Dissertations / Theses on the topic 'Virus encephalitis'

ABSTRACT Eastern Equine Encephalitis virus (EEEV) is an alphavirus with high pathogenicity in both humans and horses. Florida continues to have the highest occurrence of human cases in the USA, with four fatalities recorded in 2010. Unlike other states, Florida supports year-round EEEV transmission. This research uses Geographic Information Science (GIS) to examine spatial patterns of documented sentinel seroconversions and horse cases in order to understand the relationships between habitat and transmission intensity of EEEV in Florida. Sentinel sites were categorized as enzootic, periodically enzootic, and negative based on the amount of chicken seroconversions to EEEV. Sentinel sites were analyzed based on land classification data d using the Kruskal-Wallis test to determine which habitats were associated with disease transmission. Cluster analyses were performed for the horse cases using density-based spatial clustering of applications with noise (DBSCAN). Ecological associations of EEEV were examined using compositional analysis and Euclidean distance analysis to determine if the proportion or proximity of certain habitats played a role in transmission. The research in these studies provides evidence of ecological associations for EEEV transmission in Florida that hasn't been previously analyzed. Furthermore, these studies provide the groundwork for better understanding of why there is a disproportionate number of horse and human cases of EEEV in Florida than in any other state.

Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia, the Muar strain, is thought to represent a putative fifth genotype. I have determined the complete nucleotide and amino acid sequence of the Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of the Muar strain revealed that it does represent a distinct fifth genotype of JEV. I elucidated the Muar signature amino acids in the envelope (E) protein, including E327 Glutamine on the exposed lateral surface of the putative receptor binding domain of the E protein, which distinguishes the Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean (range) evolutionary rate is 4.35 x 10-4 (3.4906 X 10-4 to 5.303 x 10-4) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s. It is postulated to have originated in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection. The ability of intravenous immunoglobulins (IVIGs) which sometimes are used as supportive treatment for JEV infection to protect against strains of JEV representing the five major genotypes was assessed. Neutralization assays showed IVIGs appear cross-reactive across the five JEV genotypes with effective but lower titers for the Muar strain as well as representatives from genotype IV. Whether there are other strains from genotype V, and what happened to them remains unknown.

Ticks are blood-sucking parasites that are an inconvenience for both humans and animals. The tick by itself is normally harmless unless they attack in excessive numbers. The harm from ticks stems from them being excellent vectors for other parasites, in the form of bacteria and virus that via the ticks are provided a bridge to move across the blood streams of different animals, including humans. One of the most pathogenic tick-borne disease for humans is caused by a flavivirus, the tick-borne encephalitis virus (TBEV). Each year approximately 10 000 individuals on the Eurasian continent develop neurological disease, in the form of meningitis, encephalitis, myelitis and radiculitis, following a bite by a TBEV infected tick. To evaluate the risk of TBEV infection after a tick-bite, we have developed a study to investigate ticks that have bitten humans and to follow up the tick-bitten humans to investigate if they get infected, and if they develop symptoms, and further trace the virus back to the tick that is infected with TBEV. Ticks, blood samples, and questionnaires were collected in collaboration with 34 primary health care centers in Sweden and on the Åland Islands during 2008 and 2009. Several demographical and biological factors were investigated regarding the interaction between ticks and humans. The main finding was that men removed the ticks later than women, and that both older men and older women removed the ticks later than younger individuals. This could in part explain why older individuals in general, and men in particular, are at greater risk of acquiring tick-borne encephalitis (TBE). Furthermore, the prevalence of TBEV in ticks that have bitten humans were investigated, in order to correlate the copy number of TBEV in the tick and the tick feeding-time to the risk of developing symptomatic and asymptomatic infection. This entailed the development of new methodology for tick analysis and TBEV real-time PCR. The result showed a very low risk of TBEV infection in the studied areas, only 5 of 2167 investigated ticks contained TBEV. Three of the individuals bitten by TBEV infected ticks were vaccinated and did not develop symptoms of TBEV infection. One unvaccinated individual got bitten by a tick containing 1800 virus copies, with a feeding-time of 12-24h, and interestingly showed no signs of infection. Another unvaccinated individual got bitten by a tick containing 7.7 million virus copies, with a feedingtime of >60h. This individual developed symptoms consistent with a 1st phase of TBE, including fever and headache, but did not develop the 2nd neurological phase of TBEV infection. Despite only  finding 5 ticks infected with TBEV, a correlation between the virus load in the tick and the tick feeding-time was observed. In 2 other individuals, TBEV antibody seroconversion was detected during the 3 month study period, one without symptoms, while the other experienced symptoms consistent with the 1st phase of TBE. These observations support the hypothesis that a higher virus amount in the tick and a longer feeding time increases the risk of TBEV infection. To further examine TBEV in ticks that have bitten humans and find factors that may predict the risk of human infection and development of TBE, we characterized several TBEV strains genetically. Including TBEV strains isolated from ticks that have bitten human, from questing field-collected ticks, and TBEV strains isolated from patients with TBE. In one of the ticks detached from a human after >60h of feeding, there was a heterogeneous population of TBEV quasispecies with varying poly(A) length in the 3’ untranslated region of the genome was observed. These variations might have implications for differences in virulence between TBEV strains, and the heterogeneous quasispecies population observed could be the virus adapting from replication in tick cells to mammalian cells. We also investigated the response to TBEV vaccination in relation to 14 health-related factors in a population of older individuals on the Åland Islands. Blood samples, questionnaires, and vaccination records were collected from 533 individuals. Three different serological assays to characterize antibody response to TBEV vaccination were used. The main finding was that the number of vaccine doses in relation to age was the most important factor determining successful vaccination. The response to each vaccination dose declined linearly with age, and as much as 47%  of individuals 50 years or older that had taken 3 vaccine doses were seronegative, compared to 23% that had taken 4 doses and 6% with 5 doses. Comparison between the serological assays revealed that the cutoffs determining the balance between sensitivity and specificity differed, but not the overall accuracy. Taken together, these results contribute to a better understanding of the TBEV epidemiology and can provide tools in the prevention of TBE.

This thesis explores the importance of IFN in SFV encephalitis. A quantitative PCR assay for IFN-β and IFN-α transcripts and a quantitative IFN bioassay were developed to determine differences in IFN expression under different infection conditions. Mouse models and primary cell lines were used to establish the importance of PKR for IFN=β expression during SFV infection and to determine whether SFV nsP2 has a role in modulating IFN responses. In the absence of PKR, at early times post-infection, cultured cells reproducibly produced significantly lower levels of IFN-β transcripts. Reduced levels of functional IFN were also demonstrated by bioassay. Previous data has shown that PKR is not required for IFN-β induction. The sensitivity of the qPCR assay has allowed the demonstration that PKR, although not critical for IFN induction, is involved in IFN-β induction and is particularly important at early time points post infection. SFV-nsP2 has been postulated to be involved in IFN interference. Comparing SFV4 to SFV4-nsP2-RDR (a mutant virus with a single amino acid change within the nuclear localisation signal of nsP2, which prevents its translocation into the nucleus) demonstrated that relative to the number of infected cells, the SFV4nsP2-RDR mutant induced over ten –fold more IFN-β transcripts than the wildtype SFV4 strain; this upregulation was specific to IFN-β. The IFN bioassay results supported this data; SFV4-nsP2-RDR induced higher functional IFN levels in comparison to wt SFV4. Both viruses grew to similar titres and at similar rates. In the mutant and wt infections both NF-κB and IRF-3 translocated into the nucleus; however, preliminary EMSA data has suggested that the amount of NF-κB and IRF-3 translocated into the nucleus; however preliminary EMSA data has suggested that the amount of NF-κB bound to the IFN-β promoter is reduced during a wt infection. This suggests a possible mechanism for the differential IFN expression and represents the first IFN evasion mechanism described for an alphavirus.

