Accepted Manuscript

Norovirus contamination and the glycosphingolipid biosynthesis pathway in Pacificoyster: A transcriptomics study

Liping Ma, Laijin Su, Hui Liu, Feng Zhao, Deqing Zhou, Delin Duan

PII: S1050-4648(17)30229-2

DOI: 10.1016/j.fsi.2017.04.023

Reference: YFSIM 4548

To appear in: Fish and Shellfish Immunology

Received Date: 4 January 2017

Revised Date: 20 March 2017

Accepted Date: 26 April 2017

Please cite this article as: Ma L, Su L, Liu H, Zhao F, Zhou D, Duan D, Norovirus contamination andthe glycosphingolipid biosynthesis pathway in Pacific oyster: A transcriptomics study, Fish and ShellfishImmunology (2017), doi: 10.1016/j.fsi.2017.04.023.

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Norovirus Contamination and the Glycosphingolipid 1

Biosynthesis Pathway in Pacific Oyster: A 2

Transcriptomics Study 3

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Liping Maa,b , Laijin Sua, Hui Liua , Feng Zhaoa , Deqing Zhoua∗ , Delin Duanb∗∗ 7

8

a Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry 9

of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery 10

Sciences, Qingdao, Shandong, 266071,China 11

b Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese 12

Academy of Sciences, Qingdao, Shandong, 266071, China 13

∗ Corresponding authors 14

E-mail address: zhoudq@ysfri.ac.cn (Zhou); dlduan@qdio.ac.cn (Duan) 15

Tel: +86 0532-85819337(Zhou); +86 0532-82898556 (Duan) 16

Fax: +86 0532-85819337(Zhou); +86 0532-82898556 (Duan) 17

∗ Corresponding author

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong, 266071, China. ∗∗

Corresponding author Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, 266071, China.

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ABSTRACT 18

Noroviruses are the primary pathogens associated with shellfish-borne 19

gastroenteritis outbreaks. These viruses remain stable in oysters, suggesting an active 20

mechanism for virus concentration. In this study, a deep RNA sequencing technique 21

was used to analyze the transcriptome profiles of Pacific oysters at different time 22

points after inoculation with norovirus (GII.4). We obtained a maximum of 23

65,294,698 clean sample reads. When aligned to the reference genome, the average 24

mapping ratio of clean data was approximately 65%. In the samples harvested at 12, 25

24, and 48 h after contamination, 2,223, 2,990, and 2,020 genes, respectively, were 26

differentially expressed in contaminated and non-contaminated oyster digestive 27

tissues, including 500, 1748, and 1039 up-regulated and 1723, 1242, and 981 28

down-regulated genes, respectively. In particular, FUT2 and B3GNT4, genes encoding 29

the signaling components of glycosphingolipid biosynthesis, were significantly 30

up-regulated in contaminated samples. In addition, we found up-regulation of some 31

immune- and disease-related genes in the MHC I pathway (PA28, HSP 70, HSP90, 32

CANX, BRp57, and CALR) and MHC II pathway (GILT, CTSBLS, RFX, and NFY), 33

although NoVs did not cause diseases in the oysters. We detected two types of 34

HBGA-like molecules with positive-to-negative ratios similar to type A and H1 35

HBGA-like molecules in digestive tissues that were significantly higher in norovirus- 36

contaminated than in non-contaminated oysters. Thus, our transcriptome data analysis 37

indicated that a human pathogen (GII.4 Norovirus) was likely concentrated in the 38

digestive tissues of oysters via HBGA-like molecules that were synthesized by the 39

glycosphingolipid biosynthesis pathway. The identified differentially expressed genes 40

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also provide potential candidates for functional analysis to identify genes involved in 41

the accumulation of noroviruses in oysters. 42

Keywords: Crassostrea gigas, Norovirus, Transcriptome, Glycosphingolipid 43

biosynthesis pathway 44

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1. Introduction 64

Noroviruses (NoVs), which belong to the family Caliciviridae [1], are the major 65

causative agents of water- and food-borne acute nonbacterial gastroenteritis in 66

humans. They are often transmitted by the consumption of contaminated shellfish. 67

The strain-specific binding of NoVs to carbohydrate antigens of the ABH blood group 68

