Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

DNA vaccination protects mice against Zika virus-induced damage to the testes.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Claudin 1 Expression Levels Affect miRNA Dynamics in Human Basal-Like Breast Cancer Cells.

Deemed a putative tumor suppressor in breast cancer, the tight junction protein claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype. Moreover, recent in vitro studies show that claudin 1 can regulate breast cancer cell motility and proliferation. Herein, we investigated whether microRNA (miRNA) dysregulation is associated with alterations in the level of claudin 1. Using next-generation sequencing (NGS), we identified seven miRNAs (miR-9-5p, miR-9-3p, let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) that were deregulated as a consequence of claudin 1 overexpression in the MDA-MB231 human breast cancer (HBC) cell line. Most of these miRNAs have been associated with tumor suppression in a variety of cancers, including breast cancer. Moreover, through gene expression profiling analysis, we identified epithelial-mesenchymal transition-related genes, including platelet-derived growth factor receptor-beta (PDGFRB) and cadherin 1 (CDH1, E cadherin), whose downregulation correlated with claudin 1 overexpression. Collectively, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Our data suggest that the dysregulated expression of these miRNAs, in conjunction with the high claudin 1 levels, could serve as a useful biomarker that identifies a subset of tumors within the poorly characterized basal-like subtype of breast cancer. Further studies are warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer.

Activation of pro-survival CaMK4ß/CREB and pro-death MST1 signaling at early and late times during a mouse model of prion disease.

BACKGROUND: The signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo. METHODS: Mice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155-190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states. RESULTS: Hierarchical and functional clustering identified CaMK4ß and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4ß and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4ß/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi. CONCLUSION: Pro-survival CaMK4ß/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.