Discovery of insect-specific flaviviruses in Culex spp.
In 2006, ~1,300 Culex mosquitoes from northeastern Colorado were tested for viral RNA by RT-PCR using universal flavivirus primers. Unexpectedly, numerous Culex pools testing positive for flavivirus RNA were negative for the flaviviruses that most commonly are found in Culex in Colorado: WNV and SLEV. To rule out contamination, 80 pools (89 mosquitoes) of Culiseta spp. mosquitoes and 64 pools (1,997 mosquitoes) of Ae. vexans Meigen were tested by RT-PCR with the universal flavivirus primers. All of these pools tested negative.
PCR products from flavivirus-positive Culex
pools were then sequenced, revealing similarity in some cases to a previously described insect-specific flavivirus, CxFV. In addition, we discovered sequences from a novel insect-specific flavivirus referred to as Calbertado virus (CLBOV), because this virus has been found in California, Alberta, Canada,32,33
and Colorado. Specific primers were designed to detect these insect-specific flaviviruses in mosquito pools.
In 2007, we collected ~43,000 Culex spp. mosquitoes. All Culex pools were first tested with the universal flavivirus primers. Thereafter, all flavivirus-positive pools were tested with WNV-specific, CxFV-specific, and CLBOV-specific primers.
Trends for insect-specific flaviviruses in Culex spp. in time and space.
Infection rates for WNV, CxFV, and CLBOV in Cx. tarsalis and Cx. pipiens were calculated by month () and site () for both 2006 and 2007. Infection rates include male and female pools of mosquitoes combined, because viral RNA was detected in both. The seasonal patterns of infection rates are shown for 2007 () when Culex abundance was higher and virus detection was based on larger numbers of mosquitoes than in 2006.
Monthly infection rates with West Nile virus (WNV), Culex flavivirus (CxFV), and Calbertado virus (CLBOV) for Cx. tarsalis and Cx. pipiens in Colorado from 2006 to 2007
Infection rates by trapping site with West Nile virus (WNV), Culex flavivirus (CxFV), and Calbertado virus (CLBOV) for Cx. tarsalis and Cx. pipiens in Colorado from 2006 to 2007
Figure 2. Monthly infection rates from June to September 2007 for Cx. tarsalis (A–C) and Cx. pipiens (D–F) with WNV (A and D), CxFV (B and E), and CLBOV (C and F). Error bars indicate 95% skewness-corrected confidence intervals. Estimates for infection (more ...)
In the case of the 2006 collections, most CxFV-positive pools (36/37) came from Cx. pipiens, with only a single Cx. tarsalis pool positive for CxFV. Conversely, all (34) CLBOV-positive pools were from Cx. tarsalis (). This pattern was less clear in 2007, when both CxFV and CLBOV were found in Cx. tarsalis as well as Cx. pipiens. However, there still was a trend for most CxFV-positive pools being recorded from Cx. pipiens (contributing 125 of 178 CxFV-positive pools) and most CLBOV-positive pools coming from Cx. tarsalis (contributing 113 of 121 CLBOV-positive pools).
The overall infection rates per 1,000 Cx. tarsalis in 2006 were 5.47 for WNV, 0.83 for CxFV, and 40.13 for CLBOV. The corresponding infection rates for Cx. tarsalis in 2007 were 2.65 for WNV, 1.34 for CxFV, and 2.95 for CLBOV. Differences between years are likely related, in part, to the fact that different sites were sampled in 2006 and 2007. The overall infection rates per 1,000 Cx. pipiens in 2006 were 0 for WNV, 462.42 for CxFV, and 0 for CLBOV. The corresponding infection rates for Cx. pipiens in 2007 were 2.41 for WNV, 72.09 for CxFV, and 3.18 for CLBOV. Notably, CxFV infection rates in Cx. pipiens were very high compared with the other two viruses.
In 2007, the WNV and CxFV infection rates in Cx. tarsalis increased gradually from June to September, whereas the CLBOV infection rates were highest in June (3.28) and then decreased slightly from July to September ( and ). WNV and CLBOV infection rates in Cx. pipiens followed similar seasonal patterns, with the highest infection rates occurring in June when mosquito counts were low ( and ).
