A Human Skin Model for Assessing Arboviral Infections.

Arboviruses such as flaviviruses and alphaviruses cause a significant human healthcare burden on a global scale. Transmission of these viruses occurs during human blood feeding at the mosquito-skin interface. Not only do pathogen immune evasion strategies influence the initial infection and replication of pathogens delivered, but arthropod salivary factors also influence transmission foci. In vitro cell cultures do not provide an adequate environment to study complex interactions between viral, mosquito, and host factors. To address this need for a whole tissue system, we describe a proof of concept model for arbovirus infection using adult human skin ex vivo with Zika virus (flavivirus) and Mayaro virus (alphavirus). Replication of these viruses in human skin was observed up to 4 days after infection. Egressed viruses could be detected in the culture media as well. Antiviral and proinflammatory genes, including chemoattractant chemokines, were expressed in infected tissue. Immunohistochemical analysis showed the presence of virus in the skin tissue 4 days after infection. This model will be useful to further investigate: (i) the immediate molecular mechanisms of arbovirus infection in human skin, and (ii) the influence of arthropod salivary molecules during initial infection of arboviruses in a more physiologically relevant system.

In vitro and in vivo characterization of erythrosin B and derivatives against Zika virus.

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B-NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure-activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B-NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Association between brain morphology and electrophysiological features in Congenital Zika Virus Syndrome: A cross-sectional, observational study.

BACKGROUND: Intrauterine infection with the Zika virus (ZIKV) has been connected to severe brain malformations, microcephaly, and abnormal electrophysiological activity. METHODS: We describe the interictal electroencephalographic (EEG) recordings of 47 children born with ZIKV-derived microcephaly. EEGs were recorded in the first year of life and correlated with brain morphology. In 31 subjects, we tested the association between computed tomography (CT) findings and interictal epileptiform discharges (IED). In eighteen, CTs were used for correlating volumetric measurements of the brainstem, cerebellum, and prosencephalon with the rate of IED. FINDINGS: Twenty-nine out of 47 (62%) subjects were diagnosed as having epilepsy. Those subjects presented epileptiform discharges, including unilateral interictal spikes (26/29, 90%), bilateral synchronous and asynchronous interictal spikes (21/29, 72%), and hypsarrhythmia (12/29, 41%). Interestingly, 58% of subjects with clinical epilepsy were born with rhombencephalon malformations, while none of the subjects without epilepsy showed macroscopic abnormalities in this region. The presence of rhombencephalon malformation was associated with epilepsy (odds ratio of 34; 95% CI: 2 - 654). Also, the presence of IED was associated with smaller brain volumes. Age-corrected total brain volume was inversely correlated with the rate of IED during sleep. Finally, 11 of 44 (25%) subjects presented sleep spindles. We observed an odds ratio of 0·25 (95% CI: 0·06 - 1·04) for having sleep spindles given the IED presence. INTERPRETATION: The findings suggest that certain CT imaging features are associated with an increased likelihood of developing epilepsy, including higher rates of IED and impaired development of sleep spindles, in the first year of life of CZVS subjects. FUNDING: This work was supported by the Brazilian Federal Government through a postdoctoral fellowship for EBS (Talented Youth, Science without Borders), an undergraduate scholarship for AJR (Institutional Program of Science Initiation Scholarships, Federal University of Rio Grande do Norte, Brazil), by International Centre for Genetic Engineering and Biotechnology (CRP/BRA18-05_EC) and by CAPES (Grant number 440893/2016-0), and CNPq (Grant number 88881.130729/2016-01).

Modelling of pathogen-host systems using deeper ORF annotations and transcriptomics to inform proteomics analyses.

The Zika virus is a flavivirus that can cause fulminant outbreaks and lead to Guillain-Barré syndrome, microcephaly and fetal demise. Like other flaviviruses, the Zika virus is transmitted by mosquitoes and provokes neurological disorders. Despite its risk to public health, no antiviral nor vaccine are currently available. In the recent years, several studies have set to identify human host proteins interacting with Zika viral proteins to better understand its pathogenicity. Yet these studies used standard human protein sequence databases. Such databases rely on genome annotations, which enforce a minimal open reading frame (ORF) length criterion. An ever-increasing number of studies have demonstrated the shortcomings of such annotation, which overlooks thousands of functional ORFs. Here we show that the use of a customized database including currently non-annotated proteins led to the identification of 4 alternative proteins as interactors of the viral capsid and NS4A proteins. Furthermore, 12 alternative proteins were identified in the proteome profiling of Zika infected monocytes, one of which was significantly up-regulated. This study presents a computational framework for the re-analysis of proteomics datasets to better investigate the viral-host protein interplays upon infection with the Zika virus.