Short Communication: Mosquito Histone 2A Protein Facilitate Japanese Encephalitis Virus Infection in the Mosquito

RNAi and infection

For in vitro RNAi, small interfering RNA (siRNA) (Supplementary Table S1) was transfected into C6/36 cells with Cellfectin II reagent (Invitrogen). JEV was used to inoculate the cells at 24 h posttransfection. After infected cells were collected, total RNA was isolated, and the viral or gene load was determined by RT-qPCR. In vivo RNAi was performed as described previously (Liu et al., 2017). siRNAs targeting genes in the Culex mosquito were synthesized by GenePharma (Shanghai, China), and their sequences are shown in Supplementary Table S1. For RNAi and virus challenge, female mosquitoes 5 days after eclosion were injected with 2 μg of dsRNA in 250 nL of PBS into the thorax (Liu et al., 2020). This study was approved by the institutional review board of Academic Committee of Shanghai Veterinary Research Institute.

RNA isolation and real-time RT PCR

For RT-qPCR, RNA was extracted from cell suspensions or mosquito samples with a Qiagen Total RNA Isolation Kit according to the manufacturer's instructions. All experiments were performed in triplicate, and gene expression levels are presented relative to those of β-actin. The fold change in relative gene expression compared with the control value was determined with the standard 2^−ΔΔCt method.

Statistical analyses

All experiments were carried out in triplicate. Mean values with standard deviations were calculated, and statistical analysis were performed using Student's t-test and Fisher's methods. p-Values upon the comparison of data from each independent experiment were calculated by Student's t-test. Fisher's method was used to test the difference among the groups.

Anti-JEV E monoclonal antibody 2H4

To precisely study the interaction of JEV particles with mosquito vector proteins, we first developed a monoclonal antibody against the JEV E protein, 2H4. Validation of the monoclonal antibody for western blot (WB) and immunocoprecipitation (IP) demonstrated that 2H4 was specific in WB (Fig. 1A). 2H4 could also be used in IP experiments (Fig. 1C, D).

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FIG. 1.

Mosquito H2A facilitates JEV infection in vitro and in vivo. (A) Validation of 2H4 monoclonal antibody. (B) Experimental flow of JEV IP/MS. (C) Silver staining result of 2H4-IP/MS. (D) Validation of 2H4-IP by western blot. (E, G) H2A upregulated after JEV infection in C6/36 cell (E) and in Culex quinquefasciatus (G). (F, H) H2A RNAi impeded JEV infection in C6/36 cells (F) and C. quinquefasciatus (H). (I) Efficiency of RNA interference in C6/36 cell and C. quinquefasciatus. IP/MS, immunoprecipitation/mass spectrum; RNAi, RNA interference.

In vivo and in vitro IP/MS analysis of JEV

To analyze the binding of mosquito vector salivary gland secretory proteins to JEV particles, we first infected Culex quinquefasciatus mosquitoes and C6/36 cells with JEV and collected samples on the showed time points (C6/36 samples were collected at day 6 postinfection, mosquito samples were collected at day 7 and 14 postinfection) as shown in Fig. 1B. Samples were subjected to IP with the 2H4 monoclonal antibody, and the immunoprecipitated proteins were analyzed by MS as presented in Fig. 1C. Mock infection or IgG was used as a control. The results showed that there were 41 proteins captured in vitro and 24 proteins captured in vivo. Two proteins (60S ribosomal protein and histone H2A) were captured both in the mosquito salivary gland and cell supernatant. The mass spectral density of extracellular and intracellular H2A was 726340000 and 299310000, respectively (Supplementary Files S1 and S2). The mass spectral density of salivary gland H2A on Day 7 was 2787500, and on Day 14 was 23879000 (Supplementary Files S1 and S2). This indicates that the binding of H2A protein to JEV may be associated with the viral infection.

To verify the IP/MS results, we prepared antibodies specific to mosquito H2A. The same IP steps were used to precipitate JEV and bound proteins, and then WB was performed with the anti-H2A antibody. The results showed that H2A could bind to JEV as presented in Fig. 1D.

H2A promotes JEV infection in vivo and in vitro

To further analyze the effect of H2A on JEV infection, mosquitoes exposed to JEV via blood meal and C6/36 cells were inoculated with JEV. H2A mRNA levels were then measured at indicated time points postinfection (Fig. 1B). RT-qPCR data indicated the H2A upregulation in both JEV infected mosquitoes and C6/36 cells (Fig. 1E, G). Mock infection of mosquitoes and cells were used as controls. Next, we designed and synthesized siRNA oligos against mosquito H2A and introduced the siRNA into cells by transfection or in mosquitoes through intrathoracic injection. All siRNA and primer sequences are lists in Supplementary Table S1. Mock infection or negative siRNA was used as a control.

We then performed viral infection in both cell culture and mosquitoes as stated above and evaluated the viral replication by RT-qPCR. In C6/36 cells, H2A interference significantly reduced the level of JEV replication at 6- and 9-days postinfection (dpi) (Fig. 1F), with the JEV mRNA level in the H2A-interference group being ∼1.5-fold less than that in the control group. In mosquitoes, interference with H2A expression also significantly reduced JEV mRNA levels in infected mosquitoes, and the JEV mRNA in the H2A-interference group was ∼1.5-fold less than the control group at seven dpi (Fig. 1H). These results suggest that H2A might promote JEV infection of mosquito vectors. All infection experiments were based on effective RNAi (Fig. 1I).