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

Chen R, Wang E, Tsetsarkin KA, Weaver SC - PLoS Pathog. (2013)

Bottom Line: 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.Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

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.

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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).
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ppat-1003591-g003: 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).

Mentions: Due to the intrinsic experimental error caused by 1) small variations in the viral titer of initial inocula, 2) variation in cell density among triplicate test samples, and 3) RNA quantification, competition tests were conducted to more sensitively compare fitness levels between virus pairs. To determine the underlying reason for the fixation of Asian lineage CHIKV 3′UTR, we evaluated its fitness for infection and dissemination in the main Asian mosquito vector (1950s–2007 during evolution of the Asian lineage), A. aegypti, and viremia in the surrogate vertebrate host, CD1 mice infected at 11–12 days of age [37] using competition experiments. The Mal06 variants containing a deletion of one copy of either DR(1+2) or DR3 were competed against the genetically marked wt strain, and the results are shown in Fig. 3. Control competitions indicated that the synonymous genetic marker did not significantly influence viral fitness in mosquitoes [1] or CD1 mice (Fig. 3B).


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

Chen R, Wang E, Tsetsarkin KA, Weaver SC - PLoS Pathog. (2013)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757053&req=5

ppat-1003591-g003: 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).
Mentions: Due to the intrinsic experimental error caused by 1) small variations in the viral titer of initial inocula, 2) variation in cell density among triplicate test samples, and 3) RNA quantification, competition tests were conducted to more sensitively compare fitness levels between virus pairs. To determine the underlying reason for the fixation of Asian lineage CHIKV 3′UTR, we evaluated its fitness for infection and dissemination in the main Asian mosquito vector (1950s–2007 during evolution of the Asian lineage), A. aegypti, and viremia in the surrogate vertebrate host, CD1 mice infected at 11–12 days of age [37] using competition experiments. The Mal06 variants containing a deletion of one copy of either DR(1+2) or DR3 were competed against the genetically marked wt strain, and the results are shown in Fig. 3. Control competitions indicated that the synonymous genetic marker did not significantly influence viral fitness in mosquitoes [1] or CD1 mice (Fig. 3B).

Bottom Line: 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.Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

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.

Show MeSH
Related in: MedlinePlus