Sequential adaptive mutations enhance efficient vector switching by Chikungunya virus and its epidemic emergence

Abstract

The adaptation of Chikungunya virus (CHIKV) to a new vector, the Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics.

Figure 1. Effect of the E2-L210Q substitution on dissemination of CHIKV in A. albopictus mosquitoes (Galveston and Thailand colonies).

Above each figure is a schematic representation of the viruses used in the competition assay. Asterisks indicate authentic (w.t.) residues for the SL07 strain at the indicated positions. A 1∶1 mixture of viruses [SL07-226V-Apa and SL07-226V-210Q] (A) and [SL07-226V and SL07-226V-210Q-Apa] (B) was orally presented to A. albopictus and at 10 dpi, the presence of disseminated E2-210L and E2-210Q CHIKV infection was assayed as described in the Materials and Methods. Graphs show numbers and proportions of mosquitoes containing virus populations expressing leucine (210L), glutamine (210Q) or containing both residues (210L/210Q) in mosquitoes heads and legs (representing disseminated infections). The difference in number of mosquitoes with E2-210L versus E2-210Q residues was tested for significance with a one-tailed McNemar test. BM indicates combined titers of competitors in blood meals used for mosquito infection.

Figure 2. Effect of the E2-L210Q substitution on positive selection of a mutant CHIKV strain within a wild-type population during alternating passaging in A. albopictus mosquitoes and Vero cells.

A. Schematic representation of the alternating passage experiment. The SL07-226V-210Q virus was mixed with 100-fold excess of SL07-226V-Apa virus and presented orally to A. albopictus (Galveston). At 10 dpi CHIKV was extracted from combined head and leg homogenates derived from 50 individual mosquitoes and used for Vero cells infection. The cycle was repeated a total of 3 times. At 10 dpi of third mosquito passage, heads and legs of individual mosquitoes were processed as described in the Materials and Methods. B. Graph shows numbers and proportions of mosquitoes containing virus populations expressing leucine (210L), glutamine (210Q) or both residues (210L/210Q) in mosquito heads and legs after the third passage in A. albopictus (representing disseminated infections). The original mixture used to initiate the infections was not quantified because the PCR-restriction digest assay cannot detect a minority population present at only 1% frequency.

Figure 3. The effect of the E2-L210Q substitution on CHIKV fitness in A. aegypti mosquitoes and 293 cells.

Above each figure is a schematic representation of the viruses used in the competition assay. Asterisks indicate authentic (w.t.) residues for the SL07 strain at the indicated positions. A and B. The effect of the E2-L210Q substitution on CHIKV fitness in A. aegypti. Graphs show numbers and proportions of mosquitoes containing virus populations expressing leucine (210L), glutamine (210Q) or both residues (210L/210Q) in the background of E1-226A (A) and E1-226V (B) viruses in heads and legs of A. aegypti (Thailand colony) assayed at 10 dpi. BM indicates combined titers of CHIKV (E2-210L and E2-210Q) in blood meals used for mosquito infection. C and D. The effect of the E2-L210Q substitution on CHIKV fitness in 293 cells. 293 cells were infected at multiplicity of 0.1 pfu/cell in triplicate with 1∶1 mixture of [SL07-226V-Apa and SL07-226V-210Q] (C) and [SL07-226V and SL07-226V-210Q-Apa] (D). At 2 dpi, supernatants were collected for RNA extraction and RT-PCR analysis. The relative fitness (RF) within a given competition was determined as the average ratio between E2-210L and E2-210Q bands in the sample (r), divided by the starting ratio of E2-210L and E2-210Q bands in the inoculum (i) used for infection.

