Preparation and application of chikungunya pseudovirus containing double reporter genes

Chikungunya virus (CHIKV), a highly infectious and rapidly spread viral pathogen, is classified as a pathogenic agent at the biosafety level 3. Operation of live authentic CHIKV needs a specific laboratory with the P3 or above containment, which greatly confines the CHIKV-associated studies. To establish an evaluation system of CHIKV that can be utilized in a BSL2 laboratory, we constructed a pseudovirus (PsV) system of CHIKV containing double reporter genes (ZsGreen1 and luciferase). The fluorescent ZsGreen1 is a convenient and cheap reporter for monitoring the efficiency of transfection and titration of PsV. The enzyme luciferase is a sensitive reporter for the application of PsV to neutralization assay or drug screening. The CHIKV PsV produced in this study, with a titer of up to 3.16 × 106 TU/ml, was confirmed by Western blotting and transmission electronic microscopy (TEM). Finally, we developed a microneutralization assay with the CHIKV PsV produced in this study, which was successfully applied to evaluate neutralizing activities of convalescent sera from CHIKV-infected patients. In summary, we have established a convenient and sensitive double-reporter CHIKV pseudovirus system, which provides a safe and effective platform for screening anti-CHIKV drugs and evaluating vaccines against CHIKV.

Production of CHIKV PsV. The three-plasmid system was used to produce CHIKV PsV, including the CHIKV envelope proteins expressing plasmid pMD2.G-CHIKV-env, the packaging plasmid PsPAX2 and the transfer plasmid pLVX-IRES-ZsGreen1-Luc. At 24 hours post co-transfection, cells were in good conditions and 70% of them expressed the ZsGreen1 fluorescent protein. At 48 hours post co-transfection, there were about 90% of cells expressing fluorescent protein (Fig. 2a), suggesting that these plasmids were efficiently transduced into 293T cells. Furthermore, most fluorescent cells began to shrank, fall off and even die with a grape-cluster-like appearance indicating the release of PsV, which is the possible reason that cells at 24 hpt appeared to be more fluorescent than those at 48 hpt. The results of immunoblotting analysis confirmed the expression of CHIKV envelope proteins in the PsV particles (Fig. 2b). Finally, we observed the formation of CHIKV PsV particles with the size of 80-140 nm and typical enveloped structure under transmission electronic microscope (Fig. 2c). These results suggest that we have successfully produced CHIKV PsV particles.

Transduction of CHIKV PsV in 293T cells. The transduction of CHIKV PsV was further evaluated in
293T cells. Expression of the reporter ZsGreen1 was monitored under the fluorescence microscope or by flow cytometric analysis. The results showed that CHIKV PsV could transduce and release the reporter ZsGreen1 into 293T cells (Fig. 3a). The titer of CHIKV PsV in this study was 3.16×10 6 TU/ml, similar to that of the positive control VSV-G PsV (3.29×10 6 TU/ml) (Fig. 3b).

Development of CHIKV PsV micro-neutralization assay.
To validate the applicability of the constructed CHIKV PsV in this study, we developed a microneutralization assay using these PsV and serially diluted rabbit serum sample that were known to contain anti-CHIKV antibodies. 500 TU of CHIKV PsVs were used for each neutralization reaction with VSV-G PsV as a control (non-CHIKV envelope proteins control) for the specificity of neutralization. The activity of luciferase was measured on the microplate reader to evaluate the neutralizing ability of antibodies. Compared with the VSV-G PsV, the CHIKV PsV could be specifically neutralized by the antibody in the known CHIKV serum sample. CHIKV PsV transduction was inhibited by the serum sample in a dose-dependent manner. The neutralization titer of serum (50% inhibitory dose, ID 50 ) against CHIKV PsV was 117.4 ( Fig. 4), indicating that PsV is potentially applicable in the researches of CHIKV.
CHIKV pseudovirus transduction is inhibited by sera from infected individuals. CHIKV convalescent serum samples were firstly analyzed via ELISA. The human serum samples were then divided into three groups according to ELISA-OD 450 (Table 1). Microneutralization assay was conducted on these samples using CHIKV PsV produced in this study. Through measurement of luciferase activity, most of the CHIKV convalescent human serum samples showed strong neutralization activity in a dose dependent manner (Fig. 5a). Negative control serum samples showed certain neutralization at the first several concentrations, which decreased rapidly and kept at a low level. This result suggested that the neutralization is specifically against CHIKV (Fig. 5a). The CHIKV PsV neutralization titers (PsV NT) were shown in Table 1. In order to analyze the relationship between neutralizing antibody and total IgG antibodies against CHIKV, we made a plot for these convalescent serum samples using the values of ELISA-OD 450 as x-axis and PsV NTs as y-axis. Through this comparison, we found there was a significantly positive correlation between neutralizing antibody and total IgG antibodies (Fig. 5b).

