Orientador: Ana Carolina Gomes Jardim / Resumo: Nas últimas décadas, diversos vírus que tinham sua ocorrência limitada a pequenas regiões se espalharam pelo globo, causando epidemias e preocupação entre as autoridades de saúde. Apesar dos inúmeros avanços no tratamento das infecções virais, vários destes vírus ainda não possuem tratamento especifico e eficaz. Adicionalmente, a alta taxa de resistência e o surgimento de novas mutações, torna a busca por novos antivirais desafiadora e de extrema importância. Desta forma, o presente trabalho teve como objetivo investigar a atividade antiviral de compostos de origem natural ou sintética contra os vírus da Chikungunya (CHIKV) e Enterovirus A71 (EV-A71). Contra o CHIKV, 48.750 compostos sintéticos foram inicialmente avaliados in silico por docking molecular, dos quais 12 compostos demonstraram apresentar interação com a região de ligação a ADP-ribose do dominío macro da proteína viral não estrutural 3 (nsP3), e foram selecionados para ensaios in vitro. Ensaios de viabilidade celular foram realizados para determinar a máxima concentração não tóxica de cada composto, que foi utilizada nos ensaios anti-CHIKV em células de hepatocarcimona humano Huh-7, transfectadas com os replicons subgenômicos do CHIKV. Os resultados demonstraram que os compostos C5 e C13 na concentração de 20 µM inibiram 53 e 76% da replicação do CHIKV em células Huh-7, respectivamente. Contra o EV-A71, 6 proteínas isoladas da peçonha de serpentes foram testadas em concentrações não tóxicas em células Vero infect... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last decades, several viruses that had their occurrence limited to small regions spread through the globe, causing epidemics and concern among health authorities. Despite the numerous advances in the treatment of viral infections, several of these viruses have no specific and effective treatment yet. In addition, the high rate of resistance and the emergence of new mutations, makes the search for new antivirals challenging and extremely important. The present work aimed to investigate the antiviral activity of compounds from natural or synthetic origin against Chikungunya virus (CHIKV) and Enterovirus A71 (EV-A71). Against CHIKV, 48,750 synthetic compounds were initially evaluated in silico by molecular docking, of which 12 compounds demonstrated to be interacting with the ADP-ribose binding region of viral non- structural protein 3 (nsP3) macro domain and were selected for in vitro assays. Cell viability assays were performed to determine the maximum non-toxic concentration of each compound and used in anti-CHIKV assays in human hepatocarcinoma cells (Huh-7) transiently transfected with the CHIKV subgenomics replicons. The results demonstrated that the C5 and C13 compounds at 20 µM inhibited 53 and 76% of CHIKV replication in Huh-7 cells, respectively. Against EV-A71, 6 proteins isolated from snake venom were tested at non-toxic concentrations in infected Vero cells, and the virucidal, protective and anti-EV-A71 replication activity was evaluated. From the tested toxi... (Complete abstract click electronic access below) / Doutor

Enterovirus D68 (EV-D68) virus has become more prevalent over the last 15 to 20 years. EV-D68 attacks the respiratory system and can cause severe disease in individuals who have underlying respiratory problems. There have also been reports of individuals with EV-D68 showing signs of neurological system problems and acute flaccid paralysis. Because of the increase in patients with EV-D68 and also the potential for neurological disease, an animal model is needed to study the disease and to evaluate experimental therapies for EV-D68 infection. To develop the animal model, 4-week old AG129 mice that lack alpha and beta interferon receptors, making them immunosuppressed, were used. The mice were infected with EV-D68 by the intranasal route to allow infection of the lungs. On day-3 post-infection the mice were euthanized and lungs were removed and homogenized for collection of virus. The newly collected virus was then used to infect another set of mice. This procedure was repeated 30 times. As passage number increased so did the amount of virus that was collected from the lungs of mice. The virus titer increased 320-fold between mouse passage 0 to 30. At the end of the thirtieth passage, multiple tissues (lungs, liver, kidney, spleen, blood, brain, spinal cord and leg muscle) were collected from infected mice over several days and titered to demonstrate how quickly the virus spread to various tissues within the mouse. The virus replicated most rapidly in the lungs and remained in the lungs longer than the other tissues evaluated. However, large quantities of virus were found in all tissues evaluated. Finally, several experimental antiviral compounds were evaluated: rupintrivir, pleconaril, ribavirin, enviroxime and guanidine, all of which showed therapeutic potential in cell culture. In the animal model rupintrivir, pleconaril, ribavirin and enviroxime did not show any therapeutic effect. Only guanidine reduced the amount of virus that was found in the lungs as well as in whole blood.

