Journal of Medical Sciences

: 2021  |  Volume : 41  |  Issue : 3  |  Page : 134--139

Production of mosquito cell-derived Zika virus-like particles using BacMos system

Hui-Tsu Lin1, Der-Jiang Chiao1, Szu-Cheng Kuo2,  
1 National Defense Medical Center, Institute of Preventive Medicine, Taipei, Taiwan
2 National Defense Medical Center, Institute of Preventive Medicine; Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan

Correspondence Address:
Dr. Szu-Cheng Kuo
Institute of Preventive Medicine, National Defense Medical Center, Taipei 11490


Background: Zika virus (ZIKV) is a mosquito-borne flavivirus which has been conclusively linked to Guillain-Barré syndrome and microcephaly. The worldwide emergence of ZIKV has greatly increased the demand for vaccines that reduce or prevent disease transmission. Neutralizing human antibodies which target ZIKV E proteins have been shown to prevent ZIKV replication. Virus-like particles (VLPs) lacking viral genetic material comprise self-assembled multi-subunit protein structures that are capable of strongly activating humoral and cellular immunity. Flavivirus prM and E proteins are both necessary and sufficient for the production of VLPs. Thus, it appears that ZIKV VLPs are an ideal target for vaccine design and serological detection. Methods: In this study, the BacMos (baculovirus/mosquito) method was used to introduce the ZIKV prME gene into mosquito cells. Immunofluorescence assays (IFAs), dot blot (DB) analysis, and Western blot (WB) analysis were used to evaluate the expression and secretion of ZIKV glycoproteins. VLP formation was confirmed using transmission electron microscopic (TEM) and dynamic light scattering (DLS) analysis. Results: IFA presented intense signals from ZIKV E-positive cells in BacMos-ZIKV prME-transduced cells. DB and WB detected abundant ZIKV glycoproteins in the culture medium of BacMos-ZIKV prME-transduced cells. TEM observation and DLS analysis revealed that ZIKV VLPs comprised spherical particles, with an average diameter of 30 nm. Conclusions: Mosquito cell-derived ZIKV VLPs are promising candidates for the development of safe, efficacious vaccines and diagnostic antigens in the future.

How to cite this article:
Lin HT, Chiao DJ, Kuo SC. Production of mosquito cell-derived Zika virus-like particles using BacMos system.J Med Sci 2021;41:134-139

How to cite this URL:
Lin HT, Chiao DJ, Kuo SC. Production of mosquito cell-derived Zika virus-like particles using BacMos system. J Med Sci [serial online] 2021 [cited 2021 Jun 22 ];41:134-139
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Full Text


Zika virus (ZIKV) is a mosquito-borne flavivirus associated with Guillain-Barré syndrome and microcephaly.[1],[2],[3],[4] The worldwide emergence of ZIKV has increased the demand for vaccines which reduce or prevent disease transmission.[5] VLPs comprise self-assembled multi-subunit protein structures that are configurative and antigenic to their corresponding native viruses.[6] VLPs induce adaptive immunity by activating the innate immune system.[7],[8],[9],[10] They also display repeated high-density viral antigens in an authentic conformation, which promotes B-cell activation and results in a robust humoral immune response as well as cellular-mediated immunity.[11],[12] VLPs are therefore highly effective as subunit vaccines and as antigens for serological detection. When expressed together in a cell, flavivirus prM and E proteins can self-assemble into VLPs.[13],[14] Previous studies have demonstrated the efficacy of using baculoviruses (1) as vectors for gene delivery into mosquitoes and (2) in the production of mosquito cell-derived Japanese encephalitis VLPs using the BacMos system.[15],[16] The current study used the BacMos system to produce flavivirus ZIKV VLPs.


Cell and viral cultures

C6/36 (Aedes albopictus) cells were cultured in RPMI 1640 medium (GIBCO, Invitrogen, CA, USA) containing 10% fetal bovine serum and 1x antibiotic-antimycotic solution (GIBCO, Invitrogen, CA, USA) at 28°C under 5% CO2. AP-61 (Aedes pseudoscutellaris) cells were cultured in L-15 medium (GIBCO, Invitrogen, CA, USA) containing 10% fetal bovine serum and 1x antibiotic-antimycotic solution at 28°C. Viral titers of ZIKV (ATCC® VR-1843™) propagated in the Vero cells were determined via plaque assays in Vero cells.

