Publications by authors named "Xianwen Zhang"

80 Publications

The N501Y spike substitution enhances SARS-CoV-2 transmission.

bioRxiv 2021 Mar 9. Epub 2021 Mar 9.

Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.
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http://dx.doi.org/10.1101/2021.03.08.434499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986995PMC
March 2021

A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence.

Cell 2021 04 23;184(8):2229-2238.e13. Epub 2021 Feb 23.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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http://dx.doi.org/10.1016/j.cell.2021.02.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901297PMC
April 2021

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

JACC Basic Transl Sci 2021 Apr 26;6(4):331-345. Epub 2021 Feb 26.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

There is ongoing debate as to whether cardiac complications of coronavirus disease-2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology and provides a model system to study this emerging disease.
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http://dx.doi.org/10.1016/j.jacbts.2021.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909907PMC
April 2021

Visualization and Analysis in the Field of Pan-Cancer Studies and Its Application in Breast Cancer Treatment.

Front Med (Lausanne) 2021 17;8:635035. Epub 2021 Feb 17.

School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, China.

Although all cancers are molecularly distinct, many share common driver mutations. Pan-cancer analysis, utilizes next-generation sequencing (NGS), pan-cancer model systems, and pan-cancer projects such as The Cancer Genome Atlas (TCGA), to assess frequently mutated genes and other genomic abnormalities that are common among many cancer types, regardless of the tumor origin, providing new directions for tumor biology research. However, there is currently no study that has objectively analyzed the results of pan-cancer studies on cancer biology. For this study, 999 articles on pan-cancer published from 2006 to 2020 were obtained from the Scopus database, and bibliometric methods were used to analyze citations, international cooperation, co-authorship and keyword co-occurrence clusters. Furthermore, we also focused on and summarized the application of pan-cancer in breast cancer. Our result shows that the pan-cancer studies were first published in 2006 and entered a period of rapid development after 2013. So far, 86 countries have carried out international cooperation in sharing research. Researchers form the United States and Canada have published the most articles and have made the most extensive contribution to this field, respectively. Through author keyword analysis of the 999 articles, TCGA, biomarkers, NGS, immunotherapy, DNA methylation, prognosis, and several other keywords appear frequently, and these terms are hot spots in pan-cancer studies. There are four subtypes of breast cancer (luminalA, luminalB, HER2, and basal-like) according to pan-cancer analysis of breast cancer. Meanwhile, it was found that breast cancer has genetic similarity to pan-gynecological cancers, such as ovarian cancer, which indicates related etiology and possibly similar treatments. Collectively, with the emergence of new detection methods, new cancer databases, and the involvement of more researchers, pan-cancer analyses will play a greater role in cancer biology research.
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http://dx.doi.org/10.3389/fmed.2021.635035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926202PMC
February 2021

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies.

Nat Med 2021 04 4;27(4):717-726. Epub 2021 Mar 4.

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.
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http://dx.doi.org/10.1038/s41591-021-01294-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058618PMC
April 2021

Thioredoxin-1 Rescues MPP/MPTP-Induced Ferroptosis by Increasing Glutathione Peroxidase 4.

Mol Neurobiol 2021 Feb 26. Epub 2021 Feb 26.

Laboratory of Molecular Neurobiology, Medical School, Kunming University of Science and Technology, No.727 Jingming South Road, Kunming, 650500, China.

Parkinson's disease (PD), a common neurodegenerative disease, is typically associated with the loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc). Ferroptosis is a newly identified cell death, which associated with iron accumulation, glutathione (GSH) depletion, lipid peroxidation formation, reactive oxygen species (ROS) accumulation, and glutathione peroxidase 4 (GPX4) reduction. It has been reported that ferroptosis is linked with PD.Thioredoxin-1 (Trx-1) is a redox regulating protein and plays various roles in regulating the activity of transcription factors and inhibiting apoptosis. However, whether Trx-1 plays the role in regulating ferroptosis involved in PD is still unknown. Our present study showed that 1-methyl-4-phenylpyridinium (MPP) decreased cell viability, GPX4, and Trx-1, which were reversed by Ferrostatin-1 (Fer-1) in PC 12 cells and SH-SY5Y cells. Moreover, the decreased GPX4 and GSH, and increased ROS were inhibited by Fer-1 and Trx-1 overexpression. We further repeated that behavior deficits resulted from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were improved in Trx-1 overexpression transgenic mice. Trx-1 reversed the decreases of GPX4 and tyrosine hydroxylase (TH) induced by MPTP in the substantia nigra pars compacta (SNpc). Our results suggest that Trx-1 inhibits ferroptosis in PD through regulating GPX4 and GSH.
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http://dx.doi.org/10.1007/s12035-021-02320-1DOI Listing
February 2021

Chemical Constituents and their Antioxidant, Anti-Inflammatory and Anti-Acetylcholinesterase Activities from Pholidota cantonensis.

