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. 2018 Aug 22;3(4):e00230-18.
doi: 10.1128/mSphere.00230-18.

Differential Transmission of Antiviral Drug-Resistant Chikungunya Viruses by Aedes Mosquitoes

Leen Delang  1 , Pei-Shi Yen  2 , Thomas Vallet  3 , Marie Vazeille  2 , Marco Vignuzzi  3 , Anna-Bella Failloux  2
Affiliations

Differential Transmission of Antiviral Drug-Resistant Chikungunya Viruses by Aedes Mosquitoes

Leen Delang et al. mSphere. .

Abstract

The chikungunya virus (CHIKV) is transmitted by female Aedes aegypti and Aedes albopictus mosquitoes, mostly present in (sub)tropical regions. No antivirals are available to treat CHIKV infections. If antiviral drugs are proven efficient to treat CHIKV-infected patients, it will be pivotal to determine whether drug-resistant viruses can be transmitted from one human to another by their mosquito vectors. We orally infected Aedes aegypti mosquitoes with a blood meal containing wild-type or drug-resistant CHIKV variants (i.e., MADTPres CHIKV, with mutation in the nsP1 gene, and T-705res CHIKV, with mutation in the RNA-dependent RNA polymerase [RdRp] gene). Viral loads were quantified in bodies (infection), heads (dissemination), and saliva (transmission) of individual mosquitoes. The infection rate of the resistant viruses was similar to that of the wild-type virus. However, the dissemination of T-705res CHIKV was markedly decreased compared to wild-type and MADTPres CHIKV. Furthermore, T-705res CHIKV was only transmitted in the saliva at day 20 postinfection (p.i.), whereas transmission of wild-type CHIKV was observed at day 3 p.i. The attenuated phenotype of the T-705res virus was confirmed in mosquito cell culture, whereas the replication fitness in Vero cells was similar to that of the wild type. In bodies and heads of mosquitoes infected with the resistant variants, the resistant phenotype and genotype were retained. Also in the saliva, the resistant genotype of MADTPres CHIKV was maintained. Our results illustrate that the fitness of drug-resistant variants should be evaluated in both hosts to be able to select antiviral drugs with a limited risk for the spread of drug resistance by mosquitoes.IMPORTANCE Because of its global reemergence and unusual morbidities associated with infection, the chikungunya virus (CHIKV) has become a substantial public health problem. However, no antivirals are currently available to treat CHIKV infections. If antiviral drugs will prove to be efficient to treat CHIKV-infected patients, it will be essential to understand if drug-resistant viruses can be transmitted from one human to another by the mosquito. We therefore orally infected Aedes mosquitoes with drug-resistant CHIKV variants and determined the replication and transmission levels. One of the antiviral drug-resistant CHIKV variants could efficiently replicate and disseminate in both laboratory and field-collected mosquitoes. In addition, this variant retained its drug-resistant genotype in the saliva. In contrast, the other drug-resistant variant was markedly attenuated in mosquitoes. Our results illustrate that extra caution for drug resistance should be considered when developing an antiarbovirus antiviral in order to minimize the risk of spreading drug resistance by mosquitoes.

Keywords: Aedes; antiviral agents; chikungunya virus; drug resistance; mosquito; transmission.

