Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes - PubMed (original) (raw)

Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes

Jianying Liu et al. Nat Microbiol. 2016.

Abstract

The arbovirus life cycle involves viral transfer between a vertebrate host and an arthropod vector, and acquisition of virus from an infected mammalian host by a vector is an essential step in this process. Here, we report that flavivirus nonstructural protein-1 (NS1), which is abundantly secreted into the serum of an infected host, plays a critical role in flavivirus acquisition by mosquitoes. The presence of dengue virus (DENV) and Japanese encephalitis virus NS1s in the blood of infected interferon-α and γ receptor-deficient mice (AG6) facilitated virus acquisition by their native mosquito vectors because the protein enabled the virus to overcome the immune barrier of the mosquito midgut. Active immunization of AG6 mice with a modified DENV NS1 reduced DENV acquisition by mosquitoes and protected mice against a lethal DENV challenge, suggesting that immunization with NS1 could reduce the number of virus-carrying mosquitoes as well as the incidence of flaviviral diseases. Our study demonstrates that flaviviruses utilize NS1 proteins produced during their vertebrate phases to enhance their acquisition by vectors, which might be a result of flavivirus evolution to adapt to multiple host environments.

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Figures

Figure 1

Figure 1. DENV sNS1 facilitates DENV acquisition via membrane blood feeding

(a–c) The presence of recombinant DENV2 sNS1 in blood increased DENV2 acquisition by A. aegypti. (a) Recombinant DENV2 sNS1 protein was expressed and purified from Drosophila S2 cells. A total of 10 μg of purified sNS1 was incubated with fresh human blood (500 μl) and supernatant from DENV2-infected Vero cells (500 μl) to feed A. aegypti via an in vitro membrane blood meal. (b,c) A concentration of 1x105 pfu/ml DENV2 was used for mosquito oral infection. Mosquitoes fed the same amount of BSA served as negative controls. (d–f) Immunoblockade of DENV sNS1 in supernatant from infected Vero cells reduced DENV acquisition by A. aegypti. (d) Serially diluted anti-DENV2 NS1 antisera were mixed with supernatant from DENV2-infected Vero cells (500 μl) and fresh human blood (500 μl) for in vitro membrane feeding of A. aegypti. (e,f) A concentration of 1x106 pfu/ml DENV2 was used for oral infection. As a mock control, mosquitoes were fed the same dilution of pre-immune sera. (g–i) The presence of purified native sNS1 (nNS1) in viremic human blood increased DENV2 acquisition by A. aegypti. Infectious DENV2 particles and native sNS1 protein were purified from the supernatant of DENV2-infected Vero cells. Following this, native sNS1 and 1x106 pfu purified DENV2 virions were incubated with human blood (1 ml) for mosquito oral feeding. The equivalent amount of BSA was used as a negative control. (a–i) The DENV2 NGC strain was used for mosquito oral infection. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. (b,e,h) The number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. (c,f,i) The data are represented as the percentage of mosquito infection. Differences in mosquito infective ratio were compared using Fisher’s exact test. (i) The p values were adjusted using Bonferroni correction to account for multiple comparisons. Differences were considered significant if _p<_0.017. (a–i) The experiments were biologically repeated at least 3 times with similar result.

Figure 2

Figure 2. Passive transfer of DENV2 sNS1 antibodies into infected AG6 mice prevents DENV acquisition by mosquitoes

(a) Schematic representation of the study design. AG6 mice were intraperitoneally infected with 1x106 pfu of the DENV2 43 strain. Subsequently, 100 μl of a DENV2 NS1 antibody was intraperitoneally inoculated into the mice at 12 hr post infection. An equivalent quantity of untreated serum was inoculated as a mock control. After allowing 12 hr for antibody dissemination, the infected mice were subjected to daily mosquito biting from day 1 to day 4 post mouse infection. The mouse blood-fed mosquitoes were reared for an additional 8 days for DENV detection. (b,c) DENV2 infection of AG6 mice. (b) Measurement of DENV2 sNS1 concentration. Mouse sera were used to determine the amounts of DENV2 sNS1 present from 0 to 5 days post infection by ELISA. (c) Detection of DENV2 viremia in the blood of infected AG6 mice. The presence of infectious viral particles in the blood plasma was assessed using a plaque assay. (b,c) The data are representative of at least five infected AG6 mice. The values in the graph represent the mean ± SEM. A non-parametric Mann-Whitney test was used for statistical analysis. (d–g) Immunoblockade of DENV2 sNS1 in the infected AG6 mice reduced the infection of fed A. aegypti (d,e) and A. albopictus (f,g). (d,f) The number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. (b–g) The DENV2 43 strain was used for animal infections. (e,g) The data are represented as the percentage of mosquito infection. Differences in mosquito infective ratios were assessed using Fisher’s exact test. “*”, “**” and “***” represent _p<_0.05, _p<_0.01, and _p<_0.001, respectively. (b–g) The experiments were biologically reproduced at least 3 times.

