O Zika vírus (ZIKV) é um vírus RNA de fita única, pertencente à família Flaviviridae. É transmitido entre os humanos geralmente pelos mosquitos da espécie Aedes, mas transmissão via sexual, perinatal e por transfusão sanguínea também foram relatadas. Os sintomas aparecem em 20% dos indivíduos infectados e incluem febre, dor de cabeça, rash cutânea, conjuntivite, mialgia e artralgia. Em 2016, durante a grande epidemia do ZIKV pelas Américas, o interesse pelo seu diagnóstico rápido se intensificou, devido a relação do vírus com o aumento da incidência de casos da síndrome de Guillain-Barré em adultos e da microcefalia em recém nascidos de mulheres grávidas infectadas. De acordo com o CDC (Center for Disease Control and Prevention) o diagnóstico dos pacientes sintomáticos deve ser realizado através da detecção dos ácidos nucleicos do vírus por PCR (Polymerase chain reaction) em amostras pareadas de sangue e urina. Estudos recentes têm postulado que a saliva é uma alternativa importante para detecção do ZIKV. A saliva requer menor complexidade no processamento quando comparada ao sangue, simplificando a reação. A amplificação Isotérmica mediada por Loop (LAMP) é um teste sorológico de alta sensibilidade e especificidade para detectar rapidamente DNA ou RNA de patógenos, incluindo o ZIKV. O fato de não requerer ciclos térmicos como o PCR, faz do LAMP uma reação mais simples, rápida e mais econômica por exigir menos energia. O objetivo deste estudo foi de avaliar e comparar a eficácia da saliva e da urina em diagnosticar a infecção pelo Zika vírus em indivíduos na fase aguda da doença, através da detecção do RNA viral por meio do LAMP. Ao todo, 131 amostras (68 saliva e 63 urina) de 69 indivíduos brasileiros apresentando sinais e sintomas específicos e confirmados positivamente para o ZIKV através da análise do sangue por PCR, foram coletadas e analisadas por LAMP. A média de idade dos indivíduos foi de 34,7 (±13,6), sendo 46 (66,7%) do sexo feminino. Das 68 amostras de saliva analisadas por LAMP, 45 (66,2%) foram positivas para o ZIKV com o Tempo de positividade (Tp) médio de 13,5 minutos. Enquanto que das 63 amostras de urina, 25 (39,7%) foram positivas com o Tp médio de 15,8 minutos. A saliva pôde diagnosticar mais indivíduos (p=0.0042) e em menor Tp (p=0.0176) quando comparada à urina. A saliva demonstrou ser uma alternativa viável no diagnóstico da infecção do ZIKV, em indivíduos na fase aguda da doença, através do LAMP. Nossos achados contribuem para o conhecimento do comportamento do Zika vírus no organismo, uma vez que pouco se conhece em relação à excreção do ZIKV na saliva. / Zika virus (ZIKV) is a single-stranded RNA virus, member of the Flaviviridae family. It is transmitted among humans usually by Aedes mosquito species, but sexual transmission, perinatal and blood transfusion have also been reported. Symptoms appear in 20% of infected individuals and include fever, cutaneous rash, headache, conjunctivitis, myalgia and arthralgia. In 2016, during the Americas ZIKV outbreak, the interest in a rapid diagnosis intensified due to a sudden increase in cases of Guillain-Barré syndrome in adults and microcephaly in newborns of infected pregnant women related with ZIKV. According to CDC (Center for Disease Control and Prevention) the diagnosis of symptomatic patients should be done through nucleic acid detection by PCR (Polimerase Chain Reaction) in paired samples of blood and urine. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood, which greatly simplifies the assay process. Loop-mediated Isothermal Amplification (LAMP) is a molecular test with high sensibility and specificity for rapid detection of DNA or RNA of pathogens, including ZIKV. The fact that LAMP does not require thermal cycling makes the assay simple, fast and cost effective compared to PCR assay. The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. A total of 131 samples (68 saliva and 63 urine) from 69 subjects in the acute phase of ZIKV infection and confirmed positive for ZIKV by blood analysis through PCR were collected and analyzed by LAMP. The mean age of the individuals was 34.7 (±13,6) years old, of whom 46 (66.7%) were females. From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 minutes, and from the 63 urine samples, 25 (39.7%) were positive with an average Tp of 15.8 minutes. Saliva detected more samples (p=0.0042) and had faster Tp (p=0.0176) as compared to urine. Thus, saliva proved to be a feasible alternative for diagnosis of ZIKV infection during the acute phase by LAMP. The findings of this study can contribute to the knowledge of the Zika virus behavior in the human organism, since this issue is not totally understood.

A dengue é doença causada pelo vírus dengue (DENV), que acomete cerca de 390 milhões de pessoas no mundo, representando uma ameaça à saúde pública mundial. Até o momento não se dispõe de tratamento específico ou de vacinas para a maioria da população. Um dos maiores obstáculos para o desenvolvimento de vacinas ou para a compreensão da biologia do vírus é a falta de modelos animais que mimetizem a doença vista em humanos. Neste trabalho, o principal objetivo foi a busca de modelos alternativos de infecção com um isolado clínico (JHA1) de DENV sorotipo 2 (DENV2) capaz de infectar camundongos imonocompetentes após administração pela via intracraniana. Foram testadas duas vias alternativas de inoculação viral (intraperitoneal e intravenosa) em camundongos adultos imunocompetentes, além da via intracraniana em camundongos C57BL/6. Por fim, tentativas de mapeamento de mutações relacionadas à neurovirulência do JHA1 em camundongos imunocompetentes. Os resultados obtidos contribuem para a caracterização do JHA1 e para a busca de modelos experimentais alternativos de DENV. / Dengue fever is a disease caused by the dengue virus (DENV), which affects around 390 million people worldwide, posing a threat to global public health. To date, there is no specific treatment or vaccination for the majority of the population. One of the major obstacles to vaccine development or understanding the biology of the virus is the lack of animal models that mimic the disease seen in humans. In this work, the main objective was to search for alternative models of infection with a clinical isolate (JHA1) of DENV serotype 2 (DENV2) capable of infecting immunocompetent mice after administration by the intracranial route. Two alternative routes of viral inoculation (intraperitoneal and intravenous) were tested in immunocompetent adult mice, in addition to the intracranial route in C57BL/6 mice. Finally, attempts to map mutations related to JHA1 neurovirulence in immunocompetent mice. The results obtained contribute to the characterization of JHA1 and to the search for alternative experimental models of DENV.

A Febre Amarela (FA) é uma doença viral infecciosa, não contagiosa que pode se manifestar desde um quadro febril até a forma clássica íctero-hemorrágica, cujo agente etiológico é o vírus da febre amarela (VFA). A doença tem avançando para as regiões Sudeste e Sul com um aumento de 4 vezes no número de vítimas fatais. O presente trabalho teve como objetivos avaliar a possível circulação do vírus da febre amarela silvestre, através da detecção de anticorpos IgM por meio da técnica de MAC-ELISA, e anticorpos IgG, pelas técnicas de Inibição da hemaglutinação e Neutralização; detecção do genoma viral utilizando a técnica de RT-PCR no soro de indivíduos assintomáticos; bem como avaliar a proteção vacinal, por meio da técnica de neutralização em soros de indivíduos das zonas rurais do município de Jacupiranga e Teodoro Sampaio. Um total de 238 soros foram coletados, 152 (Jacupiranga) e 86 (Teodoro Sampaio). Foram detectados um total 15,9% (38/238) de anticorpos IH contra o vírus selvagem e/ou vacinal. Destes 13,16% (5/38) anticorpos IH foram provenientes dos soros de Jacupiranga e 86,84 (33/38) de Teodoro Sampaio. Anticorpos neutralizantes foram detectados em 34% (13N/38IH) dos IH reativos, destes 15,38% (2/13) foram oriundos deJacupiranga e 84,62% (11/13). A detecçção de anticorpos neutralizantes nos indivíduos de Jacupiranga levantam a necessidade de pesquisas futuras na busca do vírus da febre amarela nos seus reservatórios selvagens e vetores. Não houve a detecção do genoma viral por RTPCR nas duas populações, bem como a detecção de anticorpos IgM específicos, por meio das reações de MACELISA, o que evidencia a não circulação recente deste vírus na população estudada. O grau de imunizaçao na população de Teodoro Sampaio, com histórico de vacinação, por meio das reações de IH e Neutralização foi baixo, uma vez que somente 42,30% da população apresentou anticorpos inibidores da hemaglutinação e o grau de proteção pela reação de neutralização foi de apenas 33,33%. Este trabalho aponta não só para a necessidade de monitoramento pós-vacinal em áreas endêmicas de febre amarela, com indicação de vacinação, como também a necessidade de estudos epidemiológicos, uma vez que se detectou reação monotípica no teste do IH, além de anticorpos neutralizantes para o vírus selvagem da febre amarela em um indivíduo residente em município de risco para a febre amarela. / Yellow fever (YF) is an non-contagious infection disease. The clinical signs can be a nonspecific fever or the classical ictero-hemorrhagic form as a result of the Yellow Fever virus (YFV) infection. Considering