Our results indicate a wide distribution of a novel insect-specific flavivirus. Calbertado virus was identified in Cx. tarsalis
pools obtained throughout western Canada and from Cx. tarsalis
and Cx. pipiens
obtained in California and Colorado.19
Despite its extensive geographic range, this flavivirus has highly conserved NS5 nucleotide and amino acid sequences.
Phylogenetic analysis of a 270-amino acid sequence in the NS5 protein separated the viruses in this study into 3 main clades. Although the greatest divergence was between the Clade 3 viruses and all other Calbertado genotypes, Clade 3 nucleotide sequence similarity with other viruses in the complex was still almost 90%, and the amino acid sequence divergence was approximately 3%.
The NS5 nucleotide sequences in California viruses harbored a few single nucleotide polymorphisms that made them distinct from the others in Clade 1, but overall they were more similar to this group than they were to the members of Clades 2 or 3 (). It is interesting to note that although California genotype AFSB 5050 clearly clustered with Clade 1 on the basis of nucleotide identity, it had the same polymorphic amino acid sites as Alberta sequences 11, 1024 and 1055, which placed it in Clades 1 and 2.
In general, the sequences appeared to cluster on the basis of geographic boundaries. However, there were a number of notable exceptions. One Manitoba and one Colorado CLBOV NS5 sequence (44-13 MB, 5901 CO) exhibited distinct nucleotide and amino acid polymorphisms and were the only representative genotypes in Clade 3, and other sequences from these locations were situated within Clades 1 and 2. Sequence similarity between the three CLBOV sequences from Saskatchewan was quite high but specific single nucleotide polymorphisms and amino acid polymorphisms corresponded with the placement of 90 SK in Clade 2 and 42 SK and 43 SK in Clade 1. The sequences from the Alberta pools are also of interest. Two of the nucleotide sequences (10 and 95) were virtually identical to the 52–48 sequence from Manitoba and one (27) appeared most similar to those found in British Columbia. Although there was clustering of similar CLBOVs in various provinces or regions, the results also show evidence of co-circulation of distinct genotypes within the same general area from which mosquitoes were obtained.
Although CLBOV is now added to the rapidly increasing list of insect-specific flaviviruses,3,4
it exhibits only 60–70% NS5 amino acid similarity with other members of the complex. Further analysis of genomic regions encoding the NS3 gene indicated similar phylogenetic grouping (Drebot MA, unpublished data). On the basis of these observations and the recent isolation of this virus from mosquitoes obtained in California and Colorado (Bolling and others19
and Brault AC, unpublished data), it is likely that CLBOV will be recognized as a new species within the genus Flavivirus
This study and other recent publications indicated that CLBOV is associated primarily with Cx. tarsalis
Real time RT-PCR testing of 400 pools of Cx. pipiens
from the province of Quebec provided no evidence for CLBOV RNA. However, preliminary studies using CxFV-specific primers indicated that a related virus may be present in eastern Canada (Drebot MA, unpublished data), as observed in Cx. p. pipiens
and Cx. p. quinquefasciatus
from the United States and other countries.4,9,12–16
In addition, Aedes
and other genera of mosquitoes obtained in Canada and the United States should be assayed for viruses similar to Aedes
flavivirus, which was recently identified in Japan.11
The ever widening geographic range of these viruses is an interesting finding and should be explored further.
Our study also demonstrates that infection rates for CLBOV virus are variable across geographic areas. For example, we detected CLBOV RNA in more than 40% of Cx. tarsalis
pools obtained from sites in southern Alberta (minimum infection rate = 16.5). However, mosquitoes were obtained in Alberta during 2003 and 2005, and Cx. tarsalis
from other western provinces in Canada were obtained in 2007. Seasonal and yearly prevalence and infection rates of CLBOV in Cx. tarsalis
at various sites or regions in Canada warrant further study. Recent studies in Colorado indicate that there can be differences in the infection rates of mosquitoes obtained from the same site at various times during the spring and summer.19
Similar changes in virus prevalence may occur in western Canada and other geographic areas that could be influenced by factors such as climate, environmental conditions, and ecosystems.
A number of questions remain regarding the evolutionary origins of these viruses, the mechanism for their transmission, the extent of arthropod taxa for which these virus–host relationships exist, and the specificity of these associations. For example, some of the insect-specific flaviviruses appear to be associated with Aedes
spp. mosquitoes, and the CxFVs have been linked with the Cx. pipiens
The CLBOV agent described in our study has been associated with Cx. tarsalis
mosquitoes from western Canada and the western United States in California and Colorado. This geographic pattern is paralleled by the distribution of Cx. tarsalis
mosquitoes in western North America and in conjunction with the virus only being identified in this one species could indicate that a highly specific virus–host association has evolved between these mosquitoes and CLBOV.
Wider surveillance testing for CLBOV and other insect-specific viruses will be needed to test this hypothesis further. However, it is interesting that CxFVs have been identified in primarily Cx. pipiens
complex mosquito pools from Asia and Central and North America. More restricted geographic distribution of Cx. tarsalis
mosquitoes compared with that of the Cx. pipiens
complex could indicate restricted gene flow and consequently enhanced evolutionary rates of these viruses compared with that of the CxFV complex. Close genetic identity of CxFVs from Texas with sequence data generated from CxFV detected in Cx. pipiens
mosquitoes from Japan indicate the potential for exchange of these CxFV genotypes through large ports such as Houston by which Cx. pipiens
mosquitoes are likely imported. The limited distribution of Cx. tarsalis
mosquitoes could preclude such global gene flow of CLBOV genotypes. However, it should also be noted that studies using microsatellite markers have indicated extensive gene flow between Cx. tarsalis
populations in North America.24,25
This finding could explain why similar CLBOV genotypes have been identified in Manitoba and Colorado. Detailed genetic studies of CLBOVs from geographically isolated Cx. tarsalis
mosquitoes will need to be assessed over time to fully evaluate this hypothesis.
It should also be determined whether infection of a mosquito with CLBOV alters the susceptibility for superinfection with an alternative human disease flavivirus.3
During the screening of Cx. tarsalis
mosquitoes from Manitoba, we observed that a significant proportion (30%) of pools containing CLBOV RNA also contained WNV RNA (Drebot MA, unpublished data). However, this number was reduced (1.6%) in Cx. tarsalis
pools from California 2008 (Brault AC and others, unpublished data). Co-infection of Cx. quinquefasciatus
with CxFV and WNV indicated that overall there was little impact on the infectivity or transmissibility of WNV in these mosquitoes.4
However, it was noted that the level of transmission of WNV could be enhanced depending on the origin of the mosquito colony used.
Recently, a second study characterizing co-infections of Culex
spp. with WNV and CxFV in Chicago implied a positive ecological association between the two viruses.26
Further studies are required to characterize possible interference or perhaps augmentation of arbovirus co-infection that may be conferred by persistent infection with these viruses. Co-infections of mosquitoes by various combinations of medically important flaviviruses and insect-specific flaviviruses such as CLBOV will shed further light on the role these agents may play in arboviral ecology, prevalence, vector competence, and vertical transmission of other flaviviruses.3
Studies are also warranted with respect to how insect-specific flaviviruses might affect the biology of the arthropod host, including reproductive patterns, longevity, sensitivity to environmental conditions such as weather and climate changes, and other characteristics that might be artificially manipulated to interrupt or modulate arthropod breeding or reproductive capacity. Also, the study of molecular determinants of these viruses that encode altered mosquito tropism, mosquito transmission mechanisms, and loss of vertebrate infectivity will provide fundamental insights into virus–host interactions.