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Infection Experiments (Hepatitis C Virus)
感染实验(丙型肝炎病毒)   

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Abstract

The establishment of a cell culture system for hepatitis C virus based on the JFH-1 strain and human hepatoma cell lines has been instrumental for the study of the viral replication cycle. The robustness of the JFH1-based cell culture models enabled many laboratories around the world to perform HCV infections in cell culture, accelerating the identification of cellular and viral targets to develop novel antiviral compounds. Although other robust infection systems based on different molecular clones and different cell lines have been developed since then, here we describe the protocols corresponding to infections with JFH-1 and JFH1-derived viruses carried out in our laboratory to produce virus stocks and persistently infected cell cultures. We also describe the experimental setups used to determine virus spreading capacity (multiple cycle infections) as well as to dissect early and late aspects of HCV infection (single cycle infections).

Materials and Reagents

  1. Biological materials
    1. Human hepatoma cells Huh-7 (Nakabayashi et al., 1982) and derived subclone Huh-7.5.1 clone 2, hereafter clone 2 (Pedersen et al., 2007)
    2. Viruses: Genotype 2a JFH-1 strain (Zhong et al., 2005) and a cell culture-adapted JFH1 variant D183 (Zhong et al., 2006)
    3. Human monoclonal anti-E2 (AR3A) (antibody provided by Mansun Law, The Scripps Research Institute) (Law et al., 2008)
    4. Alexa Fluor 555 Goat Anti-Human IgG (H+L) (Alexa 555-conjugated antibody) (Life Technologies, InvitrogenTM, catalog number: A21433 )
    5. Fetal bovine serum (FBS) (LINUS, catalog number: 501805 )
    6. BSA (Roche Diagnostics, catalog number: 10 735 086 001 )

  2. Reagents
    1. 1 M HEPES (pH 7.4) (Sigma-Aldrich, catalog number: H3375-500G )
    2. 100x MEM nonessential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 )
    3. Penicillin/streptomycin (10,000 U/ ml) (Life Technologies, Gibco®, catalog number: 15140-122 )
    4. 0.5% trypsin/EDTA solution (10x) (Life Technologies, Gibco®, catalog number: 15400-054 )
    5. Prolong (Life Technologies, catalog number: P-36930 )
    6. Triton X-100 (Sigma-Aldrich, catalog number: T-8787 )
    7. Formaldehyde solution (37% wt% in H2O) (Sigma-Aldrich, catalog number: 252549 )
    8. 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: 32670 )
    9. Dulbecco´s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 ) (see Recipes)
    10. 10x phosphate buffered saline (PBS) (see Recipes)
    11. Immunofluorescence (IF) buffer (see Recipes)
    12. 4% formaldehyde solution (see Recipes)

Equipment

  1. CO2 incubator
  2. 1.5 ml safe-lock PCR clean microtubes (Eppendorf, catalog number: 0030 123 328 )
  3. Falcon 50 ml tubes (Falcon®, catalog number: 352098 )
  4. 75 cm2 cell culture flask (canted neck, 0.2 µM vent cap) (Corning, catalog number: 430641 )
  5. 162 cm2 traditional straight neck cell culture flask with vent cap (corning, catalog number: 3151 )
  6. 6 well culture cluster (flat bottom) (Corning, Costar®, catalog number 3506 )
  7. 12 well culture cluster (flat bottom) (Corning, Costar®, catalog number 3513 )
  8. 24 well culture cluster (flat bottom) (Corning, Costar®, catalog number 3527 )
  9. 96 well culture cluster (flat bottom) (Corning, Costar®, catalog number 3599 )
  10. 5 ml stripette (Corning, Costar®, catalog number 4487 )
  11. 10 ml stripette (Corning, Costar®, catalog number 4488 )
  12. 25 ml stripette(Corning, Costar®, catalog number 4489 )
  13. Pipet aid (IBS INTEGRA Biosciences, catalog number: 155000 )
  14. Pipetman p200 micropipette (GILSON®, catalog number: F123601 )
  15. Pipetman p1000 micropipette (GILSON®, catalog number: F123602 )
  16. Centrifuges (Hettich Zentrifugen, catalog number: EBA 12R )
  17. -20 °C freezer
  18. -80 °C freezer
  19. Fluorescence microscope (inverted fluorescence microscope with long distance objectives)
  20. Microscope slides and coverslips (Thermo Fisher Scientific, catalog number: 5425508 )
  21. Filter tips 10 µl (Sorenson Bioscience, catalog number: 35200 )
  22. Filter tips 20 µl (Sorenson Bioscience, catalog number: 35220 )
  23. Filter tips 200 µl (Sorenson Bioscience, catalog number: 35230 )
  24. Filter tips 1,000 µl (Sorenson Bioscience, catalog number: 35260 )

Procedure

Part I. Multiple cycle infections
Multiple cycle infections are also defined as low multiplicity of infection (MOI, i.e. number of infectious particles/cell) experiments. They are generally used to produce virus stocks and to generate persistently infected cell cultures. They also constitute a first approach to characterize the capacity of a virus to spread under different experimental conditions or to compare the spreading capacity of different viruses.

