Elsevier

Fish & Shellfish Immunology

Volume 66, July 2017, Pages 26-34
Fish & Shellfish Immunology

Full length article
Norovirus contamination and the glycosphingolipid biosynthesis pathway in Pacific oyster: A transcriptomics study

https://doi.org/10.1016/j.fsi.2017.04.023Get rights and content

Highlights

  • RNA-seq proved to be an effective method to explore the expression of genes associated with viral infection.

  • The comparison at the transcriptome level contributed to our understanding of the related genes expression changes and KEGG pathways involved in the process of GII.4 NoV accumulated in oysters' digestive tissue.

  • The glycosphingolipid biosynthesis pathway likely contributed to the concentration of NoVs in the oyster digestive tract.

Abstract

Noroviruses are the primary pathogens associated with shellfish-borne gastroenteritis outbreaks. These viruses remain stable in oysters, suggesting an active mechanism for virus concentration. In this study, a deep RNA sequencing technique was used to analyze the transcriptome profiles of Pacific oysters at different time points after inoculation with norovirus (GII.4). We obtained a maximum of 65, 294, 698 clean sample reads. When aligned to the reference genome, the average mapping ratio of clean data was approximately 65%. In the samples harvested at 12, 24, and 48 h after contamination, 2,223, 2,990, and 2020 genes, respectively, were differentially expressed in contaminated and non-contaminated oyster digestive tissues, including 500, 1748, and 1039 up-regulated and 1723, 1242, and 981 down-regulated genes, respectively. In particular, FUT2 and B3GNT4, genes encoding the signaling components of glycosphingolipid biosynthesis, were significantly up-regulated in contaminated samples. In addition, we found up-regulation of some immune- and disease-related genes in the MHC I pathway (PA28, HSP 70, HSP90, CANX, BRp57, and CALR) and MHC II pathway (GILT, CTSBLS, RFX, and NFY), although NoVs did not cause diseases in the oysters. We detected two types of HBGA-like molecules with positive-to-negative ratios similar to type A and H1 HBGA-like molecules in digestive tissues that were significantly higher in norovirus-contaminated than in non-contaminated oysters. Thus, our transcriptome data analysis indicated that a human pathogen (GII.4 Norovirus) was likely concentrated in the digestive tissues of oysters via HBGA-like molecules that were synthesized by the glycosphingolipid biosynthesis pathway. The identified differentially expressed genes also provide potential candidates for functional analysis to identify genes involved in the accumulation of noroviruses in oysters.

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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