搜索

Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates
感染血清1型和2型登革热病毒临床分离株的小鼠模型   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated with secondary heterotypic infections through a phenomenon that is described as antibody-dependent enhancement of infection (ADE). There are an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly. At present, however, there are no approved antiviral drugs against DENV infection. The lack of a suitable animal model has hampered the evaluation of novel antiviral candidates for DENV infection. Since DENV poorly establishes infection in immunocompetent mice, AG129 mice (lacking type I and II IFN [interferon] receptors) and mouse-adapted DENV2 strains have been applied to dengue animal models that enable to reproduce several of the major pathologies of human infection. Recently, we developed new mouse models with clinical isolates DENV1 and DENV2 that would be useful for drug testing and dengue pathogenesis studies (Watanabe et al., 2016). Here we describe the details to establish dengue mouse models of clinical isolates; from in vitro preparation of the materials to in vivo virus infection. Of note, since infectivity of DENV in mice differs among virus strains, not all clinical isolates can induce severe dengue.

Keywords: Dengue virus(登革热病毒), Lethal mouse model(致死性小鼠模型), Clinical virus(临床病毒), Antibody-dependent enhancement of virus infection(病毒感染的抗体依赖性增强), Drug testing(药物试验)

Background

To overcome the drawback that DENV does not replicate well in rodent cells, many efforts have been made over the years to develop small animal models that mimic human dengue infection. The inbred mouse model system allows experimental variability to be minimized, and genetically engineered mouse models enable to reproduce some aspects of dengue clinical symptoms in the animals. A past study showed that AG129 mice (lacking type I and II IFN receptors) infected with a DENV2 clinical isolate succumbed to infection with signs of paralysis, a condition of central nervous system involvement that is rare in human cases (Shresta et al., 2004). Alternatively, mouse-adapted DENV2 strains that can induce human DHF/DSS-like diseases in AG129 mice were generated and have been used for dengue research (Shresta et al., 2006; Zellweger et al., 2010). Although the use of the mouse-adapted strains is valuable for some aspects of DENV pathogenesis studies and potential therapeutic drug testing, one considerable limitation is the variable pathogenesis depending on the serotype/genotype, and adaptation of virus in mouse might alter tissue tropism. Recently, we developed new mouse models of clinical isolates DENV1 and DENV2; the mice succumbed to infection with signs of severe dengue symptoms (Watanabe et al., 2016). Non-lethal infection with clinical isolates becomes lethal accompanied with high levels of viremia and cytokine production in the presence of DENV antibodies (Abs) (ADE condition) in AG129 mice, suggesting that this system enables to extend the use of clinical DENV isolates for the study of Ab-mediated DENV pathogenesis and the evaluation of anti-dengue candidates.

Materials and Reagents

  1. 50 ml centrifuge tubes (Corning, Falcon®, catalog number: 357550 )
  2. 0.45 μm membrane filter (Sartorius, Minisart®, catalog number: 16537 )
  3. 0.2 μm membrane filter unit for bulk culture (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 567-0020 )
  4. HiTrap Protein G HP-5 ml (GE Healthcare, catalog number: 170-0405-01 )
  5. 96-well PCR plate (Bio-Rad Laboratories, catalog number: HSP9601 )
  6. SnakeskinTM dialysis tubing 10 kDa (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 68100 )
  7. 30 G insulin syringe (BD, catalog number: 328818 )
  8. 27 G needle (BD, catalog number: 305109 )
  9. 1.5 ml Eppendorf tubes (Corning, Axygen, catalog number: MCT-150-c )
  10. Plate cover seal (Bio-Rad Laboratories, catalog number: MSB1001 )
  11. CryoTubes vials for freezing viruses (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 368632 )
  12. Dengue virus: DENV-2 mouse-adapted S221 strain (Zellweger et al., 2010), DENV-1 clinical isolate (EDEN1: GenBank accession
    EU081230.1) (Low et al., 2006), DENV2 clinical isolate (EDEN2: GenBank accession EU081177.1) (Low et al., 2006)
  13. Cells of the Aedes albopictus C6/36 line (clone C6/36) (ATCC, catalog number: CRL-1660TM )
  14. Cells of the baby hamster kidney cell line (BHK-21 [C-13]) (ATCC, catalog number: CCL-10TM )
  15. Hybridoma cells (D1-4G2-15) (ATCC, catalog number: HB-112TM )
  16. Sv/129 mice deficient in type I and II IFN receptors (AG129 mice) (B&K Universal)
  17. RPMI1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  18. Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  19. Liquid nitrogen
  20. 3.7% formaldehyde (Sigma-Aldrich, catalog number: F1635 )
  21. Crystal violet (Sigma-Aldrich, catalog number: C3886 )
    Note: This product has been discontinued.
  22. Protein-Free hybridoma medium (PFHM-II medium) (Thermo Fisher Scientific, GibcoTM, catalog number: 12040077 )
  23. 1x PBS (Lonza, catalog number: 17-516Q )
  24. Glycine (Sigma-Aldrich, catalog number: G7126 )
  25. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  26. Ethylenediaminetetraacetic acid (EDTA) (EMD Millipore, catalog number: 819040 )
  27. QIAamp Viral RNA Mini Kit (Qiagen, catalog number: 52906 )
  28. qScript One-Step qRT-PCR Kit (Quantabio, catalog number: 95057 )
  29. Methyl-cellulose powder (EMD Millipore, catalog number: 17851 )
  30. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  31. Penicillin and streptomycin (PenStrep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  32. 7.5% sodium bicarbonate solution (Thermo Fisher Scientific, GibcoTM, catalog number: 25080094 )
  33. 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  34. Ethanol (EtOH) (EMD Millipore, catalog number: 1009832511 )
  35. RPMI 1640 powder (Thermo Fisher Scientific, GibcoTM, catalog number: 31800022 )
  36. 1 N HCl (Sigma-Aldrich, catalog number: 258148 )
  37. Tris (First BASE Laboratories Sdn Bhd, catalog number: BIO-1400 )
  38. Primers and probes
    DENV1 forward primer: 5’-ACACCAGGGGCTGTACCTTGG-3’
    DENV1 reverse primer: 5’-CATTCCATTTTCTGGCGTTCT-3’
    DENV1 taqman probe: FAM-5’-CTGTCTCTACAGCATCATTCCAGGCA-3’-TAMRA
    DENV2 forward primer: 5’-CATATTGACGCTGGGAAAGA-3’
    DENV2 reverse primer: 5’-AGAACCTGTTGATTCAAC-3’
    DENV2 taqman probe: FAM-5’-CTGTCTCCTCAGCATCATTCCAGGCA-3’-TAMRA
  39. Standard for realtime RT-PCR: plasmids containing whole genome sequences of DENV-1 (EDEN1: EU081230.1) or DENV-2 (EDEN2: EU081177.1)
  40. 0.8% methyl-cellulose medium (see Recipes)
  41. 1% Crystal violet (see Recipes)
  42. 0.1 M glycine (pH 2.7) (see Recipes)
  43. 1 M Tris- HCl (pH 9.0) (see Recipes)