  1. Virus stock production
    Clone 2 cells are used to produce virus stocks because they yield higher infectivity titers in shorter incubation periods, as compared with parental Huh-7 cells.
    1. Seed 4.5 x 106 Clone 2 cells in 15 ml of complete DMEM in a T162 flask the day before infection.
    2. Incubate for 24 h in 5% CO2 at 37 °C.
    3. Dilute the starting virus (JFH-1 or D183) stock in complete DMEM (2 ml/well) to obtain a MOI of 0.01 FFU/cell. [Starting virus stocks are generated by electroporation of in vitro transcribed RNA as described in Zhong et al. (2005).]
    4. Inoculate by adding the virus dilution (2 ml/well) into the T162 flask.
    5. Incubate cells in 5% CO2 at 37 °C for 72 h.
    6. For D183 virus stocks proceed directly to step A13.
    7. For JFH-1 stocks, wash the cells once with warm PBS and discard.
    8. Add 1 ml of trypsin/EDTA solution per flask.
    9. Incubate for 3-5 min at 20-25 °C.
    10. Add 9 ml of complete DMEM and carefully resuspend the cells.
    11. Add 50 ml of complete DMEM and split cells into three T162 flasks (20 ml per flask).
    12. Incubate the cells for additional 72 h in 5% CO2 at 37 °C when producing a JFH-1 stock.
    13. Collect cell supernatants, centrifuge 1 min at 4,000 rpm at 20-25 °C. Collect supernatants and store at -80 °C in aliquots.
    14. Replenish cells with complete DMEM (20 ml per T162 flask).
    15. Incubate for 24 h in 5% CO2 at 37 °C.
    16. Repeat step A13 through 15 for two-three consecutive days.
    17. Determine infectivity titer of the samples collected in step A13 (see Part II).

  2. Generation of persistently infected cell cultures
    Persistently HCV infected Huh-7 cells are characterized by constant production of viral RNA, proteins and progeny infectious virus for prolonged periods of time. Although persistently infected cells can be cultured for several months, we recommend limiting the culture passages to less than 8 weeks to reduce the risk for undesired genetic drift of the viral and cell populations (Zhong et al., 2006). Persistent infections can only be established in Huh-7, as more permissive cell lines display severe cytopathology at the peak of infection (Zhong et al., 2006).
    1. Seed 2 x 106 Huh-7 cells in 10ml of complete DMEM in a T75 flask.
    2. Incubate for 24 h in 5% CO2 at 37 °C.
    3. Inoculate cells with JFH-1 at a MOI of 0.01 FFU/cell in a final volume of 10 ml per flask.
    4. Incubate for a minimum of 12-14 days in 5% CO2 at 37 °C.
    5. Maintain subconfluent cultures at all times by splitting the cultures every 3-4 days:
      1. Wash cells once with warm PBS and discard.
      2. Add 1 ml of trypsin/EDTA solution per flask.
      3. Incubate for 3-5 min at 20-25 °C.
      4. Add 5 ml of complete DMEM and resuspend cells in a final volume of 6 ml.
      5. Discard 4 ml of cell suspension and plate 2 ml into a T75 flask. Add 8 ml of complete DMEM.
    6. At days 12-14 post-inoculation, the cells will display a marked reduction in their proliferation rate. This occurs at the peak of infection but subsides in the following passages, where persistently infected Huh-7 cells are fully viable but display a slightly longer doubling time as compared with their uninfected counterparts (Zhong et al., 2006).
    7. Detach the cells from the flask as described in step B5.
    8. Collect 200 µl of cell suspension and plate it onto a sterile glass coverslip in a M12 plate well. Add 800 µl of complete DMEM and incubate for 24 h. Proceed with this sample to step B10.
    9. Seed the rest of the cells in a T162 flask. Proceed to step B15.
    10. Remove supernatant from step 8 and add a 4% formaldehyde solution in PBS (500 µl) onto the glass coverslip.
    11. Incubate for 20 min at 20-25 °C.
    12. Wash the cells twice with PBS (1 ml).
    13. Process the sample to perform immunofluorescence staining (see Part II, step 16).
    14. Determine the percentage of HCV antigen-positive cells by immunofluorescence microscopy. At day 12-14 post-inoculation nearly 100% of the cells should be HCV antigen-positive.
    15. Once infection reaches 100% of the cells, the persistently infected cell cultures are maintained subconfluent for a maximum of 8-10 weeks.
      In order to maintain subconfluent cultures, split them every 3-4 days as follow:
      1. Wash cells once with warm PBS and discard.
      2. Add 1 ml of trypsin/EDTA solution per flask.
      3. Incubate for 3-5 min at 20-25 °C.
      4. Add 5 ml of complete DMEM and resuspend cells in a final volume of 6 ml.
      5. Discard 4 ml of cell suspension and plate 2 ml into a T75 flask. Add 8 ml of complete DMEM.
    16. Samples of the cells and supernatants are collected according to the experimental design of each experiment (Gastaminza et al., 2008).

