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Flaviviruses replicate in membrane factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus viral protein accumulation contribute to ER stress. As a consequence, the host cell exhibits an Unfolded Protein Response (UPR), subsequently stimulating appropriate cellular responses such as adaptation, autophagy or apoptosis. The correct redox conditions of this compartment are essential to form native disulfide bonds in proteins. ZIKA virus (ZIKV) has ability to induce persistent ER stress leading to activation of UPR pathways. In this study, we wondered whether ZIKV affects the redox balance and consequently the oxidative protein folding in the ER. We found that ZIKV replication influences redox state leading to aggregation of viral envelope protein as amyloid-like structures in the infected cells.

International audience; The development of new diagnostic assays become a priority for managing COVID-19. To this aim, we presented here an in-house ELISA based on the production of two major recombinant and high-quality antigens from SARSCoV-2. Full-length N and S-RBD fragment proteins fused to mouse IgG2a-Fc were produced in the CHO cell line. Secreted recombinant proteins were easily purified with standard Protein A chromatography and were used in an in-house ELISA to detect anti-N and anti-RBD IgGs in the plasma of COVID-19 RTPCR-positive patients. High reactivity against recombinant antigens was readily detected in all positive plasma samples, whereas no recognition was observed with control healthy subject’s plasmas. Remarkably, unpurified recombinant N protein obtained from cell culture supernatant was also suitable for the monitoring by ELISA of IgG levels in positive patients. This work provides an early prospection for low price but high-quality serological kit development.

International audience; Zika virus (ZIKV) is an emerging mosquito-borne flavivirus considered as a threat to human health due to large epidemics and serious clinical outcomes such as microcephaly in new-borns. Like all flaviviruses, ZIKV relies on the cellular machinery to complete its viral cycle, with the endoplasmic reticulum (ER) being the critical site of viral replication factories. The sudden high protein load in the ER induces an ER stress to which the cell responds with an appropriate unfolded protein response (UPR) in an attempt to restore its disturbed homeostasis. When the restoration fails, the cell signalling leads to a programmed cell death by apoptosis with the upregulation of the UPR-induced C/EBP homologous protein (CHOP) which acts as the main trigger for this fatal outcome. Our previous studies have shown the ability of ZIKV to manipulate various cellular responses in order to optimize virus production. ZIKV is able to delay apoptosis to its benefit and although ER stress is induced, the UPR is not complete. Here we discovered that ZIKV impairs the expression of CHOP/DDIT3, the main factor responsible of ER-stress driven apoptosis. Surprisingly, the mechanism does not take place at the transcriptional level but at the translational level.

International audience; Flaviviruses replicate in membranous factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus polyprotein integration contribute to ER stress and the host cell may exhibit an Unfolded Protein Response (UPR) to this protein accumulation, stimulating appropriate cellular responses such as adaptation, autophagy or cell death. These different stress responses support other antiviral strategies initiated by infected cells and can help to overcome viral infection. In epithelial A549 cells, a model currently used to study the flavivirus infection cycle and the host cell responses, all three pathways leading to UPR are activated during infection by Dengue virus (DENV), Yellow Fever virus (YFV) or West Nile virus (WNV). In the present study, we investigated the capacity of ZIKA virus (ZIKV) to induce ER stress in A549 cells. We observed that the cells respond to ZIKV infection by implementing an UPR through activation of the IRE1 and PERK pathway without activation of the ATF6 branch. By modulating the ER stress response, we found that UPR inducers significantly inhibit ZIKV replication. Interestingly, our findings provide evidence that ZIKV could manipulate the UPR to escape this host cell defence system by downregulating GRP78/BiP expression. This subversion of GRP78 expression could lead to unresolved and persistent ER stress which can be a benefit for virus growth.

Both cellular and humoral immunities are critically important to control COVID19 infection but little is known about the kinetics of those responses and, in particular, in patients who will go on to develop a severe form of the disease over several weeks. We herein report the first set of data of our prospective cohort study of 90 hospitalized cases. Serological surveys were thoroughly performed over 2 month period by assessing IgG and IgM responses by immunofluorescence, immunoblot, Western blot and conventional ELISA using clinical RUN isolates of SARS-CoV-2 immobilized on 96 well plates. While the IgM and, unexpectedly, the IgG responses were readily detected early during the course of the disease (5-7 days post-first symptoms), our results (n=3-5 and over the full dilution set of the plasma 1/200 to 1/12800) demonstrated a significant decrease (over 2.5-fold) of IgG levels in severe (ICU) hospitalized patients (exemplified in patient 1) by WB and ELISA. In contrast, mild non-ICU patients had a steady and yet robust rise in their specific IgG levels against the virus. Interestingly, both responses (IgM and IgG) were initially against the nucleocapsid (50kDa band on the WB) and spreading to other major viral protein S and domains (S1 and S2. In conclusion, serological testing may be helpful for the diagnosis of patients with negative RT-PCR results and for the identification of asymptomatic cases. Moreover, medical care and protections should be maintained particularly for recovered patients (severe cases) who may remain at risk of relapsing or reinfection. Experiments to ascertain T cell responses but although their kinetics overtime are now highly warranted. All in all, these studies will help to delineate the best routes for vaccination.