The flavivirus genus is of major concern for world morbidity and mortality and includes viruses causing both encephalitic as well as hemorrhagic diseases. The incidence of Tick-borne encephalitis is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains. In this study we have generated strategies for detection of broad types of tick-borne flaviviruses in pools of I. ricinus sampled in Sweden. The positive collection on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self-replicating DNA based sub-genomic TBEV replicon capable of expressing reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/βPIX protein complex, impairing neurite outgrowth in neuronal growth factor induced PC12 cells. We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins. These interactions might affect cellular pathways and might have a role in flavivirus replication. We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in sampled ticks.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Das Frühsommer-Meningoezephalitis-Virus (FSMEV) ist eines der wichtigsten vektorübertragenen Viren in Europa und Asien. Die häufigste Übertragung erfolgt durch den Stich einer infizierten Zecke, gelegentlich werden FSME Infektionen auch durch den Genuss von Rohmilchprodukten infizierter Tiere verursacht. Die Pathogenese von Caco-2 Monolayer Epithelzellen zeigten nach Infektion mit FSMEV morphologische Änderungen mit signifikanter Vakuolisierung. Ultrastrukturanalysen zeigten eine Ausdehnung des rauen ER und das Auftreten FSMEV haltiger Kavernen. Monolayer von Caco-2 Zellen bildeten eine Barriere mit stabilem transepithelialem elektrischem Widerstand (TEER). Auch traten Viren im basolateralen Medium auf, die über einen Tanscystose pathway (PW) aufgenommen wurden. Der Zelleintritt von FSMEV konnte durch verschiedene Inhibitoren wirksam blockiert werden, was darauf hinweist, dass Aktinfilamente und Mikrotubuli wichtig für die PI3K-abhängige Endozytose sind. Die experimentelle Flüssigkeitsaufnahme zeigte erhöhte intrazelluläre Ansammlungen von FITC-Dextran haltigen Vesikeln und die Co-Lokalisation von FSME-Viren mit frühem Endosom Antigen-1 und mit sorting nexin-5. Was auf die Makropinozytose als Transportmechanismus hinweist. Während der Infektion wurden weitere Hinweise für die Virustranslokation über den parazellulären Weg gefunden. Das konnte den FSMEV Pathomechanismus in humanen Intestinalepithelzellen über Nahrungsmittel näher aufklären. Die Untersuchung der zwei UPR „signaling PWs“ während der FSMEV Infektion in VeroE6 Zellen zeigte, dass die Menge von „heat shock protein“ 72 im Verlauf der FSMEV Infektion ansteigt, und eine FSMEV Infektion den „IRE1- und den ATF6 PW“ aktiviert. Auch die Hemmung des „IRE1 PW“ wirkt auf die FSMEV Infektion, was darauf hinweist, dass eine FSMEV Infektion die beiden „UPR signaling PWs“ aktiviert. Diese Hemmung der FSMEV Replikation durch UPR Inhibitoren könnte ein neuer Ansatz für spezifische Therapien gegen FSME sein.
Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. The transmission mainly occurs by the bite of an infected tick. Consuming of rough milk products from infected livestock animals also occasionally cause TBE cases. Human intestinal Caco-2 cells were used to investigate the pathogenesis caused by TBEV. During TBEV infection Caco-2 monolayers showed morphological changes with significant vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers showed an intact epithelial barrier with stable transepithelial electrical resistance (TER). Concomitantly, viruses were detected in the basolateral medium, taken up via a transcytosis pathway. TBEV cell entry was efficiently blocked with different inhibitors, suggesting that actin filaments and microtubules are important for PI3K-dependent endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles and co-localization of TBEV with early endosome antigen-1 and with sorting nexin-5 could confirm macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Thus, TBEV pathomechanisms in human intestinal epithelial cells and its transmission via the alimentary route were enlightened. In addition, I investigated the effects of the two unfolded protein response (UPR) signaling pathways upon TBEV infection in Vero E6 cells. I showed that the amount of heat shock protein 72 increased in the course of TBEV infection. I then confirmed that TBEV infection activates the IRE1 pathway and ATF6 pathway. These findings provide the first evidence that TBEV infection activates the two UPR signaling pathways. Moreover, inhibition of UPR may provide a novel therapeutic strategy against TBE.

