Toscana Virus RNA in Sergentomyia minuta Flies
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《传染病的形成》
Universite de la Mediterranee, Marseille, France
Faculte de Medicine Bobigny, Paris, France
In July 2005, a total of 123 Sergentomyia minuta were collected in a 4-day period near Marseille, southeastern France. This work was part of a larger collaborative study, the results of which will be published separately. CDC miniature light traps (John W. Hock Co., Gainesville, FL, USA) were adapted to sandflies with an ultra-fine mesh. Traps were hung 1–2 m above the ground. They were placed in the late afternoon inside or near animal housing facilities (for chickens, rabbits, goats, or horses) in the suburbs of Marseille for 4 successive nights. In these areas, large numbers of geckos were noticed. Each morning, sandflies were collected, identified morphologically, and placed in 1.5-mL Eppendorf tubes. S. minuta flies were identified by appearance, and genus was confirmed by sequence analysis, as previously reported (7).
Five pools of the captured S. minuta were prepared with a maximum of 30 flies per pool. They were ground in 20% fetal bovine serum–enriched phosphate-buffered saline in a Mixer Mill MM300 (Qiagen, Courtaboeuf, France) with one 3-mm tungsten bead and clarified by low-speed centrifugation. We used 200 μL supernatant for total RNA purification onto the MagNAPure platform with the MagNA Pure LC RNA High Performance Kit (Roche Diagnostics, Meylan, France). We used 10 μL RNA suspension for reverse transcription PCR, with primers targeting either a consensus sequence for the phlebovirus polymerase gene (L RNA segment) or Toscana virus (8) and the nucleoprotein (N) gene (S RNA segment) specifically (9).
Only 1 TOSV-positive pool was observed with primers specific to TOSV polymerase and N genes, respectively. A positive result was observed with primers NPhlebo2+ and ATos2, previously found to target polymerase genes of a range of phleboviruses (8). This result was confirmed by sequence analysis (GenBank accession no. DQ195277), which showed 82.8% and 96% identity at the nucleotide and amino acid level, respectively, with a TOSV isolate from Italy (GenBank accession no. X68414). The same pool also tested positive with primers (5′-CGTRGCAGCCACYTCATTAG-3′ and 5′-GTGTCGGCYGCSTTTGTTCC-3′) designed in this study from the alignment of the 13 sequences of TOSV retrieved from GenBank (accession nos. are shown in the Figure). Comparing the sequence of this 272-bp PCR product with homologous sequences of selected phleboviruses available in the GenBank database showed 97.4%, 87.1%–88.2%, and 78.7% identity at the nucleotide level with TOSV strains isolated in Italy, TOSV isolated in Spain, and sandfly fever Naples virus (Sabin strain), respectively. Phylogenetic analyses of the N gene indicated that this virus clustered with TOSV strains circulating in Italy and Spain but is most closely related to isolates from Italy (Figure). Comparative analysis within the polymerase gene confirmed these data, but distance analysis with sequences of Spanish TOSV was not possible because genetic data were lacking in public databases. The remaining 400-μL volume of sandfly material was used to attempt virus isolation in Vero cells and by intracerebral injection of 2-day-old suckling mice, but no virus was recovered.
To our knowledge, this is the first time that TOSV has been detected in phlebotomine flies other than P. perniciosus and P. perfiliewi. S. minuta was identified with morphologic keys and confirmed by sequencing a portion of the 28S gene (7). Sergentomyia spp. have been reported to be infected by a variety of different RNA viruses, such as Chandipura (10), Saboya (11), Tete, and 2 unclassified viruses (ArD 95737 and ArD 111740). However, S. minuta feed on reptiles but not on humans, which may prevent them from being vectors of human infection. Additional studies are needed to better understand the role of Sergentomyia spp. and other arthropods in the ecology of TOSV. Whether TOSV also circulates in Phlebotomus spp. in France remains to be determined, but the evidence for human infections with this virus shows that more extensive investigations are needed to understand the role of this arbovirus in neurologic diseases in the Mediterranean.
This work was supported in part by Vizier, a European project of the Sixth Framework Programme for Research and Technological Development (contract no. LSHG-CT-2004-511960, http://www.vizier-europe.org).
References
Charrel RN, Gallian P, Navarro-Mari J-M, Nicoletti L, Papa A, Sanchez-Seco MP, et al. Emergence of Toscana virus in Europe. Emerg Infect Dis. 2005;11:1657–63.
