Antiviral Activity of Olanexidine-Containing Hand Rub against Human Noroviruses

Abstract

Human norovirus (HuNoV) is the leading cause of epidemic and sporadic acute gastroenteritis worldwide. HuNoV transmission occurs predominantly by direct person-to-person contact, and its health burden is associated with poor hand hygiene and a lack of effective antiseptics and disinfectants. Specific therapies and methods to prevent and control HuNoV spread previously were difficult to evaluate because of the lack of a cell culture system to propagate infectious virus. This barrier has been overcome with the successful cultivation of HuNoV in nontransformed human intestinal enteroids (HIEs). Here, we report using the HIE cultivation system to evaluate the virucidal efficacy of an olanexidine gluconate-based hand rub (OLG-HR) and 70% ethanol (EtOH_70%) against HuNoVs. OLG-HR exhibited fast-acting virucidal activity against a spectrum of HuNoVs including GII.4 Sydney[P31], GII.4 Den Haag[P4], GII.4 New Orleans[P4], GII.3[P21], GII.17[P13], and GI.1[P1] strains. Exposure of HuNoV to OLG-HR for 30 to 60 s resulted in complete loss of the ability of virus to bind to the cells and reduced in vitro binding to glycans in porcine gastric mucin. By contrast, the virucidal efficiency of EtOH_70% on virus infectivity was strain specific. Dynamic light scattering (DLS) and electron microscopy of virus-like particles (VLPs) show that OLG-HR treatment causes partial disassembly and possibly conformational changes in VP1, interfering with histo-blood group antigen (HBGA) binding and infectivity, whereas EtOH_70% treatment causes particle disassembly and clumping of the disassembled products, leading to loss of infectivity while retaining HBGA binding. The highly effective inactivation of HuNoV infectivity by OLG-HR suggests that this compound could reduce HuNoV transmission. IMPORTANCE Human noroviruses (HuNoVs) are highly contagious and cause nonbacterial acute gastroenteritis in all age groups worldwide. Since the introduction of rotavirus vaccines, HuNoVs have become the leading cause of diarrheal illness in children. These viruses are very stable in the environment and resistant to common disinfectants. This study evaluated the virucidal efficacy of a new disinfectant, olanexidine-based hand rub (OLG-HR), against HuNoV strains in an ex vivo human intestinal stem cell-derived enteroid (HIE) cultivation system. Exposure of multiple HuNoV strains to OLG-HR for 30 to 60 s resulted in complete loss of infectivity and binding to HBGAs, possibly due to partial disassembly and conformational changes in the major virus capsid (VP1). By comparison, the virucidal efficiency of EtOH_70% was strain specific, leading to loss of infectivity while retaining HBGA binding. These findings show the utility of the ex vivo HIE cultivation system to test the effectiveness of disinfectants and report a highly effective product.

Keywords: HIEs; HuNoV; IntestiCult media; OLG-HR; antiviral; noroviruses; olanexidine; organoids; virucidal activity.

FIG 1

Virucidal agents, OLG-HR and EtOH, are not cytotoxic to jHIE monolayers. (A) Schematic experimental design. Cell viability of J2 (B) or J4^Fut2 (C) HIE monolayers, expressed as percentage relative to PBS-treated monolayers, was determined by MTT assay. PBS and indomethacin treatments were used as negative and positive controls, respectively. Each experiment was performed twice. Compiled data represent the mean of 6 wells for each treatment. Error bars denote standard deviation. Asterisks indicate significant difference in percentage of viable cells at the indicated treatment compared to those of the PBS control. Significance was determined using one-way ANOVA with Dunnett multicomparison test. *, P < 0.05; ***, P < 0.001; n.s., not significant.

FIG 2

OLG-HR neutralizer does not affect HuNoV replication. Neutralizer efficiency was evaluated with GII.4 variants and GII.3 in J2 and with GII.17 and GI.1 in J4^Fut2 HIE culture monolayers. (A) Schematic experimental design. (B to G) Viral replication of HuNoV strains after each treatment. Compiled data from two experiments are presented. Error bars denote standard deviation (n = 12). Values above the bars represent log₁₀ difference in net replication between conditions ([Δ24 hpi–1 hpi with treatment) − (Δ24 hpi–1 hpi with PBS)]. Significance was determined using one-way ANOVA followed by Dunnett’s test (*, P < 0.05; n.s., not significant).

FIG 3

Virucidal agents, OLG-HR and EtOH, inhibit HuNoV replication. jHIE monolayers were inoculated with each virus previously exposed for 30 s or 1 min to the test substance and diluted with neutralizer. (A) Schematic experimental design. (B to G) Viral replication of HuNoV strains after each treatment. Compiled data of two experiments represent the geometric mean of 12 wells. Error bars denote standard deviation (n = 12). Values represent percentage of fold reduction in net viral replication in each condition relative to PBS control. Significance of net replication (Δ24 hpi–1 hpi) was determined using Dunnett’s multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).

FIG 4

VLP-glycan binding assay. PGM binding to VLPs was determined by incubating OLG-HR-treated GII.4 Sydney, GII.3, GII.17, or GI.1 VLPs with PGM-coated plates, followed by VLP detection with guinea pig polyclonal antiserum to GII.4_Sydney 2012. Values on bars represent the glycan binding percentage relative to PBS control. Error bars denote standard deviation (n = 3). Asterisks indicate significant difference between treatment conditions and PBS (***, P < 0.001; *, P < 0.05).

FIG 5

Dynamic light scattering of VLPs after OLG-HR or ethanol exposure. Average diameters of treated GII.4 Sydney, GII.3, GII.17, and GI.1 VLPs for each condition were calculated using Zetasizer software. VLP samples in PBS were incubated at a ratio of 1:9 with the indicated treatment for 1 min at room temperature and then diluted 1:10 in PBS to get a final VLP concentration of 200 nM in PBS. All experiments were performed in triplicates (n = 3) using standard settings (refractive index, 1.335; viscosity, 0.9; temperature, 25°C).

FIG 6

Negative staining electron microscopy. Each HuNoV VLP sample was mixed at a 1:9 ratio of VLP with the indicated treatment for 1 min at room temperature. A 3-μL aliquot of each sample mixture was applied onto a glow discharged 200-mesh 2/2 Quantifoil holey carbon grid containing an 8-nm layer of carbon and incubated for 3 min. Grids were then blotted, washed with Milli-Q water and then 2% uranyl acetate was applied to the grids for 1 min. Finished grids were stored in a dehumidifier. Images were collected at 200 kV on a JEM-2100 electron microscope with a LaB6 filament and a 3k × 4k direct electron detector camera (DE12). Images were collected manually by SerialEM 9 at a ×25,000 magnification. Scale bar, 50 nm.