Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are major human pathogens. HSV establishes latency in the nervous system and reactivates to cause recurrent disease, resulting in transmission of progeny virions to naïve individuals. Though HSV-1 and HSV-2 share similar structure and genes, they have distinctive recurrence profiles. Generally, HSV-1 reactivation is associated with disease 'above the waist' and HSV-2 reactivation is associated with disease 'below the waist'. This phenomenon was described decades ago but still remains unexplained. The mechanism of HSV latent infection in the peripheral nervous system (PNS) has been extensively investigated, especially with in sensory neurons. Another component of the peripheral nervous system (PNS), autonomic neurons, were also known to be infected with HSV productively and latently, but largely ignored because of the assumption that there is no difference in the pathogenesis of HSV in the neurons and that both HSV-1 and HSV-2 behave in the same way in different types of neurons. However, autonomic neurons differ in physiological function compared to sensory neurons. Activation factors of autonomic neurons, such as emotional stress, trauma and hormonal fluctuation, are also known HSV reactivation triggering factors. Therefore, I hypothesized that autonomic neurons innervating the site of HSV infection are responsible the different reactivation frequencies of HSV-1 and HSV-2 after peripheral invasion. In this report, the role of autonomic neurons in HSV pathogenesis were examined using the female guinea pig reactivation model. Major findings of this report are that 1) parasympathetic ganglia innervating the ocular region support latent infection of HSV-1 selectively, thus contributing the more frequent HSV-1 reactivation, 2) mixed autonomic ganglia in the genital area support HSV-2 latent infection selectively, and 3) sympathetic neurons in the genital region supported productive and latent infection of HSV-1 and HSV-2 differently. All of the results in this report indicate that autonomic neurons play a distinctive role in HSV pathogenesis compared to the sensory neurons and are responsible for the different reactivation frequencies of HSV-1 and HSV-2. This report raises the importance of autonomic neurons in HSV pathogenesis and challenges the paradigm of HSV pathogenesis. / Ph. D.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-­‐virus interaction. This study made use of next-­‐generation sequencing to investigate the effect of virus-­‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐derived sRNAs and vsiRNAs. Broad and narrow size-­‐range datasets were generated using sRNA libraries prepared from total and size-­‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-­‐ and trans-­‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-­‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-­‐halves. Reads associated with tRNA-­‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-­‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple. / AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-­‐virus interaksie is beskikbaar. Hierdie studie het van volgende-­‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl. miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐afkomstige sRNAs en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-­‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte. Deur middel van bioinformatiese data-­‐ontleding is 130 genomiese loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-­‐beskikbare degradoom datastel. Cis-­‐ en trans-­‐natuurlike antisense transkripte (NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook geïdentifiseer. Vir 25 van hierdie loci is 'n in-­‐fase miRNA teiken geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs het ooreengestem met tRFs en tRNA-­‐helftes. Volgordes geassosieer met tRNA-­‐helftes was prominent in die breë grootte-­‐verspreiding datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek voorbereiding op die interpretasie van resultate. Ontleding van die differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies het geen verskil tussen gesonde en geïnfekteerde plante getoon nie, behalwe vir die tRNA-­‐afkomstige sRNAs, wat wel verandering die vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat in hierdie studie geïdentifiseer is, het die bestaande kennis van appel sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige funksionele studies in appel. Hierdie studie bied ook die eerste, en mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in appel.

