Dissertations / Theses on the topic 'Native virus'

We sought to develop an inactivated nasal mumps virus (MuV) vaccine combined with the Protollin (Prl) adjuvant/delivery system. Antigen based on split Jones MuV was produced and characterized.
Eight-week old BALB/c female mice were vaccinated with two or three doses of MuV antigen (4 or 8 micrograms) with or without 4 micrograms of Prl. Weight and behaviours were monitored to assess safety, and serum, respiratory secretions and splenocytes were obtained at study termination to assess MuV-specific immunity.
All vaccines were well-tolerated. Administration of 8 micrograms of MuV-Prl induced greater serum and mucosal antibodies than MuV antigen alone. MuV-Prl vaccines seemed to favour a Th1-type immune environment. Serum antibodies induced were capable of neutralizing MuV in vitro.
The intranasal MuV-Prl vaccine was safe and immunogenic. Future work will focus on the development of a trivalent MMR-Prl vaccine. Such a vaccine will be of great interest to the global health community.
Nous avons voulu explorer la faisabilité d'un vaccin contre le virus des oreillons (VdO) inactivé et administré par voie intra-nasale, et combiné avec l'adjuvant Protollin (Prl). Notre laboratoire a généré et a caractérisé des antigènes de virion entier désintégré utilisant un détergent. Des souris femelles de souche BALB/c âgées de huit semaines ont été vaccinées avec deux ou trois doses de VdO désintégré en antigène (4 ou 8 µg), avec ou sans 4 µg Prl. Les souris ont été suivies afin d'évaluer l'innocuité; des sérums et des sécrétions des muqueuses ont été obtenus à des intervalles afin d'évaluer l'immunité spécifique de VdO.
Tous les vaccins ont été bien tolérés chez les souris. Les vaccins VdO-Prl ont produit un plus grand taux d'IgG sériques et IgA au niveau de la muqueuse comparés aux vaccins VdO utilisés seuls. Les vaccins VdO-Prl ont tendance à générer une réponse immunitaire déviée sur Th1. Les anticorps sériques étaient capables de neutraliser le VdO.
Nous avons démontré que l'ensemble des vaccins de virus inactivés VdO-Prl administrés par voie intra-nasale est sans danger est immunogénique. Nous voulons générer un vaccin inactivé trivalent rougeole-oreillons-rubéole combiné avec le Prl. Un tel vaccin serait utile.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of a pandemic that has been devastating the U.S. and global swine industry for more than twenty years. PRRSV vaccine development is challenging due to virus heterogeneity. Evidence indicates that the major envelope protein, GP5, is the primary target for a subunit vaccine. In native virions GP5 primarily exists as a disulfide linked complex with the membrane protein, M, which also possesses immunogenic properties. Recent studies report that the GP5/M complex is a more significant vaccine candidate. Currently, no bulk purification methods have been reported for PRRSV proteins. The objective of this research was to develop a purification process for GP5 or GP5/M from native virions. PRRS virions were isolated and concentrated through sucrose cushion ultracentrifugation and target envelope proteins were solubilized with Triton X-100 detergent for further processing. GP5/M was not consistently identified in samples and was therefore abandoned. GP5 was identified by Western blot throughout processing with a αORF5 antibody. Cation exchange chromatography (CEX) was utilized for partial fractionation of GP5, although the viral nucleocapsid protein, N, was a major impurity in CEX elution fractions. As a second chromatographic step, hydrophobic interaction chromatography (HIC) further purified GP5 by means of a two-stage elution scheme. Pure GP5 was eluted from the HIC resin in the second HIC elution stage by Triton X-100 displacement; however the protein is present as a homodimeric/tetrameric aggregate. This process will be useful in PRRSV vaccine development and the purified GP5 product could be used as much needed positive controls in animal studies.
Master of Science

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases.
Master of Science in Life Sciences

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.

