Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. Because the effectiveness of seasonal vaccines is highly variable and the currently available drugs are limited in their efficacy because of the emergence of drug resistance, there is an urgent need to develop novel antivirals. In this study, we characterized a recombinant IAV-carrying Gaussia luciferase (Gluc) gene and determined its potential as a tool for evaluating therapeutics. We demonstrated that this recombinant IAV is replication-competent in tissue culture and pathogenic in mice, although it is slightly attenuated compared to the parental virus. Luciferase expression correlated well with virus propagation both in vitro and in vivo, providing a simple measure for viral replication in tissue culture and in mouse lungs. To demonstrate the utility of this virus, ribavirin and oseltamivir phosphate were used to treat the IAV-infected cells and mice, and we observed the dose-dependent inhibition of viral replication by a luciferase assay. Moreover, the decreased luciferase expression in the infected lungs could predict the protective efficacy of antiviral interventions as early as day 2 post virus challenge. In summary, this study provides a new and quantitative approach to evaluate antivirals against IAV.

Influenza A viruses (IAV) carrying reporter genes provide a powerful tool to study viral infection and pathogenesis in vivo, however, incorporating a non-essential gene into the IAV genome often results in virus attenuation and genetic instability. Very few studies have systematically compared different reporter IAVs, and most optimization attempts seem to lack authentic directions. In this study, we evaluated the ratio of genome copies to the number of infectious unit of two reporter IAVs, PR8-NS1-Gluc and PR8-PB2-Gluc. As a result, PR8-NS1-Gluc and PR8-PB2-Gluc produced 41.4 and 3.8 genomes containing noninfectious particles respectively for every such particle produced by parental PR8 virus. RdRp assay demonstrated that modification of segment NS by inserting reporter genes can interfere with the replication competitive property of the corresponding vRNAs, and the balance of the 8 segments of the reporter IAVs were drastically impaired in infected cells. As a consequence, large amounts of NS-null noninfectious particles were produced during the PR8-NS1-Gluc packaging. In summary, we unravel a mechanism underlying attenuation of reporter IAVs, which suggests a new approach to restore infectivity and virulence by introducing extra mutations compensating for the impaired replication property of corresponding segments.

Influenza A virus infections cause significant morbidity and mortality, and novel antivirals are urgently needed. Influenza RNA-dependent RNA polymerase (RdRp) activity has been acknowledged as a promising target for novel antivirals. In this study, a phenotypic versus target-based screening strategy was established to identify the influenza A virus inhibitors targeting the virus RNA transcription/replication steps by sequentially using an RdRp-targeted screen and a replication-competent reporter virus-based approach using the same compounds. To demonstrate the utility of this approach, a pilot screen of a library of 891 compounds derived from natural products was carried out. Quality control analysis indicates that the primary screen was robust for identification of influenza A virus inhibitors targeting RdRp activity. Finally, two hit candidates were identified, and one was validated as a putative RdRp inhibitor. This strategy can greatly reduce the number of false positives and improve the accuracy and efficacy of primary screening, thereby providing a powerful tool for antiviral discovery.

Reporter influenza A viruses (IAVs) carrying fluorescent or luminescent genes provide a powerful tool for both basic and translational research. Most reporter IAVs are based on the backbone of either subtype H1N1 viruses, A/Puerto Rico/8/1934 (PR8) or A/WSN/1933, but no reporter subtype H3N2 virus is currently available to our knowledge. Since the IAV subtype H3N2 co-circulates with H1N1 among humans causing annual epidemics, a reporter influenza A subtype H3N2 virus would be highly valuable. In this study, the segments of A/Wyoming/3/03 (NY, H3N2) virus encoding hemagglutinin and neuraminidase, respectively, were reassorted with the six internal genes of PR8 where the NS gene was fused with a Gaussia luciferase (Gluc) gene. Using reverse genetics, NY-r19-Gluc, a replication competent reassortant influenza A subtype H3N2 virus expressing reporter Gluc was successfully generated. This reporter virus is stable during replication in Madin-Darby canine kidney (MDCK) cells, and preliminary studies demonstrated it as a useful tool to evaluate antivirals. In addition, NY-r19-Gluc virus will be a powerful tool in other studies including the application of diagnostic and therapeutic antibodies as well as the evaluation of novel vaccines.

The human immune system has evolved to cope with various virus infections using two major strategies: host defense and viral tolerance. The host defense relies on the innate and adaptive immune responses to control virus replication, while the viral tolerance allows sufficient virus replication in the host with minimal or no clinical signs. Generally, a balanced host defense and viral tolerance can guarantee health from infections, while disturbed immune homeostasis usually results in diseases. It is desirable to develop drugs/therapeutics to enhance the invulnerability of host immune equilibrium to combat viral infections. In this review, we summarize the advanced understanding on mechanisms of both the host defense against and tolerance to virus infections, and therapeutic agents/approaches that work by modulating the balance between host defense and tolerance.

Targeting the crosstalk between histone methyltransferases G9a and EZH2 represents a promising strategy for cancer therapy. To improve upon our previously reported dual inhibitor SU08, we rationally designed and synthesized five series (SU, UA, UB, N, and L) of novel G9a/EZH2 dual inhibitors, incorporating diverse pyridone, urea, and thienopyrimidine moieties, yielding a total of 43 compounds. In vitro enzymatic screening revealed that compound SU101 against both EZH2 (IC₅₀ = 4.08 nM) and G9a (IC₅₀ = 2.92 nM) was comparable to SU08. Western blot analysis confirmed that SU101 and SU103 effectively suppressed both H3K27me3 and H3K9me2 expression in rhabdomyosarcoma RD cells. In cellular antiproliferative assays, SU101 showed the strongest activity (IC₅₀ = 15.33 μM). Molecular docking and dynamics simulations verified stable binding interactions between SU101 and both target proteins. Pharmacokinetic studies in rats demonstrated that SU101 possesses excellent oral bioavailability (94.3%), moderate plasma protein binding (84.6%), and an acceptable acute toxicity profile (LD₅₀ = 31.02 mg/kg in mice). Overall, these results suggest that SU101 may serve as a promising lead compound for further optimization and support the continued exploration of dual G9a/EZH2 inhibitors as a potential therapeutic approach for rhabdomyosarcoma.