Vector-borne diseases are an increasing global threat to humans due to climate changes, elevating the risk of infections transmitted by mosquitos, ticks, and other arthropod vectors. Ixodes ricinus, a common tick in Europe, transmits dangerous tick-borne pathogens to humans. Tick-borne encephalitis (TBE) is a vector-borne disease caused by TBE virus (TBEV). Climate change has contributed to increased tick abundance and incidence of tick-borne diseases, and between 10,000 and 15,000 human TBE cases are reported annually in Europe and Asia. TBEV shows a patchy geographical distribution pattern where each patch represents a natural focus. In nature, TBEV is maintained within the tick-rodent enzootic cycle. Co-feeding is the main route for TBEV transmission from infected to uninfected ticks and for maintenance within the natural foci. The increasing number of TBE cases in Scandinavia highlights the importance of characterizing additional TBEV sequences and of identifying novel natural foci, and in this work we sequenced and phylogenetically characterized four TBEV strains: Saringe-2009 (from a blood-fed nymph), JP-296 (from a questing adult male), JP-554 (from a questing adult male), and Mandal-2009 (from a pool of questing nymphs, n = 10). Mandal-2009 represents a TBEV genome from a natural focus in southern Norway. Saringe-2009 is from a natural endemic focus in northern Stockholm, Sweden, and JP-296 and JP-554 originate from a natural focus “Torö” in southern Stockholm. In addition, we have studied the effect of different biotic and abiotic factors on population dynamics of I. ricinus in southern Stockholm and observed significant spatiotemporal variations in tick activity patterns. Seasonal synchrony of immature stages and total tick abundance are important factors for the probability of horizontal transmission of TBEV among co-feeding ticks. We found that the probability of co-occurrence of larvae, nymphs, and female adults was highest during early summer whereas increasing vegetation height and increasing amounts of forest and open water around the study sites had a significant negative effect on co-occurrence of larvae, nymphs, and female adults. The proximal part of the 3 ́non-coding region (3 ́NCR) of TBEV contains an internal poly(A) tract, and genomic analysis of Saringe-2009 revealed variability in the poly(A) tract indicating the existence of different variants within the TBEV pool of Saringe-2009. Like other RNA viruses, TBEV exists as swarms of unique variants called quasispecies. Because Saringe-2009 came from an engorged nymph that had been feeding on blood for >60 h, we propose that Saringe-2009 represents a putative shift in the TBEV pool when the virus switches from ectothermic/tick to endothermic/mammalian environments. We investigated the role of poly(A) tract variability in replication and virulence of TBEV by generating two infectious clones of the TBEV strain Toro-2003, one with a short/wild-type (A)3C(A)6 poly(A) tract and one with a long (A)3C(A)38 poly(A) tract. The infectious clone with the long poly(A) tract showed poor replication in cell culture but was more virulent in C57BL/6 mice than the wild-type clone. RNA folding predictions of the TBEV genomes suggested that insertion of a long poly(A) tract abolishes a stem loop structure at the beginning of the 3 ́NCR. Next generation sequencing (NGS) analysis of the TBEV genomes after passaging in cell culture and/or mouse brain revealed molecular determinants and quasispecies structure that might contribute to the observed differences in virulence. Our findings suggest that the long poly(A) tract imparts instability to the TBEV genome resulting in higher quasispecies diversity that in turn contributes to TBEV virulence. Phylogenetic analysis of Saringe-2009, JP-296, JP-554, and Mandal-2009 predicted a strong evolutionary relationship among the four strains. They clustered with Toro-2003, the first TBEV strain from Torö, demonstrating a Scandinavian clade. Except for the proximal part of the 3 ́NCR, TBEV is highly conserved in its genomic structure. Genomic analysis revealed that Mandal-2009 contains a truncated 3 ́NCR similar to the highly virulent strain Hypr, whereas JP-296 and JP-554 have a genomic organization identical to Toro-2003, the prototypic TBEV strain from the same natural focus. NGS revealed significantly higher quasispecies diversity for JP-296 and JP-554 compared to Mandal-2009. In addition, single nucleotide polymerphism (SNP) analysis showed that 40% of the SNPs were common between quasispecies populations of JP-296 and JP-554, indicating the persistence and maintenance of TBEV quasispecies within the natural focus. Taken together, these findings indicate the importance of environmental factors for the occurrence pattern of the different life-stages of the tick vector, which are important for the persistence of TBEV in nature. Our findings also show that the selection pressure exerted by specific host also affects the population structure of the TBEV quasispecies. In addition, our results further demonstrate that the evolution of quasispecies has effect on TBEV virulence in mice.
Vektorburna sjukdomar är ett växande globalt hot mot både människor och djur. De pågående klimatförändringarna kan leda till förhöjda risker för infektioner överförda av myggor, fästingar och andra leddjursvektorer. Ixodes ricinus är en vanlig fästing i Europa som överför fästingburna patogener som är farliga för människor. Fästingburen encefalit (TBE) är en vektorburen sjukdom som orsakas av TBE-virus (TBEV). De pågående klimatförändringarna har bidragit till en ökning både av vektorn och sjukdomsfrekvensen. Mellan 10 000 och 15 000 mänskliga TBE-fall rapporteras årligen i Europa och Asien. Den geografiska fördelningen av TBEV visar ett ojämnt fördelningsmönster där viruset är koncentrerat till vissa fokusområden. TBEV återfinns i naturen i en livscykel där viruset hela tiden överförs mellan fästingar och däggdjur. Spridningen sker dels från en infekterad fästing till ett ryggradsdjur när fästingen äter på värddjuret. Spridning mellan fästingar sker troligen främst genom så kallad “co-feeding”, det vill säga att flera fästingar suger blod samtidigt från samma värddjur. Viruset kan då passera från en infekterad fästing, genom värddjuret till oinfekterade fästingar. Virus kan identifieras och studeras med genetiska metoder. Det ökande antalet TBE-fall i Skandinavien styrker vikten av att hitta och karakterisera ytterligare TBEV-stammar och identifiera nya naturliga fokusområden. Vi har sekvenserat och fylogenetiskt beskrivit fyra TBEV-stammar: Saringe-2009 (blodfylld nymf), JP-296 (födosökande vuxen hane), JP-554 (födosökande vuxen hane) och Mandal-2009 (födosökande nymfer, n = 10). Mandal-2009 är ett TBEV från ett naturligt fokusområde i södra Norge. Saringe-2009 kommer från ett naturligt fokusområde i norra Stockholms län, Sverige. JP-296 och JP-554 härstammar från Torö som är ett naturligt fokusområde i södra Stockholms län, Sverige. Förutom den genetiska sekvenseringen av TBEV har vi också studerat effekten av olika biotiska och abiotiska faktorer på populationsdynamik av I. ricinus i södra Stockholm och observerade variation i fästingsaktivitetsmönster både temporalt och spatialt. Förekomstmönster av fästinglarver, nymfer och vuxna honor, och det totala antalet fästingar är viktiga faktorer för sannolikheten för horisontell överföring av TBEV mellan fästingar. Vi fann att sannolikheten för synkron förekomst av larver, nymfer och honor var högst under försommaren. Vegetationshöjd, mängden skog och mängd öppet vatten runt undersökningsområden hade signifikanta negativa effekter på sannolikheten för att larver, nymfer och honor skulle förekomma samtidigt. Den variabla delen av den icke-kodande 3 ́regionen (3'NCR) av TBEV-genomet innehåller ofta en intern poly(A)-sekvens. Liksom andra RNA-virus, förekommer TBEV som så kallade ”quasispecies” vilka definieras som grupper av olika genetiska varianter av virus. Genom analysen av TBEV-stam Saringe-2009 avslöjades variation i poly(A)-sekvensen vilket indikerar förekomst av ”quasispecies”. Eftersom Saringe-2009 kom från en blodfylld nymf som hade sugit blod i > 60 timmar, föreslår vi att Saringe-2009 visar en förändring i ”quasispecies”-poolen när viruset överförs från exoterm fästingmiljö till endoterm däggdjursmiljö. Vi undersökte poly(A)-ekvensens variabilitet och dess roll vid replikering och för virulens hos TBEV, genom att skapa två infektiösa kloner av Torö-2003 stammen; en med en kort/vild-typ (A)3C(A)6 poly(A)-sekkvens, och en med en lång (A)3C(A)38 poly(A)-sekvens. Den infektiösa klonen med lång poly(A)-sekvens replikerade sämre än vildtypklonen i cellkultur, men (A)3C(A)38 poly(A) var mer virulent i C57BL/6-möss än (A)3C(A)6 poly(A). Datasimulering av TBEV-genomets sekundär-RNA-struktur visade att de längre poly(A)-sekvenserna påverkar veckningen av en specifik sekundärstruktur (SL14) i början av 3 ́NCR. Djupsekvenseringsanalys av TBEV-gnomen avslöjade skillnader för specifika gener och ”quasispecies”-strukturen efter passering i cellkultur och/eller mushjärna. Dessa förändringar föreslås bidra till de observerade skillnaderna i virulens. Våra resultat indikerar att den långa poly(A)-sekvensen ger instabilitet i TBEV-genomet, vilket resulterar i ökad mångfald av ”quasispecies”-populationen som i sin tur kan bidra till TBEV-virulens. Fylogenetisk analys av Saringe-2009, JP-296, JP-554 och Mandal-2009 visade på ett nära släktskap mellan de fyra skandinaviska TBEV-stammarna. De nya stammarna formerade ett kluster med en tidigare TBEV-stam identifierad på Torö (Toro-2003), vilket skapade ett skandinaviskt klad. Genetisk analys visade att Mandal-2009 innehåller en trunkerad 3 ́NCR som liknar den högvirulenta stammen HYPR. JP-296 och JP-554 hade däremot samma genetiska struktur som den längre Torö-2003 stammen från samma fokusområde. Djupsekvensering visade höge mångfald av ”quasispecies”-populationen för JP-296 och JP- 554 jämfört med Mandal-2009. Analys av enkel nukleotid polymorfism (SNP) visade att 40 % av alla SNP var gemensamma mellan ”quasispecies”-populationen för JP-296 och JP-554. Detta indikerar att TBEV-”quasispecies”-strukturen kan vara konserverad för närbesläktade virus vilken kan leda till att den bevaras inom specifika fokusområden. Sammantaget så visar dessa studier att miljöfaktorer påverkar förekomsten av fästingvektorn och dess olika livsstadier, vilket är en bakomliggande faktor för utbredning av TBEV i naturliga fokusområden. Det visar även på att värdmiljön påverkar strukturen för ”quasispecies”-populationen. Dessutom visar våra studier att evolution och utveckling av ”quasispecies”-strukturen kan påverka virulensen för TBEV i möss.

