Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-03-15T12:49:24Z No. of bitstreams: 2 Dissertação - Danilo Rezende e Silva - 2014.pdf: 795804 bytes, checksum: 70571f234568a148006646c85383db05 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-16T11:26:10Z (GMT) No. of bitstreams: 2 Dissertação - Danilo Rezende e Silva - 2014.pdf: 795804 bytes, checksum: 70571f234568a148006646c85383db05 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-16T11:26:10Z (GMT). No. of bitstreams: 2 Dissertação - Danilo Rezende e Silva - 2014.pdf: 795804 bytes, checksum: 70571f234568a148006646c85383db05 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-04 / Identification of herpesvirus in specimes fixed in formalin and paraffin embedded has been used in experimental analysis. Several molecular techniques have been used in research and in diagnosing of herpesviral meningoencephalitis. 80 cases of routine in veterinary pathology were reviewed on the Department of Animal Pathology of Veterinary and Science Animal School Medicine of Federal University of Goias, Goiania, GO (EVZ/UFG) performed between January 2007 to December 2012. Were used 18 cases with a total of 44 paraffin blocks (Group I - 14 blocks, Group II - 17 blocks and Group III - 13 blocks), which were grouped according to histopathological diagnosis, in Group I - samples without histological changes and no clinical history of neurological disease, Group II - samples with nonspecific encephalitis, Group III - samples with rabies encephalitis. Were extracted DNA fragments, from cuts blocks, using a commercial extraction kit of formalin and paraffin embedded tissue. Afterwards the technique standardization of conventional PCR for bovine herpesvirus-1 and bovine herpersvírus type-5 was performed. Were positive for BoHV-5 28% (5/18) of cases, with 11% (2/18) of the group with nonspecific encephalitis and 17% (3/18) of the group with encephalitis and positive for the rabies virus. In the test for checking of the bands for the BoHV-1, all samples, 100% (18/18) were negative. These results demonstrate that the conventional PCR technique is helpful in aiding the definitive diagnosis of encephalitis bovine herpesvirus type-5. / A extração do DNA de herpesvírus em amostras fixadas em formol, assim como amostras emblocadas em parafina, tem sido utilizada em análises experimentais, permitindo o emprego de técnicas moleculares em pesquisas e no diagnóstico das meningonencefalites por herpesvírus. Foram revisados 80 casos da rotina anatomopatológica do Setor de Patologia Animal da Escola de Veterinária e Zootecnia da Universidade Federal de Goiás, Goiânia, GO (EVZ/UFG) realizados entre janeiro de 2007 à dezembro de 2012. Foram utilizados 18 casos com um total de 44 blocos (Grupo I – 14 blocos, Grupo II – 17 blocos e Grupo III – 13 blocos), os quais foram agrupados, de acordo com diagnóstico histopatológico, em Grupo I – amostras sem alterações histológicas e sem histórico clínico de doença neurológica, Grupo II – amostras com encefalite inespecífica, Grupo III – amostras com encefalite por Raiva. DNA dos fragmentos foram obtidos a partir dos cortes dos blocos, foi extraído com kit comercial de extração de material parafinizado. Posteriormente foi realizada a padronização da técnica de PCR convencional para herpesvírus bovino tipo-1 e herpersvírus bovino tipo-5. Foram positivos para BoHV-5 28% (5/18) dos casos, sendo 11% (2/18) do grupo com encefalite inespecífica e 17% (3/18) do grupo com encefalite e positivos para o vírus da raiva. No teste para verificação das bandas para o BoHV-1, todas as amostras, 100% (18/18), foram negativas. Esses resultados comprovam que a técnica de PCR convencional é útil no auxílio do diagnóstico definitivo de encefalite por herpesvírus bovino tipo-5.

The lack of vaccines for emerging and re-emerging diseases highlights technical gaps and indicates a need for innovative approaches to produce new vaccines. Vaccines may be improved by knowledge of host responses to vaccination, disease pathogenesis, and the effect of age and genetics on vaccine outcome. This study's purpose was to quantitatively assess the molecular epidemiology of Francisella tularensis (Ft) and Venezuelan Equine Encephalitis Virus (VEEV). Study results support the Epidemiology Nexus model which holds that association of changes in gene expression to vaccination facilitate understanding the mechanisms of immune development and link public health and disease epidemiology. My research questions assessed the relationship between gene expression following vaccination, the relationship between age and vaccine response, and the association between Human Leukocyte Antigen (HLA) allele and vaccine response. The study was a novel secondary analysis of human data subjected to ANOVA to measure association between treatment and outcome, correlation to measure association of age with vaccine outcome, and Mann-Whitney U tests to measure association of HLA allele with vaccine outcome. Both Ft and VEEV vaccination elicited significant changes in gene expression. A highly positive relationship between age and vaccine outcome was shown for VEEV. The results may affect positive social change by contributing to a growing compendium of evidence of vaccine efficacy mechanisms that may function to assure the public of vaccine safety, combat vaccine hesitancy, and promote vaccine acceptance, as well as contribute mechanistic knowledge to reduce developmental costs of novel vaccines.

