Chang Ho Ho Lee
  • Dankook University
Research fields
  • Molecular biology
Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks
Authors:  Yan Sun and Carolina B. López, date: 05/20/2016, view: 17055, Q&A: 0
Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). Here we discuss in detail how to prepare RSV stocks with a high or low content of DVGs, and how to purify defective viral particles containing DVGs from an RSV stock enriched in defective viral particles. These procedures are useful for the preparation of viral stocks and defective viral particles necessary for laboratory research. In brief, the different RSV stocks are produced in HEp2 cells, which are commonly used to amplify this virus in the laboratory. To generate an RSV stock with a high content of DVGs, HEp2 cells are sequentially infected with a high multiplicity of infection (MOI) multiple times followed by purification of the viral particles containing DVGs using gradient centrifugation. The procedure describe here has four parts: 1. Amplification of seed RSV stock with a low DVG content (RSV-LD), 2. Generation of a stock with a high DVG content (RSV-HD), 3. Purification of DVGs by gradient centrifugation, 4. Characterization of purified DVGs.
Respiratory Syncytial Virus Infection in Mice and Detection of Viral Genomes in the Lung Using RT-qPCR
Authors:  Yan Sun and Carolina B. López, date: 05/20/2016, view: 10183, Q&A: 0
Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated with the development and exacerbation of chronic lung conditions including asthma, and it is a major cause of hospitalizations in infants. Because of its clinical relevance, experimental animal models to study RSV in vivo are needed. The most common and accessible animal model in research laboratories is the mouse. However, commonly use RSV strains poorly establish infection in mice and thus titration of the virus from mouse lungs to confirm infection is not sensitive enough to detect early viral infection. Here we discuss in detail how to infect BALB/c mice with RSV and how to detect RSV genomes in the lung using reverse transcription quantitative PCR (RT-qPCR). This method allows detection of viral genomes as early as day 1 post-infection (shown in Figure 2), whereas traditional TCID50 fails to detect significant virus until after day 2 post-infection. Of note, despite of higher sensitivity, genome RT-qPCR only shows the production of viral genomes and thus positive results for this assay are not proof of production of infectious viral particles.
Primer Extension Reactions for the PCR- based α- complementation Assay
Authors:  Vasudevan Achuthan and Jeffrey J. DeStefano, date: 06/20/2015, view: 10206, Q&A: 0
The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.
Mismatched Primer Extension Assays
Authors:  Vasudevan Achuthan and Jeffrey J. DeStefano, date: 06/20/2015, view: 7220, Q&A: 0
Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+. The relative fidelity of the polymerase can then be estimated using the standard extension efficiency. The assay described here is based on the method published in Mendelman et al. (1990).
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