Volume 85, Issue 1
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



Mosquitoes were collected in Colorado during 2006 and 2007 to examine spatial and seasonal patterns of risk for exposure to vectors and West Nile virus. We used universal flavivirus primers to test pools of mosquitoes for viral RNA. This led to the detection and subsequent isolation of two insect-specific flaviviruses: flavivirus (CxFV), which was first described from Japan, and a novel insect flavivirus, designated Calbertado virus (CLBOV), which has also been detected in California and Canada. We recorded both viruses in and from Colorado. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of CxFV RNA in eggs and larvae from a laboratory colony established in 2005 and naturally infected with CxFV, suggesting vertical transmission as a means of viral maintenance in natural populations. Finally, we present phylogenetic analyses of the relationships between insect-specific flaviviruses and other selected flaviviruses.


Article metrics loading...

Loading full text...

Full text loading...



  1. Cook S, Holmes EC, , 2006. A multigene analysis of the phylogenetic relationships among the flaviviruses (Family: Flaviviridae) and the evolution of vector transmission. Arch Virol 151: 309325.[Crossref]
  2. Stollar V, Thomas VL, , 1975. An agent in the Aedes aegypti cell line (Peleg) which causes fusion of Aedes albopictus cells. Virology 64: 367377.[Crossref]
  3. Crabtree MB, Sang RC, Stollar V, Dunster LM, Miller BR, , 2003. Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus. Arch Virol 148: 10951118.[Crossref]
  4. Sang RC, Gichogo A, Gachoya J, Dunster MD, Ofula V, Hunt AR, Crabtree MB, Miller BR, Dunster LM, , 2003. Isolation of a new flavivirus related to cell fusing agent virus (CFAV) from field-collected flood-water Aedes mosquitoes sampled from a dambo in central Kenya. Arch Virol 148: 10851093.[Crossref]
  5. Hoshino K, Isawa H, Tsuda Y, Yano K, Sasaki T, Yuda M, Takasaki T, Kobayashi M, Sawabe K, , 2007. Genetic characterization of a new insect flavivirus isolated from Culex pipiens mosquito in Japan. Virology 359: 405414.[Crossref]
  6. Crabtree MB, Nga PT, Miller BR, , 2009. Isolation and characterization of a new mosquito flavivirus, Quang Binh virus, from Vietnam. Arch Virol 154: 857860.[Crossref]
  7. Hoshino K, Isawa H, Tsuda Y, Sawabe K, Kobayashi M, , 2009. Isolation and characterization of a new insect flavivirus from Aedes albopictus and Aedes flavopictus mosquitoes in Japan. Virology 391: 119129.[Crossref]
  8. Junglen S, Kopp A, Kurth A, Pauli G, Ellerbrok H, Leendertz FH, , 2009. A new flavivirus and a new vector: characterization of a novel flavivirus isolated from Uranotaenia mosquitoes from a tropical rain forest. J Virol 83: 44624468.[Crossref]
  9. Huhtamo E, Putkuri N, Kurkela S, Manni T, Vaheri A, Vapalahti O, Uzcátegui NY, , 2009. Characterization of a novel flavivirus from mosquitoes in Northern Europe related to mosquito-borne flaviviruses of the tropics. J Virol 83: 95329540.[Crossref]
  10. Cook S, Moureau G, Harbach RE, Mukwaya L, Goodger K, Ssenfuka F, Gould E, Holmes EC, de Lamballerie X, , 2009. Isolation of a novel species of flavivirus and a new strain of Culex flavivirus (Flaviviridae) from a natural mosquito population in Uganda. J Gen Virol 90: 26692678.[Crossref]
  11. Cook S, Bennett SN, Holmes EC, De Chesse R, Moureau G, de Lamballerie X, , 2006. Isolation of a new strain of the flavivirus cell fusing agent virus in a natural mosquito population from Puerto Rico. J Gen Virol 87: 735748.[Crossref]
