Interplay Between Tick-Borne Encephalitis and the Host Innate Immunity

Chaitanya Kurhade

Department of Clinical Microbiology Umeå 2017

This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7601-821-7 ISSN: 0346-6612 Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU Print Service, Umeå University Umeå, Sweden 2017

Table of Contents

Abstract ...... iii Enkel sammanfattning på svenska ...... v List of Publications ...... vii Abbreviations ...... viii Introduction and background ...... 1 1. ...... 1 1.1 Tick-borne encephalitis virus ...... 2 1.2 Langat virus ...... 3 1.3 ...... 3 1.4 ...... 4 1.5 virus ...... 5 2. Virion structure, genome organization, and viral proteins of ...... 6 3. The life cycle of flaviviruses ...... 8 4. Pathogenesis of neurotropic flaviviruses ...... 10 5. Innate immune recognition ...... 13 5.1 Toll-like receptors ...... 13 5.2 RIG-I-like receptors ...... 14 5.3 Innate recognition of flaviviruses ...... 14 6. IFN signalling ...... 16 7. Interferon-stimulated genes ...... 17 7.1 Viperin ...... 19 7.2 Interplay of viperin with the immune system ...... 20 8. Brain heterogeneity and the innate immune response in the CNS ...... 21 Aims ...... 23 Methodological considerations ...... 24 1. Quantification of virus burden ...... 24 2. Studies in mice ...... 24 3. Primary cells ...... 25 4. Detection and quantification of protein ...... 25 5. Protein-protein interaction studies ...... 26 6. Gene expression and quantification ...... 26 7. Fluorescence-based quantification and localization ...... 26 8. Immunohistochemistry ...... 27 9. Flow cytometry ...... 27 Results and discussion ...... 28 1. To investigate the role of IPS-1 in tick-borne flavivirus infection and immunity (Paper I) ...... 28

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2. To understand the in vivo role and mechanism of action of the antiviral protein viperin in flavivirus infection (Papers II and III) ...... 30 2.1 To understand the in vivo role of the antiviral protein viperin in flavivirus infection (Paper II) ...... 30 2.2 To understand the mechanism of action of the antiviral protein viperin in flavivirus infection (Paper III) ...... 32 3. To investigate a possible correlation between the disease severity of TBEV strains and their pathogenesis in mice (Paper IV) ...... 34 Concluding remarks ...... 36 Acknowledgements ...... 38 References ...... 40

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Abstract

Flaviviruses are important emerging and re-emerging arthropod-borne pathogens that cause significant morbidity and mortality in humans. It consists of globally distributed human pathogens such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), virus (YFV), (DENV), and Zika virus (ZIKV). Depending on type, flaviviruses can cause a variety of symptoms ranging from haemorrhage to neurological disorders.

Virus infection is detected by host pattern recognition receptors (PRRs), and through downstream signalling it leads to the production of interferons (IFNs). These IFNs then act in an autocrine or paracrine manner on the cells to induce various IFN-stimulated genes (ISGs), which have antiviral roles. However, the amount of IFN produced depends on the nature of the PRRs used by host cells to detect a particular virus. Although there are many PRRs present in the host cells, their relative contribution in different cell types and against a specific virus may vary. In the first study, we determined the importance of IPS-1 signalling in immunity and pathogenicity of tick-borne flaviviruses. This is an adaptor protein for cytoplasmic RIG-I-like receptors. Using IPS-1-deficient mice, we showed its importance against TBEV and Langat virus (LGTV) infection (the LGTV model virus belongs to the TBEV serogroup). Absence of IPS-1 leads to uncontrolled virus replication in the central nervous system (CNS), but it has only a minor role in shaping the humoral immune response at the periphery. LGTV-infected IPS- 1-deficient mice showed apoptosis, activation of microglia and astrocytes, an elevated proinflammatory response, and recruitment of immune cells to the CNS. Interestingly, we also found that IFN-b upregulation after viral infection was dependent on IPS-1 in the olfactory bulb of the brain. Thus, our results suggest that local immune microenvironment of distinct brain regions is critical for determination of virus permissiveness.

Interferons can upregulate several ISGs. Viperin is one such ISG that has a broad- spectrum antiviral action against many . However, the importance of cell type and the significance of viperin in controlling many flavivirus infections in vivo is not known. Using viperin-deficient mice, we found that viperin was necessary for restriction of LGTV replication in the olfactory bulb and cerebrum, but not in the cerebellum. This finding was also confirmed with primary neurons derived from these brain regions. Furthermore, we could also show the particular importance of viperin in cortical neurons against TBEV, WNV, and ZIKV infection. The results suggested that a single ISG can shape the susceptibility and immune response to a flavivirus in different regions of the brain.

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Although viperin is such an important ISG against flaviviruses, the exact molecular mechanism of action is not known. To understand the mechanism, we performed co-immunoprecipitation screening to identify TBEV proteins that could interact with viperin. While viperin interacted with the prM, E, NS2A, NS2B, and NS3 proteins of TBEV, its interaction with NS3 led to its degradation through the proteosomal pathway. Furthermore, viperin could reduce the stability of other viperin-binding TBEV proteins in an NS3-dependent manner. We screened for viperin activity regarding interaction with NS3 proteins of other flaviviruses. Viperin interacted with NS3 of JEV, ZIKV, and YFV, but selectively degraded NS3 proteins of TBEV and ZIKV, and this activity correlated with its antiviral activity against these viruses.

The last study was based on in vivo characterization of the newly isolated MucAr HB 171/11 strain of TBEV which caused unusual gastrointestinal and constitutional symptoms. This strain was compared with another strain, Torö- 2003, of the same European subtype of TBEV but isolated from the different focus. Here we found unique differences in their neuroinvasiveness and neurovirulence, and in the immune response to these two strains.

In summary, my work shed some light on the interplay between tick-borne flavivirus and the innate immune system. I have shown two examples of CNS region-specific differences in innate immune response regarding both in IFN induction pathways and antiviral effectors. Furthermore, we have investigated the in vivo pathogenesis of a strain of TBEV that caused unusual gastrointestinal and constitutional symptoms.

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Enkel sammanfattning på svenska

Flavivirus finns spridda över hela världen och orsakar miljontals infektioner varje år. Några av de medicinsk mest viktiga flavivirusen är fästingburen encefalit virus (TBEV), West Nile virus (WNV), Japansk encefalit virus (JEV), gula febern (YFV) och Zika virus (ZIKV). Dessa virus kan orsaka olika komplikationer till exempel blödarfeber och hjärninflammation.

Vid en infektion så upptäcker värdcellen virusinfektionen med hjälp av speciella receptorer, så kallade PRRs. Dessa finns i alla celler och känner igen viruskomponenter som normalt inte finns i en oinfekterad cell. När PRRs detekterar en virusinfektion svarar cellen med att tillverka ett signal protein interferon (IFN). IFN skickas ut ur cellen och hämmar virusinfektioner genom att sätta igång ett försvarsprogram i andra celler bestående av hundratals försvarsproteiner som kan motverka virusinfektionen. Vilka PRRs som behövs för att detektera ett virus är olika vid olika virusinfektioner. I första studien fann vi att IPS-1 är av yttersta vikt för skydda mot fästingburna flavivirus. IPS-1 är ett så kallat adapter protein som behövs för att två PRRs, RIG-I och MDA-5, ska kunna förmedla signaler som leder till IFN tillverkning. Med hjälp av möss som saknar IPS-1 fann vi att IPS-1 behövs för att tillverka IFN protein och skydda mot fästingburna flavivirus. IPS-1 var särskilt viktigt för interferon produktion inom luktloben i hjärnan. Därför kunde vi dra slutsatsen att immunresponsen regleras olika inom olika delar av hjärnan.

Ett försvarsprotein som visat sig vara särskilt viktig vid virusinfektion är viperin. Viperin har visat sig kunna hämma en rad olika virus men den specifika rollen av viperin in vivo vid flavivirus infektion var inte fullt känd. Vi fann att viperin behövs för att hämma LGTV i lukloben och storhjärnan men inte i lillhjärnan. Vi kunde bekräfta detta med hjälp av primära nervceller isolerade från dessa hjärnregioner. Vi fann även att viperin var av yttersta vikt för att kontrollera TBEV, WNV och ZIKV infektion i nervceller från hjärnbarken (del av storhjärnan). Därför kunde vi dra slutsatsen att ett enskilt försvarsprotein kan avgöra mottagligheten mot flavivirus inom olika hjärnregioner.

Trots att viperin är så viktig för att skydda mot flavivirus så vet vi inte hur viperin åstadkommer detta. Därför ville vi undersöka hur viperin kan förmedla sin antivirala effekt. Vi fann att viperin kan binda till flera TBEV proteiner, men att viperin specifikt kan bryta ner ett virusprotein som heter NS3. NS3 är väldigt viktigt för att flavivirus ska kunna etablera en infektion och kunna föröka sig. Eftersom vi visste att viperin kan hämma andra flavivirus ville vi veta om viperin även förstör NS3 från JEV, ZIKV och YFV. Vi upptäckte att viperin kunde binda till NS3 hos alla dessa flavivirus men att viperin specifikt förstörde TBEV och

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ZIKV NS3, intressant nog så kunde viperin endast hämma dessa virus infektioner men inte JEV och YFV.

I den sista studien ville vi karaktärisera en ny TBEV stam som bara orsakar mag- och tarmbesvär men inga neurologiska symptom. TBEV har aldrig tidigare visat sig kunna orsaka detta och därför ville vi undersöka saken vidare. Vi fann att denna TBEV stam skiljde sig mot en närbesläktad stam genom att orsaka en starkare immunrespons men mildare sjukdomsförlopp.

Sammanfattningsvis har jag undersökt samspelet mellan fästingburna flavivirus och det medfödda immunförsvaret. Jag har även visat att immunresponsen regleras olika inom olika hjärnregioner, både beträffande IFN inducering och antivirala proteiner. Vidare har jag hittat mekanismen för hur viperin proteinet hämmar TBEV och ZIKV, vilket var genom att förstöra NS3. Dessutom har jag karaktäriserat sjukdomsförloppet hos möss efter infektion med en ovanlig TBEV stam som orsakar mag och tarm besvär.

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List of Publications

I. Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1. Chaitanya Kurhade, Loreen Zegenhagen, Elvira Weber, Sharmila Nair, Kristin Michaelsen-Preusse, Julia Spanier, Nelson O Gekara, Andrea Kröger, Anna K Överby. Journal of Neuroinflammation 2016 13:22. doi: 10.1186/s12974-016-0487-9.

II. Viperin restrict neurotropic flavivirus infection in cell type and region-specific manner. Richard Lindqvist*, Chaitanya Kurhade*, Jonathan Gilthorpe, Anna K Överby. Manuscript submitted to Journal of Neuroinflammation.