Equipment

  1. NuncTM 175 cm2 angled-neck easy flasks (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 159920 )
  2. Cell scraper (Corning, catalog number: 3010 )
  3. Incubator (SANYO, model: MIR-262 ) without CO2 atmosphere at 28 °C
  4. Humidified incubator (NuAire, model: NU5500 ) with 5% CO2 atmosphere at 37 °C
  5. Swinging rotor centrifuge (for cells) (Thermo Fisher Scientific, model: Heraeus Multifuge 3S-R )
  6. Benchtop fixed-angle rotor centrifuge (for serum) (Eppendorf, model: 5424 )
  7. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: Forma 900 Series )
  8. Autoclave (TOMY DIGITAL BIOLOGY, model: SX-700 )
  9. AKTApurifierTM UPC 10 (GE Healthcare, catalog number: 28406268 )
  10. pH meter (Sartorius, model: pH Basic Series )
  11. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ND-2000 )
  12. Real-time thermal cycler (Bio-Rad Laboratories, model: CFX96 )
  13. Mouse restrainer (Plas-labs, catalog number: 551-BSRR )
  14. Olympus inverted fluorescence microscope (Olympus, model: IX71 )

Software

  1. GraphPad Prism software

Procedure

  1. Generation of DENV stocks
    Note: A high virus titer of clinical DENV strains is required to induce severe diseases in AG129 mice. The virus titer obtained from C6/36 cells differs among virus strains. Thus, optimization of culture condition such as multiplicity of infection (MOI) of virus inoculum or timing of virus collection is needed to obtain enough virus titer for each virus strain.
    1. Maintain C6/36 cells in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin and 25 mM HEPES. Incubate cells at 28 °C under non CO2 atmosphere condition.
      Note: Growth media supplemented with 20% FBS improves cell growth if C6/36 cells do not proliferate desirably.
    2. Grow C6/36 cells in a 175 cm2 flask in culture medium until ~90% confluency (see Figure 1).
    3. Thaw virus stock and dilute with serum-free RPMI 1640 medium. Discard medium from culture flask and add 5 ml of virus inoculum at MOI of 0.1 (for EDEN1 or S221) or 1 (for EDEN2) into 175 cm2 flask.
      Note: At confluency, there are approximately 3 x 107 cells in a T175 flask, hence for an MOI 0.1-1, 3 x 106-3 x 107 pfu are needed (refer to Procedure B on how to determine virus titers).
    4. Incubate for 1 h at 28 °C under non CO2 atmosphere condition.
    5. Remove virus inoculum and add 25 ml RPMI 1640 medium (2% FBS) to the 175 cm2 flask.
    6. Incubate for 4-7 days at 28 °C under non CO2 atmosphere condition.
      Note: Cytopathic effect is observed depending on virus strain (seen in EDEN2 infection but not in EDEN1 or S221 infection). Check virus titer in the supernatant at different time point to determine the optimal day to obtain high virus titer (refer to Procedure B on how to determine virus titers). Incubation periods are 5 days for EDEN2 and S221, and 7 days for EDEN1.
    7. Scrape cells and transfer into a centrifuge tube, then spin down the cells at 1,800 x g for 10 min at 4 °C.
    8. Collect supernatant and transfer into a fresh tube through a 0.45 μm membrane filter.
    9. Aliquot virus into cryotubes and store in a -80 °C freezer or liquid nitrogen until use.
      Note: It is recommended to store virus stock in liquid nitrogen for long-term storage.

