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 are 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 a 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.
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