  3. Determining virus spreading capacity
    The following experimental setup enables determining the relative virus spread capacity under different experimental conditions, or to compare the overall growth capacity of different viruses (e.g. mutant viruses). In contrast to single cycle infections (see Part III), this experimental setup does not allow discriminating different aspects of the infection. Thus, determining extracellular progeny virus production is sufficient to characterize the virus spreading capacity. However, we propose collection of additional samples to monitor virus growth using alternative readouts.
    1. Seed 2 x 105 Huh-7 cells/well in a final volume of 2 ml of complete medium in a 6-well plates one day before inoculation.
    2. Incubate for 24 h in 5% CO2 at 37 °C.
    3. Inoculate cells with JFH-1 or D183 virus at a MOI of 0.01 FFU/ cell in a final volume of 2 ml per well.
    4. Incubate for 2-3 days in 5% CO2 at 37 °C until they reach 90% confluency.
    5. Collect supernatants and transfer to 1.5 ml tubes. Centrifuge 1 minute at 13,000 rpm. Transfer supernatants to clean 1.5 ml tubes. This sample is used to determine extracellular infectivity (see Part II).
    6. Wash cells once with warm PBS and discard.
    7. Add 300 µl of trypsin/EDTA solution per well.
    8. Incubate for 3-5 min at 20-25 °C.
    9. Add 600 µl of complete DMEM and resuspend the cells in a final volume of 900 µl.
    10. Plate 1:3 of the cells (300 µl) in a final volume of 2 ml/well (add 1.7 ml of complete DMEM/well to prepare the next time point of the experiment).
    11. Transfer 1:3 of the cells (300 µl) to a 1.5 ml tube. Pellet the cells at 3,000 rpm for 5 min at 4 °C. Extract total cellular RNA to determine intracellular HCV RNA levels (Mingorance et al., 2014).
    12. Transfer 1:3 of the cells (300 µl) to a 1.5 ml tube. Pellet the cells at 3,000 rpm for 10 min at 4 °C. Extract total cellular protein to determine viral antigen levels by western-blot (Mingorance et al., 2014).
    13. Incubate the cells from step C10 for 3 days in 5% CO2 at 37 °C.
    14. Repeat as in steps C5-13 until the end of the experiment. Typically sequences of days 2, 4, 6, 8 and 10 or days 3, 6, 9 and 12 should be sufficient to characterize JFH-1 virus spread in Huh-7 cells. In the case of D183, maximum titers are typically reached after day 5-6.

Part II. Virus titration by endpoint dilution and immunofluorescence
This procedure is based on the detection of infection foci (infected cell clusters) by immunofluorescence microscopy. Each infection focus is attributed to one infectious unit, defined as focus forming unit (FFU). The number of infection foci obtained by inoculation of naive cells with serial sample dilutions is used to determine the infectivity titer.

  1. Seed 104 Clone 2 cells/well in a final volume of 100 µl of complete DMEM in 96-well flat bottom plates. Four to eight wells/sample are required, depending on the expected infectivity titer. Sample replicates are strongly encouraged.
  2. Incubate for 24 h in 5% CO2 at 37 °C.
  3. Thaw the infectious samples at 20-25 °C.
  4. Add 200 µl of complete DMEM to a clean 96-well plate, using 4-8 wells per sample. Set the wells sequentially in a lane to accommodate the different sample dilutions.
  5. Add 50 µl of the sample onto the first well.
  6. Discard the tip.
  7. Homogenize virus-medium dilution with a new filter tip, being careful not to make bubbles.
  8. Take 50 µl of the mixture and carefully transfer into the next well.
  9. Repeat steps C5-7 until reaching the desired dilution factor.
  10. Remove the supernatant of the cells from step C1.
  11. Add 100 µl of each of the different virus dilutions onto each well.
  12. Incubate for 72 h in 5% CO2 at 37 °C.
  13. Discard cell supernatants.
  14. Add 150 µl of a 4% formaldehyde solution in PBS.
  15. Incubate for 20 min at 20-25 °C.
  16. Wash twice with PBS (200 µl).
  17. Add 30 µl/well of IF buffer.
  18. Incubate for 1 h at 20-25 °C.
  19. Prepare a 1 µg/ml dilution of the anti-E2 antibody AR3A in IF buffer.
  20. Discard IF buffer from the 96-well plate.
  21. Add 25 µl/well of the anti-E2 antibody dilution.
  22. Incubate for 1 h at 20-25 °C.
  23. Prepare a 1:500 dilution of the goat anti-human IgG-Alexa 555 antibody in IF buffer. Add DAPI (1 µg/ml in IF buffer).
  24. Wash the 96-well plate twice with PBS (200 µl).
  25. Add 25 µl/well of the secondary antibody dilution prepared as described in step 23.
  26. Incubate for 1 h at 20-25 °C.
  27. Wash the 96-well plate twice with PBS (200 µl).
  28. Add 50 µl of PBS to the test wells. In case of using a glass coverslip, mount onto a microscope slide using ProlongTM.
  29. Observe under the fluorescence microscope. Count infection foci only in dilutions where they are discrete and well separated from one another, until reaching negative wells (endpoint dilution).
  30. Calculate the infectivity titer in FFU/ml by, multiplying the number of infection foci found in the different dilutions by the corresponding dilution factor. Multiply by 10 to obtain the foci number per ml (100 µl were used for inoculation). Average the infectivity titers estimated in the different dilutions (typically discrete foci are only observed in 2-3 wells). It is highly recommended to repeat the experiment with different dilutions if the infectivity titer is not calculated out of 2-3 wells or if the endpoint dilution has not been reached.

Part III. Single cycle infection experiments
Single cycle infection experiments, also termed high multiplicity infections (MOI of 10 FFU/cell), attempt modeling synchronic infections of one virion infecting one target cell and are generally used to dissect early and late aspects of the hepatitis C virus life cycle. In contrast to multiple cycle infections, we recommend measuring different parameters such as intracellular and extracellular infectivity and RNA. Simultaneous analysis of these parameters in this experimental setup will enable determining the efficiency of different aspects of the infection. Since high infectivity titer virus stocks are required to perform these experiments and sufficient titers are difficult to reach with the parental JFH-1 strain, we typically use D183 virus stocks for these experiments (see Part I, section A).