Japanese Encephalitis (JE) is the most common form of viral encephalitis in the world, caused by the Japanese Encephalitis virus (JEV), it is responsible for around 10,000 deaths a year whilst many more are left with long term neurological sequelae and disability. This work sought to use small-scale development techniques alongside high-throughput methodologies to explore and develop selected processing techniques. Formaldehyde inactivation of JEV was characterised and optimised through the use of Design of Experiments screening techniques where temperature, time and formaldehyde concentration were found to be key factors in antigen loss. Glycine and to a lesser extent sorbitol were found to have positive effects as stabilisers during inactivation at different stages of the process. Four anion exchange resins were screened at micro-scale, with the help of an ELISA method evaluated for high-throughput screening, for their potential to replace sucrose gradient purification as the principle purification step of the process. Although Q Sepharose FF was eventually chosen for scale-up studies, the transition of method and recovery rates from batch bind micro-plate studies to 1 mL column scale proved difficult. Yet it was observed that pre-treatment of feed with formaldehyde and glycine could increase JEV antigen recovery rates in flow-through mode chromatography, thought to be due to enhanced stability of the virus particles.

Japanese encephalitis virus (JEV), a member of the family flaviviridae, is one of the most important pathogens of the developing countries, causing high mortality and morbidity amongst children. The present study is aimed at the development of a DNA vaccine for Japanese Encephalitis (JE). As a first step towards developing a DNA vaccine for JE, an eukaryotic expression plasmid encoding the envelope (E) glycoprotein of Japanese Encephalitis Virus (pCMXENV) was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. Several independent immunization and intracerebral (i.e.) JEV challenge experiments were carried out and the results indicate that 51% and 59% of the mice are protected from lethal i.e. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV, which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-y (EFN-y) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Thl sub type. These results demonstrated that immunization with a plasmid DNA expressing intracellular form of JEV E protein confers significant protection against i.e. JEV challenge even in the absence of detectable antiviral antibodies. We then examined the potency of JEV DNA vaccines as well as that of the inactivated mouse brain derived BIKEN vaccine in the i.e. challenge model. The results indicate that all the mice immunized with BIKEN JE vaccine were protected against i.e. JEV challenge while 50% protection was observed in case of mice immunized with pJME or pJNSl and 38% protection was observed in pCMXENV-immunized mice. Immunization with both pJME and pJNSl resulted in 66% protection. These results indicate that the BIKEN JE vaccine confers better protection against i.e. JEV challenge than DNA vaccines. The fact that the BIKEN vaccine conferred better protection against i.e. JEV challenge than DNA vaccines indicated that the i.e. JEV challenge model can be exploited further to examine the potency of different DNA vaccine constructs. Towards this goal, we constructed plasmids that encode secretory or nonsecretory forms of JEV E protein and examined their potency in the i.e. JEV challenge model. Our results indicate that i.m. immunization of mice with plasmid encoding secretory form of JEV E protein confers higher level (75%-80%) protection than those encoding nonsecretory forms. Cytokine analysis of splenocytes isolated from DNA immunized mice after stimulation in vitro with JEV revealed that immunization with plasmid encoding secretory form of JEV E protein induces both Thl and Th2 responses while those encoding nonsecretory forms induce only Thl type of response. Thus, synthesis of secretory form of JEV E protein results in an altered immune response leading better protection against i.e. JEV challenge. Based on our studies, we propose that both cellular and humoral immune responses play a key role in protective immunity against i.e. JEV challenge and DNA vaccines that can induce higher levels of neutralizing antibodies will be as efficient as the BIKEN vaccine in conferring protection against i.e. JEV challenge.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.

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), yellow fever virus (YFV), dengue virus (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 viruses. 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. 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.
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 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 magoch 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.

To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

Within the state of Florida, there are three arboviruses of public health importance that can cause neuroinvasive disease in humans: West Nile Virus, Saint Louis Encephalitis Virus, and Eastern Equine Encephalitis Virus. Mosquitoes (Diptera: Culicidae) within the genus Culex are known and suspected vectors of these diseases. The vectors of these diseases can be present in urban wetland habitats that allow for exposure to residential communities. Vector ecology must be investigated in order to understand the dynamics of disease transmission. In Hillsborough County, Florida the spatial and temporal distribution of these vectors are not well established. An ecological study was conducted in the University of South Florida's Ecopreserve using trapping methodologies to sample the adult and gravid females as well as collect the egg population. Collections were made at three spatial points for the duration of July through December 2013 and compared to meteorological variables. Culex erraticus, a proposed bridge vector of Eastern Equine Encephalitis, was the most abundant adult species and gravid female captured. Culex nigripalpus, primary Floridian vector of Saint Louis Encephalitis and bridge vector of West Nile Virus, was the second most abundant adult species caught as well as the majority of eggs collected. Based on the results collected, the presence of Culex erraticus and Culex nigripalpus was confirmed. The majority of Culex erraticus adults were collected in September and October and Culex nigripalpus adults were the highest in July and August. The results of the gravid and egg collection generated crucial insight regarding methodology for studying vector ecology within this urban wetland habitat. However, modeling at spatial points based on meteorological variables yielded inconsistent results that illicit further investigation regarding these arboviral vectors of disease.

The genomic RNA encoding the structural proteins of louping ill, a tickborne flavivirus, was cloned and sequenced. Sequence comparisons of louping ill envelope protein showed greater homology with tick-borne than mosquito-borne flaviviruses and greater homology with the western than the far eastern subtype of tick-borne encephalitis virus. Louping ill and tick-borne encephalitis viruses are probably varieties of a common tick-borne ancestral virus. The average amino acid sequence diversity between members of the tick-borne serogroup was significantly lower than that of mosquito-borne serogroups, suggesting that tick-borne flaviviruses have been subjected to different evolutionary immune selection pressure from the mosquito-borne viruses. Using the published model of tick-borne encephalitis envelope protein and the derived sequence data on louping ill virus, three discontinuous peptides (amino acids 81-88, 207-212 and 230-234) which may represent critical molecular determinants within the receptor binding site of tick-borne flaviviruses, were identified. These peptides may provide a specific genetic marker for these viruses. Recombinant baculoviruses and vaccinia viruses containing cloned DNA, encoding either the envelope protein or the structural proteins of louping ill virus, were constructed. Glycosylated envelope protein, presented both inside and on the surface of insect and mammalian cells, was expressed by all four recombinant viruses. Differences in antigenic presentation of envelope protein were observed between envelope protein and structural protein constructs as well as between insect cell and mammalian cell expression systems. Despite the expression of epitopes known to elicit neutralizing and protective antibodies when present in authentic antigen, the recombinant envelope protein expressed by either baculovirus or vaccinia virus failed to induce, under the experimental conditions employed, either neutralizing or protective antibodies in both mice and rabbits against louping ill virus. Hence, louping ill envelope protein expressed by baculoviruses and vaccinia viruses was antigenically reactive but immunogenically inert.

Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.

Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis (JE), the leading cause of vaccine-preventable neurological disease. JEV is a flavivirus that is primarily transmitted through the bite of infected mosquitoes, similar to dengue virus (DENV), St. Louis encephalitis virus (SLEV), West Nile virus (WNV), and Zika virus (ZIKV). The two viral characteristics that dictate virulence are (1) neuroinvasiveness, the ability of the virus to invade the central nervous system(CNS), and (2) neurovirulence, the capacity of the virus to kill resident cells in the CNS. The clinically proven live-attenuated JEV vaccine, SA14-14-2, lacks both pathogenic characteristics unlike its virulent parental virus, SA14. Previous work has revealed the viral E gene as the main determinant of these two pathogenic properties, though the molecular mechanisms behind their attenuation remain unclear. The E gene encodes for the viral envelope glycoprotein that is involved in viral entry into susceptible host cells. The E protein of SA14-14-2 differs from SA14 by nine amino acids. To investigate the role of these mutations in JEV virulence, we created a series of SA14E mutants using infectious cDNA technology. Here, we report the independent function of domains I (DI) and II (DII) of the viral E protein in JEV neurotropism. We reveal that an individual mutation in DI, E138K,and synergism between two mutations in DII, E244G and K279M,are independently sufficient for the attenuation of JEV neuroinvasion. Also, we report that multiple E mutations are required for full attenuation of JEV neurovirulence. Overall, our findings show the direct relationship between genetic factors and JEV neuroinvasion. These results provide a solid foundational base for the logical development of other, currently non-existing, live-attenuated neurotropic flavivirus vaccines and antivirals.

Japanese encephalitis (JE) is a brain infection caused by JE virus (JEV). It has an estimated worldwide incidence of 68,000 cases and 10,000 to 15,000 deaths annually. Despite large effective immunization campaigns, Japanese encephalitis remains a disease of global health concern, because the virus is spreading. There are five genotypes of JEV (genotype I – V), each associated with different geographical areas and associated epidemiology. The Muar strain of JEV, the fifth genotype, is believed to represent the oldest lineage from which genotype I - IV evolved. Muar was isolated in Singapore in 1952. At the same time as Muar was isolated; the Tengah strain of JEV was also isolated from a nearby location. However, Tengah and the characteristics of Tengah have largely remained unknown. Muar was considered the only known representative of genotype-V prior to 2009. Vector competence studies have examined genotype-I, II and III of JEV. However, genotype-IV and genotype-V have never been investigated in vector competence studies. Therefore, the infectivity of these viruses to mosquitoes is unknown. The competence of non-Asian mosquitoes to JEV has been demonstrated suggesting potential for emergence in some other regions. In Great Britain, JEV is considered a potential threat to animals and public health. However, the level of competence of British mosquitoes to any arbovirus is not known. The overall objective of this thesis was to characterise Tengah, investigate molecular and mosquito factors that might relate to the lack of circulation of genotype-V isolates and assess the potential of arbovirus (JEV) emergence in Great Britain. Molecular characterization of Tengah strain showed that it is another isolate of genotype-V, with 99% sequence similarity to Muar. Evolutionary analysis performed using the Bayesian Evolutionary analysis of Sampling Trees (BEAST) program estimated that JEV is evolving at a rate of 3.53 × 104 nucleotide substitution per site per year. Vector competence studies demonstrated Culex quinquefasciatus mosquitoes are able to transmit Muar with transmission rates of 23% at 21-days post infection. Comparison of transmission between Muar (genotype V) and Nakayama (genotype III) found no significant difference between the two genotypes. Ochlerotatus detritus, a British mosquito, was susceptible to JEV at both 23°C and 28°C as determined by the detection of virus in the saliva 7 days post infection. The overall transmission rate was 13% at 23°C and 25% at 28°C. There was no significant difference between the two temperatures. Infection rates for Ochlerotatus detritus and Culex quinquefaciatus were similar. This thesis has shown that on account of its similarity to the Muar isolate, Tengah represents a variant of Muar and is another genotype-V isolate. Evolutionary analysis showed that JEV originated from its ancestral virus in the year 1120 while the Time to Most Recent Common Ancestor for genotype V was in the year 1840. Muar has the ability to infect, replicate and be transmitted in Culex quinquefasciatus, suggesting that the limited distribution, isolation and circulation of genotype-V is probably not explained solely by mosquito factors. Ochlerotatus detritus is competent to transmit JEV and would therefore pose a threat should this virus occur in Great Britain.

Background: West Nile Virus (WNV) is a serious mosquito-borne disease that can potentially lead to death. The purpose of this study is to spatially examine known risk factors for WNV within Georgia at the county level. The study produces maps that relate known WNV cases to high, medium, and low risk factor areas for additional analyses. Methodology: Cartographic visualization and statistical analysis software was used to examine the relationships between: the geographical distribution of age, race, gender, urbanicity, and population density of Georgians in relation to WNV cases by county. Chi-square analysis and odds rations were calculated to determine whether or not associations of risk and the likelihood of WNV case reports were significant. Results: Gender was found to be significantly associated with the distribution of reported WNV cases. Identification of high risk areas throughout the state was determined through the use of Geographic Information System software. Conclusion: Insights into the visual distribution of WNV risk factors throughout the state of Georgia can assist policy makers and public health planners to optimize resources in WNV transmission and prevention abatement and education efforts. This exploratory study provides a critical first glimpse into the distribution of WNV risk factors throughout the state.

博士
長庚大學
生物醫學研究所
98
Due to the lack of a proofreading function and error-repairing ability of the genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor involved in genomic variations of these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using 2 local pure clones (T1P1-S1 and CJN-S1) derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/prM junction comprising a fragment of 868 nucleotides (nt 10~877), the recombinant progeny virus was formed primarily in BHK-21 cells that had been co-infected with the 2 clones. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123~323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, 3 stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that RNA recombination generates genetic diversity in the JE virus; which thus must be considered in studies on viral evolution, epidemiology, and possible vaccine safety.

碩士
國防醫學院
微生物及免疫學研究所
87
The portion of this research is studying the expression of p35 gene will make Japanese encephalitis virus (JEV) persistently infect the baby hamster kidney fibroblast (BHK-21) cells or not. Persistent JEV-infection can be readily established in murine N18 and astrocytoma DBT cells, but not in BHK-21 and CHO cells. BHK-21 cells were killed upon JEV infection for JEV induced apoptosis. However, over-expression of human bcl-2 in BHK-21 cells delayed JEV-induced apoptosis and rendered cells to be persistently infected with JEV. The results of this study suggest that p35 and bcl-2 have similar fuctions in BHK-21 cells. p35 is an anti-apoptotic viral protein of baculovirus. Expression of p35 in BHK-21 cells (B-p35) delayed JEV-induced apoptosis and rendered a portion of the cells to be persistently infected with virus (B-p35/RP9). The results of the studies above in our laboratory suggested that cells would express the truncated viral nonstructural protein-1 (NS1) upon JEV persistent infection. For the experiments, we got the same suggestion that truncated viral NS1 as well as its derivative NS1' were also detected in B-p35/RP9 cells. Besides, CHO cells are similar to BHK-21 cells that would not be persistently infected with JEV. Expression of p35 in CHO cells also rendered cells to be persistently infected with JEV and to express the truncated viral NS1. We proved that was persistent infection. The other portion of this research is studying JEV persistent infection in human myeloid leukemia cells (K562). The different cell lines were dead upon JEV infection and JEV-induced apoptosis, and little survival cells would be persistently infected with virus. K562 is a human myeloid leukemia cell line. The results of our research suggested that the cell line could be persistently infected with dengue virus and cells had no CPE. The same as dengue virus, the results of this research suggested that the cell line would be infected with JEV and had no CPE. The cells almost be persistently infected under culturing for long time. We could detect the truncated viral NS1 by western blot. And K562/JEV or DEN could interfere K562/DEN or JEV super-infection. K562/JEV grew in serum starvation is better than K562/DEN. Each K562/DEN cell could release 3 virus particles, and the K562/JEV cell could release 500 virus particles. The results suggested that the K562 cells prefer to JEV infect.

碩士
國防醫學院
微生物及免疫學研究所
92
The role of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) on viral life cycle largely remains unknown. Previously, we found that fusion of JEV NS2A with a Gal-4 DNA binding domain could trans-activate the Gal-4 dependent reporter gene expression. In the present study, a series of JEV NS2A deletion clones have been constructed to map the transactivation domain of NS2A. It appears that the N-terminal residues 1 to 68 of NS2A possess the transactivation activity. To further reveal the role of this transactivation domain on JEV replication, we constructed a JEV infectious clone with a deletion on the 5-68 residues of NS2A. After in vitro transcription and RNA transfection, we found that the wild-type but not the NS2A-partial-deleted JEV infectious clone readily expressed JEV proteins and produced infectious viral progeny. Our results support an essential role of this transactivation domain of NS2A on JEV replication. In order to further study the role of NS2A, we intended to overexpress NS2A for immunization and antiserum production. Since overexpression of NS2A in bacteria has been shown to be toxic, we used a baculovirus expression system in this study. JEV NS2A has been cloned downstream of a strong polyhedrin promoter and in-framed fused with human immunoglobin G (IgG). This recombinant baculovirus could express the protein with expected size, recognized by anti-human IgG antibody and purified by protein A sepharose beads. However, the protein expression level was still very low and would be difficult for further usage. We also adapted a mammalian matchmaker two-hybrid assay system to investigate the interaction between JNS2A with other JEV viral proteins. Cotransfection of individual JEV proteins in-framed fused with Gal-4 DNA binding domain, NS2A fused with VP16 activation domain and reporter plasmids, did not reveal any significant interaction of NS2A with other viral proteins. Overall, we suggest that JEV NS2A might be a transcriptional activator, and is involved in the modulation of JEV RNA replication.

碩士
中興大學
生物醫學研究所
95
Japanese encephalitis virus (JEV), a member of the family Flaviviridae, which causes acute encephalitis in human. JEV targets the CNS, clinically manifesting with fever, headache, vomiting, signs of meningeal irritation, and altered consciousness, leading to high mortality and neurological sequelae in some of those who survive. Though neurological disorder caused by conventional viruses are often characterized by evidence of immune system recognition and the presence of inflammatory components among the neuropathological changes, the mechanisms by which these viruses cause neurological disease are not fully understood. In many cases, the virus is probably not directly involved in the destruction of brain tissue but many cause damage indirectly by triggering cell mediated immune response by activating glial cells. In our studies, morphological change and pro-inflammatory cytokine production were found in JEV-infected glial cells. In addition, JEV-infected microglia significantly elevated extracellular level of glutamate, a major excitotoxic neurotransmitter in the CNS. We have demonstrated that TNF-α, NO and PKC might involve in regulating glutamate release. JEV-infected microglia elevated intracellular glutamate level through the up-regulating glutaminase. Generally, cytokine production and glutamate release are well recognized events in glial activation. Our findings suggest that JEV infection induces glial activation. In conclusion, the relationship of JEV infection, neuroglia activation, and inflammatory responses are highly relative. The inflammatory responses of activated neuroglia might play a role in JEV-induced neurotoxicity.

碩士
國立東華大學
生命科學系
100
Autophagy is a process to maintain cellular homeostasis by degrading unfolded proteins and injured organelles in the cytosol, and it is originally considered to participate in defending the invading microorganisms and viruses. In this study, we found that infection of Japanese encephalitis virus (JEV), a major encephalitis-causing agent in endemic Asia, on mouse embryonic fibroblast (MEF) cells may inhibit autophagic process as revealed by the decreased expression of microtubule-associated protein 1 light chain 3 (LC3) detected by western blot and the reduction of the puncta-formation of LC3 in the cytosol by immunofluorescence assay. The degradation of p62, an adaptive protein containing an ubiquitin-binding association (UBA) domain and a LC-3-interacting region (LIR) involved in targeting cargos for selective autophagy was evident in mock-infected cells, but was defective in JEV-infected ones. Blockade of autophagy by treatment of mock-infected or JEV-infected cells with autophagy inhibitors, bafilomycin A1 or E-64d, that one can inhibit the fusion between autophagosome and lysosome, and the other could inhibit enzymatic activities of lysosomal proteases, respectively, resulted in the accumulation of both LC3-II and p62. As cells infected by JEV had lower LC3 intensity and fewer numbers of autophagosomes, the area of lipid droplets was reversely correlated with the area of autophagosomes. In addition, JEV did not replicate within autophagosome, as neither autophagosome nor p62 protein was colocalized with JEV replicating double stranded RNA (dsRNA) by immunofluorescence assay. Although JEV infection may inhibit autophagy, one key regulator in autophagy signaling pathway, autophagy-related gene 5 (Atg5), was activated as it was processed and conjugated with Atg12 to become the Atg5-Atg12 complex that can proceed the flowing signaling to regulate LC3-II formation. Infection of Atg5 deficient MEF cells with JEV produced less infectious viral particles than did that of wild type MEF cells. In addition, treatment of cells with autophagy inducer, rapamycin, could increase JEV production, in the contrary, treatment with autophagy inhibitor, 3-methyladenine (3-MA), would reduce the viral titer. Thus, JEV infection may interfere with the autophagic process and take advantage of the important regulators in autophagy to facilitate its own viral production.

碩士
國立中興大學
生物科技學研究所
93
Mosquito-borne Japanese Encephalitis Virus (JEV) is the leading cause of viral encephalitis. Since the outbreak in 1924 in Japan, JEV has transmitted seasonally and become an endemic disease in Taiwan. The prevailing area spreads all over the whole province every year. Swine is the most important vertebrate-amplifying host. When more than 50 % of swine gave positive antibody reactions, the encephalitis case reports would reach to the peak within 2-3 weeks and maintained for a month. An inactivated JEV vaccine derived from infected mouse brain has been licensed in Japan since 1956. Although the vaccine has effectively decreased the morbidity of JEV, it is not recommended by the physicians that the adults receive the vaccination, due to the related allergic reactions. Because of the ability to express large amout inability to infect animals, plant viruses may be developed and utilized as the alternative vectors for vaccines. In this study, the coding sequence of JEV envelope protein domain III (JEV EDIII) was expressed using a vector based on the recombinant Bamboo mosaic virus (BaMV). The Foot-and-mouth disease virus (FMDV) 2A sequence was used to bridge the antigen and coat protein of viral vector. By utility the recombinat virus to infect the plants, JEV antigen was produced. The recombinant viruses were single-lesion passaged testing through 10 generations to test the stability. It was found that recombinant viruses can still display the antigen of JEV steadily. As revealed by immuno electron microscopy, the recombinant viruses can express JEV antigen on the surface of the virions. To test the feasibility of the JEV EDIII vaccine produced by BaMV-based vector, the immunogenic effects of the recombinant viruses were evaluated using the mouse model. After parenteral injections, mice produced antibodies against JEV EDIII. Autopsie analyses of the immunized mice revealed no obvious pathological change, demonstrably the safty in using the recombinant viruses as vaccines. In order to increase the antigen expression levels of the chimeric virus, the 2A peptide sequence of vectors was modified. In addition, modification of RGD motif of JEV antigen was performed to increase the antigenecity. The re-engineered viruses produced different quantities of JEV antigens in infected plants. This research demonstrated that utilizations of BaMV vecters in successful production of JEV antigens in plants for the first time. Therefore, BaMV-based vector is an ideal candidate for the development of animal vaccines.

碩士
中國醫藥大學
醫學檢驗生物技術學系碩士班
95
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes severe neurological diseases with a high fatality rate. In this study, the goal of this study is to characterize JEV-induced apoptosis, to identify the dominant viral protein involved in apoptosis, and to investigate the molecular pathways of JEV protein-induced apoptosis. Flow cytometric analysis revealed apoptosis of HL-CZ and TE-671 cells infected with JEV at a MOI of 1 after 2-day infection. Propidium iodide staining, Annexin-V staining and fluorimetric assay of caspase-3 indicated that JEV proteins E, NS2B, NS3, and NS2b-NS3 protease induced apoptosis of HL-CZ and TE-671 cells, respectively. Western blotting showed that single expression of NS2B, NS3-180, and NS2BNS3 protease activated caspases 3 and 8 in HL-CZ cell. Active form of caspases 3 and 9 were detected in JEV E, NS3-180, and NS2BNS3 protease -expressing TE-671 cells, while active form of caspase 9 was only found in NS2b-NS3 protease-expression HL-CZ cell. In addition, the increase of ROS was measured in NS2B-, and NS2b-NS3 protease –expressing TE-671 cells. The decrease of MMP was measured in E –expressing HL-CZ cells and NS2b-NS3 protease-expression TE-671 cells. The increase of Ca2+. was measured in NS2b-NS3 protease –expressing HL-CZ cells. This study provides more information on the mechanism of the JEV induced apoptosis.

