Dissertations / Theses on the topic 'Enteroviru'

Human enterovirus 71 (HEV71) is a member of the Human Enterovirus A species within the Family Picornaviridae. Since 1997, HEV71 has emerged as a major cause of epidemics of hand, foot and mouth disease (HFMD) associated with severe neurological disease in the Asia-Pacific region. At the present time, little is known about the pathogenesis of acute neurological disease caused by HEV71. The major aim of this study was to generate infectious cDNA clones of HEV71 and use them as tools for investigating the biology of HEV71 and molecular genetics of HEV71 virulence and pathogenesis. Two infectious cDNA clones of HEV71 clinical isolates, 26M (genotype B3) and 6F (genotype C2) were successfully constructed using a low copy number plasmid vector and an appropriate bacterial host. Transfection of cDNA clones or RNA transcripts derived from these clones produced infectious viruses. Phenotypic characterisation of clone-derived viruses (CDV-26M and CDV-6F) was performed, and CDV-26M and CDV-6F were found to have indistinguishable phenotypes compared to their wild type viruses. Strains HEV71-26M and HEV71-6F were found to have distinct cell culture growth phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains a series of chimeric viruses were constructed by exchanging the 5„S untranslated region (5„S UTR), structural protein (P1), and nonstructural protein (P2 and P3) gene regions using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5„S UTR of both strains were compatible but not responsible for the observed phenotypes. Both the P1 and P2-P3 genome regions influence the HEV71 growth phenotype in cell culture, phenotype expression is dependent on specific P1/P2-P3 combinations and is not reciprocal. In the previous study, in order to investigate the pathogenesis of HEV71 infection, a mouse HEV71 model was developed using a mouse-adapted variant of HEV71-26M. Mouse-adapted strain MP-26M caused fore- and/or hindlimb paralysis in mice, whereas HEV71-26M-infected mice did not develop clinical signs of infection at any virus dose or route of inoculation tested. In this study, the molecular basis of mouse adaptation by HEV71 was identified. Nucleotide sequence analysis of HEV71-26M and MP-26M revealed three point mutations in the open reading frame, each resulting in an amino acid substitution in the VP1, VP2 and 2C proteins; no mutations were identified in the untranslated regions of the genome. To determine which of the three amino acid mutations were responsible for the adaptation and virulence of HEV71-26M in mice, recombinant cDNA clones containing one, or a combination of two or three mutations, were constructed. Mouse virulence assays of the mutated viruses clearly demonstrated that a non-conservative amino acid substitution (G710„_E) in the capsid protein VP1 alone was sufficient to confer the mouse virulence phenotype on HEV71. In addition, a mouse oral infection model was established in this study. Oral inoculation with the mouse-adapted HEV71 virus, MP-26M, induced fore-or hindlimb paralysis in newborn mice in an age- and dose-dependent manner. As oral transmission is the natural route of HEV71 infection, this murine HEV71 oral infection model will provide a suitable tool for studying HEV71 pathogenesis, for defining neurological determinants, and for testing vaccine efficacy and immunogenicity in the future.

Human enterovirus 71 (HEV71) is a member of the Human Enterovirus A species within the Family Picornaviridae. Since 1997, HEV71 has emerged as a major cause of epidemics of hand, foot and mouth disease (HFMD) associated with severe neurological disease in the Asia-Pacific region. At the present time, little is known about the pathogenesis of acute neurological disease caused by HEV71. The major aim of this study was to generate infectious cDNA clones of HEV71 and use them as tools for investigating the biology of HEV71 and molecular genetics of HEV71 virulence and pathogenesis. Two infectious cDNA clones of HEV71 clinical isolates, 26M (genotype B3) and 6F (genotype C2) were successfully constructed using a low copy number plasmid vector and an appropriate bacterial host. Transfection of cDNA clones or RNA transcripts derived from these clones produced infectious viruses. Phenotypic characterisation of clone-derived viruses (CDV-26M and CDV-6F) was performed, and CDV-26M and CDV-6F were found to have indistinguishable phenotypes compared to their wild type viruses. Strains HEV71-26M and HEV71-6F were found to have distinct cell culture growth phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains a series of chimeric viruses were constructed by exchanging the 5„S untranslated region (5„S UTR), structural protein (P1), and nonstructural protein (P2 and P3) gene regions using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5„S UTR of both strains were compatible but not responsible for the observed phenotypes. Both the P1 and P2-P3 genome regions influence the HEV71 growth phenotype in cell culture, phenotype expression is dependent on specific P1/P2-P3 combinations and is not reciprocal. In the previous study, in order to investigate the pathogenesis of HEV71 infection, a mouse HEV71 model was developed using a mouse-adapted variant of HEV71-26M. Mouse-adapted strain MP-26M caused fore- and/or hindlimb paralysis in mice, whereas HEV71-26M-infected mice did not develop clinical signs of infection at any virus dose or route of inoculation tested. In this study, the molecular basis of mouse adaptation by HEV71 was identified. Nucleotide sequence analysis of HEV71-26M and MP-26M revealed three point mutations in the open reading frame, each resulting in an amino acid substitution in the VP1, VP2 and 2C proteins; no mutations were identified in the untranslated regions of the genome. To determine which of the three amino acid mutations were responsible for the adaptation and virulence of HEV71-26M in mice, recombinant cDNA clones containing one, or a combination of two or three mutations, were constructed. Mouse virulence assays of the mutated viruses clearly demonstrated that a non-conservative amino acid substitution (G710„_E) in the capsid protein VP1 alone was sufficient to confer the mouse virulence phenotype on HEV71. In addition, a mouse oral infection model was established in this study. Oral inoculation with the mouse-adapted HEV71 virus, MP-26M, induced fore-or hindlimb paralysis in newborn mice in an age- and dose-dependent manner. As oral transmission is the natural route of HEV71 infection, this murine HEV71 oral infection model will provide a suitable tool for studying HEV71 pathogenesis, for defining neurological determinants, and for testing vaccine efficacy and immunogenicity in the future.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
Os enterovírus humanos (HEV) são espécies do gênero Enterovirus, família Picornaviridae. Existem cerca de 120 sorotipos de HEV que são divididos em quatro espécies, designadas de HEV-A a D. Estes agentes infectam anualmente, milhões de pessoas no mundo, resultando em uma grande variedade de quadros clínicos que vão desde infecções inaparentes à febres inespecíficas, resfriado comum, à doenças graves, tais como meningite e poliomielite paralítica. As crianças são mais susceptiveis à infecção. A transmissão ocorre tanto pela via entérica e por via respiratória. O vírus pode ser excretado nas fezes por várias semanas. Este estudo teve como objectivo isolar e identificar os sorotipos de HEVs circulantes, a partir de amostras de fezes de crianças menores de 15 anos de idade, com quadros compatíveis a infecção por esses agentes, no Hospital Geral de Mavalane na Cidade de Maputo, em Moçambique. Neste trabalho, foram utilizadas 178 amostras de fezes obtidas entre novembro de 2011 a fevereiro de 2012. As amostras foram inoculadas em culturas de células e os enterovírus isolados foram identificados através de metódos moleculares, nas amostras negativas foi pesquisado o adenovírus. Das 45 amostras positivas em cultivos celulares, os enterovírus foram isolados e identificados em 26 (14,6 %). A proporção sexo masculino e feminino foi de 1,8: 1. O isolamento dos enterovírus diminuiu à medida que a idade aumentou. O sequenciamento gênomico revelou uma grande diversidade de enterovírus humanos. Entre os 26 enterovírus isolados, o Echovírus 29 foi o agente mais identificado com 19,2 %, seguido pelo Enterovírus 99 (11,5%). Foram identificados também Coxsackievírus A5, Echovírus sorotipos 11, 13 e Enterovírus C com 7,7 % de cada ; Coxsackievírus sorotipos A10, A13, A20, B4 e B6 com 3,85 % cada; Echovírus sorotipos 7, 21 e 25, com 3,85 % cada um, e Poliovírus sorotipos 2 e 3 com 3,85 %, respectivamente. Adenovírus foram isolados em 20 amostras, representando 11,2 % do total (20/178). Duas amostras apresentaram co-infecção enterovírus/adenovírus. Os resultados deste trabalho evidenciam a circulação de uma grande diversidade enterovírus humanos na cidade de Maputo, sendo os echovírus mais frequentes, mas também mostra a circulação de adenovírus humanos. Outros testes laboratoriais seriam necessários, para se relacionar inequivocamente a participação desses agentes virais na etiologia dos quadros clínicos observados.
The human enteroviruses (HEV) are species of the genus Enterovirus, family Picornaviridae. There are about 120 serotypes of HEV divided into four species, designated HEV- A to D. These agents infect millions of people worldwide each year, resulting in a wide variety of clinical conditions ranging from unapparent infection, undifferentiated fevers, and common cold to serious diseases such as meningitis and paralytic poliomyelitis. Children are more susceptible to infection. Transmission occurs by the fecal-oral and respiratory tract. The virus can be excreted in the feces for several weeks. The aim of this study was to isolate and identify human enteroviruses from stool samples of children less than 15 years of age presenting enterovirus compatible symptoms in Mavalane General Hospital in Maputo City, Mozambique. In this study, we used 178 stool samples from children under 15 years of age, obtained from November, 2011 to February, 2012. Samples were inoculated onto cell culture and the enterovirus isolates were identified by molecular methods, the negative samples was screened adenovirus. Twenty-six out of the 45 cell-culture positive samples were constituted by enteroviruses (14.6 %). The ratio between male and female was 1.8:1. Isolation of enterovirus decreases as the age increased. The genomic sequencing showed a diversity of human entrovirus. Among the 26 isolates, Echovirus serotype 29 was the most identified with 19.2 %; Coxsackievirus 99 was identified in 11.5 %, while Coxsackievirus A5, Echovirus serotypes 11, 13 and Enterovirus C, were identified in 7.7 % each. Coxsackievirus serotypes A10, A13, A20, B4 and B6 were present in 3.85 % each; Echovirus serotypes 7, 21 and 25, at 3.85 %, and poliovirus serotypes 2 and 3 in 3.85 %, respectively. Adenoviruses were isolated from 20 samples, 11.2 % (20/178). Two samples showed were co-infected with both enterovirus and adenoviruses. The results of this study showed a great diversity of enterovirus serotypes in the city of Maputo and echovirus was the most prevalent enterovirus found. We also showed the circulation of adenoviruses. However other laboratorial tests would be necessary in order to unequivocally correlate the participation of these viral agents in the etiology of the observed clinical findings.