[2,3] serves as a striking example of viral glycan specificity [4]. In addition to 69

glycoproteins, naturally occurring histo-blood group antigens (HBGAs) are also 70

present on glycosphingolipids (GSLs), which are particularly abundant in the 71

epithelial cells of the gastrointestinal tract [5,6]. Glycosphingolipids are ubiquitous 72

molecules composed of a lipid and a carbohydrate moiety, and they function as 73

antigen/toxin receptors in cell adhesion/recognition processes. They are also involved 74

in the initiation/modulation of signal transduction pathways. HBGAs are generated 75

through the ordered addition of monosaccharides by glycan-modifying enzymes. The 76

antigens that produce polymorphic ABH, Lewis, and secretor phenotypes can be 77

found on a variety of N- and O-linked glycoproteins, as well as on the lacto-, 78

neolacto-, ganglio-, and globoseries GSLs [7,8]. 79

Oysters are known to be common vectors for NoV contamination, which is 80

responsible for outbreaks of acute gastroenteritis in humans. As people observed, 81

NoVs may bind specifically to oyster tissues through carbohydrates, which might 82

facilitate the bioaccumulation of NoV and increase its persistence in oysters. It has 83

been demonstrated using immunohistochemistry that NoV particles can bind 84

specifically to the digestive ducts (such as the midgut, main and secondary ducts, and 85

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tubules) of oysters via carbohydrate structures with a terminal N-acetylgalactosamine 86

residue through an α linkage, which is the same binding site that recognizes human 87

HBGAs [9]. It has also been verified that multiple HBGAs are expressed in oyster 88

gastrointestinal tissues, which might be the major mechanism for the bioaccumulation 89

of NoVs [10]. However, the molecular mechanisms underlying the role of the HBGA 90

ligand in NoV bioaccumulation in oysters is still poorly understood. 91

The whole genome sequencing of the Pacific oyster was completed in 2012, which 92

provided information regarding stress adaptation and the complexity of shell 93

formation in this organism [11]. However, systematic analysis of the Pacific oyster 94

genes involved in NoV contamination has not been performed. In the present study, 95

we analyzed the transcriptome of wild Pacific oysters after contamination with GII.4 96

NoV at different time points. We obtained and functionally annotated a large number 97

of genes that were differentially expressed upon NoV pollution, and verified the gene 98

expression patterns for some of these using quantitative reverse 99

transcription-polymerase chain reaction (qRT-PCR) analyses. For simplicity, we 100

focused further analysis on the GSL biosynthesis pathways, as they are representative 101

and relevant examples of functional differentially expressed gene networks potentially 102

related to NoV contamination and maintenance. Our results offered an insight into the 103

molecular mechanism of the synthesis of HBGA-like molecules in oysters and likely 104

shed new light on the concentration and elimination of NoVs in shellfish. 105

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2. Materials and methods 107

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2.1. Pacific oysters and GII.4 NoVs 108

Wild Pacific oysters (Crassostrea gigas), harvested from the clean sea area of 109

Aoshanwei, Qingdao, China, were kindly provided by Professor Li Li of Institute of 110

Oceanology, Chinese Academy of Sciences. Oysters of similar size and strong vitality 111

were scrubbed, rinsed, and bred in large tanks of seawater. Environmental data such 112

as water temperature and salinity were monitored on a daily basis at exactly the same 113

location as the oysters. 114

Fecal concentrate samples containing NoV genogroup II (GII.4, Norovirus 115

Hu/GII/Beijing/361/2007/CHN, GenBank Accession No. EU839594) were kindly 116

provided by the Chinese Center for Disease Control and Prevention (CDC, Beijing, 117

China). NoV purification was performed using a modified version of the cesium 118

chloride (CsCl) ultracentrifugation method, as described in a previous study [12]. 119

Fecal concentrates of NoVs (2 mL) were mixed with 8 ml of a saturated CsCl solution. 120

Ultracentrifugation was performed in 15-mL centrifuge tubes at 100,000 ×g and 15 °C 121

for 24 h with a Hitachi centrifuge CP100wx (Hitachi, Tokyo, Japan). The fractions 122

containing NoV were collected by bottom puncture, dialyzed against 123

phosphate-buffered saline (PBS), and then stored at 4 °C until further use. The NoV 124

RNA copies were quantified using real-time RT-PCR, as descried by Ma et al. [13]. 125

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2.2. NoV pollution and sample collection 127

The oysters were bred in the laboratory purification pool for one week. During 128

this time, NoV detection was performed every day using the methods described by 129

