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Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo.
Virol J. 2009 Aug 24; 6:131.VJ

Abstract

During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells). In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.

Authors+Show Affiliations

Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. pfefferle@bni-hamburg.deNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19698190

Citation

Pfefferle, Susanne, et al. "Reverse Genetic Characterization of the Natural Genomic Deletion in SARS-Coronavirus Strain Frankfurt-1 Open Reading Frame 7b Reveals an Attenuating Function of the 7b Protein In-vitro and In-vivo." Virology Journal, vol. 6, 2009, p. 131.
Pfefferle S, Krähling V, Ditt V, et al. Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. Virol J. 2009;6:131.
Pfefferle, S., Krähling, V., Ditt, V., Grywna, K., Mühlberger, E., & Drosten, C. (2009). Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. Virology Journal, 6, 131. https://doi.org/10.1186/1743-422X-6-131
Pfefferle S, et al. Reverse Genetic Characterization of the Natural Genomic Deletion in SARS-Coronavirus Strain Frankfurt-1 Open Reading Frame 7b Reveals an Attenuating Function of the 7b Protein In-vitro and In-vivo. Virol J. 2009 Aug 24;6:131. PubMed PMID: 19698190.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. AU - Pfefferle,Susanne, AU - Krähling,Verena, AU - Ditt,Vanessa, AU - Grywna,Klaus, AU - Mühlberger,Elke, AU - Drosten,Christian, Y1 - 2009/08/24/ PY - 2009/07/30/received PY - 2009/08/24/accepted PY - 2009/8/25/entrez PY - 2009/8/25/pubmed PY - 2010/2/5/medline SP - 131 EP - 131 JF - Virology journal JO - Virol J VL - 6 N2 - During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells). In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic. SN - 1743-422X UR - https://www.unboundmedicine.com/medline/citation/19698190/Reverse_genetic_characterization_of_the_natural_genomic_deletion_in_SARS_Coronavirus_strain_Frankfurt_1_open_reading_frame_7b_reveals_an_attenuating_function_of_the_7b_protein_in_vitro_and_in_vivo_ L2 - https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-6-131 DB - PRIME DP - Unbound Medicine ER -