Enteroviruses are associated with various diseases accompanied by rare but severe complications. In recent years, outbreaks of enterovirus D68 and enterovirus A71 associated with severe respiratory infections and neurological complications have been reported worldwide. Since information on molecular epidemiology in respiratory samples is still limited, the genetic diversity of enteroviruses was retrospectively analysed over a 4-year period (2013–2016) in respiratory samples from paediatric patients. Partial viral major capsid protein gene (VP1) sequences were determined for genotyping. Enteroviruses were detected in 255 (6.1%) of 4187 specimens. Phylogenetic analyses of 233 (91.4%) strains revealed 25 different genotypes distributed to Enterovirus A (39.1%), Enterovirus B (34.3%), and Enterovirus D (26.6%). The most frequently detected genotypes were enterovirus D68 (26.6%), coxsackievirus A6 (15.9%), and enterovirus A71 (7.3%). Enterovirus D68 detections were associated with lower respiratory tract infections and increased oxygen demand. Meningitis/encephalitis and other neurological symptoms were related to enterovirus A71, while coxsackievirus A6 was associated with upper respiratory diseases. Prematurity turned out as a potential risk factor for increased oxygen demand during enterovirus infections. The detailed analysis of epidemiological and clinical data contributes to the non-polio enterovirus surveillance in Europe and showed high and rapidly changing genetic diversity of circulating enteroviruses, including different enterovirus D68 variants.

BACKGROUND:In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.RESULTS:Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected.CONCLUSIONS:This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.

Enterovírus (HEV), herpesvírus 1 e 2 (HHV-1 e HHV-2) e adenovírus (HAdV) são importantes agentes de infecções do SNC. Neste trabalho, técnicas moleculares foram aplicadas para a detecção destes vírus em quadros de infecção do SNC. Amostras de líquor foram colhidas de pacientes atendidos no HU-USP entre agosto e novembro/2010 e fevereiro/2012 a janeiro/2013. Através da Nested-PCR HEV foram detectados em 9,8% das amostras, HAdV em 2,5% e HHV-1 e 2 em 1,1%, além de 3 casos de coinfecção, 2 entre HEV e HHV, e 1 entre HEV e HAdV. O material genético viral foi extraído através dos métodos Qiaamp DNA Blood (Qiagen®) e MagMAXTM Viral RNA Isolation (Ambiom), e este último pareceu mais adequado à aplicação na rotina clínica. A análise quimiocitológica do líquor mostrou-se importante no direcionamento da conduta clínica, mas a detecção do vírus é fundamental para a conclusão do diagnóstico. A PCR em tempo real, cuja padronização foi iniciada neste trabalho, consiste em importante ferramenta para a utilização futura no diagnóstico complementar das infecções virais do SNC. / Enteroviruses (HEV), herpesviruses 1 and 2 (HHV-1 and HHV-2) and adenoviruses (HAdV) are important causative agents of infections of the CNS. In this study, molecular techniques were applied to the detection of these viruses. CSF samples were collected from patients treated at the University Hospital of USP, between August and November, 2010, and February 2012 and January 2013. By the Nested-PCR reaction, HEV were detected in 9.8% of the samples, HAdV in 2.5% and HHV-1 and 2 in 1.1%. There were 3 cases of coinfection: 2 with HEV and HHV and other with HEV and HAdV. The viral genetic materials were extracted by QIAamp DNA Blood kit (Qiagen®) and MagMAXTM Viral RNA Isolation (Ambiom), and the second one showed to be more suitable for the application in clinical diagnosis. The CSF chemocytologic analysis proved to be important in directing the clinical conduct, but the detection of viruses is essential for the diagnosis. The real time PCR, which standardization was initiated in this work, will be an important tool for complementary diagnosis of viral infections of the CNS.