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Ma et al. [13]. No NoVs were detected in any of the oyster samples. We then selected 130

20 Pacific oysters in a 10-L beaker with artificial seawater at 20 ± 1 °C. The 131

experiment included 4 groups: three of them were inoculated with 2 mL GII.4 NoV at 132

a concentration of (8.31 ± 0.56) × 105 genomic copies/100 µL and the fourth was a 133

control without NoVs. Pacific oysters polluted with NoV were harvested at 12, 24, 134

and 48 h after contamination. The digestive tissues were used for RNA extraction. At 135

each time point, the mRNA from 5 individuals was pooled as a sample to construct 136

the cDNA library. 137

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2.3. RNA extraction, mRNA purification, and cDNA sequencing 139

Total RNA was extracted from oyster digestive tissue with or without NoV 140

pollution using Tripure reagent (Roche Diagnostics, Roswell, GA, USA) according to 141

the manufacturer's instructions. The total RNA was then treated with RNase-free 142

DNase I (Takara Bio, Shiga, Japan) for 30 min at 37 °C to remove residual DNA. 143

Quantification and quality of the RNA were verified using an Agilent 2100 144

Bio-analyzer (Agilent Technologies, Santa Clara, CA, USA) and the samples were 145

checked by RNase-free agarose gel electrophoresis. Poly (A) mRNA was then isolated 146

using oligo-dT beads (Qiagen, Venlo, Netherlands). The mRNA was broken into short 147

fragments using a fragmentation buffer, after which first-strand cDNA was generated 148

using random hexamer-primed reverse transcription. The second-strand cDNA was 149

then synthesized using RNase H and DNA polymerase I. The cDNA fragments were 150

purified using a QIAquick PCR extraction kit (Qiagen, Venlo, Netherlands). These 151

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purified fragments were then washed with EB buffer for end reparation and poly (A) 152

addition and then ligated to sequencing adapters. Following agarose gel 153

electrophoresis and extraction of the cDNA from gels, the cDNA fragments were 154

purified and enriched by PCR to construct the final cDNA library. The cDNA library 155

was sequenced on the Illumina sequencing platform (Illumina HiSeq™ 2500, 156

Illumina, San Diego, CA, USA) using paired-end technology by Gene Denovo Co. 157

(Guangzhou, China). A Perl program was written to select clean reads by removing 158

low quality sequences (i.e., > 50% bases with quality lower than 20 in one sequence), 159

reads with more than 5% N bases (unknown bases), and reads containing adaptor 160

sequences. 161

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2.4. Transcript assembly and expression value estimation 163

Sequencing reads in FASTQ format were mapped to Crassostrea gigas v9 164

GCA_000297895 from NCBI and splice junctions were identified using TopHat [14]. 165

The Cufflinks package [15] was used for performing genome guided transcript 166

assembly and expression abundance estimates. First, Cufflinks was used to 167

reconstruct transcripts based on genome annotation. The transcripts from each sample 168

were then merged using Cuffmerge. Novel transcripts were extracted from the result 169

using the threshold, “length ≥ 200 bp and exon number ≥ 2,” and compared with 3 170

protein databases to obtain functional annotation using blastx [16] with an E-value 171

cut-off of 1e-5. The databases used for comparison were NCBI non-redundant protein 172

database (Nr) (http://www.ncbi.nlm.nih.gov/), KEGG (http://www.kegg.jp/), and GO 173

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(http://geneontology.org/). Next, the novel transcripts were integrated with the 174

existing transcripts in genome annotation to construct a new .gft file. Finally, 175

Cuffquant and Cuffnorm were used to the estimate the transcript expression values in 176

terms of fragments per kilobase of transcript per million mapped reads with the 177

parameters: library normalization methods, classic-fpkm; library types, frunstranded. 178

All raw data obtained in this study have been uploaded to the SRA database of 179

NCBI, and the corresponding accession numbers are listed in Table 1. 180

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2.5. Statistics of alternative splicing and determination of gene boundaries 182

ASprofile [17] was used to extract, compare, and classify different alternative 183

splicing events from the results of Cufflinks. The gene structures were optimized 184

according to the results of transcript assembly from Cufflinks. The extensions of the 5′ 185

and 3′ boundaries were determined by comparing the potential gene model with the 186

existing gene annotations. 187

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2.6. Differentially expressed genes (DEGs) and function enrichment analyses 189

