Recent advances of phenotypic screening strategies in the application of anti-influenza virus drug discovery.

Seasonal and pandemic influenza virus infections not only pose a serious threat to human health but also cause tremendous economic losses and social burdens. However, due to the inherent high variability of influenza virus RNA genomes, the existing anti-influenza virus drugs have been frequently faced with the clinical issue of emerging drug-resistant mutants. Therefore, there is an urgent need to develop efficient and broad-spectrum antiviral agents against wild-type and drug-resistant mutant strains. Phenotypic screening has been widely employed as a reliable strategy to evaluate antiviral efficacy of novel agents independent of their modes of action, either directly targeting viral proteins or regulating cellular factors involved in the virus life cycle. Here, from the point of view of medicinal chemistry, we review the research progress of phenotypic screening strategies by focusing direct acting antivirals against influenza virus. It could provide scientific insights into discovery of a distinctive class of therapeutic candidates that ensure high efficiency but low cytotoxicity, and address issues from circulation of drug-resistant influenza viruses in the future.

Discovery of antiviral SARS-CoV-2 main protease inhibitors by structure-guided hit-to-lead optimization of carmofur.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) has been targeted for the development of anti-SARS-CoV-2 agents against COVID-19 infection because Mpro processes essential viral polyproteins and plays a key role in SARS-CoV-2 replication. In this study, we report the development of novel SARS-CoV-2 Mpro inhibitors derived from carmofur, a previously identified compound that has shown moderate potency as a covalent inhibitor of SARS-CoV-2 Mpro. To employ a structure-guided drug design strategy, a putative intact binding mode of carmofur at catalytic active site of Mpro was initially predicted by docking simulation. Based on the predicted binding mode, a series of carmofur derivatives aiming to occupy the Mpro substrate binding regions were investigated for structure-activity relationship analysis. As a result, an indole-based derivative, speculated to interact with the S4 binding pocket, 21b (IC50 = 1.5 ± 0.1 µM) was discovered. Its structure was further modified and evaluated in silico by combining docking simulation, free energy perturbation calculation and subpocket interaction analysis to optimize the interactions at the S2 and S4 binding pockets. Among the newly designed novel derivatives, 21h and 21i showed the best inhibitory potencies against Mpro with IC50 values of 0.35 and 0.37 µM, respectively. Moreover, their antiviral activities were confirmed with EC50 values of 20-30 µM in the SARS-CoV-2-infected cell-based assay, suggesting that these novel Mpro inhibitors could be applied as potential lead compounds for the development of substantial anti-SARS-CoV-2 agents.

Intracellular Dynamics-Resolved Label-Free Scattering Reveals Real-Time Metabolism of Single Bacteria.

Nanoscopic investigation of bacterial cells is essential to reveal their physiological status, impacting all cellular functions. Currently, this requires labeled probes or targeted staining procedures. Herein, we report a new bacterial feature, intracellular dynamics-resolved Rayleigh scattering (IDRS), that visualizes spatiotemporal cytoplasmic transitions in unlabeled bacteria and characterizes their real-time physiological status in 10 s. From single-bacterium IDRS signals, we discovered unique spatial patterns and their multiple transitions in Gram-negative and Gram-positive bacteria. The magnitude of IDRS signal variation highly correlated with the metabolic status of bacteria, differentiating persistent subpopulations. This is also the first report demonstrating distinct real-time metabolic conditions of unlabeled drug-resistant bacteria that are exposed to different doses of antibiotics. Our strategy opens up a way to simultaneously trace in situ metabolic and antibiotic resistance statuses, which can be applied in single-cell level control of bacterial metabolism and efficacy with a heterogeneous nature.

Identification of broad-spectrum neutralizing antibodies against influenza A virus and evaluation of their prophylactic efficacy in mice.