At the most basic level viruses are biological nano-containers constituted by genetic material enclosed in a protein shell, capsid. A peculiar feature of viruses, both bacterial and some eukaryotic viruses, lies in the high packaging density of the genome in order to fit itself in the small capsid and hence the high internal osmotic pressure. Virus is a relatively stable particle equipped with fascinating mechanical properties of the capsid that are crucial for the virus lifecycle. Viruses have only one purpose: infect a host cell for reproducing themselves in order to generate new viral progeny (Roos et al. 2007). Therefore, the first and foremost consideration arising from the concept of virus reflects its pathogenesis and virulence that can ultimately result in many important infectious diseases such as common cold, influenza, hepatitis, rabies, measles, cancer and AIDS. As a consequence, pathogenic viruses represent a heavy hurdle for the global health and there is a strong need for developing robust strategies such as vaccines or antiviral drugs against virus infections (Baram- Pinto et al. 2010). On the other hand, viruses in the course of evolution have become efficient specialized gene delivery agents. Therefore they represent powerful tools in biomedicine for gene therapy and vaccine purposes (Schaffer et al. 2008). For successful gene therapy and immunization programs, the efficiency and stability of viral vectors are fundamental aspects (Jorio et al. 2006). To address this challenge, in the present research project we have investigated the interaction between viruses and nanomaterials. In the last years materials on the nanoscale for their unique properties have provided a broad range of potential biomedical uses (Verma et al. 2008) and for that reason we decided to explore their application with viruses. More specifically, we have examined three types of sulfonate- functionalized gold nanoparticles (AuNPs), namely, MUS:OT, MUS and MUS:brOT NPs, which are less than 5 nm in size, negatively charged and poorly cytotoxic (Verma et al. 2008). The NPs are coated with self-assembled monolayer (SAM) of thiolated organic molecules and one of the ligand is a sulfonated molecule, MUS (Verma et al. 2008). The MUS ligand itself was tested in our experiments as well. As virus models we focused on human recombinant adenovirus type 5 (Ad), one of the most promising viral vector as vaccine and gene therapy carrier and two picornaviruses of the genus enterovirus, namely, EV1 and CVB3, important human pathogens associated with several infectious diseases (e.g. myocarditis, aseptic meningitis, encephalitis, paralysis)(Kossila et al. 2002)(Marjomäki et al. 2014a). In spite of their medical impact, there are no therapeutic treatments available against picornavirus infections and the only vaccine products are against three types of poliovirus and hepatitis A virus (Merilahti et al. 2012). Two sets of experiments were carried out: (1) Short-term incubation of Ad with nanomaterials for 1 h at 37°C prior transducing HeLa cells or before in vivo administration in zebrafish and mice. The results demonstrated that Ad shortly pre-treated with nanomaterials showed a significant increase in the gene expression in vitro and in vivo The NPs’enhanced adenovirus transduction aims to reduce Ad vector doses in vivo thereby minimizing the adverse reactions of the immune response due to high vector dosage; (2) Long-term thermostabilization studies of Ad, EV1 and CVB3 in vitro in the presence and in the absence of our nanomaterials and other substances such as sugars (sucrose, glucose, glycerol) and Polyethylene glycol (PEG) molecules at 37°C or room temperature for extensive periods of time. Our results showed the capability of the nanomaterials and sucrose to increase substantially the heat stability of the viruses. In order to elucidate the thermal inactivation mechanism of viral particles and the stabilizing effect provided by some compounds on viruses we set out to formulate an analytical theory. This line of research fits in the context of developing more thermo-stable viral vector preparations for vaccine purposes that do not require the maintenance of the challenging cold chain system in order to preserve the effectiveness of viral vaccines during the storage, shipment and administration to the patients and hence to ensure the success of global immunization programs (Alcock et al. 2010).

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Introduction Childhood acute infectious diarrhea is one of the biggest health problems faced by developing countries and its incidence has been increased in children who attend daycare. Objective To evaluate the possible relation between bacterial and viral enteropathogens with diarrhea in a children population of a public daycare in São José do Rio Preto city São Paulo state. Material and methods The group of Microorganisms Investigation Center (CIM) of the Medicine College of São José do Rio Preto (FAMERP) collected and processed 100 fecal samples of 50 healthy children (control group) and other 50 children who presented fecal material with compatible aspect to diarrheic clinic. Stool samples were transported in Cary Blair transport media for bacterial analysis. All specimens were examined on the day of collection according to standard bacteriologic procedures. Briefly, suggestive bacterial colonies were isolated from McConkey, Salmonella Shigella, brilliant green (after enrichment in tetrathionate broth), and Columbia agar. Isolates identified by biochemical tests were serotyped by standard techniques (EPM-Milli and Oxidase stripes plus commercially available antisera; PROBAC, Brazil). For a viral analysis, an aliquot of the obtained fecal material was frozen under -70 degrees Celsius and, afterwards, conducted to the Virology Section of the Institute Evandro Chagas, Ananindeua, Pará state. The identification of the astrovirus and calicivirus was done by RT-PCR (Polymerase chain reaction, through reverse transcriptase). Polyacrylamide gel electrophoresis (PAGE) was carried out in Tris glycine buffer and rotavirus genome profile was defined following electrophoresis of extracted dsRNA through vertical 5% acrylamide bisacrylamide gels. Results and discussion There was no difference concerning the gender between the two groups, with a slight higher representation of female 52 (52,0%). The age group ranged from 6 months to 7 years old (an average of 1,6 years). The most frequent bacteria in the population was 38 strains of E.coli (38%), distributed like this: EPEC (12%), EIEC (3%), Pseudomonas spp. (2%) and E.coli O157 (1%). Fourteen children presented mixed colonization of Enterobacter and E.coli (14,1%). The circulating of enteric viruses in the children population are the Norovirus (2%) and Astrovirus (1%). The presence of Norovirus and Astrovirus is traditionally associated with the urban area inhabitants. The food intake out of the daycare and home indicated the presence of enteropathogens. The bacterial and viral agents detected are not associated with the diarrhea occurrence in the studied population. Conclusion: The results obtained in this study demonstrated that the children who attend daycare are asymptomatic carriers of potential pathologic agents, this fact deserves further investigation in this area, as well as in other country areas. This study will be useful for creating effective strategies of prevention, control and treatment, in order to improve the life condition of the group in this work.
Introdução: A diarreéia infecciosa aguda infantil é um dos maiores problemas de saúde enfrentado pelos países em desenvolvimento e tem sua incidência aumentada em crianças que frequentam creches. Objetivo: Avaliar a possível associação de enteropatógenos bacterianos e virais com a diarreéia em uma população infantil de uma creche pública do município São José do Rio Preto SP. , pela equipe do Centro de Investigação de Microrganismos (CIM) da Faculdade de Medicina de São José do Rio Preto (FAMERP). Material e Método: A equipe do Centro de Investigação de Microrganismos (CIM) da Faculdade de Medicina de São José do Rio Preto (FAMERP) efetuou a coleta e o processamento de Foram analisadas 100 amostras fecais, provenientes de sendo 50 crianças sadias no (grupo controle) e de outras 50 crianças que apresentaram material fecal com aspecto compatível à clínica diarreéica. Para análise bacteriológica, parte do material fecal foi utilizado meio detransportadoenviado em meio de transporte Cary Blair, com imediata semeadura as amostras foram semeadas em meio Ágar MacConkey (DIFCO), Ágar SS (DIFCO), em caldo Tetrationato, anterior à semeadura em em Ágar Verde Brilhante (DIFCO) e em Àgar Columbia (DIFCO) com carvão ativado. A técnica de aglutinação a partir de uma suspensão bacteriana foi utilizada para a identificação tipagem sorológica das enterobactérias. Para análise viral, uma alíquota do material fecal obtido foi congelada a -70 graus Ccelsius e, posteriormente, encaminhada ao Setor de Virologia do Instituto Evandro Chagas, Ananindeua, Estado do Pará. APara detecção dos Astrovírus e Calicivírus foram foi realizadas por RT-PCR (Reação em Cadeia da Polimerase via transcriptase reversa). Já Aa detecção dos rotavírus foi realizada efetuada por meio de eletroforese em gel de poliacrilamida (PAGE) em tampão Tris-glicina, e oseu perfil do genômico do rotavírus foi definido após eletroforese do RNA fita dupla (dsRNA) extraído em géis verticais de bisacrilamida-acrilamida a 5%. Resultados e Discussão?: Não houve diferença quanto ao gênero entre os dois grupos, com ligeira maior representação maior frequencia do sexo feminino 52 (52,0%). A faixa etária variou de seis meses a sete anos de idade (média de 1,6 anos). As bactérias mais frequentes na população são foram 38 cepascasos de E. coli (38%), assim distribuídas: sendo EPEC (- 12%), EIEC - (3%), Pseudomonas spp. - (2%) e E. coli O157 (- 1%). Houve tambémCatorze 14 crianças apresentaram casos de colonizaçãoção mista por mista de Enterobacter e E. coli (14,1%). Os vírus entéricos circulantes nessas população infantil crianças são o Norovírus (2%) e o Astrovírus (1%). A presença de Norovírus e Astrovírus está tradicionalmente associada com àa população residente em área urbana. O consumo de alimentos fora da creche e do domicílio foi indicativo dae presença de enteropatógenos. Os agentes bacterianos e virais detectados não estão associados aos casos de diarréeia na população estudada. Conclusão: Os dados obtidos neste estudo demostram que as crianças que frequentam creches são portadores assintomáticas de potenciais agentes patogênicos, fato este merece investigação adicional nesta região área, bem como em outras regiõesdo país. Este estudo contribuirá para a criação de estratégias efetivas de prevenção, controle e tratamento, melhorando assim a condição de vida do grupo em estudo.

