Análise transcriptômica da interação mamoeiro-Papaya Meleira Virus

O mamoeiro (Carica papaya L.) é uma das fruteiras mais cultivadas nas regiões tropicais e subtropicais do mundo. O Brasil faz parte do grupo dos países que mais produzem e exportam mamão no mundo. O Espírito Santo e a Bahia são responsáveis por mais de 70% da área brasileira produtora deste fruto. Porém, doenças causadas por microrganismos infecciosos afetam de modo considerável sua produção. Entre as principais doenças, destaca-se a meleira do mamoeiro, causada pelo Papaya meleira virus (PMeV), que ainda não possui uma cultivar resistente. Interessantemente os sintomas somente são desencadeados após a frutificação. Os mecanismos moleculares envolvidos no desenvolvimento dos sintomas e na resposta de defesa da planta ao PMeV ainda não foram esclarecidos. Para entender os pontos chaves desta interação, que permitam o desenvolvimento de metodologias de melhoramento genético, um estudo transcriptômico foi abordado. A tecnologia RNA-seq foi usada para o sequenciamento do transcriptoma a partir de plantas com 3, 6 e 8 meses de idade após plantio, inoculadas e não inoculadas com o PMeV. Os genes diferencialmente expressos nos 3 tempos e nas duas condições foram preditos e analisados. Estas análises revelaram um padrão de expressão geral dos genes envolvidos nesta interação. Foram encontrados 21 genes com o perfil de expressão alterado nas plantas inoculadas exclusivamente nos seis meses de idade. Destes, 8 genes envolvidos em processos de respostas de defesa e morte celular, resposta ao estresse e resposta ao estímulo biótico e abiótico foram reprimidos; enquanto os demais (13 genes), envolvidos principalmente em processos metabólicos primários, biogêneses, diferenciação e ciclo celular, comunicação e crescimento celular, bem como processos envolvidos em reprodução, e desenvolvimento da floração, foram superexpressos. Estes resultados sugerem que, aos seis meses de idade, a planta é obrigada a alterar seu programa de expressão gênica, direcionando a resposta para os processos próprios do desenvolvimento, requeridos nesse estádio fisiológico, que primam sob a resposta ao estresse, fato que finalmente leva ao desenvolvimento dos sintomas.

Preliminar characterization of a new virus isolated from the spinal fluid of patients with meningitis in São Paulo, Brazil

A total of 138 patients with the age of 4 months to 57 years were attended in different hospitals of São Paulo State with aseptic meningitis. A probable new agent was isolated from the cerebrospinal fluid of 35 of 53 specimens examined. Replication of the agent with similar characteristics was detected by CPE produced in the MDCK cell line. Virus-like particles measuring about 40 nm in diameter were observed by negative staining electron microscopy. No hemaglutinating activity was detected at pH 7.2 by using either human, guinea pig, chicken and at pH ranged 6.0 - 7.2 with goose red blood cells. The agent was not pathogenic to newborn or adult mice. Virus infectivity as measured by CPE was sensitive to chloroform and not inhibited by BuDR, suggesting that agent is an enveloped virus with RNA genome.

Occurrence of influenza B/Hong Kong-Like strains in Brazil, during 2002

Through the influenza virus surveillance from January to October 2002, influenza B/Hong Kong-like strains circulating in the Southeast and Centre East regions of Brazil have been demonstrated. This strain is a variant from B/Victoria/02/88 whose since 1991 and until recently have been isolated relatively infrequently and have been limited to South-Eastern Asia. A total of 510 respiratory secretions were collected from patients 0 to 60 years of age, with acute respiratory illness, living in the Southeast and Centre East regions of Brazil, of which 86 (17.13%) were positive for influenza virus. Among them 12 (13.95%) were characterized as B/Hong Kong/330/2001; 3 (3.49%) as B/Hong Kong/1351/2002 a variant from B/Hong Kong/330/2001; 1 (1.16%) as B/Sichuan/379/99; 1 (1.16%) as B/Shizuoka/5/2001, until now. The percentages of cases notified during the surveillance period were 34.88%, 15.12%, 15.12%, 4.65%, 15.12%, 13.95%, in the age groups of 0-4, 5-10, 11-15, 16-20, 21-30, 31-50, respectively. The highest proportion of isolates was observed among children younger than 4 years but serious morbidity and mortality has not been observed among people older than 65 years, although B influenza virus component for vaccination campaign 2002 was B/Sichuan/379/99 strain. This was probably due to the elderly protection acquired against B/Victoria/02/88. In addition, in influenza A/Panama/2007/99-like (H3N2) strains 22 (25.58%) were also detected, but influenza A(H1N1) has not been detected yet.

