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

Abstract

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...

Replication-competent fluorescent-expressing influenza B virus

“…However, similar methods have not been used for the generation of recombinant fluorescent-expressing IBVs. While preparing this manuscript, Fulton et al described the generation of replication competent IBVs expressing reporter genes from the viral polymerase PB2, PB1 or PA viral segments using the backbone of influenza B/Yamagata/16/1988 (B/Yamagata lineage) (Fulton et al, 2015). Here, we produced and characterized replication-competent reporter-expressing viruses in the backbone of the more recent influenza B/Brisbane/60/2008 (B/Victoria lineage) using a strategy similar to the one that we previously used to generate fluorescent IAV (Nogales et al, 2014a).…”

A Recombinant Antibody-Expressing Influenza Virus Delays Tumor Growth in a Mouse Model

“…Moreover, the tumor-targeting ability of IAV has been enhanced through the inactivation of the IAV protein responsible for interferon antagonism, NS1 (Bergmann et al, 2001;Efferson et al, 2006;Muster et al, 2004), and through the manipulation of the host immune response via cytokine expression (Hock et al, 2017;van Rikxoort et al, 2012). Influenza viruses expressing transgenes from the polymerase segments have been described previously (Fulton et al, 2015;Heaton et al, 2013;Pena et al, 2013;Tran et al, 2013), although this strategy has not been employed to express more than one transgene from the same virus. As a proof of concept, this report describes a replication-competent IAV that utilizes the PB1 and PA viral polymerase segments to express a well-characterized antibody that targets group 2 IAV hemagglutinins.…”

Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

“…Gaussia and firefly luciferase insertions into the NA, PB2, or PA gene of A/PR/8/34 (H1N1) caused 10-to 100-fold lower replication in vitro and in mice, resulting in 50% mouse lethal dose (MLD 50 ) values Ն50-fold higher than those of the corresponding wild-type virus (10,14,17). Finally, the replication rate of B/Yamagata/16/1988 in MDCK cells was reduced 5to 10-fold; in vivo properties were not reported (11).…”

“…Influenza A viruses, from the family Orthomyxoviridae, contain 8 gene segments (9). Luciferase insertions have been introduced into PB2 (7, 10), PB1 (11,12), PA (13)(14)(15)(16), and NA (17) genes, in addition to an NS-PB1 rearrangement (18,19). Influenza reporter viruses must retain gene-packaging sequences at the termini and need to be relatively small due to the relatively small size (ϳ0.9 to 2.4 kb) of the gene segments (20).…”

“…Limitations on insertion size can be addressed by selecting a relatively small reporter gene, such as the NanoLuc (Nluc) luciferase gene (NanoLuc [Nluc]) (15), which is relatively small and has ϳ150-fold greater light output than firefly and Renilla luciferases (34). Of course, gene-packaging sequences need to be added to insertions at the 3= and 5= termini (10,11,(13)(14)(15)(16)(17)35). Multiple reporter gene candidates can be created and compared in parallel or in an iterative manner to counteract attenuation (14,15).…”