Human enteroviruses (HEVs), particularly Coxsackie B viruses (CVBs), might trigger the onset of type 1 diabetes (T1D), either by direct infection of the insulin-producing beta-cells or by an indirect inflammatory response. The overall aim of this thesis was to study the tropism of HEVs in isolated human pancreatic cell clusters in vitro including virus effects on islet function, gene-expression and ultrastructure. Furthermore, the expression of the major CVB-receptor, CAR, was investigated in pancreatic tissue from T1D-related subjects and CVB-infected islets. Also, tissues and isolated islets from two adult organ-donors who died close to disease onset were studied.The results showed that beta-cells were destroyed through lytic infections with different strains of CVBs and that islets function did not depend on replication per se but on the degree of islet destruction. Virus particles were observed in beta-cells in association with insulin granules, however no virus replication or particles could be observed in the exocrine cell clusters, as opposed to in mice models. The virus-infected islets had a decreased expression of insulin mRNA and CAR mRNA/protein, possibly reflecting virus-killed beta-cells. Infected beta-cells contained a high number of insulin granules, which might indicate an impaired function.The in vivo studies showed presence of virus proteins in the islets of both donors who died close to onset of T1D and elevated expression of innate immunity genes, potentially indicating viral infection, but direct evidence is lacking. Both donors were immune-reactive for insulin but the isolated islets had an impaired or completely lacking glucose response. Ultrastructural analysis showed both damaged beta-cells and normal-looking beta-cells, indicating that the latter might still have the potential to function but were blocked. CAR-expression was significantly increased in T1D-related subjects which might indicate tissue damage and/or inflammation in these subjects.To conclude, these results showed that CVBs could infect human primary beta-cells, likely by binding to CAR and lead to functional abnormalities, indicating that they could cause T1D in vivo. Exocrine cells were not permissive to CVB, which raises the question if mice-models should be used to study human pancreatitis. Also, unique materials from two T1D organ-donors were described.

Enteroviruses belong to the Picornaviridae family and are small icosahedral viruses with RNA genomes of positive polarity, containing a single open reading frame. They mostly cause mild or asymptomatic infections, but also a wide array of diseases including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis, myocarditis, hand-foot-and-mouth disease, hepatitis and respiratory diseases, ranging from severe infections to the common cold. The projects described in this thesis have been carried out through reverse genetic studies of Enterovirus B and Rhinovirus C.                   In Papers I and II, a cassette vector was used to study recombination and translation of the RNA genome. It was found that the non-structural coding region could replicate when combined with the structural protein-coding region of other viruses of the same species. Furthermore, the genome could be translated and replicated without the presence of the structural protein-coding region. Moreover, it was found that when two additional nucleotides were introduced, shifting the reading frame, the virus could revert to the original reading frame, restoring efficient replication. In Paper III, a vector containing the genome of echovirus 5 was altered to produce an authentic 5’end of the in vitro transcribed RNA, which increased efficiency of replication initiation 20 times. This result is important, as it may lead to more efficient oncolytic virotherapy. An authentic 5’end was further used in Paper IV, where replication of Rhinovirus C in cell lines was attempted. Although passaging of the virus was unsuccessful, the genome was replicated and cytopathic effect induced after transfection. The restriction of efficient replication was therefore hypothesized to lie in the attachment and entry stages of the replication cycle. In Paper V, a cytolytic virus was found to have almost 10 times larger impact on gene expression of the host cell than a non-cytolytic variant. Furthermore, the lytic virus was found to build up inside the host cell, while the non-cytolytic virus was efficiently released.                   As a whole, this thesis has contributed to a deeper understanding of replication of enteroviruses, which may prove important in development of novel vaccines, antiviral agents and oncolytic virotherapies.

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.

Introduction: Hand, foot and mouth disease (HFMD) is a childhood exanthema caused by enteroviruses, such as coxsackie virus A (CV A) 16. However since 1997 large epidemics ofHFMD caused by human enterovirus (EV) 71 and associated with severe and sometimes fatal neurological complications have occurred across Asia. Aims: To examine: (i) the diagnostic approach for detection ofEV71, (ii) the clinical and molecular epidemiology of the virus in Sarawak, (iii) the clinical predictors for neurological involvement, and (iv) the viral determinants for clinical phenotype of EV71 infection. Methods: A prospective study was set up to examine children with HFMD presenting to Sibu Hospital, Sarawak, Malaysia between January 2000 and December 2006. Detailed history and clinical examination was performed and recorded on standardised forms. Throat and rectal swabs, and swabs from skin vesicles and mouth ulcers, if present, were taken from every patient. Lumbar puncture was performed in patients with suspected neurological involvement. Virus isolation and RT-PCR for enteroviruses were performed on all specimens. Isolated enteroviruses were typed by nucleotide sequencing of VP 1 and VP4 genes and genogrouped by pbylogenetic analysis. Results: Throat and vesicle swabs were the most useful samples for detection of EV71. Using virus culture results as the reference, an EV71-specific assay originally developed for molecular typing ofEV71 clinical isolates had a sensitivity of76.9% (258/337), specificity of 82.6% (133/161), positive predictive value of90.2% (259/287) and negative predictive value of63.0% (l33/211) when evaluated with 337 EV71-positive, 161 non-EV71 culture-positive clinical specimens. Epidemics of EV71-associated HFMD occurred every 3 years in Sarawak, and were caused by genogroups B4 and B5, and Cl. The genogroups of EV71 differ in their risk of causing neurological disease and family clusters. Total duration of fever 23 days, peak temperature 238SC and history oflethargy were identified and validated as independent risk factors for neurological involvement. EV71-positive children were more likely to have neurological disease when compared to CVAl6-positive children. Discussion: EV71 has become a major public health problem in Asia and may continue to spread globally. The transmission dynamic of the virus is poorly understood. The public health intervention measure to date has been empirical and generic, but they have considerable socioeconomic implication. There is neither specific antiviral nor vaccine for EV71. Intravenous immunoglobulin is now used presumptively for severe EV71 infection in many Asian countries, although there are little data on its efficacy. Early diagnosis of neurological involvement may help reduce the mortality. A better understanding on diagnosis and management of this neurological infectious disease can help public health doctors and clinicians manage the epidemic caused by the virus when it spread to a new territory.

The use of the neutralisation assay in the serotyping of unknown enterovirus isolates has been successfully applied for years since its description by Lim and Benyesh-Melnick1. However, the procedure is labour-intensive and time consuming. In addition, the continuous depletion of the Benyesh-Melnick sera makes it prohibitive for most diagnostic laboratories to continue using the assay. Owing to these factors, a majority of laboratories only type for the polioviruses and report any other enterovirus isolate as “a nonpolio enterovirus”. With this approach, however, important findings such as the isolation of a new enterovirus or association of a known enterovirus with a new clinical syndrome will remain unidentified. In this study, a multiple-serum-pools approach similar to that described by Lim and Benyesh-Melnick1 (LBM) for the neutralisation test, was applied in an immunofluorescence (IMF) test for the rapid serotyping of enteroviruses. This test was able to type any unknown enterovirus isolate belonging to the different enterovirus serotypes tested in this study, in about three hours as compared to the eight days currently taken by the LBM neutralisation test1.

Enterovirus 70 (EV70), the causative agent of acute haemorrhagic conjunctivitis (AHC) is a picornavirus with a number of unique biological and pathogenic properties. In vivo, EV70 shows tropism for the conjunctiva and in rare cases, can infect the central nervous system, while in vitro, it has the ability to infect a wide range of mammalian cells in culture. The main objective of this study was to identify sites associated with neutralization of EV70. Identification of neutralization-associated sites is an initial stage for producing subunit vaccines, and neutralizing monoclonal antibodies produced against these sites may have direct application as therapeutic agents. Several different monoclonal antibodies which have neutralizing activity against the prototype EV70 J670 were tested. Two antibodies yielded resistant virus plaques. The nucleotide sequence of the capsid protein-coding region of each mutant was determined following cDNA synthesis, PCR amplification and cloning. The deduced amino acid sequences of the capsid protein-coding regions were compared with the corresponding sequences from the EV70 prototype and several wild isolates of EV70. Amino acid residues that correlated with escape from neutralization were identified. Two neutralization associated determinants were identified on VP1 and VP2. The locations of these sites were compared with neutralization associated sites of other picornaviruses.