Plant Foods Hum Nutr 2021 Mar 23;76(1):105-110. Epub 2021 Feb 23.

Clinical Medical College, Yangzhou University, Yangzhou, 225001, China.

Alzheimer's disease (AD) has the third highest health expenditures after heart disease and cancer. It has emerged as a serious global health issue. The discovery of new drugs to prevent and treat AD is of utmost importance. Pholidota cantonensis is an edible medicinal plant consumed in China. It is widely used in traditional Chinese medicine to treat various diseases. P. cantonensis has been reported to have antioxidant, anti-inflammatory, antitumor and antibacterial activities. Among these properties, its potent antioxidant activity has attracted our attention, since oxidative stress is one of the important pathological mechanisms involved in AD. This study aimed to isolate the compounds from the active extract and evaluate their bioactivities. Fifteen compounds, including one new compound, were obtained. The isolates were tested for 2,2'-diphenyl-1-picrylhydrazyl (DPPH)/2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, anti-acetylcholinesterase (anti-AChE) activities and inhibitory effects on nitrogen monoxide (NO) release in the BV-2 cells. Compounds 1, 2, 4, 6, 8, and 13-15 exhibited two kinds of AD-associated bioactivities. More importantly, compound 13 showed more potent NO inhibitory activity (IC = 0.72 ± 0.08 μM) than the positive control quercetin (IC = 12.94 ± 0.08 μM). Compound 13 also had a higher inhibitory rate (99.59 ± 0.43%) on AChE than that of the positive control galantamine (78.32 ± 1.16%) at the concentrate of 50 μg/mL. Our studies provide new insights into this plant in terms of its potential in the development of new multi-target anti-Alzheimer's disease (anti-AD) drugs.
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http://dx.doi.org/10.1007/s11130-020-00874-4DOI Listing
March 2021

SARS-CoV-2 variants show resistance to neutralization by many monoclonal and serum-derived polyclonal antibodies.

Res Sq 2021 Feb 10. Epub 2021 Feb 10.

The University of Texas Medical Branch at Galveston.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 106 million people and causing 2.3 million deaths. The rapid deployment of antibody-based countermeasures has provided hope for curtailing disease and ending the pandemic . However, the emergence of rapidly-spreading SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), and elsewhere with mutations in the spike protein has raised concern for escape from neutralizing antibody responses and loss of vaccine efficacy based on preliminary data with pseudoviruses . Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera, and human sera from recipients of the Pfizer-BioNTech (BNT162b2) mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, a chimeric Washington strain with a South African spike gene (Wash SA-B.1.351), and isogenic recombinant variants with designed mutations or deletions at positions 69-70, 417, 484, 501, and/or 614 of the spike protein. Several highly neutralizing mAbs engaging the receptor binding domain (RBD) or N-terminal domain (NTD) lost inhibitory activity against Wash SA-B.1.351 or recombinant variants with an E484K spike mutation. Most convalescent sera and virtually all mRNA vaccine-induced immune sera tested showed markedly diminished neutralizing activity against the Wash SA-B.1.351 strain or recombinant viruses containing mutations at position 484 and 501. We also noted that cell line selection used for growth of virus stocks or neutralization assays can impact the potency of antibodies against different SARS-CoV-2 variants, which has implications for assay standardization and congruence of results across laboratories. As several antibodies binding specific regions of the RBD and NTD show loss-of-neutralization potency against emerging variants, updated mAb cocktails, targeting of highly conserved regions, enhancement of mAb potency, or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection .
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http://dx.doi.org/10.21203/rs.3.rs-228079/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885928PMC
February 2021

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera.