  1. Seed 5 x 104 Huh-7 cells/well in a final volume of 500 µl of complete DMEM in 24-well plates the day before infection. Prepare a different plate per time point (see step 9).
  2. Incubate for 24 h in 5% CO2 at 37 °C.
  3. Remove medium and inoculate the cells with D183 virus at MOI of 10 FFU/cell in a final volume of 500 µl per well.
  4. Keep a sample of the inoculum in order to confirm the multiplicity of infection by infectivity titration (see Part II).
  5. Incubate for 5 h in 5% CO2 at 37 °C.
  6. Remove the inoculum and wash cells twice with warm PBS (1 ml).
  7. Add 500 µl per well of complete DMEM.
  8. Incubate in 5% CO2 at 37 °C. Typically peak titers are reached around 40 h post-infection. Thus, we recommend collecting samples at 24, 48 and 72 h post-infection. Collecting an additional sample at 5 h, will provide background levels of infectivity and HCV RNA that can be used as a baseline reference for the rest of the time points.
  9. Collect samples at 5, 24, 48 and 72 h:
    1. Collect supernatants to determine extracellular infectivity and HCV RNA. Store at -80 °C until analysis (Mingorance et al., 2014).
    2. Wash cells once with PBS (1 ml) and discard.
    3. Add trypsin/EDTA solution (100 µl per well).
    4. Incubate for 3-5 min at 20-25 °C.
    5. Resuspend cells in 500 µl of complete DMEM per well. Carefully homogenize to prepare a single-cell suspension.
    6. Transfer 500 µl of the cell suspension to a 1.5 ml tube to determine intracellular infectivity as described in (Gastaminza et al., 2008). Store at -80 °C until analysis.
    7. Collect 100 µl of the cell suspension in a 1.5 ml tube and process for RNA extraction. Alternatively, store at -80 °C.

Representative data



Figure 1. Immunofluorescence microscopy of Huh-7 cells infected with serial dilutions of infected cell supernatant and visualization of individual infection foci in cells inoculated with 1:10 diluted samples. Red channel anti-E2 antibody, blue channel cell nuclei stained with DAPI.

The typical yield of virus stocks should be around 104 FFU/ml for stocks generated with JFH-1 virus and >106 FFU/ml with D183 virus.
Persistently infected cell cultures should produce consistently titers >103 FFU/ml for the first month after reaching 100% infection.
Single cycle infection experiments should display peak extracellular infectivity titers >104 FFU/ml, typically 48 h post-infection.

Notes

  1. Manipulation of infectious recombinant hepatitis C virus requires specific biosafety conditions and infrastructure. Researchers are advised to revise the degree of biocontainment required by law in the country/region where research will be conducted.

Recipes

  1. Complete Dulbecco´s Modified Eagle´s Medium (complete DMEM)
    DMEM supplemented with 10 mM HEPES
    1x non-essential amino acids
    100 U/ ml penicillin/streptomycin and 10% fetal bovine serum (heat-inactivated at 56 °C for 30 min)
  2. 10x phosphate buffered saline (PBS)
    Dissolve 80 g NaCl, 2 g KCl, 26.8 g Na2HPO4-H2O and 2.4 g KH2PO4 in 800 ml H2O Adjust to pH 7.4 with HCl
    Adjust volume to 1 L with H2O
  3. Immunofluorescence (IF) buffer
    3% BSA
    0.3% TritonX-100 in PBS
  4. 1x trypsin/EDTA (10x) solution
    Dissolve 100 ml of 0.5% trypsin/EDTA (10x) solution in 900 ml 1x PBS
  5. 4% formaldehyde solution
    10.8 ml formaldehyde
    80 ml H2O and 10 ml 10x PBS

Acknowledgments

This protocol is based on work initially developed at Dr. Francis V. Chisari´s laboratory at The Scripps Research Institute (La Jolla, CA) in collaboration with Dr. Takaji Wakita´s group at the Tokyo Metropolitan Institute of Medical Science (Tokyo, Japan) and was originally generated and improved with the essential contribution of Jin Zhong (currently at Pasteur Institute in Shangai), Sharookh Kapadia (currently at Genentech), Guofeng Cheng (currently at Gilead), Susan Uprichard (currently at U. of Illinois, Chicago) and specially Stefan Wieland (currently at Basel University Hospital Basel).
L.M. was funded by a JAE-Pre fellowship from Consejo Superior de Investigaciones Científicas and CIBERehd (Instituto de Salud Carlos III). C.V. is funded by a Fundación “La Caixa”/CNB fellowship. This work was supported by the grants Plan Nacional De Investigación Científica, Desarrollo e Innovación Tecnológica from the Spanish Ministry of Science and Innovation (SAF2010-19270) and a Marie Curie Career Integration Grant (PCIG-9-GA-2011-293664) from the European Union 7th Framework Programme for Research.

References

  1. Friesland, M., Mingorance, L., Chung, J., Chisari, F. V. and Gastaminza, P. (2013). Sigma-1 receptor regulates early steps of viral RNA replication at the onset of hepatitis C virus infection. J Virol 87(11): 6377-6390.
  2. Gastaminza, P., Cheng, G., Wieland, S., Zhong, J., Liao, W. and Chisari, F. V. (2008). Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 82(5): 2120-2129.
  3. Gastaminza, P., Dryden, K. A., Boyd, B., Wood, M. R., Law, M., Yeager, M. and Chisari, F. V. (2010). Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture. J Virol 84(21): 10999-11009.
  4. Law, M., Maruyama, T., Lewis, J., Giang, E., Tarr, A. W., Stamataki, Z., Gastaminza, P., Chisari, F. V., Jones, I. M., Fox, R. I., Ball, J. K., McKeating, J. A., Kneteman, N. M. and Burton, D. R. (2008). Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 14(1): 25-27.
  5. Mingorance, L., Friesland, M., Coto-Llerena, M., Perez-del-Pulgar, S., Boix, L., Lopez-Oliva, J. M., Bruix, J., Forns, X. and Gastaminza, P. (2014). Selective inhibition of hepatitis C virus infection by hydroxyzine and benztropine. Antimicrob Agents Chemother 58(6): 3451-3460.
  6. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. and Sato, J. (1982). Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer research 42(9): 3858-3863.
  7. Pedersen, I. M., Cheng, G., Wieland, S., Volinia, S., Croce, C. M., Chisari, F. V. and David, M. (2007). Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 449(7164): 919-922.
  8. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Wieland, S. F., Uprichard, S. L., Wakita, T. and Chisari, F. V. (2005). Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102(26): 9294-9299.
  9. Zhong, J., Gastaminza, P., Chung, J., Stamataki, Z., Isogawa, M., Cheng, G., McKeating, J. A. and Chisari, F. V. (2006). Persistent hepatitis C virus infection in vitro: coevolution of virus and host. J Virol 80(22): 11082-11093.

