IntroductionHuman noroviruses (HuNoVs) are genetically diverse RNA viruses that cause acute gastroenteritis, with genogroup II (GII) accounting for over 90% of global infections. Glycans, particularly histo-blood group antigens (HBGAs), have been identified as attachment factors or receptors for HuNoVs infection. However, the glycan-binding receptors of the later-identified GII genotypes GII.23/24/25 remain elusive.MethodsWe used saliva- and glycan-based ELISA assays to identify the binding spectra of GII.23/24/25 strains. We also solved the crystal structures of their P domains, including the GII.25 P domain in complex with the H disaccharide. Single-point mutagenesis was performed to identify key residues involved in glycan binding.ResultsThe P domains of GII.24 and GII.25 can recognize multiple types of saliva samples, including both A/B/O secretor and nonsecretor individuals. In contrast, GII.23 primarily binds to B secretor saliva samples. Furthermore, GII.23/24 P domains are able to interact with the H disaccharide, whereas GII.25 exhibits binding affinity for both H disaccharide and B trisaccharide. Crystal structures of GII.23/24/25 P domains revealed high structural similarity, and the complex of GII.25 P domains with H disaccharide was resolved. Single-point mutagenesis identified N352, R353, D382, G443, G444, and H445 as critical residues for H disaccharide binding in GII.25 P domain, while A351 determines glycan-binding specificity.DiscussionOur findings demonstrate that GII.23/24/25 exhibit glycan-binding patterns similar to most other GII HuNoV genotypes. The structural insights provide a better understanding of virus-host evolution and inform the development of therapeutic strategies against HuNoVs.

Objective:To investigate the glycan binding specificity of the P protein in GII.8 and GII.9 noroviruses.Methods:The P particle protein of GII.8 and GII.9 noroviruses was expressed by the Escherichia coli system. The oligosaccharide binding assay and saliva binding assay were carried out to explore the interactions between P particles and the histo-blood group antigens (HBGAs). Sequence alignment and structural superimposition were used to analyze the characteristics of the sequence and structure of the glycan binding sites.Results:The P protein of GII.8 showed no obvious binding to the tested glycans while it bound to some saliva samples of A/B/O types. The P protein of GII.9 recognized H disaccharide and interacted with most of the saliva samples. The P protein of GII.8 possessed the similar potential glycan binding site as that of GII.9 according to the sequence and structural analysis. The site was close to that of prevalent GII.4 strains.Conclusions:The non-dominant strains, GII.8 and GII.9 strains showed different glycan binding ability to different HBGAs, indicating that there may be discrepancy in prevalence. It is necessary to continually monitor the prevalence of non-dominant noroviruses.

Noroviruses (NoVs) are the primary cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) are receptors or attachment factors that affect the prevalence and host susceptibility of NoVs. GII.6 NoV is one of the predominant genotypes in humans, which recognizes the type ABO secretor of HBGAs. However, the structural basis of GII.6 NoV's interaction with HBGAs receptors remains elusive. In this study, we investigated the binding features of the GII.6 strain to HBGAs using saliva- and glycan-ELISA assays and characterized the molecular basis of the GII.6 virus that recognizes H disaccharide. We showed that the GII.6 ​P domain recognized some A and O secretor's saliva samples, most B secretor's saliva samples, and H disaccharide antigen, but did not bind non-secretors' saliva. Further, we determined the crystal structures of GII.6 and its complex with H disaccharides at 1.7 ​Å, revealing that the P domain of GII.6 shares the conventional binding interface and mode of GII HBGAs. Single residue mutations at the GII.6-H binding sites could inhibit the binding of GII.6 to HBGAs, demonstrating that the interaction residues were crucial in maintaining NoV-glycan integrity. Finally, structural and sequence analyses showed that the major residues of the GII.6-H interaction were conserved among NoVs in the GII genogroup. Taken together, our study characterized the functional and structural features of GII.6 that allow it to interact with HBGAs, and shed light on NoV evolution, epidemiology, and anti-viral drug development.