Introdução: O Vírus da Hepatite B (HBV) é um vírus de DNA com tropismo por hepatócitos, que apresenta um genoma circular parcialmente dupla fita. Baseado na divergência de sequência do genoma completo, dez linhagens evolutivas, denominadas “genótipos” de HBV, foram descritas (A-J), sendo F e H considerados como “indígenas” da América. Os genótipos de HBV apresentam uma forte estruturação geográfica, o que pode refletir padrões das migrações humanas. Na América do Sul, áreas de alto endemismo incluem a região amazônica, e as maiores taxas de infecção têm sido observadas em populações Nativas Americanas. Embora a forte estruturação geográfica seja indicativa de uma origem antiga, a maioria das análises visando datar a origem dos genótipos “americanos” F e H resulta em datações extremamente recentes que não condizem com eventos históricos relacionados ao HBV. Objetivo: Os objetivos desse trabalho compreendem avaliar o impacto de diferentes taxas evolutivas e da seleção purificadora sobre as estimativas de datação molecular a fim de inferir quais taxas são mais condizentes com a origem do HBV na América; caracterizar o HBV circulante em uma amostra histórica de Nativos Americanos, e discutir os processos históricos que possam ser relevantes para entender os padrões observados. Material e Métodos: Nós realizamos análise Bayesiana utilizando sequências disponíveis dos genótipos F e H e diferentes taxas evolutivas previamente reportadas, e comparamos a ocorrência de mutações sinônimas e não-sinônimas em ramos da filogenia classificados como “antigos” ou “recentes” a fim de inferir a atuação da seleção purificadora ao longo do tempo. Para caracterização do HBV presente nas populações Nativas Americanas, detecção e amplificação do DNA viral foi obtida através de PCR seguido de sequenciamento e análise filogenética. Análise Bayesiana de Skyline Plot foi realizada para comparar a dinâmica populacional do subgenótipo A1 presente na amostra de Nativos Americanos e em outras cepas isoladas no Brasil. Resultados e conclusão: Nossos resultados mostram que as estimativas de datação molecular são fortemente influenciadas pelas taxas evolutivas utilizadas na análise. Além disso, foi observado excesso de mutações não-sinônimas nos ramos recentes da filogenia, o que é compatível com a ocorrência de seleção purificadora, e pode gerar um viés sobre as estimativas, produzindo datações recentes demais. Na amostra de Nativos Americanos, nós constatamos o predomínio do subgenótipo A1, relacionado com populações africanas. Análise de Skyline Plot mostrou que a expansão populacional nas cepas isoladas de Nativos Americanos é mais recente que aquela inferida para outras cepas brasileiras, sugerindo que os processos históricos que contribuíram para a formação do subgenótipo A1 dos Nativos Americanos são relacionados com ondas migratórias mais recentes em direção à região amazônica.
Introduction: Hepatitis B virus (HBV) is a hepatotropic DNA virus that presents a partially double-stranded circular genome. Based on sequence divergence of the complete genome, ten HBV evolutionary lineages, called “genotypes” have been described (A-J), with F and H being considered as indigenous from the Americas. HBV genotypes present a remarkable geographic structure which may reflect historic patterns of human migrations. In South America, areas of high endemism include the Amazon basin, and high prevalence rates have been observed in Native American populations. Although the strong geographical structure indicates an ancient origin, most analysis trying to date the origin of the “American” genotypes F and H result in extremely recent dates that disagree with historical events related with HBV. Objective: The aims of this study comprise evaluate the impact of different evolutionary rates and of the purifying selection on molecular dating estimates in order to infer which rates are in better agreement with the origin of HBV in the Americas; to characterize the HBV circulating in a historical sample of Native Americans, and discussing the historical processes that might be relevant to understand the observed patterns. Materials and Methods: We performed a Bayesian analysis using the available sequences of F and H genotypes and different evolutionary rates previously reported, and compared the occurrence of synonymous and non-synonymous mutations in branches of phylogeny classified as “old” or “young” in order to infer the effects of purifying selection over time. For the characterization of HBV from Native American populations, detection and amplification of viral DNA were obtained through PCR followed by sequencing and phylogenetic analysis. Bayesian Skyline Plot analysis was performed to compare the population dynamics of the A1 subgenotype present in the sample of Native American and in other strains isolated from Brazil. Results and Conclusion: Our results show that molecular dating estimates are strongly influenced by the evolutionary rate assumed in the analysis. In addition, we observed an excess of non-synonymous mutations in recent branches of phylogeny, which is compatible with the occurrence of purifying selection and may create a bias on the estimates, producing too recent datings. In the sample of Native Americans, we observed a predominance of the A1 subgenotype, related with African populations. Skyline Plot analysis showed that population expansion in strains isolated from Native Americans is more recent than that inferred from other Brazilian strains, suggesting that the historical processes that contributed to the presence of A1 subgenotype A1 Native Americans are related with more recent migratory waves towards the Amazon region.

ABSTRACTSweetpotato virus disease (SPVD) is a devastating disease due to the dual infection and synergistic interaction of sweetpotato feathery mottle potyvirus (SPFMV) and sweetpotato chlorotic stunt crinivirus (SPCSV). This study was conducted to: 1) determine the inheritance of resistance to SPVD in sweetpotato; 2) estimate the nature of genetic variance; and 3) evaluate methods for screening large populations for resistance to SPVD. The genetic basis of resistance to SPVD was investigated in three studies. The first genetic study consisted of a randomized block design at two sites in Uganda, during 1998-2000, using 45 full-sib diallel (half) families of 10 parental clones varying in SPVD resistance. The second study also conducted in Uganda, examined progeny from 15 promising sweetpotato diallel families (1352 genotypes), while the third examined two of the most promising families (294 genotypes) from the same diallel at the International Potato Center (CIP), Lima, Peru. Genetic component analysis of the 45 diallel families showed significant general combining ability (GCA) and specific combining ability (SCA) effects for resistance to SPVD. GCA to SCA variance components ratios were large (0.51-0.87) and resistant parents exhibited high GCA, indicating that additive gene effects were predominant in the inheritance of resistance to SPVD and recovery. Use of a suitable sweetpotato genotype for increase of SPVD inoculum and modified cleft graft inoculation led to rapid progress in screening large populations for SPVD resistance. The distribution of SPVD scores in the promising families was skewed toward highly susceptible categories, in Uganda and Peru. Inoculation of the two families at CIP with either SPCSV or SPFMV, and Mendelian segregation analysis for resistant versus susceptible categories for the two viruses suggest that resistance to SPCSV and SPFMV is conditioned by two, separate recessive genes. In the proposed model for inheritance, the two genes are unlinked and they are inherited in a hexasomic or tetradisomic manner. Based on amplified fragment length polymorphism (AFLP) and quantitative trait (QTL) loci analyses we identified two AFLP unlinked markers associated with loci conferring resistance to SPCSV and SPFMV in these progenies. We propose spcsv1 and spfmv1 to be the names of the genes.

