key: cord-0808183-vn6kjy2z
authors: Pu, Tao; Ding, Chen; Li, Yadong; Liu, Xiaojuan; Li, Haiwei; Duan, Jinmei; Zhang, Heng; Bi, Yanwei; Cun, Wei
title: Evaluate severe acute respiratory syndrome coronavirus 2 infectivity by pseudoviral particles
date: 2020-04-25
journal: J Med Virol
DOI: 10.1002/jmv.25865
sha: 983333f01e1dc23ff905c88edc3ac9ef87a984ad
doc_id: 808183
cord_uid: vn6kjy2z

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection in humans in late 2019, it has rapidly spread worldwide. To identify the biological characteristics of SARS‐CoV‐2 in a normal laboratory environment (biosafety level 2 [BSL‐2]), a lentiviral‐based nucleocapsid was used to carry the spike protein of SARS‐CoV‐2 onto the surface of pseudoviral particles as a surrogate model to evaluate the infective characterization of SARS‐CoV‐2. This study indicated that SARS‐CoV‐2 has extensive tissue tropism for humans and may infect monkeys and tree shrews but not rodents. More importantly, the use of pseudoviral particles in this study allows rapid assessment of neutralizing antibodies in serum in a BSL‐2 laboratory. This study will provide a quick and easy tool for evaluating neutralizing antibodies in the serum of recovering patients and assessing the potency of candidate vaccines.

In December 2019, a novel pulmonary cluster infection was found in Wuhan, China. 1 The infected person developed fever, dry cough, dyspnea, gastrointestinal upset, and other severe respiratory syndromes. 2, 3 It was caused by a novel coronavirus infection, and the pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 4 SARS-CoV-2 can be transmitted through saliva, droplets or close contact, and many of its biological characteristics are currently unknown. 2 Both SARS-CoV-2 and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) can cause severe respiratory syndrome.

The SARS-CoV-1 epidemic infection occurred in 2003, and then quickly spread to many regions around the world, causing global public health crisis with strong infectiousness and high mortality. 7, 8 SARS-CoV-2 and SARS-CoV-1 have similarities in structure and bioinformatics, but their homology at the genome level is less than 80%. 9 Some studies have shown that intermediate hosts may be wild animals such as pangolins, 10, 11 and then humans can be infected. The spike protein on the surface of the coronavirus plays a key role in the process of invading cells. Compared with SARS-CoV-1, an important variation of SARS-CoV-2 is the introduction of a furin protease site in the spike protein. 12, 13 It greatly increases the infection efficiency of SARS-CoV-2. With the analysis of the spike protein structure of SARS-CoV-2 by cryoelectron microscopy, it was found that SARS-CoV-2 had stronger binding ability to ACE2 than SARS-CoV-1, 14 and the serum cross-reactivity between SARS-CoV-1 and SARS-CoV-2 is still unknown. Since there are currently no drugs and vaccines that inhibit these viral infections, it is necessary to establish effective systems to assess their effectiveness. Tao Pu and Chen Ding contributed equally to this study.

Both SARS-CoV-1 and SARS-CoV-2 induce highly pathogenic infectious diseases, which need to be operated in a biosafety level 3 (BSL-3) laboratory for biosafety consideration. In this study, SARS-CoV spike protein coated pseudoviral particles (saCoV2pp) were established, which have similar infection characteristics to the virus but no amplification ability. saCoV2pp were used to infect cells derived from different tissues and animal species to evaluate its infective characterization and was successfully used to evaluate neutralizing antibodies in serum.

The Huh7.5 cell line was a gift from the laboratory of Charles M.

Rice; RH35 was obtained from the Institute of Zoology, Chinese Academy of Sciences; immortalized tree shrew liver cells X9.0 and X9.5 and monkey liver cells RHT6.0 were isolated by our laboratory;

Vero, A357, Caco-2, KMB17, Hep2, Hacat, NIH3T3, CHO-K1, HEK293, HEK293T, HL7702, HepG2, Hep1-6, and Huh7 cells were preserved by our laboratory. All the cells were cultured at 37°C with 5% CO 2 in flasks with Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS; Gibco), penicillin, and streptomycin.

We transfected plasmids encoding wild-type or codon-optimized SARS-CoV-2 spike protein into HEK293T cells using jetPRIME transfection reagent. Cells were incubated for 4 hours at 37°C with transfection medium. Then, the transfection medium was replaced with DMEM containing 10% FBS, and the supernatant was removed after 48 hours. The cells were lysed with radioimmunoprecipitation assay, at 10 µg/well; samples were analyzed by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes by the Trans-Blot Turbo TM Transfer System (Bio-Rad). The membrane was blocked with 5% nonfat milk for 1 hour at room temperature and incubated with the primary antibody (cat: 40150-T52; Sino Biological) (1:1000 dilution) for 1 hour at room temperature and washed five times with trisbuffered saline with Tween 20 (TBST) buffer. The goat anti-rabbit secondary antibody (1:5000 dilution) was incubated at room temperature for 1 hour and washed five times with TBST buffer. Finally, signals were visualized using Western Detection Reagent (GE Healthcare).