Construction of transfer vectors

Generation of recombinant baculoviruses followed previously described protocols.[17] In brief, synthetic 1464-bp SpeI-XhoI DNA fragments containing modified IRES and DsRed gene were subcloned into SpeI-XhoI sites of pFastBac1 vectors, and the resulting plasmids were named pFastBacT1-Ph-MCS-LIR-DsRed2. Synthetic 981-bp SacI-SpeI DNA fragments containing hr1-pag1,[16] cecropin B1 genes (sequence ID: KJ439044.1), and poly A signal sequences were subcloned into SacI-SpeI sites of pFastBacT1-Ph-MCS-LIR-DsRed2, and the resulting plasmids were named pFastBacT1-Ph-MCS-LIR-hr1pagcecrob. Finally, synthetic 2397-bp DNA fragments encoding ZIKV prME (105–794th amino acid residues, GenBank: ANO46310.1) were subcloned into SmaI-NotI sites of pFastBacT1-Ph-MCS-LIR-DsRed2-hr1pag1-cecroB1 vectors, and the resulting plasmids were named pFastBacT1-Ph-MCS-LIR-DsRed2-hr1pag1–ZIKV prME of the transfer vector of BacMos-ZIKV prME [Figure 1].{Figure 1}

Immunofluorescence assays

Cells were transduced using BacMos-ZIKV-prME at a multiplicity of infection (MOI) of 5 or infected with ZIKV at an MOI of 0.1. At 3 days posttransduction (dpt) or 3 days postinfection, cells were fixed and incubated with monoclonal antibody (6B6C mAb) anti-ZIKV E (1:100)[16] for 1 h. Cells were subsequently washed three times with PBS and incubated with Alexa Fluor 488-conjugated secondary antibodies for 1 hour. After a final wash with PBS, images of cells were captured using an inverted fluorescence microscope.

Immunoblot analysis

Mosquito cells were seeded at 8 × 104 (AP-61) or 4 × 105 (C6/36) cells/well in a 24-well plate and transduced with recombinant viruses at an MOI of 5 or infected with ZIKV at an MOI of 0.1. Culture supernatant of the transduced cells at 3 dpt was harvested and centrifuged at 12,000 rpm for 5 min. Samples were prepared in nonreducing lysis buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], and 1% Triton 100) at 4°C. Proteins (20 μl/well) were separated using SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes (PROTRAN, Schleicher and Schuell), blocked using Tris-buffered saline (TBS; 100 mM Tris, pH 7.4, 100 mM NaCl) containing 5% (v/v) nonfat dry milk, and then detected using 6B6C (1:4000) against ZIKV E or anti-Zika prM (GTX 133305; 1:1000). For dot blot (DB) analysis, harvested culture medium was centrifuged at 12,000 rpm for 5 min, and 100 μl samples were applied to nitrocellulose membranes (PROTRAN, Schleicher and Schuell) using a bio-dot microfiltration apparatus. Membranes were subsequently blocked using TBS (100 mM Tris, pH 7.4, 100 mM NaCl) containing 5% (v/v) nonfat dry milk, and detected using 6B6C mAb against ZIKV E rabbit (R) anti-ZIKV E antibodies (GeneTex, GTX133314). SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, USA) was used to determine the presence of HRP on membranes.

Production, purification, and analysis of mosquito cell-derived virus-like particles

Culture supernatant was harvested and passed through a 0.45-μm filter to remove debris. Filtered culture medium was 10-fold concentrated, diafiltrated using a 10-fold volume of high salt buffer (1X PBS and 0.5 M NaCl), and then 5-fold concentrated using the MidiKros Module Tangential Flow Filtration (TFF) System (300,000 molecular weight cutoff, Spectrum Repligen, USA). VLP proteins were analyzed using SDS-PAGE and stained using Coomassie blue.

Transmission electron microscopic observation

Partial purified ZIKV VLPs were deposited on a copper grid and negatively stained using 2% uranyl acetate. Transmission electron microscopic (TEM) observation was performed using a Hitachi HT7700.

Dynamic light scattering

Particle size was determined using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK), whereby each sample was measured via two series of 15 runs (10 s per run). The size distribution of the particles was calculated using the Zetasizer software suite.


Expression of Zika virus structural proteins in mosquito cells

Immunofluorescence assay results [Figure 2] showed intense ZIKV E glycoprotein signals in the transduced C6/36, transduced AP-61, and ZIKV-infected C6/36 cells. However, weak signals of ZIKV E glycoprotein were detected in the ZIKV-infected AP-61 cells. This indicates that the level of ZIKV E glycoprotein expression was comparable between transduced and infected cells. To assess the intracellular localization of expressed E glycoproteins, we examined infected and transduced mosquito cells using immunofluorescence microscopy. E glycoprotein signals were observed exclusively in the cytoplasm of transduced and infected cells. The secretion of ZIKV E glycoproteins in the BacMos-ZIKV prME-transduced mosquito cells was detected by dot immunoblotting assays [Figure 3]a on culture media harvested at various time points at 2–9 dpt. Immunoblot results revealed the accumulation of secreted ZIKV E glycoproteins [Figure 3]b in the transduced mosquito cells following incubation at 2–6 dpt, and secreted ZIKV prM glycoprotein [Figure 3]c was also detected in both BacMos-ZIKV-prME-transduced C6/36 and AP-61 cells. These results confirm the secretion of ZIKV prM and E glycoproteins by BacMos-ZIKV-prME-transduced mosquito cells.{Figure 2}{Figure 3}

Secreted Zika virus envelope proteins display variant antigenic structures in different mosquito cell lines