International audience; Accumulation of misfolded proteins within the endoplasmic reticulum (ER) induces an unfolded protein response (UPR) that either restores homeostasis or triggers apoptosis in case of adaptation failure. The three activated branches of UPR lead to IRE1-, PERK- and ATF6- dependent transcriptional induction of the gene encoding the transcription factor C/EBP homologous protein (CHOP) which plays an important role in apoptosis induction. In conventional immunoblotting conditions, detection of CHOP is a difficult task. Using a fixation step, we have optimized the detection of CHOP and this method provides a valuable tool to decipher CHOP involvement in UPR.

Flaviviruses replicate in membranous factories associated with the endoplasmic reticulum (ER). The replication rate generates a high polyprotein integration that can contribute to ER stress. Therefore, the host cell can develop an Unfolded Protein Response (UPR) to this protein accumulation which will stimulate appropriate cellular responses in the infectious context (Adaptation, autophagy or cell death). These different stress responses can help to overcome the virus and usually support antiviral strategies initiated by infected cells. In the present study, we investigated the capacity of ZIKA virus (ZIKV) to induce ER stress in epithelial A549 cells with a special focus on the causal factors behind it. We observed that the cells respond to ZIKV infection by implementing an UPR through activation of the IRE1 and PERK pathway but surprisingly without activation of the ATF6 branch. When we examined the effects of modulating the ER stress response we found that UPR inducers significantly inhibit ZIKV replication. Interestingly, our findings provide evidence that ZIKV can altered the UPR in order to escape to the host cell defense system.

International audience; Zika virus (ZIKV) is an emerging human mosquito-transmitted pathogen of global concern, known to be associated with complications such as congenital defects and neurological disorders in adults. ZIKV infection is associated with induction of cell death. However, previous studies suggest that the virally induced apoptosis occurs at a slower rate compared to the course of viral production. In this present study, we investigated the capacity of ZIKV to delay host cell apoptosis. We provide evidence that ZIKV has the ability to interfere with apoptosis whether it is intrinsically or extrinsically induced. In cells expressing viral replicon-type constructions, we show that this control is achieved through replication. Finally, our work highlights an important role for anti-apoptotic Bcl-2 family protein in the ability of ZIKV to control apoptotic pathways, avoiding premature cell death and thereby promoting virus replication in the host-cell.

Zika virus (ZIKV) is an emerging human mosquito-transmitted pathogen of global concern, known to cause severe complications such as congenital defects and neurological disorders in adults. ZIKV infection is associated with cell death. However, previous studies suggest that the virally-induced apoptosis occurs at a slower rate compared to the course of viral production. In this present study, we investigated the capacity of ZIKV to delay host cell apoptosis. We provide evidence that ZIKV has the ability to control programmed cell death whether it is intrinsically or extrinsically induced. In cells expressing viral replicon-type constructions, we show that this control is achieved through replication. Finally, our work highlights an important role for anti-apoptotic Bcl-2 family protein in the ability of ZIKV to control apoptotic pathways, avoiding premature cell death and thereby promoting virus replication in the host-cell.

The neurological complications of infection by the mosquito-borne Zika virus (ZIKV) include Guillain–Barré syndrome (GBS), an acute inflammatory demyelinating polyneuritis. GBS was first associated with recent ZIKV epidemics caused by the emergence of the ZIKV Asian lineage in South Pacific. Here, we hypothesize that ZIKV-associated GBS relates to a molecular mimicry between viral envelope E (E) protein and neural proteins involved in GBS. The analysis of the ZIKV epidemic strains showed that the glycan loop (GL) region of the E protein includes an IVNDT motif which is conserved in voltage-dependent L-type calcium channel subunit alpha-1C (Cav1.2) and Heat Shock 70 kDa protein 12A (HSP70 12A). Both VSCC-alpha 1C and HSP70 12A belong to protein families which have been associated with neurological autoimmune diseases in central nervous system. The purpose of our in silico analysis is to point out that IVNDT motif of ZIKV E-GL region should be taken in consideration for the development of safe and effective anti-Zika vaccines by precluding the possibility of adverse neurologic events including autoimmune diseases such as GBS through a potent mimicry with Heat Shock 70 kDa protein 12A (HSP70 12A).

