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. 2024 May 14;98(5):e0195923.
doi: 10.1128/jvi.01959-23. Epub 2024 Apr 18.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection

Yi He #  1 , Chang Miao #  1 , Shiping Yang  1 , Changhao Xu  1 , Yuwei Liu  1 , Xi Zhu  1 , Yiping Wen  1   2   3 , Rui Wu  1   2   3 , Qin Zhao  1   2   3 , Xiaobo Huang  1   2   3 , Qigui Yan  1   2   3 , Yifei Lang  1   2   3 , Shan Zhao  1   2   3 , Yiping Wang  1   2   3 , Xinfeng Han  2   3 , Sanjie Cao  1   2   3 , Yajie Hu  4 , Senyan Du  1   2   3
Affiliations

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection

Yi He et al. J Virol. .

Abstract

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Keywords: Japanese encephalitis virus; attachment; infection in mosquitoes; sialic acids.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
JEV infection was inhibited by blocking sialic acid on C6/36 cells and mosquito. (A–F) C6/36 cells were pretreated with increasing concentrations of neuraminidase from C. perfringens or 1 µM/mL lectins (ConA, SNA, and WGA), followed by JEV infection at a multiplicity of infection (MOI) of 0.5. (A and D) The infected cells were collected at 24 h post-infection for detection of viral genomes by RT-qPCR. (B, C, E, and F) C6/36 cells were pretreated with 1 U/mL neuraminidase or 1 µM/mL lectins, and the cell culture supernatant and infected cells were collected at 36 h post-infection. (B and E) Viral titers in the supernatant were determined by plaque assay. (C and F) JEV E protein abundance in infected cells was assessed by western blot assays using JEV E polyclonal antibody and GAPDH monoclonal antibody. (G and H) Increasing concentrations of neuraminidase or 2 µM/mL lectins were microinjected into C. quinquefasciatus thoraxes 1 day before 10 50% mosquito infectious dose (MID50) JEV infection. The JEV burden in entire mosquitoes was determined by RT-qPCR at 3 days post-infection. (I) Schematic of the study design. First, 1 U/mL neuraminidase, 2 µM/mL SNA, or 10 µM/mL WGA mixed with fresh mice blood and supernatant from JEV-infected C6/36 cells was used to feed C. quinquefasciatus via an in vitro blood feeding system. The JEV infection of entire mosquitoes was determined by RT-qPCR 8 days after a blood meal. (J, L, and N) The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. Each dot represents a mosquito. The horizontal line represents the mean value of the group. The limit of detection for the viral genome/actin mRNA ratio was 0.001. Gene expression was normalized to the C. quinquefasciatus actin gene. The data are presented as the mean ± SEM. (A, B, D, E, G, and H) The nonparametric Mann-Whitney test was used for statistical analyses. (K, M, and O) The data at the top of each column represent the percentage of infected mosquitoes. Differences in the infectivity ratio were compared using Fisher’s exact test. *P < 0.05 and **P < 0.01. The experiment was repeated three times with similar results.
Fig 2
Fig 2
The sialic acid biosynthetic pathway was correlated with JEV infection. (A) Schematic of the sialic acid biosynthetic pathway in insects. (B) Schematic representation of the study design. The CSAS, ST, or SAS gene was silenced by thoracic microinjection of dsRNA in C. quinquefasciatus. The mosquitoes were inoculated with GFP dsRNA as mock controls. After 3 days, the post-dsRNA treatment mosquitoes were microinjected with 10 MID50 JEV. The entire mosquitoes were killed for JEV detection at 3 days post-infection. (C–E) The JEV burden in microinjected mosquitoes was determined at 3 days post-infection. Each dot represents the mRNA level in one mosquito. The horizontal line represents the median. The data are presented as the mean ± SEM. The nonparametric Mann-Whitney test was used for statistical analysis. (F–I) After 3 days of dsRNA treatment, the mosquitoes were infected with JEV by membrane feeding. The infection rate of mosquitoes was determined after 8 days. (F and H) The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. Each dot represents a mosquito. The horizontal line represents the mean value of the group. (G and I) The data at the top of each column represent the percentage of infected mosquitoes. Differences in the infectivity ratio were compared using Fisher’s exact test. *P < 0.05. The data from two independent experiments were combined.
Fig 3
Fig 3
Sialic acids promote JEV attachment. (A) Schematic representation of the study design. For the attachment assay, C6/36 cells were incubated with JEV at a MOI of 5 and 1 µM/mL lectins (ConA or SNA) for 1 h at 4°C. The unbound virions were removed by washing with PBS. (C) The cell extracts were subjected to RT-qPCR. For the internalization assay, after JEV virion adsorption at 4°C for 1 h, the cells were washed with PBS and subsequently incubated to 30°C for 1 h with 1 µM/mL lectins (ConA or SNA) to allow virion internalization. After washing with PBS, the cells were treated with sodium citrate buffer to remove noninternalized virions. (B) Viral internalization into cells was assessed by RT-qPCR. (D and E) After treatment with 1 U/mL neuraminidase, C6/36 cells were infected with JEV at a MOI of 5 at 4°C for 1 h. Unbound viruses were removed by washing with PBS. (D) Viral attachment was assessed by RT-qPCR. (E) Immunofluorescence analysis. Cells were stained with an anti-JEV E polyclonal antibody (red) and biotin-labeled SNA (green), and the nuclei were stained with DAPI. Scale bars, 40 µm. (F and G) The relative fluorescence intensities of sialic acids and JEV were calculated by ImageJ. (B–D) The data are presented as the mean ± SEM. The nonparametric Mann-Whitney test was used for statistical analysis. **P < 0.01; n.s., not significant.
Fig 4
Fig 4
Sialic acids interact with the JEV E protein at the last α-helix in domain III. (A and B) C6/36 cells were seeded into 96-well plates, and cells were incubated with virions or protein. Sialic acids on the C6/36 cell surface were bound to biotinylated SNA followed by HRP-conjugated streptavidin. The interaction of JEV virions or purified JEV E protein with sialic acids on the C6/36 cell surface was analyzed by cell-based ELISA. (C) Purified JEV E proteins were coated on an enzyme-labeled plate and incubated with different concentrations of sialic acid. The interaction between these proteins was measured by ELISA using biotinylated SNA followed by HRP-conjugated streptavidin. (D, F, G, I, L, and N) Purified JEV E protein or truncated E proteins from S2 cells that were transfected with recombinant plasmid (tagged with V5) were incubated with sialic acids. The protein complex was pulled down with biotinylated SNA. Bound proteins were eluted and analyzed by immunoblotting with a V5-tag antibody. (E) Schematic representation of the three domains of the JEV E protein. (H) Schematic representation of the truncations at the N-terminal and C-terminal of JEV E DIII; the dashed line means delete the N-termina of E DIII (Δ312–369 amino acids). (J) Schematic diagrams displaying the structure of the C-terminal of JEV E DIII (370–427 amino acids). (K) Schematic diagram showing the truncations of JEV E DIII (370–427 amino acids). (M) Schematic diagrams displaying the structure of JEV E (Δ370–401 amino acids); the dashed line means delete 370–401 amino acids. The data are presented as the mean ± SEM. The nonparametric Mann-Whitney test was used for statistical analysis. **P < 0.01.
Fig 5
Fig 5
Alignment of the E DIII protein sequences of flaviviruses and the physical structure of the JEV E protein. (A) Alignment of the E DIII protein sequences of JEV, WNV, ZIKV, DENV, and YFV. The yellow line marks the last helix in DIII. (B) Model of the location of the last helix (402–426 amino acids) of the JEV E protein.
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