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Hepatitis E virus (HEV) is one of the main causes of acute hepatitis worldwide. Infections are particularly severe in pregnant women and chronic hepatitis E is known to occur in immunocompromised patients. Current therapy (ribavirin or pegylated alpha interferon) has severe side effects and cannot be employed in all patients. In order to evaluate potential new inhibitors of HEV replication, a virus yield assay can be employed in which the amount of viral RNA progeny released into the culture medium is quantified by reverse-transcription quantitative PCR (RT-qPCR) (Debing et al., 2014).

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Infectious Virus Yield Assay for Hepatitis E Virus
戊型肝炎病毒的感染病毒产量分析

微生物学 > 微生物-宿主相互作用 > 病毒
作者: Yannick Debing
Yannick DebingAffiliation: Microbiology and Immunology Department, University of Leuven, Leuven, Belgium
Bio-protocol author page: a1544
Kai Dallmeier
Kai DallmeierAffiliation: Microbiology and Immunology Department, University of Leuven, Leuven, Belgium
Bio-protocol author page: a1545
 and Johan Neyts
Johan NeytsAffiliation: Microbiology and Immunology Department, University of Leuven, Leuven, Belgium
For correspondence: johan.neyts@rega.kuleuven.be
Bio-protocol author page: a1546
Vol 4, Iss 15, 8/5/2014, 3527 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1195

[Abstract] Hepatitis E virus (HEV) is one of the main causes of acute hepatitis worldwide. Infections are particularly severe in pregnant women and chronic hepatitis E is known to occur in immunocompromised patients. Current therapy (ribavirin or pegylated alpha interferon) has severe side effects and cannot be employed in all patients. In order to evaluate potential new inhibitors of HEV replication, a virus yield assay can be employed in which the amount of viral RNA progeny released into the culture medium is quantified by reverse-transcription quantitative PCR (RT-qPCR) (Debing et al., 2014).

Keywords: Hepatitis E virus(戊型肝炎病毒), Antiviral assay(抗病毒活性测定), RT-qPCR(RT-qPCR), Inhibitor(抑制剂), Cell culture(细胞培养)

[Abstract]

Materials and Reagents

  1. HepG2/C3A human hepatoma cell line (ATCC, catalog number: CRL-10741 )
  2. Huh7 human hepatoma cell line (Japanese Collection of Research Bioresources, catalog number: JCRB0403 )
  3. HEV Kernow-C1 p6 plasmid (genotype 3 full-length genome; a kind gift from Suzanne Emerson, NIH) (Shukla et al., 2012)
  4. MluI restriction endonuclease with accompanying 10x buffer D (Promega Corporation, catalog number: R6381 )
  5. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
  6. T7 RiboMAX large scale RNA production system (Promega Corporation, catalog number: P1300 )
  7. RNeasy mini kit (QIAGEN, catalog number: 74104 )
  8. ScriptCap m7G capping system (CELLSCRIPTTM, catalog number: C-SCCE0610 )
  9. Anti-fungal agent (2.5 µg/ml, Fungizone) (Life Technologies, catalog number: 15290-018 )
  10. Dulbecco’s modified Eagle’s medium with high glucose (DMEM) (Life Technologies, catalog number: 41965-039 )
  11. Fetal bovine serum (FBS) (not heat-inactivated) (Life Technologies, catalog number: 10270-106 )
  12. Opti-MEM I reduced serum medium (Life Technologies, catalog number: 31985-062 )
  13. Lipofectin transfection reagent (Life Technologies, catalog number: 18292-011 )
  14. Dulbecco’s phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Life Technologies, catalog number: 14190-094 )
  15. Penicillin/Streptomycin (PS) (10,000 IU/ml) (Life Technologies, catalog number: 15140-148 )
  16. CellTiter 96 AQueous MTS reagent powder [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega corporation, catalog number: G1111 )
  17. Phenazine methosulphate (PMS) (Sigma-Aldrich, catalog number: P9625 )
  18. Minimum essential medium (MEM) (no glutamine, no phenol red) (Life Technologies, catalog number: 51200-046 )
  19. NucleoSpin RNA virus kit (MACHEREY-NAGEL, catalog number: 740956 )
  20. One step qRT-PCR MasterMix Low Rox for probe assays (Kaneka Corporation, Eurogentec, catalog number: RT-QPRT-032XLR )
  21. Forward primer (5’-GGTGGTTTCTGGGGTGAC-3’) and reverse primer (5’- AGGGGTTGGTTGGATGAA-3’) (custom order) (Integrated DNA Technologies) (dissolved to a final concentration of 10 µM in buffer TE)
  22. Fluorescent probe (5’-FAM-TGATTCTCAGCCCTTCGC-MGBNFQ-3’ with FAM, 6-carboxyfluorescein; MGBNFQ, minor groove binder non-fluorescent quencher) (Life Technologies, custom order) (dissolved to a final concentration of 10 µM in buffer TE)
  23. qPCR DNA standard
    The cloned cDNA target sequence is ligated into a plasmid (e.g. with the CloneJet PCR cloning kit, Thermo Fisher Scientific, catalog number: K1231 ) and miniprepped.
    Note: The concentration is determined by spectrophotometry and a logarithmic dilution series is made in buffer TE over 6 orders of magnitude. Alternatively, the original Kernow-C1 p6 plasmid can be used to make such a dilution series.
  24. Tris (hydroxymethyl) aminomethane powder (Trizma base) (Sigma-Aldrich, catalog number: T1503 ) (dissolved to 1 M and pH 8.0 in H2O)
  25. Ethylenediaminetetraacetic acid disodium salt solution (EDTA, 0.5 M) (Sigma-Aldrich, catalog number: E7889 )
  26. MTS/PMS solution (see Recipes)
  27. Buffer TE (see Recipes)