Arthritogenic alphaviruses are emerging arthropod-borne viruses that occasionally cause sporadic to global outbreaks all over the world. Many environmental factors including xenobiotics have been identified as capable of influencing the spread, the susceptibility and the outcome of viral infection. Among them cadmium is a toxic non-essential heavy metal and a prevalent environmental contaminant. In the present study we evaluated the effect of cadmium exposure on alphavirus infection in vitro. We infected Human Embryonic Kidney (HEK) 293 cells in the presence of cadmium chloride (CdCl2) with Sindbis virus. Cell viability, apoptosis and viral growth were then examined. Our data show that effective doses of cadmium decreased the virus mediated-cell death by inhibition of apoptosis. Moreover, virus growth in HEK 293 cells was also reduced by CdCl2 treatment. Altogether our results demonstrate that cadmium triggers a protective response which renders HEK 293 cells resistant against Sindbis virus infection., Highlights • Cd treatment impairs the efficiency of SINV growth in HEK 293 cells. • Cd could rescue cells from SINV-mediated cell death. • Cd inhibits SINV-induced apoptosis. • Cd reduced etoposide-mediated cell death through reduction of apoptosis.

International audience; Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and its associated pathology, COVID-19, have been of particular concerns these last months due to the worldwide burden they represent. The number of cases requiring intensive care being the critical point in this epidemic, a better understanding of the pathophysiology leading to these severe cases is urgently needed. Tissue lesions can be caused by the pathogen or can be driven by an overwhelmed immune response. Focusing on SARS-CoV-2, we and others have observed that this virus can trigger indeed an immune response that can be dysregulated in severe patients and leading to further injury to multiple organs. The purpose of the review is to bring to light the current knowledge about SARS-CoV-2 virologic and immunologic features. Thus, we address virus biology, life cycle, tropism for many organs and how ultimately it will affect several host biological and physiological functions, notably the immune response. Given that therapeutic avenues are now highly warranted, we also discuss the immunotherapies available to manage the infection and the clinical outcomes.

Zika virus (ZIKV) is an emerging arthropod-borne virus of major public health concern. ZIKV infection is responsible for congenital Zika disease and other neurological defects. Antibody-mediated virus neutralization is an essential component of protective antiviral immunity against ZIKV. In the present study, we assessed whether our GFP reporter ZIKV derived from African viral strain MR766 could be useful for the development of a flow cytometry neutralization test (FNT), as an alternative to the conventional plaque-reduction neutralization test (PRNT). To improve the efficacy of GFP-expressing MR766, we selected virus variant MR766GFP showing a high level of GFP signal in infected cells. A MR766GFP-based FNT was assayed with immune sera from adult mice that received ZIKBeHMR-2. The chimeric ZIKV clone ZIKBeHMR-2 comprises the structural protein region of epidemic strain BeH819015 into MR766 backbone. We reported that adult mice inoculated with ZIKBeHMR-2 developed high levels of neutralizing anti-ZIKV antibodies. Comparative analysis between MR766GFP-based FNT and conventional PRNT was performed using mouse anti-ZIKBeHMR-2 immune sera. Indistinguishable neutralization patterns were observed when compared with PRNT50 and FNT50. We consider that the newly developed MR766GFP-based FNT is a valid format for measuring ZIKV-neutralizing antibodies in serum specimens.

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

About 1 million people in the world die each year from diseases spread by mosquitoes, and understanding the mechanism of host identification by the mosquitoes through olfaction is at stake. The role of odorant binding proteins (OBPs) in the primary molecular events of olfaction in mosquitoes is becoming an important focus of biological research in this area. Here, we present a comprehensive comparative genomics study of OBPs in the three disease-transmitting mosquito species Anopheles gambiae, Aedes aegypti ,a nd Culex quinquefasciatus starting with the identification of 110 new OBPs in these three genomes. We have characterized their genomic distribution and orthologous and phylogenetic relationships. The diversity and expansion observed with respect to the Aedes and Culex genomes suggests that the OBP gene family acquired functional diversity concurrently with functional constraints posed on these two species. Sequences with unique features have been characterized such as the “two-domain OBPs” (previously known as Atypical OBPs) and “MinusC OBPs” in mosquito genomes. The extensive comparative genomics featured in this work hence provides useful primary insights into the role of OBPs in the molecular adaptations of mosquito olfactory system and could provide more clues for the identification of potential targets for insect repellants and attractants.

Zika virus (ZIKV) is an emerging flavivirus since the first epidemics in South Pacific in 2007. The recent finding that ZIKV is now circulating in Western Hemisphere and can be associated to severe human diseases, warrants the need for its study. Here we evaluate the susceptibility of human lung epithelial A549 cells to South Pacific epidemic strain of ZIKV isolated in 2013. We showed that ZIKV growth in A549 cells is greatly efficient. ZIKV infection resulted in the secretion of IFN-β followed by the expression of pro-inflammatory cytokines such as IL-1β, and transcriptional activity of IFIT genes. At the maximum of virus progeny production, ZIKV triggers mitochondrial apoptosis through activation of caspases-3 and -9. Whereas at early infection times, the rapid release of IFN-β which exerts an antiviral effect against ZIKV might delay apoptosis in infected cells.

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.