This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herfst, S.
Right arrow Articles by Fouchier, R. A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herfst, S.
Right arrow Articles by Fouchier, R. A. M.

 Previous Article  |  Next Article 

Journal of Virology, August 2004, p. 8264-8270, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8264-8270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Recovery of Human Metapneumovirus Genetic Lineages A and B from Cloned cDNA

Sander Herfst,1,{dagger} Miranda de Graaf,1,{dagger} Jeanne H. Schickli,2 Roderick S. Tang,2 Jasmine Kaur,2 Chin-Fen Yang,2 Richard R. Spaete,2 Aurelia A. Haller,2,{ddagger} Bernadette G. van den Hoogen,1 Albert D. M. E. Osterhaus,1 and Ron A. M. Fouchier1*

Department of Virology, Erasmus Medical Center, Rotterdam, The Netherlands,1 MedImmune Vaccines, Inc., Mountain View, California 940432

Received 28 November 2003/ Accepted 17 March 2004

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 107.2 and 105.7 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

* Corresponding author. Mailing address: Department of Virology, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: (31) 10 4088066. Fax: (31) 10 4089485. E-mail: r.fouchier{at}erasmusmc.nl.

{dagger} S.H. and M.D.G. contributed equally to the results of this study.

{ddagger} Present address: GlobeImmune Inc., Aurora, CO 80010.

Journal of Virology, August 2004, p. 8264-8270, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8264-8270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • de Graaf, M., Schrauwen, E. J. A., Herfst, S., van Amerongen, G., Osterhaus, A. D. M. E., Fouchier, R. A. M. (2009). Fusion protein is the main determinant of metapneumovirus host tropism. J. Gen. Virol. 90: 1408-1416 [Abstract] [Full Text]  
  • de Graaf, M., Osterhaus, A. D. M. E., Fouchier, R. A. M., Holmes, E. C. (2008). Evolutionary dynamics of human and avian metapneumoviruses. J. Gen. Virol. 89: 2933-2942 [Abstract] [Full Text]  
  • Derdowski, A., Peters, T. R., Glover, N., Qian, R., Utley, T. J., Burnett, A., Williams, J. V., Spearman, P., Crowe, J. E. Jr (2008). Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation. J. Gen. Virol. 89: 2698-2708 [Abstract] [Full Text]  
  • Herfst, S., de Graaf, M., Schrauwen, E. J. A., Sprong, L., Hussain, K., van den Hoogen, B. G., Osterhaus, A. D. M. E., Fouchier, R. A. M. (2008). Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters. J. Gen. Virol. 89: 1553-1562 [Abstract] [Full Text]  
  • de Graaf, M., Herfst, S., Schrauwen, E. J. A., Choi, Y., van den Hoogen, B. G., Osterhaus, A. D. M. E., Fouchier, R. A. M. (2008). Specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses. J. Gen. Virol. 89: 975-983 [Abstract] [Full Text]  
  • Ling, R., Sinkovic, S., Toquin, D., Guionie, O., Eterradossi, N., Easton, A. J. (2008). Deletion of the SH gene from avian metapneumovirus has a greater impact on virus production and immunogenicity in turkeys than deletion of the G gene or M2-2 open reading frame. J. Gen. Virol. 89: 525-533 [Abstract] [Full Text]  
  • Liu, L., Bastien, N., Li, Y. (2007). Intracellular Processing, Glycosylation, and Cell Surface Expression of Human Metapneumovirus Attachment Glycoprotein. J. Virol. 81: 13435-13443 [Abstract] [Full Text]  
  • Herfst, S., de Graaf, M., Schrauwen, E. J. A., Ulbrandt, N. D., Barnes, A. S., Senthil, K., Osterhaus, A. D. M. E., Fouchier, R. A. M., van den Hoogen, B. G. (2007). Immunization of Syrian golden hamsters with F subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains. J. Gen. Virol. 88: 2702-2709 [Abstract] [Full Text]  
  • Biacchesi, S., Murphy, B. R., Collins, P. L., Buchholz, U. J. (2007). Frequent Frameshift and Point Mutations in the SH Gene of Human Metapneumovirus Passaged In Vitro. J. Virol. 81: 6057-6067 [Abstract] [Full Text]  
  • van den Hoogen, B. G., Herfst, S., de Graaf, M., Sprong, L., van Lavieren, R., van Amerongen, G., Yuksel, S., Fouchier, R. A. M., Osterhaus, A. D. M. E., de Swart, R. L. (2007). Experimental infection of macaques with human metapneumovirus induces transient protective immunity. J. Gen. Virol. 88: 1251-1259 [Abstract] [Full Text]  
  • Kahn, J. S. (2006). Epidemiology of Human Metapneumovirus. Clin. Microbiol. Rev. 19: 546-557 [Abstract] [Full Text]  
  • Herd, K. A., Mahalingam, S., Mackay, I. M., Nissen, M., Sloots, T. P., Tindle, R. W. (2006). Cytotoxic T-Lymphocyte Epitope Vaccination Protects against Human Metapneumovirus Infection and Disease in Mice. J. Virol. 80: 2034-2044 [Abstract] [Full Text]  
  • Schickli, J. H., Kaur, J., Ulbrandt, N., Spaete, R. R., Tang, R. S. (2005). An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters. J. Virol. 79: 10678-10689 [Abstract] [Full Text]  
  • Buchholz, U. J., Biacchesi, S., Pham, Q. N., Tran, K. C., Yang, L., Luongo, C. L., Skiadopoulos, M. H., Murphy, B. R., Collins, P. L. (2005). Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity. J. Virol. 79: 6588-6597 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»