The majority of Hepatitis C Virus (HCV) infections result in chronic infection, which can ultimately, cause hepatocellular carcinoma and liver failure. Within the infected host, the virus exists as a population of heterogeneous viral variants called a quasispecies. The envelope region of HCV genome is the most heterogeneous among the whole genome. The envelope proteins mediate viral entry and are also the target of the host immune response. Studying envelope gene evolution can provide important molecular insights into. entry and antibody escape, which ultimately can inform vaccine and therapy developments. Despite this, the influence of host antibody response in driving envelope gene evolution and its role in immune evasion is unclear. To address this shortfall we studied the evolution of HCV envelope genes, over a period of 7-15 years, in treatment naive chronically infected patients. The pattern of HCV evolution was patient specific and clustering of quasispecies was not dependant on the sampling time, which is in stark contrast with other related viruses. The hyper variable region I (HVR-l) was under strong selective pressure. The residues from HVR-l and downstream region, in and around receptor and antibodies binding sites were positively selected during viral evolution. The isolated envelope genes from only two patients (40%) were functional in the retroviral pseudo-particle assay (HCVpp). The functional envelope genes isolated from sequential samples showed variations in infectivity, which could be attributed to substitutions at multiple residues acquired during the course of evolution. Construction of El E2 chimeras derived from infectious and non-infectious clones showed that the region downstream of HVR-l was an important determinant of in vitro infectivity. The patients' isolated HCVpp functional envelope genes were efficiently neutralised by autologous IgGs, and there was no obvious evidence of antibody escape. One of the EIE2 chimeras showed differential sensitivity to well-defined CD81 binding-site-specific neutralising mono clonal antibody, demonstrating the existence of complex mechanisms underpinning antibody resistance. Finally, studies of quasi species transmission in a xenomouse model showed the occurrence of a transmission bottleneck. In multiple transmission experiments a minor variant within the inoculum consistently became established within the recipient mouse host. The established variant harboured amino acid substitutions at amino acid residues 198, 448 and 474 of the virus polyprotein, including loss of a predicted glycosylation site (N448D). It was not possible to define the phenotypic effects of the substitutions as the EIE2 clones were non-infectious in the HCVpp assays. Defining viral determinants involved in successful transmission will help the design of vaccines.

This thesis aims to investigate the nature and aetiology of Bell's palsy by studying its natural history and epidemiology in general practice, and by means of virological studies. This thesis is concerned principally with testing the first part (sentence) of this hypothesis. A new hypothesis is outlined of the aetiology of Bell's palsy which provides a framework for the investigations:- "Bell's palsy is due to a reactivation of HSV in the geniculate ganglion. During this process, neurotransmitters (opioid peptides) and interferon are produced. These cause local vaso-dilation and damage, particularly to the suprageniculate part of the facial nerve." This thesis is concerned principally with testing the first part (sentence) of this hypothesis. The virological studies set out to examine a possible role for HSV in Bell's palsy, which is contingent on the belief that HSV is normally resident or resident to some degree in the geniculate ganglia of the general population. The evidence of the DNA/DNA hybridization study suggests that HSV may be ubiquitously present in human cadaveric geni? culate ganglia. From these a substantial proportion might be expected to reactivate. In contrast the observed incidence of Bell's palsy in the descriptive study of 16.4 per 100,000 per year suggests that if HSV is a cause the mere occurrence of reactivation is an inadequate explanation of the disease mechanism. The epidemiological studies describe Bell's palsy in British general practice where cases are less strongly selected than in hospital studies. By means of a case-control study and match-pair analysis further inves? tigations are made as to the effect of different factors including various types of stress in the aetiology of Bell's palsy. The results of these studies suggest numerous aetiological agents,of particular relevance to the hypothesis are genetic factors, states of increased "stress" and opioid "sensivity", which are discussed. In conclusion the balance of evidence is compatible with the proposed hypothesis, which in the author's opinion justifies further research, especially since it carries treatment implications.

Cette these comporte d'une part la synthese et la caracterisation de derives n-undecenyl-o et s glucopyranoside comportant en position 6 une fonction sulfate ou carboxylate de sodium. Le caractere tensio-actif de ces molecules a permis, par irradiation gamma d'une solution micellaire, d'obtenir des polyanions de masse elevee. D'autre part a ete realise la perallylation du glucose, du o-methyl glucose et du saccharose, sur les doubles liaisons ont ete additionnes des mercaptoacides et des mercapto-sulfonates de sodium. Les polyanions obtenus ont ete caracterises par les techniques spectroscopiques classiques. Le pouvoir inhibiteur vis-a-vis du vih de ces deux familles de molecules a ete teste. Quelques unes ont presente des activites inhibitrices interessantes

Le projet de thèse consistait à étudier le mouvement du Virus de la jaunisse du navet (TuYV) dans le système vasculaire. Le premier objectif était d’identifier la nature du complexe viral cheminant dans les tubes criblés : virions et/ou complexes ribonucléoprotéiques. L’analyse du mouvement de mutants viraux dans différentes espèces végétales, en absence ou en présence de protéines de capside de type sauvage apportées en trans, a permis de démontrer une étroite relation entre la formation de virions et le transport à longue distance. Le second objectif de cette étude portait sur l’identification de partenaires cellulaires de la protéine P4 du TuYV. Deux protéines ont été identifiées par un criblage de banques d’ADNc d’A. thaliana par le système du double hybride dans la levure, et l’analyse de leur implication dans le cycle viral a été amorcée par des expériences de localisation subcellulaire et de validation fonctionnelle in planta
In the project, Turnip yellows virus (TuYV) transport in the phloem was analysed. The first objective was to identify the nature of the viral complex involved in vascular movement: virions and/or ribonucleoprotein complexes. Mutant viruses were modified in the capsid protein gene to inhibit formation of virions. By analyzing their movement in different host plants, in the absence or in the presence of the wild-type capsid proteins brought in trans, we demonstrated a strong relation between virion formation and virus long-distance movement. The second objective was to identify cellular partners of the TuYV-P4 protein, a putative movement protein which is host-specific. Two proteins were identified by screening a cDNA library of A. thaliana using the yeast two hybrid technique, and their function in the virus cycle was assessed by performing sub-cellular localizations and infection of A. thaliana KO mutants