碩士
國立臺灣海洋大學
機械與機電工程學系
98
In Southeast Asia, Taiwan is the major epidemic for the Japanese encephalitis diseases. In the current, vaccine is an effective method to against Japanese encephalitis diseases. But also because of the vaccine is used today have concerns about it side effects, so that disease prevention is more difficult. Therefore, understanding the pathogenesis of Japanese encephalitis virus (JEV) infection processes in the human body, which provide advice to judge the timing of medication and help development of new vaccine. Common virus detection and analysis experiment, most of all have to use large detection equipment in the laboratory for analysis. Although it has accurate detection performance, but is accompanied by disadvantage of required long detection time, most take 48 hours or even more, wasting a lot quantity of testing sample and can not be monitoring and measurement in real time, this results of increased human cost of detection and waste of resources. In this study, we provide a chip with low sample demand, our system can be analyzed the pathogenesis of cells infected with virus, and also could monitoring virus infection in real time. This study has produced a microcantilever sensor chip using MEMS technology. The chip size is 3.15 x 3.15 cm, and integrated microfluidic system, optical measurement system, and a biocompatible hydrogel material, producing a micro-biological detection system to detect Japanese encephalitis virus on the cell infection process. First, the PDMS microfluidic system and produced the microcantilever sensor chip using oxygen plasma bombardment for surface modification and bonding, then injected hydrogel solution into microfluidic channel and exposure UV light to curing, after washing the residual hydrogel and sterilization procedures for the chip, which leads to culture medium containing cells, the cells will attached to gel block and adhesion on surface, and finally penetrating the JEV pass microfluidic system for cell infection, and measurement microcantilever sensor deflection by using optical detection system.

碩士
中國醫藥大學
醫學檢驗生物技術學系碩士班
100
Japanese encephalitis virus (JEV) is a member of mosquito-borne Flaviviridae that cause Japanese encephalitis with a high fatality rate. JEV genome is an approximately 11kb single-stranded positive sense RNA, containing a type I cap and a single large open reading frame (ORF) flanked by 5’and 3’ untranslated regions (UTRs). This ORF encodes a single polyprotein as three structure proteins and seven nonstructural (NS) proteins by the proteolytic process. Viral infectious clones and replicons are powerful tools for studying the viral replication and pathogenesis. However, constructing JEV infectious clones is difficult due to recombination and deletion events. The study aimed to construct novel JEV infectious clones and replicons, JEV infectious clones and replicons fused with CMV promoter at 5’end and HDV ribozyme at 3’ end were constructed based on high-copy number vector (pcDNA3.1/HisC) and low-copy number vector (pBR322) and transformed in E.coli DH5α and BD1528. Firstly, three fragments(F1、F2、F3) of the full-length JEV genome were cloned into pcDNA3.1/HisC vector, respectively. Up to now, F2 and F3 fragments have been in-frame cloned into pcDNA3.1/HisC. However, the JE-F1(1-3600) occurred a deletion in 574-1936nt. This deleted clone supply as template for further construction of JEV infectious clones and replicons. On the other hands, a modified pBR322 vector with restriction enzyme site linker and SV40 polyA was used for constructing pBR322-SV40pA plasmid. The JEV F2-F3fragment fused with 67bp HDV ribozyme sequence at 3’-end is successfully inserted into this vector and grown in E.coli BD1528. But, JE-F1(1-3600 nt) clone appeared a nonsense mutation in the prM protein. For constructing replicons, most of structure protein gene was deleted, the gene order in replicons was 5’-CMV promoter-JEV5’-UTR-8 amino acids of Capsid protein (nucleotides 96-119)-DsRed2 fluorescent protein-foot and mouth disease virus 2A self-cleaving protease (FMDV2A)- retain the 30 amino acids of Envelope protein (nucleotides 2388-2477), NS1-NS5 proteins- 3’-UTR and 67bp HDVr sequence. We expect that these JEV replicons exhibit a significant biological function.

碩士
國防醫學院
微生物及免疫學研究所
93
In recent years, vectors based on self-replicating RNA (replicon) of positive-strand RNA viruses are becoming powerful tools for virus study. Subgenomic replicons, which contain deletions in the genome have been successfully generated for several flaviviruses, such as Kunjin virus, west nile virus and yellow fever virus, etc. A lack of Japanese encephalitis virus (JEV) replicon has prompted us to initiate this study. The full-length JEV cDNA was RT-PCR from a Taiwanese isolate RP-9 and cloned under a SP6 promoter. Transfection of the in vitro transcribed RNA readily established a complete JEV life cycle, as evident by immunofluorescent staining and viral plaque formation in BHK-21 cells. We selected Renilla luciferase (RLuc) as a reporter due to its relatively small size (936 bp) and its robust enzyme activity. Two types of replicon plasmids were constructed in this study. Type I replicons contained the RLuc reporter fused in-frame with the JEV open reading frame in the position where the viral structural region was deleted, resulting in the JEV-RLuc replicon. Type II replicon contained an insertion of IRES-RLuc fragment at the upstream end of the JEV 3’UTR, in which the expression of RLuc was under the control of an internal ribosome entry site (IRES). Transfection of BHK-21 cells with the in vitro transcribed replicon RNA only yielded one RLuc peak between 3 to 10 hours post transfection. This reporter activity was similar to that obtained from a replication-defective replicon, which contains a mutation in the nonstructural NS5 GDD RNA polymerase motif, indicating that translation but not replication might have occurred. Using this JEV-RLuc system to study viral translation, we found that the reporter activity was greatly dependent on the 5’ cap structure on the viral RNA. Furthermore, viral nonstructural genes seem to play a role in enhancing viral translation, since deletion of the NS1 to NS5 region of the replicon greatly reduced the reporter activity. These replicons will be further modified for viral replication study and might be used to dissect the viral sequences and cellular factors required for JEV translation and replication.