Discussion
Adaptive mutation of A226V facilitates CHIKV to infect Ae. Albopictus, contributing to the transmission of mutant CHIKV to broader region than the prototypic viruses 16 . Within last two decades, CHIKV spread rapidly from African continent to Asia, America and Europe, leading to millions of infectious cases and thousands of deaths, which becomes a global concern of public health [16][17][18][19] . Therefore, we should make full preparation for this potential outbreak, including elucidation of pathogenesis, drug screening and vaccine development, and so on. However, dependence of CHIKV operation on P3 containment hinders the progress of CHIKV researches due to the limitation of the high-level biosafety facility. In the present study, we constructed CHIKV PsV particles  www.nature.com/scientificreports/ containing CHIKV envelope and two reporters. This system is potentially applicable in studying the interaction between CHIKV and host cell, drug screening, evaluation of vaccine efficacy at the P2 laboratory.
PsV is a kind of virus-like particle with capability of one-time transduction, and made of viral capsid protein and envelope proteins packing non-viral nucleic acid inside. Therefore, PsV is commonly used as a safe alternative for authentic live virus, particularly for highly pathogenic viruses, in studies of viral serology [20][21][22] , anti-viral strategy 13,23,24 , and viral pathogenesis [24][25][26] . The reporter in PsV is designed for evaluation of the interaction between host cell and PsV. The most common reporter is luciferase because it is very sensitive. However, measurement of luciferase activity is complicate and expensive. Dual reporters (ZsGreen1 and luciferase) have been increasingly engineered into PsV of this study and other reports 27,28 . The fluorescent reporter ZsGreen1 in www.nature.com/scientificreports/ PsV can be used to monitor efficiency of transfection and titration, which is more convenient and cheaper than the reporter luciferase.
In this study, we constructed PsV particles, where CHIKV envelope was inserted into HIV capsid. Therefore, the formed PsV particle owned the morphology and size of HIV particle (80-140 nm) (Fig. 2c). It is generally believed that the capsid is an essential element for CHIKV to form infectious virus particles [29][30][31][32] . Actually, even though capsid is completely deficient in the mutant CHIKV, infectious virus can still be formed although some of morphological characteristics are different from naïve CHIKV 33 .
Prior to the utilization of PsV, it is necessary to determine the titer of viral particle. The copy number of viral genome or concentration of viral protein (such as p21 of HIV-1) is usually used in the titration experiments. However, these methods are not only time-consuming and laborious, but also cannot exclude the non-infectious viral particles, which leads to the higher titer than actual value 4,25,34,35 . In contrast, PsV constructed in this study contained the reporter ZsGreen1. Compared with the traditional titration method on basis of the virus particle itself (qPCR detection of the copy number of viral genome or ELISA measurement of viral protein concentration), ZsGreen1 is expressed only in infected cells, which effectively excludes the interference of non-infectious virus particles, making the titration more accurate. In addition, as a fluorescent signal, ZsGreen1 can be easily captured by flow cytometry, which is easier than the traditional method of titration.
The plaque reduction neutralization test (PRNT) is considered as a gold standard for neutralization assays. However, PRNT needs the use of hazardous live CHIKV and BSL3 facility, which impedes the application of PRNT to CHIKV-related studies. In addition, visualization and counting of plaques in PRNT are time-consuming and laborious procedures. Many studies have demonstrated that the PsV neutralization test could be a good substitute for the plaque reduction test 27,34 . In order to overcome the limitations of PRNT mentioned above, here we developed a PsV neutralization assay. In this assay, luciferase activity was used to quantify the neutralization www.nature.com/scientificreports/ activity, which had been considered to be semi-automatic, high throughput, sensitive and accurate 27,34,36 . As compared with the neutralization activity of VSV-G PsV, our result showed that neutralization assay of CHIKV PsV is dose dependent and CHIKV specific (Fig. 4). Previous studies have reported that neutralizing antibodies represent a constant proportion of total antibodies 34 . In this study, we found that the ratio of neutralizing antibodies was positively correlated with the total IgG of CHIKV antibodies from infected individuals. This result is consistent with the conclusions of previous studies and further illustrated that CHIKV PsV produced in this study might be a reliable and efficient alternative system for CHIKV research. In summary, the results of this study demonstrate that CHIKV PsV containing double reporters were successfully produced. These PsV, with one-time transduction of 293T cells, could be potentially used to evaluate   Fig. 5b). A total of 20 human serum samples (17 positive samples from CHIKV convalescent individuals, 3 negative samples from healthy individuals were used as controls), were divided into three groups according to ELISA-OD 450 . www.nature.com/scientificreports/ the interaction between CHIKV and host cells, which provides a safe and effective method for drug screening and vaccine evaluation in BSL2 laboratories. Preparation of CHIKV PsV. Three-plasmid system was used to construct CHIKV PsV, in which the HIVbased lentivirus packaging vector PsPAX2, which contains lentiviral Gag/Pol, was used to provide the capsid for PsV. And the plasmid pLVX-IRES-ZsGreen1-Luc that contained two reporter genes, luciferase and ZsGreen1, was used to provide RNA genome for PsV. The third plasmid pMD2.G-CHIKV-env containing CHIKV envelope fragment, was used to provide spike glycoprotein for PsV. A schematic diagram of the construction of CHIKV PsV is shown in Fig. 1a. These three plasmids were co-transfected into 293T cells (kindly provided by Dr. Longding Liu, the Institute of Medical Biology, China) using the FuGENE HD Transfection Reagent. The VSV-G PsV system that consists of three plasmids, PsPAX2, pLVX-IRES-ZsGreen1-Luc and pMD2.G for vesicular stomatitis virus (VSV-G) spike glycoprotein, was used as a positive control for transfection and production of PsV. The reporter ZsGreen1 was use to evaluate the transfection efficiency. The culture supernatant was harvested at 48 h post transfection, followed by centrifugation at 2000 g for 5 min. After passing through 0.45 μm membrane, the supernatant PsV particles was aliquoted and stored at − 80 °C for further analysis. www.nature.com/scientificreports/ Identification of CHIKV PsV. The filtered supernatant containing PsV was added to the top of 20% sucrose solution in the ultracentrifuge tube, followed by ultracentrifugation at 25,000 rpm for 2.5 h with the setting of acceleration 9 and deceleration 3 (Beckman Coulter, Optima MAX-XP) [37][38][39] . After the supernatant was discarded, 1% volume of DMEM was used to resuspend the pellet of PsV particles that was then kept at 4 °C overnight. Next day, the suspension of PsV particles was ready for further analysis. In western blotting, mouse anti-CHIKV E1 mAb (0.5 μg/ml) and HRP-conjugated anti-mouse IgG antibody were used to detect CHIKV envelope protein. Transmission electron microscopy (TEM, HITACHI H-7650) was utilized to confirm the formation of PsV particles. Ten microliter of filtered supernatant was added to the Copper mesh, followed by absorption for 2 min at room temperature. Excessive water in the Copper mesh was then removed. Phosphotungstic acid counterstaining was conducted on samples. After removal of excessive water, stained samples were kept in a dish for 30 min and observed under TEM.