Fulminant Myocarditis Associated with Pandemic H1N1 Influenza A Virus

Fulminant myocarditis associated with influenza A virus is exceedingly rare, with only a few cases reported in the literature. We describe a previously healthy 10-year-old boy, with a three-day history of flu-like symptoms without antiviral treatment. He was hospitalized with dehydration and hypothermia in the context of persistent vomiting, when he suddenly developed heart failure secondary to fulminant myocarditis. Despite aggressive management, including circulatory support and cardiopulmonary resuscitation measures, the patient died of cardiogenic shock. The postmortem histopathology was compatible with a multisystem viral infection with myocarditis and pulmonary involvement, and H1N1v polymerase chain reaction was positive. The prevalence of influenza-associated fulminant myocarditis remains unknown. Findings reported in the literature raise the possibility that the novel H1N1 influenza A virus is more commonly associated with a severe form of myocarditis than previously encountered influenza strains.

Refractory pemphigus vulgaris associated with herpes infection: case report and review

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by blistering of the skin and mucosa, which develops due to the interaction between predisposing genetic and environmental factors. Infections caused by members of the Herpesviridae family have been suggested as a possible triggering factor for PV. OBJECTIVE AND METHODS: In this report, we investigate the presence of herpesviruses in refractory lesions on the right upper eyelid. The lesion has persisted despite the treatment with corticosteroids. Polymerase chain reaction (PCR) and DNA sequence analysis have been used to detect the DNA of HSV 1/2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8. RESULTS: The sample collected from the right upper eyelid has tested positive for HSV 1/2. Sequence analysis has confirmed the PCR results and allowed the identification of the HSV strain as belonging to type 1. After treatment with acyclovir, the lesion of the right upper eyelid has cleared and not relapsed. CONCLUSION: When patients present PV lesions which are refractory to corticosteroid therapy, herpetic infection should be considered.

Integrated functional genomics for improved manufacture of recombinant enveloped virus

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences-Biotechnology

Cytokine serum levels in patients infected by human immunodeficiency virus with and without Trypanosoma cruzi coinfection

This study assessed the number of CD4 T lymphocytes, the parasitemia and serum levels of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-4 and IL-10 of patients infected by human immunodeficiency virus (HIV) and human immunodeficiency virus/Chagas' disease coinfection. CD4 T lymphocytes were low in the two groups of patients, although significantly lower in patients without Chagas' disease. Serum levels of IFN-gamma, IL-4 and TNF-alpha were significantly higher in patients with HIV/Chagas' disease. IL-4/IFN-gamma ratios were higher in patients with HIV/Chagas' disease, which showed a clear balance in favor of Th2-like cytokines in this group of patients. This Th2 balance was higher in patients with detectable parasitemia. We conclude that, although immunosuppression was observed, with CD4 T lymphocytes bellow 200/µm³, these patients did not display reactivation of T. cruzi infection and that a balance favorable to Th2 was associated with the presence of parasitemia.

Investigation of measles IgM-seropositive cases of febrile rash illnesses in the absence of documented measles virus transmission, State of São Paulo, Brazil, 2000-2004

INTRODUCTION: To review measles IgM-positive cases of febrile rash illnesses in the State of São Paulo, Brazil, over the five-year period following interruption of measles virus transmission. METHODS: We reviewed 463 measles IgM-positive cases of febrile rash illness in the State of São Paulo, from 2000 to 2004. Individuals vaccinated against measles < 56 days prior to specimen collection were considered to be exposed to the vaccine. Serum from the acute and convalescent phases was tested for evidence of measles, rubella, parvovirus B19 and human herpes virus-6 infection. In the absence of seroconversion to measles immunoglobulin-G, measles IgM-positive cases were considered false positives in individuals with evidence of other viral infections. RESULTS: Among the 463 individuals with febrile rash illness who tested positive for measles IgM antibodies during the period, 297 (64%) were classified as exposed to the vaccine. Among the 166 cases that were not exposed to the vaccine, 109 (66%) were considered false positives based on the absence of seroconversion, among which 21 (13%) had evidence of rubella virus infection, 49 (30%) parvovirus B19 and 28 (17%) human herpes virus-6 infection. CONCLUSIONS: Following the interruption of measles virus transmission, thorough investigation of measles IgM-positive cases is required, especially among cases not exposed to the vaccine. Laboratory testing for etiologies of febrile rash illness aids interpretation of these cases.