目的 体外培养GⅠ.1型人札如病毒(human sapovirus,HuSaV),并制备其衣壳蛋白VP1多克隆抗体.方法 将中国疾病预防中心病毒病预防控制所腹泻室保存的G Ⅰ.1型HuSaV阳性粪便标本接种至添加不同胆酸盐[甘氨鹅脱氧胆酸(GCDCA)、甘氨胆酸(GCA)]的人十二指肠腺癌细胞(HuTu-80)中,利用PCR和RT-qPCR法确定病毒感染、增殖及传代情况.PCR扩增VP1基因,克隆入原核表达载体pGEX-6P-1,构建重组表达质粒pGEX-6P-1-VP1,转化E.coliBL21(DE3),IPTG诱导表达.表达的重组VP1蛋白纯化后免疫2只雌性新西兰大耳白兔,共免疫4次,免疫后18、28、38和48 d采血,ELISA法检测血清效价.结果 G Ⅰ.1型HuSaV在胆酸盐GCA存在下可有效感染HuTu-80细胞,增殖后的病毒在HuTu-80细胞中能够稳定连续传3代.表达的重组GST-VP1蛋白相对分子质量约86 000,纯化后约60 000(切除GST标签).制备的抗HuSaV VP1蛋白多克隆抗体效价可达1:12 800以上.结论 利用HuTu-80细胞添加胆酸盐成功实现了 HuSaV的体外分离培养,并制备了 HuSaV VP1蛋白高效价多克隆抗体,为深入开展HuSaV的鉴定、感染及致病机制等奠定了基础.

诺如病毒(Noroviruses,NoVs)是引起全球急性胃肠炎的常见病原.组织血型抗原(Histo-blood groups antigens,HBGAs)是NoVs黏附因子(受体),能促进病毒感染宿主细胞.NoVs主要衣壳蛋白突出(Protruding,P)区是与HBGAs结合的关键结构域.本研究构建了非流行毒株GII.26型NoVsP区的原核表达重组质粒,以谷胱甘肽巯基转移酶(Glutathione s-transferase,GST)亲和层析纯化P蛋白,人鼻病毒的3C蛋白酶去掉GST标签,通过酶联免疫吸附实验探索P蛋白与HBGAs相互作用的特点,借助同源结构模拟以及结构重叠分析其与相应糖分子之间可能存在的对接位点.结果 表明,P蛋白可与包括A型、B型、AB型、O型和非分泌型的215种唾液中的大部分发生结合,但只与19种寡糖中的H双糖结合;模拟的GII.26P单体的空间构象与GII.17类似,可通过糖结合位点的5个氨基酸与H双糖特异性结合.本研究阐明了GII.26 P蛋白与HBGAs的结合特征及潜在分子机制,为进一步揭示GII.26 NoVs可能的流行趋势及研发潜在抗病毒药物奠定一定的基础.

诺如病毒(Noroviruses,NoVs)是导致人急性胃肠炎的最重要病原体之一,也是引起食源性疾病暴发的首要病原体.组织血型抗原(Histo-blood groups antigens,HBGAs)是NoVs的受体或宿主易感因子.已有研究表明HBGAs与NoVs的感染和流行高度相关.G Ⅱ.23是最近报道的NoVs新基因型.为了研究G Ⅱ.23与HBGAs的结合特征,表达纯化G Ⅱ.23基因型的P蛋白之后,通过唾液和寡糖结合实验研究其与HBGAs的结合特性,并通过同源结构模拟探索G Ⅱ.23 P蛋白与糖抗原潜在的对接分子机制,与已经解析的G Ⅱ.10的P蛋白与岩藻糖的复合物结构进行重叠.结果发现,G Ⅱ.23 P蛋白可以与B型唾液结合,但不结合A、O+和O-非分泌型唾液;P蛋白与H双糖抗原发生结合;分子模拟显示G Ⅱ.23 P蛋白具有与岩藻糖环结合的类似特征.本研究首次揭示了G Ⅱ.23 P蛋白与HBGAs受体的结合特征,为深入探索G Ⅱ.23基因型NoVs的进化、感染以及流行的具体机制提供了基础资料.