Reagents
Titration of PsV particle (flow cytometry). The titer of the PsV was determined by transduction of HEK 293T with serial ten-fold dilutions of PsV particles. 0.5-1 × 10 5 cells were seeded per well in a 24-well plate (500 μl) 37  Micro-neutralization assay. Neutralization assay was performed in the 96-well plate as described previously 40 . The CHIKV PsV were diluted to 1×10 4 TU/ml and 50 µl of them were mixed with a 2-fold serial dilution ( Fig. 4 and Fig. 5a) of serum samples from CHIKV-immunized rabbits or CHIKV convalescent patients (kindly provided by Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research, Yunnan Institute of Parasitic Diseases). The mixture was incubated at 37 ℃ for 1 to 2 h and then added to the cells (50,000/well) in each well. After 72 h of incubation, the expression of the reporter ZsGreen1 was observed under the fluorescence microscope (Leica, DMIL LED) and the activity of luciferase was measured via Bright-Glo™ Luciferase Assay System according to the protocol recommended by the manufacture. The neutralization curve was made on basis of the activity of luciferase. And neutralization titer of serum samples against CHIKV PsV was expressed as the 50% inhibitory dose (ID 50 ) that was calculated via the inhibition curve.
Statistical data analysis. Determination of ID 50 values were performed using the GraphPad Prism 8.0.2 software (La Jolla, CA, USA) as nonlinear regression, which is expressed as log(inhibitor) vs. normalized response-Variable slope. The statistical significance analysis was performed by Student's t test. p values of<0.05 (*) were considered as statistical significance.