Orientador: Tereza Cristina Cardoso da Silva / Coorientador: Wagner Luis Ferreira / Banca: Vera Cláudia Lorenzetti Magalhães Curci / Banca: Flávia de Rezende Eugênio / Resumo: 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+ / Abstract: 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 + / Mestre

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-06-15T12:04:27Z No. of bitstreams: 1 Salome Goncalves Simoes.pdf: 981710 bytes, checksum: bba8ce6b1296a67fe6adae0958a19b21 (MD5) / Made available in DSpace on 2016-06-15T12:04:27Z (GMT). No. of bitstreams: 1 Salome Goncalves Simoes.pdf: 981710 bytes, checksum: bba8ce6b1296a67fe6adae0958a19b21 (MD5) Previous issue date: 2015-09-11 / The knowledge of diseases that affect cattle is extremely important, the neurological manifestation of disease are frequent and caused by various agents , the Bovine herpesvirus type 5 ( BoHV - 5 ) is a virus that causes neuropathy in cattle , latency in the central nervous system ( SNC) and clinical manifestation in periods of stress. The object of the study was to research the DNA presence of Herpesvirus Type 5 in cattle through the Real Time Polymerase Chain Reaction (qPCR) based upon the utilization of two protocols and analyze the histopathological findings in samples of the cattle central nervous system. Forty-eight (48) cattle samples of the central nervous system samples were obtained comprised of brain, cerebellum, brain stem and spinal cord derived from three slaughterhouses located in the cities of Ouricuri, Bodocó and Exú, Araripe Wilderness in the state of Pernambuco. Under molecular analysis the efficiency of the protocols was compared using the following primers. With the primers used in protocol 1 there was obtained 29.16% (14/48) of the positive samples, however, the utilized primers in protocol 2 resulted in well defined peaks with regard to positivity of the positive controls and all of the samples were negative toward the qPCR. Based on histopathology analysis of central nervous system samples from Ouricuri-PE the following lesions became evident: lymphocytic infiltrate 90% (18/20), gliosis 80% (16/20), neuronophagia 80% (16/20), edema 70% (14/20), macrophages 65% (13/20), congestion 65% (13/20), demyelination 45% (9/20), bleeding 35% (7/20), necrosis 15% (3/20), gitter cell 5% (1/20), spongiosis 5% (1/20) and perivascular cuff 5% (1/20). As a result of the diversity of pathogens and similarity of neurological diseases in cattle the use of primers that present few crossed reactions (dimers) is fundamental for the efficiency and success of the qPCR against BoHV – 5. / O conhecimento das enfermidades que acometem bovinos é de extrema importância, as doenças de manifestação neurológica são frequentes e causadas por vários agentes, o Herpesvírus bovino tipo 5 ( BoHV – 5 ) é um vírus que provoca neuropatia em bovinos, latência no sistema nervoso central (SNC) e manifestação clínica em períodos de estresse. Objetivou-se com este estudo pesquisar a ocorrência da infecção pelo BoHV-5 em bovinos pela reação em cadeia da polimerase em tempo real (qPCR) com base na utilização de dois protocolos e analisar os achados histopatológicos de amostras de sistema nervoso central de bovinos. Foram obtidas 48 amostras de SNC de bovinos, constituídas por encéfalo, cerebelo, tronco encefálico e medula, procedentes de três matadouros localizados nas cidades de Ouricuri, Bodocó e Exu, Sertão do Araripe, Pernambuco. Na análise molecular foram utilizados dois protocolos com diferentes primers. Com os iniciadores utilizados no protocolo 1 obteve-se 29,16% (14/48) das amostras positivas, no entanto, os iniciadores utilizados no protocolo 2, resultaram em picos bem definidos no controle positivo e todas as amostras resultaram negativas pela qPCR. Com base na análise histopatológica do SNC das amostras provenientes de bovinos do matadouro de Ouricuri-PE evidenciou-se as seguintes lesões: infiltrado linfocitário 90% (18/20), gliose 80% (16/20), neuronofagia 80% (16/20), edema 70% (14/20), macrófagos 65% (13/20), congestão 65% (13/20), desmielinização 45% (9/20), hemorragias 35% (7/20), necrose 15% (3/20), células gitter 5% (1/20), espongiose 5% (1/20) e manguito perivascular 5% (1/20). Em decorrência da diversidade de patógenos e similaridade das enfermidades neurológicas em bovinos, o uso de primers que apresentem poucas reações cruzadas (dímeros) é fundamental para a eficiência e o êxito do diagnóstico na qPCR frente ao BoHV-5.

