Influenza, commonly known as flu, is caused by influenza viruses and poses a persistent global health threat, with the extreme example of the 1918 flu pandemic, the first documented influenza outbreak in history [1,2]. For over a century, influenza has continued to cause substantial morbidity and mortality, imposing heavy social and economic burdens [3,4]. In recent years, the World Health Organization (WHO) [5] and the Centers for Disease Control and Prevention (CDC) have regularly reported influenza case numbers across many countries [6]. For instance, the China CDC reported 2 988 914 hospitalizations and 3 deaths during January 2024 alone [7]. During the influenza season in winter and spring, influenza virus subtypes with pandemic potential are of particular concern due to their significant implications for vaccine development and preparedness [8].

It is widely accepted that the development of new antiviral agents [9], effective vaccines [10] and accurate diagnostic methods [11] are critical for the control of influenza outbreaks to avoid a pandemic. To date, several drugs targeting different stages of the influenza infection cycle have been developed [12]. Among them, oseltamivir (OS, also called Tamiflu), a potent inhibitor of influenza virus neuraminidase (NA), is widely used clinically to treat influenza [13]. However, many OS-resistant strains have emerged in recent years, many due to the H275Y mutation in NA which reduces the binding of OS to NA, thus rendering the drug ineffective [14]. More importantly, OS is only effective when administered early (<48 h) [15] as it blocks the release of newly formed virion particles, a process facilitated by NA [16]. Similar to NA mutations resulting in OS-resistant strains, antigen drift and shift in influenza hemagglutinin (HA) can lead to a mismatch between the annually produced vaccine and pandemic strains [17]. Finally, the similarity of symptoms between viral and bacterial infections or inflammation underscores the importance of the accurate diagnosis of influenza [18]. On the other hand, the surveillance and the 48-h window for effective OS administration also require accurate and early diagnosis for the timely initiation of antiviral therapy. Currently, cost-effective, point-of-care, and easy-to-use influenza diagnostic kits are a highly sought-after global public health requirement.

In terms of the development of new antiviral agents, one effective strategy is the focus on evaluating the anti-influenza potential of natural products with novel chemical structures [19,20]. Pentacyclic triterpenes, secondary metabolites found in plants, fungi, marine invertebrates, and other organisms, have been isolated or semisynthesized and found to exhibit various biological activities [21]. Recently, one pentacyclic triterpene, oleanic acid (OA) and its derivatives, has shown broad anti-influenza activities with reduced induction of drug resistance [22]. Further mechanistic studies have indicated that OA can wrap around the heptad repeat-2 (HR2) domain of HA, leading to competitive inhibition of the binding of the virus to sialic acid moieties on host cell membranes, thus preventing viral infection [23]. However, the binding affinity of monomeric OA to HA is low, with a KD value in the micromolar range [24], limiting its further medicinal application. A solution to this problem is the use of modifications to enhance the binding affinity, leveraging the “cluster effect” [25,26] based on natural principles. Structural analysis of influenza virions has shown that they display hundreds of clustered HA trimers on their surfaces [27], which can bind strongly to the abundant polymeric sialyl oligosaccharides on the host cell membranes. Indeed, the multivalent presentation of OA on proper scaffolds mimicking natural cell membranes has been found to bind to HA monomers within a single HA trimer or across different HA trimers on a virion, leading to increased avidity for HA. For instance, the conjugation of multivalent OA to cyclodextrin (CD) [28,29] and small organic molecular backbones [30] enhanced both HA binding and subsequent antiviral activity. However, the complex synthesis required and the low biocompatibility have hindered large-scale applications. Our previous work introduced a novel strategy involving polyethylene glycolation (PEGylation) of OA, in which the PEGylated OA was subsequently attached to bovine serum albumin (BSA) [24] or poly (methyl vinyl ether-alt-maleic anhydride) [31] to improve the biocompatibility as well as the antiviral activity. However, these multivalent platforms do not contribute to further diagnostic applications.

In recent decades, gold nanoparticles (AuNPs) have emerged as a new form of scaffold for biomedical applications due to their excellent optical and plasmon properties, biocompatibility, low toxicity, high specific surface areas, and other desirable characteristics after surface functionalization [[32], [33], [34]]. In terms of influenza control and prevention, AuNP can be used for the development of multivalent anti-influenza agents, vaccines, and virus detection. For instance, phospha-oseltamivir [35] and thiosialosides [36,37] stabilized gold nanoparticles have been designed and prepared as potent multivalent inhibitors of NA and HA, respectively. As the aggregation of modified AuNPs in the presence of specific ligands causes color changes, sialic acid-terminated glycan [[38], [39], [40]] or antibody [41] functionalized AuNPs have been used as colorimetric detection of influenza A virus. Moreover, AuNPs can also be used as adjuvants and antigen carriers for the preparation of vaccines against influenza infections [[42], [43], [44]].

Inspired by the use of sialoside-decorated AuNPs as multivalent HA inhibitors and detection sensors, herein, a novel oleanic acid-modified gold nanorod (OA-AuNP) was prepared. Since OA can bind specifically to influenza HA, it was hoped that the resulting OA-AuNP conjugate would serve not only as a multivalent HA inhibitor for anti-influenza therapy but also as a colorimetric diagnostic sensor. The present study aimed to investigate the antiviral activity and underlying mechanisms of this dual-function platform.