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. 2017 May 8;13(8):1811–1817. doi: 10.1080/21645515.2017.1325050

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system

Li Zhang 1,†, Qianqian Li 1,†, Qiang Liu 1, Weijin Huang 1, Jianhui Nie 1, Youchun Wang 1,✉
PMCID: PMC5557217  PMID: 28481728

ABSTRACT

Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

KEYWORDS: animal model, bioluminescent imaging, marburg virus, passive protection, pseudovirus

Introduction

Marburg virus (MARV), the first filamentous virus to be discovered, together with the well-known Ebola virus, constitute the Filoviridae family. Members of the Filoviridae can cause deadly hemorrhagic fever in humans and non-human primates.1 Marburg hemorrhagic fever is considered a zoonotic disease. Infections with MARV have been linked to human entry into caves inhabited by Egyptian fruit bats (Rousettusaegyptiacus).2,3 After its first appearance in 1967, several sporadic outbreaks of Marburg hemorrhagic were reported across sub-Saharan Africa.1 To date, there have been 2 big outbreaks of Marburg hemorrhagic fever, the 1998–2000 outbreak in the Democratic Republic of the Congo and the 2004–2005 outbreak in Angola. The mortality rate for the Angola outbreak was 90%, making it one of the deadliest outbreaks on record.4,5 Although outbreaks have predominantly occourred in central Africa to date, the potential for imported cases or bioterrorism in non-African countries cannot be ignored.6

Compared with the research focused on Ebola virus, MARV has received much lesss attention and the mechanism of MARVpathogenicity remains largely unknown. MARV is classified as a risk group 4 agent due to its extremely high virulence, the potential for aerosol transmission, and the lack of prophylaxis or treatment. Handling MARV is restricted to high-containment biosafety 4 facilities, which greatly impedes the development of vaccines and drugs against this virus.7

MARV is a filamentous, enveloped, non-segmented, negative-sense RNA virus. Its genome is about 19kb in length and encodes 7 structural proteins. Glycoprotein (GP) is the sole envelope protein of MARV, and is responsible for both receptor binding and virus host membrane fusion.8

In this study, a human immunodeficiency virus (HIV)-based firefly luciferase reporter protein (Fluc) expressing MARV pseudovirus was established by the replacement of HIV envelope protein with MARV GP. This pseudovirus is replication defective, being only capable of one-round of replication, and can therefore be used safely in non-BSL-4 laboratories. Using this pseudovirus, we optimized a sensitive in vitro neutralization assay system and a convenient in vivo bioluminescent imaging mouse model; we also evaluated a MARV-immunized guinea pig serum. This work provides a useful tool for further studies exploring the mechanism of MARV infection and developing vaccines or therapeutics to prevent or treat this serious infection.

Results

Generation and packaging optimization of pseudotyped MARV

 The MARV GP pseudovirus was generated by co-transfection of an envelope-deleted HIV backbone vector and MARV GP expression vector (Fig. 1A).To obtain a high titer of pseudotyped MARV virus, 3 modified SG3 HIV vectors9 and the NL4–3 HIV backbone vectors were compared in 2 packaging cell lines. The pSG3.ΔEnv.CMV.fluc transfected 293T cells showed the highest relative light units (RLU), which indicated the best packing efficiency (Fig. 1B). We then optimized the ratio of HIV backbone vector to MARV GP expression vector, and found that a 1:2 ratio of pCDNA3.1–MARV GP to pSG3.ΔEnv.CMV.fluc was the most suitable (Fig. 1C). To further improve the p/HIV/MVGP/Fluc yields, we tested several commercial transfection reagents, including Lipofectamine 2000, Lipofectamine 3000, PEI, Neofect, Turbofect, and Vigofect. The results suggested that Lipofectamine 3000 was the ideal transfection reagent (Fig. 1D).

Figure 1.

Figure 1.

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Pseudotyped MARV construction and packaging condition optimization. (A) Schematic diagram of the packing plasmid modification and pseudotyped MARV generation. Briefly, the pseudotyped HIV backbone vector was first modified and co-transfected with the MARV glycoprotein (GP) expression plasmid into 293T cells. After 48 hours, the cell supernatant was harvested, purified, and concentrated. Several types of HIV backbone vectors, that we had modified previously, were compared.9 (B) Optimization of the HIV backbone vectors and packaging cell lines. Four envelope-lacking HIV plasmids were tested in 2 cell lines. The HIV SG3 pseudovirus was modified by deletion of the envelope and insertion of the Fluc reporter with or without nef deletion and CMV promoter addition as described previously. 9 The packaging plasmid and pCDNA3.1–MARV-GP were co-transfected into 293 or 293T cells at a ratio of 1:1 using Lipofectamine 2000. After 48 hours, the bioluminescence was detected and relative light units (RLU) were calculated. (C) Optimization of the transfection ratio of backbone vectors versus GP-expressing plasmid. 293T cells were transfected with pCDNA3.1–MARV-GPand pSG3.ΔEnv.CMV.fluc at different ratios as indicated. Lipofectamine 2000 was used in these tests. The RLU were examined 48 hours after transfection. (D) Optimization of the transfection reagents. pSG3.ΔEnv.CMV.fluc and pCDNA3.1–MARV-GP were transfected into 293T cells at a ratio of 1:2 using different transfection reagents according to their instructions. After 48 hours,the RLU were tested.

Establishment of an in vitro neutralization assay

To choose a sensitive cell line for the neutralization assay, we tested the cellular tropism of p/HIV/MVGP/Fluc. All of the cell lines tested could be infected by p/HIV/MVGP/Fluc, which indicated that the pseudovirus had a wide cell tropism. This was consistent with the wild type virus. HEK 293T cells were the most susceptible cells, and therefore the best substrate for the neutralization assay (Fig. 2A). We further optimized the assay by adjusting the 293T cell numbers from 5,000 to 100,000 per well, and found that 50,000/well was the most suitable cell density (Fig. 2B). Although the 80,000/well and 100,000/well groups also showed high bioluminescence, the cell state was not as healthy as in the 50,000/well groupdue to the high cell density. The RLU was detected every 12hours after virus infection up until 84hours. The RLU peaked at 72 hours, indicating that this may be the optimal time point to carry out the test (Fig. 2C). To obtain neutralizing serum, guinea pigs were immunized with p/HIV/MVGP/Fluc and serum was collected (Fig. 2D). The neutralization titer of the serum (50% inhibitory dilution, ID50) was examined by a pseudovirus-based neutralizing assay (Fig. 2E). The immunized serum with a high titer was mixed and used in the following study. The virus dose used in the neutralizing assay was further optimized considering the ID50 and the coefficient of determination (R2),and a 50% tissue culture infectious dose (TCID50) value of 100–400 TCID50 was recommended as the standard virus dose to ensure both sensitivity and accuracy of the neutralizing assay (Fig. 2F). The specificity of the high titer serum was investigated through immunoblotting. The guinea pig serum recognized specific bands in the MARV GP-expressing cell lysate, but not in the HIV SG3-expressing cell lysate. Furthermore, this serum could also recognize the GP of purified pseudotyped MARV, but not GP of pseudotyped Ebola virus (Fig. 2G). Moreover, the purified GP of Marburg virus was used to competitively bind to neutralizing antibodies in the serum. The inhibition rate of serum decreased as the concentration of MARV GP increased. The competition of MARV GP and MARV pseudovirus also indicated the successful induction of neutralizing antibodies by p/HIV/MVGP/Fluc (Fig. 2H). These results confirmed the specific MARV neutralizing ability of the guinea pig serum and the sensitivity of the neutralization assay. Taken together, these data provided evidence that a sensitive and specific in vitro neutralizion assay for MARV had been successfully established.

Figure 2.

Figure 2.

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Establishment and optimization of the in vitro neutralization assay. (A) Cell tropism of pseudotyped MARV. Different cells were seeded at 50,000/well in 96-well plates and infected with p/HIV/MVGP/Fluc at a MOI of 200 TCID50 as indicated. After 48 hours, the bioluminescence was tested and RLU were calculated. (B) Cell inoculum density of 293 T cells. 293 T cells were seeded from 5,000/well to 10,000/well in 96-well plate and infected with p/HIV/MVGP/Fluc at a MOI of 200 TCID50. After 48 hours, the bioluminescence was tested and RLU were calculated. (C)293T cells were seeded at 50,000/well in 96-well plates and infected with p/HIV/MVGP/Fluc at a MOI of 200 TCID50.The bioluminescence was examined every 12 hours until 84 hours after infection. (D) The flowchart of guinea pig immunization. Guinea pigs were immunized by IP injection with 1.95 × 108 TCID50 of p/HIV/MVGP/Fluc 3 times at 2 week intervals. Serum samples were collected from each week from 3 to 7 weeks after immunization. (E) The neutralization titer of the immunized serum at different time points. Serially diluted guinea pig serum was incubated with pseudotyped MARV, and used to infect 293T cells. The bioluminescence was measured 72 hours later. (F) Optimization of the pseudovirus dosage. Serially diluted guinea pig serum was incubated with different doses of pseudotyped MARV, and then mixed with 293 T cells. The bioluminescence was measured 72 hours later. (G) Western blotting of MARV GP using pseudovirus immunized guinea pig serum. Different doses of purified pseudotyped MARV or EBOV were loaded and then blotted with guinea pig serum (105 TCID50/line, Left panel). MARV GP was overexpressed in 293 T cells by transfection with pCDNA3.1–MARV-GP, or co-transfection with pCDNA3.1–MARV-GP and pSG3.ΔEnv.CMV.fluc. Single transfection of pSG3.ΔEnv.CMV.fluc or mock cells with transfection reagents only were included as controls. The cell lysates were loaded and then blotted with guinea pig serum (Right panel). HIV p24 protein was blotted as a loading control. (H) Competitive inhibition of purified MARV GP. The purified MARV GP (0.855 mg/ml) was serially diluted as indicated, mixed with 100 times diluted guinea pig serum, incubated at 37°C for 1 hour, and then mixed with 293 T cells. The bioluminescence was measured 72 hours later.

Construction and optimization of a bioluminescent imaging mouse model

To establish a bioluminescent imaging MARV mouse model, 4 different mouse strains were tested for their susceptibility to p/HIV/MVGP/Fluc. Both Kunming and Balb/c mice showed strong signals. Balb/c mice were selected for our mouse model because of the homogeneity observed for this strain in the test (Fig. 3A). Two days after pseudovirus infection, the bioluminescent signal started to appear, and reached a peak at 5 days, then faded after 10 d. Three to 5 d post-infection was therefore selected as the optimal time point to carry out the assays (Fig. 3B). Organ anatomy studies revealed that p/HIV/MVGP/Fluc was mainly distributed in the spleen, liver, thymus, lungs, intestine, muscle, and skin, which roughly reflected the wide spread organ distribution of MARV during infection (Fig. 3C). Finally, the 50% animal infectious dose (AID50) of the pseudovirus was determined to be 2.6 × 106 TCID50 in Balb/c mice (Fig. 3D). We selected an AID50 of 7.5 to 15 as the infective dose for the following experiments.

Figure 3.

Figure 3.

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Construction and optimization of the bioluminescent imaging mouse model. Five-week-old female mice were used in the experiments. Mice were injected with p/HIV/MVGP/Fluc via IP (200μl per mouse or as indicated). Bioluminescence was visualized in pseudocolor. The values of the total flux were analyzed and are shown on the right of each figure. (A) Comparison of the different mouse strains. The bioluminescence was examined 4 d after pseudovirus infection. (B) Dynamic observation of MARV pseudovirus infection in Balb/c mice. The bioluminescence was observed at the indicated time points. (C) The dissection of pseudotyped MARV-infected mouse. The bioluminescence of the MAR- infected mice was observed at 5 d post infection. Mice were dissected and the bioluminescence of each organ was analyzed. The names of the organs are shown on the right. The virus-enriched organs are highlighted in red. (D) AID50 of pseudotyped MARV. Balb/c mice were inoculated with p/HIV/MVGP/Fluc from 1.6 to 1000 × 105 TCID50 per mouse. The bioluminescence was captured 4 d later. The total flux was analyzed and the AID50 was determined using the Reed-Muench method.

Evaluation of serum passive protection in the mouse model

This bioluminescent imaging mouse model was used to evaluate the protectiveness of p/HIV/MVGP/Fluc-immunized guinea pig serum (ID50 = 1954) in vivo. Our study showed that pre-incubation of p/HIV/MVGP/Fluc with immunized guinea pig serum could significantly decrease the bioluminescence in both 4-week-old (p = 0.0050) and 8-week-old (p = 0.0113) mice, which indicated that the immunized serum could protect both young and adult mice from MARV infection (Fig. 4A). Furthermore, the pre-injection of immunized guinea pig serum in vivo decreased the bioluminescence of virus-challenged mice, slightly at a low dose (p = 0.1356) and significantly at a high dose (p = 0.0001). This result suggested that the immunized serum could also protect mice from MARV infection in vivo (Fig. 4B). Taken together, these data demonstrated that a convenient bioluminescent imaging MARV mouse model had been successfully established.

Figure 4.

Figure 4.

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Evaluation of serum passiveprotection. Balb/c mice were injected with p/HIV/MVGP/Fluc via IP (7.5 AID50). All of the signals were collected at 4 d post injection and visualized in pseudocolor. Values of the total flux were analyzed and are shown on the right. (A) In vitro serum protection. In the serum group, p/HIV/MVGP/Fluc was pre-incubated with 100μl of the immunized guinea pig serum one hour before injection. (B) In vivo serum protection. Balb/c mice were injected with the immunized serum one hour before virus challenge. 100μl and 600μl of serum were used in the low dose and high dose group respectively. (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant).

Discussion

MARV is one of the most virulent and lethal viruses, causing severe hemorrhagic fever in humans and non-human primates. The mortality associated with MARV infection is as high as 90%, with no effective treatments or vaccines available to date.1

A major obstacle to research on MARV is the requirement for BSL-4 containment and procedures.1 The advent of pseudoviruses, which package the GP to the surface of replication-incomplete HIV,10-13 vesicular stomatitis virus,14 and feline immunodeficiency virus,15 allows researchers to safely work with the virus in a BSL-2 environment.

In previous MARV studies, the lentiviral NL4–3.Luc.R-E- was the major pseudovirus system used.10-13 SG3 is another widely used HIV based pseudovirus system.16-18 However, the requirement of special cells containing reporter genes greatly limits its application. In our laboratory, several backbone vectors have been constructed by the addition of Fluc reporter genes and the CMV promoter, which enhance protein expression in mammalian cells.9 The packaging efficiency of each backbone vector varies among different viruses. In this study, we found that the packaging efficiency of pseudotyped MARV was significantly improved by the modified SG3 backbone vector compared with the classical NL4–3.Luc.R-E- system. We also optimized other key steps in virus packaging, including the ratio of backbone vector to MARV GP plasmid, as well as the transfection reagents, which further improved MARV pseudovirus production. This study provided a new high-yield pseudovirus system, which would be a convenient tool for future studies of MARV.

Although the pseudotyped systems have been widely used to screen entry inhibitors, such as drugs and antibodies, as well as cellular receptors and co-receptors that regulate MARV infection in vitro,10-13 they have not previously been used for in vivo studies. Non-human primates and rodents are the most commonly used animal models for MARV.19,20 Non-human primates are the most reliable model because the clinical symptoms and pathophysiology are quite similar to those observed in humans.21 However, the cost and time associated with model are prohibitive for the high-throughput screening of drugs and the early evaluation of vaccines. Serial passages of the original MARV endue it lethal infection capacity to rodents, whereas the disease course still differs from that of humans.22 The major limitation of all of these models is their dependence on BSL-4 environments. In our MARV bioluminescence imaging mouse model, we used pseudovirus only capable of single round replication that lacked any virulent viral components other than GP, ensuring it is safe for use in BSL-2 laboratories. The bioluminescence imaging mouse model is sensitive, easy to handle, and convenient for real-time observations of virus infection both spatially and temporally. We previously confirmed that bioluminescence intensity correlated well with viral load.23 The distribution of virus could be easily observed in live animals dynamically, and could be further analyzed by organ dissection. In this study, the pseudotyped MARV was enriched in the spleen, liver, thymus, lung, intestine, muscle, and skin, which was analogous to the organ distribution of wild-type MARV.24

There are some limitations of this model that should be noted. The sensitivity of the bioluminescence may be influenced by the pigmentation of the fur and tissue, and the depth of the organs. Since most genetically engineered mice are from a C57BL/6 background, the black coat of the animal may significantly decrease the signal. Removal of the fur or breeding of the mice into an albinovariety may be a way to minimize this problem.25,26 The bioluminescence signal started to appear at day 2 and disappeared after day10 due to the one-round replication defect and the clearance of infected cells. The model is also limited to MARV GP related studies because of the deficiency in replication-related proteins. However, this model is still a good choice for preliminary studies to explore drug and vaccine candidate that are based on MARV GP, and its benefit are safety and ease to operate.

Using p/HIV/MVGP/Fluc, we induced neutralizing antibodies in guinea pigs, confirmed the specificity of the antibody, and evaluated the neutralizing titer using our optimized in vitro neutralizing assay. We further examined the passive immunity of the serum in vivo. The p/HIV/MVGP/Fluc-immunized serum showed protective effect not only on in vitro mixing, but also by in vivo pre-injection. This protection was not only evident in virus-sensitive young animals, but also in adult mice.

In addition to the applications discussed above, thus system could also be used to evaluate the active immunization of different types of vaccines, to screen entry inhibitors against MARV, and ti search for key mutations associated with immune evasion. As a complement of the classical in vitro neutralization system, which was widely used to examine candidate vaccine-induced neutralization antibodies, the bioluminescence imaging system can be used to evaluate the protective effect of candidate vaccines conveniently in vivo. Instead of wild-type MARV, p/HIV/MVGP/Fluc could be used to infect immunized mice at different time points. The relative intensity of floresence may indicate the degree of virus infection and further reflect the prophylactic efficacy of the candidate vaccines. Moreover, the GP of MARV incorporated in the pseudovirus can be easily mutated or modified using genetic approaches. The in vitro neutralization assay may therefore be used to identiy neutralization escape mutations. In addition, the mutated MARV pseudovirus can be evaluated in vivo through the bioluminescence imaging mouse model. This may be applied to confirm mutations contributing to the evasion of the virus from vaccine immunization or therapeutic monoclonal antibodies.

In summary, we have established a bioluminescence imaging mouse model for MARV, which is easy to operate, convenient to observe and analyze in a real-time manner, and most importantly, negates the limitations imposed by BSL-4classification. This model provides a powerful tool with which to rapidly advance the research on MARV.

Materials and methods

Cells

HEK 293(American Type Culture Collection [ATCC], CRL-1573), HEK 293T (ATCC, CRL-3216), HEK 293FT (Invitrogen, Carlsbad, CA), Vero (ATCC,CCL81),Vero E6 (ATCC, CRL-1586), HeLa (ATCC, CCL−2), A549 (ATCC, CCL−185), HepG2 (ATCC, HB-8065), and BHK21cells (ATCC, CCL−10) were cultured in Dulbecco's modified Eagle's medium (HyClone, SH30243.01B), and K-562 cells (ATCC, CCL−243) were grown in RPMI 1640 (HyClone, SH30809.01B). Both medium were supplemented with 10% fetal bovine serum (Gibco, 106000644, 1% penicillin–streptomycin solution (Gibco, 15140163) and 2% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Gibco, 15630080). All cells were grown at 37 in a humidified 5% CO2 atmosphere.

Pseudotyped virus

The MARV GP gene (Marburg virus isolate Mbg-422–2012, Uganda) was cloned into the eukaryotic expression vector pCDNA3.1. The HIV-based MARV pseudovirus (p/HIV/MVGP/Fluc), expressing the MARV GP and firefly luciferase reporter protein (Fluc), was generated by co-transfection of 293 or 293T cells with pCDNA3.1–MARV-GP and envelope-defective HIV-1 SG3 or NL4–3 Fluc R-E- in a 1:2 ratio or as described, using Lipofectamine 2000 (Invitrogen, 11668019), Lipofectamine 3000 (Invitrogen, L3000015), PEI(Alfa Aesar, 43896), Neofect (Neofectbiotech Co), TurboFect (Thermo Fisher Scientific, R0531), or VigoFect(Vigrous Biotechnology, T001).The p/HIV/MVGP/Fluc containing culture supernatant was harvested after 48 hours incubation, and centrifuged at 210 × g for 5 min. The supernatant was then filtered through a 0.45 μM pore-size filter, and concentrated in a 30-kDa ultrafiltration centrifugal tube (Millipore, UFC903096). The TCID50 was calculated using the Reed-Muench method as described previously.27