Nat Med 2021 04 8;27(4):620-621. Epub 2021 Feb 8.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

We engineered three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion + N501Y + D614G from UK; and E484K + N501Y + D614G from SA. Neutralization geometric mean titers (GMTs) of 20 BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.
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http://dx.doi.org/10.1038/s41591-021-01270-4DOI Listing
April 2021

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K, and N501Y variants by BNT162b2 vaccine-elicited sera.

bioRxiv 2021 Jan 27. Epub 2021 Jan 27.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston TX, U.S.A.

We engineered three SARS-CoV-2 viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion+N501Y+D614G from UK; and E484K+N501Y+D614G from SA. Neutralization geometric mean titers (GMTs) of twenty BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.
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http://dx.doi.org/10.1101/2021.01.27.427998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852264PMC
January 2021

Engineering SARS-CoV-2 using a reverse genetic system.

Nat Protoc 2021 03 29;16(3):1761-1784. Epub 2021 Jan 29.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.
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http://dx.doi.org/10.1038/s41596-021-00491-8DOI Listing
March 2021

A -complementation system for SARS-CoV-2.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a -complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The -complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the -complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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http://dx.doi.org/10.1101/2021.01.16.426970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836106PMC
January 2021

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

Nature 2021 03 25;591(7849):293-299. Epub 2021 Jan 25.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
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http://dx.doi.org/10.1038/s41586-021-03237-4DOI Listing
March 2021

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape.

Nat Commun 2021 01 20;12(1):469. Epub 2021 Jan 20.

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, 77030, USA.

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.
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http://dx.doi.org/10.1038/s41467-020-20789-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817669PMC
January 2021

Bibliometric Analysis of ATAC-Seq and Its Use in Cancer Biology via Nucleic Acid Detection.

Front Med (Lausanne) 2020 3;7:584728. Epub 2020 Nov 3.

Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Disease, Department of Spine Surgery, The Third Affiliated Hospital of Southern Medical University (Academy of Orthopedics of Guangdong Province), Guangzhou, China.

Assay for transposase-accessible chromatin using sequencing (ATAC-seq) is associated with significant progress in biological research and has attracted increasing attention. However, the impact of ATAC-seq on cancer biology has not been objectively analyzed. We categorized 440 ATAC-seq publications according to the publication date, type, field, and country. R 3.6.2 was used to analyze the distribution of research fields. VOSviewer was used for country co-authorship and author co-authorship analyses, and GraphPad Prism 8 was used for correlation analyses of the factors that may affect the number of articles published in different countries. We found that ATAC-seq plays roles in carcinogenesis, anticancer immunity, targeted therapy, and metastasis risk predictions and is most frequently used in studies of leukemia among all types of cancer. We found a significantly strong correlation between the top 10 countries in terms of the number of publications and the gross expenditure on research and development (R&D), the number of universities, and the number of researchers. At present, ATAC-seq technology is undergoing a period of rapid development, making it inseparable from the emphasis and investment in scientific research by many countries. Collectively, ATAC-seq has advantages in the study of the cancer mechanisms because it can detect nucleic acids and thus has good application prospects in the field of cancer, especially in leukemia studies. As a country's economic strength increases and the emphasis on scientific research deepens, ATAC-seq will definitely play a more significant role in the field of cancer biology.
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http://dx.doi.org/10.3389/fmed.2020.584728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670091PMC
November 2020

Spike mutation D614G alters SARS-CoV-2 fitness.

Nature 2021 04 26;592(7852):116-121. Epub 2020 Oct 26.

Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
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http://dx.doi.org/10.1038/s41586-020-2895-3DOI Listing
April 2021

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

Nat Commun 2020 10 15;11(1):5214. Epub 2020 Oct 15.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-020-19055-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567097PMC
October 2020

Exploring Heat-Response Mechanisms of MicroRNAs Based on Microarray Data of Rice Post-meiosis Panicle.

Int J Genomics 2020 17;2020:7582612. Epub 2020 Sep 17.

Key Laboratory for Crop Gene Engineering of Hunan Province, Hunan Agricultural University, Changsha 410128, China.

