Bunyaviruses: From Transmission by Arthropods to Virus Entry into the Mammalian Host First-Target Cells

Tétard

Youness

et al. 2016

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Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry recep- The Bunyaviridae is a large family of RNA viruses, which comprises five genera (Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus and Tospovirus) (1). With over 350 isolates distributed worldwide, these viruses represent a global threat to livestock, agricultural productivity and human public health (1). Many cause serious diseases with high mortality rates in domestic animals and humans, such as fatal hepatitis, encephalitis and haemorrhagic fever. With † These authors contributed equally to this work.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Differential Use of the C‐Type Lectins L‐SIGN and DC‐SIGN for Phlebovirus Endocytosis

Tétard

Youness

et al. 2016

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“…Antisera were prepared in guinea pigs n X-100-inactivated purified viruses or soluble ectodomains of GN and GC ant. The priming was followed by three booster injections of 100 µg at 4e in Freund's incomplete adjuvant, and the others in phosphate-buffered e bled before the first immunization (control pre-immune serum) and 9 The purified rabbit polyclonal antibodies against the TOSV nucleoprotein l protein NSs (T5) were developed by GenScript (Piscataway, The e immunized with either a peptide corresponding to the C terminal 14 Ss or the full-length N protein with a C-terminal His-tag. The mouse nst all TOSV structural proteins was a generous gift from R.B.…”

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confidence: 99%

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

Woelfl

Oreshkova

et al. 2020

Viruses

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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...

Deciphering Virus Entry with Fluorescently Labeled Viral Particles

Hoffmann

Mazelier

Methods in Molecular Biology

et al. 2018

To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.