Diagnóstico, epidemiologia e caracterização molecular do Herpesvírus humano 2 (HHV-2) em mulheres profissionais do sexo e gestantes

Made available in DSpace on 2018-01-30T16:39:40Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) lyana_lima_ioc_dout_2017.pdf: 4370983 bytes, checksum: 5f713a2a13522b266d5053290d59d78d (MD5)Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.O Herpesvirus humano 2 é transmitido por via sexual e destaca-se como o principal causador do herpes genital no mundo, uma infecção altamente prevalente em mulheres. O herpes genital é caracterizado pelo desenvolvimento de vesículas bolhosas e dolorosas na região genital, porém a maioria dos indivíduos infectados não apresenta manifestações e não tem conhecimento sobre sua infecção. As mulheres desempenham um papel relevante na epidemiologia do HHV-2, pois além de serem capazes de transmitir o vírus sexualmente, elas podem transmitir o vírus de forma vertical para o feto, levando a um quadro de herpes neonatal. Diante disso e atrelado a falta de estudos que investiguem o HHV-2 no Brasil, esta tese teve como objetivo realizar o diagnóstico, estudar a epidemiologia e a diversidade molecular do HHV-2 em diferentes populações de mulheres profissionais do sexo e gestantes positivas e negativas para o HIV. Esta tese resultou em cinco artigos científicos, totalizando a análise de 646 amostras e envolvendo três populações distintas: mulheres profissionais do sexo, gestantes negativas para o HIV e gestantes positivas para o HIV. Para otimizar o diagnóstico, oligonucleotídeos sintéticos desenhados para quantificação absoluta do HHV-1 e HHV-2 por PCR em tempo real foram utilizados como curvas padrão para quantificação viral, e os resultados obtidos foram semelhantes ao padrão ouro utilizado no estudo (curva de DNA). Para avaliar o papel da mulher na transmissão sexual do HHV-2, 376 mulheres profissionais do sexo do Mato Grosso do Sul foram investigadas Os resultados mostraram alta prevalência (47,3%) e presença de infecção primária com viremia detectada. As variáveis idade e número de parceiros sexuais se apresentaram como fatores de risco para a infecção causada pelo HHV-2. O uso de preservativo e o consumo diário de álcool diminuíram significativamente a chance de infecção. Para investigar o risco de transmissão vertical, 270 gestantes HIV negativas e positivas do Rio de Janeiro foram investigadas. Os resultados mostraram que a prevalência do HHV-2 em gestantes HIV-positivas foi quase 3 vezes maior (59,7%) que nas HIV-negativas (20,6%). A viremia do HHV-2 foi detectada em ambos os grupos durante quadros de infecção primária. As gestantes HIV positivas também apresentaram o DNA viral detectado no sangue durante casos de recorrência e eliminação assintomática. As variáveis estudadas que foram estatisticamente (p<0,05) associadas ao HHV-2 foram idade, relação CD4/CD8. O sequenciamento da glicoproteína B (gB) das primeiras cepas brasileiras revelaram que as mesmas pertencem ao clado B, porém duas cepas sequenciadas se destacaram por apresentar mais de 1% de divergência com as cepas previamente descritas, sugerindo a existência de um novo clado. Os resultados dessa tese construirão para facilitar o diagnóstico da infecção e a implantação de medidas de prevenção e controle do herpes genital em mulheres em idade sexualmente ativaHuman Herpesvirus 2 (HHV-2) is sexually transmitted and is the main cause of genital herpes in the world, and it is highly prevalent among women. Genital herpes is characterized by the development of bullous and painful vesicles in the genital region, but a majority of infected patients has no symptoms and is unaware of their infection. Women play a relevant role in HHV-2 epidemiology because they can pass the virus on to their sexual partners and to their babies through vertical transmission, causing neonatal herpes. In view of this and linked to the lack of studies that investigate HHV-2 in Brazil, this thesis aimed to perform the diagnosis, to study the epidemiology and molecular epidemiology of HHV-2 among female sex workers and pregnant women with and without HIV. This thesis results in five articles, envolving three diferent populations and 646 samples were analyzed. To optimize the diagnosis, synthetic oligonucleotides designed for absolute quantification of HHV-1 and HHV-2 by real-time PCR were used as standard curves for viral quantification, and the study results were similar to the gold standard used in the study (DNA curve). To evaluate the role of women in the HHV-2 sexual transmission, 376 female sex workers from Mato Grosso do Sul were investigated. The results shown high prevalence (47.3%) and presence of primary infection with detectable levels of viremia.The variables age and number of sexual partners were the risk factors for the HHV-2 infection Use of condoms and alcohol consuming decrease the chance of HHV-2 infection.To investigate the risk of vertical transmission, 270 HIV negative and positive pregnant from Rio de Janeiro were investigated. The results showed that the prevalence of HHV-2 among HIV-positive pregnant women was 3 times higher (59.7%) than among HIV-negative pregnant women (20.6%). HHV-2 viremia was detected in both groups during primary infection. HIV-positive pregnant had viral DNA detected in blood during recurrent herpes and asymptomatic shedding.The studied variables that were associated to HHV-2 with statistically significant (p < 0.05) were age, CD4/ CD8 ratio. Glycoprotein B (gB) sequencing of the first Brazilian strains revealed that they belonged to clade B, but two strains were highlighted because they showed more than 1% divergence when compared with previously described strains, suggesting a new clade. The results of this thesis will contribute to facilitate the diagnosis of the infection and the implantation of the measures of prevention and control for genital herpes among women of sexually active ag