Valassina M, Meacci F, Valensin PE, Cusi MG. Detection of neurotropic viruses circulating in Tuscany: the incisive role of Toscana virus. J Med Virol. 2000;60:86–90.
Hemmersbach-Miller M, Parola P, Charrel RN, Paul Durand J, Brouqui P. Sandfly fever due to Toscana virus: an emerging infection in southern France. Eur J Intern Med. 2004;15:316–7.
Peyrefitte CN, Devetakov I, Pastorino B, Villeneuve L, Bessaud M, Stolidi P, et al. Toscana virus and acute meningitis, France. Emerg Infect Dis. 2005;11:778–80.
Verani P, Ciufolini MG, Caciolli S, Renzi A, Nicoletti L, Sabatinelli G, et al. Ecology of viruses isolated from sand flies in Italy and characterization of a new Phlebovirus (Arbia virus). Am J Trop Med Hyg. 1988;38:433–9.
Sanbonmatsu-Gámez S, Pírez-Ruiz M, Collao X, Sánchez-Seco MP, Morillas-Márquez F, de la Rosa-Fraile M, et al. Toscana virus in Spain. Emerg Infect Dis. 2005;11:1701–7.
Depaquit J, Perrotey S, Lecointre G, Tillier A, Tillier S, Ferte H, et al. Molecular systematics of Phlebotominae: a pilot study. Paraphyly of the genus Phlebotomus. C R Acad Sci III. 1998;321:849–55.
Sánchez Seco MP, Echevarría JM, Hernández L, Estívez D, Navarro Marí JM, Tenorio A. Detection and identification of Toscana and other phleboviruses by RT-PCR assays with degenerated primers. J Med Virol. 2003;71:140–9.
Valassina M, Cusi MG, Valensin PE. Rapid identification of Toscana virus by nested PCR during an outbreak in the Siena area of Italy. J Clin Microbiol. 1996;34:2500–2.
Geevarghese G, Arankalle VA, Jadi R, Kanojia PC, Joshi MV, Mishra AC. Detection of Chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India. J Med Entomol. 2005;42:495–6.
Ba Y, Trouillet J, Thonnon J, Fontenille D. Phlebotomus of Senegal: survey of the fauna in the region of Kedougou. Isolation of arbovirus [article in French]. Bull Soc Pathol Exot. 1999;92:131–5.(Remi N. Charrel, Arezki I)
Faculte de Medicine Bobigny, Paris, France
In July 2005, a total of 123 Sergentomyia minuta were collected in a 4-day period near Marseille, southeastern France. This work was part of a larger collaborative study, the results of which will be published separately. CDC miniature light traps (John W. Hock Co., Gainesville, FL, USA) were adapted to sandflies with an ultra-fine mesh. Traps were hung 1–2 m above the ground. They were placed in the late afternoon inside or near animal housing facilities (for chickens, rabbits, goats, or horses) in the suburbs of Marseille for 4 successive nights. In these areas, large numbers of geckos were noticed. Each morning, sandflies were collected, identified morphologically, and placed in 1.5-mL Eppendorf tubes. S. minuta flies were identified by appearance, and genus was confirmed by sequence analysis, as previously reported (7).
Five pools of the captured S. minuta were prepared with a maximum of 30 flies per pool. They were ground in 20% fetal bovine serum–enriched phosphate-buffered saline in a Mixer Mill MM300 (Qiagen, Courtaboeuf, France) with one 3-mm tungsten bead and clarified by low-speed centrifugation. We used 200 μL supernatant for total RNA purification onto the MagNAPure platform with the MagNA Pure LC RNA High Performance Kit (Roche Diagnostics, Meylan, France). We used 10 μL RNA suspension for reverse transcription PCR, with primers targeting either a consensus sequence for the phlebovirus polymerase gene (L RNA segment) or Toscana virus (8) and the nucleoprotein (N) gene (S RNA segment) specifically (9).
