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      Applications of Replicating-Competent Reporter-Expressing Viruses in Diagnostic and Molecular Virology

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          Abstract

          Commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. Notably, the development of replicating-competent reporter-expressing viruses (RCREVs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. This article reviews the applications of RCREVs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. However, there remain various challenges associated with RCREVs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. Despite all these limitations, RCREVs have become powerful tools for both basic and applied virology with the development of new technologies for generating RCREVs, the inventions of novel reporters and the better understanding of regulation of viral replication.

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          Most cited references89

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          The green fluorescent protein.

          R Tsien (1998)
          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

            Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI: http://dx.doi.org/10.7554/eLife.00049.001
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              Human occludin is a hepatitis C virus entry factor required for infection of mouse cells

              Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells1,2, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp)3 or HCV produced in cell culture (HCVcc)(unpublished data), suggesting a block at the level of entry. Through an iterative cDNA library screening approach we have identified human occludin (OCLN) as an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for HCV-susceptibility of human cells, since its overexpression in uninfectable cells specifically enhanced HCVpp uptake while its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors, CD814, scavenger receptor class B type I (SR-BI)5, and claudin-1 (CLDN1)6. While the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins for promoting HCV entry; both OCLN and CD81, however, must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process and provides a major advance towards efforts to develop small animal models for HCV.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Viruses
                Viruses
                viruses
                Viruses
                MDPI
                1999-4915
                06 May 2016
                May 2016
                : 8
                : 5
                : 127
                Affiliations
                [1 ]State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, China; 15204662904@ 123456126.com (Y.L.); lilianfeng124603@ 123456163.com (L.-F.L.); yushaoxiong1987@ 123456hotmail.com (S.Y.); wangxiao2037@ 123456126.com (X.W.); tdzlk1991@ 123456163.com (L.Z.); yujiahui2014@ 123456126.com (J.Y.); xielibao2015@ 123456163.com (L.X.)
                [2 ]Department of Chemistry, College of Arts and Sciences, Georgia State University, Atlanta, GA 30302, USA; wli22@ 123456gsu.edu
                [3 ]University of Karachi, Karachi 75270, Pakistan; razimalikhan@ 123456gmail.com
                Author notes
                [* ]Correspondence: huajiqiu@ 123456hvri.ac.cn or qiuhuaji@ 123456163.com ; Tel.: +86-189-4606-6041; Fax: +86-451-5199-7170
                Article
                viruses-08-00127
                10.3390/v8050127
                4885082
                27164126
                eb3f7ee3-1bba-43b5-8e78-a45589152353
                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 18 December 2015
                : 29 April 2016
                Categories
                Review

                Microbiology & Virology
                replicating-competent virus,reporter,high-throughput screening,molecular virology

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