Toscana virus inhibits the interferon beta response in cell cultures

Toscana virus (TOSV) is a Phlebotomus-transmitted RNA virus and a frequent cause of human meningitis and meningoencephalitis in Southern Europe during the summer season. While evidence for TOSV-related central nervous system (CNS) cases is increasing, little is known about the host defenses against TOSV. We evaluated innate immune response to TOSV by analyzing frequency and activation of blood antigen-presenting cells (APCs) and cytokine levels in plasma and cerebrospinal fluid (CSF) from patients with TOSV neuroinvasive infection and controls. An altered frequency of different blood APC subsets was observed in TOSV-infected patients, with signs of monocytic deactivation. Nevertheless, a proper or even increased responsiveness of toll-like receptor 3 and 7/8 was observed in blood APCs of these patients as compared to healthy controls. Systemic levels of cytokines remained low in TOSV-infected patients, while levels of anti-inflammatory and antiviral mediators were significantly higher in CSF from TOSV-infected patients as compared to patients with other infectious and noninfectious neurological diseases. Thus, the early host response to TOSV appears effective for viral clearance, by proper response to TLR3 and TLR7/8 agonists in peripheral blood and by a strong and selective antiviral and anti-inflammatory response in the CNS.

Section: Discussionmentioning

confidence: 99%

Meningitis Caused by Toscana Virus Is Associated with Strong Antiviral Response in the CNS and Altered Frequency of Blood Antigen-Presenting Cells

Varani

Gelsomino

Bartoletti

et al. 2015

“…More recently, the NSs protein of TOSV was also identified as an interferon antagonist [62,63]. Thus, even though NSs is not essential for viral replication per se , it is important in the context of virus/host interactions.…”

Section: Genus Phlebovirusmentioning

confidence: 99%

Non-Structural Proteins of Arthropod-Borne Bunyaviruses: Roles and Functions

Eifan

Schnettler

Dietrich

et al. 2013

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod.

“…From earlier studies, it was not clear if TOSV counteracts type I IFN responses. The virus was shown to both suppress and induce the IFN responses in cell cultures [40,41]. The reason may be the different cell model systems used in those studies.…”

Section: Discussionmentioning

confidence: 99%

Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses

Woelfl

Léger

Oreshkova

et al. 2020

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSV Viruses 2019, 11, x FOR PEER REVIEW and Proteomics Core Facility of the German Cancer Research Center, Heidelberg (Table 1). B viruses were produced and purified, as described in Section 2.5. Soluble ectodomains of GN a were stably expressed in drosophila S2 cells and purified according to a standard procedure Using these protocols, the protein purity reached over 90%. Antisera were prepared in guinea by injecting 100 µg of Triton X-100-inactivated purified viruses or soluble ectodomains of GN a in Freund's complete adjuvant. The priming was followed by three booster injections of 100 µg week intervals, the first one in Freund's incomplete adjuvant, and the others in phosphate-bu saline (PBS). Animals were bled before the first immunization (control pre-immune serum) days after the last injection. The purified rabbit polyclonal antibodies against the TOSV nucleop N (T4) and non-structural protein NSs (T5) were developed by GenScript (Piscataway Netherlands). Rabbits were immunized with either a peptide corresponding to the C termin amino acid residues of NSs or the full-length N protein with a C-terminal His-tag. The m immune ascitic fluid against all TOSV structural proteins was a generous gift from R.B. (University of Texas, Galveston, Texas, USA). s were constructed by GenScript. Briefly, the cDNAs encoding the full-length S, M, and f TOSV, flanked by an upstream T7 polymerase promoter sequence and a downstream ta virus (HdV) ribozyme sequence ( Figure S1), were synthesized. The gene synthesis e subjected to blunt-end ligation into EcoRV-linearized pUC57 plasmid, or alternatively, into the pCC1 plasmid, using the CloneEZ PCR Cloning Kit (Genscript), resulting in C1-M, and pUC57-L. In addition, a pUC57-SɸNSs was created in which the S segment mRNA, with the 18 first AUG codons replaced by UAG stop codons ( Figure S2). Viruses from Plasmid DNAscells expressing T7 polymerase were seeded in 6-well plates (2.5 × 10 5 cells per well). g day, rTOSV was rescued from cells by transfection with 1 µg each of the plasmids C1-M, and pUC57-L. Alternatively, the recovery of rTOSVɸNSs was achieved following thod but using the plasmid pUC57-SɸNSs instead of the pUC57-S. Transfection was ith Lipofectamine 2000 (Thermo Fisher Scientific), using a ratio of 1 µL Lipofectamine of plasmids in 400 µL of complete GMEM without antibiotics. Supernatants were fresh culture medium containing antibiotics and 2% serum 4 h post-transfection. Five nsfection, supernatants were harvested, c...