Influenza A virus continuously infects humans and the antigenic shifts of this respiratory virus enable it to cross the species barrier, threatening public health with the risk of pandemics. Broadly neutralizing antibodies (bnAbs) that target the antigenic surface glycoprotein, hemagglutinin (HA), of influenza A virus protect against various subtypes of the virus. Here, we screened a human scFv library, through phage display and panning against recombinant HA proteins, to discover human monoclonal antibodies (mAbs) that are broadly active. Consequently, two human mAbs, named G1 and G2, were identified, which target the HA proteins of the H1N1 and H3N2 subtypes, respectively. G1 was shown to have broad binding ability to different HA subtypes of group 1. By contrast, G2 had higher binding affinity but sensed exclusively H3 subtype-derived HAs. In a cell culture-based virus-neutralizing assay, both G1 and G2 efficiently suppressed infection of the parental influenza A viruses of H1N1 and H3N2 subtypes. Mode-of-action studies showed that the G1 antibody blocked HA2-mediated membrane fusion. Meanwhile, G2 inhibited HA1-mediated viral attachment to host cells. It is noteworthy that both antibodies elicited antibody-dependent cellular cytotoxicity (ADCC) activities by recruiting FcγRIIIA-expressing effector cells. In mouse challenge models, single-shot, intraperitoneal administration of chimeric G1 and G2 antibodies with the mouse IgG constant region completely protected mice from viral infections at doses above 10 and 1 mg/kg, respectively. The newly identified bnAbs, G1 and G2, could provide insight into the development of broad-spectrum antivirals against future pandemic influenza A virus involving group 1- or H3-subtyped strains.

A Natural Virucidal and Microbicidal Spray Based on Polyphenol-Iron Sols.

Numerous disinfection methods have been developed to reduce the transmission of infectious diseases that threaten human health. However, it still remains elusively challenging to develop eco-friendly and cost-effective methods that deactivate a wide range of pathogens, from viruses to bacteria and fungi, without doing any harm to humans or the environment. Herein we report a natural spraying protocol, based on a water-dispersible supramolecular sol of nature-derived tannic acid (TA) and Fe3+, which is easy-to-use and low-cost. Our formulation effectively deactivates viruses (influenza A viruses, SARS-CoV-2, and human rhinovirus) as well as suppressing the growth and spread of pathogenic bacteria (Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Acinetobacter baumannii) and fungi (Pleurotus ostreatus and Trichophyton rubrum). Its versatile applicability in a real-life setting is also demonstrated against microorganisms present on the surfaces of common household items (e.g., air filter membranes, disposable face masks, kitchen sinks, mobile phones, refrigerators, and toilet seats).

Evaluation of Antiviral Activity of Gemcitabine Derivatives against Influenza Virus and Severe Acute Respiratory Syndrome Coronavirus 2.

Gemcitabine is a nucleoside analogue of deoxycytidine and has been reported to be a broad-spectrum antiviral agent against both DNA and RNA viruses. Screening of a nucleos(t)ide analogue-focused library identified gemcitabine and its derivatives (compounds 1, 2a, and 3a) blocking influenza virus infection. To improve their antiviral selectivity by reducing cytotoxicity, 14 additional derivatives were synthesized in which the pyridine rings of 2a and 3a were chemically modified. Structure-and-activity and structure-and-toxicity relationship studies demonstrated that compounds 2e and 2h were most potent against influenza A and B viruses but minimally cytotoxic. It is noteworthy that in contrast to cytotoxic gemcitabine, they inhibited viral infection with 90% effective concentrations of 14.5-34.3 and 11.4-15.9 µM, respectively, maintaining viability of mock-infected cells over 90% at 300 µM. Resulting antiviral selectivity was comparable to that of a clinically approved nucleoside analogue, favipiravir. The cell-based viral polymerase assay proved the mode-of-action of 2e and 2h targeting viral RNA replication and/or transcription. In a murine influenza A virus-infection model, intraperitoneal administration of 2h not only reduced viral RNA level in the lungs but also alleviated infection-mediated pulmonary infiltrates. In addition, it inhibited replication of severe acute respiratory syndrome virus 2 infection in human lung cells at subtoxic concentrations. The present study could provide a medicinal chemistry framework for the synthesis of a new class of viral polymerase inhibitors.

Label-free detection and discrimination of respiratory pathogens based on electrochemical synthesis of biomaterials-mediated plasmonic composites and machine learning analysis.

Seasonal outbreaks of respiratory viral infections remain a global concern, with increasing morbidity and mortality rates recorded annually. Timely and false responses contribute to the widespread of respiratory pathogenic diseases owing to similar symptoms at an early stage and subclinical infection. The prevention of emerging novel viruses and variants is also a big challenge. Reliable point-of-care diagnostic assays for early infection diagnosis play a critical role in the response to threats of epidemics or pandemics. We developed a facile method for specifically identifying different viruses based on surface-enhanced Raman spectroscopy (SERS) with pathogen-mediated composite materials on Au nanodimple electrodes and machine learning (ML) analyses. Virus particles were trapped in three-dimensional plasmonic concave spaces of the electrode via electrokinetic preconcentration, and Au films were simultaneously electrodeposited, leading to the acquisition of intense and in-situ SERS signals from the Au-virus composites for ultrasensitive SERS detection. The method was useful for rapid detection analysis (<15 min), and the ML analysis for specific identification of eight virus species, including human influenza A viruses (i.e., H1N1 and H3N2 strains), human rhinovirus, and human coronavirus, was conducted. The highly accurate classification was achieved using the principal component analysis-support vector machine (98.9%) and convolutional neural network (93.5%) models. This ML-associated SERS technique demonstrated high feasibility for direct multiplex detection of different virus species for on-site applications.

