Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus
Open accessAbstract
Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating…
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In animal models of other coronaviruses and prior inactivated RSV or measles vaccines, VAERD is usually linked to Th2-biased CD4+ responses with elevated IL-4, IL-5, or IL-13.
“In animal models of infections with other coronaviruses and for previous whole-cell inactivated vaccines developed for respiratory syncytial virus and measles virus, VAERD has usually been associated with the development of T helper 2 cell (TH2 cell)-biased CD4+ T cell responses, with significantly increased levels of interleukin-4 (IL-4), IL-5 and/or IL-13 (refs12,126,165–174).”
From Immunological mechanisms of vaccine-induced protection against COVID-19 in humans (2021) · cites this paper as [167]
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- Authors
- Scobey, T., Yount, B. L., Sims, A. C., Donaldson, E. F., Agnihothram, S. S., Menachery, V. D., Graham, R. L., Swanstrom, J., Bove, P. F., Kim, J. D., Grego, S., Randell, S. H., & Baric, R. S.
- Affiliations
- Departments of aEpidemiology,
- University of North Carolina at Chapel Hill (US)
- Cell Biology and Physiology, and
- RTI International (US)
- Corresponding author
- Scobey, Trevor
- Yount, Boyd L.
- Donaldson, Eric F.
- Agnihothram, Sudhakar S.
- Menachery, Vineet D.
- Graham, Rachel L.
- Swanstrom, Jesica
- Baric, Ralph S.
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2013-09-16
- Volume
- 110
- Issue
- 40
- Pages
- 16157-16162
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3791741
- OpenAlex
- W1984494056
- Cited by
- 283
- References
- 1
- Citations by year
- 2026: 8 · 2025: 10 · 2024: 10 · 2023: 20 · 2022: 18 · 2021: 35 · 2020: 66 · 2019: 17 · 2018: 14 · 2017: 19 · 2016: 20 · 2015: 20 · 2014: 23 · 2013: 3
- FWCI
- 16.22
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
- Funders
- National Institutes of Health
- Defense Threat Reduction Agency (HDTRA1-12-, HDTRA1)
- National Institute of Allergy and Infectious Diseases (R01AI108197, AI085524, AI108197, R01AI085524, U19AI100625, U19AI107810)
- Topics
- Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
- Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
- Health Sciences › Medicine › Epidemiology › Virology and Viral Diseases
- Keywords
- MERS-CoV, reverse genetics, recombinant virus, ORF5, accessory genes, dipeptidyl peptidase IV, tissue tropism, Betacoronavirus
- MeSH terms
- Red Fluorescent Protein, Cells, Cultured, Humans, Luminescent Proteins, Middle East, Polymorphism, Restriction Fragment Length, Virus Replication, Blotting, Northern, Blotting, Western, Gene Expression Regulation, Viral, DNA Primers, Coronavirus, DNA, Complementary, Dipeptidyl Peptidase 4, Communicable Diseases, Emerging, Severe Acute Respiratory Syndrome, Virus Attachment, Real-Time Polymerase Chain Reaction, Spike Glycoprotein, Coronavirus
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