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Fitness Determinations in Vesicular Stomatitis Virus
水泡性口炎病毒中的整体复制能力的测定   

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参见作者原研究论文

本实验方案简略版
Journal of Virology
May 2013

Abstract

Fitness is defined as the overall replicative ability. Testing whether a mutation (or combination of mutations) has an effect on fitness often relays on determining virus production as a surrogate measurement. However, viruses do not usually replicate in a void, and evolutionary speaking, it is key to determine replicative ability compared to other viruses, e.g. the relative fitness. John Holland developed a method for vesicular stomatitis virus based on the use of a neutral genetic marker that allows to distinguish two competitors and to measure accurately the relative ratio between the two during competition (Holland et al., 1991). The marker is a mutation in the external G glycoprotein that has no effect on the virus other than conferring resistance to a monoclonal antibody, I1. To measure fitness a marked test strain is mixed with a reference unmarked strain and the mixture is allowed to infect a cell monolayer. Ratios before and after competition are measured by plaque assay in the presence and absence of I1 antibody, and changes in ratio give the fitness value.

Materials and Reagents

  1. Test and reference VSV strains
    Any neutral mutation that confers resistance to the monoclonal antibody used in the test is appropriate. For I1, which recognizes the A1 epitope of the G glycoprotein, mutation 3853 is our preferred choice. Mutation 3846 is not a good choice because it seems to be beneficial. Mutations indicate the nucleotide number of wild type Indiana serotype, Mudd-Summers strain. Mutant can be isolated by plaque-picking or by reverse genetics. Either way, the resulting population must be fully sequenced to ensure that no other mutations have been incorporated during the process.
  2. Baby hamster kidney cells (BHK-21)
  3. 10x Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 15400 )
  4. I1 Monoclonal antibody (I1Mab) hybridoma (ATCC, catalog number: CRL-2700 )
  5. Proteose peptone No. 3 (PP3) (BD DifcoTM, catalog number: 0 12207 ) (12 g/L in dH2O, autoclaved)
  6. Bovine Calf Serum (BCS) (Life Technologies, Gibco®, catalog number: 16170-078 )
  7. Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 10437 )
  8. NaCl
  9. 10% CO2
  10. Agarose (Lonza, SeqplaqueTM GTGTM, catalog number: 50111 ) (40 g/L in dH2O, autoclaved)
  11. Glutamine (Life Technologies, InvitrogenTM, catalog number: 25030-081 )
  12. Minimal Essential Medium with Hanks salts (MEM-H) (Mediatech, Cellgro®, catalog number: 50-019-PB )
  13. Sodium bicarbonate (4.2 g/L in dH2O, autoclaved)
  14. Penicillin/streptomycin solution (Mediatech, Cellgro®, catalog number 30-004-Cl )
  15. Saline solution (see Recipes)
  16. Minimal Essential Medium with Earl’s salts (MEM-E) (Mediatech, Cellgro®, catalog number: 51-10-PB ) (see Recipes)
  17. Crystal violet solution (see Recipes)

Equipment

  1. 1.5 ml tubes
  2. T25 filtered-cap flasks (Cyto-One®, catalog number: CC7682-4825 )
  3. T-25 plug seal (if MEM-Hanks is used) (Cyto-One®, catalog number: CC7682-4325 )
  4. 5 ml and 25 ml pipettes
  5. Pasteur pipettes
  6. Type II biosafety hood
  7. 37 °C cell culture incubator
  8. Transilluminator (optional)