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Figures

FIG 1
FIG 1
Infection, dissemination, and transmission of WT and drug-resistant CHIKV by A. aegypti Paea mosquitoes. Infection rates (A), dissemination efficiencies (B), transmission efficiencies (C), and viral titers in saliva samples (D) for A. aegypti Paea mosquitoes orally infected with WT, T-705res, and MADTPres CHIKV. (A to C) Data (infection, disseminated infection, and transmission) are described using median values and interquartile ranges (IQR) and were analyzed using the Stata software. The number of mosquitoes tested is shown in parentheses. *, P < 0.05 (Fisher’s exact test). (D) Virus titers in saliva are shown as individual values; the line represents the mean value ± standard deviation (SD). The dotted line represents the detection limit. Data were analyzed with the Mann-Whitney test. The number of mosquitoes tested is shown in parentheses.
FIG 2
FIG 2
Infection, dissemination, and transmission of WT and drug-resistant CHIKV by field-collected mosquitoes. (A) Infection rates (IR), dissemination rates (DIR) and efficiencies (DE), and transmission rates (TR) and efficiencies (TE) detected at 7 dpi for A. aegypti Pazar from Turkey (F3) orally infected with the WT CHIKV 899 strain and the resistant CHIKV strains T-705res and MADTPres. Data (infection, disseminated infection, and transmission) are described using median and interquartile range (IQR) and were analyzed using the Stata software. The number of mosquitoes tested is shown in parentheses. (B) Infection rates (IR), dissemination rates (DIR) and efficiencies (DE), and transmission rates (TR) and efficiencies (TE) detected at 7 dpi for A. albopictus from France (F10) orally infected with the WT CHIKV 899 strain and the resistant CHIKV strains T-705res and MADTPres. Data (infection, disseminated infection, and transmission) are described using the median and interquartile range (IQR) and were analyzed using the Stata software. The number of mosquitoes tested is shown in parentheses.
FIG 3
FIG 3
Evolution of the antiviral resistance in CHIKV populations in mosquito bodies. EC50s of (A) T-705 and (B) MADTP-372 for CHIKV variants in the inoculum and in individual mosquito body and head homogenates at 3, 7, and 20 dpi. The data shown are individual EC50s; the line represents the mean ± SD value for each condition. The number of mosquitoes tested is shown in parentheses. Data were analyzed with the Mann-Whitney test using Graphpad Prism software: *, P < 0.05; **, P < 0.001; ***, P < 0.0005; ****, P < 0.0001. (C) Percentage of serine at position 34 in the nsP1 gene in the saliva. Virus populations in the saliva collected at 7 dpi of WT- and MADTPres CHIKV-infected mosquitoes were deep sequenced. The percentage of the virus population in individual saliva samples carrying a serine at position 34 in the nsP1 gene is depicted. The line represents the mean value for each group of samples.
FIG 4
FIG 4
Transmission of WT and T-705res CHIKV after intrathoracic injection. (A) Transmission efficiencies detected at 3 and 7 dpi for A. aegypti Paea after intrathoracic injection with 1,000 PFU of the WT CHIKV 899 strain or the resistant CHIKV strain T-705res. Data are described using median and interquartile range (IQR) and were analyzed using the Stata software. The number of mosquitoes tested is shown in parentheses. *, P < 0.05 (Fisher’s exact test). (B) Virus titers of saliva samples of individual mosquitoes. The titers are shown as individual values; the line represents the mean value ± SD. The dotted line represents the detection limit. Data were analyzed with the Mann-Whitney test. The number of mosquitoes tested is shown in parentheses.
FIG 5
FIG 5
Replication fitness in mosquitoes and cell culture. (A and B) Viral titers of WT, T-705res and MADTPres CHIKV in (A) bodies and (B) heads of A. aegypti Paea mosquitoes detected at 3, 7, and 20 dpi. The virus titers are shown as individual values; the line represents the mean value ± SD. Data were analyzed with the Mann-Whitney test. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. n = 27, 13, and 28 at 3 dpi, n = 17, 15, and 16 at 7 dpi, and n = 20, 13, and 15 at 20 dpi for bodies of WT, T-705res, and MADTPres CHIKV-infected mosquitoes. n = 22, 0, and 11 at 3 dpi, n = 20, 1, and 17 at 7 dpi, and n = 20, 6, and 15 at 20 dpi for heads of WT, T-705res and MADTPres CHIKV-infected mosquitoes. (C) Replication kinetics of WT and drug-resistant CHIKV in mosquito and mammalian cells. In vitro growth curves of WT (circles), T-705res (triangles), and MADTPres (squares) CHIKV were determined on C6/36, Aag2, Vero, and CRL-2522 cells. The data shown are mean values ± SD from duplicates of two independent experiments. Data were analyzed by the Mann-Whitney test. The dotted line shows the detection limit. *, P < 0.05.
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