After the expression level of each transcript and gene was calculated, differential 190

expression analysis was conducted using edgeR [18]. The false discovery rate (FDR) 191

method was used to determine the threshold of the p value in multiple tests; for the 192

analysis, a threshold of FDR ≤ 0.05 and an absolute value of log2Ratio ≥ 1 were 193

used to judge the significance of the differences in gene expression. 194

The DEGs were used for GO and KEGG enrichment analyses, according to a 195

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method similar to that described by Zhang et al. [19]. Both GO terms and KEGG 196

pathways with a Q-value ≤ 0.05 were considered significantly enriched in DEGs. 197

All expression data statistics and visualizations were conducted using the R package 198

software (http://www.r-project.org/). 199

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2.7. Gene expression pattern analysis 201

The union set of all of the DEGs was used for further analysis of expression 202

patterns. Short Time-series Expression Miner v1.3.8 (STEM) [20] was used to cluster 203

the genes into different expression patterns, wherein DEGs belonging to the same 204

cluster exhibited similar expression patterns. Clusters with specific expression 205

patterns were then selected for further GO and KEGG enrichment analysis [21]. 206

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2.8. Verification of transcriptomics data by qRT-PCR 208

We selected 16 representative genes for qRT-PCR analysis to evaluate the 209

expression patterns deduced from the RNA sequencing data. The sequences of the 210

selected DEGs were deposited in NCBI GenBank; the accession numbers are shown 211

in Table S1. Primers for qRT-PCR were designed using Primer-BLAST 212

(http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome), 213

based on the target sequences (Table S1). All reactions were performed with a 25-µL 214

reaction mixture containing 12.5 µL of SYBR Premix Ex Taq II (Takara Bio, Shiga, 215

Japan) and 200 nM each of forward and reverse primer. The reaction cycle parameters 216

were as follows: 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. All 217

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reactions were performed in triplicate. Dissociation curve analysis was also performed 218

after each assay to determine target specificity. β-actin was used as the internal 219

control as it was stably expressed throughout the experiments. The relative expression 220

ratio of the target gene versus β-actin was calculated using the 2–∆∆CT method. All data 221

obtained were presented in terms of relative mRNA expression. The correlation 222

between the data from RNA sequencing and qRT-PCR was expressed as a scatter 223

diagram generated using the R package (version 3.3.0, http://cran.r-project.org/). 224

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2.9. Enzyme-linked immunosorbent assay (ELISA) detection of HBGA-like molecules 226

present in NoV infected or non-infected oyster digestive tissues. 227

The Pacific oysters (Crassostrea gigas), GII.4 NoV, and oyster breeding 228

condition were the same as described in sections 2.1 and 2.2. The breeding 229

experiment included two groups; one was inoculated with 2 mL GII.4 NoV and the 230

other was a control and had no NoV. Pacific oysters with or without NoV 231

inoculation were harvested at 24 h after inoculation. Three grams of the oyster 232

digestive tissue was homogenized in 9 mL phosphate buffered saline (PBS) (pH 7.4) 233

using a handheld homogenizer (Prima, PB100, UK). The homogenized tissues were 234

heated for 10 min at 95 °C and then centrifuged at 3,000 ×g for 15 min at 4 °C. The 235

supernatant was recovered and its protein concentration was estimated using a BCA 236

protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The 237

concentration was adjusted to 40 µg/mL using PBS (pH 7.4). We utilized eight 238

monoclonal antibodies (MAbs; Covance, Dedham, MA, USA) to detect the 239

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expression of various HBGAs and precursor antigens in the oyster digestive tissues 240

and homogenate samples. The MAbs used were specific for blood group antigen 241

precursor (BG-1), type A HBGA (BG-2), type B HBGA (BG-3), type H1 HBGA 242

(BG-4), Lewis a HBGA (BG-5), Lewis b HBGA (BG-6), Lewis x HBGA (BG-7), 243

and Lewis y HBGA (BG-8). The detection of HBGA-like molecules was performed 244

as described by Tian et al. [10]. Briefly, 96-well microplates (Corning, NY, USA) 245

were incubated with 100 µL of tissue supernatant at 4 °C overnight. Each sample 246

had two replicates. The unbound material was removed by washing thrice with 300 247

µL of PBS, and the wells were blocked with 250 µL of 10% skim milk (Yili, Beijing, 248

