VIROLOGY 180, 738-753 (199 I) Sequences and Coding Strategies of the S RNAs of Toscana and Rift Valley Fever Viruses Compared to Those of Punta Toro, Sicilian Sandfly Fever, and Uukuniemi Viruses COLOMBA GIORGI,” LUISA ACCARDI,* LOREDANA NICOLEITI,” MARIA CRISTINA GRO,* KAZUAKI TAKEHARA,?’ CORINNE HILDITCH,t SHIGERU MORIKAWA, AND DAVID H. L. BISHOPt,2 *Laboratory of Virology, lstituto Superiore di San@, Via/e Regina Elena, 229, 0016 1 Rome, ltaly; and tNERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX 1 3SR, United Kingdom Received July 20, 1990; accepted October 12, 1990 The sequences and coding strategies of the S RNAs of two viruses, Toscana (TOS) and the Ml 2 derivative of Rift Valley fever ZH-548 (RVF, Phlebovirus genus, Bunyaviridae) have been determined from cDNA clones and compared to the previously published sequences of Punta Toro (PT), Sandfly fever Sicilian (SFS), and Uukuniemi (UUK) viruses. All five viruses exhibit an ambisense coding strategy for their small (S) RNA species, i.e., one gene product (the NSs protein) is encoded in the 5’half of the viral RNA, a second (the N protein) is encoded in the sequence complementary to the 3’ half. The terminal nucleotides of the S RNAs of the five viruses are comparable through 13-14 residues. The 3 and 5’ ends of these S RNAs have inverted complementary compositions. Three phleboviruses (TOS, SFS, and RVFV) exhibit comparable G-rich, centrally located intergenic sequences, albeit of different lengths. These sequences have a number of similar motifs at, or immediately following, the end of the coding regions, motifs that may be involved in their S mRNA transcription termination processes. The other two viruses (UUK, PT) have AT-rich intergenic sequences that have the potential to form secondary structure. They lack the G-rich sequences or particular sequence motifs recog- nized in the other three virus RNAs. The deduced sizes of the TOS and RVFV N proteins are 27,704 and 27,430 kDa (respectively). Their NS, proteins are 36,677 and 29,903 kDa (respectively). When aligned, the deduced sequences of the N proteins of the five viruses exhibit homologies ranging from 54 to 30%. The order of homology to RVFV N protein is PT > TOS > SFS > UUK; to TOS N protein it is PT 3 RVF > SFS > UUK. The sequences of the NS, proteins are less similar, with values ranging from 30 to ~17%. The order of homology to RVFV NSs is SFS > PT 1 TOS > UUK. Due to these more distant relationships, the homologies to TOS NSs protein are less clear. Q 1991 Academic Press. Inc. INTRODUCTION More than 300 viruses isolated from all over the world are classified as belonging to the Bunyaviridae family (Karabatsos, 1985). They have spherical and en- veloped virions (90-l 00 nm in diameter) and a single- stranded RNA genome consisting of three noncova- lently closed, circular RNA species, designated small, medium, and large (S, M, and L). On the basis of sero- logical relationships the majority of viruses of the Bun- yaviridae have been classified into five genera: Bunya- virus, Hantavirus, Nairovirus, Phlebovirus, and Uuku- virus (Bishop, 1990). Although most of the viruses have come from hematophagous arthropods and/or verte- brates, recent analyses indicate that the thrip-transmit- ted plant virus, tomato spotted wilt, and Rice stripe virus should be considered as members of the family (Milne and Francki, 1984; de Haan el a/., 1989, 1990; Kakutani et a/., 1990). ’ Present address: School of Veterinary Medicine and Animal Sciences. Towadashi, Aornori 034, Japan. ’ To whom requests for reprints should be addressed. 0042.6822/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights ot reproduction in any tarm resewed. The Phlebovirus genus is composed of at least 39 viruses (Karabatsos, 1985; Tesh er al., 1982; Travas- SOS da Rosa et a/., 1983), most of which have been isolated from phlebotomines (sandflies). On occasion they have come from vertebrates, mosquitoes, or cera- topogonids in the genus Culicoides. The prototype virus of the group is Sandfly fever Sicilian (SFS) virus. Several phleboviruses have been associated with hu- man disease (Tesh, 1988). Toscana (TOS) virus has been isolated in Italy from Phlebotomus perniciosus and P. perfiliewi sandflies (Verani et a/., 1980). The virus has been associated with cases of human ascep- tic meningitis in Italy and Portugal (Balducci et a/., 1985; Ehrnst et a/., 1985). By means of complement fixation and neutralization tests TOS virus has been classified as belonging to the Sandfly fever Naples (SFN) virus group of phleboviruses (Verani et al., 1984). Other members of this group (or so-termed “com- plex”) include SFN, Karimabad, and Tehran viruses (Bishop, 1990). Another phlebovirus, Rift Valley fever virus (RVFV), was originally isolated from the blood of a newborn lamb at the Veterinary Research Laboratory in Kabete, Kenya, in 1930 (Daubney and Hudson, 1931). It has been isolated frequently from ruminants (e.g., 73x