NUS Levan Compound Breakthrough: Probiotic Shield Against Norovirus

Understanding Norovirus: A Persistent Global Threat

Norovirus belongs to the Caliciviridae family of viruses and is highly contagious, spreading through contaminated food, water, surfaces, or direct contact with infected individuals. Globally, it accounts for approximately 685 million cases of acute gastroenteritis annually, leading to over 200,000 deaths, particularly among young children and the elderly. In the United States alone, it triggers around 20 million illnesses and 2,500 reported outbreaks each year, imposing a staggering economic burden exceeding $60 billion.

In Singapore, outbreaks frequently disrupt schools, hospitals, and military camps, with recent incidents affecting hundreds in primary schools and food establishments. The GII.4 strain, responsible for the majority of epidemics, evades immune detection easily, making prevention challenging. Common sources include raw oysters, salads, and poor hand hygiene, highlighting the need for accessible interventions beyond rigorous sanitation.

• Incubation period: 12-48 hours
• Symptoms last 1-3 days but shedding persists up to two weeks
• Highly stable in environments, surviving on surfaces for days

The NUS Team Behind the Innovation

Led by Associate Professor Li Dan from NUS's Department of Food Science and Technology within the Faculty of Science, the research team built on prior work establishing a zebrafish embryo model for cultivating human norovirus in 2023. This model overcame longstanding barriers in studying the virus, which previously required scarce human biopsy samples or surrogate animal models with limited relevance.

Assoc Prof Li's group screened exopolysaccharides from various probiotic bacteria, identifying levan from Bacillus subtilis CU1 as exceptionally potent. Bacillus subtilis, a gram-positive soil bacterium commonly used in fermented foods like natto, produces levan—a fructose-based polymer—naturally during growth. The team's expertise in food microbiology positioned them perfectly to explore microbial products for antiviral applications.

Levan: The Probiotic-Derived Polysaccharide Explained

Levan, short for levulosan, is a naturally occurring exopolysaccharide—a long-chain sugar secreted by certain bacteria outside their cells. Unlike living probiotics, levan is inert, posing no risk of overgrowth or side effects in vulnerable populations. Bacillus subtilis CU1 generates high yields of levan when fermented in nutrient-rich media, making it scalable for commercial use.

Structurally, levan consists of β-2,6-linked fructose units with branch points, giving it a unique high-avidity binding capability. This allows it to adhere strongly to viral protrusions, such as the protruding (P) domains on norovirus capsids, without triggering inflammation or immune overreaction. Step-by-step, the process involves the bacterium metabolizing sugars into levan via levansucrase enzyme, which polymerizes fructose from sucrose substrates.

Mechanism of Action: Blocking Infection at the Source

The magic lies in levan's ability to form a physical blockade. Norovirus initiates infection by binding its capsid proteins to histo-blood group antigens (HBGAs) on gut epithelial cells. Levan intercepts this by attaching to the virus's VP1 protein, particularly the P2 subdomain, with such affinity that it prevents HBGA recognition.

In vitro assays confirmed dose-dependent inhibition, where increasing levan concentrations correlated with fewer infectious particles. Unlike chemical disinfectants that inactivate post-contamination, levan acts preventively inside the body, ideal for oral supplements. This non-lytic approach preserves gut microbiota balance while neutralizing threats.

Photo by Hoyoun Lee on Unsplash

Experimental Validation Using Zebrafish Model

Leveraging their 2023 innovation, the NUS team infected zebrafish embryos with GII.4 norovirus, mimicking human gut infection. Embryos pre-treated with levan showed significantly reduced viral replication, measured via qRT-PCR for viral RNA loads. Controls without levan exhibited high infection rates, underscoring the compound's specificity.

Binding assays using virus-like particles (VLPs) visualized via transmission electron microscopy revealed levan-coated virions unable to adhere to cellular mimics. No cytotoxicity was observed even at high doses, confirming safety. These results position levan as superior to surrogates like murine norovirus, which respond differently to inhibitors.

Scalable Production: Fermenting Carrot Juice

A game-changer for accessibility: the team fermented carrot juice with Bacillus subtilis CU1, yielding high levan concentrations without purification hassles. Carrots provide natural sucrose and nutrients, supporting bacterial growth while infusing the product with vitamins. Post-fermentation, the juice retained full antiviral potency, suggesting ready-to-drink functional beverages.

This method bypasses costly extraction, aligning with Singapore's food innovation ecosystem. Pilot batches produced levan levels comparable to lab media, stable for months under refrigeration. Future optimization could target everyday foods like yogurt or juices for mass prevention.

Public Health Implications in Singapore and Beyond

Singapore faces recurrent norovirus spikes in dense settings like hawker centers and dormitories. Levan-enriched products could safeguard high-risk groups: infants, seniors, and immunocompromised patients. In hospitals, where outbreaks cost thousands in isolation and treatment, prophylactic use might curb spread.

Globally, in developing regions with poor sanitation, affordable supplements address the 200,000 annual deaths. Economic savings from reduced outbreaks—hospital stays average $1,000 per case—could reach billions. Integration into national health campaigns, like Singapore's Health Promotion Board initiatives, seems feasible.Learn more from NUS announcement

Advantages Over Existing Strategies

• No living microbes: Safer than probiotics for neonates or transplant patients.
• Targeted prevention: Acts early, unlike rehydration therapies.
• Natural and scalable: Food-grade production lowers costs vs. synthetic drugs.
• Broad potential: May inhibit other caliciviruses pending tests.

Compared to bleach disinfection (environmental only) or alcohol sanitizers (ineffective on enveloped viruses), levan offers internal protection.

Future Directions and Challenges

Next steps include human clinical trials for safety and efficacy, formulation into gummies or drinks, and strain surveillance for emerging variants. Assoc Prof Li emphasized: “Our findings show that it is possible to block the virus before it can establish an infection. This could be especially useful for people who cannot rely on strong immune responses.”

Challenges: Regulatory approval for novel foods, stability in gastric acid, and cost-effectiveness studies. Collaborations with A*STAR and SFA could accelerate commercialization. Long-term, levan exemplifies microbiome engineering for antivirals.View the full study in Food Microbiology

Photo by Hanna Lazar on Unsplash

NUS's Role in Food Science Innovation

NUS consistently leads in applied microbiology, from zebrafish norovirus models to gut-brain axis therapies. The Faculty of Science invests in interdisciplinary labs, fostering talents like PhD student Siyu Chen, first author. This positions Singapore as a biotech hub, attracting global partnerships.

For aspiring researchers, NUS offers robust programs in food safety and antimicrobials, aligning with national priorities like food security.

Broader Impacts on Global Health Security

Beyond norovirus, levan's binding mechanism could inspire defenses against other non-enveloped viruses like rotavirus or adenovirus. In an era of antimicrobial resistance, natural polysaccharides reduce reliance on antibiotics for secondary infections. Singapore's research ecosystem, blending academia and industry, exemplifies agile responses to pandemics.WHO facts on norovirus burden

This discovery underscores higher education's pivotal role in translating lab insights to societal benefits, inspiring similar efforts worldwide.