Genome sequencing and genetic characterization of Culex Flavirirus (CxFV) provides new information about its genotypes

Flaviviruses are well known for causing important mosquito-borne human diseases. They are transmitted between arthropods and vertebrates, and are capable of replicating in both hosts. In 1975, a new member of this genus, incapable of replicating in vertebrate cells, was isolated from mosquito cell cultures and named Cell Fusing Agent Virus (CFAV) [1]. More recently, the isolation of other insect-specific viruses has been reported [2‐5]. One of these insect-specific viruses is Culex Flavivirus (CxFV), an insect-specific virus that was first identified in 2007 in Culex pipiens mosquitoes [6]. Other hosts include Culex tritaeniorhynchus, Culex quinquefasciatus, and Anopheles sinensis mosquitoes [5, 7, 8].

Similar to other flaviviruses, CxFV encodes a polyprotein from a single-strand positive RNA open reading frame (ORF), flanked by 3′ and 5′-untranslated regions (UTR). The polyprotein of 3,364 aa is cleaved during and after translation into structural and non-structural proteins in the following order: C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5 [6].

Since its identification, Culex Flavivirus has been reported in the United States, Mexico, Guatemala, Trinidad, Uganda, Indonesia, Japan, China, and Brazil, indicating that it is widely distributed [5, 6, 9‐14]. Despite the increasing number of CxFV sequences deposited in GenBank, it is not clear if the phylogenetic relationships between the strains are influenced by the host species or geographic location. Moreover, little is known about the genetic profile of the CxFV genome.

Previously, we reported the first identification of CxFV in Brazil [14]. Here, we perform a genetic and phylogenetic analysis of the complete ORF of the Brazilian CxFV strain. In addition, we perform the genetic characterization of CxFV based on the ORF sequences described so far, and we propose a novel genotype classification.

Culex Flavivirus primarily infects globally distributed mosquito species of the genus Culex, which are vectors for pathogenic flaviviruses like WNV, SLEV, and JEV [27‐29]. The most represented hosts in this work, Culex pipiens and Culex quinquefasciatus, are members of the Culex pipiens complex that consists of closely related species that are difficult to distinguish morphologically [30]. Studying the geographical distribution of the host is important to understand the evolutionary relationships between the identified CxFV strains. Two main monophyletic branches, Clade 1 and Clade 2, can be observed in the phylogenetic tree with high branch support. Regarding countries, all Asian strains are grouped in Clade 1 along with some sequences from USA. Clade 2 is composed by sequences from different countries with only one sequence each. When considering host species, Clade 2 is composed only of sequences derived from Culex quinquefasciatus while Clade 1 as sequences from different hosts. This scenario prevents a clear association by simply analyzing the ancestry. Statistical analysis however revealed significant association between phylogeny and climate (temperate x tropical). Clade 1 consists of virus from temperate climate regions while Clade 2 is composed from sequences from region of tropical climate. Climate association is related with host species distribution. Although Culex pipiens occurs in temperate regions and Culex quinquefasciatus in tropical and subtropical regions their range overlaps and it has been proven they can hybridize [30‐32]. When testing for host-phylogeny association only Culex quinquefasciatus was significantly associated. Moreover, country association was only found for Japan and China. Considering that both country and host are related to climate these factors may also be associated with CxFV ancestry, however the limited number of sequences from some hosts and geographic regions is probably preventing a more consistent conclusion.

We analyzed the genetic distance between the main branches of the phylogenetic tree that was reconstructed based on all CxFV ORF sequences described so far, including the Brazilian strain described in this work. Clades 1 and 2 consist of strong groups with robust branch support, which are 10.4 % genetically distant. These observations sustain the idea that they could be two different genotypes of CxFV. For Japanese Encephalitis Virus, another Culex borne virus, genotypes are defined by 12 % difference in nucleotide composition of the highly divergent PrM gene [33, 34]. Genotypes of DENV, also a flavivirus, are defined by 6 % difference within a serotype based on the E region [35, 36]. In both cases, the limits were determined arbitrarily but supported by strong phylogenetic evidence such as ours. Other researchers have previously suggested that CxFV should be classified into different genotypes, based on the phylogenetic analysis of the E genomic region [37]. According to those authors, Clade 1 described here would consist of Genotype 1 and Clade 2 of Genotype 2. Our analysis also evidenced that the two main branches, with strong branch support (1000), in Clade 1 diverge 5.1 %. These two branches could be considered different subtypes of CxFV Genotype 1 (1a and 1b). Similar results were obtained using the E region (Additional file 5: Table S5), suggesting it is a suitable region for CxFV genotyping and subtyping. We propose that groups of CxFV that are more than 9 % genetically distant could be classified as different genotypes and that differences higher than 4 % within these groups could be considered different subtypes. Although the cut-off values are arbitrarily defined, based on the genetic data available so far, they seem suitable.

Overall, selective pressure analysis indicates that the entire ORF, as well as specific regions, are undergoing purifying selection. Accordingly, site-by-site analysis found no positively selected sites and 106 sites under negative selection. This suggests that the virus is well adapted to its host and evolutionary forces are working against amino acid change. The infection of Culex mosquitoes by CxFV apparently does not result in disease. This observation is supported by the fact that the infection of C6/36 and AeAl-2, two Aedes albopictus cell lines, results only occasionally in moderate cytopathic effects [5, 6, 12]. If this is the case, any change in the genomic composition could disrupt the fine balance between what is advantageous for the virus and at the same time does not negatively affect the host, supporting the purifying selection.

Although the differences are not substantial, the NS2A region presents higher ω values and lower percentages of negatively selected sites. It is interesting, however, to notice that the same region also showed the lowest genetic distance. At first, these data might seem in disagreement. However, it suggests that NS2A displays low genetic variability in general, but a more relaxed pressure against amino acid change when compared to other regions. CxFV and other Flaviviruses from the insect-specific group present a frameshift signal that produces an alternate reading frame called fifo [38]. The signal consists of a stem-loop structure in the RNA genome that is harbored inside the NS2A region [39]. In this case, the selection is acting not only on the protein, but also on the RNA structure to avoid disturbing the frameshift signal. It is important to highlight that different amino acids might have similar chemical properties and a change would not necessarily disrupt the tertiary structure. This could explain why, despite the low genetic distance, this region is more permissive to amino acid change.

There are still many aspects of Culex Flavivirus and other insect-specific flaviviruses that needs to be clarified. Culex ssp. mosquitoes are distributed worldwide suggesting that CxFV should be present in more places than currently reported. Here we report an association between the ancestry of CxFV and climate. The identification of new strains from different countries and hosts is important for elucidating the ecology and evolutionary history of this virus. Based on the available sequences, we propose the classification of Culex Flavivirus into two genotypes (1 and 2) and the existence of two subtypes within genotype 1.