the recent spread of the disease to the South and Southeast of Brazilian regions and the increased number of fatal cases, the aim of this study was to evaluate the possible circulation of the YFV, using the IgM enzyme-linked immnunosorbent assay (MACELISA) and Reverse transcriptase polymerase chain reaction (RT-PCR), as well as, to conduct a serological inquire and evaluate the protective vaccine response, by the hemagglutination inhibition test (HI) and serum neutralization test (SN), in the rural population of Jacupiranga and Teodoro Sampaio, located in the southern region of Brazil. A total of 238 serum samples were tested, 152 from Jacupirang and 86 from Teodoro Sampaio. Of the serum collected, 15,9% (38/238) were positive by HI, using wild and vaccine strains of YF, and out of these 13,16% (5/38) were from Jacupiranga and 86,84% (33/38) were from Teodoro Sampaio. Neutralizing antibodies were detected in 34% of the HI positive samples (13SN/38HI) and out of these 15,38% (2/13) were from Jacupiranga and 84,62% (11/13) were from Teodoro Sampaio. None of the samples were positive by MACELISA and RT-PCR indicating no evidences of recent virus circulation in the population analyzed in this study. However, the YF neutralizing antibodies detection in samples from Jacupiranga indicates the necessity of further researches in order to detect the YFV in its wild reservoirs and vectors. On the other hand, considering that 42,30% of the Teodoro Sampaio samples were positive by HI and out of these only 33,33% had neutralizing antibodies to the YFV, our results also pointed out to the importance of pos-vaccination protective immunity surveys, in order to evaluate the efficiency of the vaccines in endemic areas.

<p dir="ltr">It is with great pleasure that I present the culmination of my exploration into the process of flavivirus assembly, with particular emphasis on the envelope glycoproteins and C protein of ZIKV and DENV2, within the subsequent four chapters of this dissertation.</p><p dir="ltr">Beginning in Chapter 2, we describe findings from a structure-function study of the ZIKV prM and E transmembrane helices (TMHs) and their role in virus assembly. Using a mutagenesis approach in a ZIKV reporter virus particle (RVP) system to increase throughput and discovery, substantial information was obtained showcasing a novel function for specific residues located within a short (4 residue) connecting region between the two TMHs of prM protein – denoted as the prM TMH “turn” residues. During translation of the prM and E proteins, these TMH “turn” residues face towards the cytosolic side of the ER membrane. This orientation has been hypothesized to possibly play a role during viral assembly interactions between the envelope glycoproteins and the nucleocapsid core of flaviviruses. However, no information to date has supported or refuted this theory. Overall, a single amino acid change within the prM TMH “turn” residues was found to be highly detrimental to viral assembly, ultimately leading to the loss of capsid integration into released sub viral particles and the alteration of the lipid membrane architecture. We surmised that lipid interactions around the region of the mutation were perturbed, leading to a loss of assembly capabilities but interestingly maintaining the budding mechanisms. The work of Chapter 2 will be submitted for publication to a peer reviewed journal shortly after the submission of this dissertation.</p><p dir="ltr">Chapter 3 expands on the ZIKV RVP results described in Chapter 2 by detailing a series of mutagenesis experiments into the role of the prM and E TMHs in the fully infectious ZIKV and DENV2 systems. Mutations within the prM TMH “turn” residues of DENV2 were found to also perturb virus infectivity, with two mutations within prM completely eliminating infectivity. The two mutants were found to be capable of producing NS5 and intracellular E protein that had been glycosylated, indicating that translation was intact and that E protein trafficking into the trans-Golgi network still occurred. However, unlike the results discussed in Chapter 2, the DENV2 mutants did not release any detectable E protein into their supernatants. This suggested that while the mutants could generate viral proteins and somehow undergo protein trafficking into the Golgi (signifying potential particle maturation), no particles were released. The DENV2 results were supported by reciprocal mutations in the prM proteins of ZIKV using fully infectious cDNA clones. The ZIKV prM mutants also eliminated virus infectivity and prevented the release of the E protein into the supernatant, indicating no release of viral particles, infectious or otherwise. Overall, the mutations in the fully infectious DNEV2 and ZIKV systems add further support for a novel role of the prM TMHs in flavivirus assembly.