Both insect-specific flaviviruses occurred widely in the five-county study area. CxFV was detected from all 21 sites in the plains sampled in 2006 and 2007, and CLBOV was recorded from 20 of these sites. The viruses also occurred in foothills sites but were not recorded from montane sites where mosquito counts are very low (). Site-specific infection rates for WNV were compared with site-specific CxFV and CLBOV infection rates by graphing, but there were no apparent associations (data not shown).
Bayesian analysis was conducted using a 1-kb region of the NS5 gene sequence to assess phylogenetic relationships between insect-specific flavivirus isolates from Culex
spp. mosquitoes in Colorado and other selected flaviviruses (). Neighbor-joining, maximum parsimony, and maximum likelihood analyses resulted in similar tree topologies (data not shown). The CxFV isolate from this study was most similar to CxFV isolates from Texas and was grouped with other CxFV isolates from Iowa, Japan, Mexico, and Guatemala (). The Colorado CLBOV isolate shared the closest phylogenetic relationships with the CLBOV sequences detected in California and Alberta, Canada,32,33
encoding 97% NS5 amino acid sequence similarity.
Figure 3. Phylogenetic tree inferred from Bayesian analysis and midpoint rooted, showing relationships between insect-specific flaviviruses from Culex species mosquitoes collected in Colorado (shaded) and other flaviviruses based on a 1-kb segment of the NS5 gene. (more ...)
Based on Bayesian analysis, the insect-specific flavivirus clade contains two subclades, which correspond with insect host genus. The first subclade contains CLBOV, Quang Binh virus, and the CxFV isolates, which have all been detected in Culex
mosquitoes. The second subclade contains Kamiti River virus, cell fusing agent virus, and Aedes
flavivirus, which have all been described in Aedes
mosquitoes. Interestingly, Nounané virus, which was recently isolated from Uranotaenia
mosquitoes in Côte d'Ivoire,8
seems to only replicate in insect cells and based on our phylogenetic analysis, groups with the arthropod-borne flaviviruses.
Isolation of insect-specific flaviviruses from Culex
in Colorado was attempted by blind passages in Vero, DF-1, and C6/36 cell cultures. Vero (mammalian) and DF-1 (avian) cells inoculated with mosquito homogenate supernatants did not exhibit any CPE after one to four passages. RNA extractions were performed on Vero and DF-1 cell culture medium after each passage, and these tested negative by RT-PCR using virus-specific primers. In contrast, CxFV-infected Cx. pipiens
homogenate supernatants caused minor growth inhibition for C6/36 (mosquito) cells after several passages. RNA extracted from cell culture medium was CxFV-positive by RT-PCR after each passage. CLBOV-infected Cx. tarsalis
homogenate supernatants caused apparent CPE in C6/36 cells on 5 dpi only after 10 passages. RNA extractions of C6/36 cell culture medium for passages one to three were negative by RT-PCR using virus-specific primers, but after the fourth passage, they were positive. Spot slides of CxFV- and CLBOV-infected C6/36 cells were tested by IFA with JE, SLE, and WNV antibodies based on a previous report,14
but antigens were undetectable. IFA using virus-specific antibodies for CxFV and CLBOV produced positive results, confirming C6/36 cell infections.
CxFV infection in a Cx. pipiens laboratory colony.
After detection of CxFV from adult mosquitoes collected in 2006 and 2007, our Cx. pipiens laboratory colony, established during the summer of 2005 from egg rafts collected in Fort Collins, was tested in March of 2007 and found to be infected with CxFV by RT-PCR and virus isolation. A subset of mosquitoes had been taken from the colony in September of 2005 and stored at −80°C. These stored specimens also tested positive for CxFV, indicating that the mosquitoes were infected at the time that the colony was established. Our initial RNA extractions were performed using a column-based method that is used for screening field-collected pools; however, we compared this with a Trizol extraction method for individual colony mosquitoes and found that Trizol extraction provided more sensitive detection. Briefly, males and females of various ages were aspirated from the colony, and RNA was extracted from individual mosquitoes using the column-based method and the Trizol method. Samples were tested by standard RT-PCR, with 1 of 28 mosquitoes testing positive for CxFV using the column-based RNA extraction method and 6 of 16 mosquitoes testing positive for CxFV using the Trizol RNA extraction method. To examine viral maintenance within this naturally infected colony, total RNA was extracted from individual egg rafts, individual fourth instar larvae, and individual adult (male and female) mosquitoes using Trizol and tested by a qRT-PCR assay with virus-specific primers. All life stages were found to be positive for CxFV RNA.