Figure 4. Effect of

the E2-L210Q substitution on CHIKV fitness in A. albopictus bodies, carcasses and midguts after oral or intrathoracic infection. A, B and C. A. albopictus were fed blood meals containing 1∶1 mixes of [SL07-226V-Apa and SL07-226V-210Q] (A and B) and [SL07-226V and SL07-226V-210Q-Apa] (C) viruses. At 1, 2, 3 (B and C) and 7 dpi (A) whole mosquito bodies (A), carcasses without midguts (A), or midguts (A, B and C) were collected in pools of ten and processed as described above. BM indicates combined titers of competitors in blood meals used for mosquito infection. D. SL07-226V-Apa and SL07-226V-210Q were mixed at a 1∶1 ratio (total concentration of 5×10⁴ pfu/mL), and 0.5 µL was used to infect A. albopictus intrathoracically. At 1 and 2 dpi whole mosquitoes were collected in pools of 5 and processed as described above. The relative fitness (RF) for viruses during competition was determined as the average ratio between E2-210Q and E2-210L bands in the sample, divided by the starting ratio of E2-210Q and E2-210L bands in the BM (A, B and C) or inoculum (D) used for mosquito infections.

Figure 5. Effect of the E2-L210Q substitution on CHIKV infectivity for A. albopictus.

Mosquitoes (Thailand) were orally infected with serial 10-fold dilutions of SL07-226V or SL07-226V-210Q viruses in infectious blood meals (BM). At 10 dpi CHIKV infection in individual mosquito was detected by observing virus-induced CPE in Vero cells inoculated with mosquito homogenates. The OID₅₀ values were calculated using the PriProbit program (version 1.63) and expressed as Log₁₀(pfu)/mL (A). The difference in the infection rates between SL07-226V and SL07-226V-210Q viruses was tested for significance with a two-tailed Fishes exact test (B).

Figure 6. Effect of the E2-L210Q and E1-A226V substitutions on infectivity of CHIKV VLPs for midguts of A. albopictus (Thailand).

A. Schematic representation of VLP production and the experimental design used. At 1 and 2 dpi, mosquito midguts were analyzed by fluorescent microscopy to determine the number of cells expressing GFP and CFP in the same field of vision. B. Representative image showing number of GFP- and CFP-expressing cells in individual midguts infected with GFP/210Q/226V and CFP/210L/226V VLPs at 1 and 2 dpi. C. Representative image showing number of GFP- and CFP-expressing cells in individual midguts infected with CFP/210L/226V and GFP/210L/226A VLPs at 1 and 2 dpi. D and E. Each dot corresponds to a fold-difference in the number of cells expressing GFP vs. CFP [E2-210 (Q/L)] or CFP vs. GFP [E1-226 (V/A)] in individual mosquito midguts at 1 dpi (D) and 2 dpi (E). Red horizontal line represents the mean fold-difference for 5–10 individual midguts. The difference between strains expressing E2-L210Q and E1-A226V residues on VLP infectivity was compared for significance with a two-tailed Studens t-test.

Figure 7. Effect of the E2-L210Q substitution, expressed in the background of E1-226A, on infectivity of CHIKV VLPs for midguts of A. albopictus (Thailand).

A. Schematic representation of VLP production and the experimental design. At 1 and 2 dpi, mosquito midguts were analyzed by fluorescent microscopy to determine the number of cells expressing GFP and CFP in the same field of vision. B. Representative image showing number of GFP- and CFP-expressing cells in individual midguts infected with GFP/210L/226V and CFP/210Q/226A VLPs. C. Each dot corresponds to the fold-difference in the number of cells expressing CFP vs. GFP [E2-210 (Q/L)] in individual mosquito midguts at 1 dpi and 2 dpi. Red horizontal line represents the mean fold-difference for 10 individual midguts.

Figure 8. Atomic structure of the CHIKV E2 glycoprotein demonstrating positions in domain B involved in regulation of the alphavirus host range.

A. Trimeric form of E2 protein, view from the top. B. Domain B of CHIKV E2 protein with positions involved in CHIKV adaptation to A. albopictus [green (E2-210) and cyan (E2-211)]. Positions involved in modulation of VEEV host range are in magenta , , . Image is constructed based on atomic structure of CHIKV E2 protein [PDB ID:3N44, [39]]. The 3-D model was analyzed using the PyMol molecular viewer .