Equipment

  1. Spectrophotometer to determine RNA concentrations (e.g. Thermo Fischer Scientific, NanoDrop, model: ND-1000 )
  2. Falcon transparent 6-well plate (Corning Incorporated, catalog number: 353046 )
  3. Falcon transparent 96-well plate (Corning Incorporated, catalog number: 353072 )
  4. 37 °C and 35 °C 5% CO2 cell culture incubators
  5. Centrifuge with temperature control
  6. Saphire² microplate reader for absorbance (Tecan Trading AG)
  7. PCR plate 96-well (SARSTEDT AG, catalog number: 72.1981.202 )
  8. MicroAmp optical adhesive film (Life Technologies, catalog number: 4311971 )
  9. ABI 7500 fast real-time PCR system (Life Technologies, catalog number: 4351107 )

Procedure

  1. Production of an infectious HEV stock from plasmid
    1. Five µg of the Kernow-C1 p6 plasmid is linearized in a 100 µl reaction containing 10 µl of 10x buffer D and 2.5 µl of MluI (10 units/µl) which is incubated at 37 °C for 2 h.
    2. The digested DNA is purified using the Qiaquick gel extraction kit according to the manufacturer’s instructions with final elution in 30 µl of buffer EB.
      Note: To increase the yield, add the recommended amount of isopropanol to the mixture of the reaction and buffer QG. Other methods of DNA purification should be suitable as well.
    3. An in vitro transcription reaction is prepared with the T7 RiboMAX large scale RNA production system:
       - 10 µl of 5x T7 transcription buffer
       - 15 µl of NTPs (ATP, GTP, CTP, UTP; each at 25 mM)
       -  20 µl of DNA template
       -  5 µl of enzyme mix
      Reaction is incubated at 37 °C for 5 h. Afterwards, 5 µl of the supplied RQ1 RNase-free DNase solution is added and incubated at 37 °C for 15 min.
    4. The produced RNA is purified with the RNeasy mini kit according to the manufacturer’s instructions with elution in 60 µl of RNase-free H2O (in 2 elutions of 30 µl each). RNA concentration is determined by spectroscopy.
    5. Sixty µg of RNA is capped with the ScriptCap m7G capping system according to the manufacturer’s instructions.
    6. The produced RNA is purified with the RNeasy mini kit according to the manufacturer’s instructions with elution in 60 µl of RNase-free H2O (in 2 elutions of 30 µl each). RNA concentration is determined by spectroscopy.
    7. Huh7 cells are seeded into a transparent 6-well plate at 2 x 105 cells per well in 2 ml of DMEM supplemented with 10% FBS.
    8. Plate is incubated in a 5% CO2 incubator at 37 °C for 24 h.
    9. To transfect one well, 1 µg of capped viral RNA is added to 100 µl of Opti-MEM. In another tube, 10 µl of Lipofectin transfection reagent is added to 100 µl of Opti-MEM. Both tubes are incubated at room temperature for 30 min. Then, the contents of both tubes are combined and incubated for another 10 min at room temperature. An additional 800 µl of Opti-MEM is added and the resulting solution is mixed well.
    10. The culture medium is carefully removed from cells seeded earlier by aspiration with a pipette and each well is washed once with 2 ml of DMEM (i.e. DMEM is added to the well, incubated for a few seconds while gently rocking the plate and removed by aspiration).
    11. To each well, 1 ml of transfection mixture is added.
    12. Plate is incubated in a 5% CO2 incubator at 37 °C for 5 h.
    13. Transfection medium is removed from each well and cell layers are washed once with 2 ml of PBS (see step A10).
    14. To each well, 2.5 ml of DMEM supplemented with 10% FBS and 1% PS is added.
      Note: Optionally, amphotericin B can be added to the culture medium as an anti-fungal agent.
    15. Plate is incubated in a 5% CO2 incubator at 35 °C.
    16. Every 2-3 days (3x per week), 1 ml of culture medium is removed from each well and replaced with 1 ml of fresh DMEM supplemented with 10% FBS and 1% PS.
      Note: These partial changes of the medium are essential to maintain cell viability over the extended incubation period.
    17. Twenty days post transfection, the culture medium is collected and centrifuged for 10 min at 1,000 x g at 4 °C. The supernatant is aliquoted and stored at -80 °C.
      Note: The supernatant can be used directly as a viral stock or can be passaged once on HepG2/C3A cells for upscaling (similar to the protocol below). If required, the number of HEV RNA copies can be quantified by RT-qPCR as described below. In this case, a short RNase treatment is recommended prior to RNA extraction to remove remaining input RNA (the progeny viral RNA will be protected by the viral capsid).