Only 1 TOSV-positive pool was observed with primers specific to TOSV polymerase and N genes, respectively. A positive result was observed with primers NPhlebo2+ and ATos2, previously found to target polymerase genes of a range of phleboviruses (8). This result was confirmed by sequence analysis (GenBank accession no. DQ195277), which showed 82.8% and 96% identity at the nucleotide and amino acid level, respectively, with a TOSV isolate from Italy (GenBank accession no. X68414). The same pool also tested positive with primers (5′-CGTRGCAGCCACYTCATTAG-3′ and 5′-GTGTCGGCYGCSTTTGTTCC-3′) designed in this study from the alignment of the 13 sequences of TOSV retrieved from GenBank (accession nos. are shown in the Figure). Comparing the sequence of this 272-bp PCR product with homologous sequences of selected phleboviruses available in the GenBank database showed 97.4%, 87.1%–88.2%, and 78.7% identity at the nucleotide level with TOSV strains isolated in Italy, TOSV isolated in Spain, and sandfly fever Naples virus (Sabin strain), respectively. Phylogenetic analyses of the N gene indicated that this virus clustered with TOSV strains circulating in Italy and Spain but is most closely related to isolates from Italy (Figure). Comparative analysis within the polymerase gene confirmed these data, but distance analysis with sequences of Spanish TOSV was not possible because genetic data were lacking in public databases. The remaining 400-μL volume of sandfly material was used to attempt virus isolation in Vero cells and by intracerebral injection of 2-day-old suckling mice, but no virus was recovered.
To our knowledge, this is the first time that TOSV has been detected in phlebotomine flies other than P. perniciosus and P. perfiliewi. S. minuta was identified with morphologic keys and confirmed by sequencing a portion of the 28S gene (7). Sergentomyia spp. have been reported to be infected by a variety of different RNA viruses, such as Chandipura (10), Saboya (11), Tete, and 2 unclassified viruses (ArD 95737 and ArD 111740). However, S. minuta feed on reptiles but not on humans, which may prevent them from being vectors of human infection. Additional studies are needed to better understand the role of Sergentomyia spp. and other arthropods in the ecology of TOSV. Whether TOSV also circulates in Phlebotomus spp. in France remains to be determined, but the evidence for human infections with this virus shows that more extensive investigations are needed to understand the role of this arbovirus in neurologic diseases in the Mediterranean.
This work was supported in part by Vizier, a European project of the Sixth Framework Programme for Research and Technological Development (contract no. LSHG-CT-2004-511960, http://www.vizier-europe.org).
References
Charrel RN, Gallian P, Navarro-Mari J-M, Nicoletti L, Papa A, Sanchez-Seco MP, et al. Emergence of Toscana virus in Europe. Emerg Infect Dis. 2005;11:1657–63.
Valassina M, Meacci F, Valensin PE, Cusi MG. Detection of neurotropic viruses circulating in Tuscany: the incisive role of Toscana virus. J Med Virol. 2000;60:86–90.
Hemmersbach-Miller M, Parola P, Charrel RN, Paul Durand J, Brouqui P. Sandfly fever due to Toscana virus: an emerging infection in southern France. Eur J Intern Med. 2004;15:316–7.
Peyrefitte CN, Devetakov I, Pastorino B, Villeneuve L, Bessaud M, Stolidi P, et al. Toscana virus and acute meningitis, France. Emerg Infect Dis. 2005;11:778–80.
Verani P, Ciufolini MG, Caciolli S, Renzi A, Nicoletti L, Sabatinelli G, et al. Ecology of viruses isolated from sand flies in Italy and characterization of a new Phlebovirus (Arbia virus). Am J Trop Med Hyg. 1988;38:433–9.
Sanbonmatsu-Gámez S, Pírez-Ruiz M, Collao X, Sánchez-Seco MP, Morillas-Márquez F, de la Rosa-Fraile M, et al. Toscana virus in Spain. Emerg Infect Dis. 2005;11:1701–7.
Depaquit J, Perrotey S, Lecointre G, Tillier A, Tillier S, Ferte H, et al. Molecular systematics of Phlebotominae: a pilot study. Paraphyly of the genus Phlebotomus. C R Acad Sci III. 1998;321:849–55.
Sánchez Seco MP, Echevarría JM, Hernández L, Estívez D, Navarro Marí JM, Tenorio A. Detection and identification of Toscana and other phleboviruses by RT-PCR assays with degenerated primers. J Med Virol. 2003;71:140–9.
Valassina M, Cusi MG, Valensin PE. Rapid identification of Toscana virus by nested PCR during an outbreak in the Siena area of Italy. J Clin Microbiol. 1996;34:2500–2.
Geevarghese G, Arankalle VA, Jadi R, Kanojia PC, Joshi MV, Mishra AC. Detection of Chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India. J Med Entomol. 2005;42:495–6.
Ba Y, Trouillet J, Thonnon J, Fontenille D. Phlebotomus of Senegal: survey of the fauna in the region of Kedougou. Isolation of arbovirus [article in French]. Bull Soc Pathol Exot. 1999;92:131–5.(Remi N. Charrel, Arezki I)