to answer complex questions. In this study we developed a workflow using directional analysis of HTS data to gain a unique opportunity to simultaneously analyse the kinetics of virus transcription and replication for PIV5 strain W3, PIV2, MuV and PIV3. The workflow could be used for the study of all negative strand viruses. The developed workflow was used to investigate a number of characteristics of paramyxovirus transcription including quantification of the transcription gradient, RNA editing resulting in the generation of non-templated mRNAs and the production of read-through mRNAs. Interestingly, the processivity of the RNA polymerase during transcription was shown to remain consistent throughout the infection amongst all of the viruses analysed. Additionally, virus replication and the generation of antigenomes were found to occur at early times post infection. This was surprising, as the current model for virus replication requires sufficient levels of NP to be present in the cytoplasm before the virus can enter replicative mode. These results suggest a revision of this model in which the virus produces local sites of virus transcription and replication in the cytoplasm known as foci and it is the level of NP surrounding the virus genomes at these local sites that dictates the virus ability to enter a replicative mode. PIV5 strain W3 was shown to supress virus gene expression at late times post infection resulting in the establishment of a persistent infection. The developed workflow was used to analyse the infection dynamics of PIV5. There were no changes in the RNA polymerase processivity of transcription that could account for the suppression of protein synthesis. A comparative analysis of PIV5 strains W3 and CPI+ identified a mutation of a serine to a phenylalanine at position 157 of the P protein in CPI+, a phosphorylation site that when phosphorylated by polo-like kinase 1 (PLK-1) was previously shown to play a role in the inhibition of virus RNA synthesis, that abolished the virus ability to supress protein synthesis and establish a persistent infection. This indicates that phosphorylation of serine at position 157 is responsible for the inhibition of virus gene expression and the establishment of persistence.

As the predominant etiological agent of the “common cold,” human rhinoviruses (HRVs) have a substantial economic impact and contribute to severe respiratory complications in immune compromised and asthmatic individuals. While zinc (Zn) ions have been previously shown to have an inhibitory effect upon HRVs, clinical trials using Zn products have produced conflicting results, and the lack of a known mechanism of Zn inhibition has stymied therapeutic development. Previous research on the potential anti-rhinoviral mechanism of Zn compounds focused upon intracellular processes. My research has demonstrated that extracellular exposure of both major and minor group HRVs to Zn chloride (ZnCl) and Zn gluconate (ZnG) is sufficient to profoundly decrease the infectivity of the viral population. The infectivity of other representatives of the picornavirus family is not decreased in the presence of Zn compounds, suggesting that Zn-mediated virus inhibition is HRV specific. Other metal cations similar to Zn have not demonstrated HRV inhibition. Zn-based inhibition of HRVs is independent of pH, is effected within minutes and is dampened at lower temperatures. Furthermore, whereas EDTA can chelate Zn to prevent inhibition of HRVs, it cannot reverse the Zn-based inhibition after it has occurred. In addition, infectious center plaque assay (ICPA) and competition assay data suggest that this mechanism is not related to the virus-cellular receptor interaction, and that Zn-treated viral capsids are still able to interact with receptor binding sites. Moreover, cultivation and analysis of Zn-resistant HRV1A isolates suggests that genomic discrepancies in the VP1 capsid protein play a role in the mechanism of Zn inhibition. 3 distinct point mutations that conferred amino acid substitutions were found in multiple Zn-resistant isolates. Located in the exterior B-C loop and the interior viral-genome neighboring region of VP1, 2 out of the 3 mutations resulted in a dramatic amino-acid polarity change which likely has the chemical consequence of effectively repelling 2+ cations such as Zn. Furthermore, northern blot analysis reveals that Zn-treated HRVs exhibit an increased susceptibility to genomic RNA degradation, a phenomena that may be facilitated by a Zn-mediated cleavage of viral RNA within the viral capsid. Zn complexed to chelating compounds Hinokital (HK), Pyrithione (PT) and Pyrrolidine Dithiocarbamate (PDTC) also demonstrated extracellular HRV inhibition, with the sequence and ratio of exposure, or precipitate formation, modulating the outcome. It is not clear if these compounds augment the antiviral mechanism of Zn alone, or initiate a distinct antiviral mechanism unrelated to that performed by Zn in isolation. Based upon the data presented here, Zn mediated inhibition of HRVs can occur in a cell-independent, extracellular manner to degrade viral RNA and thereby abrogate viral infectivity. This mechanism may provide novel insight into further therapeutic development of these compounds or template the design of future small molecule therapeutics against these and similar viruses.