INTRODUCCIÓ. Les infeccions neonatals son una patologia molt prevalent i representen una de les principals causes de mortalitat durant aquest període de la vida. Gran part dels nadons que ingressen durant el primer mes de vida amb sospita de sèpsia bacteriana (SB) són diagnosticats d’infeccions produïdes per virus. Els virus més implicats en la infecció neonatal són els enterovirus (EV) amb una incidència de 7 casos per cada 1000 nadons. L’objectiu d’aquest estudi és demostrar la importància dels EV com a agents etiològics de la infecció neonatal per les seves diferències pel que fa al maneig clínic i les mesures terapèutiques a seguir. MATERIAL I MÈTODES. El present treball es un estudi retrospectiu amb recollida prospectiva de les dades. Es van incloure un total de 332 nadons d’edat gestacional > 34 SG atesos a la Unitat de Neonatologia de l’Hospital de la Santa Creu i Sant Pau entre gener de l’any 2002 fins a desembre del 2017 i amb el diagnòstic inicial de sèpsia neonatal (SN). RESULTATS. El percentatge d’infeccions per EV va ser superior al de SB amb un 34,6% i 32,5% respectivament del total de nadons estudiats. Els nadons amb infecció per EV presenten menor patologia materna a excepció de la malaltia hipertensiva de l’embaràs (p <0,001). Es tracta de nadons sans amb més edat gestacional, majors puntuacions en el test d’Apgar i major pes al naixement (p <0,001). En la meitat dels casos hi ha antecedents d’ambient epidèmic familiar (p <0,001). La febre es va presentar en el 72% dels nadons amb infecció per EV i la meningitis va ser la forma clínica més freqüent afectant el 61,7% dels casos d’infecció per EV. Les característiques del LCR dels pacients amb meningitis van ser similars en ambdós grups i només van existir diferències significatives en la concentració de proteïnes, superior en el grup de SB (p> 0,001). Les formes greus de la malaltia van ser poc freqüents (1,7%). El 51,4% dels pacients amb infecció per EV va ser sotmès a tractament antibiòtic (ATB). La detecció d’EV en mostres de LCR mitjançant RT-PCR va mostrar una elevada sensibilitat, especificitat i un elevat valor predictiu positiu i negatiu en el diagnòstic de la infecció per EV. L’evolució clínica dels pacients amb infecció per EV va ser favorable amb resolució sense seqüeles en la majoria dels casos. La letalitat de el quadre en els nens estudiats va ser del 0,9%. Els EV més freqüentment implicats en la malaltia neonatal per EV van ser E11, E6, E7 i CVB5. Es va obtenir un model predictiu que permet calcular la probabilitat de risc de presentar SB o infecció per EV i classificar correctament el 99% dels RN amb una precisió del 95,6%. Les variables que van mostrar una associació amb la infecció per EV van ser la major edat en dies de vida en el moment de patir la malaltia, majors puntuacions en el test d’Apgar als 5 minuts, el major pes al naixement, l’ambient epidèmic i l’alteració del sensori. CONCLUSIONS. Cal la inclusió dels EV en l’estudi inicial dels nadons amb sospita de sèpsia neonatal ja que aquestes infeccions es presenten amb una elevada prevalença en el nostre estudi. Els nadons amb infecció per EV tenen similituds clíniques amb els nadons amb SB, però hi ha factors que poden diferenciar les infeccions per EV de les infeccions bacterianes. La troballa d’aquests factors ha permès elaborar un model predictiu que al junt a la positivitat de les tècniques ràpides per a la detecció d’EV permeten diferenciar aquestes dos infeccions i retirar un tractament antibiòtic innecessari.
INTRODUCCIÓN. Las infecciones neonatales constituyen una de las patologías más prevalentes y representan una de las principales causas de mortalidad durante este periodo de la vida. Gran parte de los recién nacidos (RN) que ingresan durante el primer mes de vida con sospecha de sepsis bacteriana (SB) son diagnosticados de infecciones producidas por virus. Los virus mayormente implicados en la infección neonatal son los enterovirus (EV) con una incidencia de 7 casos por cada 1000 RN. El objetivo de este estudio es demostrar la importancia de los EV como agentes etiológicos en la infección neonatal por sus diferencias en cuanto al manejo clínico y las medidas terapéuticas a seguir. MATERIAL Y METODOS. El presente trabajo consiste en un estudio retrospectivo con recogida prospectiva de los datos. Se incluyeron un total de 332 RN de edad gestacional > 34 semanas gestacionales (SG) atendidos en la Unidad de Neonatología del Hospital de la Santa Creu i Sant Pau entre enero del 2002 hasta diciembre del 2017 y con el diagnóstico inicial de sepsis neonatal (SN). RESULTADOS. El porcentaje de infecciones por EV fue superior al de SB con un 34,6% y 32,5% respectivamente del total de RN estudiados. Los RN con infección por EV presentan menor patología materna a excepción de la enfermedad hipertensiva del embarazo (p<0,001), son RN sanos con mayor edad gestacional, mayores puntuaciones de Apgar y mayor peso al nacimiento (p<0,001) y en la mitad de los casos existen antecedentes de ambiente epidémico familiar (p<0,001). La fiebre se presenta en el 72% de los RN con infección por EV y la meningitis es la forma clínica más frecuente afectando al 61,7% de los casos. Las características del líquido cefalorraquídeo (LCR) de los pacientes con meningitis fueron similares en ambos grupos y solo encontramos diferencias significativas en la concentración de proteínas, superior en el grupo de SB (p>0,001). Las formas graves de la enfermedad fueron poco frecuentes (1,7%). El 51,4% de los pacientes con infección por EV fue sometido a tratamiento antibiótico (ATB). La detección de EV en muestras de LCR mediante reacción en cadena de la polimerasa-transcriptasa inversa (RT-PCR) mostró una elevada sensibilidad, especificidad y un elevado valor predictivo positivo y negativo en el diagnóstico de la infección por EV. La evolución clínica de los pacientes con infección por EV fue favorable con resolución sin secuelas en la mayoría de los casos. La letalidad del cuadro en nuestros niños fue del 0,9%. Los EV más frecuentemente implicados en la enfermedad neonatal por EV fueron E11, E6, E7 y CVB5. Se obtuvo un modelo predictivo que permite calcular la probabilidad de riesgo de presentar SB o infección por EV y clasificar correctamente el 99% de los RN con una precisión del 95,6%. Las variables que mostraron una asociación con la infección por EV fueron la mayor edad en días de vida en el momento de padecer la enfermedad, mayores puntuaciones en el test de Apgar a los 5 minutos, el mayor peso al nacimiento, el ambiente epidémico y la alteración del sensorio. CONCLUSIONES. Es necesario la inclusión de los EV en el estudio inicial de los RN con sospecha de sepsis neonatal ya que estas infecciones se presentan con una elevada prevalencia en nuestro estudio. Los RN con EV comparten similitudes clínicas con los RN con SB, pero existen factores que permiten diferenciar las infecciones por EV de las infecciones bacterianas. El hallazgo de estos factores ha permitido elaborar un modelo predictivo que junto a la positividad de las técnicas rápidas para la detección de EV permiten diferenciar estas 2 infecciones y retirar un tratamiento antibiótico innecesario.
INTRODUCTION. Neonatal infections are one of the most prevalent pathologies and represent one of the main causes of mortality during this period of life. Most of the newborns who are admitted during the first month of life with suspected Bacterial Sepsis (BS) are diagnosed with infections caused by viruses. The viruses most involved in neonatal infection are enteroviruses (EV), with an incidence of 7 cases per 1000 newborns. The objective of this study is to demonstrate the importance of EV as etiological agents in neonatal infection due to their differences in terms of clinical management and the therapeutic measures to be followed. MATERIAL AND METHODS. The present work consists of a retrospective study with prospective data collection. A total of 332 NBs of gestational age> 34 SG attended in the Neonatology Unit of the Hospital de la Santa Creu i Sant Pau between January 2002 and December 2017 and with the initial diagnosis of Neonatal Sepsis (NS) were included. RESULTS. The percentage of infections by EV was higher than that of BS with 34,6 and 32,5% respectively of the total of newborns studied. Newborns with EV infection present less maternal pathology except for hypertensive disease of pregnancy (p <0.001). Newborns with EV infection present less maternal pathology except for hypertensive disease of pregnancy (p <0.001), they are healthy newborns with a higher gestational age, higher Apgar scores and higher birth weight (p <0.001) and in the middle of the cases there is a history of a family epidemic environment (p <0.001). Fever occurs in 72% of newborns with EV infection and meningitis is the most frequent clinical form, affecting at 61.7% of EV infection cases. The CSF characteristics of patients with meningitis were similar in both groups and we only found significant differences in protein concentration, higher in the BS group (p> 0.001). Severe forms of the disease were rare (1.7%). 51.4% of the patients with EV infection underwent ATB treatment. The detection of EV in CSF samples by RT-PCR showed high sensitivity, specificity, and high positive and negative predictive values in the diagnosis of EV infection. The clinical evolution of patients with EV infection was favorable with resolution without sequelae in most cases. The case fatality rate in our children was 0.9%. The EV types most frequently implicated in neonatal EV disease were E11, E6, E7 and CVB5. A predictive model was obtained that allows calculating the risk probability of presenting BS or EV infection and correctly classifying 99% of the newborns with a precision of 95.6%. The variables that showed an association with EV infection were an older age in days of life at the time of suffering from the disease, higher scores in the Apgar test at 5 minutes, higher birth weight, an epidemic environment, and the alteration of the sensorium. CONCLUSIONS. The inclusion of EV in the initial study of newborn with suspected neonatal sepsis is necessary because these infections present with a high prevalence in our study. Newborns with EV share clinical similarities with newborns with BS, but we found factors that allow us to differentiate EV infections from bacterial infections. The finding of these factors has made it possible to develop a predictive model that, together with the positivity of the rapid techniques for the detection of EV, allows us to differentiate these 2 infections and withdraw unnecessary antibiotic treatment.
Universitat Autònoma de Barcelona. Programa de Doctorat en Pediatria, Obstetrícia i Ginecologia