简介

基于JFH-1毒株和人肝癌细胞系的丙型肝炎病毒的细胞培养系统的建立已经有助于研究病毒复制周期。 基于JFH1的细胞培养模型的鲁棒性使世界上许多实验室在细胞培养物中执行HCV感染,加速细胞和病毒靶标的鉴定以开发新的抗病毒化合物。 虽然基于不同的分子克隆和不同的细胞系的其他强大的感染系统已经发展到今天,在这里我们描述了对应的感染JFH-1和JFH1衍生的病毒在我们的实验室生产病毒库存和持续感染细胞 文化。 我们还描述了用于确定病毒传播能力(多周期感染)以及解剖早期和晚期HCV感染(单周期感染)的实验设置。

材料和试剂

  1. 生物材料
    1. 人肝癌细胞Huh-7(Nakabayashi et al。,1982)并衍生 亚克隆Huh-7.5.1克隆2,以下称为克隆2(Pedersen等人,2007)
    2. 病毒:基因型2a JFH-1株(Zhong等人,2005)和细胞培养物适应的JFH1变体D183(Zhong et al。,2006) >
    3. 人单克隆抗E2(AR3A)(抗体由Mansun Law,The Scripps Research Institute提供)(Law等人,2008)
    4. Alexa Fluor 555山羊抗人IgG(H + L)(Alexa 555-缀合的 抗体)(Life Technologies,Invitrogen TM ,目录号:A21433)
    5. 胎牛血清(FBS)(LINUS,目录号:501805)
    6. BSA(Roche Diagnostics,目录号:10 735 086 001)

  2. 试剂
    1. 1 M HEPES(pH7.4)(Sigma-Aldrich,目录号:H3375-500G)
    2. 100x MEM非必需氨基酸(Life Technologies,Gibco ,目录号:11140-050)
    3. 青霉素/链霉素(10,000U/ml)(Life Technologies,Gibco ,目录号:15140-122)
    4. 0.5%胰蛋白酶/EDTA溶液(10x)(Life Technologies,Gibco ,目录号:15400-054)
    5. Prolong(Life Technologies,目录号:P-36930)
    6. Triton X-100(Sigma-Aldrich,目录号:T-8787)
    7. 甲醛溶液(37%wt%,在H 2 O中)(Sigma-Aldrich,目录号:252549)
    8. (DAPI)(Sigma-Aldrich,目录号:32670)
    9. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:41965-039)(参见Recipes)
    10. 10x磷酸盐缓冲盐水(PBS)(见Recipes)
    11. 免疫荧光(IF)缓冲液(参见配方)
    12. 4%甲醛溶液(见配方)

设备

  1. CO <2>孵化器
  2. 1.5ml安全锁PCR干净微管(Eppendorf,目录号:0030 123 328)
  3. Falcon 50ml管(Falcon ,目录号:352098)
  4. 75cm 2细胞培养瓶(斜面颈部,0.2μM通气帽)(Corning,目录号:430641)
  5. 具有通气帽(corning,目录号:3151)的162cm 2传统直颈细胞培养瓶
  6. 6孔培养物簇(平底)(Corning,Costar ,目录号3506)
  7. 12孔培养物簇(平底)(Corning,Costar ,目录号3513)
  8. 24孔培养物簇(平底)(Corning,Costar ,目录号3527)
  9. 96孔培养物簇(平底)(Corning,Costar ,目录号3599)
  10. 5ml条带(Corning,Costar ,目录号4487)
  11. (Corning,Costar ,目录号4488)
  12. 25ml条带(Corning,Costar ,目录号4489)
  13. 吸移助剂(IBS INTEGRA Biosciences,目录号:155000)
  14. Pipetman p200微量移液管(GILSON ,目录号:F123601)
  15. Pipetman p1000微量移液管(GILSON ,目录号:F123602)
  16. 离心机(Hettich Zentrifugen,目录号:EBA 12R)
  17. -20°C冰箱
  18. -80°C冰箱
  19. 荧光显微镜(具有长距离物镜的倒置荧光显微镜)
  20. 显微镜载玻片和盖玻片(Thermo Fisher Scientific,目录号:5425508)
  21. 过滤嘴10μl(Sorenson Bioscience,目录号:35200)
  22. 过滤嘴20μl(Sorenson Bioscience,目录号:35220)
  23. 过滤嘴200μl(Sorenson Bioscience,目录号:35230)
  24. 过滤嘴1000μl(Sorenson Bioscience,目录号:35260)

程序

多发性周期感染
多循环感染也定义为低感染复数(MOI,即感染性颗粒/细胞数)实验。 它们通常用于产生病毒储液和产生持续感染的细胞培养物。 它们还构成了表征病毒在不同实验条件下扩散的能力或比较不同病毒的扩散能力的第一种方法。

