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mBio. Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate
[Source: mBio, full page: (LINK). Abstract, edited.]
Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate
Fernando Almaz?n<SUP>a</SUP>, Marta L. DeDiego<SUP>a</SUP>, Isabel Sola<SUP>a</SUP>, Sonia Zu?iga<SUP>a</SUP>, Jose L. Nieto-Torres<SUP>a</SUP>, Silvia Marquez-Jurado<SUP>a</SUP>, German Andr?s<SUP>b</SUP>, Luis Enjuanes<SUP>a</SUP>
<SUP></SUP>
Author Affiliations: Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog?a (CNB-CSIC), Campus Universidad Aut?noma de Madrid, Cantoblanco, Madrid, Spain<SUP>a </SUP>Centro de Biolog?a Molecular Severo Ochoa (CBMSO-CSIC-UAM), Campus Universidad Aut?noma de Madrid, Cantoblanco, Madrid, Spain<SUP>b</SUP>
<SUP></SUP>
Address correspondence to Luis Enjuanes, L.Enjuanes@cnb.csic.es.
F.A. and M.L.D. contributed equally to this work.
Editor Michael Buchmeier, University of California, Irvine
ABSTRACT
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection.
IMPORTANCE
Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.
Footnotes
Citation Almaz?n F, DeDiego ML, Sola I, Zu?iga S, Nieto-Torres JL, Marquez-Jurado S, Andr?s G, Enjuanes L. 2013. Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. mBio 4(5):e00650-13. doi:10.1128/mBio.00650-13.
Received 9 August 2013 Accepted 12 August 2013 Published 10 September 2013
Copyright ? 2013 Almaz?n et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate
Fernando Almaz?n<SUP>a</SUP>, Marta L. DeDiego<SUP>a</SUP>, Isabel Sola<SUP>a</SUP>, Sonia Zu?iga<SUP>a</SUP>, Jose L. Nieto-Torres<SUP>a</SUP>, Silvia Marquez-Jurado<SUP>a</SUP>, German Andr?s<SUP>b</SUP>, Luis Enjuanes<SUP>a</SUP>
<SUP></SUP>
Author Affiliations: Department of Molecular and Cell Biology, Centro Nacional de Biotecnolog?a (CNB-CSIC), Campus Universidad Aut?noma de Madrid, Cantoblanco, Madrid, Spain<SUP>a </SUP>Centro de Biolog?a Molecular Severo Ochoa (CBMSO-CSIC-UAM), Campus Universidad Aut?noma de Madrid, Cantoblanco, Madrid, Spain<SUP>b</SUP>
<SUP></SUP>
Address correspondence to Luis Enjuanes, L.Enjuanes@cnb.csic.es.
F.A. and M.L.D. contributed equally to this work.
Editor Michael Buchmeier, University of California, Irvine
ABSTRACT
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection.
IMPORTANCE
Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.
Footnotes
Citation Almaz?n F, DeDiego ML, Sola I, Zu?iga S, Nieto-Torres JL, Marquez-Jurado S, Andr?s G, Enjuanes L. 2013. Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. mBio 4(5):e00650-13. doi:10.1128/mBio.00650-13.
Received 9 August 2013 Accepted 12 August 2013 Published 10 September 2013
Copyright ? 2013 Almaz?n et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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