A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis. METHOD: Human neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype. RESULTS: We showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV. CONCLUSION: Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.

Virus-host interactions during tick-borne bunyavirus infection.

The Bunyavirales order is the largest grouping of RNA viruses, comprising emerging and re-emerging human, plant and animal pathogens. Bunyaviruses have a global distribution and many members of the order are transmitted by arthropods. They have evolved a plethora of mechanisms to manipulate the regulatory processes of the infected cell to facilitate their own replicative cycle, in hosts of disparate phylogenies. Interest in virus-vector interactions is growing rapidly. However, current understanding of tick-borne bunyavirus cellular interaction is heavily biased to studies conducted in mammalian systems. In this short review, we summarise current understandings of how tick-borne bunyaviruses utilise major cellular pathways (innate immunity, apoptosis and RNAi responses) in mammalian or tick cells to facilitate virus replication.

Exploration of immunological responses underpinning severe fever with thrombocytopenia syndrome virus infection reveals IL-6 as a therapeutic target in an immunocompromised mouse model.

Dabie bandavirus (previously severe fever with thrombocytopenia syndrome virus; SFTSV), is an emerging tick-borne bunyavirus responsible for severe fever with thrombocytopenia syndrome (SFTS), a disease with high case fatality that is characterized by high fever, thrombocytopenia, and potentially lethal hemorrhagic manifestations. Currently, neither effective therapeutic strategies nor approved vaccines exist for SFTS. Therefore, there remains a pressing need to better understand the pathogenesis of the disease and to identify therapeutic strategies to ameliorate SFTS outcomes. Using a type I interferon (IFN)-deficient mouse model, we investigated the viral tropism, disease kinetics, and the role of the virulence factor nonstructural protein (NSs) in SFTS. Ly6C+ MHCII+ cells in the lymphatic tissues were identified as an important target cell for SFTSV. Advanced SFTS was characterized by significant migration of inflammatory leukocytes, notably neutrophils, into the lymph node and spleen, however, these cells were not required to orchestrate the disease phenotype. The development of SFTS was associated with significant upregulation of proinflammatory cytokines, including high levels of IFN-γ and IL-6 in the serum, lymph node, and spleen. Humoral immunity generated by inoculation with delNSs SFTSV was 100% protective. Importantly, NSs was critical to the inhibition of the host IFNɣ response or downstream IFN-stimulated gene production and allowed for the establishment of severe disease. Finally, therapeutic but not prophylactic use of anti-IL-6 antibodies significantly increased the survival of mice following SFTSV infection and, therefore, this treatment modality presents a novel therapeutic strategy for treating severe SFTS.

Transcriptomic Studies Suggest a Coincident Role for Apoptosis and Pyroptosis but Not for Autophagic Neuronal Death in TBEV-Infected Human Neuronal/Glial Cells.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, Flavivirus genus, is responsible for neurological symptoms that may cause permanent disability or death. With an incidence on the rise, it is the major arbovirus affecting humans in Central/Northern Europe and North-Eastern Asia. Neuronal death is a critical feature of TBEV infection, yet little is known about the type of death and the molecular mechanisms involved. In this study, we used a recently established pathological model of TBEV infection based on human neuronal/glial cells differentiated from fetal neural progenitors and transcriptomic approaches to tackle this question. We confirmed the occurrence of apoptotic death in these cultures and further showed that genes involved in pyroptotic death were up-regulated, suggesting that this type of death also occurs in TBEV-infected human brain cells. On the contrary, no up-regulation of major autophagic genes was found. Furthermore, we demonstrated an up-regulation of a cluster of genes belonging to the extrinsic apoptotic pathway and revealed the cellular types expressing them. Our results suggest that neuronal death occurs by multiple mechanisms in TBEV-infected human neuronal/glial cells, thus providing a first insight into the molecular pathways that may be involved in neuronal death when the human brain is infected by TBEV.