Lodos de esgoto são resíduos do tratamento do esgoto doméstico, considerados ricos em macronutrientes e matéria orgânica, podendo também apresentar contaminação por substâncias químicas e patógenos. Sua utilização na agricultura é uma das alternativas interessantes para sua disposição final. Entretanto, a presença de contaminantes, dentre eles microrganismos patogênicos podem limitar e orientar sua aplicação. Os vírus entéricos, dentre os quais se inclui o gênero Enterovirus, são potenciais contaminantes microbianos presentes no lodo de esgoto. Esses organismos são capazes de sobreviver por meses em águas e solos e sua presença no ambiente pode trazer prejuízos à saúde da população exposta aos mesmos. O objetivo do presente estudo foi de analisar a ocorrência de Enterovirus em amostras de lodo de esgoto de seis diferentes ETEs do Estado de São Paulo, avaliar o desempenho do método analítico para a detecção desses organismos e verificar se as concentrações médias obtidas atendem à legislação. Foram coletadas um total de 35 amostras no período de fevereiro de 2009 a dezembro de 2009. As análises dos Enterovirus foram realizadas pelo ensaio de plaqueamento em cultura de células RD segundo método EPA /625/R-092/013. Os resultados obtidos foram que Enterovirus estiveram presentes em 83 por cento das amostras analisadas, com concentrações que variaram de não detectado (ND) a 12,50 UFP/gST. As taxas de recuperação obtidas variaram de 0,20 por cento a 68,50 por cento . Conclui-se que das amostras analisadas 31 por cento atendem o estabelecido pela Resolução CONAMA 375/2006
Sewage Sludge is a waste generated from wastewater treatment and is considered besides rich in macronutrients and organic matter, contaminated with pathogen and chemical substances. The usage of sludge in agriculture has been considered an interesting option. Nevertheless, the presence of contaminates such as pathogenic organisms can lead to usage limitations. Enteric viruses in wich are included enterovirus genera, a potential sludge contaminants. These organisms are able to survive for months in water and soil and their presence can bring public health concerns. The aim of this study was to analyse the occurrence of Enterovirus in samples of sewage sludge from six sewage plant treatment located in São Paulo State, to assess the method performance (recovery rate) in detecting these organisms and to verify the results obtained meet the standard established by the law. A total of 35 samples were collected spanning February to December 2009. The analyses were carried out according to method EPA/625/R-092/013. Enterovirus were present in 83 per cent of samples examined with concentration varied from not detected (ND) to 12.5 PFU/gTS. The recovery rate varied from 0,2 per cent to 68,50¨ per cent . This study showed that 31 per cent of analyzed samples met the standard established by Resolução CONAMA 375/2006

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 126-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Les @entérovirus (Picornaviridae) sont des agents infectieux communs divisés en 5 espèces (Poliovirus et Entérovirus humains A à D) qui regroupent actuellement 89 sérotypes. Ces virus à ARN positif simple brin, sont responsables de syndromes infectieux variés incluant des infections des voies respiratoires hautes ou basses chez l'adulte ou l’enfant. Actuellement l’importance épidémiologique, les caractéristiques cliniques ainsi que les mécanismes physiopathologiques des infections respiratoires par les entérovirus restent à préciser. La première partie de notre travail a eu pour objectif d’évaluer le rôle étiologique potentiel des picornavirus à tropisme respiratoire chez 192 enfants âgés de moins de 36 mois et hospitalisés pour bronchiolite. Un agent viral a été identifié chez 138 (72. 5%) des 192 enfants étudiés. Les virus les plus fréquemment détectés étaient respectivement le Virus Respiratoire Syncytial (VRS) (30%), les Rhinovirus (RVH) (21%), les entérovirus (EV) (9%), et les Métapneumovirus humains (MPVh) (4%). Les RVH et les EV sont apparus comme étant la seule cause de l'infection virale de l’arbre respiratoire dans 57 (30%) des 192 jeunes enfants, tandis qu’une co-infection avec du RVH ou du EV a été détectée dans 25 (13%) des 192 jeunes enfants (30 vs. 13%, P<10-3). Ces données suggèrent que les picornavirus (RVH et EV) à tropisme respiratoire seraient l'une des principales causes virales de bronchiolite en France. Dans une seconde étude, nous avons analysé 252 cas d’infections pédiatriques à EV diagnostiqués chez 11509 enfants. Les souches d’EV ont été isolées dans des échantillons naso-pharyngés grâce à la culture virale, et identifiées par séroneutralisation. Les syndromes respiratoires (79 (31%) des 252 infections à EV) associés à une infection par un EV sont apparus comme étant la deuxième plus fréquente pathologie pédiatrique après la méningite (111 (44%) des 252 cas) (44 vs 31%, p <10-3). Les EVs ont contribué aux infections respiratoires bases dans 54 % des 79 cas d’infection à EV. La bronchiolite a été la pathologie la plus souvent diagnostiquée dans les infections respiratoires à EV (34 (43%) des 79 cas, p <10-3), survenant le plus souvent chez les enfants âgés de 1-12 mois (P = 0. 0002). Les echovirus 11, 6 et 13 ont été les souches les plus fréquemment identifiées dans les infections respiratoires (24, 13 et 11%, respectivement). L’analyse phylogénétique du gène codant pour la protéine de capside VP1 a révélé la circulation concomitante ou successive de souches distinctes EV à tropisme respiratoire au cours du même mois ou de la période épidémique. Ces résultats indiquent que les infections des voies respiratoires représentent 30% des cas des infections pédiatriques à entérovirus. De plus, la circulation concomitante ou successives de souches génétiquement distinctes d’EV indique la possibilité d’infections respiratoires répétées au sein de la même saison épidémique, et suggère la possibilité de mécanismes de recombinaison génétique entre des souches d’EV d’espèces A ou B. Afin d’identifier les mécanismes qui peuvent réguler le développement de l’inflammation des muqueuses respiratoires au cours de l’infection des voies aériennes basses par les EVs, nous avons étudié les profils et les niveaux de production de « CC et CXC chimiokines » de cellules épithéliales pulmonaires humaines primaires (SAEC), infectées par deux souches sauvages d’EV à tropisme respiratoire. L’exposition des SAEC à l'interféron gamma (INF-γ) et aux virus sauvages de type Coxsackie B5 ou ECHO 30 induit une augmentation significative de la production de RANTES qui est synergique par rapport celle obtenue par l’infection virale ou par l’INF-γ seul. Nous avons observé que l'infection réplicative des entérovirus dans les SAEC induisait une augmentation dose et temps-dépendante des ARNm, et des protéines RANTES, MCP-1 et l'IL-8. La sécrétion de ces chimiokines est significativement augmenté à 48 ou 72 heures suivant l’infection dans les cultures traitées par de faibles doses d’INF-γ, et ceci comparativement aux cellules non infectées (P <0,001). Les chimiokines produites par les SAEC en réponse à l’infection virale ont montrés une forte activité chimiotactique pour les éosinophiles humains du sang périphérique. En outre, nous avons observé une infection productive par les entérovirus à tropisme respiratoire dans les éosinophiles. Ceux-ci ont spécifiquement sécrété des niveaux significatifs de RANTES et MCP-1,et ceci 24 heures après l'infection. Par conséquent, le processus inflammatoire induit par les entérovirus semble être déclenché par l'infection de cellules épithéliales, et amplifié par des mécanismes déclenchés par l’INF-γ ainsi que par la sécrétion de chimiokines par les éosinophiles recrutés dans la lumière bronchique. En conclusion, nos travaux indiquent que les EVs sont une cause fréquente d'infection des voies respiratoires chez les enfants, et qu’ils sont capables d’induire au cours de l’infection des cellules de l'épithélium bronchique, une sécrétion spécifique de chimiokines de type RANTES, MCP-1 et IL-8. Ces résultats suggèrent l’importance du rôle des entérovirus dans le développement de pathologies respiratoires chez les enfants immunocompétents
Enteroviruses (EV) (Picornaviridae) are among the most common viruses infecting human beings worldwide. These viral agents are associated with a wide range of human pathologies, including upper respiratory but also lower respiratory tract infections resulting in bronchitis, pneumonia or bronchiolitis in adults or in infants. In the first study, we assessed the potential role of the respiratory picornaviruses as causative agents of bronchiolitis in 192 infants ≤36 months of age and hospitalized for acute bronchiolitis. The detection of common respiratory viruses (respiratory syncytial virus, influenza virus A and B, parainfluenza virus I, II, III, and adenovirus) was performed using classical immunofluorescence antigens and cell culture detection assays in nasopharyngeal aspirates whereas the detection of human metapneumovirus (HMPV) rhinoviruses and enteroviruses was performed by molecular techniques. A potential causative virus was detected in 72. 5 % of the 192 study infants. RSV (30%), rhinovirus (21%), enterovirus (9%), influenza virus A (6%) and human metapneumovirus (4%) were the most frequent causative agents detected. Rhinoviruses or enteroviruses were detected as the only evidence of respiratory viral tract infection in 57 (30%) of 192 infants, whereas rhinovirus or enterovirus occurred in mixed viral infection detected in 25 (13%) of 192 study cases (30 vs. 13%, p<10-3). Our data suggest that respiratory picornaviruses are one of the leading etiological causes of bronchiolitis in French infants. In the second part our investigations, we analysed 252 EV-related infection cases (median age, 5. 1 years) diagnosed among 11,509 consecutive children visiting emergency departments within a 7-year period in the North of France. EV strains were isolated from nasopharyngeal samples by viral cell culture, identified by seroneutralization assay and genetically compared by partial amplification and sequencing of the VP1 gene. The respiratory syndromes (79 (31%) of 252 EV infections) appeared as the second more frequent EV induced pediatric pathologies after meningitis (111 (44%) of 252 cases) (44 vs. 31%, P<10-3), contributing to lower respiratory tract infection (LRTI) in 43 (54%) of 79 EV respiratory infection cases. Bronchiolitis was the most frequent EV induced LRTI (34 (43%) of 79 cases, P<10-3) occurring more often in infants aged 1-12 months (P=0. 0002) with spring-fall seasonality. Viruses ECHO 11, 6 and 13 were the more frequently identified respiratory strains (24, 13 and 11%, respectively). The VP1 gene phylogenetic analysis showed the concomitant or successive circulation of genetically distinct EV respiratory strains (species A or B) during the same month or annual epidemic period. Our findings indicated that respiratory tract infections accounted for appreciatively 30% of EV-induced paediatric pathologies, contributing to LRTIs in 54% of these cases. Moreover, the concomitant or successive circulation of genetically distinct EV strains indicated the possibility of paediatric repeated respiratory infections within the same epidemic season. To identify the mechanisms that can regulate the development of airway mucosa inflammation during EV respiratory lower tract infection, we investigated the production of chemokines by EV-infected bronchial epithelial cells. Cultures of primary human small airway epithelial cell (SAEC) were infected by wild-type respiratory EV strains, demonstrating a replicative and productive infection by Coxsackievirus B5 and Echovirus 30 strains. Exposure of SAEC to gamma interferon (INF-γ), in combination with Coxsackievirus B5 and Echovirus 30 infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by EV-infection or INF-γ treatment alone. We observed that the replicative infection of the SAEC by Coxsackievirus B5 and Echovirus 30 wild-type viruses induced dose and time-dependent increases in mRNA and protein secretion for RANTES, MCP-1 and IL-8. The protein secretion of these chemokines appeared to be significantly increased at 48 or 72 hours post-infection in cultures treated by low-doses of INF-γ comparatively to mock-infected cells (P<0. 001), and was correlated to the viral replication activity. SAEC-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, we observed an EV productive infection in eosinophils, which specifically released significant levels of RANTES and MCP-1, 24 hours post-infection. Therefore, the inflammatory process in EV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by INF-γ and by the secretion of eosinophil chemokines. Altogether, our findings suggest that EVs are a common cause of respiratory tract infections in paediatric patients, where they can induce the release of chemokines by bronchial epithelial cells, which may significantly contribute to the various histologic and inflammatory features of EV-induced airway disease