acase@tulane.edu / Lassa virus (LASV) is the causative agent of Lassa Fever (LF), an acute and occasionally fatal disease with hemorrhagic features. Current treatments for LF are limited to ribavirin and supportive care. The objective of this dissertation project was to characterize a human monoclonal antibody (mAb) with the potential to treat LASV infections. This antibody, mAb 8.9F, was isolated from the serum of a convalescent patient and has been shown to protect guinea pigs from lethal LASV challenge. Our approach focused on confirming that mAb 8.9F bound a quaternary neutralizing epitope (QNE), determining which residues comprised the epitope, and examining how mutations to critical residues would impact viral fitness. Our results showed that mAb 8.9F shared a number of features similar to previously characterized QNEs including broad neutralization of viral subtypes and high sensitivity to epitope disruption by detergents. We found that mAb 8.9F recognizes sites on both the GP1 and GP2 subunits of the LASV glycoprotein. Both subunits dissociate from the antibody at similar rates under chaotropic conditions, suggesting that these subunits have equal binding affinity with mAb 8.9F. To identify amino acids important to the epitope, we used a method of knockout site-directed mutagenesis that replaced sequences of LASV glycoprotein with homologous sequences from lymphocytic choriomeningitis virus. We created single mutations for those regions unreactive with mAb 8.9F and identified H124F, P145R, and F147N as mutations capable of abrogating mAb 8.9F reactivity. Viral fitness of these mutants was evaluated by measuring glycoprotein processing and viral infectivity. Our results showed that mutations abrogating mAb 8.9F binding did not significantly inhibit glycoprotein production, processing, or surface transport; neither was pseudovirus formation affected. However, these mutations did significantly reduce pseudovirus infectivity suggesting that mutants escaping mAb 8.9F neutralization may be less fit. The role of N-linked glycosylation in mAb 8.9F recognition was also examined but results proved inconclusive. In summary, this work provides a detailed analysis of the first documented QNE found on the Lassa virus glycoprotein. This work will help direct rational vaccine design and post-exposure antibody therapy. / 0 / Benjamin Bradley

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Ljungan virus är ett virus som isolerades i Sverige i mitten på nittiotalet. Under perioden 1989-1992 avled flera svenska elitorienterare plötsligt i hjärtmuskelinflammation. Man misstänkte att orienterarna kunde ha utsatts för en vektorburen infektion eftersom de exponeras för djur som finns i skog och mark under träning och tävling. Det är sedan tidigare känt att sorkar är den naturliga reservoaren för ett annat virus (Puumala virus) som kan orsaka njurskada hos människor. Sorkantalet i vissa delar av Sverige varierar kraftigt från år till år i ett cykliskt förlopp. Man fann ett samband mellan antalet sorkar och förekomsten av hjärtmuskelinflammation, typ 1 diabetes och Guillain-Barre's syndrom vilket ledde till att ett tidigare okänt virus, Ljungan virus, kunde isoleras från sorkar. Detta virus är ett litet RNA-virus som tillhör familjen Picornavirus. Till denna familj hör också flera kända virus såsom många av våra vanligt förekommande förkylningsvirus, men också virus som kan orsaka svåra sjukdomar, till exemel poliovirus och mul- och klövsjukevirus. Ljungan virus är ett nyupptäckt virus och därför är kunskapen om viruset begränsad. För att öka vår förståelse om viruset så har arvsmassan för tre svenska isolat (87-012, 174F och 145SL) av Ljungan virus kartlagts (artikel IV). Denna studie visade att Ljungan virusets arvmassa har flera unika egenskaper. Släktskapstudier visade att Ljungan virus är endast avlägset släkt med redan kända picornavirus och