Procedure

  1. Make and titer I1 antibody; use enough concentration to produce full inhibition of wild type. Alternatively the antibody can be purchased from Kerafast https://www.kerafast.com/p-171-hybridoma-8g5f11-i1.aspx.
  2. On day 1 BHK-21 stock cells are washed twice with saline solution, trypsinized and ~0.8-1.0 x 105 cells are seeded in T25 flasks with MEM-E supplemented with 7% BCS and 0.06% PP3. The flasks are gassed for 2 sec with 10% CO2 using a plugged Pasteur pipette.  Caps are locked.
  3. The cells are incubated for 24 h to produce monolayers 90% confluent.
  4. On day 2 virus mixtures are prepared. If the test virus is expected to be low fitness, the test: reference ratio should be close to 1:1. If the test virus is expected to be neutral of high fitness, ratio should be 10:1 or higher. The final viral concentration should be 106 PFU/ml (virus stocks should be titrated in advance).
    Note: A number of other neutral markers can be used for these assays. Any neutral single-nucleotide substitution may be monitored by qRT-PCR. Some nucleotide substitutions can be engineered to create or destroy restriction sites that can be identified in agarose gels and quantified by densitometry.
  5. Aspire growth medium in a T-25 flask using a Pasteur pipettedand use 200 μl of virus mixture to infect a monolayer. Incubate 10 min at room temperature (RT) and 40 min at 37 °C.  Add 5 ml of MEM-E + FBS. This is the competition passage.
    Note: This will result in the determination of fitness in BHK-21 cells under the incubation conditions specified here. Virus fitness can be determined under other kind of conditions of interest (different cell types, temperatures, presence/absence of antiviral molecules, etc) by changing the conditions of this passage.  The rest of the procedure remains the same after adjusting for dilutions so one can count 20-200 plaques/flask.
  6. Dilute a sample of the virus mixture to 1,000-fold and infect 3 BHK-21 monolayers with 200 μl to carry out plaque assays in the absence of I1Mab. This will produce approximately 200 plaques. Incubate 10 min at room temperature (RT) and 40 min at 37 °C. Melt agarose solution and add to prewarmed MEM-E+BCS to a final concentration of 0.2%. Add 5 ml to each flask.
  7. Infect 3 BHK-21 monolayers with 200 μl of the appropriate dilution to carry out plaque assay in the presence of I1Mab. If ratios are close to 1:1, 1,000-fold dilution a good choice.  If there is excess of 10:1 or more reference virus, a 100-fold dilution should produce the desired results.  Do not neutralize the mixture with I1Mab, as it will result in incorrect data due to phenotypic mixing and hiding (Valverde and Ortin, 1989; Holland et al.,1989). Incubate 10 min at room temperature (RT) and 40 min at 37 °C.  Prewarm a mixture of MEM-E + BCS and I1Mab at enough concentration to achieve complete neutralization of wild type, add agarose to a final concentration of 0.2%, and add mixture to flasks.
    Note: I1Mab needs to be titrated in advance.  Different batches, particularly if produce in the laboratory from the hybridoma, may differ greatly in their final I1 activity.
  8. Incubate competition passage and plaque assays for 24 h at 37 °C.
  9. Develop all plaque assays. Just let the overlay slide out and add 2-3 ml of crystal violet solution. Wait for 5 min at RT and rinse with tap water.
    Note: Because of the low agarose concentration there is no need to fix the cells.
  10. Count plaques and calculate initial ration (P0): (total virus – I1-resistant virus)/I1-resistant virus. The transilluminator is helpful for plaque counting.
  11. Take the yield from the competition passage yield and carry out triplicate plaque assays in the presence and absence of I1Mab as described (except that dilutions will need to be adjusted).
  12. Calculate the ratio after competition (P1) as described in step 10.
  13. Fitness is calculated as P1/P0.
  14. To increase the accuracy of the determination and increase the statistical power for subsequent analyses.
    1. If fitness values are closed to neutrality and the changes in ratio are small, use the yield of the first competition passage to carry out a second competition passage as described in step 5 and continue the process for up to 6-7 passages. Calculate fitness as the antilogarithm of the slope of log-ratio changes.
    2. If fitness values are very large or very small, carry out multiple independent competitions.


    Figure 1. Determinations of relative fitness. A test virus (white particles) is mixed with a reference virus (grey particles) and the mixture is used to infect BHK-21 cells. The viruses will compete during replication, and the virus that is best adapted will produce a higher relative number of progeny virions. When compared to a reference, strains whose relative numbers increase are high fitness; strains whose relative numbers don’t change are neutral; strains whose relative numbers decrease are low fitness.

