published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

This document describes an evaluation of acute (meningitis)-encephalitis syndrome (AES/AMES) surveillance established in India, Bangladesh and China. The key objectives of the project included 1) building on existing networks for syndromic surveillance and laboratory confirmation, 2) establishing laboratory-based surveillance for vaccine-preventable causes of encephalitis and meningitis, 3) enhancing capacity to use data to guide disease control and prevention programs, and 4) improving capacity to recognize new or emerging diseases. The syndromes encompass several diseases, including Japanese encephalitis (JE), pneumococcal meningitis, Haemophilus influenzae type b (Hib), and meningococcal meningitis. The purpose of the evaluation is to assess the extent to which the key objectives were met in the three project countries, compare and contrast the experiences among the countries, document the strengths and weaknesses, and make recommendations. The indicators used in the evaluation include feasibility of integration, availability of country protocols, appropriate training, data quality, sensitivity, specificity, positive predictive value, negative predictive value, representativeness, timeliness, integration with AFP surveillance, simplicity and efficiency, acceptability, usefulness, flexibility, stability, and sustainability. The criteria and standards are based on WHO recommendations. Data sources include AES/AMES epidemiologic and laboratory data sets, trip reports, country reports, field observations, and published bulletins. All countries made substantial progress in a relatively short period of time using the infrastructure and technical tools of existing surveillance and laboratory networks for acute flaccid paralysis. After one year, India and Bangladesh collects and maintains high quality epidemiologic data, exceeds targets for timeliness of reporting, and has quality-assured capacity for laboratory confirmation of Japanese encephalitis (JE) virus infection. India now has regional laboratory capacity for reference testing on virology and bacteriology. After two years of operations, China has population-based surveillance data for JE that meets targets for timeliness. Several levels have well-established capacity for laboratory confirmation of JE virus infection. The national level has the technical ability to provide proficiency testing for virology and to provide reference testing for bacteriology. In all countries, challenges in building capacity for basic bacteriology, quality control and quality assurance for all laboratory testing, and management of laboratory data.

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

Thesis (M.S.)--University of Tennessee, Knoxville, 2004. / Title from title page screen (viewed Feb. 2, 2005). Thesis advisor: Reid R. Gerhardt. Document formatted into pages (xiii, 110 p. : ill., maps). Vita. Includes bibliographical references (p. 90-109).

The proposed thesis contributes to the knowledge about tick-borne encephalitis and its pathogenesis. The thesis describes pathogenesis and immunopathogenesis of tick-borne encephalitis, impact of host's genotype in clinical course determination, immune response of patients with acute tick-borne encephalitis, the mechanism of tick-borne encephalitis virus migration into central nervous system and virus interaction with cells of neurovascular unit as well as potential medical interventions.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Dana Vanlandingham / Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus maintained among swine and avian species. In infected pigs, replication of JEV leads to the onset of viremia and the development of neurological and reproductive disease in young and naïve pregnant animals. The high-titer viremia levels associated with JEV infection in pigs, whilst important to the enzootic transmission cycle responsible for viral maintenance, also have human health implications within the zoonotic cycle. Sensitive and specific veterinary diagnostic methods capable of readily detecting JEV infection are critical components of JEV surveillance programs in the Asian Pacific region. In this study, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assays were evaluated for use in veterinary diagnosis of JEV. Our hypotheses for this research project were that RT-qPCR assays with fewer oligonucleotide mismatches between the primers and probes of the assays and JEV genomes will be more sensitive for the diagnosis of JEV infection and that oral shedding of JEV in swine would allow for detection of viral RNA using oral fluids. The sensitivity and specificity of three RT-qPCR assays for the detection of JEV were determined using tissue culture fluids of five representative JEV strains belonging to four endemic genotypes. The first assay (assay #1), targeting the highly conserved NS5 gene and 3UTR regions, provided optimum detection for the current predominant genotype, GI-b. All three assays were highly specific for JEV when tested against other selected flaviviruses in the JEV serocomplex. A rope-based collection method allowed for the simplified collection of oral fluids from three-week-old piglets challenged with endemic JEV strain JE-91. These fluids were then evaluated using RT-qPCR assays for the presence of viral RNA. The results suggest that the shedding of JEV in oral fluids can be readily detected and that non-invasive oral fluid collection can serve as a novel sampling method for the diagnosis and surveillance of JEV in swine.

This study was focused on the influence of hard tick from Ixodes ricinus on the replication of tick-borne encephalitis virus in vivo and the on the accompained changes in the levels of inflammatory cytokines which were determined in serum of infected mice. Futher the effect of tick cystatins, the inhibitors of cysteine proteases, on gene expression of interferon regulated factors was investigated in dendritic cells upon stimulation with Toll-like receptor ligands.

