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A single mutation in chikungunya virus affects vector specificity and epidemic potential - PubMed

A single mutation in chikungunya virus affects vector specificity and epidemic potential

Konstantin A Tsetsarkin et al. PLoS Pathog. 2007 Dec.

Abstract

Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005-2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1-226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of E1-A226V Mutation on CHIKV-GFP Viruses Ae. albopictus and Ae. aegypti Midgut Infectivity

Percent of orally infected Ae. albopictus (A, B) and Ae. aegypti (C, D) mosquitoes presented with blood meals containing various concentration of eGFP-expressing CHIK viruses. Serial 10-fold dilutions of viruses in the backbone of Reunion (LR-GFP-226V and LR-GFP-226A) (A, C) and 37997 (37997-GFP-226A and 37997-GFP-226V) (B, D) strains of CHIKV were made in L-15 medium followed by mixing the samples with defibrinated sheep blood. Mosquitoes were dissected at 7 dpi and eGFP expression in infected midguts was analyzed by fluorescence microscopy. A mosquito was considered infected if at least one foci of eGFP-expressing cells was present in the midgut. The experiments were performed twice for each virus (I and II).

Figure 2
Figure 2. Schematic Representation of Competition Experiments (A) and Competition between LR-ApaI-226V and LR-226A Viruses for Colonization of Midgut cells of Ae. albopictus (B) and Ae. aegypti (C) Mosquitoes

107 pfu of LR-ApaI-226V and LR-226A were mixed and orally presented to Ae. albopictus (B) and Ae. aegypti (C). Viral RNAs were extracted from four pools of eight to ten midguts at 7 dpi. RT-PCR products were digested with ApaI, separated in 2% agarose gel, and gels were stained using ethidium bromide. BM - initial ratio of LR-ApaI-226V and LR-226A in blood meal samples. 1–4 ratio of LR-ApaI-226V and LR-226A RNA in four independent replicas of the eight to ten midguts per replica. Relative fitness (RF1) of LR-Apa-226V to LR-226A was calculated as a ratio between 226V and 226A bands in the sample, divided by the control ratio between 226V and 226A in the blood meal. Relative fitness (RF2) of LR-226A to LR-Apa-226V was calculated as a ratio between 226A and 226V bands in the sample, divided to the control ratio between 226A and 226V in the blood meal. Results expressed as the average of four replicas ± standard deviation (SD).

Figure 3
Figure 3. Effect of E1-A226V Mutation on CHIKV Dissemination into Salivary Glands and Heads of Ae. albopictus and Ae. aegypti Mosquitoes

Ae. albopictus (A) and Ae. aegypti (C) mosquitoes were orally infected with LR-GFP-226V and LR-GFP-226A. At the indicated time points, 16–21 mosquitoes were dissected and salivary glands were analyzed for eGFP expression. Percent of dissemination was estimated as a ratio of the number of mosquitoes with eGFP-positive salivary glands to the number of mosquitoes with eGFP-positive midguts. For Ae. albopictus, infectious blood meal titers were 5.95 and 6.52 Log10TCID50/ml for LR-GFP-226V and LR-GFP-226A, respectively. For Ae. aegypti, the infectious blood meal titer was 6.95 Log10TCID50/ml for both LR-GFP-226V and LR-GFP-226A viruses. Dissemination rates were compared statistically by Fisher's exact test using SPSS version 11.5. Asterisk indicates p < 0.05. (B and D) Competition between LR-ApaI-226V and LR-226A for dissemination into heads of Ae. albopictus and Ae. aegypti mosquitoes. 107 pfu of LR-ApaI-226V and LR-226A were mixed and orally presented to Ae. albopictus (B) and Ae. aegypti (D). Viral RNAs were extracted from four pools of five heads collected at 12 dpi. RT-PCR products were digested with ApaI, separated in 2% agarose gel, and gels were stained using ethidium bromide. BM - initial ratio of LR-ApaI-226V and LR-226A in blood meal samples. 1–4 ratio of LR-ApaI-226V and LR-226A RNA in four independent replicas of the five pooled heads per replica.

Figure 4
Figure 4. Effect of E1-A226V Mutation on CHIKV Kinetics of Viral Growth in Bodies of Ae. albopictus Mosquitoes

(A) Virus production in orally infected Ae. albopictus mosquitoes. Infected mosquitoes were sampled at 0, 1, 2, 3, 5, 7, and 14 dpi and titrated on Vero cells to estimate average titer ± standard deviation of eight whole mosquitoes. Differences in viral titers were analyzed by pairwise t-tests. Asterisk indicates p < 0.05. (B) Kinetics of competition between LR-ApaI-226V and LR-226A in bodies of Ae. albopictus mosquitoes. 107 pfu of LR-ApaI-226V and LR-226A were mixed and orally presented to Ae. albopictus. Infected mosquitoes were sampled at 1, 3, 5, 7, and 14 dpi. For each time point, viral RNA was extracted from two pools of ten mosquitoes. BM - initial ratio of LR-ApaI-226V and LR-226A in blood meal samples. RF - relative fitness of LR-Apa-226V to LR-226A was calculated as a ratio between 226V and 226A bands in the sample, divided to the control ratio between 226V and 226A in the blood meal. Results expressed as average of two replicas ± standard deviation.

Figure 5
Figure 5. Effect of E1-A226V Mutation on CHIKV Transmission by Ae. albopictus and Ae. aegypti Mosquitoes

(A) Six 2- to 3-day-old suckling mice (Swiss Webster) were subcutaneously infected with a 20-μl mixture of ≈ 25 pfu LR-Apa-226V and ≈ 25 pfu of LR-226A viruses. (B and C) Ae. aegypti and Ae. albopictus mosquitoes were presented with a blood meal containing 107 pfu/ml of LR-Apa-226V and 107 pfu/ml of LR-226A viruses. At 13 dpi, ten to 15 mosquitoes were placed in separate paper cartons and starved for 24 h. The next day, the mosquitoes in each carton were presented with a 2- to 3-day-old suckling mouse (Swiss Webster). Mice were returned to their cage and sacrificed on day 3 post-exposure. Blood from each individual mouse (≈ 50 μl) was collected and immediately mixed with 450 μl of TRIzol reagent for RNA extraction. BM and inoc. - initial ratio of LR-ApaI-226V and LR-226A in blood meal samples and inoculum for subcutaneous infection. 1–6 ratio of LR-ApaI-226V and LR-226A RNA in six individual mice.

Figure 6
Figure 6. Effect of E1-A226V Mutation on In Vitro Growth of CHIKV in Standard (A) and Cholesterol-Depleted (B) C6/36 Cells

Cholesterol-depleted C6/36 cells were produced by five passages in L-15 medium containing 10% FBS treated with 2% CAB-O-Sil for 12 h at room temperature as previously described [52]. Confluent monolayers of standard (A) and cholesterol-depleted (B) C6/36 cells were infected with LR-ApaI-226V, LR-226A, 37997–226A and 37997–226V viruses at an MOI of 1.0 (A) and an MOI of 0.1 (B). Cells were washed three times with L-15 medium, and 5.5 ml of fresh L-15 supplied with 10% of standard or CAB-O-Sil-treated FBS were added to the flask. Cells were maintained at 28 °C. At the indicated times post-infection, 0.5 ml of medium was removed and stored at −80 °C for later titration on Vero cells. Viral titers are estimated as average Log10TCID50/ml ± standard deviation of two independent experiments. hpi - hours post-infection.

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