CLONING, SEQUENCING AND EXPRESSION OF THE MEDIUM GENOMIC RNA SEGMENT OF SANDFLY FEVER SICILIAN VIRUS

Abstract

Sandfly fever virus Sicilian strain (SFS) is a member of the Phlebovirus genus in the family Bunyaviridae. This virus is the causative agent of an acute, self-limiting flu-like illness lasting 2-5 days. Serologic studies have shown that SFS virus is endemic in southern Europe, the Mediterranean, North Africa, and central Asia which coincides with the distribution of its vector, Phlebotomus papatasi. Epidemics of sandfly fever occur when nonimmune adults, such as tourists or soldiers, enter an area where the virus is endemic. Sandfly fever is of military importance due to its short incubation period, capable of rendering large numbers of nonimmune troops ineffective. The development of a vaccine against SFS virus would allow vaccination of troops prior to entering endemic areas. The phlebovirus genome contains three single-stranded RNA segments designated L (large), M (medium), and S (small) of negative or ambisense polarity. The M segment encodes two viral glycoproteins, G1 and G2, and a nonstructural protein, NSм. Studies of other phleboviruses have demonstrated that the glycoproteins are important for viral infection, pathogenesis, and are the primary targets of the immune response. Analysis of the SFS virus M genomic segment was initiated to determine the sequence and genomic organization for use in the development of both nucleic acid-based diagnostic methods and recombinant vaccines. A series of cDNA clones representing the M genome segment were produced by conventional cloning methods and by polymerase chain reaction amplification. All clones utilized for sequence determination were found to be specific for the SFS virus M RNA segment when used as hybridization probes against SFS virus RNA in northern blot analysis. The M-segment cDNA clones were sequenced by the dideoxy method utilizing both manual and automated procedures. The SFS virus M RNA segment was found to be 4402 nucleotides in length. Computer analysis of this sequence predicted a single open reading frame (ORF) 1341 amino acids in length. Based on comparison of the predicted protein sequence of the SFS M segment with other reported phlebovirus M segments, PCR primers were designed to prepare expression cassettes for producing the viral glycoproteins in recombinant baculoviruses. The downstream glycoprotein region was cloned into Autographa califomica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda (Sf9) cells. The expressed protein was characterized by immunoprecipitation with SFS virus hyperimmune mouse ascitic fluid and was found to be indistinguishable, based on electrophoretic mobilities in denaturing SDS-PAGE, from the authentic SFS virus G2 envelope glycoprotein. These results led to the conclusion that the genomic organization of the SFS M RNA segment is 5'-NSм-G1-G2-3'.