碩士
國立嘉義大學
生物科技研究所
95
Japanese encephalitis (JE) has been an important endemic zoonoses in Taiwan. Since beginning in 1968, a mass vaccination program, using mouse-brain-derived and formalin-inactivated JEV Nakayama as vaccine, against JE for children was implemented in Taiwan. Dramatic decrease of JE virus infection has been reported, but still approximately 20-30 cases annually. Approximately 25 percent of infectious patients and 50 percent of the survivals develop permanent neurologic and/or psychiatric sequelae. Recently, the anti-JEV drug was unavailable in clinical therapy. Therefore, it is important to develop a new anti-viral drug. Lactoferrin exists in breast milk and mucous secretions; it is a iron-binding protein for transportion in intestines. This protein is also associated with non-specific immune response and inhibits a variety of microbial infections including bacterial, fungous, and viral infection. Lactoferrin can inhibit viral cell entrance into cell by binding to the virus particle directly or to membrane-bound heparan sulfate. In previous reports of our laboratory and others, the heparan sulfate on cell surface is one of possible receptors for Japanese encephalitis virus (JEV). The goal of this study was to investigate the inhibitory effect of bovine lactoferrin against JEV infection. Our results were summarized as below: (1) bovine lactoferrin (bLF) inhibits the infectious JEV, including wild-type (CJN-2K、T1P1、CC27) and laboratory-adapted strains (CJN-L1、CJN-S1、T1P1-L4、T1P1-S1、CC27-L1、CC27-L3、CC27-S6、CC27-S8); (2) bLF inhibits JEV infection by interacting with cells, not viral particle; (3) the mechanism of bLF inhibition JEV infection was not only blocking viral attachment to cellular membrane, but also reducing viral endocytosis and penetration; (4) directly interaction of heparan sulfate (HS) with bLF inhibit JEV infection. In HS-dependent CJN-S1 strain and HE-expressed CHO-K1 cell line, the professional antiviral effect of bLF has been observed; (5) in BHK-21 cells, soluble heparin inhibits the anti-JEV infection activity of bLF; (6) in addition to HS, other unknown molecules may bind to bLF and then inhibit JEV infection, because the antiviral effect of bLF has been observed in HS-independent CJN-L1 strain and HE-deficient CHO-pgsA745 cell line In this study, we demonstrated that bLF can inhibit JEV infection, however, the protection effect is still needed to study in animal experiments. In the future, it is possible to identify the receptor(s) for JEV using bLF inhibits JEV infection by binding to other unknown molecules. Study of the receptor(s) for JEV is acquired to understand the inhibition mechanism of lactoferrin on JEV infection.

博士
國立中興大學
生命科學系所
101
Currently, the underlying mechanisms and the specific cell types associated with Japanese encephalitis-associated leukocyte trafficking are not understood. Brain microvascular endothelial cells represent a functional barrier and could play key roles in leukocyte central nervous system trafficking. We found that cultured brain microvascular endothelial cells were susceptible to Japanese encephalitis virus (JEV) infection with limited amplification. This type of JEV infection had negligible effects on cell viability and barrier integrity. Instead, JEV-infected endothelial cells attracted more leukocytes adhesion onto surfaces and the supernatants promoted chemotaxis of leukocytes. Infection with JEV was found to elicit the elevated production of intercellular adhesion molecule-1, cytokine-induced neutrophil chemoattractant-1, and regulated-upon-activation normal T-cell expressed and secreted, contributing to the aforementioned leukocyte adhesion and chemotaxis. We further demonstrated that extracellular signal-regulated kinase was a key upstream regulator which stimulated extensive endothelial gene induction by up-regulating cytosolic phospholipase A2, NF-κB, and cAMP response element binding protein via signals involving phosphorylation. These data suggest that JEV infection could activate brain microvascular endothelial cells and modify their characteristics without compromising the barrier integrity, making them favorable for the recruitment and adhesion of circulating leukocytes, thereby together with other unidentified barrier disrupting mechanisms contributing to Japanese encephalitis and associated neuroinflammation.

碩士
中國醫藥大學
醫學檢驗生物技術學系碩士班
101
Japanese Encephalitis Virus (JEV) belongs to Flaviviridae, spreading throughout Eastern and South-eastern Asia by culicine mosquitoes, most often Culex tritaeniorhynchus. The clinical symptoms include headache, fever, vomiting, weakness, mental status changes, neurologic symptoms, movement disorders, seizures, and even death. Except for the vaccination, Japanese encephalitis has no specific treatment, just a few supportive treatments, such as IFN treatment. JEV is a single-strand positive sense RNA enveloped virus. JEV genome encodes three structural proteins (capsid, membrane, and envelope) and seven non-structural proteins, which process virus replication and disturb inflammatory responses in host cells. Dihydrofuran carboxylate CW-33 is an intermediate synthesized derivative of furoquinolines with the antiviral and anticancer activities. This study aimed to determine anti-JEV activities of CW-33 in baby hamster syrian kidney (BHK-21) cells and human medulloblastoma (TE761) cells, such cytopathic effect (CPE) inhibition, virus yield reduction, plaque reduction and apoptosis inhibition. CW-33 exhibited the less cytotoxicity with the CC50 values of > 500 μM to BHK-21 cells and 189 μM to TE671 cells, respectively. Simultaneous- and post-treatment with CW-33 alone significantly inhibited JEV-induced CPE and virus yields in BHK-21 and TE671 cells in a concentration-dependent manner. However, CW-33 had no effect on virus attachment and virucidal activity. The plaque reduction assay demonstrated CW-33 as a moderately potential anti-JEV agent with the IC50 of 115 μM. Importantly, CW-33 treatment activated JAK/STAT1 signaling pathway via elevating the phosphorylation levels of JAK1, JAK2, Tyk2, and STAT1, as well as increasing the mRNA levels of IFN-α, IFN-β, IFNAR1, IRF-3, IRF-7, PKR and OAS. The anit-JEV action of CW-33 also correlated with apoptotic reduction of JEV-infected cells, e.g. rescuing the mitochondria membrane potential, decreasing the active form of caspase-3, and increasing the phosphorylation of Akt, mTOR, ERK, and CREB. The result demonstrated CW-33 exhibiting a significant potential on the development of anti-JEV agents.

Chapter 1: Role of JEV NS1 in protective immunity and in immunopathology. Previous studies from our laboratory revealed T cell immunodominance of non structural proteins NS3 and NS1 during natural JEV infections in humans where as the structural protein E, which is a good target for neutralizing antibody response is a poor inducer of T cells. Flavivirus NS1 is also known to induce humoral immune response. Several studies in different flaviviruses have indicated a role for NS1-specific immune responses in protection against flaviviruses. Paradoxically, studies also pointed to the contribution of NS1 in pathology and immune modulation. We screened serum samples from 72 convalescent JE patients for the presence of anti-NS1 antibodies by ELISA and radioimmunoprecipitation and found NS1 reactivity in 45 samples. These antibodies to NS1 are capable of inducing complement mediated cytolysis of cells expressing NS1 on the surface. Additionally, we demonstrated twenty two fold reduction in the infectious virus produced at 48h in SW-13 cells in the presence of human complement and NS1 antiserum compared to control serum, suggesting that complement mediated cytolytic activity of anti NS1 antibody helps the host in controlling the virus propagation. Chapter 2: Comparison of immune responses to JEV structural proteins Capsid and Envelope in human volunteers vaccinated with inactivated JE vaccine and naturally exposed to live JEV. We compared the CMI responses to structural proteins E and C in human volunteers vaccinated with commercially available killed JE vaccine and in humans naturally exposed to live JEV. The results revealed that structural proteins E and C are inherently poor inducers of T cells even in killed vaccine preparation, where there is no competition from immunodominant non structural proteins. Therefore inclusion of nonstructural proteins NS1 and NS3 along with neutralizing antibody inducing envelope should improve memory and efficacy of a JE vaccine. Chapter 3: Construction and testing in the mouse model of experimental recombinant poxvirus vaccines expressing prM, E, NS1, and NS3 of JEV. Guided by the information on immune responses to JEV in the JE endemic human cohort and volunteers vaccinated with killed JE vaccine, we designed experimental vaccines as recombinant vaccinia viruses expressing NS1, NS3, prM, and E proteins of JEV (vNS1NS3prME) or NS1, NS3, prM, and C-terminally truncated E (vNS1NS3prMΔE) and studied the immune responses elicited by these vaccines in mice. Our data showed that a recombinant vaccinia virus expressing prM, ΔE, NS1, and NS3 of JEV is superior to killed JE vaccine in eliciting long lived neutralizing antibodies as well as NS1 and NS3-specific cytotoxic T lymphocytes (CTL) in addition to NS1-specific cytolytic antibodies, resulting in long lasting and enhanced protection from lethal JEV infection in mice. Our results thus identified all B and T cell antigens whose inclusion in a live-vectored vaccine would provide a vaccine with far superior efficacy over the inactivated JE vaccine.