  2. Infectious virus yield assay
    1. HepG2/C3A cells are seeded into a transparent 6-well plate at 2 x 105 cells per well in 2 ml of DMEM supplemented with 10% FBS.
    2. Plate is incubated in a 5% CO2 incubator at 37 °C for 24 h.
    3. The HEV stock is thawed rapidly at 37 °C and diluted 1: 10 (or to the desired concentration) in DMEM supplemented with 10% FBS and 1% PS. The potential antiviral molecule is added to the inoculum, an equivalent amount of solvent (e.g. dimethylsulfoxide) is added to another well to serve as a cell control.
      Note: Since HEV replication in vitro is rather poor and a high multiplicity of infection is required for a successful infection, the virus stock is not diluted very strongly. Addition of compound to the inoculum allows to evaluate effects on early events in the viral life cycle.
    4. Medium is removed from the HepG2/C3A cells and 1 ml of diluted HEV inoculum is added.
    5. Plate is incubated in a 5% CO2 incubator at 35 °C for 5 h.
    6. Inoculum is removed from each well and cell layers are washed 3 times with 2 ml of PBS.
    7. To each well, 2.5 ml of DMEM supplemented with 10% FBS and 1% PS is added. Compound is added to the desired concentration and an equivalent amount of solvent is added to the cell control well(s).
    8. Plate is incubated in a 5% CO2 incubator at 35 °C.
      Note: Optionally, 150 µl of culture medium can be removed from each well after 1 h and stored at -80 °C for later RNA extraction and analysis. This sample allows to determine the baseline viral load.
    9. Every 2-3 days (3x per week), 1 ml of culture medium is removed from each well, transferred to a 1.5 ml tube and stored at -80 °C for later RNA extraction and analysis. The removed medium is replaced with 1 ml of fresh DMEM supplemented with 10% FBS and 1% PS and compound or control solvent. Plate is incubated further in a 5% CO2 incubator at 35 °C.
    10. Twenty days post transfection, the last medium samples are transferred to 1.5 ml tubes and the rest of the culture medium is removed.
    11. Cell layers are washed with 2 ml of PBS and 1 ml of a 1: 20 dilution of MTS/PMS solution in colorless MEM is added to each well.
    12. Plate is incubated in a 5% CO2 incubator at 37 °C for 2 h.
    13. One hundred µl of culture medium from each well is transferred to a transparent 96-well plate and absorbance read-out is performed in the microplate absorbance reader by determining the optical density (OD) at 498 nm.
    14. The remaining diluted MTS/PMS solution is removed from each well and cell layers are washed once with 2 ml of PBS.
    15. To each well, 350 µl of buffer RLT (included in the RNeasy mini kit) is added for cell lysis.
    16. Cell lysates are resuspended in buffer RLT, transferred to a 1.5 ml tube and vortexed vigorously for 1 min.
    17. RNA is extracted from cell lysates with the RNeasy mini kit according to the manufacturer’s instructions with final elution in 30 µl of RNase-free H2O.
      Note: Alternatively, cell lysates can be stored at -80 °C after vortexing for extraction at a later time point.
    18. Total RNA concentration of extracted intracellular RNA is measured spectrophotometrically.
    19. To extract viral RNA from culture medium samples, samples are thawed at room temperature and viral RNA is extracted from 150 µl of culture medium with the Nucleospin RNA virus kit according to the manufacturer’s instructions.
      Note: All culture medium samples can be extracted or only those of the preferred time points (e.g. the baseline and day 20 samples).
    20. For quantification of viral RNA, the following reaction mixture is made (for analysis of a single sample):
      -    12.5 µl of One step qRT-PCR MasterMix
      -    0.0625 µl of forward primer at 100 µM (final concentration: 250 nM)
      -    0.0625 µl of reverse primer at 100 µM (final concentration: 250 nM)
      -    0.25 µl of probe at 10 µM (final concentration: 100 nM)
      -    7.0625 µl of RNase-free H2O
      -    0.125 µl of Euroscript/RNase inhibitor
      Note: Multiply the indicated amounts and volumes by the number of samples and cDNA samples to analyze and include an additional 10% extra.
    21. Twenty µl from this mixture is dispensed into each well of a PCR plate and 5 µl of RNA extract is added.
    22. To allow for absolute quantification, 5 µl of the dilution series of cloned cDNA targets is added to wells containing 20 µl of reaction mixture. In a last well, 5 µl of H2O is added to 20 µl reaction mixture (negative control).
    23. PCR plate is covered with an adhesive transparent cover. Plate is centrifuged shortly.
    24. RT-qPCR is performed with following temperature program:
      - 30 min 48 °C
      - 10 min 95 °C
      - 15”      95 °C    |   
      - 1 min  60 °C    |  x40
      With the detector preset to FAM (520 nm) and data collection during the combined annealing-elongation step at 60 °C.

Representative data

  1. A representative example of the standard curve generated for the HEV RT-qPCR is shown below:


    Figure 1. Representative example of the HEV RT-qPCR standard curve. This standard curve was generated using a series of 10-fold dilutions of the cloned target cDNA.

  2. An example of data generated by the method described here can be found below. Known HEV replication inhibitors ribavirin (RBV) and alpha interferon (IFNα) were tested (GE = genome equivalents, adapted from Debing et al., 2014).


    Figure 2. Antiviral activities of interferon alpha (IFNα) and different concentrations of ribavirin (RBV) were assessed in Huh7 cells using the infectious virus yield assay with RT-qPCR detection of viral RNA. *, P < 0.05; **, P < 0.01. GE, genome equivalents. Adapted from Debing et al. (2014).

Notes

  1. In general, Huh7 cells are more permissive to transfection than HepG2/C3A cells, while the latter are infected more efficiently and thus result in higher yields after infection. However, it is possible to transfect and infect both cell lines with the protocol outlined above, although differences in efficiency and yield may be observed.
  2. The quantities obtained by RT-qPCR are based on the concentrations of the standard dilution series and are thus expressed in ng/µl. Using the molecular weight of the plasmid standard and Avogadros number, the number of copies can be calculated. For intracellular RNA, it is advised to normalize the obtained copy number for the total cellular RNA (as determined spectrophotometrically) or for a house-keeping gene which mRNA levels are unaffected by HEV infection or compound treatment.
  3. To calculate cell viability, the following formula can be employed:
    % Viability = ODcompound/ODCC x 100

Recipes

  1. MTS/PMS solution
    1. Two g of MTS powder is dissolved in 1 L of PBS and stirred for 15 min.
    2. Next, 46 mg of PMS powder is added (pH should be between 6-6.5.).
    3. Solution is filtered, aliquoted and stored at -20 °C.
    Note: Since MTS is light-sensitive, the powder and solution should be protected from light.
  2. Buffer TE
    To 25 ml of nuclease-free H2O, add
    250 µl of a 1 M Tris solution (pH 8.0) (final concentration: 10 mM)
    50 µl of a 0.5 M EDTA solution (final concentration: 1 mM)

Acknowledgments

This protocol was adapted from Debing et al. (2014) and is partially based on earlier work by Shukla et al. (2012). Primer and probe sequences are derived from Jothikumar et al. (2006). Yannick Debing is a fellow of the Research Foundation-Flanders (FWO). This work was supported by KU Leuven Geconcerteerde Onderzoeksacties (GOA/10/014) and by EU FP7 project SILVER (260644).