Orientador: Hélio José Montassier / Banca: Aramis Augusto Pinto / Banca: José Antonio Jerez / Resumo: A pesquisa sorológica usando Imunodifusão em Gel de Ágar (IOGA) e ELISA Bloqueio de Fase Líquida (BFL-EUSA) foram aplicadas nesse estudo no período de 1996 a 1998, para detectar ou titular, respectivamente, anticorpos contra o antígeno VIA (Virus Infection Associated) e as estirpes de referência 01 Campos, A24 Cruzeiro e C3 Indaial, em algumas espécies silvestres e domésticas susceptíveis e não vacinadas. Do total de 383 amostras sanguíneas colhidas, 78 amostras eram de bovinos de vida livre; 39 de búfalos indianos; 139 de ovinos; 63 de suínos domésticos; 49 de porcos do mato e 15 de cervídeos. Todas as espécies investigadas, exceto os cervídeos, apresentaram anticorpos contra o Vírus da Febre Aftosa (VFA). O teste de IDGA detectou 47 (12,3%) amostras positivas ao antígeno VIA, principalmente em bovinos baguás e búfalos. O BFL-ELISA identificou 175 (45,7%) soros com presença de anticorpos a uma das três estirpes virais; Foi encontrado um maior número de amostras positivas contra a estirpe C3 Indaial bem como maiores títulos, em bovinos baguás. Os bubalinos e ovinos apresentaram uma maior reatividade contra as estirpes A24 Cruzeiro e 01 Campos, respectivamente, demonstrando maior número de soros reagentes. No entanto, ambas as espécies revelaram maiores títulos para a estirpe C3 Indaial. Nos suínos domésticos e silvestres, predominou a reatividade a estirpe A24 Cruzeiro. Em bovinos, a maior ocorrência de soros positivos ao teste de IOGA foi encontrada em animais entre 12 a 36 meses de idade, e na prova BFL-ELISA, em animais acima de 36 meses. Nas demais espécies, ambos os testes laboratoriais utilizados demonstraram um maior número de soros reativos em indivíduos adultos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: A serologic survey, using Agar Gel Immunodifusion (AGID) and liquid Phase Blocking-ELISA (LPB-ELISA) were assessed in this study, in the period from to 1996 to 1998, to detect or titer, respectively, antibodies to Virus Infection Associated Antigen (VIAA) and the foot-and-mouth disease virus (FMDV) 01 Campos, A24 Cruzeiro e C3 Indaial reference strains, in non-vaccinated domestic and wild susceptible animal species. A total of 383 blood serum samples were collected from free-ranging cattle (78 samples), water buffalo (39 samples), sheep (139 samples), domestic (63 samples) and wild pigs (49 samples) and deers (15 samples), living in the Pantanal (Nhecolândia region) of Mato Grosso do Sul State, Brazil. All the investigated species, excepting deer, showed positive animais to FMDV antigens. The AGID detected a total of 47 (12,3%) positive animais for VIA antigen, mostly of them were free-living cattle and buffalo. The LPD-EUSA identified 175 (45,7%) serum samples as reactive to FMDV antigens, specially among cattle, buffalo and sheep. The predominant reactivity to FMDV antigens, characterized by the higher number of positives and the higher antibodies titers, were detected to C3 Indaial strain, particularly among the free-Iiving cattle. Buffaloes and pigs showed, in turn, a prominent reactivity to A24 Cruzeiro strain, while sheep reacted more to 01 Campos strain, though buffalo and sheep serum samples had higher antibody titers to C3 Indaial strain. The highest frequency of positive animais to VIA antigen were found in free-ranging cattle, whose age ranged from 12 to 36 months, contrasting to the results of LPB-EUSA that detected the highest rate of positives in the older bovines (~36 months). The older animais from other species demonstrated, in both serological tests, the highest frequency of positivity... (Complete abstract, access undermentioned eletronic address) / Mestre

Brain development is an exquisitely coordinated process of progenitor cell proliferation followed by the migration of progeny to their final location in the developing brain. There are a myriad of points at which this process can be disturbed, and the examination of these perturbations help us further understand basic science, as well as epidemics sweeping through the world around us. Microcephaly, which is defined as a head circumference greater than 2 standard deviations below the mean, can occur through genetic, infectious, vascular, or metabolic etiologies, and the studies herein examine two forms by which microcephaly occurs. First, we investigate the role of the dynein regulatory protein Nde1 in the development of the neocortex, which is the outer region of the forebrain. NDE1 mutations are associated with severe microcephaly, and we find that unlike most microcephaly genes whose products have one role in the cell cycle, Nde1 is required at three discrete points in neuronal progenitors, termed radial glia progenitors (RGPs). We initially find that Nde1 is required to recruit dynein to the nuclear envelope to allow for interkinetic nuclear migration (INM) during G2. Additionally, Nde1 helps to initiate primary cilia resorption at the G1-to-S transition. Finally, there is a necessity for Nde1 at the G2-to-M transition after the completion of INM and prior to nuclear envelope breakdown. These three distinct roles for Nde1 illustrate the breadth of functions that the protein has during RGP proliferation, and help to explain why patients with NDE1 mutations have such severe microcephaly. As this work was ongoing there was a global outbreak of a new pathogen that had previously been dormant throughout Africa and Asia, only to emerge at epidemic proportions in the Western Hemisphere. This pathogen, the Zika Virus (ZIKV), is particularly alarming because of its subclinical course in adults but devastating consequences for fetal development, with the hallmark symptom being microcephaly. Using our organotypic brain slice model system, we demonstrate the ability of a variety of ZIKV isolates to infect and replicate in embryonic brain tissue. All ZIKV isolates that infect the organotypic slices lead to increases in apoptosis, though these increases are particularly pronounced in isolates from the Asian/American lineages. Notably, one isolate from a patient in Nigeria (termed 30656) does not replicate in mouse neuronal tissue, but electroporation of the 30656 ZIKV genome allows for a single cycle replication, suggesting that this isolate is unable to enter RGPs. All infectious isolates are pathogenic in early- and mid- gestation embryonic tissue, but only one isolate infects and replicates in late- gestation embryonic tissue. This was the most recently isolated sample tested, and it demonstrates a predilection for neurons, suggesting that ZIKV may be mutating as it spreads. These results provide foundational insight into the pathogenesis of ZIKV- associated microcephaly, and illustrate how studies of genetic forms of microcephaly can enhance and facilitate our understanding of infectious causes of the disease.

<p>Hepatitis E virus (HEV) je uzročnik akutne hepatis E infekcije kod ljudi. HEV se prenosi putem zagaĎene vode i odgovoran je za nastanak mnogobrojnih epidemija velikih razmera u zemljama u razvoju Azije i Afrike. Hepatitis E je prvi put kod svinja izolovan 1997. godine, a kasnije je dokazan i kod ostalih ţivotinjskih vrsta, kao &scaron;to su: divlje svinje, jelen, zečevi, pacovi, ptice i ostalo.<br />Prva istraţivanja prisustva HEV infekcije kod domaćih i divljih svinja u Srbiji sprovedena su 2008. godine. HEV RNK je dokazana u 30% uzoraka fecesa i 45% uzoraka organa (Petrovic et al., 2008). Analizom uzoraka krvnih seruma svinja u individualnim gazdinstvima ustanovljena je seroprevalencija od 34,6% (Lupulovic et al, 2010). Cilj ovog istraţivanja je ispitivanje ra&scaron;irenosti HEV infekcije kod svinja na farmama na teritoriji Vojvodine, kao i ispitivanje HEV seroprevalencije kod ljudi u Vojvodini.<br />Od metoda za istraţivanje kori&scaron;ćene su: nekomercijalni ELISA test (in-house ELISA), komercijalni ELISA test, Western-blot metod, real-time RT -PCR i imunohistohemijska metoda za detekciju HEV antigena.<br />Materijal za ispitivanje su bili uzorci krvi svinja (300) sa 3 farme na teritoriji Juţne Bačke i Srema i uzorci krvi ljudi (294), kao i uzorci fecesa, ţuči, jetre i mesa sakupljeni u klanicama od 95 tovljenika i 50 prasadi.<br />Prisustvo specifičnih antitela IgG klase protiv hepatitis E virusa dokazano je kod svinja na sve tri ispitujuće farme. Primenom in house ELISA testa ustanovljena je seroprevalencija od 37% na farmi A, 31% na farmi B i 54% na farmi C, dok je primenom komercijalnog ELISA testa utvrĎeno 40% seropozitivnih svinja na farmi A, 41% na fami B i 65% na farmi C. Uporednom analizom rezultata dobijenih sa oba ELISA testa, ustanovljena je prosečna seroprevalencija od 40,66% in house ELISA testom, odnosno 48,66% komercijalnim ELISA testom.