Chikungunya virus 3' untranslated region: adaptation to mosquitoes and a population bottleneck as major evolutionary forces

Abstract

The 3' untranslated genome region (UTR) of arthropod-borne viruses is characterized by enriched direct repeats (DRs) and stem-loop structures. Despite many years of theoretical and experimental study, on-going positive selection on the 3'UTR had never been observed in 'real-time,' and the role of the arbovirus 3'UTR remains poorly understood. We observed a lineage-specific 3'UTR sequence pattern in all available Asian lineage of the mosquito-borne alphavirus, chikungunya virus (CHIKV) (1958-2009), including complicated mutation and duplication patterns of the long DRs. Given that a longer genome is usually associated with less efficient replication, we hypothesized that the fixation of these genetic changes in the Asian lineage 3'UTR was due to their beneficial effects on adaptation to vectors or hosts. Using reverse genetic methods, we examined the functional importance of each direct repeat. Our results suggest that adaptation to mosquitoes, rather than to mammalian hosts, is a major evolutionary force on the CHIKV 3'UTR. Surprisingly, the Asian 3'UTR appeared to be inferior to its predicted ancestral sequence for replication in both mammals and mosquitoes, suggesting that its fixation in Asia was not a result of directional selection. Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia. We propose that this introduction of a 3'UTR with deletions led to genetic drift and compensatory mutations associated with the loss of structural/functional constraints, followed by two independent beneficial duplications and fixation due to positive selection. Our results provide further evidence that the limited epidemic potential of the Asian CHIKV strains resulted from founder effects that reduced its fitness for efficient transmission by mosquitoes there.

Figure 1. Evolution history and lineage-specific structures of the CHIKV 3′UTR.

On the left is the MCC (Maximum Clade Credibility) tree based on the complete ORF sequences, with the branches in each lineage collapsed. The estimated year of the most recent common ancestor (MRCA: mean and the 95% HPD values) of each clade is labeled left to the node. The 3′UTR structures, based on sequence alignment, are shown next to each lineage. Direct repeats are illustrated by different colored blocks, each of the four colors represents a different homologous sequence region. Sequence gaps in the alignment are indicated by white blocks. In the Asian lineage, two distinct derived differences are observed: 1) duplication of DR3, and duplication the of DR(1+2) region. The detailed alignment can be found in Fig. S2.

Figure 2. Replication kinetics of CHIKV variants in Vero and C6/36 cells.

A. Genome structures of wt and genetic engineered CHIK viruses based on Mal06 (Asian lineage) and SL07 (ECSA lineage) used in this study. B. Replication kinetics of these CHIKV variants in Vero and C6/36 cells. Cells were infected in triplicate by different CHIKV variants in MOI = 0.1. RNA copy number at selected time points post infection was measured by Real-Time RT-PCR. Error bars show the maximum and minimum value in the triplicates.

Figure 3. Competition tests on mosquitoes and mice.

A. Experimental design. Competing viruses (one of them contains a synonymous genetic marker) were mixed in a 1∶1 initial ratio based on genome copies, and used for mosquito and mice infection. The viral RNA ratio was reflected by RT-PCR amplification of a region containing the marker in the middle, followed by thorough digestion on the digestion sites created by the genetic marker. In agarose gel analyses, the lower band reflects the level of virus with the genetic marker, whereas the upper band reflects the RNA level of virus without the genetic marker. B. Competition results between the two wt viruses (Mal06 and SL07) and their correspondent mutants in CD1 baby mice. C–F. Competition between 4 pairs of viruses (C: Mal06/ΔDR3a vs. Mal06/WT+marker; D: Mal06/ΔDR(1+2)a vs. Mal06/WT+marker; E: Mal06/SL07-3′UTR vs. Mal06/WT+marker; F: SL07/Mal06-3′UTR vs. SL07/WT+marker) on the dissemination rate in A. aegypti (Thailand) and viral RNA level in CD1 baby mice. The mosquitoes were infected through blood meal with viral titer in ∼1×10⁶ pfu/ml. On day 10 post infection, the heads of mosquitoes were dissected to study the viral dissemination. The numbers of samples infected by each virus are shown by pie graph, with statistical significance assessed using a Chi-square test. Viruses are labeled in the same colors as in Fig. 2B. CD1 baby mice were infected with initial dose of 1×10⁴ pfu, 3 or 4 of them were sacrificed each day and blood viral ratio was used to measure the fitness level of competing viruses (shown in the gel).

Figure 4. Hypothetical evolutionary pathway of CHIKV Asian lineage 3′UTR.

Color blocks in this figure correspond to those in Fig. 1.