ZIKV-Specific NS1 Epitopes as Serological Markers of Acute Zika Virus Infection.

BACKGROUND: Zika virus (ZIKV) infections have reemerged as a global health issue due to serious clinical complications. Development of specific serological assays to detect and differentiate ZIKV from other cocirculating flaviviruses for accurate diagnosis remains a challenge. METHODS: We investigated antibody responses in 51 acute ZIKV-infected adult patients from Campinas, Brazil, including 7 pregnant women who later delivered during the study. Using enzyme-linked immunosorbent assays, levels of antibody response were measured and specific epitopes identified. RESULTS: Several antibody-binding hot spots were identified in ZIKV immunogenic antigens, including membrane, envelope (E) and nonstructural protein 1 (NS1). Interestingly, specific epitopes (2 from E and 2 from NS1) strongly recognized by ZIKV-infected patients' antibodies were identified and were not cross-recognized by dengue virus (DENV)-infected patients' antibodies. Corresponding DENV peptides were not strongly recognized by ZIKV-infected patients' antibodies. Notably, ZIKV-infected pregnant women had specific epitope recognition for ZIKV NS1 (amino acid residues 17-34), which could be a potential serological marker for early ZIKV detection. CONCLUSIONS: This study identified 6 linear ZIKV-specific epitopes for early detection of ZIKV infections. We observed differential epitope recognition between ZIKV-infected and DENV-infected patients. This information will be useful for developing diagnostic methods that differentiate between closely related flaviviruses.

Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib.

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

Sero-prevalence and cross-reactivity of chikungunya virus specific anti-E2EP3 antibodies in arbovirus-infected patients.

Chikungunya virus (CHIKV) and clinically-related arboviruses cause large epidemics with serious economic and social impact. As clinical symptoms of CHIKV infections are similar to several flavivirus infections, good detection methods to identify CHIKV infection are desired for improved treatment and clinical management. The strength of anti-E2EP3 antibody responses was explored in a longitudinal study on 38 CHIKV-infected patients. We compared their anti-E2EP3 responses with those of patients infected with non-CHIKV alphaviruses, or flaviviruses. E2EP3 cross-reactive samples from patients infected with non-CHIKV viruses were further analyzed with an in vitro CHIKV neutralization assay. CHIKV-specific anti-E2EP3 antibody responses were detected in 72% to 100% of patients. Serum samples from patients infected with other non-CHIKV alphaviruses were cross-reactive to E2EP3. Interestingly, some of these antibodies demonstrated clearly in vitro CHIKV neutralizing activity. Contrastingly, serum samples from flaviviruses-infected patients showed a low level of cross-reactivity against E2EP3. Using CHIKV E2EP3 as a serology marker not only allows early detection of CHIKV specific antibodies, but would also allow the differentiation between CHIKV infections and flavivirus infections with 93% accuracy, thereby allowing precise acute febrile diagnosis and improving clinical management in regions newly suffering from CHIKV outbreaks including the Americas.

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection.

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain-binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.

Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins.

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.

A novel quantitative flow cytometry-based assay for autophagy.

Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells.