Multi-Modal Imaging with a Toolbox of Influenza AReporter Viruses

Tran, Vy M.; Poole, Daniel S.; Jeffery, Justin J.; Sheahan, Timothy P.; Creech, Donald; Yevtodiyenko, Aleksey; Peat, Andrew J.; Francis, Kevin P.; You, Shihyun; Mehle, Andrew

doi:10.3390/v7102873

Cited by 42 publications

(55 citation statements)

References 25 publications

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“…We have previously shown that this limitation can be circumvented by using replication-competent viruses harboring fluorescent reporter genes whose expression can be monitored and quantified directly (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). In addition, those reporter-expressing viruses have been demonstrated to be valuable assets for the screening and the identification of antivirals and/or host factors with antiviral activity (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Since the expression and activity of Venus and NLuc are dependent on BIRFLU infection, the presence of both reporters in infected cells (Venus) or in the culture supernatants (NLuc) can be used as a valid surrogate of viral replication.…”

Section: Birflu Reporter Gene Expression Levels Correlate With Dose-dmentioning

confidence: 99%

“…The modification of viral segments for the incorporation of reporter genes, such as fluorescent or luciferase proteins, in replication-competent IAV has been a crucial technological advance in the field. Genetically modified IAV expressing reporter genes is an excellent tool for the tracking of viral infection in vitro and in vivo, providing a robust quantitative readout of viral replication (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). In addition, this readout is compatible with high-throughput screening (HTS) technologies and useful to assess viral infection in cultured cells and animal models without the use of laborious secondary approaches to identify the presence of the virus in infected cells and/or animals (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41).…”

mentioning

confidence: 99%

“…For example, in vitro, fluorescent proteins are a better option to observe localization in cells (2,(34)(35)(36). However, for quantitative purposes, luciferases could be more convenient (32,33,40,47,48). For in vivo studies, although luciferase reporters require the inoculation of a chemical substrate, they are preferred over fluorescent proteins for whole-animal imaging.…”

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confidence: 99%

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A Novel Fluorescent and Bioluminescent Bireporter Influenza A Virus To Evaluate Viral Infections

Nogales

Ávila‐Pérez

Rangel-Moreno

et al. 2019

J Virol

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Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select a fluorescent or a luciferase reporter gene, depending on the type of study. To circumvent this limitation, we engineered a novel recombinant replication-competent bireporter IAV (BIRFLU) expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed growth kinetics comparable to those of wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows monitoring of viral inhibition by fluorescence or bioluminescence, overcoming the limitations associated with the use of one reporter gene as a readout. In vivo, BIRFLU effectively infected mice, and both reporter genes were detected using in vivo imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections. IMPORTANCE Influenza A virus (IAV) causes a human respiratory disease that is associated with significant health and economic consequences. In recent years, the use of replication-competent IAV expressing an easily traceable fluorescent or luciferase reporter protein has significantly contributed to progress in influenza research. However, researchers have been forced to select a fluorescent or a luciferase reporter gene due to the restricted capacity of the influenza viral genome for including foreign sequences. To overcome this limitation, we generated, for the first time, a recombinant replication-competent bireporter IAV (BIRFLU) that stably expresses two reporter genes (one fluorescent and one luciferase) to track IAV infections in vitro and in vivo. The combination of cutting-edge techniques from molecular biology, animal research, and imaging technologies brings researchers the unique opportunity to use this new generation of reporter-expressing IAV to study viral infection dynamics in both cultured cells and animal models of viral infection. RESULTSGeneration of a BIRFLU expressing NLuc and Venus. To generate replicationcompetent bireporter IAV (BIRFLU) (Fig. 1), the sequence of NLuc and the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site were cloned in front ...

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Section: Birflu Reporter Gene Expression Levels Correlate With Dose-dmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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A Novel Fluorescent and Bioluminescent Bireporter Influenza A Virus To Evaluate Viral Infections

Nogales

Ávila‐Pérez

Rangel-Moreno

et al. 2019

J Virol

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“…We probed each successive step that occurs early in the infectious cycle, beginning with viral attachment. Wild-type or edited cells were incubated with bioluminescent virions (PASN) that package nanoluciferase into the viral particle (Tran et al, 2015). Cells were incubated at 4˚C to enable binding but prevent internalization of virions, and luciferase activity was assayed from the bound virions.…”

Section: Eps8 Functions Post-fusion But Before Viral Gene Expression mentioning

confidence: 99%

“…The recombinant influenza A reporter viruses WSN PASTN (Tran et al, 2013), A/California/04/2009 PASTN (H1N1, CA04 PASTN) (Karlsson, 2015), WSN PASN (Tran et al, 2015), WSN with the polymerase from A/green-winged teal/ OH/175/1983 (H2N1) encoding PB2 S590/R591/K627 (S009-SRK PASTN) (Tran et al, 2015), B/Brisbane/60/2008 (B/Brisbane) PASTN, and FVG-R (Hao et al, 2008) were rescued using the influenza virus reverse genetics system and prepared as previously described. WSN PASN was further purified by centrifugation through a 20% sucrose cushion to remove contaminating luciferase present in the media (Tran et al, 2015). WSN-GFP was amplified and titered on HA-MDCK cells (Marsh et al, 2007).…”

Section: Virusesmentioning

confidence: 99%

EPS8 facilitates uncoating of influenza A virus

Larson

Tran

Yú

et al. 2019

Preprint

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All viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We used gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identified the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate. Knockout and overexpression of EPS8 confirmed its importance in enhancing FLUAV infection and titers. Loss of EPS8 did not affect virion attachment, uptake, or fusion. Rather, our data show that EPS8 specifically functions during virion uncoating. EPS8 physically associated with incoming virion components, and subsequent nuclear import of released ribonucleoprotein complexes was significantly delayed in the absence of EPS8. Our study identified EPS8 as a host factor important for uncoating, a crucial step of FLUAV infection during which the interface between the virus and host is still being discovered.

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