  1. 病毒库存生产
    克隆2细胞用于产生病毒 因为它们在更短的时间内产生更高的感染性效价 潜伏期,与亲本Huh-7细胞相比
    1. 在感染前一天,在T162烧瓶中,将4.5×10 6个克隆2细胞接种在15ml完全DMEM中。
    2. 在37℃下在5%CO 2中孵育24小时
    3. 稀释起始病毒(JFH-1或D183)股票在完全DMEM(2 ml /孔)以获得0.01FFU /细胞的MOI。 [起始病毒库存 通过体外转录的RNA的电穿孔产生   Zhong et al。(2005)。]
    4. 通过将病毒稀释液(2ml /孔)加入T162烧瓶中接种
    5. 在37℃下将细胞在5%CO 2中孵育72小时
    6. 对于D183病毒株,直接进行步骤A13
    7. 对于JFH-1股票,用温PBS洗涤细胞一次并丢弃
    8. 每瓶加入1ml胰蛋白酶/EDTA溶液
    9. 在20-25℃下孵育3-5分钟。
    10. 加入9毫升完全DMEM,仔细重悬细胞
    11. 加入50ml完全DMEM和分裂细胞到三个T162烧瓶(每个烧瓶20ml)。
    12. 当生产JFH-1原液时,在37℃下在5%CO 2中孵育细胞另外72小时。
    13. 收集细胞上清液,在20-25°C下以4000rpm离心1分钟。 收集上清液,保存于-80°C等分试样
    14. 用完全DMEM(每个T162烧瓶20ml)补充细胞
    15. 在37℃下在5%CO 2中孵育24小时
    16. 重复步骤A13至15连续三天。
    17. 确定在步骤A13中收集的样品的感染滴度(参见第II部分)

  2. 产生持续感染的细胞培养物
    持续HCV感染的Huh-7细胞的特征是恒定的 生产病毒RNA,蛋白质和后代感染性病毒 延长的时间。 虽然持续感染细胞可以 培养几个月,我们建议限制文化段落 至小于8周以降低不期望的遗传漂移的风险 病毒和细胞群体(Zhong et al。,2006)。 持续 感染只能在Huh-7中建立,作为更容许的细胞 线在感染的高峰时显示严重的细胞病理学(Zhong et al al。,2006)。
    1. 将2×10 6个Huh-7细胞在10ml的在T75烧瓶中的完全DMEM中接种。
    2. 在37℃下在5%CO 2中孵育24小时
    3. 用JFH-1以0.01FFU /细胞的MOI接种细胞,每个瓶的终体积为10ml。
    4. 在37℃下在5%CO 2中孵育最少12-14天。
    5. 通过每3-4天分割培养物,始终保持亚汇合培养物:
      1. 用温热的PBS洗涤细胞一次,并丢弃
      2. 每瓶加入1ml胰蛋白酶/EDTA溶液
      3. 在20-25℃下孵育3-5分钟。
      4. 加入5ml完全DMEM,并重悬细胞,最终体积为6ml
      5. 丢弃4毫升的细胞悬浮液和板2毫升到T75烧瓶。 加入8ml完全DMEM。
    6. 在接种后12-14天,细胞将显示标记的 降低其增殖速率。 这发生在峰的 感染,但在下面的段落,在那里坚持 感染的Huh-7细胞完全可行,但显示稍长 与其未感染的对应物相比倍增时间(Zhong et al al。,2006)。
    7. 按照步骤B5所述从烧瓶中取出细胞。
    8. 收集200微升的细胞悬浮液,并把它放在无菌玻璃上 盖玻片在M12板孔中。 加入800μl的完整DMEM孵育 24小时。 继续此示例到步骤B10。
    9. 将其余的细胞接种在T162烧瓶中。 继续执行步骤B15。
    10. 除去步骤8的上清液,并将4%甲醛的PBS溶液(500μl)加到玻璃盖玻片上
    11. 在20-25℃孵育20分钟。
    12. 用PBS(1ml)洗涤细胞两次
    13. 处理样品进行免疫荧光染色(见第二部分,第16步)
    14. 确定HCV抗原阳性细胞的百分比 免疫荧光显微镜。 在接种后第12-14天,接近100%   的细胞应该是HCV抗原阳性的
    15. 一旦感染 达到100%的细胞,持续感染的细胞培养物 维持亚汇合最多8-10周。
      为了保持亚汇合培养,每3-4天分为如下:
      1. 用温热的PBS洗涤细胞一次,并丢弃
      2. 每瓶加入1ml胰蛋白酶/EDTA溶液
      3. 在20-25℃下孵育3-5分钟。
      4. 加入5ml完全DMEM,并重悬细胞,最终体积为6ml
      5. 丢弃4毫升的细胞悬浮液和板2毫升到T75烧瓶。 加入8ml完全DMEM。
    16. 根据下述收集细胞和上清液的样品 每个实验的实验设计(Gastaminza等人,2008)。