III. Viperin targets Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation. Christakis Panayiotou, Richard Lindqvist, Chaitanya Kurhade, Kirstin Vonderstein, Arunkumar S. Upadhyay, Jenny Pasto, Karin Edlund, Anna K Överby. Manuscript submitted to Journal of Virology

IV. Correlation of disease severity in human tick-borne encephalitis and pathogenicity in mice. Chaitanya Kurhade*, Yi-Ping Lee*, Sarah Schreier*, Loreen Zegenhagen, Andrea Kröger, Anna K Överby. Manuscript submitted to Emerging Infectious Diseases (Dispatch format)

* Authors contributed equally

Articles reprinted with permission of the publisher.

Review not included in the thesis

I. Brain heterogeneity leads to differential innate immune responses and modulates pathogenesis of viral infections. Loreen Zegenhagen, Chaitanya Kurhade, Anna K Överby, Andrea Kröger. Cytokine Growth Factor Review. 2016 doi: 10.1016/j.cytogfr.2016.03.006.

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Abbreviations

APOBEC3 Apolipoprotein B editing complex BBB Blood-brain barrier BSL Biosafety level C Capsid CARD Caspase activation and recruitment domain CCR5 CC chemokine receptor type 5 CD Cluster of differentiation CHIKV virus CNS Central nervous system Co-ip Co-immunoprecipitation D.p.i. Days post infection DENV Dengue virus Ds Double-stranded E Envelope EBV Epstein-Barr virus ER Endoplasmic reticulum EV71 Enterovirus 71 FFA Focus-forming assay Ffu Focus-forming unit GAS Interferon gamma-activated sequence GBF-1 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 HCMV Human cytomegalovirus HCV Hepatitis C virus HeV HF Host factors HIV Human immunodeficiency virus H.p.i. Hours post infection HSV Herpes simplex virus HTLV Human T-lymphotropic virus I.c. Intracranial IFIT Interferon-induced protein with tetratricopeptide repeats IFITM Interferon-induced transmembrane protein IFN Interferon IFNAR Interferon-α/β receptor IFNGR Interferon Υ receptor IFNLR Interferon λ receptor IHC Immunohistochemistry IKK IkB kinase JCV JC virus

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IL Interleukin IL10RB Interleukin 10 receptor subunit beta I.p. Intraperitoneal IPS-1 IFN-β promoter stimulator-1 IRAK Interleukin-1 receptor-associated kinase Irg1 Immune responsive gene 1 IRF Interferon regulatory factor ISG Interferon-stimulated gene ISGF3 Interferon-stimulated gene factor 3 ISRE Interferon-sensitive response element JAK Janus kinase JEV Japanese encephalitis virus LGTV Langat virus LACV La Crosse virus MAV Mouse adenovirus MAVS Mitochondrial antiviral signalling protein MDA-5 Melanoma differentiation-associated protein 5 MEF Mouse embryonic fibroblast MX Myxovirus resistance MyD88 Myeloid differentiation primary response 88 NF-kB Nuclear factor kappa-light-chain enhancer of activated B cells NMJ Neuromuscular junction NS Non-structural NTPase Nucleoside triphosphatase Oas Oligoadenylate synthetase-dependent ORF Open reading frame PBMC Peripheral blood mononuclear cell pDC Plasmacytoid dendritic cell PKR Protein kinase R PNS Peripheral nervous system PrM Pre-membrane PRV Pseudorabies virus PRR Pattern recognition receptor RABV virus RC Replication complex RIG-I Retinoic acid-inducible gene-I RSAD-2 Radical S-adenosyl methionine domain containing protein 2 RSV Respiratory syncytial virus Ss Single-stranded SIV Simian immunodeficiency virus SOCS Suppressor of cytokine signalling STAT Signal transducer and activator of transcription TBE Tick-borne encephalitis

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TBEV Tick-borne encephalitis virus TBK TANK binding kinase TIR Toll/interleukim-1 receptor TLR Toll-like receptor TRAF TNF receptor associated factor TRIF TIR-domain-containing adapter-inducing interferon-β TRIM Tripartite motif Tyk Tyrosine kinase USP18 Ubiquitin specific peptidase UTR Untranslated region VISA Virus-induced signalling adapter VSV Vesicular stomatitis virus VZV Varicella zoster virus WNV West Nile virus WT Wild-type YFV Yellow fever virus ZIKV Zika virus

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Introduction and background

1. Flavivirus

The genus flavivirus belonging to the family consists of more than 70 enveloped RNA viruses. Most of them are arthropod-borne viruses and are transmitted by mosquitoes or ticks. More than 50% of the flaviviruses have been associated with disease in humans. Some of the important human pathogens including yellow fever virus (YFV), dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) belong to this genus. Based on the type of disease and the symptoms, these viruses can be classified into ones that can cause vascular leak and haemorrhage, including DENV and YFV, and neurotropic flavivirus, which can cause neurological disorders (e.g. encephalitis), including WNV, TBEV, ZIKV, and JEV [1]. Flaviviruses have a worldwide distribution, but individual species are restricted to specific endemic or epidemic areas (e.g. YFV in tropical and subtropical regions of Africa and South America; DENV in tropical areas of Asia, Oceania, Africa, Australia, and the Americas; JEV in Southeast Asia; and TBEV in Europe and Northern Asia). With the changes in climate and the socio-political scenario across the world, flaviviruses are being introduced into new geographical areas. An outbreak of WNV occurred in New York City in 1999, and from there it spread throughout much of North America and Central America. In 2015-2016, ZIKV emerged in the Americas, with its associations with Guillain–Barré Syndrome in adults and microcephaly in children (Figure 1).

ZIKV ZIKV

Figure 1. Worldwide distribution of flaviviruses. Distribution of TBEV, ZIKV, WNV, JEV, YFV, and DENV are indicated. The modified illustration was reprinted with permission of the publisher [2].

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1.1 Tick-borne encephalitis virus

TBEV is one of the most important causes of arthropod-borne viral diseases in Europe and Russia, with an annual incidence of around 13,000 [3]. TBEV was initially isolated in 1937, during the Soviet era [4]. Now TBEV is endemic in much of Europe and Russia. In Sweden, there has been a sharp increase in cases of TBEV: from January to October 2017, 362 cases were reported, as compared to 238 cases during the whole of 2016. In nature, the virus is maintained in a zoonotic cycle with ticks being the vector and reservoir of the virus and rodents being host for the ticks. Virus is transmitted to humans through tick bites or consumption of infected milk [5, 6]. The infection is characterized by a biphasic course in the primary phase after the tick bite; the incubation period is 2–14 days and patients show non-specific symptoms such as signs of fatigue; headache; and pain in the neck, shoulders, and lower back―accompanied by high fever and vomiting. After about 8‒10 days of remission, the second phase of the disease occurs in 20‒30% of patients. It consists of neurological involvement with symptoms of encephalitis (inflammation of brain parenchyma), meningitis (inflammation of the meninges), myelitis (inflammation of the spinal cord), and paralysis. 30‒60% of patients develop neurological sequelae [7-10]. A post-TBE encephalitis syndrome exists in around 46% of patients, which causes lifelong morbidity and affects quality of life. Most commonly reported are various cognitive or neuropsychiatric complaints, balance disorders, headache, dysphasia, hearing defects, and spinal paralysis [11]. Such long-lasting effects of TBEV mean long-term costs and burden on individuals and society. The seroprevalence of TBEV in European countries varies from 0% to 5%. However, it can be as high as 20‒40% in highly exposed professionals such as farmers and woodcutters. Effective vaccine is available against TBEV; however, treatment options are limited to supportive care [12]. Meningitis, encephalitis, or meningoencephalitis require hospitalization and supportive care based on the severity. Anti-inflammatory drugs, such as corticosteroids, may be considered under specific circumstances, for symptomatic relief. Intubation and ventilator support may be necessary [13].

TBEV subtypes

TBEV has been classified into three main subtypes, European (TBEV-Eur), Siberian (TBEV-Sib), and Far-Eastern (TBEV-FE), based on phylogenetic analysis, with differences of 15.2–16.4% at the nucleotide level and 6.2–6.9% at the amino acid level [14]. Two new subtypes (strain 178-19 and strain 886-84) have been claimed by Russian virologists [15]. The TBEV-Eu subtype is found in Europe and in some patches in Russia, and can cause mortality in 1‒2% of

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patients. TBEV-Sib is found in eastern Europe (including Finland), western Russia, and Siberia, and the mortality rate is the 6‒8% range, whereas TBEV-FE can be found from eastern Russia to Japan and the mortality is about 30% [14]. In nature, TBEV is maintained in Ixodes species ticks―TBEV-Eur mainly in I. ricinus and TBEV-Sib and TBEV-FE mainly in I. persulcatus. Generally speaking, these TBEV subtypes can be found according to the geographical distribution of their tick vector species [9].

The frequency and development of the clinical course of certain forms of disease varies within different subtypes. European strains usually take the biphasic form of the disease in 72‒87% of patients [16]. However, Siberian and Far-Eastern TBEV can cause prolonged or chronic forms of disease [17]. Siberian subtype strains are associated with focal encephalitic forms with an incidence of 5%, meningeal forms (almost 47%), febrile forms (40%), and biphasic forms in about (21%). However, in Far-Eastern TBE there are focal encephalitic symptoms in 31‒ 64% of cases, meningeal forms in 26%, febrile forms in 14‒16%, and biphasic forms in 3‒8% [18]. Mutational changes can influence the neuroinvasivess and neurovirulence properties, and can cause unusual symptoms [19, 20].

1.2 Langat virus

Langat virus (LGTV) is a member of the TBEV complex; it was isolated in Malaysia in 1956 from a hard tick of the genus Ixodes [21]. There are no known natural cases of human disease associated with LGTV. In the past, LGTV was tested as a live attenuated vaccine against TBEV, but it was discontinued as 1 person in 10,000 vaccinated developed encephalitis [7]. LGTV is a naturally attenuated member of the TBEV serogroup and it has a close molecular relationship (82‒88% amino acid identity) with TBEV. This makes it an ideal surrogate model for the study of TBEV pathogenesis in a biosafety-level-2 laboratory setting [22].