References

  1. Debing, Y., Emerson, S. U., Wang, Y., Pan, Q., Balzarini, J., Dallmeier, K., Neyts, J. (2014). Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon. Antimicrob Agents Chemother 58(1): 267-273.
  2. Jothikumar, N., Cromeans, T.L., Robertson, B.H., Meng, X.J., Hill, V.R. (2006) A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 131(1): 65-71.
  3. Shukla, P., Nguyen, H.T., Faulk, K., Mather, K., Torian, U., Engle, R.E., Emerson, S.U. (2012) Adaptation of a genotype 3 hepatitis E virus to efficient growth in cell culture depends on an inserted human gene segment acquired by recombination. J Virol 86(10): 5697-5707.

材料和试剂

  1. HepG2/C3A人肝癌细胞系(ATCC,目录号:CRL-10741)
  2. Huh7人肝癌细胞系(Japanese Collection of Research Bioresources,目录号:JCRB0403)
  3. HEV Kernow-C1 p6质粒(基因型3全长基因组;来自Suzanne Emerson,NIH的赠品)(Shukla等人,2012)
  4. MluI限制性内切酶与伴随的10x缓冲液D(Promega Corporation,目录号:R6381)
  5. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  6. T7 RiboMAX大规模RNA生产系统(Promega Corporation,目录号:P1300)
  7. RNeasy迷你试剂盒(QIAGEN,目录号:74104)
  8. ScriptCap m7G加盖系统(CELLSCRIPT TM ,目录号:C-SCCE0610)
  9. 抗真菌剂(2.5μg/ml,Fungizone)(Life Technologies,目录号:15290-018)
  10. 具有高葡萄糖的Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,目录号:41965-039)
  11. 胎牛血清(FBS)(非热灭活)(Life Technologies,目录号:10270-106)
  12. Opti-MEM I还原血清培养基(Life Technologies,目录号:31985-062)
  13. Lipofectin转染试剂(Life Technologies,目录号:18292-011)
  14. 不含Ca 2+和Mg 2+的Dulbecco's磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190-094)
  15. 青霉素/链霉素(PS)(10,000IU/ml)(Life Technologies,目录号:15140-148)
  16. CellTiter 96 AQueous MTS试剂粉末[3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑](Promega公司,目录号:G1111)
  17. 吩嗪硫酸甲酯(PMS)(Sigma-Aldrich,目录号:P9625)
  18. 最低必需培养基(MEM)(无谷氨酰胺,无酚红)(Life Technologies,目录号:51200-046)
  19. NucleoSpin RNA病毒试剂盒(MACHEREY-NAGEL,目录号:740956)
  20. 用于探针测定的一步qRT-PCR MasterMix Low Rox(Kaneka Corporation,Eurogentec,目录号:RT-QPRT-032XLR)
  21. 正向引物(5'-GGTGGTTTCTGGGGTGAC-3')和反向引物(5'- AGGGGTTGGTTGGATGAA-3')(定制顺序)(Integrated DNA Technologies)(在缓冲液TE中溶解至终浓度为10μM)
  22. 荧光探针(Life Technologies,定制顺序)(在缓冲液TE中溶解至终浓度为10μM)的荧光探针(具有FAM,6-羧基荧光素的5'- FAM-TGATTCTCAGCCCTTCGC-MGBNFQ-3'; MGBNFQ,小沟结合剂非荧光淬灭剂) )
  23. qPCR DNA标准
    将克隆的cDNA靶序列连接到质粒(例如,用CloneJet PCR克隆试剂盒,Thermo Fisher Scientific,目录号:K1231)中并微量制备。
    注意:通过分光光度法测定浓度,并在缓冲液TE中在6个数量级上进行对数稀释系列。或者,可以使用原始的Kernow-C1 p6质粒制备这样的稀释系列。
  24. 三(羟甲基)氨基甲烷粉末(Trizma碱)(Sigma-Aldrich,目录号:T1503)(溶解至1M,pH 8.0,在H 2 O中)
  25. 乙二胺四乙酸二钠盐溶液(EDTA,0.5M)(Sigma-Aldrich,目录号:E7889)
  26. MTS/PMS解决方案(参见配方)
  27. 缓冲TE(参见配方)