  3. 确定病毒传播能力
    以下实验设置可以确定相对病毒   在不同实验条件下的扩散能力,或比较 不同病毒(例如突变病毒)的总体生长能力。 与单周期感染(见第三部分),这个实验   设置不允许区分感染的不同方面。   因此,确定胞外后代病毒生产是足够的 以表征病毒传播能力。 但是,我们建议 收集其他样品以监测病毒生长 替代读数。
    1. 在接种前一天,在6孔板中在2ml完全培养基的终体积中接种2×10 5个Huh-7细胞/孔。
    2. 在37℃下在5%CO 2中孵育24小时
    3. 用JFH-1或D183病毒以0.01FFU /细胞的MOI接种细胞,每孔最终体积为2ml。
    4. 在37℃下在5%CO 2中孵育2-3天,直到它们达到90%汇合。
    5. 收集上清液并转移至1.5ml管中。 离心机1 分钟。 转移上清至干净的1.5ml管。 这个 样品用于测定细胞外感染性(见第二部分)
    6. 用温热的PBS洗涤细胞一次,并丢弃
    7. 每孔加入300μl胰蛋白酶/EDTA溶液
    8. 在20-25℃下孵育3-5分钟。
    9. 加入600微升完整的DMEM,并重悬细胞在最终体积为900微升
    10. 板1:3μL细胞(300μl),终体积为2ml /孔(加入   1.7 ml完全DMEM /孔,以准备下一个时间点 实验)。
    11. 转移1:3的细胞(300微升)到1.5毫升 管。 将细胞在4℃下以3,000rpm离心5分钟。 提取总计 细胞RNA以确定细胞内HCV RNA水平(Mingorance等人, al。,2014)。
    12. 转移1:3的细胞(300微升)到1.5毫升管。   将细胞在4℃下以3,000rpm离心10分钟。 提取总计 细胞蛋白通过Western印迹测定病毒抗原水平 (Mingorance等人,2014)。
    13. 将来自步骤C10的细胞在37℃下在5%CO 2中孵育3天。
    14. 重复步骤C5-13,直到实验结束。 通常   第2,4,6,8和10天或第3,6,9和12天的序列应当是 足以表征在Huh-7细胞中扩散的JFH-1病毒。 在里面 D183的情况,通常在5-6天后达到最大效价。

第二部分。 通过终点稀释和免疫荧光进行病毒滴定
该程序基于通过免疫荧光显微镜检测感染灶(感染细胞簇)。 每个感染的焦点归因于一个感染单位,定义为焦点形成单位(FFU)。 通过用系列样品稀释液接种天然细胞获得的感染灶的数量用于测定感染性滴度。

  1. 种子10 克隆2细胞/孔,在终体积为100μl的完全DMEM中   在96孔平底板中。 需要四至八个孔/样品,   取决于预期的感染性滴度。 重复样品为 强烈鼓励。
  2. 在37℃下在5%CO 2中孵育24小时
  3. 在20-25℃下解冻感染样品。
  4. 加入200微升的完整DMEM到一个干净的96孔板,使用4-8 孔。 在一个通道中顺序设置孔以容纳 不同的样品稀释液
  5. 将50μl样品加入第一个孔中。
  6. 丢弃提示。
  7. 用新的过滤嘴均匀地病毒 - 培养基稀释,小心不要产生气泡
  8. 取50μl的混合物,仔细转移到下一口井。
  9. 重复步骤C5-7,直到达到所需的稀释因子。
  10. 去除步骤C1中细胞的上清液。
  11. 每孔加入100μl不同的病毒稀释液。
  12. 在37℃下在5%CO 2中孵育72小时
  13. 弃去细胞上清液。
  14. 加入150μl的4%甲醛的PBS溶液
  15. 在20-25℃下孵育20分钟。
  16. 用PBS(200μl)洗涤两次。
  17. 加入30μl/孔的IF缓冲液
  18. 在20-25℃下孵育1小时。
  19. 在IF缓冲液中制备抗E2抗体AR3A的1μg/ml稀释液
  20. 从96孔板中丢弃IF缓冲液。
  21. 加入25μl/孔的抗E2抗体稀释液
  22. 在20-25℃下孵育1小时。
  23. 制备山羊抗人IgG-Alexa 555抗体在IF缓冲液中的1:500稀释液。 加入DAPI(1μg/ml,在IF缓冲液中)
  24. 用PBS(200μl)洗涤96孔板两次。
  25. 加入25μl/孔的如步骤23中所述制备的二次抗体稀释液
  26. 在20-25℃下孵育1小时。
  27. 用PBS(200μl)洗涤96孔板两次。
  28. 加入50微升PBS的测试孔。 在使用玻璃盖玻片的情况下,使用Prolong TM安装到显微镜载玻片上。
  29. 在荧光显微镜下观察。 计数感染灶 只有在它们是离散的并且与一个分离的稀释度 另一个,直到达到阴性孔(终点稀释)。
  30. 计算感染性滴度,单位为FFU/ml,乘以数 在不同稀释度中发现的感染灶由相应的 稀释因子。 乘以10以获得每ml的灶数(100μl   用于接种)。 平均传染性滴度估计   不同的稀释度(通常仅观察到离散的焦点) 2-3孔)。 强烈建议重复实验 如果不计算感染性滴度,则不同稀释度 2-3孔或如果未达到终点稀释。

第三部分。单周期感染实验
单周期感染实验,也称为高多重感染(MOI为10FFU /细胞),尝试建模感染一个靶细胞的一个病毒体的同步感染,并且通常用于解剖丙型肝炎病毒生命周期的早期和晚期方面。与多周期感染相反,我们建议测量不同的参数,如细胞内和细胞外感染性和RNA。在该实验装置中同时分析这些参数将能够确定感染的不同方面的效率。由于需要高感染性滴度病毒储液来进行这些实验,并且用亲本JFH-1菌株难以达到足够的滴度,所以我们通常使用D183病毒储液用于这些实验(参见第I部分,A部分)。