1.3 Zika virus

ZIKV was first isolated in the Zika forest in Uganda, in 1947, and it remained hidden for nearly 70 years [23]. A seroprevalence to ZIKV of about 6.1% was found in samples from several areas of Uganda, indicating that human infections were prevalent, but it did not cause any major outbreak [24]. In 2013‒2014, cases of ZIKV infection started in French Polynesia and then in March 2015, ZIKV was introduced into Brazil and spread rapidly throughout the Americas [25]. In African forests, ZIKV persists in the transmission cycle between Aedes mosquitoes and non-human primates, while in an urban environment Zika virus

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is transmitted in a human-mosquito-human transmission cycle. Recent studies have indicated that ZIKV can also be transmitted from mother to foetus, and also by the sexual route [26]. ZIKV symptoms start to appear after 3‒12 days from the mosquito bite, and the majority of cases are asymptomatic. Mild fever, skin rashes, conjunctivitis, muscle pain, joint pain, back pain, malaise, and headache are some of the common symptoms, which are generally indistinguishable from those caused by other flaviviruses such as DENV [27]. Recently, more severe clinical sequelae have increasingly been associated with ZIKV. ZIKV infection during pregnancy is associated with foetal microcephaly, which involves disorders of the foetal brain development and shrinking of head size, which is followed by cognitive and physical delays [28]. An astonishing 42% of foetuses have been found to show some kind of ultrasound abnormality in ZIKV-infected pregnant women in Brazil [29]. ZIKV infection in adults has been associated with severe neurological sequelae including meningitis, meningoencephalitis, and Guillain‒Barré syndrome [30]. As compared to other encephalitic flaviviruses such as TBEV and WNV, ZIKV is less neuroinvasive in adults and rarely causes encephalitis or meningitis [31]. It preferentially infects and injures neural progenitor cells, and is thus more prone to cause neurodevelopmental problems in the fetus [32]. ZIKV can also infect the eye and cause uveitis and conjunctivitis [33, 34]. There are currently no ZIKV-specific therapeutics or vaccines available [35]. Currently, vector control and travel restrictions to endemic areas are the only interventions available. However, the multiple routes of transmission (arthropod vector, sexually, via blood, and possibly via other body fluids) may increase risk of transmission of ZIKV.

1.4 West Nile virus

WNV was first isolated from a blood sample of a patient from the West Nile province of Uganda in 1937 [36]. This virus is one of the most widely distributed , with an extensive distribution throughout Africa, the Middle East, parts of Europe, South Asia, and Australia [37]. WNV is maintained in an enzootic bird-mosquito-bird cycle. It is transmitted mainly by the Culex mosquito species. Additional routes of transmission include blood transfusions, organ transplants, exposure in a laboratory setting, and from mother to baby during pregnancy, delivery, or breastfeeding [38, 39]. Birds act as the carrier of WNV, and humans and horses are the dead-end hosts―as they do not sufficiently amplify virus for transmission by mosquitoes [40]. Most of the infections are asymptomatic, but a few individuals develop symptoms such as headache, body aches, joint pains, vomiting, diarrhea, and rash. Most of the cases recover completely, but fatigue and weakness can last for weeks or months. Less than 1% of infected individuals develop neurological disease such as encephalitis, meningitis, or acute flaccid paralysis and death [41]. The fatality rate for hospitalized encephalitic cases is

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approximately 10%, but it can be higher in older age groups and in patients with a compromised immune system. There is no specific treatment available, and an approved vaccine is only available for horses and not for humans [38, 42].

1.5 Japanese encephalitis virus

Japanese encephalitis is associated with the majority of encephalitis cases in Asia, causing an estimated 50,000 clinical cases and 15,000 deaths annually in humans [43]. JEV is endemic in Southeast Asia, eastern parts of the Russian Federation, parts of Australia, and a few Western Pacific islands such as Saipan and Papua New Guinea [44]. JEV was first isolated in Japan in 1935, but its presence had been described as early as 1870 [45]. JEV is an transmitted in an enzootic cycle among mosquito vectors and vertebrate hosts. A variety of mosquito species may act as vectors in the enzootic cycle, but culicine mosquitoes (primarily Culex tritaeniorhynchus) act as the principal vector for human infection. Pigs and birds act as carrier and virus-amplifying host for transmission to humans. Ardeid birds also contribute to the long-distance dissemination of JEV into new geographic locations [46]. Humans and horses are dead-end hosts for the virus [47]. JE typically develops in patients after an incubation period of 5–15 days, followed by non-specific flu-like symptoms, including fever, headache, malaise, and vomiting, that may last for several days. This mild febrile illness is followed by the acute encephalitic phase involving neurological symptoms. For JEV, 20–30% of cases are fatal, and 30–50% of survivors have significant neurological sequelae [48]. No specific antiviral drugs are available to treat JEV infection. However, inactivated vaccine based on JEV derived from mouse brain has been available for many years (using the prototype strain). Currently, many new JEV vaccines based on live attenuated and cell culture inactivated vaccines are available on the market [49, 50].

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2. Virion structure, genome organization, and viral proteins of flaviviruses

The mature flavivirus virion is smooth and spherical, with a diameter of around 500 Å. The viral genome consists of a positive-sense single-stranded RNA genome of ~11 kb with a single open reading frame (ORF) flanked by 5´- and 3´- untranslated regions (UTR) [51]. It codes for a single polyprotein, which is co- translationally and post-translationally modified into mature proteins. The genes code for three structural proteins: capsid (C), pre-membrane (prM), and envelope (E); and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [52], many of which are multifunctional, being responsible for viral replication and perturbation of the immune system (Figure 2).

Figure 2. Schematic representation of flavivirus genome organization and polyprotein processing. Flavivirus genome which consist of single ORF is translated to polyprotein, which is then cleaved by viral and host protease.

The E protein forms the glycoprotein shell of the virus and shares ∼40% amino acid identity with other flaviviruses. This protein is important for binding of the virus to its cellular receptors and for entry into the host cell. Thus, it’s an important determinant of viral tropism, and host antibodies are directed against it [53]. E and prM form the envelope of immature virus particles, while cleavage of prM to M by host furin protein leads to maturation of virus particles [54]. Multiple C subunits bind together to form the nucleocapsid encasing the RNA genome. C protein has been found to localize to the nucleus in many flavivirus species including DENV, JEV, and TBEV, and in some cases nuclear localization of C has been shown to enhance viral replication [55-57]. During flavivirus

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infection, NS1 can exist as a monomer, as a membrane-bound dimer, or as a secreted hexamer. NS1 has an important role in RNA replication and localizes to the endoplasmic reticulum (ER), but it can also localize to the cell surface. Mutation of the N-linked glycosylation sites in NS1 have an effect on viral RNA replication [58, 59]. NS2A interacts with NS3, NS5, and the 3´-UTR of virus RNA and plays a role in replication, packaging, and assembly of the virus [51, 60]. NS2B forms a stable complex with the N-terminus of NS3, and acts as a cofactor for the NS2B-NS3 serine protease [61, 62]. NS3 is a multi-domain protein with an N-terminal NS3 protease and a C-terminal portion containing the RNA triphosphatase and RNA helicase activities involved in capping and synthesis of viral RNA [63, 64]. NS4A takes part in viral replication and co-localizes in the replication complex. During flavivirus infection, NS4A is also involved in the formation of vesicular packets on the ER [60]. NS4B co-localizes with viral RNA and NS3 in ER-derived replication complex (RC) and plays a role in viral replication [65, 66]. NS5 is the largest and most conserved viral protein. Its N- terminal domain consists of guanyltransferase and S-adenosyl methionine methyltransferase activity, which helps in the formation of the 5´ RNA cap structure. The C-terminal end contains an RNA-dependent RNA polymerase domain that mediates RNA replication [67]. The 5′-UTR and the 3′-UTR contain RNA sequence motifs that are involved in viral RNA translation, replication, and possibly packaging. The sequence composition, length, and exact location can vary considerably between different members of the genus, in particular between tick-borne and mosquito-borne flaviviruses [68, 69]. Along with these roles in the virus life cycle, viral proteins also play an important role in counteracting the host immune system―and thus enhance flavivirus infection [1].

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3. The life cycle of flaviviruses

Flaviviruses replicate in the cytoplasm of susceptible cells. Several cellular molecules capable of mediating virus attachment are known, but no specific receptor for internalization of these viruses into host cells has yet been identified [70]. The virus attaches to the cell surface, which is mediated by the E protein, and it enters the cell by receptor-mediated endocytosis [71, 72]. Following internalization, the low pH of the endosome triggers structural rearrangements in the viral glycoproteins; these drive fusions of the viral and endocytic membranes to release the viral RNA into the cytoplasm [73, 74]. The RNA is directly translated as a single polyprotein. It is then co-translationally and post- translationally processed by the virus-encoded serine protease NS2B/NS3, and by host-encoded proteases, including signalase and furin, to produce the structural proteins and non-structural proteins in the order C-prM-E-NS1-NS2A- NS2B-NS3-NS4A-NS4B-NS5 [52]. The non-structural proteins actively replicate the viral RNA in replication complexes associated with the rough endoplasmic reticulum (RER) and in Golgi-derived membranes called vesicle packets (VPs) [75]. The non-structural proteins and dsRNA are concentrated in the VPs, which are the sites of viral RNA synthesis (Figure 3). After translation of input genomic RNA, the RNA-dependent RNA polymerase NS5 copies complementary minus- stand RNA from genomic RNA, which serves as a template for the synthesis of new positive-stranded viral RNA [76].

Figure 3. Flavivirus replication vesicle. Flavivirus replicate at the ER surface in the infected cell. The non- structural proteins along with host factors (not shown) actively replicate the viral RNA in replication complexes which are formed by invaginations of the ER membranes called as vesicle packets (VPs).

The first step in the particle assembly process is coating of the newly synthesized viral RNA with the C protein. Next, E and PrM hetero-dimerize in the ER membrane and the nucleocapsid bud into the lumen of the rough ER envelope the nucleocapsid, forming an immature virus particle. Immature particles are transported via the secretory pathway to the Golgi. In the low-pH environment of the trans-Golgi, furin-mediated cleavage of prM to M drives maturation of the virus, accompanied by conformational rearrangements of E. The mature particles eventually leave the host cell by exocytosis (Figure 4) [54, 77-79].

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Figure 4. The flavivirus life cycle. Virion entry is initiated after the envelope protein, E, engages cellular receptors, followed by receptor-mediated endocytosis of the virus Within the acidic lumen of the endosome. The low-pH environment triggers conformational changes in the E protein which leads to viral fusion with the endosomal membrane, allowing the release of the virus genome into the cytoplasm. Virus positive-sense single-stranded RNA is translated into a polyprotein which is subsequently processed by virus and host cell proteases. RNA replication and virus assembly takes place at the membranes of the endoplasmic reticulum (ER) and results in the formation of immature virions. Immature virions are transported through the host secretory pathway, resulting in glycosylation of the viral E protein. At trans-Golgi network (TGN), host cell furin mediated-cleavage of the protein prM leads to the mature membrane protein, M. Mature virions are transported to the plasma membrane and released by exocytosis.

