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      Visualizing the replication of respiratory syncytial virus in cells and in living mice

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          Abstract

          Respiratory syncytial virus (RSV) is the most important cause of severe lower-respiratory tract disease in calves and young children, yet no human vaccine nor efficient curative treatments are available. Here we describe a recombinant human RSV reverse genetics system in which the red fluorescent protein (mCherry) or the firefly luciferase (Luc) genes are inserted into the RSV genome. Expression of mCherry and Luc are correlated with infection rate, allowing the monitoring of RSV multiplication in cell culture. Replication of the Luc-encoding virus in living mice can be visualized by bioluminescent imaging, bioluminescence being detected in the snout and lungs of infected mice after nasal inoculation. We propose that these recombinant viruses are convenient and valuable tools for screening of compounds active against RSV, and can be used as an extremely sensitive readout for studying effects of antiviral therapeutics in living mice.

          Supplementary information

          The online version of this article (doi:10.1038/ncomms6104) contains supplementary material, which is available to authorized users.

          Abstract

          Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Here, Rameix-Welti et al. create fluorescent and bioluminescent RSV strains that allow real-time analysis of viral replication and screening of antiviral compounds in cultured cells and in live animals.

          Supplementary information

          The online version of this article (doi:10.1038/ncomms6104) contains supplementary material, which is available to authorized users.

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          Most cited references28

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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.
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            Animal models of human respiratory syncytial virus disease.

            Infection with the human pneumovirus pathogen, respiratory syncytial virus (hRSV), causes a wide spectrum of respiratory disease, notably among infants and the elderly. Laboratory animal studies permit detailed experimental modeling of hRSV disease and are therefore indispensable in the search for novel therapies and preventative strategies. Present animal models include several target species for hRSV, including chimpanzees, cattle, sheep, cotton rats, and mice, as well as alternative animal pneumovirus models, such as bovine RSV and pneumonia virus of mice. These diverse animal models reproduce different features of hRSV disease, and their utilization should therefore be based on the scientific hypothesis under investigation. The purpose of this review is to summarize the strengths and limitations of each of these animal models. Our intent is to provide a resource for investigators and an impetus for future research.
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              Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

              Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.
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                Author and article information

                Contributors
                elyanne.gault@apr.aphp.fr
                jean-francois.eleouet@jouy.inra.fr
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                3 October 2014
                2014
                : 5
                : 5104
                Affiliations
                [1 ]GRID grid.452943.d, Unité de Virologie et Immunologie Moleculaires (UR892), INRA, ; Jouy-en-Josas, F78352 France
                [2 ]Physiopathologie et diagnostic des infections microbiennes, EA3647—EPIM, UFR des Sciences de la Santé Simone Veil—UVSQ, 2 avenue de la Source de la Bièvre, Montigny-Le-Bretonneux, 78180 France
                [3 ]GRID grid.413756.2, ISNI 0000 0000 9982 5352, AP-HP, Hôpital Ambroise Paré, Laboratoire de Microbiologie, ; Boulogne-Billancourt, 92100 France
                [4 ]Infection Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, USA, ,
                Article
                BFncomms6104
                10.1038/ncomms6104
                7091779
                25277263
                0e0c2d04-0636-4d25-8603-4d3f6675fd32
                © Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2014

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 26 February 2014
                : 29 August 2014
                Categories
                Article
                Custom metadata
                © The Author(s) 2014

                Uncategorized
                bioluminescence imaging,virology
                Uncategorized
                bioluminescence imaging, virology

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