设备

  1. 用于测定RNA浓度的分光光度计(例如Thermo Fischer Scientific,NanoDrop,型号:ND-1000)
  2. Falcon透明6孔板(Corning Incorporated,目录号:353046)
  3. Falcon透明96孔板(Corning Incorporated,目录号:353072)
  4. 37℃和35℃5%CO 2细胞培养孵化器
  5. 带温度控制的离心机
  6. Saphire²酶标仪(Tecan Trading AG)
  7. PCR板96孔(SARSTEDT AG,目录号:72.1981.202)
  8. MicroAmp光学粘合膜(Life Technologies,目录号:4311971)
  9. ABI 7500快速实时PCR系统(Life Technologies,目录号:4351107)

程序

  1. 从质粒生产感染性HEV原种
    1. 将5μgKernow-C1 p6质粒在含有10μl10×缓冲液D和2.5μlMluI(10单位/μl)的100μl反应物中线性化,其在37℃温育2小时。
    2. 使用Qiaquick凝胶提取试剂盒根据制造商的说明纯化消化的DNA,在30μl缓冲液EB中最终洗脱。
      注意:为了提高产量,向反应和缓冲液QG的混合物中加入推荐量的异丙醇。其他DNA纯化方法也应该适用。
    3. 使用T7 RiboMAX大规模RNA生产系统制备体外转录反应:
        - 10μl5x T7转录缓冲液
      - 15μlNTP(ATP,GTP,CTP,UTP;每种为25mM)
        -   20μlDNA模板
        -   5μl酶混合物
      将反应在37℃下孵育5小时。之后,加入5μl提供的RQ1 RNase-free DNase溶液,并在37℃下孵育15分钟。
    4. 根据制造商的说明书,用RNeasy微型试剂盒纯化所产生的RNA,在60μl无RNA酶的H 2 O(在2个30μl每份中的洗脱液)中洗脱。通过光谱法测定RNA浓度
    5. 根据制造商的说明书,用ScriptCap m7G帽系统覆盖60μg的RNA
    6. 根据制造商的说明书,用RNeasy微型试剂盒纯化所产生的RNA,在60μl无RNA酶的H 2 O(在2个30μl每份中的洗脱液)中洗脱。通过光谱法测定RNA浓度
    7. 将Huh7细胞以2×10 5个细胞/孔接种在2ml补充有10%FBS的DMEM中的透明6孔板中。
    8. 将板在5%CO 2培养箱中在37℃孵育24小时
    9. 为了转染一个孔,将1μg加帽的病毒RNA加入到100μlOpti-MEM中。在另一个管中,将10μl的Lipofectin转染试剂加入到100μl的Opti-MEM中。将两管在室温下温育30分钟。然后,将两个管的内容物合并,并在室温下再温育10分钟。再加入800μlOpti-MEM,所得溶液充分混合
    10. 通过用移液管抽吸而从先前接种的细胞中小心地除去培养基,并用2ml DMEM(每孔)将每个孔洗涤一次。将DMEM加入孔中,温育几秒钟,同时温和摇动板并通过吸气除去)
    11. 向每个孔中加入1ml转染混合物
    12. 将板在5%CO 2培养箱中在37℃下孵育5小时
    13. 从每个孔中除去转染培养基,并用2ml PBS将细胞层洗涤一次(见步骤A10)。
    14. 向每个孔中加入2.5ml补充有10%FBS和1%PS的DMEM 注意:任选地,可将两性霉素B作为抗真菌剂加入培养基中。
    15. 将板在35℃的5%CO 2培养箱中温育
    16. 每2-3天(每周3次),从每个孔中取出1ml培养基,并用1ml补充有10%FBS和1%PS的新鲜DMEM替换。 注意:培养基的这些部分改变对于在延长的培养期内维持细胞活力是必要的。
    17. 转染后20天,收集培养基并在4℃下以1,000×g离心10分钟。将上清液等分并储存在-80℃ 注意:上清液可以直接用作病毒原种,或者可以在HepG2/C3A细胞上传代一次用于升级(类似于以下方案)。如果需要,HEV RNA拷贝数可以通过如下所述的RT-qPCR进行定量。在这种情况下,建议在RNA提取之前进行短的RNA酶处理以除去剩余的输入RNA(后代病毒RNA将被病毒衣壳保护)。