Recipes

  1. Saline solution
    Add 7 g of NaCl to dH2O to 1,000 ml
    Autoclave 20 min
    Stored at RT
  2. MEM-E
    Mix powder with 9.55 L of dH2O
    Distribute 0.955 L in each of 10 1-L bottle and autoclave
    Let cool and add to each bottle
    1. 25 ml of 4.2% bicarbonate
    2. 70 ml of serum (FEB or BCS)
    3. 10 ml of penicillin/streptomycin mixture
    At this time the medium can be stored in the fridge.  When ready for use add
    1. 10 ml glutamine (diluted 1:10 from purchased stock)
    2. 5 ml of 12% PP3
    Notes:
    1. Making medium is worth it if there is a large volume of cell culture performed in the laboratory.  Ready-to-use MEM-E can also be purchased from several companies.
    2. MEM with Hanks salts (MEM-H) is also a good choice and only requires that at the time that cells are split each flask is gassed for 2-3 sec with 5-10% CO2 (in N2/O2).  The advantage is that there is no need for continuous supply of CO2, so any 37 °C incubator can be used.  The medium is prepared similarly, but all components except serum and PP3 are mixed together and filtered before aliquoting.  The powder has glutamine, so it does not need to be added.
  3. Crystal violet solution
    Mix 750 ml H2O, 250 ml Ethanol and 5 g crystal violet
    Stir until dissolved and stored at RT

Acknowledgments

This protocol was originally published in Holland et al. (1991).

References

  1. Holland, J. J., de la Torre, J. C., Clarke, D. K. and Duarte, E. (1991). Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses. J Virol 65(6): 2960-2967.
  2. Holland, J. J., de la Torre, J. C., Steinhauer, D. A., Clarke, D., Duarte, E. and Domingo, E. (1989). Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions. J Virol 63(12): 5030-5036.
  3. Valcarcel, J. and Ortin, J. (1989). Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus. J Virol 63(9): 4107-4109. 

简介

健身被定义为整体的复制能力。测试突变(或突变的组合)是否对适应性有影响经常决定作为替代测量的病毒生产。然而,病毒通常不在空白中复制,并且在进化论上,确定与其他病毒相比的复制能力是关键,例如相对适合度。 John Holland开发了一种基于使用中性遗传标记物的水疱性口炎病毒的方法,所述中性遗传标记物允许区分两个竞争者并且准确地测量竞争期间两者之间的相对比率(Holland等人,1991 )。标记是外部G糖蛋白中的突变,其对赋予对单克隆抗体I1的抗性以外的病毒没有影响。为了测量适应度,将标记的测试菌株与参考未标记的菌株混合,并使混合物感染细胞单层。通过在I1抗体存在和不存在下的噬斑测定来测量竞争前后的比率,比率的变化给出适合度值。

材料和试剂

  1. 测试和参考VSV毒性
    任何赋予对测试中使用的单克隆抗体的抗性的中性突变是合适的。 对于识别G糖蛋白的A1表位的I1,突变3853是我们优选的选择。 突变3846不是一个好的选择,因为它似乎是有益的。 突变表示野生型Indiana血清型Mudd-Summers菌株的核苷酸数。 突变体可以通过匾拾取或通过反向遗传学分离。 无论哪种方式,所得到的群体必须完全测序,以确保在该过程中没有引入其他突变
  2. 幼仓鼠肾细胞(BHK-21)
  3. 10x胰蛋白酶/EDTA(Life Technologies,Gibco ,目录号:15400)
  4. I1单克隆抗体(I1Mab)杂交瘤(ATCC,目录号:CRL-2700)
  5. 蛋白胨No.3(PP3)(BD Difco Corp.,目录号:012207)(12g/L,在dH 2 O中,高压灭菌)
  6. 牛小牛血清(BCS)(Life Technologies,Gibco ,目录号:16170-078)
  7. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10437)
  8. NaCl
  9. 10%CO 2
  10. 琼脂糖(Lonza,Seqplaque TM,目录号:50111)(在dH 2 O中为40g/L,高压灭菌)
  11. 谷氨酰胺(Life Technologies,Invitrogen TM,目录号:25030-081)
  12. 具有Hanks盐的最小必需培养基(MEM-H)(Mediatech,Cellgro ,目录号:50-019-PB)
  13. 碳酸氢钠(4.2g/L,在dH 2 O中,高压灭菌)
  14. 青霉素/链霉素溶液(Mediatech,Cellgro ,目录号30-004-Cl)
  15. 盐水溶液(见配方)
  16. 具有伯爵盐的最小必需培养基(MEM-E)(Mediatech,Cellgro ,目录号:51-10-PB)(参见配方)
  17. 水晶紫溶液(见配方)