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

No description available.

SUMMARY Assessment of health care services quality and accessibility for tick-borne encephalitis patients in Kaunas region Liucijus Ambraška Supervisor Janina Petkevičienė, Dr.Sc.Assoc. Prof., Department of Social Medicine. Faculty of Public Health, Kaunas University of Medicine.- Kaunas, 2005.-P.68 Key words: tick-borne encephalitis, quality and accessibility of health care services Incidence of tick-borne encephalitis (TBE) is increasing by data of Lithuanian health information center. Aim of the study – to assess accessibility and quality of health care services for TBE patients in Kaunas region. Objectives: to establish peculiarities of transmission TBE infection and opinion about TBE prophylaxis of reconvalescents; to evaluate duration of admission period of TBE patients to specialized hospital and peculiarities of monitoring after discharging and satisfaction with health care services; to determine standpoint of general practitioners (GP) on TBE prophylaxis and reconvalescents monitoring problems. Methods. Case histories of TBE patients treated in Kaunas Clinical Hospital of Infectious Diseases in years 2001-2002 (n=187) analyzed. Questionnaires about state of health and assessment of quality of health care services during period of illness and reconvalescence were posted. 104 answers received. Questionnaires about standpoint on TBE control problems handed to 150 GP and returned by 101. Original questionnaires used. Differences between groups assessed using Mann-Whitney... [to full text]

The aim of this study was to characterize the replication of tick borne encephalitis virus in mouse macrophages and investigate the influence of tick saliva derived from Ixodes ricinus on the viral replication. Moreover, the effect of interferon (the member of type I interferons) and nitric oxide on virus replication was studied.

Os herpesvírus bovino tipo 1 (BoHV-1) e 5 (BoHV-5) são alfaherpesvírus freqüentemente associados a meningoencefalites. Por outro lado, o vírus da raiva é o agente mais frequentemente identificado como causador de encefalites virais em bovinos no Brasil. O objetivo do presente estudo foi examinar a ocorrência de infecções por BoHV-1 e/ou BoHV-5 em amostras de tecido encefálico bovino submetidas ao diagnóstico de raiva e avaliar seu possível envolvimento nos quadros de encefalite que originaram a suspeita de raiva. Para tanto, 101 amostras desses tecidos, sendo 39 positivas para raiva e 62 negativas, recebidas pelo órgão oficial responsável pelo diagnóstico de raiva no Estado do Rio Grande do Sul (IPVDF) no período de 2009- 2010, foram submetidas a exames buscando o isolamento viral e amplificação de genomas de herpesvírus bovinos. Ao isolamento viral, todas as amostras foram negativas para vírus infeccioso após a realização de três passagens cegas em células MDBK. As mesmas foram submetidas à amplificação por “nested PCR” (nPCR) para a pesquisa de genomas de BoHV-1 e BoHV-5. Das 101 amostras totais analisadas esta técnica revelou que 25,7% (26/101) continham genomas de BoHV-1 e 21,8% (22/101) continham genomas de BoHV-5. Genomas de ambos os tipos foram identificadas em 30 (29,7%) amostras. Entre as amostras que foram também positivas para raiva em 23% (9/39) foram detectados genomas de BoHV-1 e em 15,4% (6/39) continham genomas de BoHV-5. Em 16 destas 39 amostras (41%) foram detectados genomas de BoHV-1 e BoHV-5. Em contrapartida, nas amostras negativas para o vírus rábico, 27,4% (17/62) também foram positivas para BoHV-1, 25,8% (16/62) foram positivas para BoHV-5. Detectaram-se os genomas de ambos BoHVs em 22,6% (14/62) dos animais. Estas diferenças não foram estatisticamente significativas, indicando não haver correlação entre a ocorrência de raiva e infecções por herpesvírus na amostragem realizada. Estes resultados indicam que, embora as infecções por BoHV-1 e BoHV-5 tenham apresentado elevada incidência nessas amostras, não havia vírus infeccioso nas mesmas, sugerindo infecções latentes sem envolvimento aparente nos quadros de encefalite que originaram a suspeita inicial de raiva. / Bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) are alphaherpesviruses associated with a number of clinical manifestations in cattle, including encephalitis. On the other hand, rabies virus is the agent most frequently identified as cause of viral encephalitis in cattle in Brazil. The aim of this study was to examine the occurrence of BoHV-1 and / or BoHV-5 in bovine brain tissue samples submitted to rabies diagnosis. The search was carried out by virus isolation and nested polymerase chain reaction (PCR) in brain tissues of cattle submitted to rabies diagnosis in the state of Rio Grande do Sul in the period 2009-2010. One hundred and one brain samples from cattle with signs of neurological disease, of which 39 were positive and 62 negative for rabies, were used in this study. At virus isolation, all samples were negative for the presence of infectious herpesviruses after three successive passages in MDBK cells. Of the 101 total samples analyzed, this test revealed that 25.7% (26/101) of cattle were infected with BoHV-1 and 21.8% (22/101) were infected with BoHV-5. Genomes of both types were detected in 29.7% (30/101) samples. With the 39 samples positive for rabies virus, BoHV-1 genome was detected in 23% (9/39) and 15.4% (6/39) were positive for BoHV-5 as well as in 41% (16/39) of these samples, which were positive for both BoHVs. On the other hand, the negative samples for rabies virus, 27.4% (17/62) also were positive for BoHV-1, as well and 25.8% (16/62) were positive for BoHV-5. Genomes of both BoHVs were detected in 22.6% (14/62) of the specimens. These differences were not statistically significant indicating no correlation between the occurrence of rabies and herpesvirus infections in the animals. These results do not imply that the herpesviruses detected, even showing a high incidence, were the causative agents of meningoencephalitis in the samples tested, once it was not possible to isolate virus in its infectious form, however it suggests a latent infection in the animals involved with neurological signs of meningoencephalitis whose primary suspicion was rabies.

