Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Gudmundsdottir, Hulda; Ólafsdóttir, Katrín; Franzdóttir, Sigríður Rut; Andrésdóttir, Valgerður

doi:10.1016/j.jviromet.2010.04.026

Cited by 3 publications

(2 citation statements)

References 17 publications

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“…Nevertheless, these tools are unable to represent the entire replication cycle. These shortcomings can be avoided by employing viruses production from full-length infectious clones that contain convenient reporters, which have been generated for various RNA viruses including Visna virus [ 22 ], Chandipura virus [ 23 ], hepatitis C virus [ 24 ], coxsackievirus B3 [ 25 ] and EV71 [ 26 ], but not for CA16. Due to the lack of a CA16 high infective cell model, full-length CA16 infectious clones are often difficult to manipulate.…”

Section: Introductionmentioning

confidence: 99%

Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

Jin

et al. 2014

Viruses

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Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

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Section: Introductionmentioning

confidence: 99%

Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

Jin

et al. 2014

Viruses

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“…In PRRSV, rescue systems to recover infectious viruses from full length complementary DNA (cDNA) clones have been established for type 1 and type 2 PRRSV to understand the viral life cycle, determine the function of viral protein, and study mutant virus pathogenesis [23][24][25][26][27][28][29][30][31][32][33][34]. Green fluorescent protein (GFP) and its variant enhanced GFP (EGFP) inserted into a number of viral genomes have become the widely used reporter genes to study viral entry mechanisms, observe the transport of viral nucleic acids in and out of the nucleus region, track the assembly and transport of newly synthesized viral components, and visualize the cell-tocell spread of newly synthesized viral particles [35][36][37][38][39][40].…”

Section: Introductionmentioning

confidence: 99%

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit

Wang

Huang

Kong

et al. 2013

Vet Res

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Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3′-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

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Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Jónsson

Andrésdóttir

2011

JoVE

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Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis 1 . The primary target cells of MVV in vivo are considered to be of the monocyte lineage 2 . Certain strains of MVV can replicate in other cell types, however 3,4 . The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV 5 . However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo 6 . MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus 7 . The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages 8 . There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo 8 . Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3483 Protocol TransfectionThe molecular clone is contained in two plasmids, p8XSp5-egfp and p67r, of 12 kb and 4.5 kb respectively. 9,10 Note: The lamb serum can be substituted by fetal bovine serum (FBS). We routinely use lamb serum, since some strains of MVV are inhibited by FBS; this MVV strain is not inhibited by FBS, however.

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Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Cited by 3 publications

References 17 publications

Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

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