Sandfly Fever Sicilian Research Articles

Analysis of 3′ and 5′ ends of N and NSs messenger RNAs of Toscana Phlebovirus

The 5′ and 3′ ends of N and NSs mRNAs, transcribed from the S segment of Toscana Phlebovirus, were analyzed by oligonucleotide primer extension and S1 nuclease mapping procedures. The results showed that both mRNAs acquired, at their 5′ end, approximately 9–15 nucleotides not present in the viral template, suggesting an initiation transcription mechanism similar to the one described for influenza virus. Furthermore, the 3′ ends of the two mRNAs were located in a sequence motif conserved in the S segment of two other Phleboviruses, the Rift Valley Fever and Sandfly Fever Sicilian viruses. This finding suggests the possible involvement of this sequence in the mechanism of transcription termination.

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

The sequences and coding strategies of the S RNAs of two viruses, Toscana (TOS) and the M12 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 NS s 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 recognized 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 s 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 ⩾ RVF > SFS > UUK. The sequences of the NS s proteins are less similar, with values ranging from 30 to <17%. The order of homology to RVFV NS s is SFS > PT . TOS > UUK. Due to these more distant relationships, the homologies to TOS NS s protein are less clear.

ELISA for detection of IgM and IgG antibodies to sandfly fever sicilian virus

• An enzyme-linked immunosorbent assay (ELISA) was developed to detect specific human immunoglobulin G and M antibodies to sandfly fever Sicilian (SFS) virus. Acute and early convalescent serum pairs with ⩾ 7 days between the 2 specimens were available from 20 patients and all showed significant optical density (OD) increase and significant titre rise (⩾ 4-fold) by IgG ELISA. However, negative or borderline-positive sera were found as late as 11 days after onset of symptoms when tested by IgG ELISA. • Specific IgM antibodies were detected during the first week of symptoms, and maximum OD values were obtained during the first 4 weeks after onset of disease. The IgM OD values declined over the following 3–9 months. All sera collected later than 14 months post-onset were negative by IgM ELISA. • The combination of early antibody response and the need to test only one serum specimen gives IgM ELISA an advantage over IgG ELISA in patient diagnosis. • The IgG ELISA was also evaluated as a seroepidemiological tool and compared to a plaque reduction neutralization test (PRNT) using sera from a normal Cypriot population. Of 183 sera tested, 34 (19%) were positive in plaque reduction neutralization tests (PRNT) and 113 (62%) by IgG ELISA. A number of PRNT-negative sera were strongly positive by IgG ELISA and also by indirect immunofluorescence test, which may suggest the presence of a virus related to SFS in Cyprus which has not yet been isolated.

Incidence of Sand Fly Fever Among Swedish United Nations Soldiers on Cyprus During 1985

Members of a Swedish United Nations (UN) battalion of 362 soldiers were bled just before and immediately after their 6 month tour of duty in Cyprus during 1985. Sera were tested for presence of specific antibodies to sand fly fever (SF) Sicilian, SF Naples, and Toscana viruses by both indirect immunofluorescence antibody test (IFT) and plaque reduction neutralization test (PRNT). Of 298 serum pairs available, seroconversion was noted in 11 soldiers: 7 cases for SF Sicilian, 3 for SF Naples, and 1 for Toscana virus. IFT and PRNT revealed identical results. Seroconversion was associated with clinical disease in 7 out of 7, 2 out of 3, and 0 out of 1 for SF Sicilian, SF Naples, and Toscana infections, respectively. Virus isolation was attempted on acute-phase sera collected from febrile patients. Of 5 such acute-phase sera from patients with serologically verified SF, virus was recovered from 2: 1 strain of SF Sicilian virus and 1 strain of SF Naples virus.

Coding strategy of the S RNA segment of dugbe virus ( Nairovirus; Bunyaviridae)

The S RNA segment of Dugbe (DUG) virus ( Nairovirus; Bunyaviridae) was sequenced from three overlapping cDNA clones and by primer extension. The S RNA is 1712 nucleotides in length and contains one large open reading frame (ORF) of 1326 nucleotides coding for a 49.4-kDa protein on viral complementary (vc) RNA. This protein in size corresponds to the DUG nucleocapsid (N) protein (P. Cash, 1985, J. Gen. Virol. 66, 141–148). The 49.4-kDa product was expressed as a fusion protein with, β-galactosidase in Escherichia coli cells and confirmed as DUG N protein by Western blotting with DUG N-specific monoclonal antibody. An additional ORF of 150 nucleotides coding for a possible 5.9-kDa protein is present in the +1 reading frame, 3′ to the N protein ORF on vcRNA. DUG S segment mRNA was found to be essentially full length. No evidence was obtained for the existence of a smaller mRNA species that could code for a 5.9-kDa protein. Comparisons of the DUG S RNA sequence and predicted N protein amino acid sequence, with the respective sequences of snowshoe hare, La Crosse (bunyaviruses), Punta Toro, Sandfly fever Sicilian (phleboviruses), and Hantaan (hantavirus) viruses, failed to detect any sequence similarity, although the genomic structure of DUG S RNA is similar to that of the S RNA segment of Hantaan (HTN) virus.

The S RNA segment of Sandfly fever Sicilian virus: Evidence for an ambisense genome

The complete nucleotide sequence of the S RNA segment of Sandfly Fever Sicilian (SFS) virus ( Phlebovirus, Bunyaviridae) was determined from overlapping cDNA clones and by primer extension. The RNA is 1746 nucleotides in length and has two large open reading frames (ORF), one of which (24.8 kDa) is viral-complementary in sense, and the other (30.4 kDa) is in the viral sense. This ambisense genome arrangement has been seen in another member of the Phlebovirus genus, Punta Toro (PT) virus ( T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293–306 ), but not in representatives of either the Bunyavirus or H antavirus genera of the Bunyaviridae. Comparison of the predicted amino acid sequences for SFS virus with the recognized products of PT S RNA ( T. Ihara, Y. Matsuura, and D. H. L. Bishop, 1985, Virology 147, 317–325 ; H. A. Overton, T. Ihara, and D. H. L. Bishop, 1987, Virology 157, 338–350 ) indicated that the 24.8-kDa ORF encodes the nucleoprotein (N) of SFS virus, and the 30.4-kDa ORF codes for a nonstructural protein (NSs). Subgenomic messenger RNAs, from which these two proteins are presumably translated, were detected in virus-infected cellS.

Experimental infection of Phlebotomus papatasi with sand fly fever Sicilian virus.

Experimental studies were conducted to evaluate humans as hosts infecting the sand fly Phlebotomus papatasi with sand fly fever Sicilian (SFS) virus. Viral antigen and infectious virus circulated in the blood of infected volunteers on days 4 and 5 after intravenous inoculation with SFS virus. Viremia levels during the latter period were high enough to infect feeding sand flies, but only 13% (9/69) of the flies became infected. One out of every 3 infected sand flies that survived to feed a second time transmitted SFS to a hamster. These results confirm a vertebrate-sand fly-vertebrate transmission cycle for SFS virus, and demonstrate that horizontal transmission may contribute to the maintenance of this virus in nature.