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: Os enterovírus são a principal causa de infeção do Sistema Nervoso Central, nomeadamente de meningite vírica, em idade pediátrica. O objetivo do estudo foi caraterizar as infeções do Sistema Nervoso Central por enterovírus, num hospital pediátrico durante 8,5 anos. Material e métodos: Estudo observacional, transversal, analítico, com colheita retrospetiva de dados de processos clínicos dos casos diagnosticados com infeção do Sistema Nervoso Central, com identificação de enterovírus por polymerase chain reaction no líquido cefalorraquidiano, no Hospital Pediátrico do Centro Hospitalar e Universitário de Coimbra, de 01/01/2011 a 30/06/2019. Foram avaliados fatores demográficos, manifestações clínicas, exames complementares de diagnóstico, tratamento, diagnóstico e evolução. A análise estatística foi realizada em SPSS 25®.Resultados: Foram diagnosticados 82 casos, 61% do sexo masculino, com idade mediana de 6,7 anos (IQR 10,71). O maior número de casos foi em 2014 e 2018 (18 e 19 casos), com um mínimo de dois em 2013. Ocorreram mais casos no verão (40,2%). A febre (87,8%) foi a manifestação clínica mais comum. No grupo A (idade 1000 células/mm3), 36,4% com predomínio de polimorfonucleares. A proteinorráquia foi elevada em 45,7% e a glicorráquia baixa em 10,9%. A média de leucócitos foi 10992±3652/µL e a mediana de proteína C-reativa 1,1mg/dL (IQR 1,99). Em 42,7% foi iniciada antibioticoterapia, com suspensão após identificação de enterovírus em 57,1%. A duração mediana de internamento foi de um dia (IQR 1). Foram diagnosticadas com meningite 76 crianças, cinco com encefalite/meningoencefalite e uma com meningite e polirradiculite aguda. Dois irmãos tiveram diagnóstico na mesma semana. A complicação mais frequente foi a síndrome pós-punção lombar.Discussão: Ocorreu grande variabilidade do número de casos ao longo dos anos. Observou-se pleocitose ligeira na maioria, mas com valores muito elevados em alguns, e um terço dos casos teve predomínio de polimorfonucleares, o que mostra que a deteção rápida do vírus é importante para as decisões terapêuticas. A identificação do vírus levou à suspensão da antibioticoterapia em mais de metade dos casos.Conclusão: A maioria das infeções por enterovírus apresenta evolução benigna. O seu diagnóstico pode diminuir a duração do internamento e da antibioticoterapia e evitar a realização de exames desnecessários. .
Introduction: Enteroviruses are the main cause of Central Nervous System infection, namely viral meningitis, in paediatrics. The aim of this study was to characterise Central Nervous System infections by enterovirus, in a paediatric hospital during 8.5 years.Material and Methods: Cross-sectional analytic study with retrospective data collection of all the clinical cases diagnosed with Central Nervous System infection and identification of enterovirus in the cerebrospinal fluid by polymerase chain reaction, in Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra from 01/01/2011 to 30/06/2019. Demographic factors, clinical features, diagnostic tests, treatment and outcome were evaluated. Statistical analysis was done using SPSS 25®. Results: Eighty-two cases were included, 61% male, with a median age of 6.7 years (IQR 10.71). The largest number of cases was observed in 2014 and 2018 (18 and 19 cases), with a minimum of two in 2013. There were more cases in the Summer (40.2%). Fever (87.8%) was the most common symptom. In group A (age 1000 cells/mm3), with predominance of polymorphonuclear cells in 36.4%. Cerebrospinal fluid protein count was elevated in 45.7% and glucose count low in 10.9%. The average leucocyte count was 10992±3652/µL and median C-reactive protein 1.1mg/dL (IQR 1.99). In 42.7% antibiotics were started, with 57.1% stopped after identification of enterovirus. The median duration of admission was one day (IQR 1). Meningitis was diagnosed in 76 children, encephalitis/meningoencephalitis in five and one with meningitis and acute polyradiculitis. Two siblings had the diagnosis in the same week. The most frequent complication was post-lumbar puncture syndrome. Discussion: There was great variability in the number of cases throughout the years. Most had slight pleocytosis, but with very high values in some, and one third had polymorphonuclear cell predominance, showing that rapid virus detection is important for therapeutic decisions. Identification of the virus lead to the suspension of antibiotics in more than half the cases.Conclusion: Most enterovirus infections are benign. Their diagnosis can reduce the length of hospital stay and of antibiotic treatment and avoid unnecessary diagnostic tests. .

