Genome rearrangement of influenza virus for anti-viral drug screening

Abstract

Rearrangement of the influenza A genome such that NS2 is expressed downstream of PB1 permits the insertion of a foreign gene in the NS gene segment. In this report, the genome rearranged strategy was extended to A/California/04/2009 (pH1N1), and Gaussia luciferase (GLuc) or GFP was expressed downstream of the full-length NS1 gene (designated GLucCa04 and GFPCa04, respectively). In growth kinetics studies, culture of amantadine sensitive GLucCa04 (Sens/GlucCa04) in the presence of amantadine significantly decreased GLuc expression and viral titers for 48 h post-infection (hpi). When Sens/GlucCa04 was subsequently used in an in vitro anti-viral screening assay, amantadine treatment significantly decreased GLuc expression from amantadine sensitive compared to amantadine resistant GLucCa04 (Res/GlucCa04) as early as 16 hpi. In in vivo screening studies, DBA mice were treated daily with amantadine from 1 day prior to infection and inoculated with either Sens/GlucCa04 or Res/GlucCa04 alone or as a co-infection with the parental strain. On days 3 and 5 post-infection, lung samples were collected and amantadine treatment was shown to decrease GLuc expression by 2 orders of magnitude (p<0.05) in Sens/GlucCa04 infected mice. Furthermore, while both Sens and Res/GlucCa04 were highly attenuated, addition of the parental strain to the inoculum yielded clinical disease indicative of GLuc expression and pulmonary viral titers. These findings indicate that the use of GLucCa04 can potentially accelerate in vitro and in vivo anti-viral screening by shortening the time required for virus detection.

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

“…10). Therefore, direct quantitation of the EGFP signal provides a valuable tool to study inhibitors of entry and replication kinetics, as has been previously reported for influenza virus using viruses that express GFP to assess antiviral efficacy (61,62).…”

Investigating Influenza A Virus Infection: Tools To Track Infection and Limit Tropism

“…Multiple groups have published positive, linear correlations of virus titers in vitro, ex vivo, and in vivo, providing a more rapid and high-throughput approach than standard plaque assays (Table 1) (7)(8)(9)(10)(11). This technique has been utilized for rapid, noninvasive determination of therapies, including amantadine, oseltamivir, zanamivir, antiviral serum, and novel antibodies or vaccinations, by detecting levels of fluorescent/luminescent signal after treatment in vitro, ex vivo, or in vivo (Table 1) (5,(7)(8)(9)(10)(11)(12)(13). These studies have added to our understanding of virus replication and spread in live animals.…”

Generation and application of replication-competent Venus-expressing H5N1, H7N9, and H9N2 influenza A viruses

“…It has since been shown that packaging signals at the 5 0 and 3 0 ends of all gene segments are required for the efficient encapsidation of viral RNA (vRNA) into virions [23][24][25][26][27][28][29]. Exploiting this requirement, researchers have manipulated the viral genome by inserting exogenous reporter genes such as luciferase, green fluorescent protein (GFP) or its variants into various viral gene segments including HA, NA, nonstructural gene (NS), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA), to generate reporter influenza viruses [25,[30][31][32][33][34][35][36][37][38][39][40][41][42][43][44]. Currently, most reporter influenza viruses have been generated in the background of a laboratory-adapted influenza virus, such as A/Puerto Rico/8/34(PR8, H1N1) or A/WSN/1933(WSN, H1N1) [25,[30][31][32][33][34][35]37,38,40,42,44].…”