Abstract
Human rhinovirus (HRV) contains a 7.2 kb messenger-sense RNA genome which is the template for reproducing progeny viruses after it enters the cytoplasm of a host cell. Reverse genetics refers to the regeneration of progeny viruses from an artificial cDNA copy of the RNA genome of an RNA virus. It has been a powerful molecular genetic tool for studying HRV and other RNA viruses because the artificial DNA stage makes it practical to introduce specific mutations into the viral RNA genome. This chapter uses HRV-16 as the model virus to illustrate the strategy and methods for constructing and cloning the artificial cDNA copy of a full-length HRV genome, identifying the infectious cDNA clone isolates, and selecting the most vigorous cDNA clone isolate to serve as the standard parental clone for future molecular genetic study of the virus.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Turner RB, Lee WM (2009) Rhinovirus. In: Richman DD, Whitley RJ, Hayden FG (eds) Clinical virology. ASM, Washington, DC, pp 1063–1082
Lee WM, Monroe SS, Rueckert RR (1993) Role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome. J Virol 67:2110–2122
Rueckert R (1996) Picornaviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM et al (eds) Fields virology, 3rd edn. Lippincott-Raven Publishers, Philadelphia, PA, pp 609–654
Taniguchi T, Palmieri M, Weissmann C (1978) QB DNA-containing hybrid plasmids giving rise to QB phage formation in the bacterial host. Nature 274:223–228
Racaniello VR, Baltimore D (1981) Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci USA 78:4887–4891
Ahlquist P, French R, Janda M, Loesch-Fries LS (1984) Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc Natl Acad Sci USA 81:7066–7070
Janda M, French R, Ahlquist P (1987) High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cdna and effects of 5’ extensions on transcript infectivity. Virology 158:259–262
Bochkov YA, Palmenberg AC, Lee WM, Rathe JA, Amineva SP, Sun X, Pasic TR, Jarjour NN, Liggett SB, Gern JE (2011) Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nat Med 17:627–632
Huang T, Wang W, Bessaud M, Ren P, Sheng J, Yan H, Zhang J, Lin X, Wang Y, Delpeyroux F et al (2009) Evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection. PLoS One 4:e6355
Palmenberg AC, Spiro D, Kuzmickas R, Wang S, Djikeng A, Rathe JA, Fraser-Liggett CM, Liggett SB (2009) Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution. Science 324:55–59
McErlean P, Shackelton LA, Lambert SB, Nissen MD, Sloots TP, Mackay IM (2007) Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis. J Clin Virol 39:67–75
Tapparel C, Junier T, Gerlach D, Cordey S, Van Belle S, Perrin L, Zdobnov EM, Kaiser L (2007) New complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features. BMC Genomics 8:224
Harris JR, Racaniello VR (2005) Amino acid changes in proteins 2B and 3A mediate rhinovirus type 39 growth in mouse cells. J Virol 79:5363–5373
Kistler AL, Webster DR, Rouskin S, Magrini V, Credle JJ, Schnurr DP, Boushey HA, Mardis ER, Li H, DeRisi JL (2007) Genome-wide diversity and selective pressure in the human rhinovirus. Virol J 4:40
Lau SK, Yip CC, Tsoi HW, Lee RA, So LY, Lau YL, Chan KH, Woo PC, Yuen KY (2007) Clinical features and complete genome characterization of a distinct human rhinovirus (HRV) genetic cluster, probably representing a previously undetected HRV species, HRV-C, associated with acute respiratory illness in children. J Clin Microbiol 45:3655–3664
Lee WM, Wang W, Rueckert RR (1995) Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group. Virus Genes 9:177–181
Hughes PJ, North C, Jellis CH, Minor PD, Stanway G (1988) The nucleotide sequence of human rhinovirus 1B: molecular relationships within the rhinovirus genus. J Gen Virol 69(Pt 1):49–58
Skern T, Sommergruber W, Blaas D, Gruendler P, Fraundorfer F, Pieler C, Fogy I, Kuechler E (1985) Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region. Nucleic Acids Res 13:2111–2126
Stanway G, Hughes PJ, Mountford RC, Minor PD, Almond JW (1984) The complete nucleotide sequence of a common cold virus: human rhinovirus 14. Nucleic Acids Res 12:7859–7875
Djikeng A, Halpin R, Kuzmickas R, Depasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson NG, Ghedin E et al (2008) Viral genome sequencing by random priming methods. BMC Genomics 9:5
Hao W, Bernard K, Patel N, Ulbrandt N, Feng H, Svabek C, Wilson S, Stracener C, Wang K, Suzich J et al (2012) Infection and propagation of human rhinovirus C in human airway epithelial cells. J Virol 86:13524–13532
Ashraf S, Brockman-Schneider R, Bochkov YA, Pasic TR, Gern JE (2012) Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells. Virology 436:143–149
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lee, WM., Wang, W., Bochkov, Y.A., Lee, I. (2015). Reverse Genetics System for Studying Human Rhinovirus Infections. In: Jans, D., Ghildyal, R. (eds) Rhinoviruses. Methods in Molecular Biology, vol 1221. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1571-2_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1571-2_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1570-5
Online ISBN: 978-1-4939-1571-2
eBook Packages: Springer Protocols