Diversos agentes virais são causadores de meningites e meningoencefalites. Neste estudo, técnicas moleculares foram utilizadas para detecção de HEV, HHV e HAdV em amostras de líquor colhidas de janeiro de 2005 a março de 2007. Dos três métodos de extração de DNA e RNA testados, o kit DNA Qiablood Qiagen® se mostrou o mais sensível e específico. A nested PCR detectou HEV em 28% das amostras, HSV em 4%, HHV-3 em 1%; HHV-4 em 0,3%, HHV-5 em 0,3%, HHV-6 em 0,7% e HAdV em 13%. Através da PCR em tempo real os HEV foram detectados em 23,3% e HSV em 5,1%. Por neutralização, somente duas amostras foram sorotipadas (Echovirus 6 e Coxsackievirus B). Os HEV detectados foram então seqüenciados para a determinação do sorotipo. Os sorotipos Echovirus 18 (53%) e Coxsackievirus B5 (26%) foram os mais freqüentes. As técnicas de biologia molecular aplicadas na detecção de HEV, HHV e HAdV no líquor trazem grandes vantagens ao diagnóstico de doenças do SNC graças à rapidez no diagnóstico, alta sensibilidade e especificidade. / Several viruses are etiological agents of meningitis and meningoencephalitis. In this study, molecular techniques were used for the detection of HEV, HHV and HAdV in liquor samples, collected from January 2005 to March 2007. Among the three methods tested for the extraction of DNA and RNA, the DNA Qiablood kit - Qiagen® was the most sensible and specific. HEV (28%), HSV (4%), HHV-3 (1%), HHV-4 (0.3%), HHV-5 (0.3%), HHV-6 (0.7%) and HAdV (13%) were detected by Nested PCR in the samples. By real time PCR, HEV were detected in 23.3% and HSV in 5.1%. Only two HEV could be serotyped by neutralization (Echovirus 6 and Coxsackievirus B). All detected HEV were then sequenced to determine the serotype. The serotypes echovirus 18 (53%) and Coxsackievirus B5 (26%) were the most frequent. Molecular biology techniques applied in the detection of HEV, HAdV and HHV in CSF bring major benefits to the diagnosis of meningitis thanks to the rapid diagnosis, high sensibility and specificity.

Les entérovirus humains (EV) représentent la première cause des méningites aseptiques chez l'enfant. Le diagnostic de certitude repose sur la détection génomique de l'entérovirus par RT-PCR dans des échantillons de liquide céphalorachidien (LCR) est recommandée pour le diagnostic. La fièvre sans point d'appel et les maladies de type "sepsis" sont également des affections fréquentes chez les nourrissons (0 à 2 ans), qui peuvent être la conséquence d'une infection virale, en particulier à EV. Actuellement, le diagnostic des EV dans le sang est rarement établi dans la pratique courante.Les données concernant 1) l’existence d’une réplication de l’EV dans les leucocytes sanguins, 2) le niveau de charge virale de l’EV dans le sang et les échantillons de LCR et sa corrélation éventuelle avec l’intensité du processus inflammatoire réactionnel sont fragmentaires. Dans la première partie de cette thèse, l'objectif était d'évaluer la détection des EV par PCR dans des échantillons de sang de nouveau-nés, de nourrissons et d'enfants hospitalisés pour une fièvre isolée, un sepsis ou un syndrome méningé. Nous avons mené une étude observationnelle prospective multicentrique nationale (étude BLEDI) dans 35 départements de pédiatrie au cours de la période estivo-automnale (augmentation des circulations d'EV) en 2015-2016. Nos résultats ont montré que le taux de détection des EV dans le sang était significativement plus élevé que dans le LCR chez les nouveaux-nés et les nourrissons hospitalisés pour une fièvre isolée, un sepsis ou un syndrome méningé.Ces données ouvrent des perspectives pour un nouvel algorithme de diagnostic des fièvres inexpliquées chez les enfants âgés de moins de 2 ans. Dans le second volet de cette thèse, nous avons étudié de manière prospective la charge virale d'EV dans le sang et dans des échantillons de LCR de patients infectés. Nos résultats ont montré que la charge virale d'EV dans le sang variait selon le groupe d'âge, la présentation clinique et le type d'EV. Trois profils de cinétique d'infection comparant la charge virale dans le sang et le LCR ont été envisagés et sont en cours d'étude. Enfin, le dernier axe était de déterminer s'il existait une réplication de l’EV dans les leucocytes sanguins. Nous avons montré à partir d'échantillons de sang infectés in vitro par EV que la réplication de ces virus variait selon les génotypes d'EV. / Human enteroviruses (EV) are the most frequent cause of paediatric aseptic meningitis. Detection of enterovirus by PCR in cerebrospinal fluid (CSF) specimens is recommended for diagnosis. Fever without source and sepsis-like diseases are frequent affections in infants (0 to 2 years) which can be the consequence of a viral infection in particular to EV. At present, the daily routin diagnosis of EV in the blood is rarely performed. The concerning data 1) existence of EV replication in the blood leukocytes, 2) the EV viral load level in blood and CSF specimens and its possible relation with the intensity of the associated inflammatory process are fragmented. In the first part of this PhD thesis, the aim was to assess detection of enterovirus by PCR in blood specimens of newbrons, infants, and children with fever without source, sepsis-like disease or suspected meningitis. We did a prospective, multicentre, observational study (BLEDI study) at 35 paediatric departements during the seasonal period of increased EV circulation in 2015-2016. Our results showed that detection of EV was significantly higher in the blood than in the CSF from newborns and infants admitted with fever without source or sepsis-like disease. These data open up the perspectives for a new diagnostic algorithm of febrile illness in patients aged 2 years or younger. We also explored prospectively EV viral load in blood and in CSF specimens of infected patients. Our results showed that EV viral load in the blood varied by age group, clinical presentation and EV type. Three profiles of infection kinetics comparing EV viral load in the blood and the CSF have been considered and are under study. Finally, we explored the hypothesis of an EV replication in the blood leukocytes. We showed from blood samples infected in vitro by EV that the replication of these viruses varied by EV genotypes.

