Uukuniemi Virus as a Tick-Borne Virus Model.

International audienceIn the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged on different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome close to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection was persistent. The glycoprotein GC was mainly highly mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases peptide-N-glycosidase F (PNGase F) and endoglycosidase H (Endo H). Treatment with β-mercaptoethanol did not impact the apparent molecular weight of GN On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F, and the electrophoretic mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per focus forming unit differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells in investigations of the cell biology of arbovirus transmission and entry into mammalian hosts. Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research on not only tick-borne phleboviruses but also all viruses and other pathogens transmitted by ticks

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SARS-CoV-2 induces transcription of human endogenous retrovirus RNA followed by type W envelope protein expression in human lymphoid cells.

Abstract Patients with COVID-19 may develop abnormal inflammatory response and lymphopenia, followed in some cases by delayed-onset syndromes, often long-lasting after resolution of the initial SARS-CoV-2 infection. As viral infections may activate human endogenous retroviral elements (HERV), we studied the effect of SARS-CoV-2 on HERV-W and HERV-K envelope (ENV) expression, known to be involved in immunological and neurological pathogenesis of human diseases. We demonstrate here that an initial exposure to SARS-CoV-2 virus activates early HERV-W and K transcription in peripheral blood mononuclear cell (PBMC) cultures from healthy donors. Within a week of primary PBMC culture, only HERV-W ENV protein expression was detected in lymphoid cells of some donors, although SARS-CoV-2 infection of PBMC was not observed. HERV activation was reproduced with UV-inactivated virus and with a recombinant spike protein. Interestingly, exposure to SARS-CoV-2 protein induced a significant production of interleukin 6 in PBMC, independently from detectable HERV expression. Altogether, these results show that SARS-CoV-2 viral protein could induce HERV-W ENV expression in lymphocytes from some individuals, underlying the importance to further address the implicated molecular pathways, to understand patients‘ genetic susceptibility associated to the activation of HERV-W and its possible relevance for targeting therapeutic intervention in COVID-19 associated syndromes

Hamster organotypic modeling of SARS-CoV-2 lung and brainstem infection

International audienceAbstract SARS-CoV-2 has caused a global pandemic of COVID-19 since its emergence in December 2019. The infection causes a severe acute respiratory syndrome and may also spread to central nervous system leading to neurological sequelae. We have developed and characterized two new organotypic cultures from hamster brainstem and lung tissues that offer a unique opportunity to study the early steps of viral infection and screening antivirals. These models are not dedicated to investigate how the virus reaches the brain. However, they allow validating the early tropism of the virus in the lungs and demonstrating that SARS-CoV-2 could infect the brainstem and the cerebellum, mainly by targeting granular neurons. Viral infection induces specific interferon and innate immune responses with patterns specific to each organ, along with cell death by apoptosis, necroptosis, and pyroptosis. Overall, our data illustrate the potential of rapid modeling of complex tissue-level interactions during infection by a newly emerged virus

A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors

International audienceOur therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development