Molluscum contagiosum virus (MCV) is a significant human pathogen causing benign tumours in the human skin. Molluscum contagiosum (MC) infection is most common in children, young adults and immunodeficient individuals. MCV replicates well in the human skin in vivo, but conditions that support MCV replication in vitro are unknown. The lack of in vitro cell culture system has significantly limited progress in MCV research. The aims of this study were, (i) To develop a reporter assay for the sensitive detection and quantitation of MCV infections in vitro, (ii) To express suitable MCV virion surface antigens to develop a sensitive MCV ELISA assay, and (iii) To raise and characterize monoclonal antibodies (mAbs) against these antigens for the detection of MCV in infected human skin. All goals were achieved. A reporter assay based on simultaneous transfection of luciferase/EGFP reporter plasmids and infection with live MCV worked well and was used to test the infectivity of MCV in several human and animal cells. A novel C-terminal MC084 ELISA was established and used to determine MCV seroprevalence in two European populations. mAbs against MC084 and MC133 were raised and partially characterized. Interesting findings were that MCV not only infects human keratinocytes, but also a wide range of animal and human cells in vitro, which, however, do not support replication. Our MCV ELISA gives seroprevalences in UK and German general populations comparable with previous Australian and Japanese studies (<40 years of age). Surprisingly, in older age groups (vaccinated against smallpox), a significantly higher MCV seroprevalence was observed. This was not due to crossreactivity with VACV. We propose this increase may be due to childhood MCV antibodies being boosted by subsequent vaccination or vice versa. Finally, the mAbs raised are unique reagents and will be used in future work to test a hypothesis that MCV replicates in human epidermal stem cells.

Le nombre de greffe de sang placentaire chez les adultes n’a cessé d’augmenter ces dernières années, exposant les patients à un risque théoriquement accru d’infections. Au cours d’une étude rétrospective, nous avons comparé l’incidence des infections virales à CMV, EBV et HHV-6 après allogreffe de cellules souches hématopoïétiques provenant soit d’un donneur non apparenté soit d’un sang de cordon. Une augmentation très significative de la fréquence et de l’intensité des infections HHV6 a été observée chez les sujets receveurs d’un greffon placentaire. Pour expliquer cet accroissement, nous avons ensuite déterminé la composition cellulaire et l’expression du CD46 (récepteur membranaire ubiquitaire de l’HHV-6) au sein de plusieurs types de greffons. Ces données, obtenues par analyse multiparamétrique en cytométrie de flux, montrent notamment un déficit quantitatif très significatif en cellules dendritiques plasmacytoïdes (pDCs, cellules impliquées de manière professionnelle dans la défense antivirale de l’organisme par production d’interféron de type 1) et une moindre expression du CD46 sur les cellules dans les greffons placentaires. Finalement, pour préciser les effets éventuels du virus sur les fonctions cellulaires, nous avons effectué des essais préliminaires d’infection in vitro de pDCs triées à partir du sang périphérique de donneur sains. Ces premières données montrent une modulation de l’expression du CD80 et du CD86 sur les pDCs et une stimulation importante de la sécrétion d’IFN alpha par ces dernières. L’influence significative de l’origine placentaire du greffon sur la réactivation HHV-6 après allogreffe reste pour le moment inexpliquée
Cord blood (CB) is increasingly used as an alternative source of graft in adults with hematologic malignancies, predisposing patients to a theoretical increased risk of infections. In a retrospective study, we compared the incidence of CMV, EBV and HHV-6 viral infections after allogeneic transplantation using stem cells issued from either unrelated donor or cord blood (CB). A very significant increase in the frequency and intensity of HHV6 infections was observed in patients receiving CB grafts. To explain this phenomenon, we then determined the cellular composition and the CD46 (ubiquitous HHV-6 membrane cell receptor) expression in several types of blood and graft sources. These data, obtained by multi-parametric flow cytometry analyses, showed a very significant deficiency in plasmacytoid dendritic cells (pDCs, cells professionally involved in antiviral defense of the organism through production of type 1 interferon) and lower CD46 cells expression in CB grafts. Finally, to clarify the possible effects of the virus on cell functions, we performed preliminary in vitro experiments consisting on HHV-6 infection of sorted human peripheral pDCs issued from healthy donors. These early data showed a modulation of CD80 and CD86 expressions on pDCs associated with a significant stimulation of type 1 IFN-alpha secretion by these cells. So far, the significant influence of cord blood graft source on HHV-6 reactivation after allogeneic transplantation remains to be elucidated