Synthesis and biological evaluation of new ß-D-N4-hydroxycytidine analogs against SARS-CoV-2, influenza viruses and DENV-2.

Drug repurposing approach was applied to find a potent antiviral agent against RNA viruses such as SARS-CoV-2, influenza viruses and dengue virus with a concise strategy of small change in parent molecular structure. For this purpose, ß-D-N4-hydroxycytidine (NHC, 1) with a broad spectrum of antiviral activity was chosen as the parent molecule. Among the prepared NHC analogs (8a-g, and 9) from uridine, ß-D-N4-O-isobutyrylcytidine (8a) showed potent activity against SARS-CoV-2 (EC50 3.50 µM), Flu A (H1N1) (EC50 5.80 µM), Flu A (H3N2) (EC50 7.30 µM), Flu B (EC50 3.40 µM) and DENV-2 (EC50 3.95 µM) in vitro. Furthermore, its potency against SARS-CoV-2 was >5-fold, 3.4-fold, and 3-fold compared to that of NHC (1), MK-4482 (2), and remdesivir (RDV) in vitro, respectively. Ultimately, compound 8a was expected to be a potent inhibitor toward RNA viruses as a viral mutagenic agent like MK-4482.

The efficacy of a 2,4-diaminoquinazoline compound as an intranasal vaccine adjuvant to protect against influenza A virus infection in vivo.

Adjuvants are substances added to vaccines to enhance antigen-specific immune responses or to protect antigens from rapid elimination. As pattern recognition receptors, Toll-like receptors 7 (TLR7) and 8 (TLR8) activate the innate immune system by sensing endosomal single-stranded RNA of RNA viruses. Here, we investigated if a 2,4-diaminoquinazoline-based TLR7/8 agonist, (S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexan-1-ol (named compound 31), could be used as an adjuvant to enhance the serological and mucosal immunity of an inactivated influenza A virus vaccine. The compound induced the production of proinflammatory cytokines in macrophages. In a dose-response analysis, intranasal administration of 1 µg compound 31 together with an inactivated vaccine (0.5 µg) to mice not only enhanced virus-specific IgG and IgA production but also neutralized influenza A virus with statistical significance. Notably, in a virus-challenge model, the combination of the vaccine and compound 31 alleviated viral infection-mediated loss of body weight and increased survival rates by 40% compared with vaccine only-treated mice. We suggest that compound 31 is a promising lead compound for developing mucosal vaccine adjuvants to protect against respiratory RNA viruses such as influenza viruses and potentially coronaviruses.

Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection.

The palatine tonsils (hereinafter referred to as "tonsils") serve as a reservoir for viral infections and play roles in the immune system's first line of defense. The aims of this study were to establish tonsil epithelial cell-derived organoids and examine their feasibility as an ex vivo model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The tonsil organoids successfully recapitulated the key characteristics of the tonsil epithelium, including cellular composition, histologic properties, and biomarker distribution. Notably, the basal layer cells of the organoids express molecules essential for SARS-CoV-2 entry, such as angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin, being susceptible to the viral infection. Changes in the gene expression profile in tonsil organoids revealed that 395 genes associated with oncostatin M signaling and lipid metabolism were highly upregulated within 72 h after SARS-CoV-2 infection. Notably, remdesivir suppressed the viral RNA copy number in organoid culture supernatants and intracellular viral protein levels in a dose-dependent manner. Here, we suggest that tonsil epithelial organoids could provide a preclinical and translational research platform for investigating SARS-CoV-2 infectivity and transmissibility or for evaluating antiviral candidates.

Current Progress in the Development of Hepatitis B Virus Capsid Assembly Modulators: Chemical Structure, Mode-of-Action and Efficacy.

Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.

Antiviral Activity of Isoquinolone Derivatives against Influenza Viruses and Their Cytotoxicity.