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4. Pathogenesis of neurotropic flaviviruses

Most of our knowledge of the pathogenesis of flavivirus comes from study of the primary human infection and laboratory infection with animal models. These arboviruses mainly spread through the bites of infected ticks or mosquitoes and virus is transmitted to the human host during the blood meal. During this process, saliva containing virus is deposited in the blood or in skin tissues. In the skin, the virus infects resident dendritic cells such as Langerhans cells, which then traffic virus to the draining lymph node. From here, the virus enters the bloodstream from the lymphatic system, which results in transient low-level viremia lasting a few days. This usually wanes with the production of antibodies. Following viremia, the virus infects many organs of the body (such as the spleen and kidney), which varies depending on the flavivirus. Virus is cleared from the peripheral system within a week or two, while in a subset of cases it enters the central nervous system (CNS). The mechanisms by which these neurotropic flaviviruses cross the blood-brain barrier (BBB) remain largely unknown, but there are many ways that a virus can cross the BBB: (1) direct axonal retrograde transport from infected peripheral neurons; (2) spread via neuromuscular junctions (NMJs) from muscles into somatic motor neurons in the spinal cord; (3) infection of olfactory neurons and spread to the olfactory bulb; (4) a “Trojan horse” mechanism in which the virus is transported by infected immune cells that traffic to the CNS; and (5) infection or passive transport through the brain microvascular endothelium cells (BMVECs) (Figure 5).

Some viruses do not require breakdown of the BBB to enter the CNS; TBEV, WNV, and JEV can enter the CNS even when the BBB is intact [80-82]. In the case of JEV and WNV, cell-free virus enters the CNS without disturbing the BBB. It is not the virus but the inflammatory cytokines/chemokines induced by JEV and WNV infection that inhibit the expression of tight junction proteins and increase in multiple matrix metalloproteinases (MMPs) between microvascular endothelial cells, which ultimately results in the enhancement of BBB permeability [81, 83]. In WNV infection, the innate sensing molecule Toll-like receptor 3 (TLR3) may play a role in invasion of WNV into the CNS by upregulating expression of tumour necrosis factor (TNF)-α, thereby resulting in capillary leakage and increased BBB permeability [84].

In the brain, neurotropic flaviviruses mainly target neurons, ultimately leading to death through apoptosis―which can be devastating, as these neurons are mostly terminally differentiated [85]. Microglia and astrocytes hardly get infected, but they are activated by virus infection and release proinflammatory cytokines, chemokines, and chemoattractants that help in recruiting peripheral leukocytes in the brain once the BBB is broken [80]. After neuroinflammation and breakage

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Figure 5. Routes of virus entry into the CNS. A. Alpha herpesviruses (e.g. HSV-1, VZV, and PRV) infect pseudo-unipolar sensory neurons of PNS ganglia. CNS spread is rare and requires anterograde axonal transport of progeny virions toward the spinal cord. B. RABV and poliovirus spread via neuromuscular junctions (NMJs) from muscles into somatic motor neurons in the spinal cord. C. Several viruses may infect receptor neurons in the nasal olfactory epithelium. Spread to the CNS requires anterograde axonal transport along the olfactory nerve into the brain. D. Infiltration through the BBB. The BBB is composed of brain microvascular endothelium cells (BMVECs) with specialized tight junctions, surrounding basement membrane, pericytes, astrocytes, and neurons. Infected leukocytes can traverse this barrier, carrying virus into the brain parenchyma. E. Alternatively, virus particles in the bloodstream can infect BMVECs, compromising the BBB. The illustration was reprinted with permission of the publisher [86]. Full forms of the viruses mentioned in the figure are listed in the abbreviations.

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of the BBB, immune cells from the periphery travel to the brain in response to the chemotactic signals. Incoming cytotoxic T cells can clear the infected cells from the brain, which can also result in the viral and immune cell-mediated cytopathology. How much cell injury can be attributed to viral cytopathology and how much to the inflammatory response is not known. An understanding of this phenomenon is crucial for the prevention and treatment of neurological sequelae mediated by TBEV [18, 87].

Both viral and host factors can affect the pathogenesis and disease outcome. Viral factors include the route of transmission, the virus load, and the neuroinvasive (ability of virus to enter the brain) and neurovirulence (ability to cause disease in the CNS) capabilities of the virus strain. The ability of a virus to infect different cell types and to avoid immune detection can also greatly influence its capacity to cause infection. Host factors including age, sex, nutritional status, immune status, and genetic factors have been linked to disease susceptibility. A non- functional chemokine receptor 5 (CCR5) genetic variant has been identified as being a risk factor for WNV and TBEV infection [88, 89]. CCR5 drives the migration of lymphocytes to the CNS in flavivirus encephalitis. Polymorphism in 2'-5'-oligoadenylate synthetases [90], CD209 [91], TLR3 [92], interleukin 28B (IL28B), and interleukin 10 (IL10) [93] genes have all been associated with predisposition to TBEV in a Russian population. During DENV infection, the presence of antibody against one strain can enhance infection with another strain during secondary infection, a phenomenon called antibody-dependent enhancement (ADE). This phenomenon is also possible during secondary ZIKV infection in DENV-endemic areas, due to serological cross reaction between the two viruses [94].

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5. Innate immune recognition

The innate immune system constitutes the first line of host defence in detecting the presence of microbes, and subsequently triggers the protective response to invading pathogens. Immune detection of microbes is achieved through germline-encoded pattern recognition receptors (PRRs), which recognize evolutionarily conserved structures on pathogens, called pathogen-associated molecular patterns (PAMPs). PRRs have been classified into five different families, which includes transmembrane proteins such as the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), and cytoplasmic proteins such as retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), NOD-like receptors (NLRs), and AIM2-like receptors (ALRs). These PRRs are expressed on macrophages, dendritic cells (DCs), mast cells, neutrophils, eosinophils, natural killer (NK) cells, and also on various non-professional immune cells. These PRR pathways induce transcription of a broad range of molecules including proinflammatory cytokines, type-I interferons (IFNs), chemokines, antimicrobial proteins, immune receptors, and many uncharacterized proteins [95]. The innate immune response through PRR activation orchestrates the early host response to infection and also acts as the link between innate and adaptive immune response.

5.1 Toll-like receptors

Among the TLRs, TLR3, TLR7 and TLR8 recognize nucleic acid motifs and are preferentially confined to intracellular compartments, such as endosomes, lysosomes, and endolysosomes, rather than being expressed at the cell surface, which is the case for the majority of the TLR family members [96]. TLRs are type- I integral membrane glycoproteins characterized by extracellular domains containing varying numbers of leucine-rich-repeat (LRR) motifs and a cytoplasmic signalling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/IL-1R homology (TIR) domain [97]. TLR3 recognizes dsRNA and TLR7/8 detects ssRNA. Viral uptake by the endocytosis deliver viral nucleic acids to endosomes, where they are detected by TLRs. In plasmacytoid DCs (pDCs), TLR7 and TLR8 signal via the adaptor MyD88 (myeloid differentiation primary response 88) and the IkB kinase alpha (IKKa) to phosphorylate and activate the transcription factor IRF7, which regulates expression of the IFN-α and IFN-β genes. TLR3 signals via TRIF (TIR-domain- containing adaptor inducing interferon-β), which couples to the TANK-binding kinase 1 (TBK-1) and IKKe and phosphorylates IRF3 to induce IFN-α and/or IFN- β [98].

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5.2 RIG-I-like receptors

The RLR family has three members: retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). These receptors are expressed in the cytoplasm of both immune and non-immune cell types and regulate signalling pathways that promote the IRF3-, IRF7-dependent expression of type-I and type-III IFNs, and the NF-kappa B-dependent expression of proinflammatory cytokines. RIG-I recognizes short dsRNAs and 5´-triphosphorylated single-stranded RNAs whereas MDA-5 recognizes long dsRNA [99-101]. Both RIG-I and MDA5 have two N-terminal caspase recruitment domains (CARDs) that mediate signalling by interacting with the CARD domain of the mitochondrial and peroxisomal membrane-associated protein interferon-beta promoter stimulator 1 (IPS-1; also known as MAVS, VISA, or CARDIF) [102-106]. IPS-1 contains an N-terminal CARD that forms homotypic interactions with the CARDs of RIG-I and MDA5. IPS-1 activates members of the IKK family, TBK-1 and IKKe, to phosphorylate IRF3 and IRF1 and induce IFN and ISG transcription. LGP2 lacks homology with CARD and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5 [107].

5.3 Innate recognition of flaviviruses

Viruses are recognized by the panel of intracellular receptors that recognize viral genomic nucleic acids and/or replication intermediates. Endosomal TLRs and cytoplasmic RLRs are essential intracellular sensors for detection of viral nucleic acid. Recent studies have also indicated that certain extracellular membrane- bound TLRs can also detect viral structural proteins. Over-expression studies in vitro as well as targeted gene depletion in vivo suggest that both the TLR and RLR pathways play vital roles in detecting and responding to flavivirus infections. However, the specific PRRs involved in mediating the antiviral response are likely to be virus- and cell type-specific. For JEV, RIG-I signalling is involved in initiating the antiviral response in neurons [108]. In DENV, both the RIG-I and MDA5 pathways are required for the induction of antiviral programmes [100]. In the case of WNV, the onset of the innate antiviral response was found to be merely delayed in RIG-I−/− cells compared to wild-type controls. This suggests that the RIG-I pathway mediates the initial activation of the antiviral response to WNV, although distinct secondary pathways are also clearly involved. Several lines of evidence indicate that MDA5 functions as the secondary receptor for sensing of both WNV and DENV [109, 110]. Most of these studies have indicated that RIG-I is involved in sensing all flavivirus infections; however, the role of MDA5 is virus- dependent. TLR3 has also been implicated in WNV infections, although its role

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in protection and pathogenesis is debatable [84, 111]. TLR7 is important for controlling virus infection in neurons in the CNS [112]. So, multiple PRRs are clearly involved in the initiation of the antiviral response to most flaviviruses; however, the pathways engaged during infection are virus- and cell type- dependent (Figure 6).

Figure 6. Interferon induction pathways. After viral infection double-stranded RNA and single-stranded RNA are recognized by TLR3 and TLR7/8 respectively, in the endosomes, which leads to the dimerization of receptors and recruitment of TRIF or MyD88. While viral RNA and intermediates of viral replication are detected by RIG-I and MDA-5 in the cytosol. Both RIG-I and MDA5 activates IPS-1 adaptor protein. All of these pattern-recognition receptors (PRRs) initiate signalling pathways that converge at the activation of the transcription factors IRF3, IRF7, and NF- kB. This leads to the expression of IFN and proinflammatory cytokines. RIG-I and MDA-5 can also signal thorough peroxisomal IPS-1 which is essential for rapid ISG expression through IRF-1 and IRF3.