Chez la guêpe parasitoïde Venturia canescens, des particules virales dépourvues d'ADN appelées VLP (pour "Virus-like Particules") sont produites spécifiquement dans les ovaires et tapissent le chorion des oeufs qui sont injectés dans la chenille hôte. Les VLP ont une fonction immunosuppressive pour l'hôte parasité et permettent ainsi la survie des oeufs du parasitoïde. Ces VLP résultent de l’intégration d’un nudivirus dans le génome de l’ancêtre de la guêpe, nudivirus qui a été ensuite domestiqué pour former des liposomes viraux capables de véhiculer dans l’hôte des protéines de virulence d'origine cellulaire. L’étude réalisée au cours de cette thèse a eu pour objet, d’une part, d'étudier les mécanismes de domestication virale qui ont conduit au virus symbiotique endogène actuel nommé VcENV (pour V. canescens endogenous nudivirus) et d’autre part, d'apporter des éléments de réponse sur le processus de morphogénèse et le mode d'action parasitaire des VLP
Viral particles devoid of DNA called VLPs (for Virus-Like Particles) are specifically produced in the ovaries of the parasitoid wasp Venturia canescens and line the chorion of the wasp’s eggs injected into the host caterpillar. VLPs are immunosuppressive and allow parasitoid eggs survival. These VLPs result from the integration of a nudivirus into the wasp ancestor genome, nudivirus which was then domesticated to form viral liposomes capable of carrying, into the host, virulence proteins of cellular origin. The aim of the study carried out during this thesis was, first, to analyze the viral domestication mechanisms that led to the current endogenous symbiotic virus called VcENV (for V. canescens endogenous nudivirus) and secondly to provide some answers on VLPs morphogenesis process and parasitic mode of action

This project is an exploration of 20th century dystopian literature through the lens of germ theory. This scientific principle, which emerged in the late 19th century, asserts that microorganisms pervade the world; these invisible and omnipresent germs cause specific diseases which are often life threatening. Additionally, germ theory states that vaccines and antiseptics can prevent some of these afflictions and that antibiotics can treat others. This concept of a pervasive, invisible, infection-causing other is not just a biological principle, though; in this paper, I argue that one can interpret it as an ideological framework for understanding human existence as a whole. Particularly, I believe that authors of prominent 20th century dystopian novels applied the tenets of germ theory in order to explore the potential “pathogens” that furtively exist within the human mind. These pseudo-germs are various human tendencies that, when left “untreated” by governments, create nonnormative members of society. In the eyes of dystopian regimes, it is precisely this nonnormativity that poses a lethal threat, in that it challenges the continued existence of society with the current ruling body at the helm. In this paper, I trace love (both sexual and familial) and individuation (as a function of social hierarchy, recreational activities, and the use of language) as social disease-causing pathogens in George Orwell’s 1984 and in Aldous Huxley’s Brave New World.

Le monde biologique est rempli de mystères. La compréhension de nombreux processus biologiques extrêmement complexes est grandement améliorée par la combinaison d’approches empruntées à différentes disciplines telles que la chimie et plus récemment la physique. La physique utilise des outils expérimentaux tels que les pinces optiques et les microscopies optique et électronique pour explorer les mécanismes à l’échelle microscopique se déroulant dans la cellule. La connaissance de la nature des interactions entre biomolécules et la possibilité de traduire ces interactions en équations ont permis à la physique de construire des modèles simples mais contenant les ingrédients suffisants à la description d’un mécanisme spécifique. La simulation numérique de tels modèles permet d’améliorer notre compréhension de la relation entre les mécanismes pertinents à l’échelle moléculaire et les observations expérimentales de phénomènes biologiques
The biological world is full of mysteries. The understanding of many extremely complex biological processes is greatly improved by the combination of approaches borrowed from different disciplines such as chemistry and more recently physics. Physics uses experimental tools such as optical tweezers and optical and electron microscopes to explore the microscopic mechanisms taking place in the cell. Knowledge of the nature of the interactions between biomolecules and the possibility of translating these interactions into equations allowed physics to construct models that are simple, but contain the ingredients sufficient to describe a specific mechanism. The numerical simulation of such models improves our understanding of the relationship between relevant molecular-scale mechanisms and experimental observations of biological phenomena. The structural organization of biomolecular complexes is a process that involves various scales of length and time

Tidigare forskning har visat att exponering för grön miljö tillhandahåller hälsofördelar. Dessa fördelar indikerar bland annat återhämtningseffekt av stress, trötthet och uppmärksamhetsutmattning, vilket är särskilt viktigt för universitetsstudenter. Därför syftade denna studie att undersöka om det finns en koppling mellan universitetsstudenters kontakt med grönstruktur i/nära studiemiljö och studenternas upplevelse av sin studiemiljö under covid-19 restriktionerna. Detta med hjälp av en dagbokundersökning som förstudie och telefonintervjuer. Analysverktyget bestod av teoretiska ramverk, Attention Restoration Theory (ART) och Stress Reduktion Theory (SRT). Resultat och analys har bekräftat Attention Restoration Theory (ART), där det uppges att gröna naturliga miljöer ger återhämtningseffekt och väcker uppmärksamhet efter en mental trötthet. Därtill bekräftade den även Stress Recovery Theory (SRT), där det uppges att gröna naturliga miljöer ger positiva effekt på stressminskning, -bearbetning och -återhämtning. Slutsatsen till denna undersökning är att grönstruktur har en stor betydelse för universitetsstudenterna, detta då alla väljer att vistas eller komma i kontakt med gröna naturen på ett eller annat sätt, till exempel gå en promenad i naturen eller åka till en stuga i skogen. Att naturen är en del av studenternas vardag verkar vara framför allt en omedveten upplevelse för de flesta och sker utan erkännande eller bearbetning av miljön. Dock framkommer det att processen kan vara medveten för vissa studenter.
Previous research has shown that exposure to the green environment provides health benefits. These benefits indicate among other things, recovery effect of stress, fatigue and attention-exhaustion, which is especially important for university students. Therefore, this study aimed to investigate whether there is a connection between university students' contact with the green structure in / near the study-environment and the students' experience of their study environment during the covid-19 restrictions. This with the help of a diary-survey as a pilot-study and telephone interviews. The analysis tool consisted of theoretical frameworks, Attention Restoration Theory (ART) and Stress Reduction Theory (SRT). Results and analysis have confirmed Attention Restoration Theory (ART), where it states that green natural environments have a recovery effect and attracts attention after a mental fatigue. In addition, it also confirmed the Stress Recovery Theory (SRT), which states that green natural environments have a positive effect on stress reduction, processing and recovery. The conclusion of this study is that green structure is of great importance to university students, as everyone chooses to reside or get in touch with green nature in one way or another, for example going for a walk in a green environment or staying in a cottage in the forest. That nature is a part of the students' everyday life seems to be above all an unconscious experience for most and takes place without recognition or processing of the environment. However, it appears that the process may be conscious for some students.

