Sicilian Virus 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.

High prevalence rates of antibody to three sandfly fever viruses (Sicilian, Naples, and Toscana) among Cypriots.

Neutralizing antibodies to sandfly fever Naples, sandfly fever Sicilian and Toscana viruses were investigated among 479 sera collected from a normal human population in Cyprus. Antibody prevalence rates of 57%, 32% and 20% were found to Naples, Sicilian and Toscana viruses, respectively. The observed frequency of dual and triple infections was higher than would be expected with a random chance of infection. Antibody prevalence rates were similar for men and women for all three viruses tested, but one of two study sites had significantly higher antibody prevalence to Naples and Sicilian viruses than the other. Individuals with antibodies to both Naples and Toscana viruses had higher antibody levels to Naples virus than those with antibodies to Naples virus only. If the antibody prevalence rates found in this study reflect a history of clinical disease as described in the literature, sandfly fever poses a significant public health problem in Cyprus.

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.

Sandfly fever among Swedish tourists.

All clinical and serologically confirmed cases of sandfly fever among Swedish tourists during 1986-1989 were investigated. A total of 37 cases of Sicilian virus sandfly fever and 1 case of Naples virus sandfly fever were recorded from Cyprus. One case of Toscana virus infection was documented from Spain. The clinical and laboratory findings did not differ from those in previous reports, except for elevated aminotransferase levels. A questionnaire, that offered a possibility to estimate the number of cases that had not been reported, was sent to 713 tourists who had visited different hotels in Cyprus and Mallorca in September 1987. Serum samples from 95 of these tourists were tested for sandfly fever virus antibodies. These tests indicated that only some 20% of the real number of sandfly fever infections had been diagnosed. Both clinical cases and cases found by the questionnaire showed a focal distribution with an overrepresentation of certain hotels in Cyprus. The results were reported to the local health officers and possibly due to the measures undertaken hereafter the number of sandfly fever cases diagnosed have decreased.

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.

Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus

We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family.

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.

Biochemical studies on the Phlebotomus fever group viruses (Bunyaviridae family)

Analyses of the virion polypeptides and genomes of several Phlebotomus fever group viruses, Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses, have established that they are biochemically similar to the accepted members of the Bunyaviridae family. Like snowshoe hare virus (a member of the California serogroup of the Bunyavirus genus of the Bunyaviridae family), Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses all have three viral RNA species, designated large (L), medium (M), and small (S). Oligonucleotide fingerprint analyses of Karimabad and Punta Toro virus RNA species indicated that their L, M, and S RNA species are unique. By polyacrylamide gel electrophoresis it was determined for Karimabad virus that the apparent molecular weights of its L, M, and S RNA species are 2.6 X 10(6), 2.2 X 10(6), and 0.8 X 10(6), respectively. For Punta Toro virus, the apparent molecular weights of its L, M, and S RNA species are 2.8 X 10(6), 1.8 X 10(6), and 0.75 X 10(6), respectively. The major internal nucleocapsid (N) protein of Karimabad virus was found to have a molecular weight of 21 X 10(3). A similar polypeptide size class was identified in preparations of sandfly fever Sicilian serotype, Chagres, and Punta Toro viruses. The Karimabad virus glycoproteins formed the external surface projections on virus particles and could be removed from virus preparations by protease treatment. The glycoproteins in an unreduced sample could be resolved into two size classes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They had apparent molecular weights of 62 X 10(3) and 50 X 10(3) in continuous polyacrylamide gels. When Karimabad virus preparations were reduced with 1% beta-mercaptoethanol, prior to resolution by continuous polyacrylamide gel electrophoresis, all the viral glycoprotein was recovered in a single size class, having an apparent molecular weight of 62 X 10(3). Two or three major virion polypeptides have been identified in preparations of Punta Toro, Chagres, and sandfly fever Sicilian serotype viruses.

