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.

Os esgotos domésticos contêm uma alta quantidade de bactérias e outros organismos patogênicos e não patogênicos que são descarregados em corpos hídricos. A contaminação da água, pela presença de bactérias e vírus patogênicos, traz conseqüências indesejáveis, principalmente de saúde pública. O objetivo deste trabalho foi avaliar a possibilidade de emprego da análise de colifagos, como indicador viral. Para isso, amostras de esgoto bruto, efluente tratado por reator UASB e efluente de lodo ativado, da Estação de Tratamento de Esgotos São João Navegantes, Porto Alegre, foram coletadas durante o período de junho/2006 a junho/2007. As análises físicas, químicas e microbiológicas, DBO5, turbidez, sólidos totais, quantificação de coliformes totais, fecais e de colifagos foram realizadas em 99 amostras, conforme APHA, 2005. Na determinação da presença de adenovírus e enterovírus foram testadas 36 amostras, utilizando-se a técnica de concentração por filtração-eluição e ultracentrifugação. Posteriormente, purificação e avaliação da infectividade. Por fim, as amostras foram submetidas a teste de nested PCR e RT-PCR, a fim de verificar a presença de adenovírus e enterovírus, respectivamente. Nas 36 amostras testadas, a presença de enterovírus foi confirmada em apenas 5,56% das amostras e, adenovírus, em 16,67% das amostras. Não foram encontradas correlações significativas entre colifagos e bactérias coliformes totais e fecais, bem como entre colifagos e os parâmetros físicos e químicos. Desta independência encontrada entre vírus e bactérias se destaca a necessidade de monitoramento conjunto de ambos os microrganismos. Verificou-se uma similaridade em relação à sazonalidade referente ao comportamento de colifagos e o descrito na literatura referente a vírus entéricos. Observou-se, também, a baixa eficiência do tratamento para remoção de microrganismos, ressaltando-se a importância de realização de um tratamento terciário para assegurar a qualidade microbiológica do efluente. As relações entre colifagos e vírus entéricos não foram possíveis de serem analisadas, devido aos dados inconclusivos obtidos no presente trabalho. / Domestic sewage contains a high amount of bacteria and other pathogens and non pathogens that are discharged into water bodies. Contamination of water by the presence of pathogenic bacteria and viruses, brings undesirable consequences, especially for public health. The objective of this work was to evaluate the possibility of using analysis of coliphages as viral indicator. Samples of raw sewage, treated effluent by UASB reactor and by activated sludge, from Wastewater Treatment Systems São João Navegantes, Porto Alegre, were collected during the period of june/2006 to june/2007. Physical, chemical and microbiological analyzes, namely BOD5, turbidity, total solids, quantification of total coliforms, fecal coliforms and coliphages were performed in 99 samples, following the techniques described in APHA 2005. The presence of enteric viruses was tested on 36 samples. The technique of concentration by filtration-elution and ultra-centrifugation was used for this purpose. Subsequently, purification and infectivity test was performed. The samples were tested to nested PCR and RT-PCR to verify the presence of adenovirus and enterovirus, respectively. In 36 samples tested, the presence of enteroviruses was confirmed in only 5.56% of the samples, and adenovirus, in 16.67% of the samples. No significant correlation was found among coliform bacteria and coliphages, and also between the presence of coliphages and physical and chemical parameters. The independence found between viruses and bacteria indicates the need for joint monitoring of both microrganisms. The behavior of coliphages was observed to follow the path described in the literature about enteric viruses in relation to seasonality. There was observed a low efficiency in terms of microorganisms removal, emphasizing the need for a tertiary treatment to ensure the microbiological quality of the effluents tested. Relations between coliphages and enteric viruses were not able to be analyzed, due to inconclusive data obtained in this work.

Os esgotos domésticos contêm uma alta quantidade de bactérias e outros organismos patogênicos e não patogênicos que são descarregados em corpos hídricos. A contaminação da água, pela presença de bactérias e vírus patogênicos, traz conseqüências indesejáveis, principalmente de saúde pública. O objetivo deste trabalho foi avaliar a possibilidade de emprego da análise de colifagos, como indicador viral. Para isso, amostras de esgoto bruto, efluente tratado por reator UASB e efluente de lodo ativado, da Estação de Tratamento de Esgotos São João Navegantes, Porto Alegre, foram coletadas durante o período de junho/2006 a junho/2007. As análises físicas, químicas e microbiológicas, DBO5, turbidez, sólidos totais, quantificação de coliformes totais, fecais e de colifagos foram realizadas em 99 amostras, conforme APHA, 2005. Na determinação da presença de adenovírus e enterovírus foram testadas 36 amostras, utilizando-se a técnica de concentração por filtração-eluição e ultracentrifugação. Posteriormente, purificação e avaliação da infectividade. Por fim, as amostras foram submetidas a teste de nested PCR e RT-PCR, a fim de verificar a presença de adenovírus e enterovírus, respectivamente. Nas 36 amostras testadas, a presença de enterovírus foi confirmada em apenas 5,56% das amostras e, adenovírus, em 16,67% das amostras. Não foram encontradas correlações significativas entre colifagos e bactérias coliformes totais e fecais, bem como entre colifagos e os parâmetros físicos e químicos. Desta independência encontrada entre vírus e bactérias se destaca a necessidade de monitoramento conjunto de ambos os microrganismos. Verificou-se uma similaridade em relação à sazonalidade referente ao comportamento de colifagos e o descrito na literatura referente a vírus entéricos. Observou-se, também, a baixa eficiência do tratamento para remoção de microrganismos, ressaltando-se a importância de realização de um tratamento terciário para assegurar a qualidade microbiológica do efluente. As relações entre colifagos e vírus entéricos não foram possíveis de serem analisadas, devido aos dados inconclusivos obtidos no presente trabalho. / Domestic sewage contains a high amount of bacteria and other pathogens and non pathogens that are discharged into water bodies. Contamination of water by the presence of pathogenic bacteria and viruses, brings undesirable consequences, especially for public health. The objective of this work was to evaluate the possibility of using analysis of coliphages as viral indicator. Samples of raw sewage, treated effluent by UASB reactor and by activated sludge, from Wastewater Treatment Systems São João Navegantes, Porto Alegre, were collected during the period of june/2006 to june/2007. Physical, chemical and microbiological analyzes, namely BOD5, turbidity, total solids, quantification of total coliforms, fecal coliforms and coliphages were performed in 99 samples, following the techniques described in APHA 2005. The presence of enteric viruses was tested on 36 samples. The technique of concentration by filtration-elution and ultra-centrifugation was used for this purpose. Subsequently, purification and infectivity test was performed. The samples were tested to nested PCR and RT-PCR to verify the presence of adenovirus and enterovirus, respectively. In 36 samples tested, the presence of enteroviruses was confirmed in only 5.56% of the samples, and adenovirus, in 16.67% of the samples. No significant correlation was found among coliform bacteria and coliphages, and also between the presence of coliphages and physical and chemical parameters. The independence found between viruses and bacteria indicates the need for joint monitoring of both microrganisms. The behavior of coliphages was observed to follow the path described in the literature about enteric viruses in relation to seasonality. There was observed a low efficiency in terms of microorganisms removal, emphasizing the need for a tertiary treatment to ensure the microbiological quality of the effluents tested. Relations between coliphages and enteric viruses were not able to be analyzed, due to inconclusive data obtained in this work.