Novel variants of human herpesvirus 2 from Brazilian HIV-1 coinfected subjects

BACKGROUND Human herpesvirus 2 (HHV-2) have DNA genome with a limited genetic variability and have been classified into two clades. OBJECTIVES To identify and characterise six HHV-2 isolates derived from Brazilian women. METHODS HHV-2 isolates were performed polymerase chain reaction (PCR) and sequencing of 2250 pb of the glycoprotein B (gB) coding regions. FINDINGS Four HHV-2 isolates were classified into clade B, while the remaining two, derived from HIV-1 co-infected women, showed a notable genetic divergence (> 1%). MAIN CONCLUSION The results reveal novel HHV-2 variants. The impact of these novel variants on HHV-2 pathogenesis and HIV/HHV-2 coinfection need to be investigated

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

Made available in DSpace on 2015-08-19T13:49:40Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) gentil_bentes_etal_IOC_2015.pdf: 573420 bytes, checksum: 9fb606d9da99892ab59cedf804d67ded (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Comparada e Ambiental. Rio de Janeiro, RJ,Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Comparada e Ambiental. Rio de Janeiro, RJ,Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Patologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies

Chloroquine and Sulfadoxine Derivatives Inhibit ZIKV Replication in Cervical Cells

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 &micro;M of C-Sds and observed CC50 values that ranged from 112.0 &plusmn; 1.8 to &gt;200 &micro;M in cervical cells and 43.2 &plusmn; 0.4 to 143.0 &plusmn; 1.3 &micro;M in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 &micro;M of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 &micro;M induced a reduction of 79.8% &plusmn; 4.2% to 90.7% &plusmn; 1.5% of ZIKV&ndash;envelope glycoprotein expression in infected cells as compared to 36.8% &plusmn; 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 &plusmn; 0.7 &times; 108 copies/mL in vehicle control. The dose&ndash;response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 &micro;M, respectively, while 25 &micro;M inhibited 100% of viral progeny. The treatment of Vero cells at 12 &micro;M led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC50 values ranged from 3.2 &plusmn; 0.1 to 5.0 &plusmn; 0.2 &micro;M, and the EC90 values ranged from 7.2 &plusmn; 0.1 to 11.6 &plusmn; 0.1 &micro;M, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity