Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

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ABSTRACT

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

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Most cited references 22

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A Novel Coronavirus from Patients with Pneumonia in China, 2019

Na Zhu, Dingyu Zhang, Wenling Wang … (2020)

Summary In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.)

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A pneumonia outbreak associated with a new coronavirus of probable bat origin

Peng Zhou, Xing-Lou Yang, Xian-Guang Wang … (2020)

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats 1–4 . Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans 5–7 . Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV.

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Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia

Qun Li, Xuhua Guan, Peng Wu … (2020)

Abstract Background The initial cases of novel coronavirus (2019-nCoV)–infected pneumonia (NCIP) occurred in Wuhan, Hubei Province, China, in December 2019 and January 2020. We analyzed data on the first 425 confirmed cases in Wuhan to determine the epidemiologic characteristics of NCIP. Methods We collected information on demographic characteristics, exposure history, and illness timelines of laboratory-confirmed cases of NCIP that had been reported by January 22, 2020. We described characteristics of the cases and estimated the key epidemiologic time-delay distributions. In the early period of exponential growth, we estimated the epidemic doubling time and the basic reproductive number. Results Among the first 425 patients with confirmed NCIP, the median age was 59 years and 56% were male. The majority of cases (55%) with onset before January 1, 2020, were linked to the Huanan Seafood Wholesale Market, as compared with 8.6% of the subsequent cases. The mean incubation period was 5.2 days (95% confidence interval [CI], 4.1 to 7.0), with the 95th percentile of the distribution at 12.5 days. In its early stages, the epidemic doubled in size every 7.4 days. With a mean serial interval of 7.5 days (95% CI, 5.3 to 19), the basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9). Conclusions On the basis of this information, there is evidence that human-to-human transmission has occurred among close contacts since the middle of December 2019. Considerable efforts to reduce transmission will be required to control outbreaks if similar dynamics apply elsewhere. Measures to prevent or reduce transmission should be implemented in populations at risk. (Funded by the Ministry of Science and Technology of China and others.)

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Author and article information

Journal

Journal ID (nlm-ta): Emerg Microbes Infect

Journal ID (iso-abbrev): Emerg Microbes Infect

Journal ID (publisher-id): TEMI

Journal ID (publisher-id): temi20

Title: Emerging Microbes & Infections

Publisher: Taylor & Francis

ISSN (Electronic): 2222-1751

Publication date Collection: 2020

Publication date (Electronic): 24 March 2020

Volume: 9

Issue: 1

Pages: 680-686

Affiliations

[a ]Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) , Beijing, People’s Republic of China

[b ]Graduate School of Peking Union Medical College , Beijing, People’s Republic of China

[c ]Wuhan Institute of Biological Products , Wuhan, People’s Republic of China

[d ]Division of Animal Model Research, Institute for Laboratory Animal Resources, National Institutes for Food and Drug Control , Beijing, People’s Republic of China

[e ]Institute for Biological Product Control, National Institutes for Food and Drug Control , Beijing, People’s Republic of China

Author notes

[CONTACT ] Youchun Wang wangyc@ 123456nifdc.org.cns Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) , No. 31 Huatuo Street, Daxing District, Beijing 102629, People’s Republic of China; Graduate School of Peking Union Medical College, No. 9 Dongdan Santiao, Dongcheng District, Beijing 100730, People’s Republic of China

Weijin Huang huangweijin@ 123456nifdc.org.cn Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) , No. 31 Huatuo Street, Daxing District, Beijing 102629, People’s Republic of China

Miao Xu xumiaobj@ 123456126.com Institute for Biological Product Control, National Institutes for Food and Drug Control , Beijing 100050, People’s Republic of China

[*]

These authors contributed equally to this work.

Article

Publisher ID: 1743767

DOI: 10.1080/22221751.2020.1743767

PMC ID: 7144318

PubMed ID: 32207377

SO-VID: 75e5b2bd-fe09-4d93-9092-d6a57c199f16

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This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 24 February 2020

Date revision received : 10 March 2020

Date accepted : 11 March 2020

Page count

Figures: 5, Tables: 0, Equations: 0, References: 27, Pages: 7

Funding

Funded by: National Science and Technology Major Projects of Drug Discovery

Award ID: 2018ZX09101001

This work was supported by National Science and Technology Major Projects of Drug Discovery [grant number 2018ZX09101001].

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

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Novel Coronavirus Disease COVID-19

Most cited references 22

A Novel Coronavirus from Patients with Pneumonia in China, 2019

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia

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