China) in PBS. The plates were then placed in a bag and incubated at 37 °C for 2 h. 249

After incubation, the wells were again washed thrice with PBS and dried. The 250

MAbs (Covance, USA) (anti-type A, -type B, -H1, -Lex, and –Ley) were diluted at 251

1:1,000; anti-Lea was used at 1:800 and anti-Leb was used at 1:600 dilution. These 252

MAbs (100 µL each) were added to each well incubated at 37 °C for 1 h. Unbound 253

MAbs were removed by washing the wells thrice with 200 µL of PBS with 0.5% 254

Tween 20 (PBS-T). Goat anti-mouse IgG (for IgG MAbs: BG-2, BG-4, or BG-5) or 255

goat anti-mouse IgM (for IgM MAbs: BG-1, BG-3, BG-6, BG-7, or BG-8) 256

conjugated with HRP (Boster, Wuhan, China) and diluted at 1:2,000 in PBS (100 257

µL each) were added to the appropriate wells. The plates were incubated at 37 °C 258

for 1 h and washed thrice with 200 µL of PBS-T. Soluble TMB substrate solution 259

(100 µL; Tiangen, Beijing, China) was added to each well, and the plate was 260

incubated for 15 min in the dark. The reaction was stopped by adding 100 µL of 1 261

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M sulfuric acid to each well. The HBGA-like molecules in Pacific oysters infected 262

with NoV (24 h) or without NoV were measured. The absorbance was read at 450 263

nm using a VICTOR™ X3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, 264

USA). The cut-off point for ELISA was calculated by positive-to-negative (P/N) 265

ratios. Samples were considered positive when P/N ratio ≥ 2. Data comparisons 266

between more than two groups were performed by one-way analysis of variance 267

(ANOVA) and the Holm-Sidak post hoc test. Associated p values were determined 268

by Spearman rank-order correlation. All samples were analyzed in triplicate and 269

represented at least two or three individual experiments to ensure assay consistency. 270

All statistical analyses were performed using Excel (Microsoft, Redmond, WA, 271

USA.). 272

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3. Results 274

3.1. Analysis of NoV-induced gene expression patterns in oyster digestive tissues. 275

To identify DEGs in response to GII.4 NoV contamination, 4 DEG libraries (S0, 276

S12, S24, S48) were generated from NoV-contaminated oyster digestive tissues at 0, 12, 277

24, and 48 h after treatment. Using paired-end sequencing, 23.01, 49.07, 65.29, and 278

31.68 million 125-bp paired-end reads were generated for the blank control, 12-, 24-, 279

and 48-h samples, respectively. All raw data obtained was uploaded to the SRA 280

database of NCBI, and the corresponding accession numbers are listed in Table 1. The 281

base statistical analysis before and after filtering is shown in Table 2. The percentage 282

of Q20 was approximately 94%, that of unassigned base “N” was 0.00%, and the 283

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average GC content was approximately 44% after filtering (Table 2). Using TopHat 284

mapping [14], approximately 65% of the clean reads were matched with the 285

reference Pacific oyster genome (Table 3), indicating adequate representation of the 286

sequenced genes within the genome. We performed a pairwise comparison using 287

un-polluted oysters as the control (marked with 0 h) against 12-, 24-, and 48-h 288

samples as the treatment groups. 289

We also identified differentially expressed unigenes with FDR ≤ 0.001 and 290

absolute value of log2 Ratio ≥ 1. The changes in gene expression were investigated 291

at each of the three time points (i.e., 48 h compared with 24 h, 24 h compared with 12 292

h, and 48 h compared with 12 h). The direction of the expression changes (up- or 293

down-regulation) of the DEGs are shown in Fig. 1. The results showed that the largest 294

number of up- and down-regulated DEGs were 3963 and 2091 at 24 h, compared with 295

12 h and 48 h, respectively. In contrast, only 500 up-regulated DEGs and 981 296

down-regulated DEGs were identified at 0 h, compared with 12 h and 48 h, 297

respectively. The high percentage of up-regulated genes in the 12-24 h period 298

suggested that these genes played important roles during NoV concentration in the 299

digestive tissue of oysters. Overall, a larger number of up-regulated (6189) genes 300

were identified than the down-regulated ones (5010). 301

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3.2. DEG GO-term analysis 303