The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.

Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.

Encephalitis is a matter for serious public health concern because of the high morbidity and mortality associated with many cases. Epidemiological studies have shown that viral encephalitis (VE) is more common than the sum of encephalitis caused by all other pathogens. However, more than 95% of cases have no known cause. Thus, there is a significant need to develop a sensitive method for the diagnosis of these unknown cases. Previous sequence independent amplification (SIA) assays have proved successful in detecting new viruses in many biological samples but not in CSF samples. This may be due to the relatively low sensitivity of most available methods as CSF usually contains lower concentrations of pathogen than most other samples. A known problem with these types of assays is the annealing of the random primers to human DNA which facilitates preferential amplification of background human DNA. Thus, large scale sequencing is usually required to detect a virus, which in turn reduces the detection sensitivity to more than 1000 viral copies/µl, a CSF concentration that is rarely seen in cases of VE.This project was designed to develop a highly sensitive SIA assay for novel nucleic acid identification that could be used in testing CSF samples obtained from patients with neurological diseases of unknown cause. The study started with evaluation of two existing SIA assays commonly used for virus discovery; whole genome amplification (WGA) and random PCR (r-PCR). Sequential modification and adaptation of these methods was carried out to increase their sensitivity. Ultimately, a novel primer (Sa primer) that showed no binding to most human DNA sequences in GenBank was designed and synthesised. Its 3' end was tagged with 6 and 7 random nucleotides generating 2 r-primers; Sa-6 and Sa-7. The sensitivity of the r-primers was checked in a novel assay developed during this project and named Sa-SIA using known concentrations of HCMV and HSV-1. CSF samples from Malawian children were then tested using the developed assay. Results showed that adaptation of the existing WGA and r-PCR assays allowed detection of up to 1300 viral copies/µl. When the novel primers developed in this project were used in a random PCR assay (Sa-r-PCR), it was found that using Sa-6 primer 130, 13, and 1.3 HCMV copies/µl could be detected with 100, 60, and 50% efficiency respectively. When using Sa-7 primer, the same concentrations of virus were detected with 100, 42, and 28.6% efficiency. DNase-1 treatment of the samples pre-extraction resulted in an improvement in viral detection sensitivity in samples with a high background of host DNA. Starting with template concentrations of 11000, 110, 11, and 1.1 HSV-1 copies/µl, viral detection efficiency was increased from 33.3, 10, 0, and 0% to 92, 55.6, 16.7, and 0% respectively when pre-extraction DNase-1 treatment preceded Sa-r-PCR using Sa-6 primer. The final developed assay (Sa-SIA) consisted of centrifugation, DNase-1 treatment, DNA extraction, Sa-r-PCR using Sa-6 and Sa primers, gel electrophoresis, band excision, cloning, small scale sequencing (sequencing of ≤ 20 positive clones from one constructed DNA library), and bioinformatics. It had a detection sensitivity of 1.3-11 viral copies/µl. When this assay was applied to stored CSF samples, one 448bp sequence was identified which gave 96% coverage with 81% identity to Torque teno midi virus-1 and 93% coverage with 81% identity to small anellovirus-2. A 236bp sequence from another CSF sample showed 66% coverage with 97% homology to an unclassified sequence previously identified in a viral genomic survey of stool sample in an earlier published study. In conclusion, the standardised method had been shown to detect 1.3 to 11 viral copies/µl of two viruses; HCMV and HSV-1. The detection of these viruses was achieved with only small scale sequencing. Application of this method to CSF samples has shown promising results. However, this method could be followed by more advanced post amplification analyses such as next generation sequencing.