碩士
國立成功大學
分子醫學研究所
93
Enterovirus 71 (EV71), a non-enveloped, positive strand RNA virus belongs to Picornaviridae family. EV71 is a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) in young children, and can cause severe brainstem encephalitis (BE) and pulmonary edema (PE) with high fatality rates. Cell death plays a central role in modulation of the inflammatory response during viral infection. In addition, activated T lymphocytes can release cytokines which result in apoptotic signaling pathways such as the Fas/ Fas ligand (FasL), TNFR/TNF-αlead to of cell death. The first part of study was designed to investigate of the cell death induced by the release FasL or TNF-αfrom the peripheral blood polymorphonuclear cells (PBMC) upon EV71 infection. The results showed that the concentrations of sFasL and TNF-α increased continouslly during 72 and 96 hours postinfection in EV71-infected PBMC by ELISA. However, the concentration of sFasL is not enough to cause cell death when compared with concentrations of TNF-α. In addition, adding neutralization antibody against TNF-α showed the reduction of cell death. Furthermore, inhibition of TNF-αproductionwas observed when inhibitor of NF-κB was pretreated with PBMC. The showing indicated that EV71-induced TNF-α production via NF-κB pathway which may resulted in cell death and tissue damage. 　In the second part, genetic recombination of EV71 was examined. genetic recombination is a common feature among positive-strand RNA viruses. Analysis of non-polio enteroviruses (NPEV) prototype strains has suggested that interserotypic recombination is a frequent event during natural transmission and that it may play a significant role in enterovirus evolution and virulence. To examine the role of genetic recombination in the evolution of the EV71, the partial sequences of EV71, including the 5'-UTR, VP4VP2, VP1, 2B and 3D regions, of EV71 were examined. Thirty EV71 clinical isolates before and after 1998 epidemic were compared with the homologous sequences from all other enterovirus by phylogenetic analysis using PHYLIP Neighbor-joining method. 　Four isolates (N0003-TW-05, S0584-TW-04, N3340-TW-02 and 236-TW-86) among 30 clinical isolates were identified to have the evidence of genetic mutation or recombination. Among VP4VP2, VP1, 2B and 3D sequences of two isolates from 1986 (236-TW-86 and 252-TW-86), they all belonged to genotype B. However, the 5'-UTR region of isolates 236-TW-86 belong to EV71 genotype C, indicating genetic recombination between genotype B and C in 5'-UTR region. In addition, three isolates from 2002 (N3340-TW-02), 2004 (S0584-TW-04) and 2005 (N0003-TW-05), respectively, were identified to have genetic recombination between 2B and 3D region. Sequence of 2B fragment of these isolates belonged to genotype B. However, the sequence of 5'-UTR, VP4VP2, VP1 and 3D belonged to genotype C. Using SimPlot program to check full-length sequence, the localization of recombination was identified between 3000~3500 and 5500~6000 in N3340-TW-02 isolate. 　Moreover, the virological properties of recombinated EV71 was examined, including plaque assay, one-step growth curve and temperature-resistant assay. In 39.5℃ temperature-resistant assay, the data showed that they all belong to temperature-resistant. Furthermore, N3340-TW-02 showed faster growth than non-recombinated EV71. Taken together, these results indicated that genetic recombination phenomenon may have alter their virological and virulence properties.

碩士
國立陽明大學
醫學生物技術研究所
88
An outbreak associated with enterovirus infection occurred in Taiwan area during the year of 1998. It was reported that there were 129,106 cases of hand-foot-and-mouth disease or herpangina in the outbreak ; among them, 405 patients suffered from severe diseases with complications including encephalitis, aseptic meningitis, pulmonary edema, hemorrhage, acute flaccid paralysis, and myocarditis. Accumulated death toll has been 78 patients ; most of them were five years old or younger. Enteroviruses belong to the Family Picornaviridae, where the picornaviral genome consists of a single strand of message-active RNA. Presently, it is known that there are 66 types that can infect human, including Polioviruses type 1-3, Coxsackieviruses A group, Coxsackieviruses B group, Echoviruses and Enteroviruses type 68-71. Most of the syndromes caused by enterovirus infections are mild, with few exceptions such as enterovirus 71 (EV71) where severe diseases even death may incur. It will be challenging to develop vaccines against enteroviruses due to the multiple serotypes. Therefore, an effort has been made to study the protease 2A that plays a major role in viral replication, and may serve as a molecular target for anti-enterovirus therapy. We started by analysis of the protease 2A in 10 EV71 isolates from the 1998 outbreak and 10 Coxsackie B1 (CVB1) strains collected during 1993-1999. Analysis sequences of the DNA nucleotide and protein amino acid showed that the variations among the EV71 are slight whereas those of CVB1 are greater. We further map the cleavage site of the protease 2A in EV71 prototypic strain (BrCr) by molecular cloning followed by site-directed mutagenesis. The cleavage site was confirmed by both prokaryotic and eukaryotic expression systems. As for the study on the CVB1, two clinical isolates, CVB1-8 and CVB1-5, with relatively greater variations in the protease 2A were used for further investigation. It was shown that the rate of viral replication and cytopathic effect is more rapid with the CVB1-8 infection than those with the CVB1-5 in the Vero and GBM cells. This is consistent with the finding that the cleavage of the host protein-eIF4G by protease 2A is more effective with CVB1-8 infection. The data indicate that the variations in the protease 2A regions between these two CVB isolates may account for the differences in their replication and pathogenesis. Experiments to clarify the mechanism are in progress.

碩士
高雄醫學大學
公共衛生學研究所
102
Background: Enterovirus infection is a common source of disease in children and infants and cause HFMD and herpangina outbreaks around the world. Enterovirus infection is usually transmitted via fecal-oral route or airborne route. Previous study had already detected enterovirus in exhaled air and indoor air of hospital. Intrafamilial and kindergarten transmissions were major modes of enterovirus transmission. However, no study evaluated concentration of enterovirus in exhaled air in household members and air samples of household and school. Aim: Detection of enterovirus in exhaled air of child, their family and school classmates. Evalution of intrafamiliy enterovirus transmission in family and school. Method: We used exhaled air sampling method and real-time qPCR for enterovirus detection of infected child, their family and school classmates. Result: There were six index cases of children who had been diagnosed of enterovirus infection, positive rate of exhaled air samples was 83.3%. Positive rate of household members was 42.9%, children of household were 63.3%, adults of household were 44.4%. The mean concentration of exhaled air of children was 9.59x 108copies/ m3, and adults were 4.12x 109copies/ m3. Possitive rate of air samples was 15.8%, the mean concentration of air samples were 4.97x 102copies/ m3.

碩士
中臺科技大學
生物科技研究所
102
Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease in children EV71 can cause severe central nervous system disease and death. Younger children are most susceptible population for EV71 infection. The clinical manifestation in EV71 patients may vary from mild and self-limited conditions to severe neurological complications, including encephalitis, meningoencephalitis, poliomyelitis-like paralysis with lethal outcome. Currently, no anti-EV71 drug or vaccines available, clinical treatment of EV71-associated complications relies on symptomatic and supportive therapies. Therefore this study focused on the identification of the virus virulence factors for live attenuated vaccines development. Full-length virus RNA with minor genetic variants was successful generated by PCR incorporation and in vitro transcription and was transfected into Vero cell to produce virus particles. Recovered viruses possess replication ability were confirmed by immunofluorescence assay (IFA) and RT-PCR after subculture. To selecte virulence variation clones, the plaque reduction neutralization test was applied to screen antibody-escaped mutants. After plague purify, we obtained 4 different clones and named Hau1, Hau2, Hau3 and Hau5. Hau1 have amino acid mutation at 141TM of the VP2 capsid protein and 53RH of the 3A protease. Adsorption assay showed that Hau1 binding ability was greater than wild type more than 10 times. Three dimensional structure modelling showed VP2:1411TM mutation may affect neutralizing antibody and receptor binding sites. In growth curve¸ Hau1 demostrated better replication efficiency than wild type. Hau2 owned two mutation sites located at 5’UTR and VP1. Hau3 with middle size plaque have mutation sites located at 53RH and 47SF of the 3A protease and one mutation site at 268GR of the 3D polymerase. It displayed more efficiency protein expression ability than wild type. The 3D polymerase mutation brings up diversity genome amplification profiles in early stage. Hau5 with small size plaque observed two mutant sites located at 5’UTR and 3D polymerase. Summary, The genomic mutations at 5’UTRC685T, VP2T141M, 3AS48F, 3AR53H and 3DpolG268R were affected virus growth properties and may determinate virulence in host.

碩士
國立成功大學
微生物及免疫學研究所
97
Enterovirus 71 (EV71) belongs to the Human enterovirus A of Picornaviridae. Hand-foot-and-mouth disease and herpangina are the most common clinical features of EV71 infection; however, some patients are complicated with brainstem encephalitis, pulmonary edema, pulmonary hemorrhage, and cardiopulmonary failure. Inflammatory cytokines and chemokines play an important role of EV71 infection. Antibody-dependent enhancement (ADE) infection has been reported in various viruses and has been shown to contribute to disease severity. An in vitro system of EV71 infection through ADE mechanism was established using the human monocytic cell line THP-1. The percentage of EV71-infected cells was significantly enhanced at the concentration (1000-4000 μg/ml) of commercial human immunoglobulin added to THP-1, in comparison with virus-infected cell line without adding commercial human immunoglobulin. EV71 infection was able to enhance the transcription of several inflammatory mediators, including interleukin (IL)-6, IL-8, IL-10, interferon (IFN)-β, tumour necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, IFN-γ inducible protein (IP)-10 and monokine induced by IFN-γ (MIG) via ADE. To further investigate EV71 ADE mechanism in vivo, 6-day-old ICR mice were pretreated with various dilution of anti-EV71 mouse antiserum or anti-EV71 IgG 24 hours before intraperitoneal infection. We found that mice significantly showed aggravated clinical symptoms and increased death at concentration of 1：2-12 of anti-EV71 IgG on the 14 days. Histopathologically, anti-EV71 IgG-added mice also revealed enhanced neuronal and muscular damage than control. Furthermore increased levels of several cytokines and chemokines (IFN-γ, TNF-α, MCP-1) were detected in the sera of anti-EV71 antiserum-added mice. In conclusion, our results demonstrated that the ADE mechanism may involve in the EV71 pathogenesis and contribute to enhance inflammation and tissue damage.

