Iron availability affects West Nile virus infection in its mosquito vector

Abstract

Background: Mosquitoes are responsible for transmission of viruses, including dengue, West Nile and chikungunya viruses. Female mosquitoes are infected when they blood-feed on vertebrates, a required step for oogenesis. During this process, mosquitoes encounter high iron loads. Since iron is an essential nutrient for most organisms, including pathogens, one of the defense mechanisms for the host includes sequestration of iron away from the invading pathogen. Here, we determine whether iron availability affects viral replication in mosquitoes.

Methods: To elucidate effect of iron availability on mosquito cells during infection, Culex cells were treated with either ferric ammonium citrate (FAC) or the iron chelator, deferoxamine (DFX). Real time RT-PCR was performed using ferritin (heavy chain) and NRAMP as a measure of iron homeostasis in cells. To determine iron requirement for viral replication, Culex cells were knocked down for NRAMP using dsRNA. Finally, the results were validated in Culex mosquito-infection model, by treating infected mosquitoes with DFX to reduce iron levels.

Results: Our results show that infection of Culex cells led to induction in levels of ferritin (heavy chain) and NRAMP mRNAs in time-dependent manner. Results also showed that treatment of cells with FAC, reduced expression of NRAMP (iron transporter) and increase levels of ferritin (heavy chain). Interestingly, increasing iron levels increased viral titers; while reducing intracellular iron levels, either by NRAMP knock-down or using DFX, reduced viral titers. The results from Culex mosquito infection showed that mosquitoes treated with DFX had reduced viral titers compared with untreated controls in midgut as well as carcass 8 days pi. Saliva from mosquitoes treated with DFX also showed reduced viral titers compared with untreated controls, indicating low viral transmission capacity.

Conclusions: Our results indicate that iron is required for viral replication in mosquito cells. Mosquitoes respond to viral infection, by inducing expression of heavy chain ferritin, which sequesters available iron, reducing its availability to virus infected cells. The data indicates that heavy chain ferritin may be part of an immune mechanism of mosquitoes in response to viral infections.

Keywords: Deferoxamine; Ferritin; Iron; Mosquito; NRAMP; West Nile virus.

Figures

Fig. 1

WNV infection changes ferritin and NRAMP expression in Culex cells. Culex (Hsu) cells were infected with WNV (MOI of 1). Total RNA was collected from cells at 0, 24, 48 and 72 hpi. Real-time RT-qPCR was performed using Culex ferritin (heavy chain) and NRAMP primers. RpL32 primers were used as an internal control. Error bars represent standard errors from means of three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing with 0 time point)

Fig. 2

Iron treatment increases viral titers in Culex cells. Culex cells were infected with WNV (MOI of 1) with/without iron (FAC, 100 uM) treatment. As a control, uninfected cells were used. Total RNA and supernatant medium were collected at 48 hpi. Real-time RT-qPCR was performed using Culex ferritin (heavy chain) (a) and NRAMP (b) primers. RpL32 primers were used as an internal control. Error bars represent standard errors from means of three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing with control untreated cells). c Viral titer estimation by plaque assays conducted on the supernatant media collected from cells treated as in (a). (Student’s t-test *p < 0.05, comparing with control infected cells)

Fig. 3

Culex NRAMP does not act as WNV receptor Culex (Hsu) cells were pretreated with FAC (100 uM) for 2h before infection with WNV. Total RNA was collected 3 hpi and real time RT-PCR was performed using Culex NRAMP (a) and WNV NS1 (b) primers. RpL32 primers were used as an internal control. Error bars represent standard errors from means of three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing with control infected cells)

Fig. 4

Reducing intracellular iron affects viral titers. Culex (Hsu) cells were transfected with dsRNA against Culex NRAMP 24 h before infection with WNV (MOI of 1). GFP-dsRNA was used as a negative control. Cells were also treated with FAC (100 uM). Total RNA and media supernatant were collected 48 hpi. Real time RT-PCR was performed using NRAMP (a) and ferritin (heavy chain) (b) primers. RpL32 primers were used as an internal control. Error bars represent standard errors from means of three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing with GFP-dsRNA cells). c Viral titer estimation by plaque assays conducted on the supernatant media collected from cells treated as in (a). (Student’s t-test *p < 0.05, comparing with control infected (GFP-dsRNA) cells)

Fig. 5

Iron chelator, deferoxamine, reduces viral titer in Culex cells Culex (Hsu) cells were infected with WNV with/without deferoxamin (DFX, 10 uM). As a control cells were either uninfected and/or treated with DMSO (diluent). Total RNA and media supernatant were collected 48 hpi. Real time RT-PCR was performed using ferritin (heavy chain) (a), NRAMP (b) and WNV NS1 (c) primers. RpL32 primers were used as an internal control. Error bars represent standard errors from means of three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing with control cells). d Viral titer estimation by plaque assays conducted on the supernatant media collected from cells treated as in (a). (Student’s t-test *p < 0.05, comparing with control infected cells). e Cell viability was measured from Culex (Hsu) cells treated with different concentrations (0, 10, 100 and 1000 uM) of DFX for 48 h using trypan blue exclusion

Fig. 6

DFX treatment reduces viral titers in Culex mosquitoes Female Culex annulirostris mosquitoes were blood-fed with WNV for 1 h, with or without deferoxamine (10 uM). Mosquito were dissected at 2 and 8 days pi to collect midgut and carcass and were processed for RNA extraction. Real time RT-PCR was performed using ferritin (heavy chain) and WNV NS1 primers on day 2 midgut (a). Real time RT-PCR was also performed using ferritin primers on day 2 carcass (b), day 8 midgut (c) and day 8 carcass (d). Real time RT-PCR was also performed using WNV NS1 primers on day 8 carcass (d). RpL32 primers were used as an internal control. Error bars represent standard errors from means of two separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing with infected control mosquitoes). e Viral titer estimation conducted by plaque assay in mosquito saliva mixed with cell culture medium. Viral titers from individual saliva samples were plotted on the graph (Student’s t-test *p < 0.05, comparing between with infected control mosquitoes). Control N=8; DFX N=10