Suppression of TGF-ß/Smad2 signaling by GW788388 enhances DENV-2 clearance in macrophages.

Dengue fever, caused by the dengue virus (DENV-1, -2, -3, and -4), affects millions of people in the tropical and subtropical regions worldwide. Severe dengue is correlated with high viraemia and cytokine storm, such as high levels of transforming growth factor-ß1 (TGF-ß1) in the patient's serum. Here, the TGF-ß1 signaling was investigated in the context of in vitro viral clearance. Macrophages were infected with DENV-2 at MOI 5 and treated with the TGF-ß receptor 1 and 2 inhibitor, GW788388. TGF-ß1 expression, signal transduction and viral load were evaluated 48 h after DENV-2 infection by enzyme-linked immunoassay, immunofluorescence, and RT-qPCR assays. Total TGF-ß1 level was reduced in 15% after DENV-2 infection, but the secretion of its biologically active form increased threefold during infection, which was followed by the phosphorylation of Smad2 protein. Phosphorylation of Smad2 was reduced by treatment with GW788388 and it was correlated with reduced cytokine production. Importantly, treatment led to a dose-dependent reduction in viral load, ranging from 6.6 × 105 RNA copies/ml in untreated cultures to 2.3 × 103 RNA copies/ml in cultures treated with 2 ng/ml of GW788388. The anti-TGF-ß1 antibody treatment also induced a significant reduction in viral load to 1.6 × 103 RNA copies/ml. On the other hand, the addition of recombinant TGF-ß1 in infected cultures promoted an increase in viral load to 7.0 × 106 RNA copies/ml. These results support that TGF-ß1 plays a significant role in DENV-2 replication into macrophages and suggest that targeting TGF-ß1 may represent an alternative therapeutic strategy to be explored in dengue infection.

ZIKV replication is differential in explants and cells of human placental which is suppressed by HSV-2 coinfection.

During the Zika fever outbreak in Brazil in 2015-2016, only some babies from infected mothers had teratogenic effects, suggesting that cofactors may influence congenital transmission. We investigated the ZIKV infection profile in explants and isolated cells from full-term human placenta to infection with the Brazilian Zika virus strain (ZIKVBR) and the effect of coinfection with the Brazilian Human alphaherpesvirus 2 strain (HSV-2BR) on ZIKV replication. We found that the ZIKVBR infect the explants of amniotic and chorionic membranes, as well as chorionic villi core, but not the trophoblasts layer. It was also observed that ZIKV replication was higher in amniotic cells than chorionic and trophoblasts cells. Upon coinfection, the replication of ZIKVBR was reduced according to exposed HSV-2BR load in trophoblasts cells and the levels of TNF-α and IL-6 cytokines were also reduced. These findings suggest that the placental cell types and HSV-2BR coinfection may impact on ZIKV replication.

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 µM of C-Sds and observed CC50 values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV-envelope glycoprotein expression in infected cells as compared to 36.8% ± 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 ± 0.7 × 108 copies/mL in vehicle control. The dose-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 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM 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 ± 0.1 to 5.0 ± 0.2 µM, and the EC90 values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, 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.

Infecção pelo vírus Zika de cultura de explantes e células primárias de placenta humana associada a terapia antiviral

Em 2015, houve um surto da febre do Zika no Brasil e de nascimento de natimorto/bebês com microcefalia e outras desordens neurológicas de mães que foram acometidas pela infecção, levando a associação entre ZIKV e síndrome congênita. Não está claro como o vírus alcança o feto, mas a placenta representa uma importante rota de transmissão, desde que o vírus foi detectado em placentas de mulheres infectadas e de infecções experimentais in vivo e in vitro. Além desta lacuna no conhecimento da transmissão, ainda não existe tratamento profilático e/ou terapêutico específico para pacientes e, em especial para gestantes infectadas. Desta forma, o objetivo do trabalho foi implementar o cultivo primário de explantes e células de placenta humana a termo, a fim de investigar a susceptibilidade e o perfil de infecção pelo ZIKV ex vivo, e utilizar este modelo experimental para investigar, no contexto de reposicionamento de drogas, a atividade antiviral do fármaco Nitazoxanida (NTZ). Para isto, foi utilizada a membrana fetal e vilosidade coriônica, marcadas por anticorpos característicos para fenotipagem de células placentárias, como a citoqueratina-7 (CK-7) em células epiteliais e vimentina que exclui este tipo celular. Foi observado que os explantes em cultivo apresentaram integridade morfológica, estrutural e fenótipo característicos. A membrana e vilosidade coriônica foram susceptíveis a infecção, sendo que nas vilosidades a infecção ocorreu somente em células vimentina+. O isolamento das células da membrana fetal revelou maior rendimento e viabilidade das células amnióticas comparada às coriônicas. As células amnióticas mantêm sua homogeneidade morfológica e fenotípica CK-7+ em 98% da cultura, enquanto, as coriônicas apresentam uma cultura mista variando de 72% a 28% de células CK-7+, de acordo com o rendimento de sua obtenção, podendo atingir 100% de vimentina+ ao longo do cultivo O isolamento das células das vilosidades coriônicas foi realizado a partir de explantes que se reorganizaram em esferóides formados por cerca de 30% de células CK-7+ e 60% vimentina+, embora em perfis diferentes, as células proliferativas que formaram a monocamada foram susceptíveis a infecção, esses resultados necessitam ser melhor explorados. Quando células aminióticas e coriônicas vimentina+ e CK+ foram infectadas com baixa multiplicidade de infecção (MOI 1) em diferentes tempos, os resultados demonstraram que amnion CK-7+ foram mais susceptíveis. Empregando alta carga viral (MOI 10 e 20) na cinética de infecção de células coriônicas foram observados elevados percentuais de infecção tempo-dependente (17% a 80%), porém, quando comparada a infecção com MOI 20 por 48h de células coriônicas vimentina + e CK-7+ os percentuais alcançados foram cerca de 56% e 77%, respectivamente, demonstrando que células CK+ foram mais susceptíveis a infecção. A análise por Microscopia Eletrônica de Transmissão das células coriônicas não infectadas demonstrou preservação ultraestrutural. No entanto, a infecção destas células levou a importantes alterações ultraestruturais, especialmente alargamento e formação de estruturas de membrana no retículo endoplasmático, com presença de partículas virais. Importantemente, foi determinada a atividade antiviral do fármaco Nitazoxanida sobre culturas de células coriônicas CK+ infectadas com alta carga viral (MOI 20) por 48h, avaliando redução de células infectadas por detecção antigênica, redução do efeito citopático por ensaio de plaque e redução de carga viral por RT-qPCR Foi observado que a concentração de 50 \03BCg/mL do fármaco levou a redução de 79% de células infectadas, 100% do efeito citopático e cerca de 93% do número de cópias de RNA viral/mL no sobrenadante das culturas tratadas. Com o desenvolvimento deste trabalho, reforçamos que este modelo experimental representa uma excelente ferramenta para explorar aspectos biológicos da transmissão congênita pelo ZIKV e identificar moléculas antivirais, incluindo reposicionamento de drogas, especialmente para o tratamento de gestantes infectadas.

CRISPR / Cas-9 Vector System: Targets UL-39 and Inhibits HSV-1 Replication in Vitro

HSV-1 affects approximately 67% of the world population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyzes revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.