scholarly journals Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles

2021 ◽  
Vol 11 ◽  
Author(s):  
Fuxiao Liu ◽  
Ning Wang ◽  
Jiahui Lin ◽  
Qianqian Wang ◽  
Yilan Huang ◽  
...  
Keyword(s):  
Canine Distemper Virus ◽  
Green Fluorescence Protein ◽  
Mutagenic Effect ◽  
High Fidelity ◽  
Canine Distemper ◽  
L Protein ◽  
Protein Functions ◽  
Fluorescence Protein ◽  
Next Generation Sequencing Ngs ◽  
In Cells

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.

scholarly journals Mutation Profiles of eGFP-Tagged Small Ruminant Morbillivirus During 45 Serial Passages in Ribavirin-Treated Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Fuxiao Liu ◽  
Yanli Zou ◽  
Lin Li ◽  
Chunju Liu ◽  
Xiaodong Wu
Keyword(s):  
Reverse Genetics ◽  
Small Ruminant ◽  
Green Fluorescence Protein ◽  
High Fidelity ◽  
Missense Mutations ◽  
The Family ◽  
Protein Functions ◽  
Fluorescence Protein ◽  
Next Generation Sequencing Ngs

Small ruminant morbillivirus (SRMV), formerly known as peste-des-petits-ruminants virus, classified into the genus Morbillivirus in the family Paramyxoviridae. Its L protein functions as the RNA-dependent RNA polymerases (RdRp) during viral replication. Due to the absence of efficient proofreading activity in their RdRps, various RNA viruses reveal high mutation frequencies, making them evolve rapidly during serial passages in cells, especially treated with a certain mutagen, like ribavirin. We have previously rescued a recombinant enhanced green fluorescence protein-tagged SRMV (rSRMV-eGFP) using reverse genetics. In this study, the rSRMV-eGFP was subjected to serial passages in ribavirin-treated cells. Due to the ribavirin-exerted selective pressure, it was speculated that viral progenies would form quasispecies after dozens of passages. Viral progenies at passage-10, -20, -30, -40, and -50 were separately subjected to next-generation sequencing (NGS), consequently revealing a total of 34 single-nucleotide variations, including five synonymous, 21 missense, and one non-sense mutations. The L sequence was found to harbor eight missense mutations during serial passaging. It was speculated that at least one high-fidelity variant was present in viral quasispecies at passage-50. If purified from the population of viral progenies, this putative variant would contribute to clarifying a molecular mechanism in viral high-fidelity replication in vitro.

scholarly journals Development of a Challenge-Protective Vaccine Concept by Modification of the Viral RNA-Dependent RNA Polymerase of Canine Distemper Virus

2007 ◽  
Vol 81 (24) ◽  
pp. 13649-13658 ◽  
Author(s):  
D. Silin ◽  
O. Lyubomska ◽  
M. Ludlow ◽  
W. P. Duprex ◽  
B. K. Rima
Keyword(s):  
Rna Polymerase ◽  
Neutralizing Antibodies ◽  
Canine Distemper Virus ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Hinge Region ◽  
Canine Distemper ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
L Protein

ABSTRACT We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a “quick response” in vaccine development against well-known and “new” viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

scholarly journals Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites

2021 ◽  
Author(s):  
Mahsa Babaei ◽  
Luisa Sartori ◽  
Alexey Karpukhin ◽  
Dmitrii Abashkin ◽  
Elena Matrosova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Green Fluorescence Protein ◽  
Cellular Growth ◽  
Green Fluorescence ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
Integration Sites ◽  
Fluorescence Protein ◽  
Integration Efficiency

Abstract Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

scholarly journals Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma

2021 ◽  
Vol 22 (7) ◽  
pp. 3578
Author(s):  
Federico Armando ◽  
Adnan Fayyad ◽  
Stefanie Arms ◽  
Yvonne Barthel ◽  
Dirk Schaudien ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Virus Infection ◽  
Tumor Growth ◽  
Canine Distemper Virus ◽  
Xenograft Model ◽  
Histiocytic Sarcoma ◽  
Oncolytic Viruses ◽  
Canine Distemper ◽  
Murine Xenograft Model ◽  
The Impact

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

scholarly journals Drivers of canine distemper virus exposure in dogs at a wildlife interface in Janos, Mexico

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Rocío Almuna ◽  
Andrés M. López‐Pérez ◽  
Rosa E. Sarmiento ◽  
Gerardo Suzán
Keyword(s):  
Canine Distemper Virus ◽  
Canine Distemper ◽  
Virus Exposure

scholarly journals Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity

Viruses ◽  
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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