Insect-Specific Flaviviruses from Culex Mosquitoes in Colorado, with Evidence of Vertical Transmission

Abstract

Mosquitoes were collected in Colorado during 2006 and 2007 to examine spatial and seasonal patterns of risk for exposure to Culex vectors and West Nile virus. We used universal flavivirus primers to test pools of Culex mosquitoes for viral RNA. This led to the detection and subsequent isolation of two insect-specific flaviviruses: Culex 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 Cx. tarsalis and Cx. pipiens from Colorado. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of CxFV RNA in Cx. pipiens 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 Culex populations. Finally, we present phylogenetic analyses of the relationships between insect-specific flaviviruses and other selected flaviviruses.

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

“…The most closely related viruses based on amino acid sequence homologies for the NS5 region (CxFV, CFAV, and KRV) exhibit less similarity to CLBOV when assessed at the nucleotide level. 19 These results indicate that although CLBOVs are related to other insect-specific flaviviruses, they may represent a distinct clade based on amino acid comparisons ( Figure 2 ) and nucleotide comparisons, respectively.…”

“…Clade 3 contains the sequences from a second Manitoba pool (44-13) and a Calbertado isolate from Colorado (5901). 19 Alberta sequences 1024, 1055, and 11 grouped outside the three clades and exhibited characteristics common to Clades 1 and 2 because of the mixture of genotypes identified ( Figure 4 ).…”

“…18 Initial screening of mosquito homogenates for flavivirus genomes was carried out by using universal flavivirus primers CFD2 and MAMD and other primer sets specific for the NS5 coding region of the RNA. [18][19][20] The RT-PCRs were performed by using the QIAGEN One-Step RT-PCR Kit in a total volume of 50 μL. Reaction mixtures followed kit instructions and contained 10 μL of 5× RT-PCR buffer, 2 μL of 10 mM dNTP, 5 μL of template RNA, 1.5 μL (20 mM) of each forward and reverse universal primers, 0.125 μL (40 units/μL of RNaseOUT (Invitrogen, Carlsbad, CA), 2 μL of One-…”

Insect-Specific Viruses Detected in Laboratory Mosquito Colonies and Their Potential Implications for Experiments Evaluating Arbovirus Vector Competence

“…Vertical transmission has been reported previously with other insect-specific flaviviruses, based on the detection of viral RNA in adults reared from field-caught immature stages 16,33 and in fieldcollected male mosquitoes. 17,19,32,35 When laboratory colonies are established from wild-caught mosquitoes or stocks are obtained from other laboratory colonies, especially eggs, it is generally assumed that the emerging adults are "pathogen-free." However, as the results of this study show, that may not be a valid assumption, because the mosquitoes may be infected with insect-specific viruses, which may not be detrimental to the mosquitoes, but may interfere with other types of viruses being tested in the colonies.…”

“…It is now apparent that these viruses are widespread in nature and that many are probably maintained by vertical transmission. [16][17][18][19] Some studies also suggest that superinfection exclusion (cells infected with one virus are refractory to infection by a second related virus) may occur between some insectspecific viruses and pathogenic arboviruses, 20,21 altering the vector competence of the mosquito host. In vivo and in vitro studies investigating interactions between two insect-specific flaviviruses (Culex flavivirus [CxFV] 20, 22 and Palm Creek virus [PCV] 21 ) with West Nile virus (WNV), have shown modulation of WNV replication and dissemination in dually infected mosquitoes, suggesting that some insect-specific viruses may alter the mosquito's ability to serve as a competent vector for certain pathogenic arboviruses.…”