An Infectious cDNA Clone of SARS-CoV-2

Xie, Xuping; Muruato, Antonio E.; Lokugamage, Kumari G.; Narayanan, Krishna; Zhang, Xianwen; Zou, Jing; Liu, Jianying; Schindewolf, Craig; Bopp, Nathen E.; Aguilar, Patricia V.; Plante, Kenneth S.; Weaver, Scott C.; Makino, Shinji; LeDuc, James W.; Menachery, Vineet D.; Shi, Pei Yong

doi:10.1016/j.chom.2020.04.004

Cited by 682 publications

(1,005 citation statements)

References 25 publications

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“…The full-length infectious cDNA clone of SARS-CoV-2-Nluc was generated by in vitro ligation of seven contiguous panel of cDNA according to a protocol as reported previously 80 . RNA transcript was in vitro synthesized by the mMESSAGE mMACHINE™ T7 Transcription Kit (ThermoFisher Scientific) and electroporated into Vero E6 cells to recover the recombinant SARS-CoV-2-Nluc by using the same protocol as described reviously 80 . The viral stock was prepared by amplifying the SARS-CoV-2-Nluc on Vero E6 cells for one round (P1).…”

Section: Methodsmentioning

confidence: 99%

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

Xie

Muruato

Zhang

et al. 2020

Preprint

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106

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1A high-throughput platform would greatly facilitate COVID-19 serological testing and 2 antiviral screening. Here we report a nanoluciferase SARS-CoV-2 (SARS-CoV-2-Nluc) that is 3 genetically stable and replicates similarly to the wild-type virus in cell culture. We demonstrate 4 block an authentic viral infection. However, the low throughput and long assay turnaround time 4 2 make PRNT impossible for large scale diagnosis, representing a critical gap for COVID-19 4 3 response and countermeasure development. 4The goals of this study were to (i) develop a rapid neutralization assay that maintains the 4 5 gold standard of PRNT for serological COVID-19 diagnosis, (ii) establish a high-throughput 4 6 assay for reliable antiviral screening, and (ii) screen exploratory and FDA-approved anti-4 7 infective drugs for potential COVID-19 repurposing. We established a nanoluciferase SARS-4 8CoV-2 (SARS-CoV-2-Nluc) as a platform for rapid serodiagnosis and high-throughput drug 4 9

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Section: Methodsmentioning

confidence: 99%

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

Xie

Muruato

Zhang

et al. 2020

Preprint

Self Cite

106

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“…Human enteric viruses that spread via the fecal-oral route typically withstand the harsh environment in the GI tract, including the low pH of gastric fluids, bile, and digestive enzymes in the small intestine, dehydration, and exposure to multiple bacterial byproducts in the colon. We investigated the stability of a recombinant SARS-CoV-2 mNeonGreen reporter virus (44) including taurocholic acid sodium salt and lecithin (Fig. 5A).…”

Section: Sars-cov-2 Is Rapidly Inactivated In the Human Gi Tractmentioning

confidence: 99%

“…SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 was obtained from N. Thornburg at the Centers for Disease Control and Prevention. A mNeonGreen SARS-CoV-2 reporter virus was used as previously reported (44). Virus was passaged once in Vero CCL81 cells (ATCC) and titrated by focus-forming assay in Vero-E6 cells.…”

Section: Plasmids Cells Reagents and Viruses Plasmidsmentioning

confidence: 99%

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

et al. 2020

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Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2 + mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with COVID-19. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression. RESULTS SARS-CoV-2 infects human intestinal enteroidsIn the intestine, ACE2 functions as a chaperone for the sodiumdependent neutral amino acid transporter B 0 AT1 (encoded by SLC6A19) on IECs and regulates microbial homeostasis (21,22). ACE2 expression is substantially higher in the small intestine than in all the other organs, including the lung, in both humans and mice (fig. S1, A and B). Given these data, we assessed whether SARS-CoV-2 can infect IECs as a first step for understanding its implications for fecal-oral transmission. We performed single-cell RNA sequencing (scRNA-seq) to capture the global transcriptomics in all IEC subsets in the mouse small intestinal epithelium (Fig. 1A, left). ACE2 mRNA was predominantly seen in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes (Fig. 1A, right) (23, 24). In addition, bulk RNA-seq revealed that primary human ileum enteroids had substantially higher mRNA levels of all known CoV receptors, including ACE2, than the of 10 that Ace2 high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 10 5 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 10 5 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 m. (E) Same as (C) except that virus titers were measured using a TCID 50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, ...

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“…To address the above gap, we developed a fluorescence-based assay that rapidly and reliably measures neutralization of a reporter SARS-CoV-2 by antibodies from patient specimens. The assay was built on a stable mNeonGreen SARS-CoV-2 where the mNeonGreen gene was engineered at the OFR7 of the viral genome 8 . Fig.…”

Section: Textmentioning

confidence: 99%

A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Muruato

Fontes-Garfias

Ren

et al. 2020

Preprint

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1Virus neutralization remains the gold standard for determining antibody efficacy.2 Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently 3 needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. 4Here we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS- 5CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to 50 µl of DMEM (Gibco) containing 2% FBS (Hyclone) and 100 U/ml Penicillium-Streptomycin

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An Infectious cDNA Clone of SARS-CoV-2

Cited by 682 publications

References 25 publications

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

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