Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States

Harcourt, Jennifer L.; Tamin, Azaibi; Lu, Xiaoyan; Kamili, Shifaq; Sakthivel, Senthil Kumar K.; Murray, Janna; Queen, Krista; Tao, Ying; Paden, Clinton R.; Zhang, Jing; Li, Yan; Uehara, Anna; Wang, Haibin; Goldsmith, Cynthia S.; Bullock, Hannah; Wang, Lijuan; Whitaker, Brett; Lynch, Brian; Gautam, Rashi; Schindewolf, Craig; Lokugamage, Kumari G.; Scharton, Dionna; Plante, Jessica A.; Mirchandani, Divya; Widen, Steven G.; Narayanan, Krishna; Makino, Shinji; Ksiazek, Thomas G.; Plante, Kenneth S.; Weaver, Scott C.; Lindstrom, Stephen; Tong, Suxiang; Menachery, Vineet D.; Thornburg, Natalie J.

doi:10.3201/eid2606.200516

Cited by 571 publications

(614 citation statements)

References 24 publications

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“…SARS-CoV-2 USA-WA1/2020, provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) and was originally obtained from the USA Centers of Disease Control as described 50 . SARS-CoV-2 and mouse-adapted recombinant SARS-CoV (MA15) 46 were titrated and propagated on VeroE6 cells, grown in DMEM with 5% fetal bovine serum and 1% antibiotic/antimytotic (Gibco).…”

Section: Methodsmentioning

confidence: 99%

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Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV

Lokugamage

Hage

Vries

et al. 2020

Preprint

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Article Summary: SARS-CoV-2 has similar replication kinetics to SARS-CoV, but demonstrates significant sensitivity to type I interferon treatment. Running title: SARS-CoV-2 sensitive to type I IFN pretreatmentAbstract 1 SARS-CoV-2, a novel coronavirus (CoV), has recently emerged causing an ongoing outbreak of 2 viral pneumonia around the world. While genetically distinct from the original SARS-CoV, both 3 group 2B coronaviruses share similar genome organization and origins to coronaviruses 4 harbored in bats. Importantly, initial guidance has used insights from SARS-CoV infection to 5 inform treatment and public health strategies. In this report, we evaluate SARS-CoV-2 relative to 6 the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral 7 replication kinetics to SARS-CoV in Vero cell, the novel coronavirus is much more sensitive to 8 type I interferon pretreatment. We subsequently examined homology between SARS-CoV and 9SARS-CoV-2 in viral proteins shown to be interferon antagonist. The absence of open reading 10 frame (ORF) 3b and significant changes to ORF6 suggest the two key IFN antagonists may not 11 maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in 12 susceptibility to the IFN response between SARS-CoV and SARS-CoV-2 that could help inform 13 disease progression, treatment options, and animal model development.

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Section: Methodsmentioning

confidence: 99%

“…Viral and host protein analysis were evaluated as previously described 50,54 . Briefly, cell lysates were resolved on 7.5% Mini-PROTEAN TGX SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes using a Trans-Blot Turbo transfer system (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV

Lokugamage

Hage

Vries

et al. 2020

Preprint

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279

268

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“…SARS-CoV-2 clinical isolate was obtained at VUMC and UNC from the CDC (2019-nCoV/USA-WA1/2020 strain, GenBank accession no. MN985325.1) and passaged twice in Vero E6 cells at each respective institution to create a passage 5 working stock (54 (55). The virus stock utilized for MERS-CoV in vivo studies was derived from a plaque-purified isolate of the mouse-adapted MERS-CoV p35C4 strain (56).…”

Section: Virus Strainsmentioning

confidence: 99%

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

et al. 2020

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Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N4-hydroxycytidine-5′-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

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“…We designed an in vitro ligation approach, similar to that used for constructing the infectious clones of SARS-CoV and MERS-CoV (Scobey et al, 2013;Yount et al, 2003), to directionally assemble the full-length complimentary DNA (cDNA) of the SARS-CoV-2 genome ( Figure 1A). Our reverse genetic system was based on the virus strain (2019-nCoV/USA_WA1/2020) isolated from the first reported SARS-CoV-2 case in the US (Harcourt et al, 2020;Holshue et al, 2020). Viral RNA, extracted from the passage 4 virus from Vero E6 cells, was used as a template for RT-PCR to produce cDNA fragments.…”

Section: Design Of a Sars-cov-2 Full-length Cdnamentioning

confidence: 99%

An Infectious cDNA Clone of SARS-CoV-2

Xie

Muruato

Lokugamage

et al. 2020

Cell Host & Microbe

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673

943

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Graphical Abstract Highlights d A reverse genetic system has been established for SARS-CoV-2 d Recombinant SARS-CoV-2 replicates as efficiently as the original clinical isolate d A stable mNeonGreen reporter SARS-CoV-2 has been developed d The mNeonGreen SARS-CoV-2 can be used to screen antiviral inhibitors SUMMARYThe ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 3 10 6 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectiousclone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures. -Y.S. have filed a provisional patent on the reverse genetic system of SARS-CoV-2. Other authors have no conflicts of interest to declare.

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Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States

Cited by 571 publications

References 24 publications

Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV

Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

An Infectious cDNA Clone of SARS-CoV-2

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