  12. Morales-Betoulle ME, Monzón Pineda ML, Sosa SM, Panella N, López MR, Cordón-Rosales C, Komar N, Powers A, Johnson BW, , 2008. Culex flavivirus isolates from mosquitoes in Guatemala. J Med Entomol 45: 11871190.[Crossref]
  13. Farfan-Ale JA, Loroño-Pino MA, Garcia-Rejon JE, Hovav E, Powers AM, Lin M, Dorman KS, Platt KB, Bartholomay LC, Soto V, Beaty BJ, Lanciotti RS, Blitvich BJ, , 2009. Detection of RNA from a novel West Nile-like virus and high prevalence of an insect-specific flavivirus in mosquitoes in the Yucatan Peninsula of Mexico. Am J Trop Med Hyg 80: 8595.
  14. Kim DY, Guzman H, Bueno R, Jr Dennett JA, Auguste AJ, Carrington CV, Popov VL, Weaver SC, Beasley DW, Tesh RB, , 2009. Characterization of Culex flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad. Virology 386: 154159.[Crossref]
  15. Blitvich BJ, Lin M, Dorman KS, Soto V, Hovav E, Tucker BJ, Staley M, Platt KB, Bartholomay LC, , 2009. Genomic sequence and phylogenetic analysis of Culex flavivirus, an insect-specific flavivirus, isolated from Culex pipiens (Diptera:Culicidae) in Iowa. J Med Entomol 46: 934941.[Crossref]
  16. Crochu S, Cook S, Attoui H, Charrel RN, De Chesse R, Belhouchet M, Lemasson JJ, de Micco P, de Lamballerie X, , 2004. Sequences of flavivirus-related RNA viruses persist in DNA form integrated in the genome of Aedes spp. mosquitoes. J Gen Virol 85: 19711980.[Crossref]
  17. Bolling BG, Barker CM, Moore CG, Pape WJ, Eisen L, , 2009. Seasonal patterns for entomological measures of risk for exposure to Culex vectors and West Nile virus in relation to human disease cases in northeastern Colorado. J Med Entomol 46: 15191531.[Crossref]
  18. Harmston FC, Lawson FA, , 1967. Mosquitoes of Colorado. Atlanta, GA: US Department of Health, Education, and Welfare, Public Health Service.
  19. Darsie RF, Jr Ward RA, , 2005. Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. Gainesville, FL: University Press of Florida.
  20. Bolling BG, Moore CG, Anderson SL, Blair CD, Beaty BJ, , 2007. Entomological studies along the Colorado Front Range during a period of intense West Nile virus activity. J Am Mosq Control Assoc 23: 3746.[Crossref]
  21. Scaramozzino N, Crance JM, Jouan A, DeBriel DA, Stoll F, Garin D, , 2001. Comparison of Flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. J Clin Microbiol 39: 19221927.[Crossref]
  22. Gubler DJ, Campbell GL, Nasci RS, Komar N, Petersen L, Roehrig JT, , 2000. West Nile virus in the United States: guidelines for detection, prevention, and control. Viral Immunol 13: 469475.[Crossref]
  23. Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, Komar N, Panella NA, Allen BC, Volpe KE, Davis BS, Roehrig JT, , 2000. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol 38: 40664071.
  24. Biggerstaff BJ, , 2006. PooledInfRate, Version 3.0: a Microsoft Excel Add-In to Compute Prevalence Estimates from Pooled Samples. Centers for Disease Control and Prevention. Available at: http://www.cdc.gov/ncidod/dvbid/westnile/software.htm.
  25. Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB, , 1998. Phylogeny of the genus Flavivirus. J Virol 72: 7383.
  26. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, , 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 33893402.[Crossref]
  27. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, , 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 48764882.[Crossref]
  28. Swofford DL, , 1991. PAUP: Phylogenetic Analysis Using Parsimony, Version 3.1 Computer Program. Champaign, IL: Illinois Natural History Survey.