Lessons from the epidemiological surveillance program, during the influenza A (H1N1) virus epidemic, in a reference university hospital of Southeastern Brazil

INTRODUCTION: The case definition of influenza-like illness (ILI) is a powerful epidemiological tool during influenza epidemics. METHODS: A prospective cohort study was conducted to evaluate the impact of two definitions used as epidemiological tools, in adults and children, during the influenza A H1N1 epidemic. Patients were included if they had upper respiratory samples tested for influenza by real-time reverse transcriptase polymerase chain reaction during two periods, using the ILI definition (coughing + temperature > 38ºC) in period 1, and the definition of severe acute respiratory infection (ARS) (coughing + temperature > 38ºC and dyspnoea) in period 2. RESULTS: The study included 366 adults and 147 children, covering 243 cases of ILI and 270 cases of ARS. Laboratory confirmed cases of influenza were higher in adults (50%) than in children (21.6%) ( p < 0.0001) and influenza infection was more prevalent in the ILI definition (53%) than ARS (24.4%) (p < 0.0001). Adults reported more chills and myalgia than children (p = 0.0001). Oseltamivir was administered in 58% and 46% of adults and children with influenza A H1N1, respectively. The influenza A H1N1 case fatality rate was 7% in adults and 8.3% in children. The mean time from onset of illness until antiviral administration was 4 days. CONCLUSIONS: The modification of ILI to ARS definition resulted in less accuracy in influenza diagnosis and did not improve the appropriate time and use of antiviral medication.

Hemophagocytic lymphohistiocytosis associated with H1N1 virus infection and visceral leishmaniasis in a 4.5-month-old infant

We present a case of a 4.5-month-old boy from Turkey with hemophagocytic lymphohistiocytosis (HLH) associated with H1N1 virus and Leishmania spp. coinfection. Because visceral leishmaniasis can mimic hematologic disorders like HLH, it is important to rule out this clinical condition before starting immunosuppressive therapy. In our case, treatment with liposomal amphotericin B resulted in a dramatic resolution of clinical and laboratory abnormalities.

Sexual transmission of human T-cell lymphotropic virus type 1

Human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in many parts of the world and is primarily transmitted through sexual intercourse or from mother to child. Sexual transmission occurs more efficiently from men to women than women to men and might be enhanced by sexually transmitted diseases that cause ulcers and result in mucosal ruptures, such as syphilis, herpes simplex type 2 (HSV-2), and chancroid. Other sexually transmitted diseases might result in the recruitment of inflammatory cells and could increase the risk of HTLV-1 acquisition and transmission. Additionally, factors that are associated with higher transmission risks include the presence of antibodies against the viral oncoprotein Tax (anti-Tax), a higher proviral load in peripheral blood lymphocytes, and increased cervicovaginal or seminal secretions. Seminal fluid has been reported to increase HTLV replication and transmission, whereas male circumcision and neutralizing antibodies might have a protective effect. Recently, free virions were discovered in plasma, which reveals a possible new mode of HTLV replication. It is unclear how this discovery might affect the routes of HTLV transmission, particularly sexual transmission, because HTLV transmission rates are significantly higher from men to women than women to men.

Modificaciones de Parámetros Inmunológicos en niños infectados por virus Varicela-Zoster

En nuestro país se infectan anualmente de varicela entre 350 y 400 mil individuos. El 90% son menores de 10 años y la mayoría de casos fatales ocurren en pacientes inmunocomprometidos. Los costos sanitarios directos causados por esta enfermedad alcanzan los 2 millones de dólares por año, además de los gastos generados por el lucro cesante de los padres de los niños afectados. Los niños alérgicos con crisis asmáticas a repetición que no realizan el tratamiento adecuado suelen recibir frecuentes dosis de glucocorticoides de difusión sistémica, con propiedades inmunosupresoras. (...) Se ha visto que existe una relación directa entre la frecuencia de neumonía por virus de varicela zoster (V-Z) e incidencia de mortalidad. (...) Aciclovir es una droga inerte para células sanas y de actividad específica contra el virus que reduce la severidad y extensión de las lesiones, favorece la curación y las posibilidades de las recurrencias. Aciclovir ha demostrado ser significativamente útil para reducir el tiempo de aparición de nuevas lesiones, su número, porcentaje de pacientes febriles en los primeros días y el número de lesiones residuales hipopigmentadas al mes. Se ha establecido que Aciclovir es un antiviral de elección en el tratamiento de las infecciones herpéticas. Sin embargo, en pacientes inmunosuprimidos infectados con virus Herpes y tratados con la droga pueden surgir mutantes virales resistentes. Se evaluará la eficiencia del antiviral Aciclovir en pruebas "in vitro". (...) Se estudiarán 60 niños alérgicos menores de 10 años infectados por virus VZ (varicela o herpes). (...)