Studies on the Epidemiology of Sandfly Fever in Iran

Human and animal sera from an endemic area of sandfly fever in Iran were tested by plaque reduction neutralization method against five different Phlebotomus fever virus serotypes (Naples, Sicilian, Karimabad, Salehabad, and I-47). The overall prevalence of Naples, Sicilian, and Karimabad virus antibodies among the human population was 17%, 25%, and 66%, respectively. All sera were negative against Salehabad and I-47 viruses. Age-specific antibody rates suggested that Sicilian and Karimabad viruses were endemic in the study area but that Naples virus activity was sporadic. These observations were confirmed by isolations of Sicilian and Karimabad viruses from sandflies collected in the study area. Among the animal sera tested, evidence of Phlebotomus fever virus infection was detected only in gerbils. Of 38 Rhombomys opimus tested, 34% had neutralizing antibodies against Sicilian virus and 32% against Karimabad. These results indicate that gerbils are infected with these two viruses and possibly might serve as reservoirs or amplifying hosts. The serologic studies also suggest that the ecology of Sicilian and Karimabad viruses involves chiefly sandflies, gerbils, and man, an epidemiologic pattern previously demonstrated for cutaneous leishmaniasis in the same region of Iran.

Studies on the Epidemiology of Sandfly Fever in Iran

A total of 62 virus isolates was obtained from 12,485 male and female sandflies (predominately Phlebotomus papatasi) collected in a village in central Iran during the summer of 1975. The overall virus isolation rate from female sandflies was 1 per 177 insects processed. Of the 62 virus strains obtained, 49 were identified as Sicilian virus, 11 as Karimabad, and 2 as a new member of the vesicular stomatitis serogroup. One isolate each of Sicilian and Karimabad virus was made from pools of male sandflies. The three virus types were active in the sandfly population simultaneously. Sicilian virus isolation rates showed little variation during the study period, suggesting continuous virus activity during the sandfly season. The implications of these findings for the epidemiology of sandfly fever are discussed.

Isolation of Phlebotomus fever virus from Phlebotomus papatasi and determination of the host ranges of sandflies (Diptera: Psychodidae) in West Pakistan.

Sandflies collected in the vicinity of Lahore and Peshawar, West Pakistan were identified to species and sex. The source of the blood-meal of freshly-engorged sandflies was determined by precipitin testing using avian, bovine, canine, equine, human, sheep, murine and reptilian antisera. Most Phlebotomus papatasi had fed on humans or cattle, but birds, dogs, and horses were also identified as hosts. P. sergenti had fed on birds, bovines, and humans. Most of the blood-engorged Sergentomyia yielded negative test results, but one S. baghdadis had fed on a human, and 2 blood smears from S. babu blood-meals reacted weakly with avian antiserum. The single virus isolate obtained was from a pool of P. papatasi and this agent was identified by neutralization tests in suckling mice as Sicilian phlebotomus fever virus.

Responses of monkeys to California encephalitis, Sicilian sand-fly, and Turlock arboviruses.

SummaryRhesus monkeys (Macaca mulatta) can be used for production of monotypic antisera against California encephalitis, Sicilian sand-fly fever, and Turlock arboviruses. Antisera produced with infected smb inoculated intramuscularly were specific in HI tests, and relatively nonspecific in CF tests. In the latter tests, the antisera cross-reacted principally because of nonspecific antibodies directed against normal mouse CNS antigens. Such cross-reactions were not encountered in sera obtained by hyperimmunization with infected cell culture-adjuvant mixtures.Intracerebral inoculation of these viruses in monkeys resulted in fever, and except in one animal, no other signs pathognomonic of CNS disease. Four of six monkeys had low levels of antibodies; all had antibodies following intramuscular boosters. The antibody levels were below those obtained by hyperimmunization with virus-adjuvant mixtures. Pathologic lesions in the CNS of monkeys strongly suggest that California virus produced mild encephalomyelitis. California encephalitis and Turlock viruses were propagated in cell cultures. Sicilian sand-fly virus did not regularly lyse cells of those cultures tried. These three arboviruses were inactivated by treatment with 0.05% beta-propiolactone.

Phlebotomus (sandfly) fever viruses in tissue culture.

Summary Propagation of the Naples and Sicilian types of Phlebotomus (sandfly) fever viruses in primary human and mouse kidney tissue cultures has been described. Plaques in human kidney cultures were delayed but were produced consistently by both Naples and Sicilian types. Cytopathogenic effects as a result of multiplication by either virus type, could be demonstrated in infant kidney cultures only after an adaptation period. In contrast, cytopathic changes could be seen in the first and subsequent virus passages when mouse kidney cultures were inoculated with either the Naples or Sicilian virus types. Mouse kidney cultures were found to be more susceptible to virus infection than human kidney monolayers.