Visualizing influenza virus infection in living mice

We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from the A/Puerto Rico/8/1934(H1N1) virus (Venus-H5N1 virus), became more lethal to mice, and the reporter gene was stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of the WT-Venus-H5N1 virus in mice and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus. IMPORTANCEThe ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and that its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help advance influenza virusrelated live-imaging studies in vitro and in vivo.

Section: Discussionmentioning

confidence: 99%

Molecular Determinants of Virulence and Stability of a Reporter-Expressing H5N1 Influenza A Virus

Zhao

Fukuyama

Yamada

et al. 2015

“…Importantly, this technique was able to detect transmission events that would otherwise have been missed by standard approaches (9). Another important attribute is the ability to use virus-driven luminescence to rapidly assess virus replication levels (7)(8)(9)(10)(11). 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).…”

Section: Influenza a Virus Tropism In Vivomentioning

confidence: 99%

“…Upon introduction of the substrate, the luciferase enzyme quickly generates a robust and easily detectable bioluminescent signal. One of the most exciting applications of this system is the noninvasive imaging of infected tissues in live mice and ferrets over time (7)(8)(9)(10). Additionally, in the ferret system, where both direct and respiratory contact infections can be tracked, the IAV-derived luciferase was used to visualize transmission to new hosts (9).…”

Section: Influenza a Virus Tropism In Vivomentioning

confidence: 99%

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

Fiege

Langlois

2015

Influenza A viruses display a broad cellular tropism within the respiratory tracts of mammalian hosts. Uncovering the relationship between tropism and virus immunity, pathogenesis, and transmission will be critical for the development of therapeutic interventions. Here we discuss recent developments of several recombinant strains of influenza A virus. These viruses have inserted reporters to track tropism, microRNA target sites to restrict tropism, or barcodes to assess transmission dynamics, expanding our understanding of pathogen-host interactions.

“…Research into IAV has been greatly enhanced by several replication-competent reporter viruses (5-17). These reporter viruses have been crucial for identifying host factors, understanding basic viral pathogenesis, screening for antiviral compounds, and characterizing broadly reactive antibodies (6)(7)(8)(9)(10)(12)(13)(14)(15)(16)(17)(18)(19). Lack of viruses encoding reporters has delayed similar progress for IBV.…”

mentioning

confidence: 99%

“…Despite the importance of this virus, far less is known about IBV relative to influenza A virus (IAV). Research into IAV has been greatly enhanced by several replication-competent reporter viruses (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). These reporter viruses have been crucial for identifying host factors, understanding basic viral pathogenesis, screening for antiviral compounds, and characterizing broadly reactive antibodies (6)(7)(8)(9)(10)(12)(13)(14)(15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

Replication-Competent Influenza B Reporter Viruses as Tools for Screening Antivirals and Antibodies

Fulton

Palese

Heaton

2015

cInfluenza B virus is a human pathogen responsible for significant health and economic burden. Research into this pathogen has been limited by the lack of reporter viruses. Here we describe the development of both a replication-competent fluorescent influenza B reporter virus and bioluminescent influenza B reporter virus. Furthermore, we demonstrate these reporter viruses can be used to quickly monitor viral growth and permit the rapid screening of antiviral compounds and neutralizing antibodies. Influenza B virus (IBV) is a common human pathogen that causes yearly epidemics and significant disease in the pediatric population (1-4). Despite the importance of this virus, far less is known about IBV relative to influenza A virus (IAV). Research into IAV has been greatly enhanced by several replication-competent reporter viruses (5-17). These reporter viruses have been crucial for identifying host factors, understanding basic viral pathogenesis, screening for antiviral compounds, and characterizing broadly reactive antibodies (6)(7)(8)(9)(10)(12)(13)(14)(15)(16)(17)(18)(19). Lack of viruses encoding reporters has delayed similar progress for IBV.Development of a fluorescent influenza B reporter virus. We have previously published a description of an IAV reporter virus that contains the Gaussia luciferase gene directly behind the PB2 open reading frame (ORF) in segment 1 of A/Puerto Rico/8/1934 virus (PR8) (7). A 2A proteolytic cleavage site inserted between the PB2 ORF and the Gaussia luciferase gene allows for the cotranslational separation of the two proteins (20).Utilizing a similar approach, an influenza virus codon-optimized ORF of mNeonGreen (mNeon), a gene encoding a bright monomeric fluorescent protein (21), was cloned behind each of the three polymerase segments of the B/Yamagata/16/ 1988 (Ya88) virus (Fig. 1A). The reporter segments were rescued individually utilizing standard protocols (22-24) to generate the PB1 mNeon, PB2 mNeon, and PA mNeon viruses. Madin-Darby canine kidney (MDCK) cells were infected at a multiplicity of infection (MOI) of 0.5 for 18 h with the recombinant wild-type (rWT) Ya88 or one of the three reporter viruses (with all experiments carried out at 33°C). Tosyl phenylalanyl chloromethyl ketone (TPCK) trypsin, which is required for spread of viral progeny, was excluded from the infection medium. The cells were imaged or subjected to flow cytometry after being labeled with Hoechst stain (Thermo Scientific) or LIVE/DEAD stain (Life Technologies), respectively. By epifluorescence microscopy, the PB1 mNeon and PB2 mNeon viruses were bright and easily observed by 18 h postinfection, but the PA mNeon virus was less detectable (Fig. 1B to E). Flow cytometry (BD LSRIIA) recapitulated the microscopy results (Fig. 1F to I). Quantification of the flow cytometry data with FlowJo indicated that both the PB1 mNeon and PB2 mNeon viruses yielded the brightest signals ( Fig. 1J and K), and the PB1 mNeon virus was chosen for further study.The PB1 mNeon virus can rapidly monitor the growth of IBV in vi...