Made available in DSpace on 2014-06-11T19:33:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-30Bitstream added on 2014-06-13T19:44:12Z : No. of bitstreams: 1 pedrazaordonez_fj_dr_jabo.pdf: 707975 bytes, checksum: 9868dbb427049fb448644457810a5e65 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O BoHV-5 é um alfa herpesvírus neurovirulento, que causa meningoencefalite fatal em bezerros. A morbidade é baixa contrastando com uma alta mortalidade, embora alguns animais possam-se recuperar. Varias pesquisas tem sido desenvolvidas para determinar a patogênese deste tipo de doença. No presente trabalho coelhos experimentalmente infectados com o Herpesvírus Bovino 5 (BoHV-5) foram submetidos a análise imuno-histoquímica e molecular, mediante a real time PCR, para descrever a resposta inflamatória em seis diferentes regiões do seu encéfalo. Todos os animais mostraram sinais neurológicos severos e foram eutanasiados vinte dias após a infecção inicial. Microscopicamente foi descrita uma meningoencefalite não supurativa, caracterizada por meningite, manguitos perivasculares mononucleares e malacia, mas não foram observados corpúsculos de inclusão viral. Os linfócitos T constituíram uma alta percentagem das células mononucleares envolvidas na neuroinflamação. Não foram encontradas diferenças significativas na resposta astrocitária quando comparados o grupo experimental e o grupo controle (p > 0,05). A quantificação viral pela qPCR permitiu achar partículas virais em todas as regiões encefálicas, de todos os animais do grupo experimental, sendo diretamente proporcional a quantidade de vírus e a visualização de lesões histológicas no encéfalo dos coelhos, assim como a imunodetecção do BoHV-5 pela imunohistoquímica. É possível suspeitar de um forte envolvimento do sistema imunitário do hospedeiro que fez a maioria dos animais reagir de maneira diferente ante a mesma quantidade do inóculo viral ministrado / The BoHV-5 is a neurovirulent alpha herpesvirus, which causes fatal meningoencephalitis in calves. The morbidity is low in contrast with a high mortality, although some animals can heal. Several surveys have been developed trying to better understand the pathogenesis of this type of disease. In this study rabbits experimentally infected with bovine herpesvirus 5 (BoHV-5) were subjected to immunohistochemical and molecular analysis by real time PCR, to describe the inflammatory response in six different regions of his brain. All animals showed severe neurological signs and were euthanized twenty days after the initial infection. Microscopically was described a meningoencephalitis characterized by nonsuppurative meningitis, mononuclear perivascular cuffs and malacia, but were not observed viral inclusion corpuscles. T lymphocytes constituted a high percentage of mononuclear cells involved in neuroinflammation and there were no significant differences in astrocyte response when comparing the experimental and control groups (p> 0.05). The viral quantification by qPCR allowed found viral particles in all brain regions of all experimental animals, being directly proportional to the amount of virus and the visualization of lesions in the brain of rabbits, as well as immunodetection of BoHV-5 by immunohistochemistry. It is possible to suspect a strong engagement of the host immune system that made the majority of animals respond differently compared to the same administered amount of viral inoculum

Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-02-18Bitstream added on 2014-06-13T19:44:14Z : No. of bitstreams: 1 vasconcelos_ro_dr_jabo.pdf: 1349333 bytes, checksum: 7eaa9b531ee75109ac659bcca081d17c (MD5) / A compreensão do comportamento da microglia frente a uma injúria viral contribui para o entendimento da dimensão da rede de comunicação celular, durante um processo degenerativo ou inflamatório no SNC. Pesquisas que utilizam modelos experimentais com o vírus da estomatite vesicular (VEV) têm colaborado com informações importantes sobre o comportamento e distribuição do agente no encéfalo, e também sobre o papel da resposta imune na resolução ou no agravamento das lesões nervosas. Neste estudo, foi comparada a evolução do quadro neurológico induzido pelo VEV, por meio de técnicas de imunoistoquímca, em cérebros de camundongos. Foi possível observar que o VEV causa severa degeneração e necrose do neurópilo, com lesão direta em neurônios, pois estes mostraram-se claramente positivos para o vírus, por meio da reação de imunoistoquímica no cérebro. A reação astrocitária foi intensa nos animais infectados, porém a densidade destas células reduziu com o aumento da gravidade das lesões. As células residentes (neurônios, astrócitos e microglia) e as células inflamatórias expressaram MIP-1a e, em menor proporção, MIP-1b. A microgliose reativa foi significativa nos animais com sintomatologia clínica. A diversidade morfológica da microglia foi grande, variando desde uma forma fusiforme a uma ramificada e a forma arredondada fagocítica das áreas necróticas. Foi possível observar que existe uma profunda interação entre as células residentes do SNC - neurônios, microglia, astrócitos, endotélio, frente ao estímulo viral. Baseado nos relatos da literatura é importante salientar que os astrócitos mantêm um controle ativo sobre a microglia tanto em repouso quanto ativada, via citocinas/quimiocinas. A densidade aumentada dessas células coincidiu com a redução do número de astrócitos, devido à necrose do neurópilo... / The comprehension of the microglial cell behavior in a viral injury colaborate for the understanding of the dimension of the cellular communication net, during a degenerative or inflammatory process in central nervous system (CNS). Experimental models with the vesicular stomatitis virus (VSV), has contributed with important information about the behavior and distribution of virus in the CNS. Such studies evaluated the role of the immune response in the resolution or in the damage of the nervous lesions. In this study, the evolution of the neurological signals and lesions induced by VSV infection in mice was studied, using imunohistochemical techniques. It was observed that VSV causes severe degeneration and necrosis of the neuropil and direct lesions to the neurons. The neurons were the most intensively stained cells for the virus in the brain. The reactive astrocitosis was intense in the infected animals, but the density of these cells reduced with the increase of the gravity of the lesions. The resident and inflammatory cells expressed MIP-1a and in smaller proportion MIP-1b, in different cellular types (neurons, astrocytes and microglia). The reactive microgliosis was significant in animals with clinical symptomatology and there was a great morphologic microglial diversity, varying from a fusyforme form and ramified form and the fagocitic round form of the around necrotics areas. It was possible to observe that a close interaction exists among the resident cells of SNC (neurons, microglia, astrocytes, endothelial cells) in face of the viral infection. Based on the reports of the literature it is important to point out that the astrocytes maintains an active control on the microglia (in resting or activated cells), through citokines/chemokines. The increased density of microglia coincided with the reduction of the astrocytes number, due to the necrosis of the neuropil... (Complete abstract, access undermentioned eletronic address)

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-22Bitstream added on 2014-06-13T20:35:29Z : No. of bitstreams: 1 bregano_lc_me_araca.pdf: 540539 bytes, checksum: 5989de4e2cf1d4911bd4e7adb7afaa77 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No presente estudo foram avaliados 30 cerebelos de cães naturalmente infectados pelo vírus da cinomose canina, confirmados por meio da reação RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) com segmentação do gene do nucleocapsídeo viral, e por análise microscópica das lesões teciduais após a coloração Hematoxilina-Eosina e coloração de Shorr. A distribuição das subpopulações de linfócitos T foi evidenciada pela técnica de imunoistoquímica, empregando-se anticorpos monoclonais CD3+, CD8+ e CD4+. Da mesma forma, a detecção da expressão das metaloproteinases (MMP) -2 e -9 também foi conduzida pela técnica de imunoistoquímica e os resultados foram associados aos encontrados na imunofenotipagem. Foi possível evidenciar marcação mais expressiva de MMP-2 (86,67% de intensidade moderada a intensa) e uma alta proporção de linfócitos T CD4+ e CD8+. Nesse sentido, pode-se concluir que o vírus da cinomose canina se dispersa no sistema nervoso central, alterando a integridade estrutural da barreira hematoencefálica, provavelmente pela ação da MMP-2; e os infiltrados inflamatórios perivasculares presentes são constituídos predominantemente por subpopulações de linfócitos T CD4+ e CD8+ / The present study evaluated 30 cerebella of dogs naturally infected with canine distemper virus, confirmed by RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) to target the viral nucleocapsid gene, and by microscopic examination of tissue lesions after hematoxylin-eosin and Shorr staining. The distribution of subpopulations of T lymphocytes was demonstrated by immunohistochemistry using monoclonal antibodies against CD3 +, CD8 + and CD4 +. Similarly, detection of the expression of matrix metalloproteinases (MMP) -2 and -9 was also conducted by immunohistochemistry and the results were related to those found in immunophenotyping. The results revealing more expressive marking of MMP-2 (86.67% moderate to severe) and a high proportion of CD4 + and CD8 +. In conclusion, it can be infer that canine distemper virus is spread in the central nervous system, altering the structural integrity of the blood-brain barrier, probably by the action of MMP-2. Moreover, perivascular inflammatory infiltrates consisted predominantly of T lymphocyte subpopulations CD4 + and CD8 +