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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

With the absence of gut microbiota, gnotobiotic (Gn) pigs are a unique animal model for studying infection and immunity, and evaluating vaccine and therapeutics for human enteric pathogens. Here, we demonstrate Gn pigs as effective large animal models for human enteric viruses, through evaluating human enterovirus 71 (EV71) infection and immunity, and vaccine and therapeutics for human rotavirus (HRV). Gn pigs could be infected via oral or oronasal route, the natural route of infection. Infected pigs developed clinical signs including fever, neurological and respiratory signs, similar to those seen in human patients. Fecal shedding up to 18 days post infection and virus distribution in intestinal, respiratory and central nervous system tissues were observed. Strong mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells) and systemic B cell responses (serum neutralizing antibodies) were also detected. The study demonstrates a novel large animal model for EV71 to investigate viral pathogenesis, immunity, and to evaluate vaccine and antiviral drugs. Using the well-established Gn pig model for HRV, the adjuvant and therapeutic effects of prebiotics rice bran (RB) and probiotics were evaluated. RB alone or RB plus probiotic Lactobacillus rhamnosus GG (LGG) and probiotic E. coli Nissle 1917 (EcN), were shown to protect against rotavirus diarrhea (80%-100% reduction in the incidence rate) significantly and display strong immune - stimulatory effects on the immunogenicity of an oral attenuated HRV (AttHRV) vaccine. Mechanisms for the adjuvant effect include stimulating the production of intestinal and systemic IFN-γ] producing T cells and promoting mucosal IgA antibody responses. The mechanisms for reducing rotavirus diarrhea include promoting LGG and EcN growth and colonization and host gut health, and maintaining gut integrity and permeability during rotavirus infection. We showed that RB plus LGG and EcN is a highly effective therapeutic regimen against HRV diarrhea. Together, these results indicated that Gn pigs may serve as an excellent animal model for the study of infection, immunity, vaccine and therapeutics for human enteric viruses. / Ph. D.

Made available in DSpace on 2016-01-26T12:51:40Z (GMT). No. of bitstreams: 1 marcelacristinabygermini_dissert.pdf: 1767156 bytes, checksum: 3ac31a5282ff9f5d13908111e0e281cb (MD5) Previous issue date: 2012-07-31 / 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.