Velvet tobacco mottle virus (VTMoV; genus Sobemovirus) infects Nicotiana velutina (Velvet tobacco), a native of the arid region of central Australia. In the field, the virus, mirid vector and native host plant together comprise a unique plant virus pathosystem which is well adapted to its ecological niche, and independent of anthropogenic influences. The purpose of this research was to describe the sequence variation amongst VTMoV isolates and relate this to ecological factors. The full genome sequence of VTMoV was obtained using a genome walking strategy with both degenerate and specific primers. Sequence and genome organisation confirm that VTMoV is a unique sobemovirus and phylogenetic analysis groups it separately from other sequenced Australian sobemoviruses. This is consistent with the hypothesis that VTMoV is not a recently introduced sobemovirus, but rather a product of evolution within a unique Australian ecosystem, representing a novel plant virus lineage. The genome sequences of two isolates of VTMoV, K1 and R17 were compared and a limited amount of variation was observed between these isolates. Sequence diversity was observed in the RNA dependent RNA polymerase (RdRp) gene from 15 isolates of the virus. Analysis determined mutations were limited to maintain protein function, which is indicative of purifying selection. In addition, the first evidence of recombination in the RdRp of a sobemovirus was detected in three VTMoV sequences. Sequence variation associated with transmission of VTMoV by the mirid Cyrtopeltis nicotianae [Hemiptera; Miridae] was also investigated. Isolates K1 and R17 were serially passaged monthly either through obligatory mirid transmission or via mechanical inoculation. After two years, sequences were compared from two regions of the VTMoV genome associated with movement (open reading frames of protein P1 and the coat protein). Several different trends were observed in the sequences, but only one difference could be associated with the mode of transmission. The coat protein region sequence from mirid transmission had a higher mutation rate that sequence from the mechanical mode of transmission. This thesis contains the first complete genome sequence of VTMoV. It describes natural variation amongst a range of VTMoV isolates, and assesses sequence variation in parts of the genome after long term mirid transmission. The sequence of VTMoV is discussed in the context of the unique nature of the virus and the evolutionary mechanisms that may have played a role the evolution of the virus.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2011

碩士
國立中興大學
獸醫病理學研究所
90
Classical swine fever (CSF) is a highly contagious viral disease affecting swine and generating leucopoenia and immunosuppression. At least two major genotypes, named native strain such as 94.4 isolate and new invaded strain such us Ping-Tung isolate of classical swine fever virus (CSFV), have been identified in Taiwan. The aim of this study was to evaluate the differences of pathogenicity and replication kinetics between two genotypes of CSFV in vivo. In order to minimize the influence of virus virulence following the passage of virus in cell culture, the new invaded Ping-Tung and native 94.4 isolates were repeatedly replicated in pigs for the recovery as well as amplification of viruses. The whole blood samples were collected at the peak of disease after inoculation. The pathological scores based on clinical signs, pathological lesions and histopathological examination were calculated to evaluate the differences of the pathogenicity between viruses. Moreover, RT-PCR, virus isolation and IFA were applied to assess the distributions and titers of viruses in organs during the course of infection. The results demonstrated that no significant hemorrhage and spleen infarct were noticed even after a four-time passage of the native 94.4 in pigs. However, the Ping-Tung isolate caused clinical signs more severe including fever duration, hemorrhage, and encephalitis during the period of infection. The results of nested RT-PCR also indicated that the new invaded Ping-Tung isolate had higher replication rate than the native 94.4 in tonsil, spleen, thymus, kidney and bone marrow in vivo. In addition, the porcine alveolar macrophages (PAM) were collected and used to evaluate virus titer and cytokine profiles after inoculation. The results revealed that CSFV could infect and replicate in PAM and also downregulate the m-RNA of TNF-a in pigs. In conclusion, the new invaded Ping-Tung isolate surpassed the native 94.4 in pathogenicity and in replication, that might contribute to the reason for why domination of new invaded strain in fields in Taiwan.

