Rift Valley fever virus (RVFV) is an arthropod-borne virus (arbovirus) responsible for a severe zoonotic disease affecting a wide range of domestic and wild ruminants as well as humans. RVFV is endemic in many African countries and has also caused outbreaks in Madagascar and Arabian Peninsula. With regard to its wide geographical distribution, its potential to emerge in a new area, and its capability to trigger major health and economic crisis, it is essential to study and better understand several aspects of its life cycle and, in particular, its interactions with mammalian hosts and arthropod vectors. To do so, it is key for researchers to be able to amplify in vitro viral strains isolated from the field and determine accurately the viral titers of RVFV stocks. In this chapter, we present protocols that can be easily implemented to produce and titrate RVFV stocks in your laboratory.

Rift Valley fever virus (RVFV) causes severe disease in humans and ungulates. The virus can be transmitted by mosquitoes, direct contact with infected tissues or fluids, or aerosol, making it a significant biological threat for which there is no approved vaccine or therapeutic. Herein we describe the evaluation of DEF201, an adenovirus-vectored interferon alpha which addresses the limitations of recombinant interferon alpha protein (cost, short half-life), as a pre- and post-exposure treatment in a lethal hamster RVFV challenge model. DEF201 was delivered intranasally to stimulate mucosal immunity and effectively bypass any pre-existing immunity to the vector. Complete protection against RVFV infection was observed from a single dose of DEF201 administered one or seven days prior to challenge while all control animals succumbed within three days of infection. Efficacy of treatment administered two weeks prior to challenge was limited. Post‑exposure, DEF201 was able to confer significant protection when dosed at 30 min or 6 h, but not at 24 h post-RVFV challenge. Protection was associated with reductions in serum and tissue viral loads. Our findings suggest that DEF201 may be a useful countermeasure against RVFV infection and further demonstrates its broad-spectrum capacity to stimulate single dose protective immunity.

The S segment of Rift Valley fever virus (Bunyaviridae, Phlebovirus) codes for two proteins, the nucleoprotein N and the nonstructural protein NSs. The NSs protein is a phosphoprotein of unknown function that is localized in the cytoplasm and the nuclei of infected cells where it forms filamentous structures. To characterize further the protein expressed in VC10 cells infected with the MP12 strain, we analyzed its phosphorylation states and showed that phosphorylated forms were found in both compartments. Cytoplasmic and nuclear NSs were phosphorylated only at serine residues. Phosphopeptide mapping and molecular analysis of mutants obtained by site-directed mutagenesis allowed us to map the major phosphorylation sites of nuclear and cytoplasmic forms of NSs to serine residues 252 and 256, located at the carboxy-terminus in consensus sequences for casein kinase II. A similar map was obtained when the protein was purified from mosquito cells infected with MP12. In addition, we showed that the purified unphosphorylated NSs protein expressed from pET-NSs plasmid in a coupled transcription-translation reaction containing Escherichia coli S30 extracts did not possess autophosphorylation activity but was phosphorylated in vitro after incubation with recombinant casein kinase II.

One of the major physiologic functions of erythrocytes is the mediation of chloride-bicarbonate exchange in the transport of carbon dioxide from the tissues to the lungs. The anion exchange is mediated by a typical polytopic transmembrane protein in the cell membrane, designated Band 3. A carboxyl-terminal peptide of Band 3 was affinity-labeled with pyridoxal phosphate, a substrate for the anion transport system, and then sequenced (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The 10th amino acid residue of the peptide could not be determined, suggesting post-translational modification of the residue. In the present communication, we have investigated the molecular structure of human Band 3 and the COOH-terminal 8500-dalton peptide using gas-liquid chromatography-mass spectrometry. Band 3 was modified covalently by fatty acids and these acids were released from Band 3 by hydroxylamine treatment at either pH 7 or 11, indicating that the linkage between Band 3 and the fatty acid is a thio ester bond. 1 mol of Band 3 interacted with 1 mol of fatty acid at a cysteine residue located 69 residues from the COOH terminus of Band 3. The fatty acids used in the modification were myristate, palmitate, oleate, and stearate, with palmitate being the major component. The esterified site is close to the site affinity-labeled with pyridoxal phosphate (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The amino acid sequence including the acylation site was Phe-Thr-Gly-Ile-Gln-Ile-Ile-Cys-Leu-Ala-Val-Leu, which is conserved in the G2 protein of Rift Valley fever virus as Phe-Ser-Ser-Ile-Ala-Ile-Ile-Cys-Leu-Ala-Val-Leu. The G2 protein, like Band 3, is a polytopic transmembrane protein. Although acylation of the cysteine residue of G2 protein has not been examined, the Phe-X-X-Ile-X-Ile-Ile-Cys-Leu-Ala-Val-Leu sequence could be a common motif for fatty acylation of certain membrane proteins.