Figure 3

Figure 3. Passive transfer of antibodies against JEV NS1 in infected AG6 mice prevents JEV acquisition by Culex pipiens pallens

(a) Schematic representation of the study design. AG6 mice were infected with the JEV SA-14 strain. Subsequently, a 100 μl aliquot of a JEV NS1 antibody stock was intraperitoneally injected per mouse 12 hr post infection. Infected mice were inoculated with an equivalent quantity of untreated serum as a mock control. After waiting an additional 12 hr for antibody dissemination, the infected mice were subjected to daily mosquito biting from day 1 to day 3 post mouse infection. The mouse blood-fed mosquitoes were reared for an additional 8 days for JEV detection. (b) Production of murine polyclonal antibodies against JEV NS1. The JEV NS1 gene was cloned into a pET-28a (+) expression vector and expressed in E. coli BL21 DE3. Recombinant JEV NS1 in inclusion bodies was dissolved in 8 M urea and purified for antibody generation. The antibodies were validated by immunostaining with S2-expressed JEV NS1 protein. (c) JEV infection in AG6 mice. Blood was collected from mouse tail veins from 0 to 3 days post JEV infection. The presence of infectious viral particles in the blood plasma was assessed using a plaque assay. The data are representative of at least five infected AG6 mice. Differences between untreated and JEV NS1 antibody-treated groups were assessed using a non-parametric Mann-Whitney test. (d,e) Immunoblockade of JEV sNS1 in JEV-infected AG6 mice reduced JEV acquisition by C. pipiens pallens. The fed mosquitoes were reared for an additional 8 days for JEV detection by TaqMan qPCR. (d) The number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. (e) The data are represented as the percentage of mosquito infection. Differences in mosquito infective ratios were assessed using Fisher’s exact test. “*”, “**” and “***” represent _p<_0.05, _p<_0.01, and _p<_0.001, respectively. (b–e) The experiments were biologically reproduced at least 3 times.

Figure 4

Figure 4. DENV2 sNS1 suppresses the expression of immune-related genes in the mosquito midgut

(a) Regulation of immune-related genes in the midguts of A. aegypti. The midguts of mosquitoes that acquired 10 μg purified DENV2 sNS1 or BSA via blood meals were dissected for RNA-Seq. Immune-related genes were clustered according to immune pathways and factors. (b) Validation of immune-related gene regulation. The expression levels of the immune-related genes listed in Supplementary Fig. 6 were assessed in the mosquito midguts by qPCR at 18 hr post DENV2 sNS1 feeding. Gene regulation is represented as the mRNA ratio between sNS1-fed and BSA-fed midguts. The primers are described in Supplementary Table 2. (c,d) ROS activities in the midguts of mosquitoes fed DENV2 sNS1. The midguts of mosquitoes fed either DENV2 sNS1 or BSA were dissected for H2O2 detection (c) and dihydroethidium (DHE) staining (d). (d) Nuclei were stained blue with To-Pro-3 iodide. Images were examined using a 10× objective lens on a Zeiss LSM 780 meta confocal microscope. The scale bars represent 100 μm. (b,c) The values in the graph represent the mean ± SEM. A non-parametric Mann-Whitney test was used to determine significant differences. (e,f) The role of the ROS system in DENV infection of mosquitoes. Either 3.3 mM vitamin C or 1 mM uracil mixed with 1x106 pfu purified DENV2 virions and human blood was used for a mosquito blood meal. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. (g,h) Genetic suppression of the JAK-STAT pathway and the ROS system enhanced DENV2 infection in mosquitoes. GFP dsRNA served as a negative control. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. (e,g) The number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. (f,h) Differences in mosquito infective ratios were compared using Fisher’s exact test. The p values were adjusted using Bonferroni correction to account for multiple comparisons. Differences were considered significant if _p<_0.017 (f) or _p<_0.01 (h). (a–h) The experiments were biologically repeated at least 3 times with similar result.