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6. IFN signalling

IFN acts as the first line of defence against viral infections. It was described almost 60 years ago as an agent produced by a virus which provided resistance to another virus [113, 114]. There are three distinct IFN families: type-I, type-II, and type-III. Type-I IFNs are the most studied and are broadly expressed. The most common type-I IFNs consists of 13 partially homologous IFN-α subtypes in humans (14 in mice) and a single IFN-β [115]. Type-II consists of IFN-γ, which is predominantly produced by T cells and NK cells, and acts on a broad range of cell types that express the IFN-γ receptor (IFN-γR) [116]. Type-III IFN consists of IFN-λ1, IFN-λ2, IFN-λ3 (also known as IL-29, IL-28A, and IL-28B, respectively), and the newly identified IFN-λ4, which have similar functions to the cytokines of the type-I IFN family. Type III IFNs are recognized by the receptors IFNLR1/IL10RB, which are mainly restricted to epithelial cell surfaces [117, 118].

Type-I IFNs do not act directly on viruses, but through the trancriptional induction of a large group of antiviral genes known as ISGs, which play a role in host resistance to viral infections. Type-I interferons also activate key components of the innate and adaptive immune systems, including antigen presentation and production of cytokines involved in activation of T cells, B cells, and NK cells [119, 120]. Type-I IFNs are produced by virtually all human cells, but are more strongly produced by certain immune cells such as macrophages, DCs, and fibroblast cells [121]. Type I IFNs are induced following virus recognition through endosomal TLRs (TLR3, TLR7, TLR8, and TLR9), or cytoplasmic sensors (RLRs). IFN-α/β binds to a heterodimeric transmembrane receptor composed of the subunits interferon-α receptor (IFNAR1 and IFNAR2). Ligation of IFNAR stimulates the receptor-associated protein tyrosine kinases Janus kinase (JAK1) and tyrosine kinase 2 (TYK2). Activated JAK1 and TYK2 phosphorylate signal transducer and activator of transcription 1 (STAT1) and STAT2 molecules, leading to dimerization. The STAT1-STAT2 dimer then binds with IRF9 to form the interferon stimulated gene (ISG) factor 3 (ISGF3) complex, leading to nuclear translocation. ISGF3 binds to IFN-stimulated response elements (ISREs) in the promoters of ISGs to regulate their expression. Among these genes is IRF7, which initiates the transcription of a second wave of type-I IFNs [122]. Thus, this autocrine and paracrine feedback allows type-I IFNs to create an antiviral state in surrounding cells (Figure 7).

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7. Interferon-stimulated genes

ISGs were first discovered almost four decades ago [123]. To date, more than 350 ISGs have been identified. With the recent advancement of genome-wide scanning, many new ISGs are being discovered. Most of these ISGs are induced through IFN signalling, but some subsets of ISGs are induced through IFN- independent pathways [124, 125]. They are present at baseline levels but their expression is increased following the IFN response [126]. ISGs can be divided into positive regulators of IFN signalling (e.g. RLRs, IRFs, and STAT1/2) or negative regulators (e.g. SOCS and USP18) and antiviral effectors (e.g. Mx1 and IFITM). Positive regulators such as IRF1 can translocate to the nucleus and enhance the production of a subset of ISGs, while negative regulators such as suppressor of cytokine signalling (SOCS) proteins can inhibit JAK-STAT signalling [126].

Many antiviral ISGs block viral replication by targeting pathways and functions that are required for the viral life cycle. Protein kinase R (PKR), MX1, and 2′-5′- oligoadenylate synthetase (OAS)/RNase L are some of the earliest identified ISGs, and their activity against many viruses has been well studied. PKR is a known inhibitor of cellular and viral mRNA translation [127]. MX1 is a dynamin- like GTPase that appears to target the viral nucleocapsids of the , preventing import of virus to the nucleus, resulting in inhibition before the establishment of replication [128]. Members of the OAS enzyme family are activated by double-stranded RNA to catalyse the formation of 2´-5´ oligo adenylates, which activate cellular RNase L to degrade viral genomes [129]. The IFITM (interferon-induced transmembrane) family of ISGs are unique because they prevent infection before a virus can traverse the lipid bilayer of the cell. IFITM proteins mediate cellular resistance to influenza A virus (H1N1), WNV, DENV, and many other viruses [130, 131]. Some retroviral restriction factors such as APOBEC3 (which encodes a cytosine deaminase that restricts HIV-1 by deleterious modification of reverse-transcribed viral DNA) and BST2/tetherin (which is a membrane-bound protein that inhibits release of virus particles from the cell surface) have also been well characterized [132]. The TRIM (tripartite motif) family of proteins, which consist of around 70 members, has antiviral activity against many viruses. TRIM79α, a rodent-specific IFN-inducible gene, was recently shown to inhibit TBEV by targeting RNA-dependent RNA polymerase NS5 for lysosomal degradation [133].

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Figure 7. Type I IFN signalling pathway. Binding of IFN to IFNAR receptors triggers phosphorylation of pre-associated Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), which leads to the recruitment and phosphorylation of STAT1 and STAT2. STAT1 and STAT2 associate to form a heterodimer, which in turn recruits the IFN9 to form the ISGF3. ISGF3 then translocate to the nucleus to induce genes regulated by ISRE promoter elements, resulting in expression of antiviral genes. These ISGs include IRFs and PRRs but also antiviral effectors such as viperin. ISGs can be divided into antiviral effectors and negative or positive regulators of IFN signalling. A special case of positive regulators is IRF1, which upon expression directly translocate to the nucleus to enhance expression of a subset of ISGs.

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7.1 Viperin

Viperin (virus inhibitory protein, endoplasmic reticulum–associated, IFN- inducible), also known as radical S-adenosyl methionine domain containing protein 2 (RSAD2), is a broad-spectrum antiviral ISG that acts against many viruses. Viperin can be induced through IFN-dependent JAK/STAT signalling [134] or through IFN-independent direct activation by IRF1 and IRF3 [125, 135]. An antiviral role of viperin has been shown against many viruses: influenza A virus [136, 137], HIV [138], Chikungunya virus (CHKV) [139], [140], and respiratory syncytial virus (RSV) [141]. It is also active against members of the Flaviviridae such as hepatitis C virus (HCV), ZIKV, DENV, WNV, and TBEV [142-147]. Although viperin functions mostly as an antiviral restriction factor, one study with human cytomegalovirus (HCMV) has shown that viperin functions as a proviral factor by aiding in the infection [148].

For many viruses, the mechanism of antiviral action of viperin is unknown. TBEV is highly sensitive to viperin, and it selectively blocks amplification of positive- sense RNA during the early stages of TBEV infection [145]. It also induces the secretion of unproductive non-infectious virus particles by targeting Golgi specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) dependent assembly of virus particles [57]. During HCV infection, viperin prevents replication of HCV RNA by interacting with HCV NS5A and host protein vesicle-associated membrane protein-associated protein A (VAP-A), thus preventing replication complex (RC) formation [146]. In DENV-2-infected cells, viperin co-localizes with both NS3 protein and viral RNA, both of which are components of the flavivirus RC [142]. Viperin inhibits the release of influenza A virus and HIV particles by disrupting lipid rafts [137, 138], while some viruses such as JEV are not sensitive to the action of viperin. JEV counteracts viperin by down-regulating its expression through proteasomal degradation [149].

In vivo studies have been done only with influenza virus, WNV, RSV, and CHKV. Viperin-/- mice were found to be more susceptible to WNV, with higher mortality rates and increased virus replication in the brain and spinal cord. For CHKV, higher virus replication in the footpad, increased viremia, and more pronounced joint swelling and subcutaneous oedema were detected after infection of viperin- /- mice compared to wild-type mice. Viperin was also highly induced in monocytes (a subset of peripheral blood mononuclear cells (PBMCs) targeted by CHKV in the blood) of CHKV-infected patients [139, 144]. While viperin-/- mice did not show any phenotype after lethal challenge with influenza A virus. Similar viral Influenza virus titres were present in the lungs as in those of WT mice, which contrasts with in vitro studies. This effect may be explained by the compensatory action of many other important ISGs in vivo [136]. Over-expression of viperin in chinchilla airway using a recombinant adeno-associated virus vector that

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encoded viperin resulted in reduced titres of RSV in nasopharyngeal lavage fluid [141].

7.2 Interplay of viperin with the immune system

Along with viperin’s role as an antiviral ISG, it can also modulate the innate immune response and the adaptive immune response. It augments TLR7- and TLR9-mediated IFN responses in pDCs by interacting with the signal mediators interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor-6 (TRAF6) and recruiting them to lipid droplets for ubiquitination, which then leads to nuclear translocation of IRF7 and IFN induction. Thus, TLR7-/9- dependent IFN production was found to be impaired in viperin-/- mice [150]. On the other hand, another report suggested that viperin is a negative regulator of IFN production through interaction with the adaptor protein IPS-1 in a macrophage cell line [151]. These effects of viperin are probably cell-specific, as we did not observe such viperin-IPS-1 interaction in the HEK-293 cell line [57]. Viperin is also involved in regulation of the TH2 response by binding with its regulatory factor GATA-3. Stimulation of T cells from viperin-/- mice showed reduced induction of TH2 cytokines (IL-4, IL-5, and IL-13) [152].

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8. Brain heterogeneity and the innate immune response in the CNS

The brain is the most complex organ in the CNS. Thus, its protection from invading pathogens is important for maintenance of normal functioning of the body. Earlier, it was thought that the brain is naive in the immune response and that the cells from the peripheral immune system travel to the brain when there is infection. However, recent studies have shown that brain cells contribute to the local protective innate immune response. The cell types in the brain can be divided into three main groups: neurons, glia, and endothelial cells. The ratio between these cells is highly variable in the different regions of the brain [153]. Endothelial cells are present on the luminal side of BBB, and thus link the periphery with the CNS. Endothelial cells act as the mediator and sensor for the peripheral immune modulators. Under physiological conditions, they down- regulate the proinflammatory mediators and the basal expression of adhesion molecules [154]. Astrocytes are specialized glial cells that outnumber neurons by over fivefold. They have a variety of neuroprotective functions, including regulation of synaptic function, neuronal repair, and maintaining BBB integrity―and they also produce neurotrophic factors and anti-inflammatory cytokines such as IL-10 [155]. Microglia are the resident macrophage cells in the CNS; they function as specialized phagocytic and antigen-presenting cells. These are the main IFN and proinflammatory cytokine-producing cells in the CNS. Activation of microglia is one of the important contributory factors in neurodegeneration [156].

Neurons, astrocytes, and microglia in different regions of the CNS show extensive functional and immunological heterogeneity, which may result in regional differences in innate and adaptive immune responses within the CNS [157]. Comparison of different regions of the brain highlights specific antiviral mechanisms [158-160]. Two neuronal subtypes, granule cell neurons of the cerebellum and cortical neurons from the cerebral cortex, have unique innate immune programmes and have shown differential permissiveness to replication of several positive-stranded RNA viruses [159]. The cerebellum, which contains granule cell neurons, has higher basal expression of ISGs such as Ifi27, Irg1, viperin, and STAT1. Thus, it is more resistant to WNV infection than the cerebrum [159]. In addition, an immune response generated in one region of the brain can have an impact on distinct regions of the brain. vesicular stomatitis virus (VSV) and cytomegalovirus (CMV) have been shown to induce long- distance interferon signalling from olfactory bulb to the distal regions of the brain, which blocks spread of virus to the other regions of the brain [161].