Influenza viruses are one of the major causative agents for human respiratory infections. Currently, vaccines and antivirals approved for preventing and treating viral infections are available. However, limited protection efficacy and frequent emergence of drug-resistant viruses stand for a need for the development of antivirals with different chemical skeletons from existing drugs. Screening of a chemical library identified an isoquinolone compound (1) as a hit with 50% effective concentrations (EC50s) between 0.2 and 0.6 µM against the influenza A and B viruses. However, it exhibited severe cytotoxic effects with a 50% cytotoxic concentration (CC50) of 39.0 µM in canine kidney epithelial cells. To address this cytotoxic issue, we synthesized an additional 22 chemical derivatives. Through structure-activity, as well as structure-cytotoxicity relationship studies, we discovered compound 21 that has higher EC50 values ranging from 9.9 to 18.5 µM, but greatly alleviated cytotoxicity with a CC50 value over 300 µM. Mode-of-action and cell type-dependent antiviral experiments indicated that it targets viral polymerase activity and functions also in human cells. Here, we present a new class of viral polymerase inhibitors with a core skeleton of isoquinolone, of which antiviral activity could be better improved through following design and synthesis of its derivatives for drug development.

Comparison of Antiviral Activity of Gemcitabine with 2'-Fluoro-2'-Deoxycytidine and Combination Therapy with Remdesivir against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2'-fluoro-2'-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 µM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 µM). In contrast, 2FdC was marginally active (EC50 = 175.2 µM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 µM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.

Sulfamoylbenzamide-based Capsid Assembly Modulators for Selective Inhibition of Hepatitis B Viral Replication.

As the spread of infections caused by hepatitis B virus (HBV) threatens public health worldwide, investigations from multiple perspectives and of various mechanisms of action are urgently required to increase the HBV cure rate. Targeting the encapsidation of the nuclear capsid protein (core protein, HBc) has emerged as an attractive strategy for inhibiting the viral assembly process; however, a drug targeting this mechanism has not yet been approved. We synthesized novel sulfamoylbenzamides (SBAs) as capsid assembly modulators of HBV and found that the effects and safety profiles of compounds 3 and 8 have potential therapeutic applicability against HBV. The formation of tubular particles was time-dependent in the presence of 3, indicating a new mode of protein assembly by SBA compounds. Our findings provide a new entity for developing safe and efficient treatments for HBV infection.

Antiviral activity of lambda-carrageenan against influenza viruses and severe acute respiratory syndrome coronavirus 2.

Influenza virus and coronavirus, belonging to enveloped RNA viruses, are major causes of human respiratory diseases. The aim of this study was to investigate the broad spectrum antiviral activity of a naturally existing sulfated polysaccharide, lambda-carrageenan (λ-CGN), purified from marine red algae. Cell culture-based assays revealed that the macromolecule efficiently inhibited both influenza A and B viruses with EC50 values ranging from 0.3 to 1.4 µg/ml, as well as currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an EC50 value of 0.9 ± 1.1 µg/ml. No toxicity to the host cells was observed at concentrations up to 300 µg/ml. Plaque titration and western blot analysis verified that λ-CGN reduced expression of viral proteins in cell lysates and suppressed progeny virus production in culture supernatants in a dose-dependent manner. This polyanionic compound exerts antiviral activity by targeting viral attachment to cell surface receptors and preventing virus entry. Moreover, its intranasal administration to mice during influenza A viral challenge not only alleviated infection-mediated reductions in body weight but also protected 60% of mice from virus-induced mortality. Thus, λ-CGN could be a promising antiviral agent for preventing infection with several respiratory viruses.

Determination of the vRNA and cRNA promoter activity by M segment-specific non-coding nucleotides of influenza A virus.

Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5' and 3' untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5' and 3' UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1-4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14th nucleotide uridine (U14') of the 5' UTR and the 13th nucleotide adenosine (A13) of the 3' UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14' and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13' and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.

TRIM Proteins and Their Roles in the Influenza Virus Life Cycle.

The ubiquitin-proteasome system (UPS) has been recognized for regulating fundamental cellular processes, followed by induction of proteasomal degradation of target proteins, and triggers multiple signaling pathways that are crucial for numerous aspects of cellular physiology. Especially tripartite motif (TRIM) proteins, well-known E3 ubiquitin ligases, emerge as having critical roles in several antiviral signaling pathways against varying viral infections. Here we highlight recent advances in the study of antiviral roles of TRIM proteins toward influenza virus infection in terms of the modulation of pathogen recognition receptor (PRR)-mediated innate immune sensing, direct obstruction of influenza viral propagation, and participation in virus-induced autophagy.