Rasmussen's encephalitis is a rare neurological disease first described in 1958 that is characterized by medico-refractory seizures, focal unilateral cerebral inflammation, and deficits such as hemiparesis. While we still do not have a full understanding of this disease, proposed theories behind its etiology include auto-immune manifestations, immune attack by T cells, and malfunctional alterations in genetic expression. It is classically considered a rare childhood malady with a median age of onset of six years, and cases in adolescents and adults are even rarer, representing up to 10% of all cases to date. In this report, we would like to share a rare case of Rasmussen's encephalitis that occurred in an adolescent. Our 17-year-old male patient presented with signs and symptoms beginning at age 14 and was initially diagnosed with cerebral palsy only to later present with additional symptoms and characteristic EEG and MRI findings that ultimately led to a diagnosis of Rasmussen's encephalitis. Thus, with this case report, our intent is twofold: to shed light on an atypical presentation of an already rare disease, even rarer in adolescents and adults, and to underscore the importance of keeping a broad differential when it comes to evaluating a patient with seizures.

No description available.

Tick-borne encephalitis is a vector-borne zoonotic disease with more than 12,000 annual clinical reported cases globally (WHO, 2021). Tick-borne encephalitis is caused by the flavivirus and transferred by Ixodes ricinus from roe deer to human and affects the central Nervous system. Climate change also increases tick-borne encephalitis incidences in Sweden and fluctuated considerably from year to year. A quantitative study design with secondary data was conducted to analyze the spatial and temporal pattern of Tick-borne encephalitis in Sweden from 1986-2020. The distribution of Tick-borne encephalitis within age and sex, along with other factors were also analyzed. The results showed that Tick-borne encephalitis with passing each year spread across the country. Male and age group, 50-59, have more incidence of Tick-borne encephalitis. Incidents were associated with climate conditions such as temperature and precipitation, which provided a favorable environment for Ixodes ricinus for its lifecycle activities, host searching, and disease transmission. Roe deer population, other vertebrates abundance, vaccination, population interest, economy, and land change are the critical factors that facilitate the disease incidence or control. People who visit forests for hunting, trekking, leisure, and professional activities without proper immunity and preventive measures are at risk to infect with Tick-borne encephalitis. The theory One health approach showed suitable performance for the control of this vulnerable climate zoonotic disease.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus is the causative agent of Japanese encephalitis (JE). The disease affects mostly children and around 30000– 50000 cases of JE and up to 15000 deaths are reported annually. No anti-viral drugs have been discovered against JE so far, but advances in our knowledge of the molecular biology of flaviviruses is propelling flaviviral drug research at an expeditious pace. Since JEV has a small genome which encodes for only ten proteins, there is dearth of potential drug targets. Researchers are now focusing on cellular interactomes, a complex and dynamic molecular biosystem which identifies host proteins which interact with either viral proteins or viral genomes, leading to the generation of an astronomical number of potential drug targets involving common cellular pathways that are required for the life cycle of different viruses. Such studies can pave way for the development of ‘broad-spectrum’, ‘silver-bullet’ anti-viral drugs for the treatment of multiple viral diseases. The cellular interactomes can be studied by Genomics tools such as microarray. Systematic profiling of genes involved in virus infection by RNAi, transcriptome sequencing, microRNA profiling and yeast two-hybrid system has allowed us to assess global gene expression changes providing an unprecedented view on the host-side of the virus–host interactions. Advent of these tools has led to identification of plethora anti-viral genes. For example, over expression of IFN-stimulated gene15 (ISG15) results in inhibition of JEV leading to significant reduction of viral titers. Chemokine profiling of JEV-infected cells by microarray can provide possible therapeutic