Figure 5

Figure 5. An antibody generated against DENV2 ΔNS1 does not cross-react with human cells and prevents DENV acquisition by mosquitoes

(a) Schematic representation of DENV2 ΔNS1. DENV2 ΔNS1 lacks the following antigenic regions from full-length DENV2 NS1 that potentially elicit the production of cross-reactive antibodies: amino acids 116–119, 221–266 and 311–330. (b,c) DENV NS1 antibody cross-reactivity with HUVEC cells. Purified antibodies against DENV2 full-length NS1 or DENV2 ΔNS1 were incubated with HUVECs. A pre-immune antibody served as a mock control. Antibody attachment to the HUVECs was quantified by flow cytometry (b) and ELISA (c). DENV2 NS1 antibodies were stained using anti-mouse Alexa 488-conjugated IgG for FACS assay and anti-mouse HRP-conjugated IgG for ELISA. The values in the graph represent the mean ± SEM. A non-parametric Mann-Whitney test was used to determine significant differences. The p values were adjusted using Bonferroni correction to account for multiple comparisons. Differences were considered significant if _p<_0.025. (d,e) An antibody generated against DENV2 ΔNS1 prevents DENV acquisition by mosquitoes. Murine antisera (1:100 dilution) against DENV2 full-length NS1 or DENV2 ΔNS1 were incubated with supernatant from DENV-infected Vero cells (50% v/v) and fresh human blood (50% v/v). The mixture was used for in vitro membrane feeding of A. aegypti. The same dilution of pre-immune serum served as a mock control. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. (d) The number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. (e) The data are represented as the percentage of mosquito infection. Differences in mosquito infective ratios were compared using Fisher’s exact test. The p values were adjusted using Bonferroni correction to account for multiple comparisons. Differences were considered significant if _p<_0.025. (b–e) The experiments were biologically repeated at least 3 times with similar result.

Figure 6

Figure 6. NS1 vaccination prevents DENV infections in mice and mosquitoes

(a–e) Active immunization with DENV2 ΔNS1 prevented DENV2 acquisition by mosquitoes. (a) Schematic representation of the study design. (b,c) DENV2 infection in immunized AG6 mice. (b) Detection of DENV2 viremia. Blood was collected from the tail veins of infected mice from 0 to 5 days post infection. The presence of infectious viral particles in the blood plasma was assessed using a plaque assay. (c) Detection of DENV2 sNS1 concentration. Mouse sera were used to determine DENV2 sNS1 quantities by ELISA. (d,e) NS1 immunization reduced DENV acquisition by A. aegypti. (d) The number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. (e) The data are represented as the percentage of mosquito infection. Differences in mosquito infective ratios were compared using Fisher’s exact test. (f–h) Vaccination with DENV2 ΔNS1 protected mice from lethal DENV-induced vascular leakage. (f) AG6 mice were immunized and infected following the same procedure as shown in Fig. 6a. “n” represents the number of mice in each group. The survival rates of the infected mice were statistically analyzed using the Log-rank (Mantel-Cox) test. (g,h) Evans blue dye was intravenously injected into the mice at 18 days post infection. The dye was extracted from various tissues using formamide, and the OD610 was measured (g). The infected mice were used to visualize DENV-induced vascular leakage in various tissues (h). (b–g) The red p value represents a comparison between full-length DENV2 NS1-immunized and control mice. The blue p value represents a comparison between DENV ΔNS1-immunized and control mice. The p values were adjusted using Bonferroni correction to account for multiple comparisons. Differences were considered significant if _p<_0.025. “*”, “**” and “***” represent _p<_0.025, _p<_0.005, and _p<_0.0005, respectively. (b,c,g) The data are representative of at least five infected AG6 mice. The values in the graph represent the mean ± SEM. A non-parametric Mann-Whitney test was used to determine significant differences. (b–h) The experiments were biologically reproduced at least 3 times.

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