Infección de macrófagos con virus encefalitis saint louis: efecto sobre el fenotipo celular y la apoptosis (Programa: enfermedades transmisibles y emergentes).

El Virus Encefalitis Saint Louis (VESL)es un virus neurotrópico que puede provocar en humanos encefalitis, meningitis y cefalea febril. Estudios epidemiológicos demostraron la circulación del virus en Argentina, reportándose el primer brote de encefalitis en Sud-América en Córdoba en el 2005. Los macrófagos tienen un rol muy importante en la patogénesis de las infecciones virales. Estas células son permisivas para la replicación y reservorio viral. Reconocen a los virus mediante receptores de reconocimiento de patrones, incluidos los receptores Toll-like, lo que genera la producción de moléculas antivirales y citoquinas pro-inflamatorias. Los macrófagos expresan diferentes fenotipos según el microambiente tisular y el estímulo externo. Se reconocen los macrófagos activados clásicamente (M1) que liberan citoquinas pro-inflamatorias y los macrófagos activados alternativamente (M2) que producen IL-10 y factor transformante del crecimiento. Como parte de la respuesta del macrófago a la infección viral, prolifera, se diferencia y muere. La apoptosis es un mecanismo de muerte que limita la actividad del macrófago activado. La interacción virus-macrófago ha sido analizada con numerosos tipos de virus. Sin embargo, existe escasa información sobre el impacto de VESL sobre la respuesta inmune innata. La emergencia de esta virosis en nuestro medio amerita abordar distintos aspectos de la respuesta inmune en esta infección. Este proyecto tiene como objetivo estudiar la interacción VESL-macrófago para esclarecer el rol del fenotipo celular y su relación con la depuración viral. Además, analizar la naturaleza y el tenor de los inmunomoduladores liberados y el papel de la apoptosis de los macrófagos en esta infección.

Infección de macrófagos con el Virus Encefalitis Saint Louis: efecto sobre el fenotipo celular y la apoptosis.

El virus Encefalitis Saint Louis (VESL) (género Flavivirus) experimenta una re-emergencia en la región central del país, con la ocurrencia de un brote en Córdoba y el aislamiento de cepas de distintos genotipos. Está demostrado que los Flavivirus neurotrópicos, como VESL, replican en macrófagos y células dendríticas, tanto en el tejido local como en nódulos linfáticos satélites, para luego llegar a torrente sanguíneo y ser transportados a sistema nervioso central. Es así que el nivel de viremia inicial es regulado por la depuración del virus que realizan los macrófagos. Estas células reconocen a los virus por medio de receptores de reconocimiento de patrones moleculares asociados a patógenos, que incluyen a los receptores Toll-like (TLR). La relación entre los TLR y los virus, se fundamenta en tres aspectos: 1) los TLR al ser estimulados por moléculas derivadas de virus activan vías de señalización que inducen la producción de citoquinas pro-inflamatorias, como TNF- , IL-1, 6, 8 y 18, INF-  y , que median la respuesta inmune antiviral; 2) las señales que dependen de los TLR median efectos inmunopatogénicos, como la apoptosis y la patogénesis del virus; 3) algunas estrategias terapéuticas o profilácticas antivirales se basan en la estimulación de los TLR mediante los respectivos agonistas. Como parte de la respuesta del macrófago a la infección viral, hay proliferación, diferenciación y muerte celular. A la hora de morir, estas células pueden seguir el camino que lleva a la necrosis o el de la apoptosis. Durante la activación de la respuesta inmune frente a antígenos extraños, la apoptosis es requerida para eliminar las células efectoras, una vez que han ejecutado su función y así evitar el desarrollo de procesos deletéreos para el huésped. Estudios realizados con distintos Flavivirus documentan el incremento de apoptosis de macrófagos durante la progresión de la infección y también su relación con la severidad de la patología. De acuerdo a los antecedentes expuestos, se formulan las siguientes hipótesis de estudio: 1-El fenotipo de activación del macrófago infectado con VESL está relacionado con el genotipo viral. 2-La clase de inmunomoduladores liberados y el grado de apoptosis de los macrófagos infectados con el VESL dependen del receptor de reconocimiento utilizado por el virus.El objetivo principal es caracterizar la respuesta inmune inducida en macrófagos infectados in vitro con diferentes genotipos de VESL. Para ello se plantean los siguientes objetivos específicos: 1-Determinar la capacidad de replicación de VESL en macrófagos.2-Evaluar la expresión de molécula de superficie, receptores y la producción de inmunomoduladores en macrófagos infectados con VESL.3-Analizar el impacto de la infección con VESL sobre la apoptosis de macrófagos.4-Correlacionar la expresión de antígenos de superficie, receptores, producción de inmunomoduladores, apoptosis y carga viral con el genotipo viral que infecta al macrófago.Se utilizará una línea línea celular mieloide U937 y cepas del VESL genotipo III, V y VII. Se estudiará la infección de las mismas y determinará la expresión de: CD14, CD16, CD54/ICAM-1, HLA-DR, Fas, R-TNF, CD86, IL4R, TLR2, TLR3, TLR4 y TLR7 por Citometría de Flujo. En el sobrenadante de los cultivos infectados se cuantificarán las concentraciones de IFN-, IFN-, TNF-, IL-1, IL-6, IL-8, IL-10, IL-12, IL-18 y TGF- por técnica de ELISA.Se determinará la apoptosis en los macrófagos infectados mediante marcación con Anexina V-Ficoeritrina y análisis de fragmentación del ADN.La emergencia de esta virosis en nuestro medio amerita abordar distintos aspectos de la respuesta inmune en esta infección. El conocimiento de las características de la activación del macrófago cuando se infecta con VESL, los inmunomoduladores liberados y el impacto de la infección sobre la apoptosis de ésta célula, aportaría posibles blancos para el diseño futuro de estrategias terapéuticas o profilácticas contra esta infección.

