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Enterovirus Competition Assay to Assess Replication Fitness

用于检测复制适应性的肠道病毒竞争实验

Valeria Lulla Valeria Lulla
AF Andrew E. Firth

发布: 2019年05月20日第9卷第10期 DOI: 10.21769/BioProtoc.3233 浏览次数: 6898

评审: Kristin L. ShinglerJean Kaoru Millet

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Abstract

In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Relative viral replication fitness is usually measured from multiple cycle growth competition assays. Competition assays provide a sensitive measurement of viral fitness since the viruses are competing for cellular targets under identical growth conditions. This protocol describes a competition assay for enteroviruses and contains two alternative formats for initial infections, which can be varied depending on specific goals for each particular experiment. The protocol involves infection of cells with competing viruses, passaging, RNA extraction from infected cells, RT-PCR and Sanger sequencing followed by comparative analysis of resulting chromatograms obtained under various initial infection conditions. The techniques are applicable to members of many virus families, such as alphaviruses, flaviviruses, pestiviruses, and other RNA viruses with an established reverse genetics system.

Keywords: Enterovirus (肠病毒) search Competition assay (竞争实验) search Virus titration (病毒滴定) search Dual infection (双重感染) search Pairwise growth competition assay (成对增长竞争测定法) search Virus passaging (病毒传代) search Sanger sequencing (Sanger 测序) search

Background

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. In our recent paper (Lulla et al., 2019) we reported that many enterovirus genomes harbor an upstream open reading frame (uORF) that encodes an additional viral protein, UP (upstream protein). Using the echovirus 7 (EV7) reverse genetics system, we created two UP knock-out mutants (EV7-Loop and EV7-PTC), which exhibited wtEV7-like growth properties. Therefore, we decided to perform a competition assay to determine possible subtle fitness defects.

Materials and Reagents

  1. Materials
    1. Pipette tips (Fisher, catalog numbers: 02-707-426, 02-707-403, 02-707-438)
    2. 1.5 ml microfuge tubes (STARLAB, catalog number: 51615-5500)
    3. T175 tissue culture flasks (TPP® tissue culture flasks, Sigma, catalog number: Z707562)
    4. 12-well polystyrene culture plates (TPP® tissue culture plates, Sigma, catalog number: Z707775)

  2. Viruses
    Echovirus 7 (EV7), derived from EV7 infectious clone
    Note: The cDNA of Echovirus 7 strain Wallace was sourced from Michael Lindberg (GenBank accession number AF465516, with the silent substitution 1687G-to-A) and was cloned downstream of a T7 RNA promoter. Mutant viruses EV7-Loop and EV7-PTC were generated by mutagenesis of the original EV7 infectious clone and prepared exactly as wt EV7 (Lulla et al., 2019).

  3. Cell lines
    RD cells (human rhabdomyosarcoma cell line, ATCC, CCL-136), verified mycoplasma-free by next-generation sequencing

  4. Reagents
    1. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, catalog number: D6546)
    2. Fetal bovine serum (FBS) (PAN Biotech, catalog number: P40-37500)
    3. Penicillin-streptomycin (10,000 U/ml) (Life Technologies, catalog number: 15140-122)
    4. 200 mM L-Glutamine (Gibco, catalog number: 25030081)
    5. Phosphate buffered saline (PBS) (Life Technologies, catalog number: 14190-144)
    6. 1 M HEPES buffer, pH range: 7.2-7.5 (Life Technologies, catalog number: 15630-080)
    7. 0.25% Trypsin (Gibco, catalog number: 15090046-100)
    8. Bovine serum albumin (BSA) (PAN Biotech, catalog number: P06-1395500)
    9. Direct-zolTM RNA MiniPrep Plus (Zymo Research, catalog number: R2072) 
    10. PhusionTM RT-PCR Kit (Thermo Scientific, catalog number: F-546S)
    11. Complete DMEM (see Recipes)
    12. Serum-free DMEM (see Recipes)

Equipment

  1. -80 °C freezer
  2. Vortexer
  3. Pipettes: 1000 µl, 200 µl, 20 µl, 2 µl
  4. CO2 incubator
  5. Haemocytometer
  6. BSL2 cell culture cabinet
  7. Rocking platform shaker
  8. Phase-contrast inverted microscope (Nikon TMS)
  9. 4 °C refrigerator
  10. Autoclave

Software

  1. BioEdit (Free software, Ibis Therapeutics, http://www.mbio.ncsu.edu/BioEdit/bioedit.html)

Procedure

English
中文翻译

文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Lulla, V. and Firth, A. E. (2019). Enterovirus Competition Assay to Assess Replication Fitness. Bio-protocol 9(10): e3233. DOI: 10.21769/BioProtoc.3233.

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分类

微生物学 > 微生物细胞生物学 > 病毒繁殖

分子生物学 > RNA > RNA 纯化

分子生物学 > RNA > RNA 测序

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