碩士
國立臺灣大學
獸醫學研究所
90
Subgroup J avian leukosis virus (ALV-J) has become more and more important because its rapid horizontal spread, and it causes body weight suppression in commercial broilers. Since Taiwan broiler breeders are imported from other countries, we would like to know the situation of ALV-J infection in Taiwan broilers as the breeding companies claim that they have controlled the ALV-J infection in their breeders. Furthermore, there was no information on ALV-J infection in native chickens in Taiwan. Thus, the objectives of this study were to investigate the situation of ALV-J infection in broilers and native chickens in Taiwan. Because commercial ELISA kit had false positive reaction, virus isolation and RT-PCR were used for identification of ALV-J infection. We had totally investigated 8 commercial broiler flocks from a slaughter house, and 5 of them were infected with ALV-J (62.5%). We had investigated 4 native chicken flocks and 1 native chicken breeder flocks, and only the native chicken breeder showed ALV-J infection. Immunotolerant infection was suspected in the native breeder chickens since they showed viremia and lack of antibody (V+A-). The sequences of the gp85 gene of ALV-J isolates from breeders, broilers and native chickens were 94-100% identity among them. Phylogenetic trees showed that Taiwan isolates fell into two lineages. And it indicated those isolates had no relation to poultry flocks and breeders. Furthermore, Phylogenetic trees showed isolates could be grouped together by time they isolated, and virus variation could be saw during past 3 years (2000-2002). Thought commercial application of the eradication program over several years has resulted in a marked reduction in the prevalence of ALV-J in overseas breeding company, according to our research, Taiwan is still highly contaminated by ALV-J. So we have to control ALV-J infection strictly in Taiwan.

碩士
國立屏東科技大學
生物科技系
106
Metarhizium anisopliae is a kind of entomopathogenic fungi on insects, it is distributed all over the world. The main application for this fungi is as a pest control for crops, acting as a biological pesticide. In previous studies, Metarhizium anisopliae was able to produce a large quantity of secondary metabolites containing anticancer and antiviral activities. In this study, Metarhizium anisopliae MA-126 was cultured into solid-state fermentation and liquid state fermentation. After partition extraction, the NS2B protein had 35% and 10% inhibition rate when both of them have 50μg/ml concentration on ethyl acetate layer. The ethyl acetate layer was purified by LH-20 column, high performance liquid chromatography (HPLC) to yield eight compounds. Compound1-4 are separation from liquid state fermentation(5S,8S,9R)-8-benzoyl-9-hydroxy-8-methoxy-3-methyl-2-(5-methylfuran-2-yl)-1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione(1), (3S,3aR,7aS)-3a,7a-dihydroxy-3-((S)-1'-hydroxy-4'-methylpent-3'-en-1'-yl)-4-methoxy-3-methylhexahydroisobenzofuran-5(3H)-(2), chlovalicin(3), (4S,4aR,5S,8aR)-4,4a,8a-trihydroxy-5-methoxy-4-methyl-3-(3'-methylbut-2'-en-1'-yl)octahydro-6H-isothiochromen-6-one(4), compound 5-8 are separation from solid state fermentation. Destruxin A(5), Destruxin B(6), Destruxin E chlorohydrin(7) and Destruxin E diol(8). 　　All structures were elucidated by NMR spectra techniques, including 1H, 13C, DEPT, COSY, HMQC, HMBC, and NOESY, with physical data(mass spectrometry, optical rotation, IR spectrometry), also compare with the compound data in the literature to establish.Compound 1 is the new compound, compound 1-4 are the first natural compounds isolated from Metarhizium anisopliae.

碩士
國立臺灣大學
獸醫學研究所
92
Control of subgroup J avian leukosis virus (ALV-J) is mainly through elimination, and the first challenge is to differentiate between the infected and noninfected chickens. Methods for direct detection of ALV-J include the detection of viral RNA and viral antigen, or indirect detection for antibodies. However all of the currently available diagnostic methods have some limitation. The specific aim of this study is to develop more useful diagnostic methods for detecting ALV-J. In this study, partial gp85 gene, which included three variable regions: vr3, hr1 and hr2, was cloned into pRSET B and expressed in BL21(DE3). The expressed protein gp85N was used to develop indirect ELISA for detecting ALV-J specific antibody but this indirect ELISA had nonspecific reaction. The same gene fragment was used to prepare monoclonal antibodies. Two monoclonal antibodies, mAb14 and mAb22, were obtained and used to detect ALV-J antigen. The results indicate that both mAb14 and mAb22 react with gp85N, but only the later could detect ALV-J virus. In order to increase economic benefits, the poultry industries hybrid broiler breeder and native chicken in Taiwan and ALV-J was introduced into Taiwan by this way. But the situation of ALV-J infection in hybrid native chickens was unclear. The serological investigation was done for hybrid native chickens. In this study, 10 flocks of breeder and 40 flocks of meat-type chicken were investigated. Breeder flocks was 100% (10/10) ALV-J antibody positive no matter the male or female, meat-type-chicken flocks was 15% (3/20) ALV-J antibody positive in male and 10% (2/ 20) ALV-J antibody positive in female. The results of serological investigation indicate ALV-J is widespread among hybrid native chickens. Furthermore, tumors induced by ALV-J were seen in broiler breeder and layer breeder, and viruses were isolated from these clinical cases during 2002-2004 reveal that ALV-J still exists in broiler breeder and layer breeder flocks.