viruset bör därför utgöra en egen undergrupp i familjen (artikel III). Med kunskap om Ljungan virusets arvsmassa så var det möjligt att visa att Ljungan virus förekommer även på andra ställen än i Sverige (artikel VI). I mitten på 60-talet isolerades ett virus, M1146, från sork som fångats i Oregon, USA. Baserat på egenskaper hos proteinhöljet (kapsiden) så antog man då att M1146 var ett picornavirus. Studier av arvsmassan för detta virus visade att M1146 är närmast besläktat med de svenska Ljungan virus isolaten och har samma unika egenskaper i sin arvsmassa (artikel VI). Dessa studier har varit möjliga eftersom vi tidigare har utvecklat en metod för att producera stora mängder av hela arvsmassan (artikel I och II). Slutligen har det proteinhölje som innesluter och skyddar Ljungan virusets arvsmassa studerats (artikel V). Dessa studier visade att kapsiden är uppbyggd av tre proteiner och inte fyra som hos de flesta picornavirus. Dessa studier underlättades av att en enkel och effektiv metod för att odla Ljungan virus i provröret har utvecklats (artikel V). Sammantaget så har dessa studier försett oss med nya kunskaper om Ljungan virus som möjliggör fortsatta studier av dess biologi och eventuella förmåga att orsaka sjukdom hos människor och djur.

It is today well known that the outcome of a certain infection depends on factors of both the host and the pathogen. Studies of host genetic susceptibility to infectious diseases aim to increase the understanding of why some individuals are more susceptible than others, to a certain infection. Knowledge of genetic susceptibility to a viral disease may be used in development of new therapeutic means, and also to recognize individuals who are at increased risk of severe symptoms if infected with a pathogen. It seems however that a risk factor for one disease may play a protective role in another situation; like a double-edged sword. In this thesis I have studied genetic factors affecting susceptibility to norovirus (NoV) and factors affecting the risk of developing tick-borne encephalitis (TBE) after infection with TBE virus (TBEV). NoV is the cause of the “winter vomiting disease”, affecting millions of people every year, and causing up to 200,000 fatalities among children in developing countries, each year. It is today recognized that the secretor status of an individual, i.e. the ability to express ABO blood groups and related antigens, in secretions and on mucosa, affect the risk of being infected by NoV. By studying authentic NoV outbreaks in Denmark, Spain and Sweden and by comparing the secretor status of affected and unaffected individuals we were able to confirm that secretor status have indeed great impact on susceptibility to some NoV strains, but also that there are strains circulating, which infect individuals regardless of secretor status. TBEV is endemic in many parts of Europe and Asia but studies have shown that 70-95% of all infections are asymptomatic or sub-clinical. Some individuals do however develop TBE, a severe disease including meningitis or encephalitis with or without myelitis. Also, many patients suffer from long-time sequelae and TBEV infections may in worst case be fatal. The reason for difference in disease outcome is not known and we have chosen to study if genetic factors affecting the immune response may play a role in disease outcome. To do this we used a prospectively collected Lithuanian material with samples from patients with TBE, AME (aseptic meningoencephalitis) and matched healthy controls. So far we have found that a deletion in chemokine receptor 5 (CCR5), a gene encoding a receptor involved in cell migration, is a risk factor for developing disease. We have also data showing that toll-like receptor 3 (TLR3), a receptor recognizing double stranded RNA (dsRNA), which is a product of TBEV replication, may instead of being protective increase the risk of TBE.