We sought to determine the antigenic structure of secreted ZIKV E glycoproteins from different mosquito cell lines by subjecting culture media harvested from BacMos-ZIKV prME-transduced-C6/36 or -AP-61 cells to DB analysis with 6B6C or R anti-ZIKV E antibodies. As shown in [Figure 4], secreted ZIKV E glycoproteins from the transduced-AP-61 cells were specifically detected by 6B6C. Conversely, secreted ZIKV E glycoproteins from the transduced-C6/36 cells were specifically bound by R anti-ZIKV E. These findings indicate that ZIKV E glycoproteins secreted from different mosquito cell lines have different antigenic structures.{Figure 4}

Partial purification and characterization of Zika virus virus-like particles

We further characterized the secretion of ZIKV glycoproteins by partially purifying these glycoproteins via TFF. Concentration and partial purification of secreted ZIKV glycoproteins was confirmed through Coomassie blue staining [Figure 5] and Western blot. In addition, spherical particles with an average diameter of 30 nm were detected using dynamic light scattering (DLS) analysis and further confirmed under EM observation [Figure 6]. Taken together, these results indicate that ZIKV VLPs were secreted from the BacMos-ZIKV-prME-transduced mosquito cells.{Figure 5}{Figure 6}


ZIKV is a serious threat to public health which will only be controlled after the implementation of an effective vaccination program.[18] The protective efficacy of the DNA–prME vaccine against ZIKV has been proven in mice[19] and monkey[20] models. Other research involving mice found the following: (1) Neutralizing human antibodies that target ZIKV E proteins prevented ZIKV replication and associated fetal disease in mice.[21] (2) Recombinant adenoviral vectors expressing the ZIKV E antigen induced protective humoral immunity against ZIKV infection.[22] (3) DNA vaccines expressing ZIKV prME proteins that elicit neutralizing antibodies appear to have protected recipients from ZIKV-mediated diseases.[19],[23] Mammalian-derived ZIKA VLPs vaccines produced from the stable cell lines have been shown to induce a robust neutralizing antibody response against ZIKV, and vaccination with mRNA vaccines that encode the ZIKV prME gene has been shown to confer protective immunity against the virus in mice.[24],[25],[26],[27] A modified dendrimer-based RNA nanoparticle vaccine bearing a VEE replicon which expresses ZIKV prME has also been found to induce cellular and humoral immunity against ZIKV.[27] Progress in the development of VLP-based vaccines is gaining traction.[28],[29] These findings clearly demonstrate that the ZIKV prME protein is an ideal target for vaccine design and the generation of VLPs. Results of the current work also demonstrate the efficacy of BacMos[15] in promoting the expression and secretion of ZIKV glycoproteins in the C6/36 and AP-61 cells [Figure 2] and [Figure 3]. Note that the maturity of flavivirus VLPs affects their antigenic structures. The 6B6C-1 (a conformationally dependent mAb) specifically binds to mature flavivirus VLPs.[30] However, the rabbit anti-Zika E antibody (GeneTex, GTX133314), which is generated by immunizing the synthetic peptides of ZIKV E proteins, preferentially recognizes the linear antigenic structures of E proteins. Our DB analysis results [Figure 4] revealed that transduced AP-61 cells secreted ZIKV E glycoproteins (detected specifically by 6B6C), which indicates that dominant mature antigenic structures of ZIKV E glycoprotein are secreted from AP-61 cell. Conversely, ZIKV E glycoproteins secreted from transduced-C6/36 cells were found to specifically bind with R anti-Zika E antibodies. This indicates that, under the BacMos system, different mosquito cell lines secrete ZIKV E glycoproteins with distinct antigenic structures. The resulting mosquito-derived ZIKA VLPs were characterized in terms of partial purification using TFF [Figure 5]. In TEM and DLS analysis [Figure 6], the average diameter of the ZIKV VLPs was found to approximate 30 nm, which is similar to the size of Lepidoptera-derived ZIKV VLPs.[31] To compare mammalian-derived ZIKA VLPs, mosquito-derived VLP with differences on lipids and carbohydrates resembles the infectious structure of early arboviruses in humans. In addition, the production of mosquito cell-derived VLPs at lower temperature could elicit high titers of neutralizing antibodies comparing mammalian-derived ZIKV VLP produced at 37°C.[32] In the future, the specific native epitopes of these mosquito cell-derived ZIKV VLPs could be characterized using MAC-ELISA in terms of their resemblance to authentic virions. These mosquito cell-derived ZIKV VLPs may be critical antigens that can serve as a candidate vaccine against ZIKV infection.


This study demonstrated a simple strategy for generating mosquito cell-derived ZIKV VLPs and provided a good target antigen for serological detection as well as potential candidate vaccine against ZIKV infection for future development

Financial support and sponsorship

This research was funded by grants NHRI-108A1-MRCO-0519191 and MR-109-CO-11 from The National Health Research Institutes.

Conflicts of interest

There are no conflicts of interest.


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