碩士
國立臺灣大學
昆蟲學研究所
97
The fire ant (Solenopsis invicta) eradication program has been carried out in Taiwan since 2004. It certainly effectively restrains the populations of fire ant on treatment areas. Unfortunately, the reinfection or new infection site by natural nuptial flight of monogyne colony are still promulgated. Current new approaches to manage fire ants are integrating biological control agents with baits applications. This study aims to investigate alternative suitable biological control agents in Taiwan, and examines how SINV-1 (Solenopsis invicta virus-1) infection affects interspecific competition between incipient S. invicta against two native ants, Pheidole fervens and Monomorium chinense, by conducting two levels of trial, colony interference and individual confrontation, in laboratory conditions. The results from colony interference study showed that both native ants owing numerical advantages were capable to kill either infected or healthy queens of S. invicta. There was a significant less time for M. chinense to eliminate SINV-1 infected S. invicta compared to healthy ones. All S. invicta could repulse the invading of equal worker numbers of P. fervens. Compared with healthy S. invicta, SINV-1 infected S. invicta spent longer time to terminate P. fervens colonies. Virus infection was observed to have significant effects on foraging behavior and invading willingness of S. invicta. SINV-1 infected S. invicta recruited lesser number of foragers than healthy one, and in particular on competitive native ants were present. In confrontation trial M. chinense caused significant greater mortality on infected S. invicta minors than did on healthy ones. However, S. invicta majors (either infected or healthy) performed high competitive abilities against M. chinense, which might account for chemical defense and/or size advantages. In dealing with P. fervens, one cannot but admit that soldiers indeed function in defense, which caused S. invicta high mortality. These data suggest that virus may somehow weaken the competitive ability of infected S. invicta and make them prone to be terminated by M. chinense but not by P. fervens. Chemical interference by M. chinense might be a more likely influencing factor on mortality of infected S. invicta than the physical combat by P. fervens. Results from this study highlight the likely success on area-wide control of S. invicta in Taiwan by an alternative strategy that involves integration of competitors and pathogen.

博士
國立臺灣大學
生化科技學系
107
Norovirus (NoV) is one of the leading causes of acute nonbacterial gastroenteritis outbreaks worldwide. Due to the lack of a reliable and efficient cell culture system for producing inactivated or attenuated whole NoV vaccines, the development of NoV vaccines relies largely on virus-like particles (VLPs) formed by the major capsid protein VP1 or subviral particles formed by the exterior protrusion (P) domain of VP1. The P particle is composed of 12 P dimers and revealed the same antigenic types as VLP. Additionally, due to the presence of three outermost surface loops in the P domain, the P particle could serve as a platform for carrying foreign antigens. The goal of this study is to provide the fundamental understanding of different constructs of P particles formed by the P domain of NoV strain GII.4 isolated from Taiwan to establish the multiple antigen presentation platform. The P domain was expressed in Pichia pastoris, a well-known expression system with several advantages including high cell density fermentation at low cost and non-risk of endotoxin. The production of NoV P protein reached 220 mg/L as a soluble form in fermentation cultures. The overexpression P protein provided the cornerstone for the development of tag-free P protein purification schemes. For purification of tag-free P protein, based on the charge and the surface histidine in native NoV, two purification schemes were developed: (1) The host cell proteins and the target P protein were separated by the difference of the binding strength to the chromatography column. Using anion-exchange and hydrophobic interaction chromatography purification schemes, NoV P protein with high purity was obtained. However, the P protein recovery was 2.5%. (2) Using the HisTrap affinity column and anion-exchange column, the native NoV P protein was purified, and recovery and purity were 28.1% and 82.1%, respectively. Besides, the NoV major capsid protein VP1 was also purified using the HisTrap affinity column and gel filtration column. The purity of NoV VP1 protein was over 90% and the recovery was 20%. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis of the purified NoV VP1 revealed that VP1 proteins were self-assembled into particles, and these particles remained HBGA binding ability as evidenced by saliva binding assay. Similar to P-His protein, the P protein also formed biologically functional small P particle composed by six of P dimer. The purified P-His and P protein, analyzed by TEM and DLS, were also revealed triangle-, square- and ring-shaped. Previous studies showed that the small P particles were only found with C-terminus modification. Without terminal modification, small P particles were formed in this study. The amino acid sequence analysis showed only four different amino acids between the P domain in this study and other investigated GII.4 strains, suggesting that these amino acids might play an important role in P particle formation. To extend the application of the small P particle, the green fluorescent protein was used to mimic the foreign antigen and inserted into NoV P loop2 distal end. From gel filtration and fluorescent microscopy, the chimera small P proteins were self-assembly into chimera small P particles and showed green fluorescence. It was indicated that these Taiwan native small P particle can be an antigen presentation platform. This study was the first report of NoV P protein covering overexpression in P. pastoris, easy handling tag-free purification schemes, and formation of small P particles without terminal modification as foreign antigen displaying platform.

El género Glandularia (familia de las Verbenáceas), está integrado por especies de ornamentales nativas con potencial para ser empleado en canteros, borduras e inclusive como planta en maceta. En nuestro país se destacan las especies G. incisa y G. peruviana por sus flores de color rojo intenso, su hábito erecto y rastrero, y su rusticidad. En un trabajo anterior se identificó Groundnut ringsopt virus (GRSV) infectando Glandularia. El objetivo del presente trabajo fue desarrollar y optimizar metodologías para la propagación in vitro de G. incisa y G. peruviana libre de GRSV. Utilizando WPM con 0,025 mg/l de TDZ junto con 0,025 mg/l de ANA se estableció el cultivo de ápices caulinares con un 70% de regeneración en ambas especies. A través del cultivo de ápices in vitro se obtuvo un 83% de plantas libres de GRSV para G. incisa y del 33% para G. peruviana. El porcentaje más alto de liberación fue del 90% para el tratamiento de termoterapia a 38ºC-32°C durante 10 días combinada con el cultivo de ápices de G. incisa. La evaluación del material saneado fue realizada por DAS-ELISA y AC-RT-PCR. La combinación de técnicas de liberación de virus mediante cultivo in vitro y métodos sensibles de diagnóstico de las virosis son la clave para la obtención en corto tiempo de grandes volúmenes de plantas de alta calidad genética y fitosanitaria necesarias para impulsar el cultivo de Glandularia en nuestro país.