  2. 传染病毒产量测定
    1. 将HepG2/C3A细胞以2×10 5个细胞/孔接种在2ml补充有10%FBS的DMEM中的透明6孔板中。
    2. 将板在5%CO 2培养箱中在37℃孵育24小时
    3. 将HEV原液在37℃下快速解冻,并在补充有10%FBS和1%PS的DMEM中以1:10(或至所需浓度)稀释。将潜在的抗病毒分子加入到接种物中,将等量的溶剂(例如二甲基亚砜)加入另一个孔中作为细胞对照。
      注意:由于HEV在体外复制相当差并且成功感染需要高的感染复数,所以病毒储液不会被非常强烈地稀释。向接种物中加入化合物可以评估对病毒生命周期中早期事件的影响。
    4. 从HepG2/C3A细胞中除去培养基,并加入1ml稀释的HEV接种物
    5. 将板在5%CO 2培养箱中在35℃温育5小时
    6. 从每个孔中取出接种物,并用2ml PBS洗涤细胞层3次
    7. 向每个孔中加入2.5ml补充有10%FBS和1%PS的DMEM。加入化合物至所需浓度,并将等量的溶剂加入到细胞对照孔中
    8. 将板在35℃的5%CO 2培养箱中温育 注意:任选地,可以在1小时后从每个孔中取出150μl培养基,并储存在-80℃下用于随后的RNA提取和分析。此样本允许确定基线病毒载量。
    9. 每2-3天(每周3次),从每个孔中取出1ml培养基,转移到1.5ml管中,并储存在-80℃用于随后的RNA提取和分析。用1ml补充有10%FBS和1%PS和化合物或对照溶剂的新鲜DMEM替换除去的培养基。将板在5%CO 2培养箱中在35℃下进一步孵育
    10. 转染后20天,将最后培养基样品转移到1.5ml管中,并除去其余培养基。
    11. 用2ml PBS洗涤细胞层,向每个孔中加入1ml 1:20稀释的MTS/PMS无色MEM溶液。
    12. 将板在5%CO 2培养箱中在37℃下孵育2小时
    13. 将来自每个孔的100μl培养基转移到透明的96-孔板中,通过测定在498nm的光密度(OD)在微孔板吸光度读数器中进行吸光度读数。
    14. 从每个孔中除去剩余的稀释的MTS/PMS溶液,并用2ml PBS洗涤细胞层一次
    15. 向每个孔中加入350μl缓冲液RLT(包含在RNeasy mini试剂盒中)用于细胞裂解。
    16. 将细胞裂解物重悬于缓冲液RLT中,转移至1.5ml管中并剧烈涡旋1分钟。
    17. 使用RNeasy mini试剂盒根据制造商的说明书从细胞裂解物中提取RNA,在30μl无RNA酶的H 2 O中最终洗脱。
      注意:或者,细胞裂解物可以在-80℃下保存,然后在以后的时间点涡旋提取。
    18. 通过分光光度法测量提取的细胞内RNA的总RNA浓度
    19. 为了从培养基样品中提取病毒RNA,将样品在室温下解冻,并根据制造商的说明书用Nucleospin RNA病毒试剂盒从150μl培养基中提取病毒RNA。
      注意:可以提取所有培养基样品,或只提取优选时间点(例如基线和第20天样品)。
    20. 为了定量病毒RNA,制备以下反应混合物(用于分析单个样品):
      -      12.5μl一步式qRT-PCR MasterMix
      -     0.0625μl100μM的正向引物(终浓度:250nM)
      -     0.0625μl100μM反向引物(终浓度:250nM)
      -    &0.25μl10μM探针(最终浓度:100nM)
      -     7.0625μl无核糖核酸酶H 2 O ub /> -      0.125μlEuroscript/RNase抑制剂
      注意:将指定的量和体积乘以要分析的样品和cDNA样品的数量,并额外增加10%。
    21. 将来自该混合物的20μl分配到PCR板的每个孔中,并加入5μlRNA提取物
    22. 为了进行绝对定量,将5μl克隆的cDNA靶标的稀释系列加入到含有20μl反应混合物的孔中。在最后一个孔中,将5μlH 2 O加入到20μl反应混合物(阴性对照)中。
    23. PCR板用粘性透明盖覆盖。板很快离心。
    24. RT-qPCR用以下温度程序进行:
      - 30分钟48℃
      - 10分钟95℃
      - 15"      95°C    |   
      - 1分钟 60°C    |  x40
      将检测器预设为FAM(520nm),并在60℃的组合退火 - 延长步骤期间收集数据。

