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      The Multiplicity of Infection of Recombinant Vaccinia Virus Expressing the T7 RNA Polymerase Determines the Rescue Efficiency of Vesicular Stomatitis Virus


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          Vesicular stomatitis virus (VSV) has a wide range of cell tropism, making it a prototype of studying the negative-strand RNA virus (NSRV), including virus–host interactions and vaccine development. Although VSV rescue systems have been progressively optimized throughout time, the T7-based expression system is the most commonly utilized to rescue VSV. However, it remains a significant barrier for many labs. In our study, we found that rescue VSV’s efficiency is associated with the various multiplicities of infection (MOIs) of recombinant vaccinia virus expressing the T7 RNA polymerase (vTF-7.3). It works at maximum efficiency while the MOI of vTF-7.3 is 5, which is analyzed by quantitative PCR, Western blot, and flow cytometry, compared to the lowest rescue level with MOI of 1. Meanwhile, our data also suggest that purification of vTF-7.3 prior to transfection is a prerequisite for VSV rescue. Overall, our study reveals for the first time a precise correlation between vTF-7.3 and rescue efficiency, which may aid in resolving the uncertainties in the quest to build the VSV reverse genetic system.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2.

            Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.
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              Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.

              In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all cis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

                Author and article information

                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                04 April 2022
                : 13
                : 846426
                [1] 1State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Lanzhou, China
                [2] 2College of Animal Science, Anhui Science and Technology University , Fengyang, China
                [3] 3Hebei Key Laboratory of Preventive Veterinary Medicine, College of Animal Science and Technology, Hebei Normal University of Science and Technology , Qinhuangdao, China
                [4] 4State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Lanzhou, China
                [5] 5Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses , Yangzhou, China
                Author notes

                Edited by: Chunfu Zheng, University of Calgary, Canada

                Reviewed by: Ting Qi, National Engineering Research Center of Veterinary Biologics, Harbin Veterinary Research Institute (CAAS), China; Xiaodong Cheng, Xiamen University, China

                *Correspondence: Weike Li, liweike@ 123456caas.cn

                This article was submitted to Virology, a section of the journal Frontiers in Microbiology

                Copyright © 2022 Yang, Tan, Fang, Chen, Zhang, Hu, Han, Liu, Fu, Jing and Li.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                : 31 December 2021
                : 10 January 2022
                Page count
                Figures: 6, Tables: 2, Equations: 0, References: 40, Pages: 8, Words: 4787
                Funded by: Natural Science Foundation of Gansu Province, doi 10.13039/501100004775;
                Original Research

                Microbiology & Virology
                vesicular stomatitis virus,vtf-7.3,reverse genetic,rescue efficiency,the multiplicity of infection


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