碩士
大同大學
應用數學學系(所)
96
In order to investigate enterovirus 71,which has the highest severe complications rate and the lethality in enterovirus syndrome, we tried to construct an epidemic model to explore possible preventions of the infectious disease. This paper refered to four traditional epidemic models and two epidemic models(SEIJR and SIR epidemic model) which were used to forecast the epidemic situation and construct a differential equation model according to the mechanism of enterovirus 71.This mathematical model was temporarily called SICR epidemic model in this paper. After we simulated the development of epidemic situation with the solution of SICR epidemic model, we came up a good plan to control epidemic situation to analyze the parameter values of model that influenced epidemic situation evolved. We discovered the quarantining parameter was the most important factor to control epidemic situation, as shown in the examples. The SICR epidemic model and the result of experiment by numerical analysis may offer some preventions with reference to controlling epidemic situation of enterovirus syndrome. Until now, we were still short of epidemic situation data, therefore we only could reorganize the estimate epidemic situation parameter value from the public information. The value of experiment in the SICR epidemic model could not only use the precise parameter value, but the precision in simulation. To improve this shortcoming, it was still necessary to depend on the assistance of the medical information.

碩士
國立成功大學
微生物及免疫學研究所
95
Enterovirus 71 (EV71) causes fatal encephalitis in infants and young children, but its pathogenesis remains unclear. Previous studies show that lymphocytes were present in the central nervous system of infected-patients. However, the types of lymphocytes have not been determined. Our autopsy data show that B, CD4+, and CD8+ T cells were all present in the central nervous system of an expired EV71-infected child. So we studied the roles of lymphocytes in EV71 infection using neonatal mice without B cells, CD4+ T cells, or CD8+ T cells. Our data show that only 6% of B cell deficient mice survived while the survival rate of wild-type mice was 75% after being infected with 1 x 107 plaque forming units of virus. The viral titers in the central nervous system and peripheral organs of B cell deficient mice were significantly higher than those of wild-type mice. After adoptive transfer of the anti-EV71 antibody, 100% of B cell deficient mice survived from EV71 infection with reduced viral titers in tissues, showing that the antibody produced by B cells protected mice against EV71 infection by reducing the viral loads in infected tissues. Under this condition, we were surprised to find that the survival rate of mice with CD4+ T cell or CD8+ T cell deficiency was comparable to that of wild-type mice after infection. The viral titers in the central nervous system and peripheral organs of them were also comparable to those of wild-type mice. Additional studies show that the survival rates of CD4+ T cell deficient mice and wild-type mice depleted with CD4+ T cells using the specific antibody were still comparable after infection, suggesting the compensation of CD4+ T-cell function by other immune cells is not likely. However, when CD4+ T cell and CD8+ T cell deficient mice were infected with a higher dose (3 x 107 plaque forming units) of virus, their survival rates were significantly lower than those of wild-type mice, and the viral titers in the central nervous system and peripheral organs of them were significantly higher than those of wild-type mice. Thus, our results show that lymphocytes protect mice from EV71 infection, and that antibody produced by B cells plays a more important role than CD4+ and CD8+ T cells.

碩士
長庚大學
基礎醫學研究所
94
Enterovirus 71 (EV71) is a member of picornaviridae family. The major symptoms of enterovirus 71 infection included hand-food-and mouth disease, herpangina, and encephalitis. The 2C protein is highly conserved among all picornaviruses. Poliovirus 2C protein protein was required for viral replication. Poliovirus 2C protein induces intracelluar membranes rearrangement and vesicles accumulation. In our laboratory, we had found some putative cellular proteins that can interact with enterovirus 71 2C protein by yeast two-hybrid system. My project is using co-immunoprecipitation assay and indirect immunofluorescence assay to confirm the interaction between the N-terminal of enterovirus 71 2C protein and the putative cellular protein, intersex like protein (IXL) and G-protein-coupled receptor 125 (GPR125). In my data, I found that IXL can not interact with N-terminal of enterovirus 71 2C protein. However, we verify that N-terminal of enterovirus 71 2C can interact with GPR125 by co-immunoprecipitation assay. We further confirmed the interaction between GPR125 and N-terminal of enterovirus 71 2C protein by immunofluorescence assay. I found that both GPR125 and enterovirus 71 2C protein were colocalized in endoplasmic reticulum.

碩士
長庚大學
基礎醫學研究所
94
Enterovirus 71 (EV71) is a positive-stranded RNA virus in the genus Enterovirus of the family Picornaviridae. EV71 infection can cause hand, foot, and mouth disease (HFMD), herpangina and associated with fatal pulmonary edema as well as severe neurological complications, including encephalitis, meningitis, and a poliomyelitis-like syndrome in children. Several major outbreaks of EV71 have occurred in recent years in Taiwan. In this study we screened 22 Chinese medicine extracts for anti-EV71 activity. We found Houttuynia cordata Thunb. can neutralize the EV71-induced cytopathic effect (CPE) in Vero cells. The 50% inhibitory concentration of H. cordata on EV71 was approximately 125.92±27.837 g/mL. A plaque reduction assay showed that H. cordata can reduce the plaque formation. H. cordata was able to reduce viral protein synthesis and also abate the apoptotic process in enterovirus 71-infected Vero cells. We concluded that H. cordata possesses antiviral activity and has a potential for development as an anti-enterovirus 71 agent.

碩士
國立陽明大學
公共衛生研究所
93
Abstract Introduction - Enterovirus 71 (EV71) is a neurotropic human pathogen, but the nature of tissue tropism and receptors facilitating cell entry is laregely unknown. Aim - The aim of this study was to study cell entry and to identify EV71 receptor. Methods & Results - By use of a virus overlay protein binding assay (VOPBA) and a cholesterol-depleting agent - methyl-β-cyclodextrin (MβCD), we showed that the cellular proteins of RD cells (EV71 permissive cell line) capable of binding to EV71 are located to the lipid raft. Using a 2-dimensional PAGE to separate plasma membrane proteins of RD cells, VOPBA, MALDI-TOF and LC-MS, one EV71-binding protein was putatively identified as osteoprotegerin (OPG). Using a recombinant human OPG (rhOPG) on VOPBA, we confirmed that human OPG could bind to EV71 or VP1 of EV71 in vitro. The expression of OPG in EV71-permissive cell lines, RD & BE(2)-C (human neuroblastoma cells), was confirmed by Western blot, cytometry, and confocal microscopy; OPG is present in the cytoplasm, on cell surface, and in culture medium as secreted homodimers. A mouse L cell line (ML cells) that is originally nonpermissive to infection by EV71 but capable of supporting EV71 replication upon transfection with EV71 genomic RNA could be rendered permissive to EV71 if transfected with human OPG cDNA. ML cell line expresses mouse OPG, 89% homologous to human OPG by amino acid sequence, showed no interaction with EV71. Confocal microscopy of ML cells transfected with OPG cDNA and infected with EV71 virus showed that only cells expressing hOPG (using a human OPG specific anti-OPG antibody) were stained positive for EV71. Furthermore, OPG-ML transfectant showed an enhanced permissiveness to infection by EV71 of different genotpes. The infectivity of EV71 could be blocked by anti-OPG antibody by up to 60% in BE(2)-C cells but not in RD cells. Our data so far indicate that OPG is an EV71 binding protein, can facilitate cell entry of EV71 to ML cells, and is probably a receptor or a coreceptor for EV71. Discussion - Identification of OPG can facilitate research in pathogenesis mechanisms of viral entry.

碩士
國立成功大學
微生物及免疫學研究所
90
Enterovirus 71 (EV71), a single positive strand RNA virus, belongs to Picornaviridae. Since the first epidemic in 1973, there have been several outbreaks of hand-foot-mouth disease (HFMD) caused by EV71 in the United States, Australia, and East Asia. An outbreak at Taiwan in 1998, caused 55 children die with central nervous system (CNS) involvement. The fecal-oral route is thought to be the predominant mode of enterovirus transmission. EV71 has been associated with an array of clinical syndromes including HFMD, aseptic meningitis, encephalitis, polio-like myelitis, and paralysis among infants and children. Its pathogenesis is not clearly understood. There is also no animal model to understand how enterovirus spreads to the central nervous system (CNS). Therefore, we established a murine model of EV71 infection by oral route to understand viral spread in vivo. EV71 can infect intestinal epithelial cell line Caco-2 and neuroblastoma cell line SK-N-SH. The cell- or mouse-adapted EV71 was used to infect murine neonatal intestine ex vivo. The EV71 viral protein 1 (VP1) was detected in intestine epithelial cells 2 days post infection, and stronger staining was observed in tissues infected with mouse-adapted strain than those infected with Caco-2 adapted or parental one. Since mouse-adapted EV71 can successfully infect murine neonatal intestine, we further explored the EV71 infection orally. Oral inoculation of 1 x 106 PFU mouse-adapted EV71 into neonate mice caused flaccid paralysis and death in 50 % of mice at 6-9 days post infection while parental EV71 only induced mild skin lesions. The mortality increased to 100 % at day 3 post infection when mice were inoculated with 1 x 107 PFU of virus. EV71 can be re-isolated from intestine, blood, spleen, brain stem, brain, spinal cord, heart, liver, lung, skin, and hind limb muscle of the infected mice. The pathological analysis found cell infiltration in brain stem, spinal cord and intestine. The kinetic study showed that two-phase viremia appeared at 6 and 24 hours post infection, respectively. The EV71 VP1 antigen first occurred in intestinal muscularis externa, then in cervical and lumbar spinal cord, and finally in the brain stem. Furthermore, EV71 can spread between littermates by fecal route or close contact. This animal model mimics the clinical EV71 infection, and can be used for further study of EV71 pathogenesis and screening of antiviral drugs.