Host immune response to mosquito-transmitted chikungunya virus differs from that elicited by needle inoculated virus

Abstract

Background: Mosquito-borne diseases are a worldwide public health threat. Mosquitoes transmit viruses or parasites during feeding, along with salivary proteins that modulate host responses to facilitate both blood feeding and pathogen transmission. Understanding these earliest events in mosquito transmission of arboviruses by mosquitoes is essential for development and assessment of rational vaccine and treatment strategies. In this report, we compared host immune responses to chikungunya virus (CHIKV) transmission by (1) mosquito bite, or (2) by needle inoculation.

Methods and findings: Differential cytokine expression was measured using quantitative real-time RT-PCR, at sites of uninfected mosquito bites, CHIKV-infected mosquito bites, and needle-inoculated CHIKV. Both uninfected and CHIKV infected mosquitoes polarized host cytokine response to a TH2 profile. Compared to uninfected mosquito bites, expression of IL-4 induced by CHIKV-infected mosquitoes were 150 fold and 527.1 fold higher at 3 hours post feeding (hpf) and 6 hpf, respectively. A significant suppression of TH1 cytokines and TLR-3 was also observed. These significant differences may result from variation in the composition of uninfected and CHIKV-infected mosquito saliva. Needle injected CHIKV induced a robust interferon-gamma, no detectable IL-4, and a significant up-regulation of TLR-3.

Conclusions: This report describes the first analysis of cutaneous cytokines in mice bitten by CHIKV-infected mosquitoes. Our data demonstrate contrasting immune activation in the response to CHIKV infection by mosquito bite or needle inoculation. The significant role of mosquito saliva in these earliest events of CHIKV transmission and infection are highlighted.

Figure 1. Comparison between uninfected mosquito (UIM) bites and CHIKV infected mosquito (CIM) bites.

Uninfected and CHIKV infected Ae. aegypti ware allowed to feed upon mouse ears, and total RNA was extracted from biopsies at the indicated times. Real-time RT-PCR was performed to measure expression of the indicated cytokine mRNAs. RNA extracted from ears of mice not exposed to mosquitoes were considered as naïve and assigned an arbitrary value of 1.0, and changes in mosquito-induced cytokine gene expression are expressed as the ratio between mosquito-fed and naïve samples. GAPDH gene was used as a normalizing control. The asterisk denotes a statistically significant difference between the means of naïve and experimental groups (*-P≤0·05; **- P≤0·001). N = 3 per group.

Figure 2. Comparison between CHIKV infected mosquito (CIM) bites and needle injected CHIKV.

CHIKV infected Ae. aegypti were allowed to feed on mouse ears and total RNA was extracted from biopsies at the indicated times. In parallel, total RNA was extracted from mouse ear biopsies at sites of needle inoculation of CHIKV or medium without virus. Real-time RT-PCR was performed to measure expression of the indicated cytokine mRNAs. RNA extracted from ears of mice not exposed to mosquitoes was considered as naïve for CHIKV infected mosquito bite tissue samples. Medium-inoculated mouse biopsy samples were considered naive for needle inoculated CHIKV samples. Naive samples were assigned an arbitrary value of 1.0, and changes in mosquito-induced cytokine gene expression were expressed as the ratio between mosquito-fed and naïve samples. GAPDH gene was used as a normalizing control. The asterisk denotes a statistically significant difference between the means of naïve and experimental groups (*-P≤0·05; **- P≤0·001). N = 3 per group.

Figure 3. Histopathological changes at the mosquito bite and CHIKV injected sites.

Biopsies obtained from mouse ear samples were fixed in 10% neutral formalin and paraffin embedded. Four to five millimeter sections were made and H&E stained. Slides were observed for cellular recruitment at the mosquito bite site or CHIKV injection site. Yellow arrows in the images point to eosinophils. A-uninfected mice (naïve); B- uninfected mosquito bite site (3hpf); C- uninfected mosquito bite site (6 hpf); D-CHIKV infected mosquito bite site (3 hpf); E-CHIKV infected mosquito bite site (6 hpf); F-medium injected site, G-needle injected CHIKV site(3 hpi); H-needle injected CHIKV site (6 hpi). N = 3 per group.