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IFNs are produced in the CNS by glial cells, mostly microglia and astrocytes, and also by neurons. A variety of mechanisms stimulate IFN production in glial cells, including innate stimuli from toll-like and other receptors, which can recognize endogenous entities as well as pathogens. Most of the TLRs (TLR1-9) are present on the microglia, while only some of the TLRs are expressed on astrocytes and neurons [162]. Many other factors linked to the IFN system are involved in this antiviral response in the CNS. The use of mice deficient in IRF-1, IFNAR, or IPS- 1 has highlighted the importance of type-I IFN in the control of virus replication in the CNS [80, 163, 164].

CNS immune reactions take place in isolation from the innate and adaptive immune interplay that characterizes peripheral immunity, as it is separated by the BBB. However, microglia and astrocytes engage in significant cross talk with CNS-infiltrating T cells and other components of the innate immune system during the infection [165]. Leukocytes in the circulation bind to endothelial cells of the BBB through adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1), sensing for distress signals from the CNS. Such a signal may be mediated by chemokines and cytokines released by microglia or astrocytes [166].

Virus infection and the resulting immune response can disturb the complex architecture and functioning of the brain, which can lead to long-term neurological problems or even death. Thus, an immune balance is required between protective and overtly active immune components within the brain, as it may affect the survival of terminally differentiated neurons.

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Aims

The overall aim of the work described in this thesis has been to study the interplay between flaviviruses and the host innate immune system, concentrating especially on TBEV.

The specific aims were:

I. To investigate the role of IPS-1 in tick-borne flavivirus infection and immunity (Paper I). II. To understand the in vivo role and mechanism of action of the antiviral protein viperin in flavivirus infection (Papers II and III). III. To investigate a possible correlation between the disease severity of TBEV strains and their pathogenesis in mice (Paper IV).

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Methodological considerations

In this section, a brief overview of the materials and methods used in this thesis is presented together with the rationale and some comments. For a more detailed description, please refer to Papers I‒IV.

1. Quantification of virus burden

Viral burden can be expressed as the amount of infectious virus particles or the amount of viral genomic material. The standard method used to determine virus concentration in terms of infectious dose is the plaque assay, and for genomic material it is real-time polymerase chain reaction (RT-PCR). Viral plaque assays determine the number of plaque-forming units in a virus sample. Viruses that can cause a cytopathic effect and kill cells will create a plaque (an area of killed infected cells surrounded by uninfected cells), which can be seen visually or with an optical microscope. We used a variant of the plaque assay called the focus- forming assay (FFA). Instead of relying on cell lysis in order to detect plaque formation, the FFA employs immunostaining techniques using horseradish peroxidase-labelled antibodies specific for a particular viral antigen, to detect infected host cells and infectious virus particles before an actual plaque is formed. This variant is more robust, reliable and take less time compared to the original plaque assay, since all virus strains does not cause cytopathic effect. To detect viral RNA, we used quantitative reverse transcription PCR (qRT-PCR). Reverse transcription refers to the synthesis of DNA from an RNA template. This cDNA template is used as a starting material to amplify a sequence using specific primers and probes attached to a fluorescent signal, to quantitate the amount of starting material. The qRT-PCR used to detect the TBEV and LGTV had a sensitivity of around 10 genomic copies.

2. Studies in mice

Animal models are vital for the study of the basic mechanisms of pathogenesis and the immune response to virus infections in a more natural context. Research using immunocompromised mice and/or mouse-adapted viruses allows investigation of questions that may be ethically difficult to address in human studies. The ability to knock out a particular gene of interest from mice gives the additional advantage of studying its function and impact on overall health and on the immune system during virus infection. C57BL6 is a good mouse model to study the pathogenesis of and the immune response to TBEV, as it can reach the

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brain after peripheral inoculation and cause disease in the CNS. All the knockout mice (IPS-1-/- and viperin-/-) used in the study had a C57BL6 background.

3. Primary cells

Serial passage of cell lines over time can cause genotypic and phenotypic variation in the cells. The type I IFN response/signalling is often disrupted in cancer cells making the results less reliable when studying innate immunity [167]. Primary cells are more biologically relevant tools than cell lines for studying human and animal biology. Primary cells are isolated directly from human or animal tissue using enzymatic or mechanical methods. With the emergence of newer technologies such as 3D culture, the use of primary cells is becoming increasingly prevalent to achieve improved results. These cells have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived than continuous (tumour-derived or artificially immortalized) cell lines. They therefore constitute a more representative model of the in vivo state. We used primary neurons, astrocytes, and embryonic fibroblast cells to study virus infection and the immune response.

4. Detection and quantification of protein

Protein detection and quantification is an important tool to answer a multitude of questions in different research fields. Western blot analysis is a traditional method for detection and analysis of proteins. This widely accepted technique is used to detect a specific protein (antigen) in a given sample. It uses SDS- polyacrylamide gel electrophoresis to separate various proteins, which are then transferred to a matrix, where they are stained with antibodies specific to the target protein. By analysing the relative intensity, details of expression of the target protein can be obtained. This method is used extensively to detect the expression of various cellular proteins under different conditions. Enzyme-linked immunosorbent assay (ELISA) is another technique that is commonly used to detect and quantify peptides, proteins, antibodies, and hormones. ELISA uses an enzyme linked to an antigen or antibody as a marker for the detection of a specific protein. This technique was used to detect IFN concentration in serum.

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5. Protein-protein interaction studies

Proteins are the working horses of the cellular machinery; they act by interacting with other proteins and macromolecules in the cell. Study of protein-protein interactions is important to understand and explain a molecular phenomenon. One of the commonly used techniques is co-immunoprecipitation (co-ip) assay. It works on the principle that when a cell is lysed under non-denaturing conditions and a protein (bait protein) is pulled out by its specific antibody (associated with agarose beads), then another protein (prey protein) or protein complexes are also fished out (precipitated) along with the bait protein. Analysis of the precipitated proteins can then be done either by mass spectroscopy (identification or screening) or by western blot analysis (to confirm a known interaction partner). We used co-ip to identify and confirm the interaction of viperin with various viral proteins. This was also confirmed with immunofluorescence and co-localization studies

6. Gene expression and quantification

Gene expression is a highly regulated mechanism that controls the function of the gene. Increased mRNA expression of genes indicates their upregulation after stimulation. Quantitative RT-PCR is the most sensitive technique available for detection and quantification of mRNA. We performed gene expression analysis to study the basal expression of some ISGs, and the expression and upregulation of proinflammatory cytokines in cells and organs of mice after infection.

7. Fluorescence-based quantification and localization

Fluorescein-labelled antibodies can be used to detect specific proteins of interest in a sample. We confocal microscopy to visualize the fluorescence-labelled cells. Confocal microscopy is a specialized form of standard fluorescence microscopy that uses particular optical components to generate high-resolution images of material stained with fluorescent probes. Confocal microscopes have become a powerful tool of choice for researchers interested in serious imaging of cell structure and function. These microscopes have revolutionized our view of cells and have been a major instrument in unravelling the complexities of the morphology and dynamics of cells and tissues. This technique was used to study the location of virus proteins in the cellular compartments. Another modification of fluorescence-based quantification is TROPHOS Plate RUNNER HD® (TROPHOS SA, Marseille, France), which can be useful for detection and quantitation of the fluorescence-based signal. This technique was used to count cells expressing particular proteins and to count virus-infected cells.

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8. Immunohistochemistry

Immunohistochemistry (IHC) combines anatomical, immunological, and biochemical techniques to image discrete components in tissues by using appropriately labelled antibodies to bind specifically to their target antigens in situ. IHC makes it possible to visualize and document the high-resolution distribution and localization of specific cellular components within cells and in their proper histological context. Compared to other bio-techniques that are based on the antigen-antibody reaction such as immunoprecipitation and western blot, immunohistochemistry provides in situ information that gives a more convincing experimental result in the tissue. This technique was used to detect virus-infected cells in mouse brain.

9. Flow cytometry

Flow cytometry is a widely used method for analysis of the expression of cell- surface and intracellular molecules, thus characterizing and defining different cell types in a heterogeneous cell population. The staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with unlabelled or fluorochrome- labelled antibodies and analysed in a flow cytometer. We used flow cytometry to analyse and count immune infiltrating cells in the brain.

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Results and discussion

1. To investigate the role of IPS-1 in tick-borne flavivirus infection and immunity (Paper I)

Type-I IFNs are well-studied cytokines that mediate many antiviral effects through production of ISGs, and they represent a first line of defence against virus infection. These IFNs can have a range of direct and indirect effects on various cell types during infection. Type-I IFNs can be produced by several different pathways, against virus infection. In the case of RNA virus infection, IFNs are produced mainly by the TLR or RLR pathways. However, the signalling pathways that are used for IFN production vary according to the virus and the cell types being infected. We have previously shown the importance of IFN against LGTV infection-induced inflammation and encephalitis in the peripheral and CNS system in vivo [168]. In the study in Paper I, we investigated the importance of IPS-1 signalling (the adaptor protein for RLR pathways) in in vivo tick-borne flavivirus infection. We observed that IPS-1 signalling was important for survival of mice against TBEV and LGTV infection. This protection against LGTV was associated with control of the virus burden in serum, in peripheral organs (the spleen and lungs), and in the CNS (the spinal cord and brain). Although there was a higher viral burden in IPS-1-/- mice, a lower level of IFN-α was observed in serum, which agrees with our previous finding that recognition of LGTV is IPS-1- dependent [169].

The brain is the final important stop during the journey of a neurotropic flavivirus in the host. Brain has distinct immune response against the virus infection, where resident brain cells can produce IFN, proinflammatory cytokines, and chemokine adhesion molecules. In the scenario of viral infection of the brain, neurons are the main target, and killing of neurons―which are terminally differentiated cells―could be detrimental to the host [170]. We observed apoptosis of brain cells in LGTV-infected IPS-1-/- mice but not in WT mice, and this increased apoptosis in the brain has been shown to be associated with the severity of encephalitic disease during many flavivirus infections [85, 171]. In the brain sections, we observed a higher degree of infection of neurons in the olfactory bulb regions of the brain in IPS-1-/- mice. Astrocytes and microglia were hardly infected, but they were activated in response to infection―or as a result of immunopathology or cytokine response. Activation of microglia and astrocytes during Murray Valley encephalitis virus (MVE) infection triggers the release of chemotactic factors for neutrophils, which can infiltrate the brain and give immunopathology [172].

