An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection

Ye, Chuangtao; Wang, Dan; Li, He; Ma, Heng; Dong, Yangchao; Yao, Min; Wang, Yuan; Zhang, Hui; Zhang, Liang; Cheng, Long; Zhang, Xu; Lei, Yingfeng; Zhang, Fanglin; Ye, Wei

doi:10.3389/fphar.2019.01203

Cited by 17 publications

(19 citation statements)

References 33 publications

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“…While plaque assays are perhaps the most commonly used technique for the quantification of infectious virus, immunohistochemical focus forming assays are also commonly used for the quantification of virus [10,[16][17][18]. We have established that SARS-CoV-2 can be reliably quantified by a 96-well plate-based focus forming assay in only 24 h. When compared to traditional plaque assays for SARS-CoV-2 which require 72 h for easily quantifiable plaques, the ability to quantify infectious SARS-CoV-2 within 24 h in a 96-well plate format represents a significant advantage for studies requiring higher-throughput.…”

Section: Discussionmentioning

confidence: 99%

Propagation, Inactivation, and Safety Testing of SARS-CoV-2

Jureka

Silvas

Basler

2020

Viruses

113

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In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.

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Section: Discussionmentioning

confidence: 99%

Propagation, Inactivation, and Safety Testing of SARS-CoV-2

Jureka

Silvas

Basler

2020

Viruses

113

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Section: Discussionmentioning

confidence: 99%

Propagation, inactivation, and safety testing of SARS-CoV-2

Jureka

Silvas

Basler

2020

Preprint

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In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-9 CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has 10 been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 11 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease 12 that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly 13 described and effective procedures that enable the propagation and quantification of infectious 14 virus. Because work with the virus is recommended to be performed at biosafety level 3, validated 15 methods to effectively inactivate the virus to enable safe study of RNA, DNA and protein from 16 infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell 17 lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline 18 cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate 19 effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat. 20 cells to characterize viral genome sequences, monitor viral gene expression and genome replication, 43 and to characterize host responses to infection. Removal of intact, virus-infected cells is critical for 44 studies involving microscopy aimed at understanding the SARS-CoV-2:host interplay at the cellular 45 level, and also for high-throughput analysis of the progression of viral replication in response to 46 antivirals. Whole inactivated virus and viral proteins are needed for the development of inactivated 47 whole-virus vaccine preparations and also as a source of antigen for immunoassays. 48To help address these needs and to facilitate SARS-CoV-2 research efforts, we describe here 49 methods for the propagation of SARS-CoV-2 in multiple cell lines. We have also determined a more 50 efficient method for quantifying virus by plaque assay and have developed a SARS-CoV-2-specific 51 focus forming assay which can enhance throughput of assays requiring quantification of viral titers. 52Additionally, we describe validation if methods for the inactivation of SARS-CoV-2 through the use 53 of TRIzol, 10% neutral buffered formalin, beta-propiolactone, and heat. Taken together, the data 54 presented here will serve to provide researchers with a helpful basis of information to aid in their 55 work on SARS-CoV-2. 56 57 2. Materials and Methods 58 2.1 Cells and Virus 59 60 Vero E6 (ATCC# CRL-1586), Calu-3 (ATCC# HTB-55), Caco-2 (ATCC# HTB-37), Huh7, A549 61 (ATCC# CCL-185), and 293T cells were maintained in DMEM (Corning) supplemented with 10% heat 62 inactivated fetal bovine serum (FBS; GIBCO). Cells were kept in a 37°C, 5% CO2 incubator without 63 antibiotics or antimycotics. SARS-CoV-2, strain USA_WA1/2020, was obtained from the World 64 Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical 65 Branch-Galveston. 6...

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“…2n also resulted in being more active in in vitro assays against HTNV than two orthohantavirus candidate drugs: ETAR (1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4] triazole) (EC50 = 10 µ M) and Favipiravir (EC50 =150.8 μM), respectively 2-and 37-fold more potent [13,23]. Compounds 1a-c, 1e-j, 2a-n, 3f, 3j-n, and 4f, 4j-n, were evaluated for their potential inhibitory activity against HTNV using a chemiluminescence focus reduction assay (C-FRA).…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, in order to obtain a titer decrease, less than 2 logs (1,78 log10) if compared to untreated control, the reference molecule ribavirin needed to be added at 50 µ M concentration. 2n also resulted in being more active in in vitro assays against HTNV than two orthohantavirus candidate drugs: ETAR (1-beta-d-ribofuranosyl-3-ethynyl- [1,2,4] triazole) (EC 50 = 10 µM) and Favipiravir (EC 50 =150.8 µM), respectively 2-and 37-fold more potent [13,23].…”

Section: Resultsmentioning

confidence: 99%

5,6-Dichloro-2-Phenyl-Benzotriazoles: New Potent Inhibitors of Orthohantavirus

Sanna

Piras

Madeddu

et al. 2020

Viruses

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Orthohantaviruses, previously known as hantaviruses (family Hantaviridae, order Bunyavirales), are emerging zoonoses hosted by different rodent and insectivore species. Orthohantaviruses are transmitted by aerosolized excreta (urine, saliva and feces) of their reservoir hosts. When transmitted to humans, they cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe and hantavirus (cardio) pulmonary syndrome (HPS) in the Americas. Clinical studies have shown that early treatments of HFRS patients with ribavirin (RBV) improve prognosis. Nevertheless, there is the need for urgent development of specific antiviral drugs. In the search for new RNA virus inhibitors, we recently identified a series of variously substituted 5,6-dichloro-1(2)-phenyl-1(2)H-benzo[d][1,2,3]triazole derivatives active against the human respiratory syncytial virus (HRSV). Interestingly, several 2-phenyl-benzotriazoles resulted in fairly potent inhibitors of the Hantaan virus in a chemiluminescence focus reduction assay (C-FRA) showing an EC50 = 4–5 µM, ten-fold more active than ribavirin. Currently, there are no FDA approved drugs for the treatment of orthohantavirus infections. Antiviral activities and cytotoxicity profiles suggest that 5,6-dichloro-1(2)-phenyl-1(2)H-benzo[d][1,2,3]triazoles could be promising candidates for further investigation as a potential treatment of hantaviral diseases.

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An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection

Cited by 17 publications

References 33 publications

Propagation, Inactivation, and Safety Testing of SARS-CoV-2

Propagation, Inactivation, and Safety Testing of SARS-CoV-2

Propagation, inactivation, and safety testing of SARS-CoV-2

5,6-Dichloro-2-Phenyl-Benzotriazoles: New Potent Inhibitors of Orthohantavirus

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