Alterações nucleares das cellulas do figado nas infecções de Macacus Rhesus e M. Cynomolgus pelo virus da febre amarella

No figado de um Macacus rhesus inoculado por BEAUREPAIRE ARAGÃO com sangue deum caso benigno de febre amarella e no qual elle descreveu symptomas e lesões typicas semelhantes ás obidas por STOKES, BAUER e HUDSON pela inoculação com o virus africano no mesmo animal, encontrámos alterações nucleares da mesma natureza das assignaladas, no herpes zoster, herpes symptomatico, varicella e virus III do coelho e descriptas ora sob o nome de "inclusões acidophilas intranucleares" (LIPSCHÜTZ, GOODPASTURE), ora sob o de "degeneração oxychromatica" (LAUDA e LUGER). Alterações nucleares semelhantes da cellula hepatica encontrámos, posteriormente em 13 M. rhesus e 2 M. cynomolgus inoculados com e virus brasileiro da febre amarella o qual fôra isolado independentemente por BEAUREPAIRE ARAGÂO e depois por A. MARQUES DA CUNHA e J. MUNIZ de dois casos benignos de febre amarella, tendo sido um dos macacos injectado directamente com o sangue do doente; dois macacos foram inoculados com Aedes aegypti infectados em homem e em macaco; os animais foram anímaes empregados em passagens em serie do virus pelo macaco, e talvez esse facto explique até certo ponto, as notaveis differenças por vezes encontradas nas alterações histopathologicas do figado, visto como, em condições naturaes, o virus nunca passa directamente de homem para homem. A intensidade com que se apresenta a degeneração oxychromatica de modo algum está na dependencia das alterações do conteúdo gorduroso, necrose e necrobiose encontradas; em um caso, ella era a unica alteração presente no figado, sendo então particularmente intensa. As inclusões acidophilas intranucleares (degeneração oxychromatica) não foram encontradas em diversos M. rhesus não inoculados e mortos por causas obscuras; no entanto, em taes figados eram presentes infiltração e degeneração gordurosas associadas a alterações de necrose e necrobiose. Alguns estadios (figuras intranucleares "em borboleta" e "em ameba", v. fig. g e h, Est. colorida) sendo abundantes, facilitam, em virtude de sua configuração especial, o reconhecimento da degeneração oxychromatica em córtes feitos segundo uma technica rapida (pequenos fragmentos de figado fixados em formol aquecido a 60°C.-trinta minutos, córtes em congelação, hematoxylina, eosina, alcool absoluto, phenol-xylol-creosoto, xylol, balsamo), não tendo, afóra isso, importancia especial. Ao passo que a degeneração e infiltração gordurosa, bem como a necrose e a necrobiose da cellula hepatica apresentam variações consideraveis em sua intensidade de um para outro animal em uma passagem em serie não interrompida do virus pelo M. rhesus, chegando mesmo a faltar interamente em dois animaes cujo figado, no entanto, mostrou-se capaz de reproduzir symptomas e lesões typicas na passagem seguinte, as inclusões acidophilas intranucleares (degeneração oxychromatica), de regra, se encontram com muito maior regularidade...