To explore heat response mechanisms of mircoRNAs (miRNAs) in rice post-meiosis panicle, microarray analysis was performed on RNA isolated from rice post-meiosis panicles which were treated at 40°C for 0 min, 10 min, 20 min, 60 min, and 2 h. By integrating paired differentially expressed (DE) miRNAs and mRNA expression profiles, we found that the expression levels of 29 DE-miRNA families were negatively correlated to their 178 DE-target genes. Further analysis showed that the majority of miRNAs in 29 DE-miRNA families resisted the heat stress by downregulating their target genes and a time lag existed between expression of miRNAs and their target genes. Then, GO-Slim classification and functional identification of these 178 target genes showed that (1) miRNAs were mainly involved in a series of basic biological processes even under heat conditions; (2) some miRNAs might play important roles in the heat resistance (such as osa-miR164, osa-miR166, osa-miR169, osa-miR319, osa-miR390, osa-miR395, and osa-miR399); (3) osa-miR172 might play important roles in protecting the rice panicle under the heat stress, but osa-miR437, osa-miR418, osa-miR164, miR156, and miR529 might negatively affect rice fertility and panicle flower; and (4) osa-miR414 might inhibit the flowering gene expression by downregulation of LOC_Os 05g51830 to delay the heading of rice. Finally, a heat-induced miRNA-PPI (protein-protein interaction) network was constructed, and three miRNA coregulatory modules were discovered.
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http://dx.doi.org/10.1155/2020/7582612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519984PMC
September 2020

Evasion of Type I Interferon by SARS-CoV-2.

Cell Rep 2020 10 19;33(1):108234. Epub 2020 Sep 19.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and host immune response determine coronavirus disease 2019 (COVID-19), but studies evaluating viral evasion of immune response are lacking. Here, we use unbiased screening to identify SARS-CoV-2 proteins that antagonize type I interferon (IFN-I) response. We found three proteins that antagonize IFN-I production via distinct mechanisms: nonstructural protein 6 (nsp6) binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, nsp13 binds and blocks TBK1 phosphorylation, and open reading frame 6 (ORF6) binds importin Karyopherin α 2 (KPNA2) to inhibit IRF3 nuclear translocation. We identify two sets of viral proteins that antagonize IFN-I signaling through blocking signal transducer and activator of transcription 1 (STAT1)/STAT2 phosphorylation or nuclear translocation. Remarkably, SARS-CoV-2 nsp1 and nsp6 suppress IFN-I signaling more efficiently than SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Thus, when treated with IFN-I, a SARS-CoV-2 replicon replicates to a higher level than chimeric replicons containing nsp1 or nsp6 from SARS-CoV or MERS-CoV. Altogether, the study provides insights on SARS-CoV-2 evasion of IFN-I response and its potential impact on viral transmission and pathogenesis.
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http://dx.doi.org/10.1016/j.celrep.2020.108234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501843PMC
October 2020

Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility.

Res Sq 2020 Sep 10. Epub 2020 Sep 10.

The University of Texas Medical Branch at Galveston.

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an "up" conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development.
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http://dx.doi.org/10.21203/rs.3.rs-70482/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491579PMC
September 2020

GPR120 promotes radiation resistance in esophageal cancer via regulating AKT and apoptosis pathway.

Anticancer Drugs 2021 01;32(1):53-60

Department of Radiation Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, P.R. China.

The aim of the study is to investigate the role of GPR120 on the biological behavior of esophageal cancer cells in the setting of radiation and explore the mechanism. GPR120 knockdown was fulfilled by siRNA-mediated effects in two esophageal cancer cell lines Eca109 and EC9706. Colony formation, survival fraction calculation, viable cell evaluation by cell counting kit-8 assay and cell apoptosis analysis by phycoerythrin annexin V and 7-amino-actinomycin (7-AAD) staining and the flow cytometry examination was evaluated in Eca109 and EC9706 under the treatment of different radiation dosage. The mechanisms were explored by the evaluation of the Akt pathway and apoptosis protein level. Significantly decreased GPR120 mRNA and protein after GPR120 siRNA treatment compared to control siRNA treatment. Significantly decreased colony formation was found in GPR120 siRNA-treated Eca109 and EC9706 cells compared to control siRNA-treated cells at the radiation dosage of 2, 4, 6 and 8 Gy. Moreover, decreased survival fraction number with increased sensitive enhancing ratio was also found in GPR120 siRNA-treated Eca109 and EC9706 cells compared to control siRNA-treated cells. Decreased cell viability and increased cell apoptosis in GPR120 siRNA-treated esophageal cancer cells. GPR120 siRNA decreased the Akt phosphorylation and anti-apoptotic Bcl-2 expression level, but increased pro-apoptotic Bim expression level in esophageal cancer cell lines. GPR120 regulated the biological behavior of the esophageal cancer cells via affecting Akt pathway and apoptosis molecules. Moreover, GPR120 siRNA combined radiation treatment could be a therapeutic choice for esophageal cancer.
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http://dx.doi.org/10.1097/CAD.0000000000000971DOI Listing
January 2021

Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility.

bioRxiv 2020 Sep 2. Epub 2020 Sep 2.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston TX, USA.