设备

  1. 1.5 ml管
  2. T25过滤盖烧瓶(Cyto-One ,目录号:CC7682-4825)
  3. T-25塞密封(如果使用MEM-Hanks)(Cyto-One ®,目录号:CC7682-4325)
  4. 5 ml和25 ml移液器
  5. 巴斯德移液器
  6. II型生物安全罩
  7. 37℃细胞培养箱
  8. 透照器(可选)

程序

  1. Make和滴度I1抗体; 使用足够的浓度产生野生型的完全抑制。 或者,抗体可购自Kerafast的 https://www.kerafast.com /p171-hybridoma-8g5f11-i1.aspx。
  2. 在第1天,用盐溶液洗涤BHK-21储存细胞两次,胰蛋白酶化,并将〜0.8-1.0×10 5个细胞接种在具有补充有7%BCS和0.06%PP3的MEM-E的T25烧瓶中。使用塞住的巴斯德移液管将烧瓶用10%CO 2鼓泡2秒。上限已锁定。
  3. 将细胞孵育24小时以产生90%汇合的单层
  4. 在第2天,制备病毒混合物。如果测试病毒预期为低适应度,则测试:参考比应接近1:1。如果测试病毒预期是高适应性的中性,比率应当为10:1或更高。最终病毒浓度应为10 6 PFU/ml(病毒储液应该预先滴定)。
    注意:许多其他中性标记可用于这些测定。任何中性单核苷酸取代可以通过qRT-PCR监测。可以设计一些核苷酸取代以产生或破坏可以在琼脂糖凝胶中鉴定并通过光密度测定法定量的限制性位点。
  5. 使用巴斯德移液器在T-25烧瓶中的Aspire生长培养基,并使用200μl病毒混合物感染单层。在室温(RT)孵育10分钟,在37℃孵育40分钟。加入5ml的MEM-E + FBS。这是比赛通道。
    注意:这将导致在此处指定的孵育条件下测定BHK-21细胞的适合度。通过改变该传代的条件,可以在其他类型的感兴趣的条件(不同的细胞类型,温度,抗病毒分子的存在/不存在等)下确定病毒适应性。在调整稀释度之后,剩余的程序保持不变,因此可以计数20-200个斑块/瓶。
  6. 将病毒混合物的样品稀释至1,000倍,并用200μl感染3个BHK-21单层以在不存在I1Mab的情况下进行噬菌斑测定。这将产生约200个斑块。在室温(RT)孵育10分钟,在37℃孵育40分钟。融化琼脂糖溶液,并加入到预热的MEM-E + BCS中至终浓度为0.2%。每个烧瓶中加入5毫升
  7. 传染3 BHK-21单层与200微升适当的稀释,以进行在IIMab存在的空斑测定。如果比率接近1:1,则1,000倍稀释是一个好的选择。如果有超过10:1或更多的参考病毒,100倍稀释应产生所需的结果。不要用I1Mab中和混合物,因为由于表型混合和隐藏将导致不正确的数据(Valverde和Ortin,1989; Holland等人,1989)。在室温(RT)孵育10分钟,在37℃孵育40分钟。将MEM-E + BCS和I1Mab的混合物预热至足以实现野生型完全中和的浓度,加入琼脂糖至终浓度为0.2%,并将混合物加入烧瓶中。 注意:I1Mab需要提前滴定。不同的批次,特别是如果在实验室中从杂交瘤产生的,其最终I1活性可能有很大不同。
  8. 在37℃下孵育竞争通道和噬菌斑测定24小时
  9. 开发所有斑块测定。只要让覆盖层滑出来,加入2-3ml的结晶紫溶液。在室温下等待5分钟,用自来水冲洗。
    注意:由于琼脂糖浓度低,不需要固定细胞。
  10. 计数噬菌斑并计算初始比例(P0):(总病毒-I1抗性病毒)/I1抗性病毒。透照器有助于斑块计数。
  11. 从竞争通道产量中获得产量,并在存在和不存在如所述的I1Mab的情况下进行一式三份的噬斑测定(除非需要调节稀释)。
  12. 按步骤10中所述计算比赛后的比率(P1)。
  13. 健身计算为P1/P0。
  14. 提高确定的准确性并增加后续分析的统计功效。
    1. 如果适合度值接近中性且比率变化小,则使用第一竞争通道的产量进行如步骤5中所述的第二竞争通道,并继续该过程达6-7代。计算适应度作为对数比变化斜率的反对数
    2. 如果适合度值非常大或非常小,执行多个独立竞赛