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With higher virus replication and proinflammatory cytokines in the brain, we speculated that it might lead to inflammatory changes in the brain. Consistent with this, we found increased infiltration of peripheral immune cells (macrophages, DCs, CD4 T cells, and CD8 T cells) in the brains of IPS-1-/- mice 7 days post infection (dpi), which was associated with BBB leakage. Although immune cells can have an advantageous effect as they help in clearing virus- infected cells, they may also contribute to immunopathology due to overt uncontrolled reaction. CD8 T+ cells have been associated with immunopathology during TBEV infection [173].

IPS-1-/- mice were more susceptible to intracranial LGTV infection, suggesting that local IPS-1 signalling is important for restriction of virus infection. Brain consists of heterogeneous subpopulations of cells and a microenvironment that might show a differential immune response. Absence of IPS-1 signalling led to higher viral replication in all parts of the brain (the olfactory bulb, cerebrum, cerebellum, and brain stem) irrespective of the route of inoculation. In the case of tick-borne flavivirus, higher virus replication in vitro leads to higher IFN-β production [169]; and this was observed in all parts of the brain after intraperitoneal infection, except for the olfactory bulb―indicating that other RLR-independent pathways can compensate for IFN-β production in the cerebrum, cerebellum, and brain stem, and that IFN-β production in the olfactory bulb is IPS-1 dependent.

Host intrinsic factors play an important role in determining the susceptibility of the host to infection. We found lower basal levels of some ISGs (viperin and IRF1) in the olfactory bulb and cerebrum in the absence of IPS-1, making them more susceptible to LGTV infection. In addition, LGTV NS5 can inhibit STAT1 phosphorylation and thus prevent downstream interferon signalling in vitro [174, 175]. In vivo, we found that a high degree of viral replication in the olfactory bulb of IPS-1-/- mice did not induce STAT1 phosphorylation at early time points; this was probably due to the IFN-antagonistic function of NS5.

Altogether, we showed that a lower basal level of some ISGs in IPS-1-/- mice, along with low IFN-β production in the olfactory bulb, leads to efficient LGTV replication and expression of IFN signalling antagonists, which then inhibit STAT1 phosphorylation early in infection. This leads to a greater proinflammatory response, greater immune cell infiltration, greater apoptosis of brain cells, and a greater degree of immunopathology―and consequently to the death of infected IPS-1-/- mice. We also proposed that different pattern recognition pathways regulate the IFN response in different regions of the brain. Thus, our study provided further insight into the newly growing concept of heterogeneity of the immune response in brain [157].

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2. To understand the in vivo role and mechanism of action of the antiviral protein viperin in flavivirus infection (Papers II and III)

2.1 To understand the in vivo role of the antiviral protein viperin in flavivirus infection (Paper II)

Viperin is a broad-spectrum antiviral ISG that is active against many viruses, but few studies have shown the in vivo significance of viperin. This might be due to redundancy of the IFN response, where other ISGs might compensate for loss of viperin. We had previously shown that over-expression of viperin inhibited TBEV replication in vitro [145]. However, the functional role of viperin against tick- borne flavivirus in vivo was not known. Using LGTV as a model for tick-borne flavivirus, we investigated the role of viperin in mice. In this study, neither WT mice nor viperin-/- mice were susceptible to peripheral LGTV infection. This could be due to redundancy among many ISGs, which might work together to impart antiviral actions. However, there could still be some subclinical infection, as we observed low levels of virus replication in the olfactory bulb of WT mice [80], but virus replicated to higher levels in the spleen, olfactory bulb, and cerebrum in the absence of viperin. The olfactory bulb is the initial site of replication of LGTV, while in the absence of viperin it can spread to the cerebrum. Interestingly, VSV replication is also restricted to the olfactory bulb in mice. The reason for this is that VSV induces long-distance IFN signalling within the brain. This activates antiviral ISG expression in distinct parts of the brain, which prevents spread of virus to other parts [161]. This phenomenon might also be valid for LGTV infection, and viperin could be one of the important ISGs that limit the spread of virus to distinct regions of the brain.

Viperin is also involved in promoting type-I IFN production in pDCs through the TLR7/9 pathways [150]. These pDCs act as one of the main sources of high levels of systemic IFN-α in serum. In line with this, less systemic IFN-α was observed in the serum of LGTV-infected viperin-/- mice. In contrast to the situation with serum, we observed increased mRNA levels of IFN-β and IFN-α2 in the olfactory bulb of LGTV-infected viperin-/- mice. This can be explained by the fact that IFN production is cell type-dependent and that upregulation of IFN in the olfactory bulb mimics more the upregulation of IFN shown in vitro where the induction of IFN-β is directly proportional to the degree of virus replication [169].

Neurotropic flaviviruses infect the brain and cause significant damage to neurons. So, effective antivirals should have a strong action in the brain―to counteract virus infection and prevent virus-associated morbidity and mortality

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[80]. Viperin-/- mice were more susceptible to intracranial infections, indicating a brain-specific role of viperin against LGTV infection. Higher expression of basal levels of viperin and early induction during infection in astrocytes could be important factors for the brain-specific function of viperin [176]. Higher susceptibility to intracranial infection was correlated with increased viral replication in olfactory bulb and cerebrum of viperin-/- mice. During WNV infection of viperin-/- mice, higher viral burdens were observed in cortex and white matter, indicating a virus-specific function of viperin in different brain regions [144]. The phenomenon of brain region-specific viral restriction has also been observed with other ISGs such as Ifi271a and IFIT2 against WNV [177, 178].

As we found that viperin was an important restriction factor within the brain, we wanted to determine the role of viperin in different brain cells. We found that the absence of viperin in primary astrocytes and cortical neurons, isolated from the cerebrum, gave a replication advantage to TBEV. This was not true in primary granule cell neurons from cerebellum, confirming our in vivo data which shows that viperin has a brain region-specific role. The IFN response leads to the release of a plethora of ISGs with concerted action against viral infection, and we found strong antiviral effects of a single ISG in cortical neurons and astrocytes. We wanted to investigate whether pre-treatment with IFN could compensate for loss of viperin in these cells. We pre-treated a subset of WT and viperin-/- cells with IFN before infection with TBEV, and found that viperin was the sole ISG responsible for protection in cortical neurons―while other ISGs could compensate to some extent in astrocytes and granule cell neurons. Viperin has been shown to be important in primary DCs and macrophages, but not in cortical neurons, during WNV infection [144], while it has been found to be effective against ZIKV in primary MEFs [147]. As viperin has been shown to have an important effect against other flaviviruses, we wanted to determine the role of viperin in the antiviral IFN response during WNV, ZIKV, and JEV infection in cortical neurons. In our studies, viperin was found to have an important effect against ZIKV in cortical neurons, but endogenous viperin had minimal effect against WNV. Although the endogenous viperin failed to inhibit WNV, we found that viperin was needed for the IFN-mediated antiviral responses against WNV. In JEV infection, viperin is not important―as JEV can degrade the viperin and is therefore resistant to its action [149]. Thus, we have shown the brain cell type- specific role of viperin against many flaviviruses and its brain region-specific role against tick-borne flaviviruses.

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2.2 To understand the mechanism of action of the antiviral protein viperin in flavivirus infection (Paper III)

Viperin has been shown to be antivirally active against many viruses, but its mechanism of action is known for only a few. Viperin acts on many stages of the virus life cycle. During influenza A virus and HIV infection, it inhibits virus budding by disrupting lipid raft formation [137, 138]. In the case of HCV, viperin binds proviral host factor VAP-A in the RC and impairs its association with NS5A, which is critical for viral replication [179]. In DENV-2 infection, viperin was found to co-localize and interact with viral proteins C and NS3, and viral RNA, but its antiviral activity against DENV-2 was found to be associated with its interaction with NS3 protein [142]. Viperin also targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles [57]. In the case of TBEV, we found that it inhibited early viral replication by inhibiting the synthesis of positive-sense RNA, suggesting the involvement of viral proteins that are targets of viperin. To investigate this, we screened for viperin’s interaction with various structural and non-structural proteins of TBEV. Viperin interacted with the prM, E, NS2A, NS2B, and NS3 proteins of TBEV. But from viperin’s point of view, interaction with NS3 had physiological impact―as NS3 was being degraded in a dose-dependent manner―while viperin’s interactions with the other viral proteins did not lead to their degradation. NS3 is a multifunctional protein in the life cycle of the virus, which interacts with many other viral proteins. It did indeed interact with prM, E, NS2A, and NS2B protein―and also helped viperin to carry out the degradation of these viral proteins. Thus, viperin could mediate NS3-dependent degradation of other viral proteins by forming a complex. The effect of viperin on prM and E, which forms the viral coat, could affect the release of progeny virus, as we detected a stronger antiviral effect on the release of infectious TBEV (as compared to an effect on RNA) late in infection [145].

Degradation of NS3 in the presence of viperin occurred through the proteosomal pathway, which is one of the major cellular protein degradation pathways. Such degradation of viral protein can be carried out directly by E3 ubiquitin ligase activity, as shown for the ISG TRIM22 [180], or indirectly, as shown for the ISG ISG12a where it binds to both the viral protein and the E3 ligase [181]. As helicase activity of NS3 is required for flavivirus positive-sense RNA synthesis, its degradation by viperin may inhibit early virus replication [145]. Such early antiviral activity against virus infection could be important in CNS cells such as astrocytes and neurons, which express high basal levels of viperin (Paper II).

Consistent with the broad antiviral activity of viperin, it interacted with NS3 from TBEV, ZIKV, YFV, and JEV but could only degrade the NS3 proteins of TBEV and ZIKV. This ability of viperin to degrade NS3 from TBEV and ZIKV correlated with

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viperin’s activity in reducing the titres of these viruses in an in vitro infection system, while it had no effect on virus titres of YFV and JEV. This indicates that YFV is not sensitive to viperin, and JEV NS3 has previously been shown to degrade viperin [149], indicating virus-specific restriction of viperin. As viperin was able to degrade the viral NS3 of TBEV and ZIKV and inhibit the life cycle of the virus, addition of exogenous NS3 prevented the antiviral action of viperin against these virus infections. As NS3 in trans cannot complement the replication cycle of NS3-deficient flaviviruses, as shown previously [182], exogenous NS3 most likely acted as decoy to rescue the virus replication. This decoy effect was further confirmed by the trans complementation experiments where NS3 from one flavivirus was able to rescue the viral replication of others.

Altogether, we found that viperin has antiviral activity against a tick-borne flavivirus (TBEV) and a mosquito-borne flavivirus (ZIKV). This antiviral activity was found to be mediated by the proteosomal degradation of NS3 and indirect degradation of other viral proteins (prM, E, NS2A, and NS2B) in the presence of NS3. Thus, this broad-spectrum, species-specific anti-flaviviral activity of viperin can be exploited for future development of antivirals.