In vitro neutralization assay

The conditions for p/HIV/MVGP/Fluc infection were optimized by the addition of virus to different cell lines in a 96-well-plate, with cell numbers ranging from 5,000 to 1,000,000 per well. The bioluminescence was tested at different time point after virus infection. Briefly, culture medium was aspirated gently to leave 100 μl in each well. The Bright-Gloluciferase reagent (Promega, E2650) was added 100 μl per well and incubated at room temperature for 2 min. The lysates were then transferred to solid black 96-well plates, and examined using a Glomax 96 microplate luminometer (Promega, Fitchburg, WI). P/HIV/MVGP/Fluc was incubated with serial dilutions of the guinea pig serum at 37°C for 1hour, and then mixed with freshly trypsinized cells in a 96-well plate. The bioluminescence was measured 72 hours post incubation or as indicated above.

Animal experiments

The animal study protocol was approved by the Animal Care and Use Committee at the National Institute for Food and Drug Control (NIFDC, Beijing, China). Five-week-old femaleBALB/c, C57BL/6, KM, and NIH mice (Institute for Laboratory Animal Resources, NIFDC), were infected with p/HIV/MVGP/Fluc via intraperitoneal (IP) injection. The bioluminescent signals were monitored at different time points from 6 hours to 14 d post infection.

Bioluminescence imaging

Bioluminescence imaging was performed with the IVIS-Lumina IIimaging system (Xenogen, Baltimore, MD) as described previously. Mice were anesthetized by IP injection of pelltobar-bitalumnatricum (240 mg/kg body weight), followed by IP injection of the substrate, d-luciferin (50 mg/kgbody weight; Xenogen-Caliper Corp., Alameda, CA,122799). Mice were imaged 10 minutes later with an exposure time of 60 seconds. The regions of interest were analyzed using Living Image software (Caliper Life Sciences, Baltimore, MD). For imaging of individual organs, mice were euthanized, dissected and imaged within 3 minutes. The relative intensities of emitted light were presented as the photon flux in photon/(s cm2sr) and displayed as pseudo-colored images, with colors ranging from red (the most intensive) to blue (the least intensive).

Serum protective test in mice

The protective serum was collected from p/HIV/MVGP/Fluc immunized guinea pigs. Guinea pigs were immunized with1.95 × 108 TCID50of p/HIV/MVGP/Fluc via IP injection on day 0, 14 and 28. The serum sample was collected on days 21, 28, 35 and 42 post immunization. The immunized guinea pig serum was pre-incubated with pseudotyped MARV (7.5 AID50) in vitro for 1 hour and used to challenge Balb/c mice via IP injection. Alternatively, Balb/c mice were first injected with immunized guinea pig serum via IP or both IP and intravenous (IV) injection 1 hour before p/HIV/MVGP/Fluc (7.5 AID50) challenge. The bioluminescence was detected 4 d after virus infection as described above.

Statistical analysis

Data were analyzed with GraphPad Prism 5.0 software (GraphPad, San Diego, CA). The results are presented as the means ± standard deviations (SD). P values of < 0.05 were considered statistically significant.

Abbreviations

Fluc

reporter gene

GP

glycoprotein proteins

HIV

backbone

MV

MARV

p

pseudovirus

Disclosure of potential conflicts of interest

The authors report no conflicts of interest.

Acknowledgment

We thank Dr. George F. Gao for the provision of purified MARV glycoproteins.

Funding

This work was supported by the National Key Research and Development Plan of China under Grant 2016YFC1200904. The funding body had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Author contributions

Youchun Wang, Li Zhang and Qiang Liu conceived and designed the experiments. Qianqian Li, Li Zhang,Weijin Huang and Jianhui Nie performed the experiments. Li Zhang and Qianqian Li analyzed the data. Li Zhang and Youchun Wang wrote the paper.

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