To examine the expression profile of the DEGs, the expression data (from 0 h to 304

48 h of treatment) were normalized to 0, log2(υ12h/υ0h), log2(υ24h/υ0h), log2(υ48h/υ0h). 305

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We were able to cluster 10,738 DEGs into 25 profiles using the STEM software, in 306

which 6293 DEGs were clustered into 9 profiles (p-value ≤ 0.05), including 4 307

up-regulated patterns (Profiles 14, 15, 8, and 16) and 5 down-regulated ones (Profiles 308

7, 5, 6, 9, and 10) (Fig. 2). To understand the functions of the DEGs and the biological 309

processes involved in NoV pollution, all the DEGs were mapped to the terms in GO 310

(Fig. 3) and KEGG databases. GO analysis showed that 4,324, 6,513, and 4,136 311

unigenes had GO annotations at 12, 24, and 72 h after contamination, respectively. 312

Among the up-regulated genes of oysters at 12 h (24 or 48 h) after contamination (Fig. 313

3), 281 (1414 or 877) unigenes were mapped to biological processes, 204 (1208 or 314

539) were mapped to cellular components, and 313 (1202 or 736) were mapped to 315

molecular functions. Under the biological process category, most were classified into 316

“cellular process” and “metabolic process.” The majority of the cellular 317

component-related genes were involved in “the cell,” “cell part,” and “organelle.” In 318

contrast, most of the molecular function-related genes were involved in “binding” and 319

“catalytic activity.” 320

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3.3. DEG KEGG pathway enrichment analysis 322

We subjected 10,101 DEGs to KEGG pathway enrichment analysis. Of these, 323

39.5% (3,991/10,101) were annotated. The 10 KEGG pathways with the highest 324

representation of DEGs are shown in Table 4. Significantly enriched pathways 325

included DNA replication (ko03030, p value = 4.94E-6), antigen processing and 326

presentation (ko04612, p value = 9.16E-6), cell cycle (ko04110, p value = 0.000184), 327

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mineral absorption (ko04978, p value = 0.000634), glycosphingolipid biosynthesis: 328

lacto and neolacto series (ko00601, p value = 0.000657), mismatch repair (ko03430, p 329

value = 0.001442), progesterone-mediated oocyte maturation (ko04914, p value = 330

0.001861), base excision repair (ko03410, p value = 0.003915), nucleotide excision 331

repair (ko03420, p value = 0.016732), and protein digestion and absorption (ko04974, 332

p value = 0.017728). Notably, 28 unigenes, among the 1222 DEGs (2.29 %) in profile 333

14, 2 (0.29 %) of the 688 in profile 15, and 2 (0.28 %) of the 688 in profile 6, were 334

annotated to the DNA replication pathway, whereas no DEGs in profiles 8 and 5 were 335

annotated to this pathway. 336

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3.4. Analysis of differently expressed unigenes related to glycosphingolipid 338

biosynthesis: lacto and neo lacto series 339

Compared to non-infected oysters, we identified 6189 up-regulated and 5010 340

down-regulated genes at 48 h after NoV contamination. Owing to the huge amount of 341

data, we focused our analysis on the major genes related to the glycosphingolipid 342

biosynthesis: lacto and neolacto series pathways, which are known to be important for 343

NoV accumulation. 344

Table 5 lists the 10 major genes related to the glycosphingolipid biosynthesis: 345

lacto and neolacto series pathways. Of these, B3GALT1 and B3GALT5 belong to the 346

galactosyltransferase family. B3GALT1 (CGI_10024598) was clustered in profile 5, 347

whereas B3GALT5 (CGI_10027819) was clustered in profile 7. These genes were 348

down-regulated at 12 h after NoV contamination (–3.8- and –1.8-fold) a nd 349

subsequently up-regulated at 48 h after contamination. In addition, two bre-4 genes 350

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(CGI_10002434 and CGI_10028074) and two GCNT3 genes (CGI_10023468 and 351

CGI_10026669) were consistently down-regulated at all 3 time points. In contrast, 352

galactoside 2-alpha-L-fucosyltransferase 2 (FUT 2) and a newly identified gene 353

(XLOC_023979), which was predicted to have the same homologous function as FUT 354