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an "up" conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development.

Importance: Understanding the evolution of SARS-CoV-2 during the COVID-19 pandemic is essential for disease control and prevention. A spike protein mutation D614G emerged and became dominant soon after the pandemic started. By engineering the D614G mutation into an authentic wild-type SARS-CoV-2 strain, we demonstrate the importance of this mutation to (i) enhanced viral replication on human lung epithelial cells and primary human airway tissues, (ii) improved viral fitness in the upper airway of infected hamsters, and (iii) increased susceptibility to neutralization. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.
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http://dx.doi.org/10.1101/2020.09.01.278689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480025PMC
September 2020

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis.

bioRxiv 2020 Aug 26. Epub 2020 Aug 26.

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT ) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays.

Importance: As COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis.

Article Summary: A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization .
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http://dx.doi.org/10.1101/2020.08.26.268854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457603PMC
August 2020

Bibliometrics Analysis of Butyrophilins as Immune Regulators [1992-2019] and Implications for Cancer Prognosis.

Front Immunol 2020 30;11:1187. Epub 2020 Jun 30.

The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan, China.

The butyrophilins (BTNs) represent a unique family of immunoglobulin. They were considered to be involved in milk lactation after their discovery in 1981. With the development of research, an increasing number of research revealed that BTNs play important roles in immune regulation [1992-2019]. Our research aimed to summarize the BTN research status and their relationship with lung cancers and breast cancers by bibliometrics and bioinformatics methods. Our results indicate that the researches on immune-regulatory functions of BTNs gradually developed from 1992 to 2006, whereas they increased quickly after 2007. There are international cooperations among 56 countries, of which the United States is the most active one with the highest number of studies as well as highest citations. By coauthorship and cocitation analysis, we showed that Adrian Hayday, who is active in γδ T-cell field, was an active author in BTN publications with average year of 2015 and led a subfield. By keywords co-occurrence analysis, we found that γδ T cell, which is an important cancer immune regulator, is one important hotspot. Finally, we found that several BTN members' expression levels were significantly correlated with prognosis of lung cancer and breast cancer patients. Thus, these BTNs might play immune regulatory effects and could serve as potential biomarkers for cancer.
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http://dx.doi.org/10.3389/fimmu.2020.01187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338374PMC
April 2021

Research on Water Cone Behavior in a Heavy Oil Reservoir with Bottom Water Considering the Starting Pressure Gradient.

ACS Omega 2020 Jul 28;5(27):16841-16847. Epub 2020 Jun 28.

School of Energy Resources, China University of Geosciences (Beijing), Beijing 100083, China.

Aiming at the unclear reorganization of the water cone shape and its flooding scope for a horizontal well in a heavy oil reservoir with bottom water, a new method was proposed in this paper to establish a numerical model with the starting pressure gradient (SPG) by commercial software to study water cone behavior. The results show that there exists SPG in heavy oil reservoirs ranging from 10 to 10 magnitude with mobility under 30 μm/mPa·s. With a mobility of 33 μm/mPa·s, the water cone and flooding scope from the model with SPG is 120 m shorter than that without SPG. Upon increasing the mobility from 11 to 90 μm/mPa·s, the flooding scope of the model with SPG changes from 125 to 315 m, showing a power exponential form. The new method proposed in this paper was significant for the water cone behavior study and has broad applications in heavy oil reservoir development in the future.
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http://dx.doi.org/10.1021/acsomega.0c02036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364788PMC
July 2020

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

bioRxiv 2020 Jun 23. Epub 2020 Jun 23.

