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Subgenomic flaviviral RNAs (sfRNAs) are small non-coding products of the incomplete degradation of viral genomic RNA. They accumulate during flaviviral infection and have been associated with many functional roles inside the host cell. Studies so far have demonstrated that sfRNA plays a crucial role in determining West Nile virus (WNV) pathogenicity. However, its modulatory role on neuronal homeostasis has not been studied in depth. In this study, we investigated the mechanism of sfRNA biosynthesis and its importance for WNV replication in neuronal cells. We found that sfRNA1 is functionally redundant for both replication and translation of WNV. However, the concurrent absence of sfRNA1 and sfRNA2 species is detrimental for the survival of the virus. Differential expression analysis on RNA-seq data from WT and ΔsfRNA replicon cell lines revealed transcriptional changes induced by sfRNA and identified a number of putative targets. Overall, it was shown that sfRNA contributes to the viral evasion by suppressing the interferon-mediated antiviral response. An additional differential expression analysis among replicon and control Neuro2A cells also clarified the transcriptional changes that support WNV replication in neuronal cells. Increased levels of translation and oxidative phosphorylation, post-translational modification processes, and activated DNA repair pathways were observed in replicon cell lines, while developmental processes such as axonal growth were deficient.

Biting midges (Culicoides) are vectors of arboviruses of both veterinary and medical importance. The surge of emerging and reemerging vector-borne diseases and their expansion in geographical areas affected by climate change has increased the importance of understanding their capacity to contribute to novel and emerging infectious diseases. The study of Culicoides virome is the first step in the assessment of this potential. In this study, we analyzed the RNA virome of 10 Culicoides species within the geographical area of Thrace in the southeastern part of Europe, a crossing point between Asia and Europe and important path of various arboviruses, utilizing the Ion Torrent next-generation sequencing (NGS) platform and a custom bioinformatics pipeline based on TRINITY assembler and alignment algorithms. The analysis of the RNA virome of 10 Culicoides species resulted in the identification of the genomic signatures of 14 novel RNA viruses, including three fully assembled viruses and four segmented viruses with at least one segment fully assembled, most of which were significantly divergent from previously identified related viruses from the Solemoviridae, Phasmaviridae, Phenuiviridae, Reoviridae, Chuviridae, Partitiviridae, Orthomyxoviridae, Rhabdoviridae, and Flaviviridae families. Each Culicoides species carried a species-specific set of viruses, some of which are related to viruses from other insect vectors in the same area, contributing to the idea of a virus-carrier web within the ecosystem. The identified viruses not only expand our current knowledge on the virome of Culicoides but also set the basis of the genetic diversity of such viruses in the area of southeastern Europe. Furthermore, our study highlights that such metagenomic approaches should include as many species as possible of the local virus-carrier web that interact and share the virome of a geographical area.

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in the proliferation, inflammation, and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of the HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh 7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only the HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of the core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal), we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing the HCV 4a core protein were more susceptible to the LPS/Dgal model, while mice expressing the HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis while linking HCV core protein genetic variability to differential pathology in vivo.

West Nile virus (WNV) is a mosquito-borne flavivirus that has emerged as a major cause of viral encephalitis and meningitis, rarely leading to death. Several risk factors have been discussed in the past concerning the severity of the disease, while few reports have focused on precipitating conditions that determine of WNV-related death. Studies on cohorts of patients suffering of West Nile disease (WND) usually encompass low numbers of deceased patients as a result of the rarity of the event. In this systematic review and critical analysis of 428 published case studies and case series, we sought to evaluate and highlight critical parameters of WND-related death. We summarized the symptoms, comorbidities, and treatment strategies related to WND in all published cases of patients that included clinical features. Symptoms such as altered mental status and renal problems presented increased incidence among deceased patients, while these patients presented increased cerebrospinal fluid (CSF) glucose. Our analysis also highlights underestimated comorbidities such as pulmonary disease to act as precipitating conditions in WND, as they were significantly increased amongst deceased patients. CSF glucose and the role of pulmonary diseases need to be revaluated either retrospectively or prospectively in WND patient cohorts, as they may be linked to increased mortality risk.