This study was focused on vector-pathogen-host interaction. Questing ticks from field were tested for presence of Borrelia burgdorferi s.l. and host DNA. Small rodents were trapped, ticks were collected from them, infestation patterns were estimated regarding the species and stage of ticks and species, sex and body weight of the host. Ticks aquired from hosts were tested for presence of Borrellia burgdorferi s.l. and tick-borne encephalitis virus. Both results from identification of hosts and from detection of pathogens were compared to be able to investigate interactions between host, vector and pathogen.

The aim of this study was to examine the effect of tick infection with tick-borne encephalitis virus on its behaviour and development. The effect of infection on feeding performance, metamorphosis, locomotion or phototaxis was analysed. Despite the fact that we were not able to demonstrate any significant effect of infection on tick behaviour, the obtained results contribute to understanding transmission dynamics of the virus during tick life cycle including co-feeding and transovarial transmission.

The presented thesis is focused on a comprehensive evaluation of situation related to the Tick-borne encephalitis on the territory of the Liberec Region in years 2001 2016. One of the main aims was to process the results of a demographic analysis of the Tick-borne encephalitis cases. These cases have been reported for 16 years within the so-called Tick-borne encephalitis surveillance to the system called EPIDAT by the Regional public health authority of the Liberec Region. Thank these analyzed data it was possible to create maps which illustrate particular focuses of the Tick-borne encephalitis in the Liberec Region and which graphically draw attention to locations threatened with infection. The KAP questionnaire study was held in a chosen Tick-borne encephalitis focus in order to improve a prevention of the Tick-borne encephalitis in the Region. This study was focused on knowledge, attitudes, opinions and practice of locals related to an occurrence of ticks and also of the Tick-borne encephalitis transmitted by them. Together with data gathering was held the distribution of the informative leaflets and also edification in this issue by a discussion about respondent´s questions. The collected data were processed by a descriptive method and also were detailed to a statistics testing. Due to an amount of analyzed information, this testing did not bring any statistic important results in most of the questions. The contribution of this thesis is especially the processed data of the Tick-borne encephalitis for those mentioned 16 years. These data could be useful for example for arguments of the public health authorities in case of discussion related to the Tick-borne encephalitis issue. The questionnaire study pointed out a problem which could cause a failure of people prevention. This problem means less of interest in health issues from their side. The question is how to learn population to perceive the risk of this disease when the half of the respondents do not feel threatened by the Tick-borne encephalitis? This fact is not certainly a problem only in the prevention of infectious diseases.