<br />Sprovedena su i istraţivanja prisustva specifičnih antitela IgG klase protiv HEV u krvnim serumima dobrovoljnih davalaca krvi i kod pacijenata. Primenom in house ELISA testa utvrĎena je<br />seroprevalencija od 15% kod dobrovoljnih davalaca krvi, dok su uzorci krvi pacijenata bili seronegativni. Testiranjem komercijalnim ELISA testom kod dobrovoljnih davalaca krvi pozitivan serolo&scaron;ki nalaz je ustanovljen kod 17,86% dobrovoljnih davalaca krvi (pregledani su serumi koji su u in house testu dali pozitivan ili sumnjiv nalaz, kao i odreĎen broj seronegativnih uzoraka), a kod pacijenata 2,12%.<br />Kao tzv. &bdquo;zlatni standard&ldquo; za definisanje rezultata sa sumnjivim serolo&scaron;kim nalazom čije su ekstinkcije bile blizu cut off vrednosti u in house ELISA testu, kori&scaron;ćen je Western blot metod. Pozitivan rezultat je potvrĎen kod 6 od ukupno pregledanih 11 uzoraka krvi svinja, odnosno od ukupno pregledanih 11 uzoraka seruma ljudi, pozitivan nalaz je ustanovljen kod 7 uzoraka.<br />Uzorci sa klanica pregledani su real-time RT- PCR metodom, a HEV RNK je dokazana u u fecesu (54%), ţuči (26%), jetri (16%) i mesu (10%) prasadi. Kod tovljenika prisustvo HEV RNK je potvrĎeno samo u fecesu (7,27%), dok su svi uzorci tkiva bili negativni.<br />Patahistolo&scaron;kim pregledom dokazane su mikroskopske lezije II stepena kod 3 uzorka (11,53%) jetri prasadi od ukupno pregledanih 26 uzoraka sa pozitivnim RT-PCR. Imunohistohemijskim pregledom uzoraka jetre prasadi nije dokazano prisustvo antigena hepatitis E virusa.<br />Definisani su protokoli laboratorijskog ispitivanja hepatitis E infekcije kod svinja i ljudi, kao i u uzorcima mesa i jetri svinja u klanicama</p> / <p>Hepatitis E virus (HEV) is the causative agent of acute hepatitis E infection in humans. HEV is transmitted through contaminated water and is responsible for the outbreaks of many large-scale epidemics in the developing countries of Asia and Africa. Swine HEV was first isolated in 1997, and was later detected in other animal species, such are: wild boar, deer, rabbits, rats, birds and more.<br />The first investigations of HEV infection in domestic and wild pigs in Serbia were carried out in 2008. HEV RNA was detected in 30% of faecal samples and 45% of the tissue samples (Petrovic et al., 2008). Analysing the blood samples of beckyard pigs, the seroprevalence of 34,6% was determined (Lupulovic et al, 2010). The aim of this study was to investigate the prevalence of HEV infection in pigs on farms in Vojvodina, as well as testing the HEV seroprevalence in humans.<br />The methods used for this study were: non-commercial ELISA (in house ELISA), the commercial ELISA, Western blot method, real-time RT-PCR and immunohistochemical method for the detection of HEV antigen.<br />Material for the study were: blood samples of pigs (300) from 3 farms on the territory of South Backa and Srem and blood samples of people (294), as well as faeces, bile, liver and meat collected in slaughterhouses from 95 fatteners and 50 piglets.<br />The presence of specific IgG antibodies against the hepatitis E virus in pigs has been detected on all three examinated farms. Upon the application of in house ELISA, the seroprevalence of 37% was establised on farm A, 31% in farm B and 54% on farm C, while using a commercial ELISA , 40% of seropositive pigs were detected on farm A, 41% of fami B and 65% Farm C. The comparative analysis of the results obtained with both ELISA, determined the average seroprevalence of 40,66% by in house ELISA and 48,66% by commercial ELISA.<br />The research of the presence of specific IgG antibodies against HEV in the serum of blood donors and patients were also conducted. Upon the application of in house ELISA, the seroprevalence of 15% were recorded in blood donors, while blood samples of patients were seronegative. Testing by commercial ELISA, positiv serological findings were diagnosed in 17,86% of blood donors (serums with positive or suspicious results in in house ELISA and a number of seronegative samples were tested), and in patients 2, 12%.<br />As so-called &quot;gold standard&quot; for defining the serological results with suspiciousserological findings, which extinctions were close to cut off values in in house ELISA, we used the Western</p>

