Additional file 1: Table S1. of Pregnant women carrying microcephaly foetuses and Zika virus contain potentially pathogenic microbes and parasites in their amniotic fluid

Similarity count table of sequences classified as Spirometra. (XLSX 14 kb

Additional file 2: Table S2. of Pregnant women carrying microcephaly foetuses and Zika virus contain potentially pathogenic microbes and parasites in their amniotic fluid

Multiple T Teste performed comparing the diversity index (Shannon and Simpson) between AF Zika samples and Controls Groups. (XLSX 11 kb

HIV-1 and HIV-1ΔNef mature particles have different protein content and distribution on a density gradient.

<p>HIV-1 and HIV-1<b>Δ</b>Nef virions were produced in Hek-293T cells and separated using a 30–70% continuous sucrose density gradient. Ten fractions were collected from top to bottom and numbered #1 through #10 accordingly. The fractions were analyzed to determine the content and distribution of IN (top panel) and CA (lower panel). (A) Protein content of soluble proteins (fractions #1–4), immature particles (#5–7) and mature particles (#8–10) for the HIV-1 (left panels) and HIV-1<b>Δ</b>Nef viruses (right panels). (B) The CA content of cell-free supernatants before fractionation. (C) Quantification of the amount of CA in fractions #8–10. (D) Quantification of the amount of IN in fraction #8–10. *<i>p</i> = 0.061, **<i>p</i> = 0.036. The WB and ELISA results presented are representative of three experiments, CA and IN values are triplicates.</p

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

<div><p>Abstract The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.</p></div

HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

<div><p>Nef is an important player for viral infectivity and AIDS progression, but the mechanisms involved are not completely understood. It was previously demonstrated that Nef interacts with GagPol through p6*-Protease region. Because p6* and Protease are involved in processing, we explored the effect of Nef on viral Protease activity and virion assembly. Using in vitro assays, we observed that Nef is highly capable of inhibiting Protease activity. The IC50 for <i>nef</i>-deficient viruses in drug susceptibility assays were 1.7- to 3.5-fold higher than the wild-type counterpart varying with the type of the Protease inhibitor used. Indicating that, in the absence of Nef, Protease is less sensitive to Protease inhibitors. We compared the protein content between wild-type and <i>nef</i>-deficient mature viral particles by gradient sedimentation and observed up to 2.7-fold reduction in the Integrase levels in <i>nef</i>-deficient mature particles. This difference in levels of Integrase correlated with the difference in infectivity levels of wild type and <i>nef</i>-deficient viral progeny. In addition, an overall decrease in the production of mature particles was detected in <i>nef</i>-deficient viruses. Collectively, our data support the hypothesis that the decreased infectivity typical of <i>nef</i>-deficient viruses is due to an abnormal function of the viral Protease, which is in turn associated with less mature particles being produced and the loss of Integrase content in these particles, and these results may characterize Nef as a regulator of viral Protease activity.</p></div

CA, MA and IN levels differ between mature HIV-1 and HIV-1ΔNef viral particles produced in MOLT cells.

<p>HIV-1 and HIV-1<b>Δ</b>Nef virions were produced in MOLT cells and separated using a 9.6–18% continuous optiprep density gradient. Twelve fractions were collected from top to bottom and numbered #1 through #12 accordingly. The fractions were precipitated with 20% TCA and analyzed by WB to determine the contents and distributions of CA (top panel), MA (middle panel) and IN (bottom panels). Left panels represent the fractions for HIV-1, and the right panels represent the fractions for HIV-1<b>Δ</b>Nef. (A) The protein contents of the mature particles for the HIV-1 (left) and HIV-1<b>Δ</b>Nef viruses (right). (B) Numbers of blue-foci of non-fractionated supernatant (sn) and non-preciptated fractions for each virus. *Denotes the detection of a nonspecific band. The result presented is the mean of three experiments.</p

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

<div><p>Abstract The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.</p></div

Increased concentrations of Protease Inhibitors are required to inhibit HIV-1ΔNef.

<p>PI – Protease Inhibitor;</p><p>NNRTI – Non Nucleosidic Reverse Transcriptase Inhibitor.</p><p>*Non-peptidomimetic protease inhibitor.</p

MicroRNA and cellular targets profiling reveal miR-217 and miR-576-3p as proviral factors during Oropouche infection

<div><p>Oropouche Virus is the etiological agent of an arbovirus febrile disease that affects thousands of people and is widespread throughout Central and South American countries. Although isolated in 1950’s, still there is scarce information regarding the virus biology and its prevalence is likely underestimated. In order to identify and elucidate interactions with host cells factors and increase the understanding about the Oropouche Virus biology, we performed microRNA (miRNA) and target genes screening in human hepatocarcinoma cell line HuH-7. Cellular miRNAs are short non-coding RNAs that regulates gene expression post-transcriptionally and play key roles in several steps of viral infections. The large scale RT-qPCR based screening found 13 differentially expressed miRNAs in Oropouche infected cells. Further validation confirmed that miR-217 and miR-576-3p were 5.5 fold up-regulated at early stages of virus infection (6 hours post-infection). Using bioinformatics and pathway enrichment analysis, we predicted the cellular targets genes for miR-217 and miR-576-3p. Differential expression analysis of RNA from 95 selected targets revealed genes involved in innate immunity modulation, viral release and neurological disorder outcomes. Further analysis revealed the gene of decapping protein 2 (DCP2), a previous known restriction factor for bunyaviruses transcription, as a miR-217 candidate target that is progressively down-regulated during Oropouche infection. Our analysis also showed that activators genes involved in innate immune response through IFN-β pathway, as STING (Stimulator of Interferon Genes) and TRAF3 (TNF-Receptor Associated Factor 3), were down-regulated as the infection progress. Inhibition of miR-217 or miR-576-3p restricts OROV replication, decreasing viral RNA (up to 8.3 fold) and virus titer (3 fold). Finally, we showed that virus escape IFN-β mediated immune response increasing the levels of cellular miR-576-3p resulting in a decreasing of its partners STING and TRAF3. We concluded stating that the present study, the first for a <i>Peribunyaviridae</i> member, gives insights in its prospective pathways that could help to understand virus biology, interactions with host cells and pathogenesis, suggesting that the virus escapes the antiviral cellular pathways increasing the expression of cognates miRNAs.</p></div