HEK293T cells were cotransfected with the spike protein-encoding or spike protein knock-out transmembrane domain (SΔTM) plasmid and the lentiviral expression vector carrying the luciferase reporter gene using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were incubated for 6 hours at 37°C with transfection medium, and then the transfection medium was replaced with DMEM containing 10% FBS. The cell supernatant was harvested after 48 hours, centrifuged at 4000 rpm for 15 minutes to remove impurities such as cell debris, and then stored at −80°C.

The cells were plated into a 48-well plate at a density 2 × 10 4 cells/well for 20 hours, the pseudovirus was removed from −80°C refrigeration, rapidly melted in a 37°C water bath, and then placed on ice, mixed with DMEM containing 2% FBS at a ratio of 1:1; then, the medium was removed from the cells, 200 µL of the pseudovirus mixture was added, and the cells were incubated at 37°C with 5% CO 2 for 48 hours. The supernatant was removed, the cells were washed with 200 µL phosphate-buffered saline (PBS), and 40 µL of lysate was lysed (Promega E1531) for 2 to 3 minutes. The lysate was removed into a tube and centrifuged at 5000 rpm for 5 minutes. The precipitate was wiped off, and the supernatant and substrate (Promega E1501) were mixed in a 1:10 ratio. Fluoroskan Ascent FL (Thermo Fisher Scientific) was used to measure the luciferase activity. The values were multiplied by a scaling factor of 10 6 .

Huh7.5 cells were plated in 96-well plates at a density of 1 × 10 4 cells/well for 20 hours. The serum was inactivated at 56°C for 30 minutes, 50 µL serum was mixed with 200 µL pseudovirus (~200 ng p24/mL), and diluted twice using the pseudovirus, making the serum and pseudovirus ratios reach 1:256. Then, the cells were incubated at 37°C with 5% CO 2 for 1 hour. The medium was removed from Huh7.5 cells, 50 µL of medium containing 2% FBS was added.

Then, adding 50 µL neutralized the virus and serum mixture, which was mixed with gentle shaking, incubated at 37°C with 5% CO 2 in an incubator for 48 hours. The supernatant was removed, the cells were washed with PBS, and 20 µL lysate was lysed (Promega E1531) for 2 to 3 minutes, then centrifuged at 12 000 rpm for 5 minutes. The supernatant and substrate (Promega E1501) were mixed at a 1:10 ratio. A Fluoroskan Ascent FL (Thermo Fisher Scientific) was used to measure the luciferase activity. The values were multiplied by a scaling factor of 10 6 .

Data were analyzed by one-way or two-way analysis of variance followed by Brown-Forsythe test or Bartlett's test where appropriate. At least three independent experiments were carried out, and the data from each experiment were expressed as the mean ± SD. P < .05 was considered significant. F I G U R E 1 Detection of the expression of wild-type and codon-optimized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins. The wild-type and codon-optimized spike protein expression plasmids were transfected into HEK293T cells. After 48 hours of transfection, Western blot analysis was performed on the transfected cell lysate to detect the protein expression levels. wt: wild-type; cop: codon optimization; con: control, HEK293T cell lysates F I G U R E 2 SARS-CoV-2 pseudoviral particle infectivity to vaccine-producing cells. The saCoV2pp infection was used in vaccine-producing cells: KMB17, NIH3T3, CHO-K1, HEK293, and Vero. After 48 hours, the supernatant was removed to detect the luciferase activity, and the pseudovirus was prepared. SΔTM in the cell lysate was used as a negative control, and saCoV1pp was used as a positive control. n = 3, P < . 

It is known that saCoV2pp has strong infectivity to human hepato- KMB17 has been successfully used in the production of polio vaccine, 15 hepatitis A vaccine, 16 and EV71 vaccine, 17 Animal models are necessary tools for studying the pathogenic mechanism of viruses. Based on the high sensitivity of SARS-CoV-2

to liver cells, we tested the sensitivity of commonly used animal In clinical tests, a method capable of detecting neutralizing antibodies in patients' serum is still lacking. In addition, the level of neutralizing antibodies in serum is also an important indicator for evaluating the vaccine protection mechanism. In this study, our results showed that the SARS-CoV-2 

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Evaluate severe acute respiratory syndrome coronavirus 2 infectivity by pseudoviral particles

The authors declare that there are no conflict of interests.

TP, CD, and YWB performed the research. JMD and YDL provided the plasmid. XJL, HZ, and HWL analyzed the data. WC designed the research. TP and WC wrote the manuscript.