A reporter system for assaying influenza virus RNP functionality based on secreted Gaussia luciferase activity.
BACKGROUND
Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP) was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc) reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc) activity was evaluated.


RESULTS
By measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min) and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system.


CONCLUSIONS
This approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.
State Key Laboratory for Molecular Virology and Genetic Engineering, Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention, China CDC, 155 Changbai Road, Beijing 102206, PR China.
Wenfei
W
Zhu
Jianfang
J
Zhou
Kun
K
Qin
Ning
N
Du
Liqi
L
Liu
Zaijiang
Z
Yu
Yun
Y
Zhu
Wenhong
W
Tian
Xiaobing
X
Wu
Yuelong
Y
Shu
D020121:5' Untranslated Regions; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D017930:Genes, Reporter; D006801:Humans; D009980:Influenza A virus / Q000254:growth & development*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014773:Virology / Q000379:methods*
D020121:5' Untranslated Regions; D012261:Ribonucleoproteins; D014764:Viral Proteins; D008156:Luciferases
Biotechniques. 2007 May;42(5):599-600, 602, 604-6
17515198
Mol Ther. 2005 Mar;11(3):435-43
15727940
Nat Methods. 2006 Dec;3(12):977-9
17099704
PLoS Pathog. 2010 Aug 05;6(8):e1001034
20700447
J Gen Virol. 2010 Oct;91(Pt 10):2485-90
20592108
J Virol Methods. 2005 Jun;126(1-2):13-20
15847914
Methods Enzymol. 1992;216:362-8
1479908
J Virol. 2009 Nov;83(21):11102-15
19692471
Nat Methods. 2008 Feb;5(2):171-3
18204457
Nat Protoc. 2009;4(4):582-91
19373229
J Virol. 1993 Apr;67(4):1761-4
8445709
Proc Natl Acad Sci U S A. 1985 Dec;82(23):7870-3
3906652
J Virol. 2009 Dec;83(24):13015-8
19793811
Proc Natl Acad Sci U S A. 2010 Mar 9;107(10):4687-92
20176961
Mol Genet Genomics. 2008 Aug;280(2):153-62
18516621
Virol J. 2009 Mar 28;6:38
19327163
Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21312-6
19995968
Virol J. 2010 Jun 01;7:112
20515447
J Nucl Med. 2008 Feb;49(2):285-94
18199619
Vaccine. 2008 Jan 24;26(4):522-31
18093698
Gene Ther. 2008 Dec;15(24):1618-22
18668144
Science. 2001 Sep 7;293(5536):1840-2
11546875
J Virol. 1999 Nov;73(11):9679-82
10516084
Virology. 2004 Mar 15;320(2):258-66
15016548