  1. 种子5×10 4个Huh-7细胞/孔,终体积为500μl完全 DMEM在24孔板中感染前一天。准备不同 板每时间点(见步骤9)
  2. 在37℃下在5%CO 2中孵育24小时
  3. 除去培养基,并以10 FFU /细胞的MOI用D183病毒接种细胞,每孔最终体积为500μl。
  4. 保存接种物的样品,以通过感染滴定(见第二部分)确认感染的多样性。
  5. 在37℃下在5%CO 2中孵育5小时
  6. 取出接种物,用温PBS(1ml)洗涤细胞两次
  7. 每孔加入500μl完全DMEM
  8. 在37℃下在5%CO 2中孵育。 通常达到峰滴度 约40小时后感染。 因此,我们建议在24,   48和72小时。 在5小时收集另外的样品, 将提供背景水平的感染性和HCV RNA 用作其余时间点的基准参考
  9. 在5,24,48和72小时收集样品:
    1. 收集上清液以确定细胞外感染性和HCV RNA。 储存于-80℃直至分析(Mingorance等人,2014年)。
    2. 用PBS(1ml)洗涤细胞一次并弃去
    3. 加入胰蛋白酶/EDTA溶液(每孔100μl)
    4. 在20-25℃下孵育3-5分钟。
    5. 重悬细胞在500微升完全DMEM每孔。 小心匀浆以制备单细胞悬浮液。
    6. 转移500微升的细胞悬浮液到1.5毫升管,以确定   细胞内感染性,如(Gastaminza等人,2008)中所述。 储存于-80℃直至分析
    7. 收集100微升的细胞悬浮液在1.5毫升管和RNA提取过程。 或者,存储在-80°C。

代表数据



图1.用连续稀释的感染细胞上清液感染的Huh-7细胞的免疫荧光显微镜检查和用1:10稀释的样品接种的细胞中个体感染灶的可视化。红色通道 抗E2抗体,用DAPI染色的蓝色通道细胞核
病毒储液的典型产率对于用JFH-1病毒产生的储液应当为约10 4 FFU/ml,对于D183病毒为约10 6 FFU/ml。 /> 持续感染的细胞培养物在达到100%感染后的第一个月应该产生≥103 FFU/ml的持续滴度。
单循环感染实验应显示峰值细胞外感染滴度> 10 4 FFU/ml,通常在感染后48小时。

笔记

  1. 感染性重组丙型肝炎病毒的操作需要特定的生物安全条件和基础设施。 建议研究人员在进行研究的国家/地区修订法律所要求的生物防护程度。

食谱

  1. 完全Dulbecco's Modified Eagle's Medium(完全DMEM)
    DMEM补充有10mM HEPES 1个非必需氨基酸
    100U/ml青霉素/链霉素和10%胎牛血清(在56℃热灭活30分钟)
  2. 10x磷酸盐缓冲盐水(PBS)
    将80g NaCl,2g KCl,26.8g Na 2 HPO 4 -H-H 2 O和2.4g KH 2 PO 4溶解, 在800ml H 2 O中用PO 4调节pH 7.4使用HCl调节pH 7.4< br /> 用H sub 2 O调整音量到1 L
  3. 免疫荧光(IF)缓冲液
    3%BSA
    0.3%TritonX-100的PBS溶液
  4. 1x胰蛋白酶/EDTA(10x)溶液
    将100ml 0.5%胰蛋白酶/EDTA(10x)溶液溶解在900ml 1×PBS中
  5. 4%甲醛溶液
    10.8ml甲醛
    80ml H 2 O和10ml 10×PBS

致谢

该协议基于最初在Scripps研究所(La Jolla,CA)的Francis V.Chisari的实验室与东京都市医学科学研究所Takaji Wakita's小组合作开发的工作(东京, (目前在上海的巴斯德研究所),Sharookh Kapadia(目前在Genentech),Guofeng Cheng(目前在Gilead),Susan Uprichard(目前在美国伊利诺斯大学)芝加哥),特别是Stefan Wieland(目前在巴塞尔巴塞尔大学医院)。
L.M.由来自Consejo Superior de InvestigacionesCientíficas和CIBERehd(Salitu Carlos III)的JAE-Pre fellowship资助。简历。由"La Caixa"基金/CNB奖学金资助。这项工作得到了来自西班牙科学和创新部(SAF2010-19270)和Marie Curie职业一体化补助金(PCIG-9-GA-2011-293664)的赠款计划国家研究计划,西班牙科学和创新部的Desarrollo eInnovaciónTecnológica欧洲联盟7 研究框架计划。

参考文献

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  2. Gastaminza,P.,Cheng,G.,Wieland,S.,Zhong,J.,Liao,W。和Chisari,F.V。(2008)。 丙型肝炎病毒装配,成熟,降解和分泌的细胞决定因素 J Virol 82(5):2120-2129。
  3. Gastaminza,P.,Dryden,K.A.,Boyd,B.,Wood,M.R.,Law,M.,Yeager,M。和Chisari,F.V。(2010)。 在细胞培养中产生的丙型肝炎病毒颗粒的超微结构和生物物理学特征。 J Virol 84(21):10999-11009。
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  8. Zhong,J.,Gastaminza,P.,Cheng,G.,Kapadia,S.,Kato,T.,Burton,D.R.,Wieland,S.F.,Uprichard,S.L.,Wakita,T.and Chisari,F.V。 体外耐药性丙型肝炎病毒感染 > Proc Natl Acad Sci USA 102(26):9294-9299。
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引用:Mingorance, L., Vasallo, C., Friesland, M. and Gastaminza, P. (2015). Infection Experiments (Hepatitis C Virus). Bio-protocol 5(3): e1392. DOI: 10.21769/BioProtoc.1392.
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