代表数据

  1. 用于HEV RT-qPCR产生的标准曲线的代表性实例如下所示:


    图1. HEV RT-qPCR标准曲线的代表性实例。使用一系列10倍稀释的克隆靶cDNA产生该标准曲线。

  2. 通过这里描述的方法产生的数据的示例可以在下面找到。已知的HEV复制抑制剂利巴韦林(RBV)和α干扰素(IFNα)(GE =基因组等同物,改编自Debing等人,2014)。


    图2.使用感染性病毒产量测定法,用病毒RNA的RT-qPCR检测,在Huh7细胞中评估干扰素α(IFNα)和不同浓度的利巴韦林(RBV)的抗病毒活性。 *,P < ; 0.05; **,P < 0.01。 GE,基因组等同物。改编自Debing 。 (2014)。

笔记

  1. 通常,Huh7细胞比HepG2/C3A细胞更容许转染,而后者被更有效地感染,因此导致感染后更高的产量。然而,可以转染和 用上述方案感染两种细胞系,尽管可以观察到效率和产量的差异
  2. 通过RT-qPCR获得的量基于标准稀释系列的浓度,因此以ng /μl表示。 使用质粒标准品的分子量和Avogadros数,可以计算拷贝数。 对于细胞内RNA,建议将所获得的总细胞RNA的拷贝数(分光光度法测定)或对于保持mRNA水平不受HEV感染或化合物处理影响的管家基因进行归一化。
  3. 为了计算细胞存活率,可以采用以下公式:
    %活力= OD化合物/OD CC×100

食谱

  1. MTS/PMS溶液
    1. 将2g MTS粉末溶解在1L PBS中并搅拌15分钟
    2. 接下来,加入46mg PMS粉末(pH应在6-6.5之间)
    3. 将溶液过滤,分装并储存在-20℃
    注意:由于MTS是光敏的,所以粉末和溶液应该避光。
  2. 缓冲区TE
    向25ml无核酸酶的H 2 O 2中加入
    250μl1M Tris溶液(pH8.0)(终浓度:10mM) 50μl0.5M EDTA溶液(终浓度:1mM)

致谢

该协议改编自Debing等人。 (2014),并且部分基于Shukla等人早期的工作。 (2012)。引物和探针序列来自Jothikumar等人。 (2006)。 Yannick Debing是研究基金会佛兰德斯(FWO)的研究员。这项工作由KU Leuven Geconcerteerde Onderzoeksacties(GOA/10/014)和欧盟FP7项目SILVER(260644)支持。

参考文献

  1. Debing,Y.,Emerson,S.U.,Wang,Y.,Pan,Q.,Balzarini,J.,Dallmeier,K.,Neyts,J。(2014)。 利巴韦林通过消耗细胞GTP库来抑制体外的艾滋病病毒复制[/em]并且与α干扰素具有中等协同作用。 Antimicrob Agents Chemother 58(1):267-273。
  2. Jothikumar,N.,Cromeans,T.L.,Robertson,B.H.,Meng,X.J.,Hill,V.R。 (2006)用于快速和灵敏检测的广泛反应性一步实时RT-PCR测定的病毒。病毒学方法 131(1):65-71。
  3. Shukla,P.,Nguyen,H.T.,Faulk,K.,Mather,K.,Torian,U.,Engle,R.E.,Emerson, (2012)基因型3戊型肝炎病毒适应于细胞培养物中的有效生长取决于插入 通过重组获得的人基因区段。 J Virol 86(10):5697-5707。
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How to cite this protocol: Debing, Y., Dallmeier, K. and Neyts, J. (2014). Infectious Virus Yield Assay for Hepatitis E Virus. Bio-protocol 4(15): e1195. DOI: 10.21769/BioProtoc.1195; Full Text



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