2 based on blastp 355

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSear356

ch&LINK_LOC=blasthome), representing functions midstream of the 357

glycosphingolipid biosynthesis pathway, were significantly up regulated at 24 h (1.68- 358

and 2.66-fold, respectively) and 48 h (3.08- and 1.25-fold, respectively) after 359

contamination. Similarly, the expression of B3GNT4, which was also in profile 15, 360

was up-regulated at 24 h (2.57-fold) and 48 h (2.73-fold). All the sequences of the 361

above genes, except for the 2 new genes, were verified and deposited in NCBI 362

GenBank; the accession numbers are shown in Table 5. 363

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3.5. Verification of transcriptomics data by qRT-PCR 365

To confirm the accuracy of the transcriptome analysis results, the mRNAs of 16 366

DEGs with different fold changes were selected and measured by qRT-PCR. The 367

results showed a strong correlation between the data from RNA sequencing and 368

qRT-PCR (r = 0.835, p < 0.001, Fig. 4). For each gene, the expression count values of 369

the transcriptome data exhibited similar expression profiles at all the four time stages, 370

compared with the results of qRT-PCR (Fig. S1). Thus, overall, the qRT-PCR analysis 371

confirmed the expression of DEGs as detected by the high-throughput sequencing 372

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analysis. 373

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3.6. Detection of HBGA-like molecules in NoV-contaminated or non-contaminated 375

oyster digestive tissue 376

We utilized eight types of HBGA-specific human MAbs to analyze the type-like 377

HBGAs in the digestive tissues of Pacific oysters. The ELISA results indicated that 378

type A and H1 HBGA-like molecules were the primary HBGA forms present in the 379

oyster digestive tissues. Conversely, the type Lewis a and Lewis y HBGA-like 380

molecules showed relatively weak positive signal (Fig. 5). Notably, the P/N ratio of 381

type A, H1, Lea, and Ley HBGA-like molecules in the digestive tissues of 382

NoV-polluted oysters was significantly higher than that in the tissues of non-polluted 383

oysters (p < 0.01; Fig. 5). However, no obvious positive signals were detected for the 384

other types of HBGAs. 385

386

4. Discussion 387

NoV-contaminated oysters are a major cause of food-related illnesses. Oysters 388

are aquatic filter feeders that rapidly concentrate enteric viruses such as poliovirus, 389

hepatitis A virus, and NoV. Viruses are stably maintained in oysters, because 390

depuration does not eliminate viral particles [22, 23]. Compared to a 95% reduction in 391

bacterial levels, only 7% of Norwalk virus was depurated after bioaccumulation [22]. 392

Long-term persistence of viruses in shellfish represents a serious public health hazard 393

and is therefore an issue of concern. As NoVs attach to carbohydrates of the ABH and 394

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Lewis histo-blood group family in humans, the potential for similar specific binding 395

in oyster tissues via related carbohydrates has been examined [9]. The results of this 396

analysis have brought new insights into the problem of virus accumulation in oysters. 397

However, the detailed molecular mechanisms behind the NoV acceptor in oysters 398

remain unclear. 399

The NoV genotype, GII.4, is the most prevalent and infectious form of the virus 400

[24]. Although only comprising approximately 10% of the body mass of the bivalve, 401

oyster digestive tissues contain a large majority of the contaminating viruses, and 402

bivalve digestive tissues have been shown to be the target of virus contamination [25]. 403

Because of their importance, we chose GII.4 NoV and Crassostrea gigas for our 404

research. We employed a deep RNA sequencing technique to analyze the 405

transcriptome profiles of oyster digestive tissue at different time points following 406

NoV contamination. From this, we identified 10,101 DEGs in non-infected (0 h) and 407

infected (12, 24, or 48 h) oyster digestive tissues, in which 3,991 unigenes were 408

annotated in 235 KEGG pathways. Overall, 6,189 unigenes were up-regulated and 409

5,010 were down-regulated between 0-48 h after contamination. Among the annotated 410

KEGG pathways, we considered the glycosphingolipid biosynthesis pathway 411

especially relevant. It is known that virus binding to the cell surface represents the 412

first step of infection and therefore, acts as a determinant for tropism. Viruses utilize 413

different receptors depending on the cell type. Molecules on the cell surface available for 414

virus binding are typically glycoconjugates (e.g., glycosphingolipids, glycoproteins, and 415

proteoglycans) [4], with HBGAs representing the receptors of most human NoVs [26, 416

