Chikungunya Virus 3′ Untranslated Region: Adaptation to Mosquitoes and a Population Bottleneck as Major Evolutionary Forces

<div><p>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.</p></div

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

<p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003591#ppat.1003591.s002" target="_blank">Fig. S2</a>.</p

Competition tests on mosquitoes and mice.

<p>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 <i>A. aegypti</i> (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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003591#ppat-1003591-g002" target="_blank">Fig. 2B</a>. 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).</p

Hypothetical evolutionary pathway of CHIKV Asian lineage 3′UTR.

<p>Color blocks in this figure correspond to those in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003591#ppat-1003591-g001" target="_blank">Fig. 1</a>.</p

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

<p>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.</p

Schematic representation of flavivirus and alphavirus recombinant crosses.

<p>A) Yellow fever virus 17D deletion mutant replicon recombinant cross, B) chikungunya virus deletion mutant replicon/defective helper recombinant cross.</p

Comparison of post-electroporation growth kinetics of yellow fever viruses (YFV) and deletion mutants in BHK-21 cells.

<p>A) YFV 17D (▪ solid line), YFV 17D GFP (▪ dashed line), and YFV 17D Cherry (○ solid line); B) YFV 17D 5′ ΔE (▴), YFV 17D 3′ ΔE (•), YFV 17D 5′ΔE Cherry (Δ), and YFV 17D 3′ ΔE GFP (○). Titers expressed as plaque forming units/mL.</p

Northern blot analysis of chikungunya virus (CHIKV) intra-genic recombinant isolates.

<p>Blot was hybridized with CHIKV-5′UTR BioProbe. Lane 1: CHIKV RNA ladder (size markers indicate CHIKV RNAs of full length genomic, replicon, full length structural gene helper, and capsid only structural helper respectively), Lane 2: CHIKV-LR-3′Δ-Replicon only electroporation, Lane 3: CHIKV-LR-5′Δ-Helper only electroporation, Lanes 4 and 8: CHIKV-LR-3′Δ-Replicon/ CHIKV-LR-5′Δ-Helper co-electroporations, and Lanes 5, 6, 7, 9, and 10: clonal recombinant isolates.</p

Schematic representation of parental replicon/defective helper recombinant cross (CHIK-LR-3′Δ-Replicon and CHIK-LR-5Δ′-Helper) along with sequence topology analysis of the cross-over region and resulting recombinant topology of clonally isolated recombinant genomes.

<p>Genes not necessarily shown to scale. aa-amino acid, E-envelope glycoprotein, ns-nonstructural, nt-nucleotide, LR-LaReunion, and UTR-untranslated region, @LR location of sequence deletion.</p

Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

<div><p>Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult <i>Aedes</i> mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.</p></div