Uma série histórica do HTLV na Bahia durante o período entre 2015 a 2019

Objetivo:  Descrever a frequência do HTLV na Bahia e no Sudoeste Baiano no período entre 2015 a 2019. Métodos: pesquisa trata-se de um estudo epidemiológico, retrospectivo, observacional de abordagem quantitativa do tipo série histórica. Os dados foram coletados do banco de dados do Departamento de Informática do Sistema Único de Saúde (DATASUS), por meio da consulta às bases de dados do Sistema de Informações de Agravos de Notificação (SINAN) e do Departamento de Doenças e Condições Crônicas e Infecções Sexualmente Transmissíveis (DCCI). Resultados e discussão: A região leste, foi a mais acometida nos anos analisados, sendo que, no ano de 2015 foram notificados 153 casos, em 2016 foram notificados 170 casos, em 2017 foram notificados 477 casos, em 2018 cerca de 390 casos e no ano de 2019 foram registradas 52 notificações de casos de HTLV. Conclusão: Os dados deste estudo sugerem que, durante os anos de 2015 a 2019 a região leste foi a mais acometida pelo HTLV na Bahia, seguido da região sul e sudeste. A região que apresentou o maior número de casos confirmados na Bahia foi a região leste, seguido da região centro-leste e da região sul. Além disso, todas as regiões de saúde apresentaram casos inconclusivos no diagnóstico, exceto a região oeste. A região leste foi a que apresentou o maior número de casos inconclusivos no período analisado

αVβ3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication

αVβ3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil

Brain natriuretic peptide based strategy to detect left ventricular dysfunction in Chagas disease : a comparison with the conventional approach.

Background: Left ventricular dysfunction (LVd) is the main predictor of mortality in Chagas disease (ChD). Aims: To compare the diagnostic performance of the conventional approach (ECG and chest X-ray) in the recognition of LVd in ChD, with a new strategy, in which BNP is measured in patients with an abnormal ECG. Methods: Consecutive ChD patients recruited at an Outpatient Reference Center in Belo Horizonte, Brazil, without other systemic diseases, in 1998–99 (sample 1, n =165) and in 2001–02 (sample 2, n =62) underwent ECG, chest X-ray, BNP measurement and echocardiography. Results: The prevalence of LVd (ejection fraction _0.40) was 9.1% in the sample 1. The conventional strategy recognized all patients with LVd (sensitivity: 100%, 95% CI: 79.6–100% and negative predictive value _PV 100%, 92.1–100%), but with low specificity (30%, 95% CI: 23.2–37.8) and +PV (12.5%, 95% IC: I7.7–19.6). The BNP/ECG strategy showed significantly better specificity (96.0%, 95% CI: 91.5–98.2, p <0.001) and +PV (66.7%, 95% CI: 43.7–83.7, p <0.001), and non-significantly lower sensitivity (80.0%, 95% CI: 54.8–93.0, p =0.25) and _PV (98.0%,95% CI: 94.2–99.3, p =0.08). Overall accuracy was improved with the new strategy. (94.5%,95% CI: 90.0– 97.1_36.4%, 95% CI: 29.4–43.9, p <0.001). Similar results were obtained for the sample 2. Conclusions: The BNP-based strategy was more accurate than the conventional approach in the detection of LVd in ChD patients and should be considered as a valid option

Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs

<div><p>Hendra virus (HeV) and Nipah virus (NiV) belong to the genus <i>Henipavirus</i> in the family <i>Paramyxoviridae</i>. Henipavirus infections were first reported in the 1990’s causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus <i>Pteropus</i> have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G) proteins as well as the full-length NiV nucleocapsid (N) protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA) were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses.</p></div

Purification and detection of ELISA antigens.

<p><b>A</b>. Immunoblot of purified his-tagged NiV N expressed in <i>Escherichia coli</i>. The identity of the NiV N protein was confirmed by anti-N monoclonal antibody F45G4 using enhanced chemiluminescence. <b>B</b>. Negative contrast electron microscopy of an inclusion body fraction of semi-purified NiV N antigen. The specimen grids were examined in a Philips CM 120 transmission electron microscope, operating at an accelerating voltage of 80 kV. Micrographs were taken between 28,000X–45,000X using Kodak Electron Microscope Film 4489. The negatives were scanned using an Epson Perfection 3200 photo scanner and enlarged 2.5X. <b>C</b>. Coomassie staining of purified recombinant sHeV G and sNiV G protein elution fractions. Purification of Strep-tagged sHeV G (A) and sNiV G (B) from <i>Leishmania tarentolae</i> cell lysates was performed via Strep-tag affinity chromatography. Elution fractions were separated by 10% SDS-PAGE under reducing conditions and visualized by Coomassie staining.</p

Immunoblot analysis of serum reactivity against plasmid derived NiV G.

<p>Serum samples from HeV or NiV infected pigs were collected at different dpi and tested for reactivity against the homologous and heterologous recombinant protein, respectively. Porcine sera from German pigs without history of henipavirus infection served as negative control (Mock 1–3). Two German pig sera (GER11 and GER34) that exceeded the calculated cut-off value in both G based ELISAs as well as one serum (GER27) that only exceeded the cut-off value in the sHeV G based ELISA were tested exemplarily for reactivity in immunoblot analysis. All sera were diluted as indicated. The monoclonal antibody 5G1B1 was utilized in a dilution of 1:100.</p

Indirect immunofluorescence analysis of cells transfected with HeV G and NiV G using mAb 5G1B1.

<p>Vero 76 cells were transfected with the indicated plasmids to express NiV and HeV G proteins. For immunostaining, the newly generated cross-reactive monoclonal antibody 5G1B1 was used as well as the respective polyclonal mice serum as positive control followed by mouse specific Alexa-Fluor 488-labeled secondary antibodies. Nuclei were stained with Hoechst. Fluorescence was visualized by a DMI7 live cell microscope (Leica), magnification 630 x.</p

Serum reactivity of experimentally henipavirus infected pigs in ELISAs based on soluble HeV and NiV glycoproteins and NiV nucleoprotein (sHeV/sNiV G; NiV N).

<p><b>A</b>. Sera from pigs (P) and minipigs (M) were collected at different days post infection (dpi) and tested for the presence of henipavirus specific IgG in the N based assay, with the red line representing the cut-off value. <b>B</b>. Serum reactivity was confirmed and specified in the sHeV G and sNiV G specific ELISAs with the white line representing the cut-off value of the sNiV G based assay and the red broken line representing the sHeV G assay cut-off value. <b>C, D</b>. One HeV IgG positive as well as one NiV IgG positive serum were serially titrated and tested for reactivity in the sHeV G based assay (C) and in the NiV based assay (D) with the red lines representing the cut-off values.</p