  2. Determination of viral titer by plaque assay
    1. Maintain BHK-21 cells in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine and 100 U/ml penicillin and streptomycin in a humidified incubator with 5% CO2 atmosphere at 37 °C.
    2. Seed cells at 2 x 105 cells per well in 500 μl into 24-well plate.
    3. Incubate cells overnight at 37 °C in 5% CO2 incubator to allow cells to adhere.
      Note: Cells become confluent on the following day. Two days incubation after seeding of 7 x 104 cells is also viable.
    4. Prepare 10-fold serial dilutions of virus in serum-free RPMI 1640.
    5. Remove culture supernatant of BHK-21 cells and add 200 μl of diluted virus into each well.
      Note: Virus should be added immediately after removing culture supernatant to avoid cells drying out.
    6. Incubate the plate for exactly 1 h at 37 °C in 5% CO2 incubator.
    7. Remove virus and add 500 μl of 0.8% methyl-cellulose medium supplemented with 2% FBS.
    8. Incubate plate for 4-5 days at 37 °C in 5% CO2 incubator.
      Note: The plaque size is affected by virus replication rate. Check the plaque size visually before fixation to obtain clear plaque morphology. Plates of EDEN1 infection are normally incubated until day 4 post-infection, whereas plates of EDEN2 or S221 are usually incubated until day 5 post-infection.
    9. Overlay 1 ml of 3.7% formaldehyde onto the cells and fix for 20 min at room temperature.
      Note: Alternatively, the plates can be put into a hermetic container containing 5 L of 3.7% formaldehyde and fixed for 20 min without removing methyl-cellulose medium. The formaldehyde solution can be kept for 3-4 months, however, longer time fixation times may be required if the 3.7% formaldehyde is not fresh. Once the solution turns brown and turbid, replace it.
    10. Rinse the plate with copious volume of water in a container. Shake the plate robustly to remove methyl-cellulose medium completely.
    11. Add 1-3 drops of 1% crystal violet into each well and stain for 2 min.
    12. Rinse the plate with copious volume of water in a container and shake the plate to remove excess water.
    13. Dry the plate out and count the number of plaques to determine virus titer.
      Caluculation of plaque forming units (pfu):
      Virus titer (pfu/ml) = average number of plaques/200 μl inoculum x 1,000 μl x dilution factor

  3. Preparation of α-DENV E protein antibodies (4G2) from hybridoma cells
    1. Culture 4G2 hybridoma cells in 50 ml of PFHM-II (Protein-Free hybridoma medium) in 175 cm2 flasks in humidified incubator with 5% CO2 atmosphere at 37 °C.
      Note: It is recommended to culture the cells in RPMI 1640 (10%) during the initial period of several days after thawing cells. Once the cells grow well, replace the media gradually by increasing the proportion of PFHM-II media in RPMI 1640 from 10% (vol/vol) to finally 100% PFHM-II media (example: 10%, 25%, 50%, 75% and 100%).
    2. Collect cell suspension into a centrifugal tube when the cells become confluent (the color of culture media turns to orange or yellow).
    3. Centrifuge cells at 900 x g for 5 min at room temperature.
    4. Collect the supernatant and stored at 4 °C without filtration until enough amount of supernatant is obtained.
    5. Continue culturing the cells and repeat steps C2-C4 if a large amount of the supernatant is required.
    6. Filter the supernatant through a 0.2 μm membrane filter unit.
    7. Load the 4G2 supernatant onto a 5 ml Protein G column pre-equilibrated in pH 7.2 PBS.
      Note: 4G2 antibody is purified using the AKTApurifier. Refer to the manufacturer’s instruction guides regarding sample loading specifications.
    8. Wash the column with PBS using 5x the column volume (i.e., 25 ml).
    9. Prepare a 96-well block containing 60 μl 1 M Tris-HCl.
      Note: The standard ratio of Tris-HCl to glycine (100:6) for neutralization is subjected to change depending on the concentration of buffers prepared. Volume of Tris-HCl to be added required for neutralization (pH 7) can be adjusted by pH paper testing.
    10. Elute antibodies using 100% 0.1 M glycine and collect 1 ml fractions into the wells of the block.
      Note: Check the purity of the antibody by running a SDS-PAGE.
    11. Select fractions of high purity and collect into a dialysis membrane, and then dialyse against PBS overnight.
    12. Quantitate the concentration of the purified antibody using NanoDrop.

  4. DENV infection in AG129 mice and measurement of viremia
    Note: Infectivity of DENV in AG129 mice differs among virus strains. DENV2 clinical strains are generally more infectious than DENV1 clinical strains in the mice (our unpublished observation). Infection in the presence of DENV-specific antibodies (ADE condition) can enhance the infection level and make it possible to induce mortality. Here we describe the methods for two successful mouse models of clinical isolates of DENV1 (EDEN1 strain) and DENV2 (EDEN2 strain) (Watanabe et al., 2016) together with the pioneering mouse model using mouse-adapted DENV2 (S221 strain) (Zellweger et al., 2010). 
    1. Prepare AG129 mice aged 7-11 weeks.
      Note: AG129 mice were purchased from B&K Universal (UK) and the breeding pairs were maintained under specific pathogen-free (SPF) conditions in the animal facility. One female AG129 mouse bears approximately 4 pups per month, therefore, maintainance of 10-breeding pairs produce about 40 mice per month.
    2. Dilute purified 4G2 with PBS to concentrations of 50 μg/100 μl for EDEN1 or EDEN2 infection and 10 μg/100 μl for S221 infection.
    3. Administer 100 μl of diluted 4G2 into AG129 mice intraperitoneally using a 30 G insulin syringe one day prior to virus infection.
    4. Dilute virus stock with PBS to concentrations of 7 x 107 pfu/200 μl for EDEN1, 1 x 108 pfu/200 μl for EDEN2 and 2 x 104 pfu/200 μl for S221. Keep the diluted virus on ice.
      Note: Inoculation of these virus titer induces lethal infection in the presence of 4G2 antibodies (ADE condition) but not in the absence of 4G2.
    5. Hold a mouse steadily in a mouse restrainer. Inoculate 200 μl of virus inoculum intravenously into mice through the tail vein using 30 G insulin syringe.
      Note:Up to 1 ml of virus can be inoculated intravenouslly into mice. Thus, as far as the titer of virus stock exceeds 1 x 108 pfu/ml, enough virus can be delivered into the mice for lethal infection.
    6. Observe mouse disease status and survival rate until day 10 post-infection.
      Note: Mice usually succumb to infection by day 5 post-infection accompanied with a severe form of human DHF/DSS-like diseases (Zellweger et al., 2010; Watanabe et al., 2016).
    7. Add 10 μl of 1% (wt/vol) EDTA (in PBS) into each 1.5 ml Eppendorf tube for blood collection. Hold the mouse vertically with the head tilted back and collect approximately 100 μl of blood from facial vein using a 27 G needle (final 0.1% EDTA).
      Note: It is possible to collect 100 μl of blood from each mouse on 10 consecutive days.
    8. Centrifuge the blood samples at 13,500 x g for 3 min.
    9. Collect serum and store at -80 °C until use.
    10. Collect 20 μl of each serum sample and mix with 120 μl PBS.
    11. Extract RNA from serum using QIAamp Viral RNA Mini Kit according to the manufacturer’s instruction.
    12. Make 20 μl reaction mixture (4 μl RNA plus 16 μl PCR reagent mixture) using the qScript One-Step qRT-PCR Kit. Prepare standard curves with plasmid DNA ranging from 107 to 101 copies/4 µl. Perform quantitative RT-PCR reactions in duplicate in Bio-Rad Real-time thermal cycler CFX96 according to the manufacturer’s instruction.
    13. Run the qPCR using the following program