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3. To investigate a possible correlation between the disease severity of TBEV strains and their pathogenesis in mice (Paper IV)

In TBEV infection, disease manifestation may vary from flu-like symptoms to severe encephalitis. However, the clinical course, severity, and frequency of disease may vary depending on strain of the virus. Recently, we isolated a strain of TBEV from ticks, from the natural focus of TBEV patients who showed gastroenteritis and constitutional symptoms [19]. These symptoms were rare for TBEV, and had not been reported earlier. This strain, MucAr HB171/11, belongs to the European subtype and was isolated from questing adult Ixodes ricinus ticks from a natural focus in south-eastern Germany, with five human infections from 2005 to 2011. The diagnosis of the disease based on symptoms becomes difficult with such unusual symptoms, which are not common for TBEV disease. To study these unusual clinical manifestations of disease, we used a mouse model to gain some insight into the pathogenesis and immune response of this strain. C57BL/6 mice are a good model system for the study of TBEV pathogenesis, as they are susceptible to infection and develop encephalitis. The pathogenesis of MucAr HB171/11 was compared with that of another European strain, Torö-2003. It was rescued from a cDNA infectious clone generated from RNA extracted from ticks collected on the island of Torö (58°49´ N, 17°50´ E) in the Stockholm archipelago in 2003. Torö-2003 cases caused mild neurological disease.

Strain Torö-2003 was more pathogenic in mice after subcutaneous infection than HB171/11. This also translated into higher viral replication in spleen and lymph node of Torö-2003 infected mice. However, we could not detect HB171/11 replication in peripheral organs. Although human infections were associated with gastrointestinal symptoms, we could not detect virus in the salivary glands, mesenchyme, colon, appendix, small intestine, or nasopharynx-associated lymphoid tissue (NALT). Although we did not specifically check for pathological changes in these organs, no visible differences were observed as compared to the mock infections. This suggests a species-specific infection pattern and tropism of these strains. As strain HB171/11 was not associated with neurological symptoms and did not replicate in the periphery, we hypothesized that it might be less neuroinvasive. We therefore isolated different parts of the brain (olfactory bulb, cerebrum, cerebellum, and brain stem) after subcutaneous infection. We observed delayed neuroinvasion with HB171/11 than with Torö-2003. The difference in the neuroinvasive capability of these strains can be attributed to molecular determinants of virulence, which are scattered throughout the genome of the viruses. For example, TBEV strain T263 has shown reduced neuroinvasiveness due to mutations in the NS2b and/or NS3 gene [20]. A variable 3´- untranslated region (UTR) of a Far-Eastern strain of TBEV has been

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shown to be an important determinant of virulence in a mouse model [183]. We have previously also shown a role for quasispecies development within the poly (A) tract as a determinant of virulence in TBEV infection in mice [184]. There are four amino acid changes in structural protein regions, and 27 amino acid changes in non-structural protein regions between the Torö-2003 and HB171/11 strains, some of which might explain these changes in the pathogenesis. A reverse genetics approach could be taken to understand the determinants of pathogenicity. We also analysed the neurovirulence capability of these strains in mice by injecting virus directly into the brain. As expected, Torö-2003 was more neurovirulent than the HB171/11 strain. Neurons are generally the target cells in the brain during TBEV infection [185]. The encephalitic nature and differential pathogenesis of these strains in vivo could also be correlated with primary cortical neuron infections, where Torö-2003 showed a replicative advantage.

The immune response against each strain varies regardless of the virulence of the virus. In one study, two TBEV strains of similar virulence in mice showed differential dynamics of appearance of virus, IFNs, and other cytokines in the blood of mice, and different ability to induce a cytokine storm at the terminal stages of disease [186]. In our study, we observed higher upregulation of proinflammatory cytokines TNFα, IL-6, and CXCL-10 in spleen and lymph nodes of HB171/11-infected mice than in Torö-2003-infected mice at early time points, whereas it reached similar levels at later time points. This suggests that HB171/11 strain triggers a response in peripheral myeloid cells such as macrophages and dendritic cells, which are potent producers of cytokines, which helps to contain its replication to the periphery. Another possibility is that Torö- 2003 might inhibit the early proinflammatory cytokine production, as TBEV encodes many countermeasures for inhibition of IFN signalling.

In summary, we compared the pathogenesis of two TBEV strains of the European subtype that cause completely different symptoms in humans. We found that strain HB171/11 was less neuroinvasive and neurovirulent than Torö-2003 but that it elicited a strong early immune response. This paves the way for the understanding of genetic factors that influences virulence of the virus. If utilized can be the base for development of a live attenuated vaccine, given the peripheral, mild nature of HB171/11 infection.

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Concluding remarks

The innate immune system is the first line of defence against viral infection and responds quickly―within minutes to hours―after infection. However, the innate immune detection pathways and their relative contribution vary according to the host cell type and the invading virus. Here we investigated some aspects of the innate immune system in flavivirus infection.

In this work, we found that IFN production through IPS-1 signalling is important in tick-borne flavivirus infection and immunity. IPS-1-deficient mice showed lower basal expression levels of antiviral ISGs, which contributed to higher viral burden. In the brain, IPS-1 was important for prevention of infection of the neurons and apoptosis of the brain cells. Absence of IPS-1 signalling also led to immune cell infiltration and development of an inflammatory state in the brain. IPS-1 signalling in the brain was more important for IFN-b upregulation in the olfactory bulb than in other regions of the brain. The study highlighted the region- specific immune response in the CNS during virus infection. Thus, we propose that various PRR pathways regulate the IFN response in different sections of the brain.

In this thesis, I have further shown the region-specific importance of viperin within the brain. Viperin is a broad-spectrum antiviral ISG that is active against many viruses. Mice lacking viperin showed high levels of tick-borne flavivirus replication in the olfactory bulb and cerebrum. Furthermore, viperin was very important in cortical neurons but not in granule cell neurons, as other IFN- induced ISGs could not compensate for the lack of viperin in cortical neurons. The results of this study suggested that a single ISG can shape the susceptibility and immune response in different regions of the brain.

We also identified the virus-specific restriction mechanism of viperin. We found that viperin interacts with NS3 proteins of different flaviviruses (JEV, YFV, TBEV, and ZIKV) but its antiviral activity correlated strictly with its ability to degrade NS3. Viperin induced degradation of the NS3 proteins of both ZIKV and TBEV via the proteasome. Viperin also reduced the stability of other viral proteins in an NS3-dependent manner. These results have led to the possibility of developing new drugs that can be used for therapeutic interventions.

We also investigated the pathogenesis of the HB 171/11 strain of TBEV, which has been associated with gastrointestinal and constitutional symptoms in patients. This strain showed a delayed neuroinvasiveness and was less neurovirulent in mice than another European TBEV strain, Torö 2003. Such unique differences

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could be attributed to differences in the genomes of these TBEV strains, which influenced both the cytokine induction profiles and the efficiency of replication in target cells.

The work described in this thesis has broadened our understanding of the heterogeneity of the innate immune response in the brain and its impact on neurotropic flavivirus infections. Further work is needed to understand the molecular differences between subtypes of cells in the brain and their specific immune responses in different parts of the brain. This knowledge may help us to understand what influences the tropism of neurotropic viruses.

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Acknowledgements

This PhD journey has taught me many things, both in my scientific life and my personal life. Here I would like to take the opportunity to thank people who have helped me in many ways throughout the journey. First of all, I would like to extend my gratitude to my supervisor, Anna. Thank you for this opportunity, which helped me to grow scientifically. For your guidance, support, and encouragement throughout my doctoral work. For giving me suggestions whenever necessary, and most importantly, for listening to my scientific ideas and giving me freedom in the lab. I would also like to thank my co-supervisor Nelson, for guidance and excellent journal club discussions. I thank our collaborator, Andrea, for teaching me the right way to do mouse studies and for her valuable guidance and scientific insight into the projects. I want to thank other people in Braunchweig―Martina, Loreen, Sharmila, and Sarah―for your help. Also, our collaborator Jonathan, for teaching me isolation of brain cells and for your valuable insights in my research.

I also thank Niklas, Magnus, Fredrik, Göran, and Ya-fang for great discussions in the virology seminars and for creating such a nice and friendly working atmosphere at the Virology Unit.

To my fellow PhD students in the lab: Richard, we have performed most of our research together. Thanks for your help in settling in Umeå, and for all the fun times in the lab and gym. Arun, we have known each other for such a long time, from being my senior during the masters to my fellow PhD student. Thanks for your valuable input in my research, for your guidance and fun times in both the lab and outside. Kirstin, I learned a lot from you about the importance of being organized in the PhD world. To the postdocs in our lab over the years. Elvira for teaching me the basic techniques, for settling me in Braunschweig, and for making me understand the difficulties ahead in PhD research already at the start. Master Lee and Christos, for all the fun times, afternoon coffee discussions, and drinking outings at Nydala Lake. Emma N., for being so easy-going in the lab and for all your help. Thanks, Naoko, Thomas, and Ebba, for being fun colleagues in the lab. Koushik, thank you for your help in the lab and fun times on our Europe trips. Naveed, thank you for being nice friend and all those dinners. Goni, thank you for your help with the thesis and fun times in the lab. To Karin, Kickan, and Katrine―for all your help and for being so nice and caring through all these years. To my office mates throughout the years, Emma H, Jenny, and Katarina, for all your help and for decorating our office. Thank you for all those nonsense discussions about life, science, and world news. I would also like to thank everyone at the Virology Unit for creating such a pleasant working environment and for the scientific discussions in the virology seminars.

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I would also like to thank Lars for all those discussions in the corridor about various topics. Lena, Maja and Veronica, thanks for your help and support with the administrative work.

To my extended family in Umeå. Chinmay and Naresh, for being such awesome friends. It will be difficult to put everything into words, so what I can say is thanks for everything. Thank you, Anubhav and Gaurao, for your help in settling in Umeå. Anandi, Mridula, Steffy, Mahesh, and Radha―for all the fun times, dancing, and parties. Rupali, thanks for all the help and for making my family’s stay more eventful during their visit to Sweden. Shrikant, Laxmi, and Sanat, thanks for all the good times. Thanks to the badminton and table tennis group for the adrenaline boost.

I have to give special mention to my masters classmates from India, who encouraged me to take this PhD journey, and they will always have made a valuable contribution in my scientific career. Rishank, Preeti, Shamik, Sindhu, Finny, and all my classmates, thank you for maintaining a competitive and yet supportive environment during those years.

Finally, thanks to my family for your love and support. Thanks for never questioning my career choices. I know you wanted me to become a real doctor, but anyway, now you can still call me “Doc”!

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