The neoagarohexaose (NA6) is an oligosaccharide that is derived from agarose, the major component of red algae cell walls, by enzymatic hydrolysis. Here we show that NA6 is a noncanonical Toll-like receptor 4 (TLR4) agonist with antiviral activity against norovirus. Its TLR4 activation was dependent on myeloid differentiation factor 2 (MD2) and cluster of differentiation 14 (CD14), leading to interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) production. This effect was abolished by TLR4 knockdown or knockout in murine macrophages. NA6 inhibited murine norovirus (MNV) replication with an EC₅₀ of 1.5 μM in RAW264.7 cells. It also lowered viral RNA titer in a human hepatocellular carcinoma Huh7-derived cell line harboring a human norovirus subgenomic replicon. The antiviral activity of NA6 was mainly attributed to IFN-β produced through the TLR4-TRIF signaling pathway. NA6-induced TNF-α, which had little effect on norovirus replication per se, primed macrophages to mount greater antiviral innate immune responses when IFN signaling was activated. NA6 boosted the induction of IFN-β in MNV-infected RAW264.7 cells and upregulated IFN-regulatory factor-1, an IFN-stimulated gene. NA6 induced IFN-β expression in the distal ileum with Peyer's patches and oral administration of NA6 reduced MNV loads through activation of TLR4 signaling, highlighting its potential contribution to protective antiviral innate immunity against norovirus.

新琼脂六糖（NA6）是一种寡糖，它是通过琼脂糖（酶法水解）衍生自红藻细胞壁的主要成分的寡糖。在这里，我们显示NA6是具有针对诺如病毒的抗病毒活性的非规范性Toll样受体4（TLR4）激动剂。它的TLR4激活取决于髓样分化因子2（MD2）和分化簇14（CD14），从而导致干扰素-β（IFN-β）和肿瘤坏死因子-α（TNF-α）的产生。通过在小鼠巨噬细胞中TLR4敲低或敲除消除了该作用。NA6以EC ₅₀抑制鼠诺如病毒（MNV）复制在RAW264.7细胞中为1.5μM。它还降低了携带人类诺如病毒亚基因组复制子的人类肝细胞癌Huh7衍生细胞系中病毒RNA的滴度。NA6的抗病毒活性主要归因于通过TLR4-TRIF信号通路产生的IFN-β。NA6诱导的TNF-α，本身对诺如病毒的复制几乎没有影响IFN信号激活后，引发的巨噬细胞会发出更大的抗病毒先天免疫应答。NA6增强了MNV感染的RAW264.7细胞中IFN-β的诱导，并上调了IFN刺激的基因IFN-regulatory factor-1。NA6诱导带有Peyer斑块的回肠末端IFN-β表达，并且通过激活TLR4信号传导，口服NA6减少了MNV负荷，突显了其对保护性抗病毒先天免疫力的潜在贡献。