Rescue of Sendai Virus from Cloned cDNA

  • Shringkhala Bajimaya
  • Tsuyoshi Hayashi
  • Toru TakimotoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1602)


Sendai virus (SeV) is a non-segment negative-sense RNA virus that naturally infects and causes pneumonia in mice. As a prototypic member of the family Paramyxoviridae, SeV has been characterized well, and these studies revealed numerous traits of paramyxovirus biology. The reverse genetics system to rescue SeV was first established in 1995. The virus was rescued from a cloned cDNA that contains full genome sequence flanked by T7 promoter and hepatitis delta virus ribozyme. To rescue SeV, it is necessary to infect cells with a recombinant vaccinia virus vTF7.3 that expresses T7 RNA polymerase, and transfect with the SeV full genome cDNAs together with supporting plasmids encoding NP, P, and L genes under the T7 promoter. Synthesized viral RNA by T7 RNA polymerase will be encapsidated with NP and associated with a polymerase complex composed of P and L. The polymerase complex transcribes and replicates the genome, and produces progeny virions. Rescued SeV needs to be plaque purified to exclude vTF7.3 from viral stock. Reverse genetics system of SeV is relatively efficient compared to other paramyxoviruses, but alternative approaches to rescue poorly growing mutant viruses are also available.

Key words

Sendai virus Rescue Parainfluenza Vaccinia virus 


  1. 1.
    Egelman EH, Wu SS, Amrein M, Portner A, Murti G (1989) The Sendai virus nucleocapsid exists in at least four different helical states. J Virol 63(5):2233–2243PubMedPubMedCentralGoogle Scholar
  2. 2.
    Calain P, Roux L (1993) The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol 67(8):4822–4830PubMedPubMedCentralGoogle Scholar
  3. 3.
    Kato A, Sakai Y, Shioda T, Kondo T, Nakanishi M, Nagai Y (1996) Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense. Genes Cells 1(6):569–579CrossRefPubMedGoogle Scholar
  4. 4.
    Garcin D, Pelet T, Calain P, Roux L, Curran J, Kolakofsky D (1995) A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus. EMBO J 14(24):6087–6094PubMedPubMedCentralGoogle Scholar
  5. 5.
    Leyrer S, Neubert WJ, Sedlmeier R (1998) Rapid and efficient recovery of Sendai virus from cDNA: factors influencing recombinant virus rescue. J Virol Methods 75(1):47–58CrossRefPubMedGoogle Scholar
  6. 6.
    Sutter G, Ohlmann M, Erfle V (1995) Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett 371(1):9–12CrossRefPubMedGoogle Scholar
  7. 7.
    Ghanem A, Kern A, Conzelmann KK (2012) Significantly improved rescue of rabies virus from cDNA plasmids. Eur J Cell Biol 91(1):10–16. doi: 10.1016/j.ejcb.2011.01.008 CrossRefPubMedGoogle Scholar
  8. 8.
    Maddon PJ, Dalgleish AG, McDougal JS, Clapham PR, Weiss RA, Axel R (1986) The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47(3):333–348CrossRefPubMedGoogle Scholar
  9. 9.
    Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83(21):8122–8126CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhan X, Slobod KS, Krishnamurthy S, Luque LE, Takimoto T, Jones B, Surman S, Russell CJ, Portner A, Hurwitz JL (2008) Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections. Vaccine 26(27–28):3480–3488CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Takahashi T, Ryan KW, Portner A (1992) A plasmid that improves the efficiency of foreign gene expression by intracellular T7 RNA polymerase. Genet Anal Tech Appl 9(3):91–95CrossRefPubMedGoogle Scholar
  12. 12.
    Takimoto T, Bousse T, Portner A (2000) Molecular cloning and expression of human parainfluenza virus type 1 L gene. Virus Res 70(1–2):45–53CrossRefPubMedGoogle Scholar
  13. 13.
    Bousse T, Matrosovich T, Portner A, Kato A, Nagai Y, Takimoto T (2002) The long noncoding region of the human parainfluenza virus type 1 f gene contributes to the read-through transcription at the m-f gene junction. J Virol 76(16):8244–8251CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kovacs GR, Vasilakis N, Moss B (2001) Regulation of viral intermediate gene expression by the vaccinia virus B1 protein kinase. J Virol 75(9):4048–4055. doi: 10.1128/JVI.75.9.4048-4055.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wyatt LS, Moss B, Rozenblatt S (1995) Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology 210(1):202–205. doi: 10.1006/viro.1995.1332 CrossRefPubMedGoogle Scholar
  16. 16.
    Sakai Y, Kiyotani K, Fukumura M, Asakawa M, Kato A, Shioda T, Yoshida T, Tanaka A, Hasegawa M, Nagai Y (1999) Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication. FEBS Lett 456(2):221–226CrossRefPubMedGoogle Scholar
  17. 17.
    Li HO, Zhu YF, Asakawa M, Kuma H, Hirata T, Ueda Y, Lee YS, Fukumura M, Iida A, Kato A, Nagai Y, Hasegawa M (2000) A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression. J Virol 74(14):6564–6569CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Tsung K, Yim JH, Marti W, Buller RM, Norton JA (1996) Gene expression and cytopathic effect of vaccinia virus inactivated by psoralen and long-wave UV light. J Virol 70(1):165–171PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Shringkhala Bajimaya
    • 1
  • Tsuyoshi Hayashi
    • 1
  • Toru Takimoto
    • 1
    Email author
  1. 1.Department of Microbiology and ImmunologyUniversity of Rochester School of Medicine and DentistryRochesterUSA

Personalised recommendations