Recovery of NanoLuc Luciferase-Tagged Canine Distemper Virus for Facilitating Rapid Screening of Antivirals in vitro

Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

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Recovery of Recombinant Canine Distemper Virus That Expresses CPV-2a VP2: Uncovering the Mutation Profile of Recombinant Undergoing 50 Serial Passages In Vitro

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.770576 ◽

2022 ◽

Vol 11 ◽

Author(s):

Fuxiao Liu ◽

Jiahui Lin ◽

Qianqian Wang ◽

Youming Zhang ◽

Hu Shan

Keyword(s):

Nonsense Mutation ◽

Canine Distemper Virus ◽

Reverse Genetics ◽

Test Strip ◽

Immunofluorescence Assay ◽

High Morbidity ◽

Missense Mutations ◽

Canine Distemper ◽

Reading Frame

Canine distemper and canine parvoviral enteritis are infections caused by the canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2), respectively. They are two common infectious diseases that cause high morbidity and mortality in affected dogs. Combination vaccines have been broadly used to protect dogs from infections of CDV, CPV-2, and other viruses. VP2 is the most abundant protein of the CPV-2 capsid. It elicits potent immunity in animals and, therefore, is widely used for designing subunit antigen-based vaccines. In this study, we rescued a recombinant CDV (QN vaccine strain) using reverse genetics. The recombinant CDV (rCDV-VP2) was demonstrated to express stably the VP2 in cells for at least 33 serial passages in vitro. Unfortunately, a nonsense mutation was initially identified in the VP2 open reading frame (ORF) at passage-34 (P34) and gradually became predominant in rCDV-VP2 quasispecies with passaging. Neither test strip detection nor indirect immunofluorescence assay demonstrated the expression of the VP2 at P50. The P50 rCDV-VP2 was subjected to next-generation sequencing, which totally identified 17 single-nucleotide variations (SNVs), consisting of 11 transitions and 6 transversions. Out of the 17 SNVs, 1 and 9 were identified as nonsense and missense mutations, respectively. Since the nonsense mutation arose in the VP2 ORF as early as P34, an earlier rCDV-VP2 progeny should be selected for the vaccination of animals in future experiments.

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Rescue of Two Recombinant Canine Distemper Viruses That Separately Express Dabie Bandavirus Gn and Gc in Vitro

10.21203/rs.3.rs-634109/v1 ◽

2021 ◽

Author(s):

Fuxiao Liu ◽

Jiahui Lin ◽

Qianqian Wang ◽

Hu Shan

Keyword(s):

Reverse Genetics ◽

Open Reading Frames ◽

Canine Distemper ◽

Significant Difference ◽

The Family ◽

Potential Vaccine ◽

Severe Fever ◽

Reading Frames

Abstract Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis with a high mortality rate in humans. Additionally, dogs are frequently reported to be infected with this disease. There has been no commercially available vaccine for humans and animals as yet. The SFTS is caused by Dabie bandavirus (DBV), formerly known as SFTS virus. The DBV is now classified into the genus Bandavirus in the family Phenuiviridae. DBV Gn and Gc can induce specific immune responses in vivo. In this study, we used reverse genetics to construct two recombinant canine distemper viruses (rCDV), rCDV-Gn and -Gc, which could express Dabie bandavirus Gn and Gc in vitro, respectively. Two foreign sequences, Gn and Gc open reading frames, were genetically stable during twenty serial viral passages in cells. Growth curve of the rCDV-Gc basically coincided with that of a wild-type CDV, but showed a significant difference from that of the rCDV-Gn. The rCDV-Gn and -Gc were derived from a common parental CDV, the virulence-attenuating QN strain. Therefore, if proven to be efficient in resisting both canine distemper and SFTS in dogs, either or both recombinant CDVs would be potential vaccine candidates.

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A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

Nucleic Acids Research ◽

10.1093/nar/gkaa1213 ◽

2020 ◽

Author(s):

Yuru Wang ◽

Christopher D Katanski ◽

Christopher Watkins ◽

Jessica N Pan ◽

Qing Dai ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Targeted Mutagenesis ◽

Rna Repair ◽

Dna And Rna ◽

Modified Dna ◽

Dna Substrates ◽

Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

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Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18073332 ◽

2021 ◽

Vol 18 (7) ◽

pp. 3332

Author(s):

Olga V. Naidenko ◽

David Q. Andrews ◽

Alexis M. Temkin ◽

Tasha Stoiber ◽

Uloma Igara Uche ◽

...

Keyword(s):

Immune System ◽

High Throughput ◽

Environmental Protection Agency ◽

High Throughput Screening ◽

Molecular Mechanisms ◽

Specific Activity ◽

Laboratory Animals ◽

In Vitro Assays ◽

Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

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High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

Nature Communications ◽

10.1038/s41467-021-23954-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhou Fang ◽

Junjian Chen ◽

Ye Zhu ◽

Guansong Hu ◽

Haoqian Xin ◽

...

Keyword(s):

Antimicrobial Activity ◽

High Throughput ◽

High Throughput Screening ◽

Rational Design ◽

Click Reaction ◽

Reaction Times ◽

Great Promise ◽

Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity

Viruses ◽

10.3390/v13010128 ◽

2021 ◽

Vol 13 (1) ◽

pp. 128

Author(s):

Neeta Shrestha ◽

Flavio M. Gall ◽

Jonathan Vesin ◽

Marc Chambon ◽

Gerardo Turcatti ◽

...

Keyword(s):

Membrane Fusion ◽

Small Molecule ◽

High Throughput Screening ◽

Measles Virus ◽

Canine Distemper Virus ◽

Rna Virus ◽

Preventive Measure ◽

Close Relative ◽

Fusion Activity ◽

Canine Distemper

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

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