    图1.相对适合度的确定。 将测试病毒(白色颗粒)与参考病毒(灰色颗粒)混合,并将混合物用于感染BHK-21细胞。 病毒在复制期间将竞争,并且最佳适应的病毒将产生更高的相对数目的后代病毒粒子。 与参考相比,相对数增加的菌株具有高适应性; 相对数目不变的菌株是中性的; 相对数目减少的菌株具有低适应性

食谱

  1. 盐溶液
    将7g NaCl加入到dH 2 O至1000ml
    中 高压灭菌器20分钟
    存储在RT
  2. MEM-E
    将粉末与9.55L dH 2 O混合 在10个1-L瓶和高压釜中分别加入0.955L 让凉爽,并添加到每个瓶子
    1. 25毫升4.2%碳酸氢钠
    2. 70ml血清(FEB或BCS)
    3. 10ml青霉素/链霉素混合物
    此时介质可以存储在冰箱中。 准备使用时添加
    1. 10ml谷氨酰胺(从购买的原液稀释1:10)
    2. 5ml 12%PP3
    注意:
    1. 如果在实验室中进行大量的细胞培养,制作培养基是值得的。准备使用的MEM-E也可以从几家公司购买。
    2. 具有汉克斯盐(MEM-H)的MEM也是一个不错的选择,并且仅需要在细胞分裂时将每个烧瓶用5-10%CO 2鼓泡2-3分钟, 2 em> 2 )。优点是不需要连续供应CO ,因此可以使用任何37℃的培养箱。类似地制备培养基,但是除了血清和PP3之外的所有组分混合在一起,并且在等分之前过滤。粉末含有谷氨酰胺,因此不需要添加。
  3. 水晶紫溶液
    混合750ml H 2 O,250ml乙醇和5g结晶紫
    搅拌直至溶解并在RT下贮存

致谢

该协议最初发表于Holland等人(1991)。

参考文献

  1. Holland,J.J.,de la Torre,J.C.,Clarke,D.K。和Duarte,E。(1991)。 克隆群体的相对适应性和适应性的定量 的RNA病毒。 Virol 65(6):2960-2967。
  2. Holland,J.J.,de la Torre,J.C.,Steinhauer,D.A.,Clarke,D.,Duarte,E.and Domingo,E。(1989)。 病毒突变频率可以通过病毒体的单克隆抗体中和而大大低估。 J Virol 63(12):5030-5036。
  3. Valcarcel,J。和Ortin,J。(1989)。 表型隐藏:RNA病毒中突变的遗留,如流感病毒中mar突变体的检测所示。 J Virol 63(9):4107-4109。 
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Novella, I. S. (2014). Fitness Determinations in Vesicular Stomatitis Virus. Bio-protocol 4(6): e1074. DOI: 10.21769/BioProtoc.1074.
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