modalities that can mitigate the morbidity associated with JEV infection. Functional classification of interferon-stimulated genes (ISG) identified using innovative methods have been the stepping stone for identification of many anti-viral genes, among them are few Broadly acting effectors like IRF1, C6orf150, HPSE, RIG-I, MDA5 and IFITM3 and some more targeted antiviral specific like DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT, OASL, RTP4, TREX1 and UNC84B. In this study, we have identified a B16F10 murine melanoma cell line that is resistant to JEV infection. DNA microarray analysis of JEV-susceptible and resistant B16F10 cell lines gave us interesting insights into JEV-induced host gene expression changes. Real time PCR validation of microarray data indicates that a number of virus and interferon inducible genes are expressed constitutively at high levels in this JEV-resistant cell line. Further, several of the mouse genes induced by JEV in B16F10 cell line were also upregulated in JEV-infected mouse brain. To understand the significance of these host gene expression changes, we attempted to generate stable murine cell lines constitutively expressing select JEV-inducible genes and study the JEV infection pattern in these cell lines. One of the JEV-inducible genes encoding thymidylate kinase (Tyki), a mitochondrial protein involved in the sysnthesis of nucleoside diphosphates, when overexpressed in NIH3T3 cells confers resistance to JEV infection as evident from reduced JEV-induced cytopathic effects and significant reduction in viral titer. Since TYKI has two distinct domains: the N-terminal domain with unknown function and the C-terminal domain with the nucleoside monophosphate kinase function, suggest that TYKI may be a bifunctional protein with other biological functions in addition to its UMP-CMP kinase activity. In order to examine whether N-terminal domain is responsible for antiviral activity of the protein, a stable cell line constitutively expressing N-terminal domain of gene was made, but the overexpression of N-terminal domain didn't confer any antiviral immunity. Thus signifying importance of kinase activity in confering antiviral immunity. Our studies indicate for the first time that Tyki may have a role in host resistance to JEV and understanding the mechanism of action Tyki may pave way for novel anti-JEV therapy. Stable cell lines constitutively expressing other JEV-inducible genes (Atf3, Gimap3, Rtp4, Glipr2, Tmem140 and Garg49) couldn't be generated. Therefore, to study the effect of overexpression of these genes on JEV infection, expression vectors encoding these genes were transfected individually to human 293T cells by nucleofection, then infected with JEV and viral titres were examined by plaque assay. Nucleofection was opted as a method of choice since it is the only non-viral method, which transfects DNA directly enter the nucleus. In contrast, other commonly used non-viral transfection methods rely on cell division for the transfer of DNA into the nucleus. Nucleofection of vectors encoding different JEV-inducible genes followed by JEV infection and assay of viral titer led to identification of one more anti-viral gene and three pro-viral genes. Garg49, an interferon and JEV inducible mitochondrial gene was identified as antiviral gene. Further studies led to the identification of GARG49 as a mitochondrial protein. Three genes, Atf3, encoding a cAMP responsive element binding protein family transcription factor, Glipr2, encoding a Glioma related pathogenesis protein and Gimap3, encoding an outer mitochondrial membrane GTPase were identified as pro viral genes. Overexpression of Tmem140, encoding a transmembrane protein and Rtp4, encoding a golgi chaperone did not significantly affect JEV titer. Conclusions: . A JEV-resistant B16F10 murine melanoma cell line was identified and several JEV-inducible genes were found to be expressed constitutively at high levels in this cell line. .We demonstrate for the first time that Tyki/Ump-Cmpk2 encoding a mitochondrial nucleoside monophosphate kinase has an anti-JEV function and the C-terminal domain is essential for anti-viral activity. .Garg49/Ifit3 encodes an interferon and JEV-inducible mitochondrial protein and it has an anti-JEV function. . Activating transcription factor 3 (ATF3), GTPase, IMAP family member 3 (GIMAP3) and GLI pathogenesis-related 2 (GLIPR2) are pro-viral proteins which facilitate virus multiplication resulting in enhanced JEV titer.

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.