Full length articleNorovirus contamination and the glycosphingolipid biosynthesis pathway in Pacific oyster: A transcriptomics study
Introduction
Noroviruses (NoVs), which belong to the family Caliciviridae [1], are the major causative agents of water- and food-borne acute nonbacterial gastroenteritis in humans. They are often transmitted by the consumption of contaminated shellfish. The strain-specific binding of NoVs to carbohydrate antigens of the ABH blood group [2], [3] serves as a striking example of viral glycan specificity [4]. In addition to glycoproteins, naturally occurring histo-blood group antigens (HBGAs) are also present on glycosphingolipids (GSLs), which are particularly abundant in the epithelial cells of the gastrointestinal tract [5], [6]. Glycosphingolipids are ubiquitous molecules composed of a lipid and a carbohydrate moiety, and they function as antigen/toxin receptors in cell adhesion/recognition processes. They are also involved in the initiation/modulation of signal transduction pathways. HBGAs are generated through the ordered addition of monosaccharides by glycan-modifying enzymes. The antigens that produce polymorphic ABH, Lewis, and secretor phenotypes can be found on a variety of N- and O-linked glycoproteins, as well as on the lacto-, neolacto-, ganglio-, and globoseries GSLs [7], [8].
Oysters are known to be common vectors for NoV contamination, which is responsible for outbreaks of acute gastroenteritis in humans. As people observed, NoVs may bind specifically to oyster tissues through carbohydrates, which might facilitate the bioaccumulation of NoV and increase its persistence in oysters. It has been demonstrated using immunohistochemistry that NoV particles can bind specifically to the digestive ducts (such as the midgut, main and secondary ducts, and tubules) of oysters via carbohydrate structures with a terminal N-acetylgalactosamine residue through an α linkage, which is the same binding site that recognizes human HBGAs [9]. It has also been verified that multiple HBGAs are expressed in oyster gastrointestinal tissues, which might be the major mechanism for the bioaccumulation of NoVs [10]. However, the molecular mechanisms underlying the role of the HBGA ligand in NoV bioaccumulation in oysters is still poorly understood.
The whole genome sequencing of the Pacific oyster was completed in 2012, which provided information regarding stress adaptation and the complexity of shell formation in this organism [11]. However, systematic analysis of the Pacific oyster genes involved in NoV contamination has not been performed. In the present study, we analyzed the transcriptome of wild Pacific oysters after contamination with GII.4 NoV at different time points. We obtained and functionally annotated a large number of genes that were differentially expressed upon NoV pollution, and verified the gene expression patterns for some of these using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. For simplicity, we focused further analysis on the GSL biosynthesis pathways, as they are representative and relevant examples of functional differentially expressed gene networks potentially related to NoV contamination and maintenance. Our results offered an insight into the molecular mechanism of the synthesis of HBGA-like molecules in oysters and likely shed new light on the concentration and elimination of NoVs in shellfish.
Section snippets
Pacific oysters and GII.4 NoVs
Wild Pacific oysters (Crassostrea gigas), harvested from the clean sea area of Aoshanwei, Qingdao, China, were kindly provided by Professor Li Li of Institute of Oceanology, Chinese Academy of Sciences. Oysters of similar size and strong vitality were scrubbed, rinsed, and bred in large tanks of seawater. Environmental data such as water temperature and salinity were monitored on a daily basis at exactly the same location as the oysters.
Fecal concentrate samples containing NoV genogroup II
Analysis of NoV-induced gene expression patterns in oyster digestive tissues
To identify DEGs in response to GII.4 NoV contamination, 4 DEG libraries (S0, S12, S24, S48) were generated from NoV-contaminated oyster digestive tissues at 0, 12, 24, and 48 h after treatment. Using paired-end sequencing, 23.01, 49.07, 65.29, and 31.68 million 125-bp paired-end reads were generated for the blank control, 12-, 24-, and 48-h samples, respectively. All raw data obtained was uploaded to the SRA database of NCBI, and the corresponding accession numbers are listed in Table 1. The
Discussion
NoV-contaminated oysters are a major cause of food-related illnesses. Oysters are aquatic filter feeders that rapidly concentrate enteric viruses such as poliovirus, hepatitis A virus, and NoV. Viruses are stably maintained in oysters, because depuration does not eliminate viral particles [22], [23]. Compared to a 95% reduction in bacterial levels, only 7% of Norwalk virus was depurated after bioaccumulation [22]. Long-term persistence of viruses in shellfish represents a serious public health
Conclusions
In this report, the transcriptome profiles of Pacific oyster after pollution with GII.4 NoV were analyzed using a deep RNA sequencing technique. In polluted and non-polluted Pacific oyster digestive tissue, DEGs were compared and their associated pathways were analyzed. The bioaccumulation process of GII.4 NoV in Pacific oyster was ascertained by detecting the regulation of a series of glycosyltransferases in the glycosphingolipid biosynthesis: lacto and neo-lacto series pathways. Consistent
Funding
This work was supported by the National Natural Science Fund of China (grant number: 31471663) and the Qingdao Postdoctoral Application Research Project.
Acknowledgments
We thank Prof. Li Li for providing Pacific oyster samples. We acknowledge Dr. Miao Jin at the China Centers for Disease Control and Prevention for providing the GII.4 Norovirus. We also thank Yaya Li and Qian Liu at Gene Denovo Co. (Guangzhou, China) for their help with the images. We would like to thank Editage [www.editage.cn] for English language editing.
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