**1. Introduction**

Influenza A viruses (IAV) are a major cause of respiratory infection in humans and are responsible for significant morbidity and mortality worldwide. Annually, seasonal influenza epidemics affect approximately 5%–15% of the global population, resulting in 290,000–650,000 deaths [1,2]. Vaccination programs are important for preventing and controlling influenza. However, the efficacy of vaccination is typically only 40%–60% and can be lower than 20% during years of vaccine mismatch [3]. Thus, antiviral therapies are a vital option for the treatment of influenza.

Until now, three types of antivirals have been approved by the FDA for influenza prevention and therapies, including M2 ion channel inhibitors (i.e., adamantanes and rimantadine) [4], neuraminidase inhibitors (NAIs, i.e., oseltamivir and zanamivir) [5], and a cap-dependent endonuclease inhibitor [6,7]. However, the M2 inhibitors are no longer used clinically as currently almost all circulating IAV strains are resistant to adamantanes [8]. Moreover, the 2008–2009 seasonal H1N1 influenza virus strain in North America presented nearly complete resistance to oseltamivir [9]. Baloxavir marboxil was approved for treating influenza last year, but recent work has shown that viral resistance is still a concern [6,10,11]. Amidst concerns about drug resistance, the development of novel antivirals with distinct mechanisms of action is necessary.

Brevilin A (chemical structure shown in Figure 1) is a sesquiterpene lactone isolated from medicinal herb *Centipeda minima*. As a major constituent of *C. minima* [12], it has been reported that brevilin A displays multiple activities such as anti-tumor [13–17], anti-bacterial [18], and antiprotozoal [19]. We previously evaluated the antiviral activity of 16 sesquiterpene lactones isolated from *C. minima* against influenza A/PR/8/34 (H1N1) virus in vitro. Eight of them showed significant antiviral activity. Among them, brevilin A exhibited the strongest antiviral effect [20], but the mechanism of this antiviral effect was not extensively studied. Here, we extend our previous findings by investigating the antiviral effects of brevilin A against various IAVs and mode of actions in vitro at a noncytotoxic concentration. We found that brevilin A exhibits significant antiviral activities against all tested IAV strains, and it inhibits the vRNA synthesis and the expression of some viral proteins. Furthermore, the anti-IAV effect of this compound in vivo was also evaluated.

**Figure 1.** The chemical structure of brevilin A.

#### **2. Materials and Methods**

#### *2.1. Compounds and Reagents*

Brevilin A (purity >95% by HPLC) was isolated from the supercritical fluid extract of *C. minima*. Ribavirin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Both compounds were dissolved in DMSO to prepare a solution with the concentration of 50 mM and stored at −20 ◦C for in vitro experiments. Brevilin A did not show cytotoxicity in Madin–Darby canine kidney (MDCK) epithelial cells up to 8 μM, which was used as the maximum concentration for in vitro antiviral assays. For in vivo experiments, brevilin A was dissolved in 10% Lipovenos containing 0.2% DMSO, 10% PEG300, and 2.5% glycerol, while oseltamivir carboxylate (Tamiflu, Roche, Basel, Switzerland), purchased from Guangzhou Overseas Chinese Hospital (Guangzhou, China), was dissolved in distilled water. Leptomycin B (LMB, a nuclear export inhibitor) solution was obtained from Beyotime Institute of Biotechnology, Shanghai, China.

Mouse anti-IAV NP (ab128193) and M2 (ab5416) antibodies, mouse anti-GAPDH antibody (ab181603), and donkey anti-mouse lgG (H + L) secondary antibody (ab150105) were purchased from Abcam Company Ltd., Shanghai, China. Mouse anti-IAV HA (GTX28262), NA (GTX629696) and M1 (GTX125928) antibodies, rabbit anti-IAV NS1 (GTX125990) and NS2 (GTX125953) antibodies were obtained from GeneTex, Alton Pkwy Irvine, CA, USA.