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-02-02Bitstream added on 2014-06-13T19:15:42Z : No. of bitstreams: 1 paes_rcs_me_jabo.pdf: 1518593 bytes, checksum: 4c347db640a4ee036b567916847c5278 (MD5) / A pesquisa sorológica usando Imunodifusão em Gel de Ágar (IOGA) e ELISA Bloqueio de Fase Líquida (BFL-EUSA) foram aplicadas nesse estudo no período de 1996 a 1998, para detectar ou titular, respectivamente, anticorpos contra o antígeno VIA (Virus Infection Associated) e as estirpes de referência 01 Campos, A24 Cruzeiro e C3 Indaial, em algumas espécies silvestres e domésticas susceptíveis e não vacinadas. Do total de 383 amostras sanguíneas colhidas, 78 amostras eram de bovinos de vida livre; 39 de búfalos indianos; 139 de ovinos; 63 de suínos domésticos; 49 de porcos do mato e 15 de cervídeos. Todas as espécies investigadas, exceto os cervídeos, apresentaram anticorpos contra o Vírus da Febre Aftosa (VFA). O teste de IDGA detectou 47 (12,3%) amostras positivas ao antígeno VIA, principalmente em bovinos baguás e búfalos. O BFL-ELISA identificou 175 (45,7%) soros com presença de anticorpos a uma das três estirpes virais; Foi encontrado um maior número de amostras positivas contra a estirpe C3 Indaial bem como maiores títulos, em bovinos baguás. Os bubalinos e ovinos apresentaram uma maior reatividade contra as estirpes A24 Cruzeiro e 01 Campos, respectivamente, demonstrando maior número de soros reagentes. No entanto, ambas as espécies revelaram maiores títulos para a estirpe C3 Indaial. Nos suínos domésticos e silvestres, predominou a reatividade a estirpe A24 Cruzeiro. Em bovinos, a maior ocorrência de soros positivos ao teste de IOGA foi encontrada em animais entre 12 a 36 meses de idade, e na prova BFL-ELISA, em animais acima de 36 meses. Nas demais espécies, ambos os testes laboratoriais utilizados demonstraram um maior número de soros reativos em indivíduos adultos... / A serologic survey, using Agar Gel Immunodifusion (AGID) and liquid Phase Blocking-ELISA (LPB-ELISA) were assessed in this study, in the period from to 1996 to 1998, to detect or titer, respectively, antibodies to Virus Infection Associated Antigen (VIAA) and the foot-and-mouth disease virus (FMDV) 01 Campos, A24 Cruzeiro e C3 Indaial reference strains, in non-vaccinated domestic and wild susceptible animal species. A total of 383 blood serum samples were collected from free-ranging cattle (78 samples), water buffalo (39 samples), sheep (139 samples), domestic (63 samples) and wild pigs (49 samples) and deers (15 samples), living in the Pantanal (Nhecolândia region) of Mato Grosso do Sul State, Brazil. All the investigated species, excepting deer, showed positive animais to FMDV antigens. The AGID detected a total of 47 (12,3%) positive animais for VIA antigen, mostly of them were free-living cattle and buffalo. The LPD-EUSA identified 175 (45,7%) serum samples as reactive to FMDV antigens, specially among cattle, buffalo and sheep. The predominant reactivity to FMDV antigens, characterized by the higher number of positives and the higher antibodies titers, were detected to C3 Indaial strain, particularly among the free-Iiving cattle. Buffaloes and pigs showed, in turn, a prominent reactivity to A24 Cruzeiro strain, while sheep reacted more to 01 Campos strain, though buffalo and sheep serum samples had higher antibody titers to C3 Indaial strain. The highest frequency of positive animais to VIA antigen were found in free-ranging cattle, whose age ranged from 12 to 36 months, contrasting to the results of LPB-EUSA that detected the highest rate of positives in the older bovines (~36 months). The older animais from other species demonstrated, in both serological tests, the highest frequency of positivity... (Complete abstract, access undermentioned eletronic address)