</p><p dir="ltr">Chapter 4 describes our efforts to reconstitute the flavivirus envelope glycoproteins into natively derived lipid nanoparticles for in vitro assembly analysis. Styrene-maleic acid copolymers (SMAs) were utilized for this study due to their ability to self-polymerize into highly hydrophobic chains in aqueous solutions. These hydrophobic chains can imbed themselves into lipid membranes to escape the aqueous environment, and in doing so “cut out” ~10nm diameter “patches” of native lipid membranes, along with any integrated membrane proteins. This “lipid/protein patch” is referred to as a styrene-maleic acid lipid nanoparticle (SMALP). Initially, attempts were made to generate SMALPs using purified Kunjin virus (KUNV) particles as the source of membrane lipids and glycoproteins due to their rapid growth rate and homogenous particle population. Unfortunately, attempts to generate SMALPs using purified KUNV were unsuccessful. It is hypothesized that the membrane curvature of purified KUNV particles generated a sterically and energetically unfavorable environment for SMALP generation, leading to the complete destruction of the particles during SMA mixing. To circumvent this issue, cells transfected with either WT or mutant ZIKV RVP cDNA were fractionated and purified ER membrane samples were mixed with SMAs to generate SMALPs. Western blot analysis suggested that the SMALP generation was successful. However, further experimentation is warranted to confirm this outcome and the structural integrity of the envelope glycoproteins within the SMALP.</p><p dir="ltr">Chapter 5 describes collaborative work on the identification of a novel compound inhibitor against flavivirus assembly, specifically targeting C protein’s interactions with RNA. This work was done in conjunction with a visiting scholar from the Indian Institute of Technology Mandi – Dr. Prateek Kumar – during his time at Purdue University from August 2022-May 2023. Much of the foundational computation work was done by Prateek prior to his arrival at Purdue University. As such, while the full context and results for the entirety of the study will be discussed, this chapter will primarily focus on the in vitro experimental results that were gathered directly by me, or results that were produced by Prateek and myself equally. This chapter demonstrates that a novel small molecule inhibitor against ZIKV C protein can, in fact, diminish ZIKV assembly by impeding C protein’s binding to RNA, prevent efficient RNA replication through binding and disruption of NS2B/3 protease, and perturb virus binding and entry prior to infection by also binding to E protein. Moreover, the novel molecule was also found to disrupt DENV2 infection as well, albeit to a lesser degree than ZIKV. This multifaceted molecule was recommended for further study in animal systems to continue testing its safety and efficacy for treatment of ZIKV and DENV2 in humans. A co-authorship manuscript has been completed on the work from this chapter and is currently awaiting submission to a peer reviewed journal.</p><p dir="ltr">Finally, Chapter 6 will combine the conclusions from the above chapters and discuss, in detail, aspects pertaining to the future of studies aiming to better understand the assembly of flaviviruses. This chapter will focus on how the link between viral assembly and membrane lipid architecture fits with previously established literature and what future directions could be employed to answer the questions proposed within.</p>

No description available.

The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.

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 &gt;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 &gt; 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.

This thesis is focused on the role of host cytoskeleton, primarily microtubules and microfilaments, during tick-borne encephalitis virus infection in human neuroblastoma cell line SK-N-SH and tick cell line IRE/CTVM19. The importance of cytoskeletal integrity and dynamics to the viral replication cycle were examined using specific chemical inhibitors showing the virus utilizes studied structures in both cell lines. Immunofluorescence microscopy revealed structural changes in the actin cytoskeleton during late infection in SK-N-SH cells. Moreover, differences in expression of cytoskeleton-associated genes in both cell lines were compared. Several genes with up-regulated expression in SK-N-SH cells were identified during late infection.