SAR study of bisamides as cyclophilin a inhibitors for the development of host-targeting therapy for hepatitis C virus infection.

The therapy of chronic hepatitis C virus infections has significantly improved with the development of direct-acting antivirals (DAAs), which contain NS3/4A protease, NS5A, and NS5B polymerase inhibitors. However, mutations in specific residues in these viral target genes are associated with resistance to the DAAs. Especially inhibitors of NS3/4A protease and NS5A, such as grazoprevir and velpatasvir, have a low barrier to resistant mutations. As a result, the mutations influence the virological outcomes after DAA treatment. CypA inhibitors, as host-targeted agents, act on host factors to inhibit HCV replication, exhibiting a high resistance barrier and pan-genotype activities against HCV. Therefore, they can be developed into alternative, more effective anti-HCV agents. However, CypA inhibitors are natural products and analogs. Based on previous studies, bisamide derivatives were designed and synthesized to develop a novel class of CypA inhibitors. Bisamide derivative 7c is a promising compound with potent anti-HCV activity at subtoxic concentrations. Surface plasmon resonance experiments revealed that 7c directly binds to CypA. All these studies indicated that the derivative 7c is a potent CypA inhibitor, which can be used as a host-targeted agent in combination with other antiviral agents for anti-HCV treatment.

In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

Influenza A virus, one of the major human respiratory pathogens, is responsible for annual seasonal endemics and unpredictable periodic pandemics. Despite the clinical availability of vaccines and antivirals, the antigenic diversity and drug resistance of this virus makes it a persistent threat to public health, underlying the need for the development of novel antivirals. In a cell culture-based high-throughput screen, a ß2-adrenergic receptor agonist, nylidrin, was identified as an antiviral compound against influenza A virus. The molecule was effective against multiple isolates of subtype H1N1, but had limited activity against subtype H3N2, depending on the strain. By examining the antiviral activity of its chemical analogues, we found that ifenprodil and clenbuterol also had reliable inhibitory effects against A/H1N1 strains. Field-based pharmacophore modeling with comparisons of active and inactive compounds revealed the importance of positive and negative electrostatic patterns of phenyl aminoethanol derivatives. Time-of-addition experiments and visualization of the intracellular localization of nucleoprotein NP demonstrated that an early step of the virus life cycle was suppressed by nylidrin. Ultimately, we discovered that nylidrin targets hemagglutinin 2 (HA2)-mediated membrane fusion by blocking conformational change of HA at acidic pH. In a mouse model, preincubation of a mouse-adapted influenza A virus (H1N1) with nylidrin completely blocked intranasal viral infection. The present study suggests that nylidrin could provide a core chemical skeleton for the development of a direct-acting inhibitor of influenza A virus entry.

Discrimination between target and non-target interactions on the viral surface by merging fluorescence emission into Rayleigh scattering.

Direct and quantitative determination of antibodies or cellular receptors dynamically binding to the surface of viral particles is the key issue for predicting the efficacy of therapeutic materials or host susceptibility to a new emerging pathogen. However, targeted visualization of infectious viruses is still highly challenging owing to their nanoscopic sizes and uncontrollable nonspecific interactions with loading molecules responsible for false signals. Here we present a multimodal single-molecule and single-particle (SMSP) visualization capable of simultaneously yet independently tracking Rayleigh scattering and fluorescence that, respectively, are generated from viruses (approximately 100 nm) and labeled interacting molecules. By analyzing real-time trajectories of fluorescent antibodies against a virus surface protein with reference to single virus-derived Rayleigh scattering, we determined heterogeneous binding stoichiometry of virus-antibody couplings irrespective of the nonspecific binder population. Therefore, our multimodal (or multi-level) SMSP assay visually identifies and selectively quantifies specific interactions between them with single binding event accuracy. As a 'specific-binding quantifier' to assess variable host susceptibility to a virus, it was further applied for distinguishing ratiometric bindings and spontaneous dissociation kinetics of synthesized isomeric receptors to influenza virus. The present framework could offer a solid analytical foundation for the development of a direct-acting antiviral agent inhibiting an integral viral enveloped protein and for nanobiological investigation for dissecting spatiotemporal nanoparticle-molecule interactions, which have been scarcely explored compared to those among plasmonic nanoparticles or among molecules only.