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-04-03T14:49:52Z No. of bitstreams: 1 HaryelleNarymaConfessorFerreira_DISSERT.pdf: 903183 bytes, checksum: 512cec0622808aed442e5d8b7f4b59a6 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-04-10T20:46:08Z (GMT) No. of bitstreams: 1 HaryelleNarymaConfessorFerreira_DISSERT.pdf: 903183 bytes, checksum: 512cec0622808aed442e5d8b7f4b59a6 (MD5) / Made available in DSpace on 2018-04-10T20:46:08Z (GMT). No. of bitstreams: 1 HaryelleNarymaConfessorFerreira_DISSERT.pdf: 903183 bytes, checksum: 512cec0622808aed442e5d8b7f4b59a6 (MD5) Previous issue date: 2018-02-26 / Introduction: The increase in the number of cases of microcephaly in Brazil and its association with the Zika virus (ZIKV) is a global public health problem. The International Classification of Functioning Disability and Health (ICF) model is a powerful tool and extremely relevant in managing disability. Objective: Describe the functioning profile of children with microcephaly associated with ZIKV in two states of northeastern Brazil. Methods: This is a descriptive cross-sectional study. The sociodemographic characteristics, head circumference and other clinical data were collected from medical charts, physical examinations, measuring instruments and interviews with the children and their parents. The Brazilian Portuguese version of the ICF core set for cerebral palsy (CP) was used. Each ICF category was assigned a qualifier, which ranged from 0 to 4 (no disability, mild disability, moderate disability, severe disability and complete disability). For environmental factors, 0 represents no barrier and 4 total barrier; +0, no facilitator +4, total facilitator. Results: A total of 34 children with microcephaly caused by ZIKV were recruited (18 girls and 16 boys) at four rehabilitation facilities in Rio Grande do Norte and Para?ba states, Brazil. The average age of the participants was 21 months and head circumference z-scores ranged from 0.92 to -5.51. The functioning profile revealed complete disability in most of the body function categories (b). The activity and participation areas (d) were highly impacted, particularly in mobility-related categories. With respect to environmental factors (e), most of the sample reported a total facilitator for the nuclear family, friends and health services, systems and policies, as well as a total barrier to social attitudes. Conclusion: This is the first study that describes the functioning profile of children with microcephaly associated with ZIKV, using a tool based on the ICF in Brazil. Our findings reinforce the need to maximize health care and access to information ? based on the ICF ? for multiprofessional teams, administrators, family members and children.

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.

Hepatitis C virus (HCV) is remarkably efficient at evading host immunity to establish chronic infection. During chronic HCV infection, interleukin-12 (IL-12) produced by monocytes/macrophages (M/Mφ) is significantly suppressed. Programmed death-1 (PD-1), an inhibitory receptor on immune cells, plays a pivotal role in suppressing T-cell responses during chronic viral infection. To determine whether PD-1 regulates IL-12 production by M/Mφ during chronic HCV infection, we examined the expressions of PD-1, its ligand PDL-1, and their relationship with IL-12 production in M/Mφ from HCV-infected, HCV-resolved, and healthy subjects by flow cytometry. Toll-like receptor (TLR) -mediated IL-12 production by M/Mφ was selectively suppressed, while PD-1/PDL-1 expressions were up-regulated, in HCV-infected subjects compared with HCV-resolved or healthy subjects. Up-regulation of PD-1 was inversely associated with the degree of IL-12 inhibition in HCV infection. Interestingly, the reduced response of M/Mφ from HCV-infected individuals to TLR ligands appeared not to be the result of a lack of the ability to sense pathogen, but to an impaired activation of intracellular janus kinase/signal transducer and activator of transfection (STAT) pathway as represented by inhibited STAT-1 phosphorylation in M/Mφ from HCV-infected individuals compared with HCV-negative subjects. Successful HCV treatment with pegylated interferon/ribavirin or blocking PD-1/PDL-1 engagement ex vivo led to reduced PD-1 expression and improved IL-12 production as well as STAT-1 activation in M/Mφ from HCV-infected individuals. These results suggest that the PD-1 inhibitory pathway may negatively regulate IL-12 expression by limiting STAT-1 phosphorylation in M/Mφ during chronic HCV infection. No claim to original US government works.

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