目的 分析诺如病毒(norovirus,NoV)G Ⅱ.13毒株09N3145与组织血型抗原(histo-blood groups antigens,HBGAs)的结合特性.方法 表达并纯化G Ⅱ.13 09N3145的P区蛋白,通过唾液及寡糖结合试验确定其与多种不同寡糖的结合特征,并以G Ⅱ.13/2010 P区蛋白与Lea寡糖复合物结构为模板,对G Ⅱ.13 09N3145 P蛋白进行同源模拟.结果 G Ⅱ.13 09N3145的P区蛋白可以与A、B、0+和非分泌型0-发生结合;可以与Lec双糖抗原和乳糖发生结合,而与A、B、H、Lewis型等寡糖无明显结合.分子模拟显示P蛋白使用一个可能的结合位点与半乳糖环结合.结论 确定了G Ⅱ.13 09N3145 P蛋白与HBGAs的结合特征,为NoV宿主相互作用、人诺如病毒(human norovirus,huNoV)进化、流行以及天然抗病毒策略的提出奠定基础.

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as host susceptibility factors. GII.13 and GII.21 huNoVs form a unique genetic lineage that emerged from mainstream GII NoVs via development of a new, nonconventional glycan binding site (GBS) that binds Lea antigen. This previous finding raised the question of whether the new GII.13/21 GBS really has such a narrow glycan binding spectrum. In this study, we provide solid phenotypic and structural evidence indicating that this new GBS recognizes a group of glycans with a common terminal β-galactose (β-Gal). First, we found that P domain proteins of GII.13/21 huNoVs circulating at different times bound three glycans sharing a common terminal β-Gal, including Lec, lactose, and mucin core 2. Second, we solved the crystal structures of the GII.13 P dimers in complex with Lec and mucin core 2, which showed that β-Gal is the major binding saccharide. Third, nonfat milk and lactose blocked the GII.13/21 P domain-glycan binding, which may explain the low prevalence of GII.13/21 viruses. Our data provide new insight into the host interactions and epidemiology of huNoVs, which would help in the control and prevention of NoV-associated diseases.IMPORTANCE Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII huNoVs in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Lea antigen, we found in this study that they can interact with a group of glycans with a common terminal β-Gal, including Lec, lactose, and mucin core 2. However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact the strategy of huNoV control and prevention.

Objective During the 2016 winter season,GⅡ.2 norovirus(NoV) suddenly emerged in China.To elucidate its mechanism of epidemic,this study focused on characteristics of binding between the P protein of capsid and histo-blood group antigens (HBGAs).Methods The research object was G Ⅱ.2 ZTX strain which had an outbreaks by the end of 2016 in Beijing.Recombinant prokaryotic expression plasmid was constructed,and the expression of virus P protein was determined and purified.The P protein characteristics of binding to HBGAs was studied through saliva and oligosaccharide binding experiments.Results Soluble P protein was successfully obtained,and combined with type A,B,AB saliva.Conclusions The result illuminate the combination with new outbreaks of NoV and salivary types,which provided a basis for its pathogenic mechanism and prevention and control measures.

ABSTRACT Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines. IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

Background:During 2016-2017, the previously rare GII.P16-GII.2 norovirus suddenly emerged as the predominant genotype causing gastroenteritis outbreaks in China and other countries. Its origin, phylodynamics, and mechanism behind the predominance remain unclear.Methods:Bayesian phylogenetic analyses were performed on 180 full capsid and 150 polymerase sequences of 2016-2017 GII.P16-GII.2 noroviruses in China, and those for all publicly available GII.P16 and GII.2 sequences. Saliva-based histo-blood group antigen (HBGA) binding assays and crystal structural analysis were conducted by using the P proteins of 2016-2017 GII.P16-GII.2 noroviruses.Results:The reemerging GII.P16-GII.2 norovirus showed a rapid genetic diversification after its emergence in 2012-2013. The antigenicity and HBGA binding profile of the early 2016-2017 and pre-2016 GII.2 noroviruses were similar. A further variant with a single Val256Ile mutation and the conventionally orientated Asp382 in the VP1 protein showed an expanded HBGA-binding spectrum. Mutations on the surface of polymerase that could alter its function were seen, which may help to accelerate the VP1 gene evolution to 5.5 × 10-3 substitutions per site per year. This virus can be traced back to Pearl River Delta, China.Conclusions:Our findings provide new insights into GII.2 norovirus epidemics and highlight the necessity of enhanced global surveillance for potential epidemics of rare-genotype noroviruses.