  29. Huelsenbeck JP, Ronquist F, , 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754755.[Crossref]
  30. Felsenstein J, , 1989. PHYLIP—Phylogeny Inference Package (Version 3.2). Cladistics 5: 164166.
  31. Page RDM, , 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357358.
  32. Tyler S, Bolling BG, Blair CD, Brault AC, Pabbaraju K, Armijos MV, Clark DC, Calisher CH, Drebot MA, , 2011. Distribution and phylogenetic comparisons of a novel mosquito flavivirus sequence present in Culex tarsalis mosquitoes from western Canada with viruses isolated in California and Colorado. Am J Trop Med Hyg 85: 162168.[Crossref]
  33. Pabbaraju K, Ho KC, Wong S, Fox JD, Kaplen B, Tyler S, Drebot M, Tilley PA, , 2009. Surveillance of mosquito-borne viruses in Alberta using reverse transcription polymerase chain reaction with generic primers. J Med Entomol 46: 640648.[Crossref]
  34. Tsai T, Smith G, Ndukwu M, Jakob W, Happ C, Kirk L, Francy D, Lampert K, , 1988. Entomologic studies after a St. Louis encephalitis epidemic in Grand Junction, Colorado. Am J Epidemiol 128: 285297.[Crossref]
  35. Lutomiah JJL, Mwandawiro C, Magambo J, Sang RC, , 2007. Infection and vertical transmission of Kamiti river virus in laboratory bred Aedes aegypti mosquitoes. J Insect Sci 7: 17.[Crossref]
  36. Coz J, Valade M, Cornet M, Robin Y, , 1976. Transmission transovarienne d'un flavivirus, le virus Koutango chez Aedes aegypti L. C R Acad Sci Hebd Seances Acad Sci D 283: 109110.
  37. Rosen L, Tesh RB, Lien JC, Cross JH, , 1978. Transovarial transmission of Japanese encephalitis virus by mosquitoes. Science 199: 909911.[Crossref]
  38. Fontenille D, Diallo M, Mondo M, Ndiaye M, Thonnon J, , 1997. First evidence of natural vertical transmission of yellow fever virus in Aedes aegypti, its epidemic vector. Trans R Soc Trop Med Hyg 91: 533535.[Crossref]
  39. Miller BR, Nasci RS, Godsey MS, Savage HM, Lutwama JJ, Lanciotti RS, Peters CJ, , 2000. First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley province, Kenya. Am J Trop Med Hyg 62: 240246.
  40. Tesh RB, Rosen L, Beaty BJ, Aitken THG, , 1977. Studies of transovarial transmission of yellow fever and Japanese encephalitis viruses in Aedes mosquitoes and their implications for the epidemiology of dengue. Dengue in the Caribbean, 179182. Washington, DC: Pan American Health Organization.
  41. Beaty BJ, Tesh RB, Aitken THG, , 1980. Transovarial transmission of yellow fever virus in Stegomyia mosquitoes. Am J Trop Med Hyg 29: 125132.
  42. Nayar JK, Rosen L, Knight JW, , 1986. Experimental vertical transmission of Saint Louis encephalitis virus by Florida mosquitoes. Am J Trop Med Hyg 35: 12961301.
  43. Shroyer DA, , 1990. Vertical maintenance of dengue-1 virus in sequential generations of Aedes albopictus . J Am Mosq Control Assoc 6: 312314.
  44. Higgs S, Beaty BJ, Marquardt WC, , 2005. Natural cycles of vector-borne pathogens. , ed. Biology of Disease Vectors, 2nd ed. Burlington, MA: Elsevier Academic Press.
  45. Kramer LD, Ebel GD, , 2003. Dynamics of flavivirus infection in mosquitoes. Adv Virus Res 60: 187232.[Crossref]
  46. Tesh RB, , 1984. Transovarial transmission of arboviruses in their invertebrate vectors. Curr Top Vector Res 2: 5776.
  47. Barker CM, Bolling BG, Black WC, IV Moore CG, Eisen L, , 2009. Mosquitoes and West Nile virus along a river corridor from prairie to montane habitats in eastern Colorado. J Vector Ecol 34: 276293.[Crossref]

Data & Media loading...

  • Received : 24 Aug 2010
  • Accepted : 31 Mar 2011

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error