A high-throughput platform would greatly facilitate COVID-19 serological testing and antiviral screening. Here we report a nanoluciferase SARS-CoV-2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. We demonstrate that the optimized reporter virus assay in Vero E6 cells can be used to measure neutralizing antibody activity in patient sera and produces results in concordance with a plaque reduction neutralization test (PRNT). Compared with the low-throughput PRNT (3 days), the SARS-CoV-2-Nluc assay has substantially shorter turnaround time (5 hours) with a high-throughput testing capacity. Thus, the assay can be readily deployed for large-scale vaccine evaluation and neutralizing antibody testing in humans. Additionally, we developed a high-throughput antiviral assay using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2). When tested against this reporter virus, remdesivir exhibited substantially more potent activity in A549-hACE2 cells compared to Vero E6 cells (EC 0.115 vs 1.28 μM), while this difference was not observed for chloroquine (EC 1.32 vs 3.52 μM), underscoring the importance of selecting appropriate cells for antiviral testing. Using the optimized SARS-CoV-2-Nluc assay, we evaluated a collection of approved and investigational antivirals and other anti-infective drugs. Nelfinavir, rupintrivir, and cobicistat were identified as the most selective inhibitors of SARS-CoV-2-Nluc (EC 0.77 to 2.74 μM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 μM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.06.22.165712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325177PMC
June 2020

Integrated transcriptome and microRNA profiles analysis reveals molecular mechanisms underlying the consecutive monoculture problem of Polygonatum odoratum.

Cell Mol Biol (Noisy-le-grand) 2020 May 15;66(2):47-52. Epub 2020 May 15.

College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China.

Polygonatum odoratum is a historically traditional Chinese medicine plant. However, the consecutive monoculture problem (CMP) widespread in other Chinese medicine limiting their cultivation on a large scale. In this study, the physiological data showed the adverse effect of CMP on the growth of P. odoratum under the consecutive cropping (CC) compared with the first cropping (FC). Then the high-throughput sequencing of miRNA and mRNA libraries of leaves and roots from FC and CC P. odoratum plants identified 671 differentially expressed genes (DEGs) and 184 differentially expressed miRNAs and revealed that the DEGs and target genes of the miRNAs were mainly involved in starch and sucrose metabolism, phenylpropanoid and brassinosteroid biosynthesis. The KEGG analysis revealed that the DEGs between CC and FC roots were enriched in the plant-pathogen interaction pathway. This study provided the expression regulation of genes related to CMP of P. odoratum but also suggested that CMP may result in the serious damage of pathogens to roots and cause the slow growth in the consecutive cropping plants.
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May 2020

Dynamic information of the time-dependent tobullian biomolecular structure using a high-accuracy size-dependent theory.

J Biomol Struct Dyn 2020 May 13:1-16. Epub 2020 May 13.

School of Mechanical Engineering, Hangzhou Dianzi University, Hangzhou, China.

As the most rigid cytoskeletal filaments, tubulin-labeled microtubules bear compressive forces in living cells, balancing the tensile forces within the cytoskeleton to maintain the cell shape. The current structure is often under several environmental conditions as well as various dynamic or static loads that can decrease the stability of the viscoelastic tubulin-labeled microtubules. For this issue, the dynamic stability analysis of size-dependent viscoelastic tubulin-labeled microtubules using modified strain gradient theory by considering the exact three-length scale parameter. Viscoelastic properties are modeled using Kelvin-Voight model to study the time-dependent tubulin-labeled microtubules structure. By applying energy methods (known as Hamilton's principle), the motion equations of the tubulin-labeled microtubules are developed. The dynamic equations are based on first-order shear deformation theory (FSDT), and generalized differential quadrature and fourth-order Runge-Kutta methods are employed to find the model for the natural frequencies. The novelty of the current study is to consider the effects of viscoelastic properties, and exact values of size-dependent parameters on dynamic behaviors of the tubulin-labeled microtubules. Considering three-length scale parameters ( = , = , = ) in this size-dependent theory leads to a better agreement with molecular dynamic (MD) simulation in comparison with other theories. The results show that when the rigidity of the edges is improved by changing the simply supported to clamped supported boundary conditions, the maximum deflection and stability of the living part would be damped much more quickly.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1760939DOI Listing
May 2020

An Infectious cDNA Clone of SARS-CoV-2.

Cell Host Microbe 2020 05 13;27(5):841-848.e3. Epub 2020 Apr 13.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA; Department of Pharmacology & Toxicology, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

The 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 × 10 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectious-clone-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.
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http://dx.doi.org/10.1016/j.chom.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153529PMC
May 2020