Made available in DSpace on 2015-04-22T22:06:36Z (GMT). No. of bitstreams: 1 Dissertacao Final Gustavo Henrique.pdf: 11557113 bytes, checksum: 738eeceeb5f826798bc9ce2b09633f72 (MD5) Previous issue date: 2009-03-06 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Arbovirosis are currently recognized by the World Health Organization as a global problem. Most arbovirosis are summarized in diseases with acute and nonspecific symptoms such as fever, headache and muscle pain. Although self limited, these symptoms create relevant social and economic impacts. In Brazil, the arbovirosis caused by viruses belonging to the genus Alphavirus, Flavivirus and Orthobunyavirus and are the main cause of outbreaks or epidemics. This study aimed to study the circulation of arbovirus mayaro, venezuelan equine encephalitis virus, yellow fever virus, Saint Louis encephalitis virus and oropouche virus in a rural and an urban area of the State of Amazonas, Brazil. Therefore, the serology for detection of immunoglobulin G was used to assess the prevalence of antibodies against these viruses in 335 residents of a rural community in the state of Amazonas and PCR was used to assess the incidence of these viruses in 250 samples collected in urban area of Manaus. The results for serology suggest the movement of mayaro virus in the rural community. The seroprevalence detected in the samples was 41.5%. There was no significant relationship to risk for mayaro infection between genders (p value = 0.7760) or between age groups (p value = 0.9422). The positive serology detected among 39 children younger than 10 years indicates a recent infection. The factors of protection against mayaro infection detected were the use of mosquito net (p value = 0.0119) and the presence of animals in surrounding (p value = 0.0407). The risk factors identified for mayaro infection were the location of residence in towns near the forest (p value <0.0001) and presence of toilet in or near the home (p value = 0.0415). The serological results suggest that infection with mayaro occurred less than 10 years in the vicinity of residences analyzed. Molecular analysis of the samples collected in the urban area of Manaus not detected genomic fragments of arboviruses. Factors such as low viremia at the time of blood collection and storage of serum samples may have contributed to the non-detection of genomic fragments. However, the protocol for the detection of genomic fragments of arboviruses based on the PCR technique is already used in research centers and surveillance of Fundação de Medicina Tropical do Amazonas FMTAM and Instituto Leônidas e Maria Deane ILMD/FIOCRUZ. / As arboviroses são atualmente reconhecidas pela Organização Mundial da Saúde como um problema global. A maioria das arboviroses resume-se em afecções com sintomatologias agudas e inespecíficas, como febre, dores de cabeça e dores musculares. Embora sejam auto limitados, tais sintomas geram impactos sociais e econômicos relevantes. No Brasil, as arboviroses provocadas pelos por vírus pertencentes aos gêneros Alphavirus, Orthobunyavirus e Flavivirus são as principais causadoras de surtos ou epidemias. O presente estudo teve como objetivo estudar a circulação dos arbovírus mayaro, vírus da encefalite eqüina venezuelana, vírus da febre amarela, vírus da encefalite de Saint Louis e vírus oropouche em uma área rural e uma área urbana do Estado do Amazonas, Brasil. Assim sendo, a sorologia para detecção de imunoglobulinas G foi utilizada para avaliar a prevalência de anticorpos contra tais vírus em 335 moradores de uma comunidade rural do Estado do Amazonas e a PCR foi utilizada para avaliar a incidência de tais vírus em 250 amostras coletadas na área urbana de Manaus. Os resultados encontrados para a sorologia sugerem a circulação do vírus mayaro na comunidade rural. A soroprevalência detectada nas amostras foi de 41,5%. Não houve relação estatisticamente significativa de risco para a infecção por mayaro entre os gêneros (p valor=0,7760) ou entre as faixas etárias (p valor=0,9422). A sorologia positiva detectada entre 39 crianças menores de 10 anos indica uma infecção recente. Os fatores de proteção contra a infecção por mayaro detectados foram o uso de mosquiteiro (p valor=0,0119) e a presença de animais no peridomicílio (p valor=0,0407). Os fatores de risco detectados para a infecção por mayaro foram a localização do domicílio em vilas próximas à floresta (p valor<0,0001) e a presença de toalete dentro ou próximo ao domicílio (p valor=0,0415). Os resultados sorológicos sugerem que a infecção por mayaro ocorreu há menos de 10 anos nas proximidades das residências analisadas. A análise molecular das amostras coletadas na zona urbana de Manaus não detectou fragmentos genômicos de arbovírus. Fatores como baixa viremia no momento da coleta de sangue e estocagem das amostras de soro podem ter contribuído para a não detecção dos fragmentos genômicos. Contudo, o protocolo de detecção de fragmentos genômicos de arbovírus baseado na técnica de PCR está em uso nos centros de pesquisa e vigilância epidemiológica da Fundação de Medicina Tropical do Amazonas FMTAM e do Instituto Leônidas e Maria Deane ILMD/FIOCRUZ.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.