Data analysis

Significant differences of viremia between data groups can be determined by a 2-tailed Student’s t-test analysis using online free software. For mouse survival, statistical analysis can be performed by the log-rank test using the GraphPad Prism software. P values less than 0.05 are considered significant.

Notes

This protocol was optimized for the two DENV clinical strains (EDEN1 and EDEN2). The protocol can be adapted to other clinical isolates including DENV serotype 3 (our unpublished data). However, not all clinical isolates can induce high mortality in AG129 mice due to their different infectivity to the mice.

Representative data



Figure 1. Bright field image of C6/36 cells showing ~90% confluency that is ready for DENV infection. The image was taken under 10x magnification using Olympus inverted fluorescence microscope.


Figure 2. Infection models of EDEN1, EDEN2 and S221 in AG129 mice. AG129 mice were inoculated intravenously with EDEN1 (7 x 107 pfu), EDEN2 (1 x 108 pfu) or S221 (2 x 104 pfu) for non-lethal infection. For the lethal ADE infection, mice were pre-injected intraperitoneally with 50 μg (for EDEN1 and EDEN2) or 10 μg (for S221) 4G2 antibody one day prior to infection. A. Mouse survival rate was monitored by day 10 post-infection. B. Blood samples were collected on days 1-8 post-infection and mixed serum from each group was subjected to real-time RT-PCR to obtain the average viral genome copy numbers. Mouse number per group is 5 (EDEN1 infection) or 6 (EDEN2 and S221 infection).
Note: Inoculation of virus alone does not induce mortality (A), whereas inoculation of virus in the presence of 4G2 causes 100% mortality (A) accompanied with enhanced peak viremia levels (B). These mouse models are applicable to in vivo evaluation of antiviral agents against not only mouse-adapted dengue virus but also dengue clinical strains (Watanabe et al., 2016).

Recipes

  1. 0.8% methyl-cellulose medium
    1. Add 8 g methyl-cellulose powder into 500 ml double distilled water, autoclave twice to dissolve the powder
    2. Prepare 500 ml 2x RPMI 1640 media by dissolving RPMI 1640 powder in double distilled water followed by supplementing with 4% heat-inactivated FBS, 4 mM L-glutamine, 200 U/ml penicillin and streptomycin, 0.075% sodium bicarbonate solution and 50 mM HEPES
    3. After filtration of 2x RPMI 1640 media through 0.2 μm membrane filter unit, mix well with 500 ml prepared methyl-cellulose
    4. Store at 4 °C
  2. 1% Crystal violet
    Add 5 g Crystal violet to 100 ml 100% EtOH and mix well to dissolve powder
    Add 400 ml double distilled water
    Store at room temperature
  3. 0.1 M glycine (pH 2.7)
    Add 3.75 g glycine into 500 ml double distilled water
    Adjust the pH to 2.7 using 1 N HCl
    Store the solution at 4 °C
  4. 1 M Tris-HCl (pH 9.0)
    Add 30.3 g of Tris into 250 ml double distilled water
    Adjust the pH to 9.0 using 1 N HCl
    Store the solution at room temperature

Acknowledgments

This work was supported by the Ministry of Health in Singapore through NMRC/MOHIAFCat1/0008/2014. The protocol described here was based on the following paper: Watanabe et al. (2016).

References

  1. Low, J. G., Ooi, E. E., Tolfvenstam, T., Leo, Y. S., Hibberd, M. L., Ng, L. C., Lai, Y. L., Yap, G. S., Li, C. S., Vasudevan, S. G. and Ong, A. (2006). Early Dengue infection and outcome study (EDEN) - study design and preliminary findings. Ann Acad Med Singapore 35(11): 783-789.
  2. Shresta, S., Kyle, J. L., Snider, H. M., Basavapatna, M., Beatty, P. R. and Harris, E. (2004). Interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas T- and B-cell-dependent immunity are less critical.. J Virol 78(6): 2701-2710.
  3. Shresta, S., Sharar, K. L., Prigozhin, D. M., Beatty, P. R. and Harris, E. (2006). Murine model for dengue virus-induced lethal disease with increased vascular permeability. J Virol 80(20): 10208-10217.
  4. Watanabe, S., Chan, K. W., Dow, G., Ooi, E. E., Low, J. G. and Vasudevan, S. G. (2016). Optimizing celgosivir therapy in mouse models of dengue virus infection of serotypes 1 and 2: The search for a window for potential therapeutic efficacy. Antiviral Res 127: 10-19.
  5. Zellweger, R. M., Prestwood, T. R. and Shresta, S. (2010). Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe 7(2): 128-139.

简介

登革热是由4种相关登革热病毒血清型(DENV1-4)的任一种感染引起的全球公共卫生威胁。临床表现的范围从自限性发热疾病,称为登革热(DF),到危及生命的严重疾病,如登革热出血热(DHF)或登革热休克综合征(DSS)。大多数DHF/DSS病例通过描述为抗体依赖性增强感染(ADE)的现象与继发性异型感染相关。估计每年有4亿人感染和数十万例严重登革热病例。然而,目前,还没有批准的抗DENV感染的抗病毒药物。缺乏合适的动物模型阻碍了DENV感染的新抗病毒候选物的评价。由于DENV在免疫活性小鼠中不良地建立感染,已将AG129小鼠(缺乏I型和II型IFN [干扰素]受体)和小鼠适应的DENV2株应用于能够繁殖人类感染的几种主要病理的登革动物模型。最近,我们开发了具有临床分离株DENV1和DENV2的新的小鼠模型,其将用于药物测试和登革热发病机理研究(Watanabe等人,2016)。在这里我们描述建立临床分离株的登革热小鼠模型的细节;从体外材料制备到体内病毒感染。值得注意的是,由于DENV在小鼠中的感染性在病毒株之间不同,不是所有临床分离株都可以诱导严重的登革热。
关键字:登革热病毒,致命的小鼠模型,临床病毒,病毒感染的抗体依赖性增强,药物测试

[背景] 为了克服DENV在啮齿动物细胞中不能很好复制的缺点,多年来已经进行了许多努力来开发模拟人类登革热感染的小动物模型。近交小鼠模型系统允许实验可变性最小化,并且遗传工程小鼠模型能够再现动物中登革热临床症状的一些方面。过去的研究显示,用DENV2临床分离物感染的AG129小鼠(缺乏I型和II型IFN受体)感染瘫痪的迹象,这是中枢神经系统受累的病症,在人类病例中是罕见的(Shresta等人。,2004)。或者,产生可在AG129小鼠中诱导人类DHF/DSS样疾病的小鼠适应的DENV2毒株,并已用于登革热研究(Shresta等人,2006; Zellweger等人。,2010)。虽然小鼠适应株的使用对于DENV发病机理研究和潜在治疗药物测试的一些方面是有价值的,但是一个相当大的限制是取决于血清型/基因型的可变发病机理,并且小鼠中病毒的适应可改变组织嗜性。最近,我们开发了新的临床分离株DENV1和DENV2的小鼠模型;小鼠死于具有严重登革热症状的体征的感染(Watanabe等人,2016)。在AG129小鼠中,在DENV抗体(Abs)(ADE条件)存在下,临床分离物的非致死性感染变得致死,伴随着高水平的病毒血症和细胞因子产生,表明该系统能够扩展临床DENV分离物研究Ab介导的DENV发病机制和抗登革热候选物的评价

关键字:登革热病毒, 致死性小鼠模型, 临床病毒, 病毒感染的抗体依赖性增强, 药物试验

材料和试剂

  1. 50ml离心管(Corning,Falcon ,目录号:357550)
  2. 0.45μm膜过滤器(Sartorius,Minisart ,目录号:16537)
  3. 用于大量培养的0.2μm膜过滤单元(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:567-0020)
  4. HiTrap Protein G HP-5ml(GE Healthcare,目录号:170-0405-01)
  5. 96孔PCR板(Bio-Rad Laboratories,目录号:HSP9601)
  6. Snakeskin TM透析管10kDa(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:68100)
  7. 30G胰岛素注射器(BD,目录号:328818)
  8. 27 G针(BD,目录号:305109)
  9. 1.5ml Eppendorf管(Corning,Axygen,目录号:MCT-150-c)
  10. 板盖密封(Bio-Rad Laboratories,目录号:MSB1001)
  11. 用于冷冻病毒的CryoTubes小瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:368632)
  12. 登革热病毒:D​​ENV-2小鼠适应的S221株(Zellweger等人,2010),DENV-1临床分离株(EDEN1:GenBank登录号
    (2006)),DENV2临床分离株(EDEN2:GenBank登录号EU081177.1)(Low等人,2006) >
  13. 白纹伊蚊白纹伊蚊C6/36细胞系(克隆C6/36)(ATCC,目录号:CRL-1660 TM )的细胞
  14. 幼仓鼠肾细胞系(BHK-21 [C-13])(ATCC,目录号:CCL-10 TM )的细胞
  15. 杂交瘤细胞(D1-4G2-15)(ATCC,目录号:HB-112 TM
  16. 缺乏I型和II型IFN受体的Sv/129小鼠(AG129小鼠)(B& K Universal)
  17. RPMI1640培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11875093)
  18. 热灭活的胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10082147)
  19. 液氮
  20. 3.7%甲醛(Sigma-Aldrich,目录号:F1635)
  21. 结晶紫(Sigma-Aldrich,目录号:C3886)
    注意:此产品已停产。
  22. 无蛋白杂交瘤培养基(PFHM-II培养基)(Thermo Fisher Scientific,Gibco TM ,目录号:12040077)
  23. 1x PBS(Lonza,目录号:17-516Q)
  24. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  25. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25200056)
  26. 乙二胺四乙酸(EDTA)(EMD Millipore,目录号:819040)
  27. QIAamp病毒RNA小试剂盒(Qiagen,目录号:52906)
  28. qScript一步qRT-PCR试剂盒(Quantabio,目录号:95057)
  29. 甲基纤维素粉末(EMD Millipore,目录号:17851)
  30. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  31. 青霉素和链霉素(PenStrep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  32. 7.5%碳酸氢钠溶液(Thermo Fisher Scientific,Gibco< sup>,目录号:25080094)。
  33. 1 M HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  34. 乙醇(EtOH)(EMD Millipore,目录号:1009832511)
  35. RPMI 1640粉末(Thermo Fisher Scientific,Gibco TM ,目录号:31800022)
  36. 1N HCl(Sigma-Aldrich,目录号:258148)
  37. Tris(First BASE Laboratories Sdn Bhd,目录号:BIO-1400)
  38. 引物和探针
    DENV1正向引物:5'-ACACCAGGGGCTGTACCTTGG-3'
    DENV1反向引物:5'-CATTCCATTTTCTGGCGTTCT-3'
    DENV1 taqman探针:FAM-5'-CTGTCTCTACAGCATCATTCCAGGCA-3'-TAMRA
    DENV2正向引物:5'-CATATTGACGCTGGGAAAGA-3'
    DENV2反向引物:5'-AGAACCTGTTGATTCAAC-3'
    DENV2 taqman探针:FAM-5'-CTGTCTCCTCAGCATCATTCCAGGCA-3'-TAMRA
  39. 实时RT-PCR的标准:含有DENV-1(EDEN1:EU081230.1)或DENV-2(EDEN2:EU081177.1)的全基因组序列的质粒
  40. 0.8%甲基纤维素培养基(参见配方)
  41. 1%结晶紫(见配方)
  42. 0.1 M甘氨酸(pH 2.7)(参见配方)
  43. 1 M Tris-HCl(pH 9.0)(参见配方)

设备

  1. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:159920)的微波反应器中进行反应。
  2. 细胞刮刀(Corning,目录号:3010)
  3. 在28℃下没有CO 2气氛的孵育器(SANYO,型号:MIR-262)
  4. 在37℃下具有5%CO 2气氛的加湿培养箱(NuAire,型号:NU5500)
  5. 摆动转子离心机(用于细胞)(Thermo Fisher Scientific,型号:Heraeus Multifuge 3S-R)
  6. 台式固定角转子离心机(用于血清)(Eppendorf,型号:5424)
  7. -80℃冷冻器(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Forma 900系列)
  8. 高压灭菌器(TOMY DIGITAL BIOLOGY,型号:SX-700)
  9. AKTApurifier TM UPC 10(GE Healthcare,目录号:28406268)
  10. pH计(Satorius,型号:pH Basic系列)
  11. NanoDrop 2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:ND-2000)
  12. 实时热循环仪(Bio-Rad Laboratories,型号:CFX96)
  13. 鼠标限制器(Plas-labs,目录号:551-BSRR)
  14. Olympus倒置荧光显微镜(Olympus,型号:IX71)

软件

  1. GraphPad Prism软件

程序

  1. 生成DENV股票
    注意:需要高病毒滴度的临床DENV毒株来诱导AG129小鼠中的严重疾病。从C6/36细胞获得的病毒滴度在病毒株中不同。因此,需要优化培养条件,例如病毒接种物的感染复数(MOI)或病毒收集的时间,以获得每种病毒株的足够的病毒滴度。
    1. 在补充有10%FBS,2mM L-谷氨酰胺,100U/ml青霉素和链霉素和25mM HEPES的RPMI 1640培养基中维持C6/36细胞。在28℃下在非CO 2气氛条件下孵育细胞。
      注意:如果C6/36细胞不期望增殖,则补充有20%FBS的生长培养基改善细胞生长。
    2. 在175cm 2烧瓶中在培养基中生长C6/36细胞直至〜90%融合(参见图1)。
    3. 解冻病毒储液,用无血清RPMI 1640培养基稀释。从培养瓶中弃去培养基,并加入5ml的病毒接种物,其MOI为0.1(对于EDEN1或S221)或1(对于EDEN2)至175cm 2烧瓶中。 注意:在融合时,在T175烧瓶中存在约3×10 7个细胞,因此对于MOI 0.1-1,3×10 6/6×3×10 7/> pfu 是必需的(有关如何确定病毒滴度,请参阅程序B)。
    4. 在28℃下在非CO 2气氛条件下孵育1小时
    5. 取出病毒接种物并向175cm 2烧瓶中加入25ml RPMI 1640培养基(2%FBS)。
    6. 在28℃下在非CO 2气氛条件下孵育4-7天。
      注意:根据病毒株观察到细胞病变效应(在EDEN2感染中观察到,但在EDEN1或S221感染中未观察到)。 在不同时间点检查上清液中的病毒滴度以确定获得高病毒滴度的最佳天数(参见如何确定病毒滴度的程序B)。 EDEN2和S221的孵育期为5天,EDEN1的孵育期为7天。
    7. 刮细胞并转移到离心管中,然后在4℃下以1,800×g离心细胞10分钟。
    8. 收集上清液并通过0.45μm膜滤器转移到新管中
    9. 将病毒等分到冷冻管中,并储存在-80°C冰箱或液氮中,直到使用。
      注意:建议将病毒储存液储存在液氮中长期储存。

  2. 通过噬斑测定确定病毒滴度
    1. 在37℃下,在具有5%CO 2气氛的潮湿培养箱中,在补充有10%FBS,2mM L-谷氨酰胺和100U/ml青霉素和链霉素的RPMI 1640中保持BHK-21细胞。 br />
    2. 将2×10 5个细胞/孔的细胞以500μl接种到24孔板中。
    3. 在37℃在5%CO 2培养箱中孵育细胞过夜,以允许细胞粘附。
      注意:细胞在第二天变得汇合。在接种7×10 4个细胞后培养两天也是可行的。
    4. 在无血清RPMI 1640中制备10倍系列稀释的病毒。
    5. 去除BHK-21细胞的培养物上清液,并向每个孔中加入200μl稀释的病毒 注意:除去培养物上清液后应立即加入病毒,以避免细胞变干。
    6. 在37℃下在5%CO 2培养箱中孵育板1小时。
    7. 取出病毒,加入500μl0.8%甲基纤维素培养基,补充2%FBS
    8. 在37℃下在5%CO 2培养箱中孵育4-5天 注意:斑块大小受病毒复制速率的影响。在固定之前目视检查斑块大小以获得清晰的斑块形态。通常孵育EDEN1感染的板直至感染后第4天,而通常孵育EDEN2或S221感染板直至感染后第5天。
    9. 覆盖1毫升3.7%甲醛的细胞,并在室温下固定20分钟 注意:或者,可以将板放入装有5L 3.7%甲醛的密封容器中,并固定2​​0分钟,而不除去甲基纤维素介质。甲醛溶液可以保持3-4个月,然而,如果3.7%的甲醛不是新鲜的,则可能需要更长的时间。一旦溶液变成棕色和浑浊,请更换。
    10. 在容器中用大量的水冲洗板。强力摇动板以完全除去甲基纤维素培养基。
    11. 向每个孔中加入1-3滴1%结晶紫,并染色2分钟
    12. 在容器中用大量的水冲洗板,摇动板以除去多余的水
    13. 干燥平板并计数噬菌斑的数目以确定病毒滴度。
      斑块形成单位(pfu)的计算:
      病毒滴度(pfu/ml)=平均噬菌斑数/200μl接种物×1,000μl×稀释因子

  3. 从杂交瘤细胞制备α-DENV E蛋白抗体(4G2)
    1. 在37℃,5%CO 2气氛的湿润培养箱中,在175cm 2烧瓶中的50ml PFHM-II(无蛋白质的杂交瘤培养基)中培养4G2杂交瘤细胞。
      注意:建议在解冻细胞后的几天的初始阶段,在RPMI 1640(10%)中培养细胞。 一旦细胞生长良好,通过将RPMI 1640中PFHM-II培养基的比例从10%[vol/vol]逐渐增加到最终100%PFHM- II媒体(例如:10%,25%,50%,75%和100%)。
    2. 当细胞变成融合时(培养基的颜色变成橙色或黄色),将细胞悬浮液收集到离心管中。
    3. 在室温下以900×g离心细胞5分钟
    4. 收集上清液并在4℃下储存,不过滤,直到获得足够量的上清液
    5. 继续培养细胞,如果需要大量上清液,重复步骤C2-C4
    6. 用0.2μm膜过滤器过滤上清液
    7. 将4G2上清液加载到在pH 7.2 PBS中预平衡的5ml蛋白G柱上 注意:使用AKTA纯化器纯化4G2抗体。有关样品加载 规格,请参阅制造商的说明指南。
    8. 使用5倍柱体积(,,25 ml),用PBS洗涤柱子。
    9. 准备含有60μl1M Tris-HCl的96孔板 注意:取决于所制备的缓冲液的浓度,用于中和的Tris-HCl与甘氨酸(100:6)的标准比例发生变化。通过pH 纸张测试可以调节中和所需的添加的Tris-HCl体积(pH7)。
    10. 使用100%0.1M甘氨酸洗脱抗体,并将1ml级分收集到块的孔中 注意:通过运行SDS-PAGE检查抗体的纯度。
    11. 选择高纯度级分,收集到透析膜中,然后用PBS透析过夜
    12. 使用NanoDrop定量纯化的抗体的浓度
  4. 在AG129小鼠中的DENV感染和病毒血症的测量 te:AG129小鼠中DENV的感染性在病毒株中不同。 DENV2临床菌株通常比小鼠中的DE1/NV1临床菌株更具感染性(我们未发表的观察)。在DENV特异性抗体(ADE阳性)存在下的感染可以增强感染水平,并使得诱导死亡成为可能。在这里,我们描述了DENV1(EDEN1毒株)和DENV2(EDEN2毒株)(EDEN2毒株)的临床分离物的两种 c 使用小鼠适应的DENV2(S221菌株)(Zellweger等人,2010),使用开创性小鼠模型,在小鼠模型中(Watanabe等人,2016)t/oeg//em>
    1. 准备年龄7-11周的AG129小鼠 注意:AG129小鼠购自B& K Universal(UK),并且在动物设施中在特定无病原体(SPF)条件下维持育种对。一个雌性AG129小鼠每月大约有4只幼仔,因此,10个育种对的保持每月产生大约40只小鼠。
    2. 用PBS稀释纯化的4G2,使EDEN1或EDEN2感染的浓度为50μg/100μl,S221感染的浓度为10μg/100μl。
    3. 在病毒感染前一天使用30 G胰岛素注射器给AG129小鼠腹膜内施用100μl稀释的4G2。
    4. 用PBS稀释病毒储液,使EDEN1浓度为7×10 7 pfu /200μl,EDEN2浓度为1×10 8 pfu /200μl,EDEN2为2×10 6 pfu/4 pfu /200μl。将稀释的病毒置于冰上。
      注意:这些病毒滴度的接种在4G2抗体(ADE条件)存在下诱导致死性感染,但不在 不存在4G2。
    5. 将鼠标稳定地放在鼠标限制器中。使用30 G胰岛素注射器通过尾静脉将200μl病毒接种物静脉内接种到小鼠中 > 病毒可以静脉内接种到小鼠中。因此,只要病毒原液的滴度超过1×10 8/pfu/pfu/ml,足够的病毒可以被递送到小鼠中用于致死感染。 br />
    6. 观察小鼠的疾病状态和存活率,直到感染后第10天 注意:小鼠通常在感染后5天感染后伴有严重形式的人类DHF/DSS样疾病(Zellweger等人,2010; Watanabe等人,2016 )。
    7. 加入10μl的1%(wt/vol)EDTA(在PBS中)到每个1.5ml Eppendorf管中以收集血液。垂直握住鼠标,头部向后倾斜,使用27 G针头(最终0.1%EDTA)从面部静脉收集大约100μl的血液。
      注意:连续10天可以从每只老鼠收集100μl血液。
    8. 将血液样品以13,500×g离心3分钟。
    9. 收集血清并储存于-80℃直至使用。
    10. 收集每个血清样品20μl,并与120μlPBS混合
    11. 使用QIAamp病毒RNA小试剂盒,根据制造商的说明从血清提取RNA
    12. 使用qScript一步qRT-PCR试剂盒制备20μl反应混合物(4μlRNA加16μlPCR试剂混合物)。用范围从10 7到10 10拷贝/4μl的质粒DNA制备标准曲线。在Bio-Rad实时热循环仪CFX96中根据制造商的说明重复进行定量RT-PCR反应。
    13. 使用以下程序运行qPCR

数据分析

数据组之间的病毒血症的显着差异可以通过使用在线自由软件的双尾学生动物试验分析来确定。对于小鼠存活,可以使用GraphPad Prism软件通过对数秩检验进行统计分析。 P 值小于0.05被认为是显着的。

笔记

该方案针对两种DENV临床菌株(EDEN1和EDEN2)进行了优化。该方案可适用于其他临床分离物,包括DENV血清型3(我们未发表的数据)。然而,不是所有的临床分离株可以诱导AG129小鼠高死亡率,因为他们对小鼠的不同的感染性。

代表数据



图1.使用Olympus倒置荧光显微镜在10x放大率下拍摄的图像。图6:显示约90%融合的C6/36细胞的明场图像,其准备用于DENV感染。

图2.AG129小鼠中EDEN1,EDEN2和S221的感染模型 AG129小鼠静脉内接种EDEN1(7×10 7 pfu),EDEN2(1×10 7 pfu)或对于非致死性感染的S221(2×10 4 pfu)。对于致死性ADE感染,在感染前一天向小鼠腹膜内注射50μg(对于EDEN1和EDEN2)或10μg(对于S221)4G2抗体。 A.通过感染后第10天监测小鼠存活率。 B.在感染后第1-8天收集血液样品,并将来自每组的混合血清进行实时RT-PCR以获得平均病毒基因组拷贝数。每组的小鼠数目为5(EDEN1感染)或6(EDEN2和S221感染)。
注意:单独接种病毒不会诱导死亡率(A),而在4G2存在下接种病毒导致100%死亡率(A)伴随增加的病毒血症峰值(B)。这些小鼠模型适用于不仅针对小鼠适应的登革热病毒而且针对登革热临床株的抗病毒剂的体内评价(Watanabe等人,2016)。

食谱

  1. 0.8%甲基纤维素培养基
    1. 将8g甲基纤维素粉末加入500ml双蒸水中,高压灭菌两次以溶解粉末
    2. 通过将RPMI 1640粉末溶解于双蒸水中,随后补充4%热灭活的FBS,4mM L-谷氨酰胺,200U/ml青霉素和链霉素,0.075%碳酸氢钠溶液和50mM HEPES
    3. 在通过0.2μm膜过滤器单元过滤2x RPMI 1640培养基之后,与500ml制备的甲基纤维素混合
    4. 存储在4°C
  2. 1%结晶紫
    加入5克结晶紫至100毫升100%乙醇,充分混合溶解粉末
    加入400 ml双蒸水
    在室温下贮存
  3. 0.1M甘氨酸(pH 2.7)
    将3.75g甘氨酸加入500ml双蒸水中 用1N HCl将pH调节至2.7 将溶液储存在4℃下
  4. 1 M Tris-HCl(pH 9.0)
    将30.3g Tris加入250ml双蒸水
    中 用1N HCl将pH调节至9.0 将溶液在室温下储存

致谢

这项工作由新加坡卫生部通过NMRC/MOHIAFCat1/0008/2014支持。本文所述的方案基于以下文献:Watanabe等人。 (2016年)。

参考文献

  1. 低,JG,Ooi,EE,Tolfvenstam,T.,Leo,YS,Hibberd,ML,Ng,LC,Lai,YL,Yap,GS,Li,CS,Vasudevan,SG和Ong, ; 早期登革热感染和结局研究(EDEN) - 研究设计和初步结果。 35(11):783-789
  2. Shresta,S.,Kyle,J.L.,Snider,H.M.,Basavapatna,M.,Beatty,P.R.and Harris,E。(2004)。 干扰素依赖性免疫对于抵抗原发性登革热病毒感染至关重要小鼠,而T和B细胞依赖性免疫不太关键。 J Virol 78(6):2701-2710。
  3. Shresta,S.,Sharar,KL,Prigozhin,DM,Beatty,PR和Harris,E。(2006)。  登革热病毒诱导的致死性疾病的鼠模型具有增加的血管通透性。 J Virol 80(20):10208-10217。 br />
  4. Watanabe,S.,Chan,KW,Dow,G.,Ooi,EE,Low,JG和Vasudevan,SG(2016)。  在抗体诱导的严重登革热病的小鼠模型中增强肝脏窦状内皮细胞的感染。细胞宿主微生物 7(2):128-139。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Watanabe, S., Chan, K. K. and Vasudevan, S. G. (2016). Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates. Bio-protocol 6(23): e2040. DOI: 10.21769/BioProtoc.2040.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。