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Influenza virus assays based on virus-inducible reporter cell lines.

Li Y, Larrimer A, Curtiss T, Kim J, Jones A, Baird-Tomlinson H, Pekosz A, Olivo PD - Influenza Other Respir Viruses (2009)

Bottom Line: A strategy for influenza A virus-induction of a reporter gene was recently described.Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains.

View Article: PubMed Central - PubMed

Affiliation: Diagnostic Hybrids Inc., Athens, OH, USA.

ABSTRACT

Background: Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5¢- and 3¢-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.

Findings: Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses.

Conclusions: These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers.

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Related in: MedlinePlus

 Photomicrographs (100×) showing GFP expression from ELVIRA® Flu‐GFP cell lines infected with influenza A or B viruses. GFP‐expressing cells were detected by fluorescent microscopy at 24 or 48 hours after infection. (A) The ELVIRA® Flu A‐GFP cell line was infected with influenza A virus strains A/Wisconsin/67/05 and A/MHIA0009, and influenza B virus strains B/Maryland/1/59 (ATCC VR‐296) and B/Massachusetts (ATCC VR‐523) at 1 MOI. Photomicrographs were taken at 24 hours after infection. (B) The ELVIRA® Flu B‐GFP cell line was infected with influenza B virus B/ODH0149 and B/GL/1739/54 at 0·5 MOI and influenza A virus influenza A virus A/MIHA0009 and A/PortChalmers/1/73(H3N2) at 1 MOI. Photomicrographs were taken at 48 hours after infection.
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f3:  Photomicrographs (100×) showing GFP expression from ELVIRA® Flu‐GFP cell lines infected with influenza A or B viruses. GFP‐expressing cells were detected by fluorescent microscopy at 24 or 48 hours after infection. (A) The ELVIRA® Flu A‐GFP cell line was infected with influenza A virus strains A/Wisconsin/67/05 and A/MHIA0009, and influenza B virus strains B/Maryland/1/59 (ATCC VR‐296) and B/Massachusetts (ATCC VR‐523) at 1 MOI. Photomicrographs were taken at 24 hours after infection. (B) The ELVIRA® Flu B‐GFP cell line was infected with influenza B virus B/ODH0149 and B/GL/1739/54 at 0·5 MOI and influenza A virus influenza A virus A/MIHA0009 and A/PortChalmers/1/73(H3N2) at 1 MOI. Photomicrographs were taken at 48 hours after infection.

Mentions: In addition to cell lines that express virus‐inducible luciferase, we generated cell lines that express GFP following influenza virus infection using the same strategy. Figure 3 shows the absence of GFP expression in mock‐infected cells and a high level of expression in the cytoplasm of influenza A virus‐infected ELVIRA® Flu A‐GFP cells and influenza B virus‐infected Flu B‐GFP cells. No cross‐induction was observed. These cells could be useful in methods for which there is an advantage to using either live or intact cells (i.e., cells that are not lysed).


Influenza virus assays based on virus-inducible reporter cell lines.

Li Y, Larrimer A, Curtiss T, Kim J, Jones A, Baird-Tomlinson H, Pekosz A, Olivo PD - Influenza Other Respir Viruses (2009)

 Photomicrographs (100×) showing GFP expression from ELVIRA® Flu‐GFP cell lines infected with influenza A or B viruses. GFP‐expressing cells were detected by fluorescent microscopy at 24 or 48 hours after infection. (A) The ELVIRA® Flu A‐GFP cell line was infected with influenza A virus strains A/Wisconsin/67/05 and A/MHIA0009, and influenza B virus strains B/Maryland/1/59 (ATCC VR‐296) and B/Massachusetts (ATCC VR‐523) at 1 MOI. Photomicrographs were taken at 24 hours after infection. (B) The ELVIRA® Flu B‐GFP cell line was infected with influenza B virus B/ODH0149 and B/GL/1739/54 at 0·5 MOI and influenza A virus influenza A virus A/MIHA0009 and A/PortChalmers/1/73(H3N2) at 1 MOI. Photomicrographs were taken at 48 hours after infection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940803&req=5

f3:  Photomicrographs (100×) showing GFP expression from ELVIRA® Flu‐GFP cell lines infected with influenza A or B viruses. GFP‐expressing cells were detected by fluorescent microscopy at 24 or 48 hours after infection. (A) The ELVIRA® Flu A‐GFP cell line was infected with influenza A virus strains A/Wisconsin/67/05 and A/MHIA0009, and influenza B virus strains B/Maryland/1/59 (ATCC VR‐296) and B/Massachusetts (ATCC VR‐523) at 1 MOI. Photomicrographs were taken at 24 hours after infection. (B) The ELVIRA® Flu B‐GFP cell line was infected with influenza B virus B/ODH0149 and B/GL/1739/54 at 0·5 MOI and influenza A virus influenza A virus A/MIHA0009 and A/PortChalmers/1/73(H3N2) at 1 MOI. Photomicrographs were taken at 48 hours after infection.
Mentions: In addition to cell lines that express virus‐inducible luciferase, we generated cell lines that express GFP following influenza virus infection using the same strategy. Figure 3 shows the absence of GFP expression in mock‐infected cells and a high level of expression in the cytoplasm of influenza A virus‐infected ELVIRA® Flu A‐GFP cells and influenza B virus‐infected Flu B‐GFP cells. No cross‐induction was observed. These cells could be useful in methods for which there is an advantage to using either live or intact cells (i.e., cells that are not lysed).

Bottom Line: A strategy for influenza A virus-induction of a reporter gene was recently described.Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains.

View Article: PubMed Central - PubMed

Affiliation: Diagnostic Hybrids Inc., Athens, OH, USA.

ABSTRACT

Background: Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5¢- and 3¢-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.

Findings: Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses.

Conclusions: These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers.

Show MeSH
Related in: MedlinePlus