International audienceAlphaviruses, including Chikungunya virus (CHIKV), produce a transient illness in humans, but severe forms leading to chronic incapacitating arthralgia/arthritis have been reported by mechanisms largely ill-characterized. The pathogenesis of CHIKV was addressed in a prospective cohort study of 49 hospitalized patients from Reunion Island subsequently categorized into two distinct groups at 12 mo postinfection. Comprehensive analyses of the clinical and immunological parameters throughout the disease course were analyzed in either the “recovered” or the “chronic” groups to identify prognostic markers of arthritis-like pathology after CHIKV disease. We found that the chronic group consisted mainly of more elderly patients (\textgreater60 y) and with much higher viral loads (up to 1010 viruses per milliliter of blood) during the acute phase. Remarkably, a rapid innate immune antiviral response was demonstrated by robust dendritic/NK/CD4/CD8 cell activation and accompanied by a rather weak Th1/Th2 cytokine response in both groups. Interestingly, the antiviral immune response witnessed by high levels of IFN-α mRNA in PBMCs and circulating IL-12 persisted for months only in the chronic group. CHIKV (RNA and proteins) was found in perivascular synovial macrophages in one chronic patient 18 mo postinfection surrounded by infiltrating NK and T cells (CD4++ but rare cytotoxic CD8). Fibroblast hyperplasia, strong angiogenesis, tissue lesions given the high levels of matrix metalloproteinase 2, and acute cell death [high cleaved poly(ADP-ribose) polymerase staining] were observed in the injured synovial tissue. These observed cellular and molecular events may contribute to chronic arthralgia/arthritis targeted by methotrexate used empirically for effective treatment but with immunosuppressive function in a context of viral persistence

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.

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Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766(NIID), the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.

Chikungunya virus (CHIKV) surprised medical workers by a massive outbreak in the Indian Ocean region, reaching Europe in 2007, with exceptional pathologies in infants and elderly patients. Although CHIKV was recently shown to persist in myoblasts, monocytes, and macrophages, we argued that robust antiviral mechanisms, including apoptosis, are essential to ward off the virus. Herein, we tested the capacity of CHIKV to mobilize the apoptotic machinery in HeLa cells as well as primary fibroblasts, making use of several inhibitors of caspases, cell blebbing, and engulfment of the apoptotic blebs by neighboring cells. CHIKV triggered apoptosis through intrinsic and extrinsic pathways. Bystander apoptosis was also evidenced in neighboring cells in a caspase-8-dependent manner. Remarkably, by hiding in apoptotic blebs, CHIKV was able to infect neighboring cells. In HeLa cells, these events were inhibited specifically by zVAD-fmk and DEVD-cho (caspase inhibitors), blebbistatin, Y-27632 (ROCK inhibitor), and genistein, annexin V, and cytochalasin B (inhibitors of blebbing and engulfment). These CHIKV-apoptotic blebs were also capable of infecting macrophages (primary cultures, MM6- and THP1-PMA differentiated cells) otherwise refractory to infection by CHIKV alone. Remarkably, viral replication in macrophages did not yield a proinflammatory response. We describe a novel infectious mechanism by which CHIKV invades host cells and escapes the host response.

BackgroundChikungunya Virus (ChikV) surprised by a massive re-emerging outbreak in Indian Ocean in 2006, reaching Europe in 2007 and exhibited exceptional severe physiopathology in infants and elderly patients. In this context, it is important to analyze the innate immune host responses triggered against ChikV. Autophagy has been shown to be an important component of the innate immune response and is involved in host defense elimination of different pathogens. However, the autophagic process was recently observed to be hijacked by virus for their own replication. Here we provide the first evidence that hallmarks of autophagy are specifically found in HEK.293 infected cells and are involved in ChikV replication.MethodsTo test the capacity of ChikV to mobilize the autophagic machinery, we performed fluorescence microscopy experiments on HEK.GFP.LC3 stable cells, and followed the LC3 distribution during the time course of ChikV infection. To confirm this, we performed electron microscopy on HEK.293 infected cells. To test the effect of ChikV-induced-autophagy on viral replication, we blocked the autophagic process, either by pharmacological (3-MA) or genetic inhibition (siRNA against the transcript of Beclin 1, an autophagic protein), and analyzed the percentage of infected cells and the viral RNA load released in the supernatant. Moreover, the effect of induction of autophagy by Rapamycin on viral replication was tested.ResultsThe increasing number of GFP-LC3 positive cells with a punctate staining together with the enhanced number of GFP-LC3 dots per cell showed that ChikV triggered an autophagic process in HEK.293 infected cells. Those results were confirmed by electron microscopy analysis since numerous membrane-bound vacuoles characteristic of autophagosomes were observed in infected cells. Moreover, we found that inhibition of autophagy, either by biochemical reagent and RNA interference, dramatically decreases ChikV replication.ConclusionsTaken together, our results suggest that autophagy may play a promoting role in ChikV replication. Investigating in details the relationship between autophagy and viral replication will greatly improve our knowledge of the pathogenesis of ChikV and provide insight for the design of candidate antiviral therapeutics.

Mosquito-borne Zika virus (ZIKV) recently emerged in South Pacific islands and Americas where large epidemics were documented. In the present study, we investigated the contribution of the structural proteins C, prM and E in the permissiveness of human host cells to epidemic strains of ZIKV. To this end, we evaluated the capacity of the epidemic strain BeH819015 to infect epithelial A549 and neuronal SH-SY5Y cells in comparison to the African historical MR766 strain. For that purpose, we generated a molecular clone of BeH819015 and a chimeric clone of MR766 which contains the BeH819015 structural protein region. We showed that ZIKV containing BeH819015 structural proteins was much less efficient in cell-attachment leading to a reduced susceptibility of A549 and SH-SY5Y cells to viral infection. Our data illustrate a previously underrated role for C, prM, and E in ZIKV epidemic strain ability to initiate viral infection in human host cells.

The medical importance of Zika virus (ZIKV) was fully highlighted during the recent epidemics in South Pacific islands and Americas due to ZIKV association with severe damage to fetal brain development and neurological complications in adult patients. A worldwide research effort has been undertaken to identify effective compounds to prevent or treat ZIKV infection. Fruits and vegetables may be sources of compounds with medicinal properties. Flavonoids are one class of plant compounds that emerge as promising antiviral molecules against ZIKV. In the present study, we demonstrated that flavonoid isoquercitrin exerts antiviral activity against African historical and Asian epidemic strains of ZIKV in human hepatoma, epithelial, and neuroblastoma cell lines. Time-of-drug addition assays showed that isoquercitrin acts on ZIKV entry by preventing the internalisation of virus particles into the host cell. Our data also suggest that the glycosylated moiety of isoquercitrin might play a role in the antiviral effect of the flavonoid against ZIKV. Our results highlight the importance of isoquercitrin as a promising natural antiviral compound to prevent ZIKV infection.