Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate
Fernando Almazán,Marta L. DeDiego,Isabel Sola,Sonia Zuñiga,Jose L. Nieto-Torres,Silvia Márquez-Jurado,Germán Andrés,Luis Enjuanes +7 more
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TLDR
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.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.read more
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Modulation of the immune response by Middle East respiratory syndrome coronavirus.
TL;DR: It is demonstrated that M, 4a, 4b proteins and Plppro of MERS‐CoV inhibit the type I interferon (IFN) and nuclear factor‐κB signaling pathways and therefore facilitate innate immune evasion, and limit early induction of IFN and cause rapid apoptosis of macrophages.
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Structural insight into the role of novel SARS-CoV-2 E protein: A potential target for vaccine development and other therapeutic strategies.
TL;DR: This work modelled the structure of the envelope (E)-protein of novel SARS-CoV-2 and proposed this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis.
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Coronaviruses: severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus in travelers
TL;DR: All physicians and travelers to the Middle East should be aware of the new threat caused by MERS-CoV and follow CDC and WHO guidelines and those who develop ill health during their trip or soon after their return should seek medical care.
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Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras
TL;DR: The results clarify the understanding of the interaction between the coronavirus M protein and the nucleocapsid protein and reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.
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Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome
Chengjin Ye,Kevin Chiem,Jun-Gyu Park,Fatai S. Oladunni,Fatai S. Oladunni,Roy N. Platt,Tim J. Anderson,Fernando Almazán,Juan Carlos de la Torre,Luis Martinez-Sobrido +9 more
TL;DR: This is the first description of a BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate and will facilitate studies addressing several important questions in the biology of SARS- CoV- 2.
References
More filters
Journal ArticleDOI
Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method
TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
Journal ArticleDOI
Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia
Ali Moh Zaki,Sander van Boheemen,Theo M. Bestebroer,Albert D. M. E. Osterhaus,Ron A. M. Fouchier +4 more
TL;DR: The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
Journal ArticleDOI
Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC
V. Stalin Raj,Huihui Mou,Saskia L. Smits,Dick H. W. Dekkers,Marcel A. Müller,Ronald Dijkman,Doreen Muth,Jeroen Demmers,Ali Moh Zaki,Ron A. M. Fouchier,Volker Thiel,Volker Thiel,Christian Drosten,Peter J. M. Rottier,Albert D. M. E. Osterhaus,Berend Jan Bosch,Bart L. Haagmans +16 more
TL;DR: Dipeptidyl peptidase 4 (DPP4; also known as CD26) is identified as a functional receptor for hCoV-EMC and will contribute critically to the understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.
Journal ArticleDOI
Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.
Hiroaki Shizuya,Bruce Birren,Ung-Jin Kim,Valeria Mancino,Tatiana Slepak,Yoshiaki Tachiiri,Melvin I. Simon +6 more
TL;DR: Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.
Journal ArticleDOI
Coronaviruses post-SARS: update on replication and pathogenesis.
Stanley Perlman,Jason Netland +1 more
TL;DR: This Review focuses on recent advances in the understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis and the recent identification of numerous novel coronaviruses.