Enterovírus (HEV), herpesvírus 1 e 2 (HHV-1 e HHV-2) e adenovírus (HAdV) são importantes agentes de infecções do SNC. Neste trabalho, técnicas moleculares foram aplicadas para a detecção destes vírus em quadros de infecção do SNC. Amostras de líquor foram colhidas de pacientes atendidos no HU-USP entre agosto e novembro/2010 e fevereiro/2012 a janeiro/2013. Através da Nested-PCR HEV foram detectados em 9,8% das amostras, HAdV em 2,5% e HHV-1 e 2 em 1,1%, além de 3 casos de coinfecção, 2 entre HEV e HHV, e 1 entre HEV e HAdV. O material genético viral foi extraído através dos métodos Qiaamp DNA Blood (Qiagen®) e MagMAXTM Viral RNA Isolation (Ambiom), e este último pareceu mais adequado à aplicação na rotina clínica. A análise quimiocitológica do líquor mostrou-se importante no direcionamento da conduta clínica, mas a detecção do vírus é fundamental para a conclusão do diagnóstico. A PCR em tempo real, cuja padronização foi iniciada neste trabalho, consiste em importante ferramenta para a utilização futura no diagnóstico complementar das infecções virais do SNC. / Enteroviruses (HEV), herpesviruses 1 and 2 (HHV-1 and HHV-2) and adenoviruses (HAdV) are important causative agents of infections of the CNS. In this study, molecular techniques were applied to the detection of these viruses. CSF samples were collected from patients treated at the University Hospital of USP, between August and November, 2010, and February 2012 and January 2013. By the Nested-PCR reaction, HEV were detected in 9.8% of the samples, HAdV in 2.5% and HHV-1 and 2 in 1.1%. There were 3 cases of coinfection: 2 with HEV and HHV and other with HEV and HAdV. The viral genetic materials were extracted by QIAamp DNA Blood kit (Qiagen®) and MagMAXTM Viral RNA Isolation (Ambiom), and the second one showed to be more suitable for the application in clinical diagnosis. The CSF chemocytologic analysis proved to be important in directing the clinical conduct, but the detection of viruses is essential for the diagnosis. The real time PCR, which standardization was initiated in this work, will be an important tool for complementary diagnosis of viral infections of the CNS.

Title from PDF of title page. / Document formatted into pages; contains 86 pages. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. / ABSTRACT: The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. / ABSTRACT: The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.