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27]. 417

Accordingly, in this report, we focused on the glycosphingolipid biosynthesis: 418

lacto and neo-lacto series pathways (p value = 0.000657; Additional file 1). Multiple 419

glycosyltransferase DEGs were found in this pathway. We also observed changes in 420

the expression of these genes upon GII.4 NoV contamination. Although the synthesis 421

pathway of HBGA-like molecules in oysters is not well known, we may provide some 422

idea of it from the oligosaccharide chain structures of different types of HBGA. For 423

example, beta-1, 3-galactosyltransferase (B3GALT), which functions upstream of the 424

glycosphingolipid biosynthesis lacto series pathway, transfers galactose from 425

UDP-galactose to substrates with a terminal beta-N-acetylglucosamine (beta-GlcNAc) 426

residue. It is also involved in the biosynthesis of carbohydrate moieties of glycolipids 427

and glycoproteins. It was down-regulated during the NoV contamination, possibly 428

because it was inactive towards substrates with terminal alpha-N-acetylglucosamine 429

(alpha-GlcNAc) or alpha-N-acetylgalactosamine (alpha-GalNAc) residues. The 430

terminal residues of type A HBGA is alpha-GalNAc. Previous studies have shown that 431

the binding of NoV to oyster tissue sections involved A/H-like HBGA [9, 10].The 432

biosynthetic pathway of HBGAs involves the linkage of glycans and oligosaccharide 433

chains to a disaccharide precursor [28-30]. The addition of alpha-L-fucose by an 434

alpha-1,2-fucosyltransferase (FUT 2) to the terminal beta-D-galactose residue of 435

glycoconjugates via an alpha-1,2-linkage generates the H antigens [31], which may be 436

followed by the biosynthesis of A or B antigens through the activity of the blood 437

group A or B transferases, respectively. In addition, in humans, there are two 438

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functional alpha-1,2-fucosyltransferases encoded by the FUT1 and FUT2 genes. 439

FUT1 is responsible for producing the H antigen in red blood cells [32], while FUT2 440

is expressed in the epithelia of secretory tissues, and individuals termed "secretors" 441

have at least one functional copy of this gene [33]. We found 3 DEGs that were 442

similar to FUT1 and FUT2, and were identified in the same pathway: CGI_10017887, 443

XLOC_023979, and XLOC_023978. The latter two are new genes in Crassostrea 444

gigas. The two genes except for XLOC_023978 were up-regulated during NoV 445

pollution. This may have been caused by the binding of NoV and H/A HBGA, 446

resulting in an increase in the expression of its synthetic gene. Further functional 447

analysis of these three genes will be helpful for understanding the synthesis of 448

HBGA-like molecules in Crassostrea gigas. Notably, the identification of these genes 449

demonstrated the existence of a biosynthetic pathway for HBGA-like molecules in 450

Crassostrea gigas, which likely participates in the GII.4 NoV pollution process. 451

To verify that successful contamination by NoV was associated with the 452

expression of HBGAs, we used ELISA to detect the changes in HBGA-like molecules 453

in NoV-infected and uninfected oysters. Notably, the expression levels of type A and 454

H1 HBGA-like ligands in the digestive tissue of oysters were markedly increased 455

after inoculation with GII.4 NoV, which was consistent with up-regulation of FUT2, 456

as revealed by RNA-seq. In our previous study, we found that the detection rate of 457

type A , H1, and Lewis y HBGA-like molecules in the digestive tissue of oysters was 458

95%, 35%, and 20%, respectively [34]. We detected all 3 of these HBGA-like 459

molecules in NoV-infected oysters. Notably, Lewis y HBGA-like ligand was not 460

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detected (P/N value < 2) in non-infected oysters. Therefore, we speculated that NoVs 461

promoted the expression of HBGA-like molecules in oysters and that GII.4 NoV 462

bound to the digestive tissues mainly through type A-like and H-like HBGA. This 463

finding was consistent with the results of previous studies on GI.1 NoV binding to the 464

digestive tissue of Crassostrea gigas. Le Guyader et al. [9] reported that Norwalk 465

virus particles specifically bound to digestive ducts through type A-like HBGA. Tian 466

et al. [10] found that the intensity of rNVLP binding was correlated with the 467

expression levels of type A and H HBGA-like molecules in the gastrointestinal tract 468

of Pacifica oysters. Maalouf et al. [35] demonstrated that unlike genogroup GI.1, 469

NoVs of the genogroup GII.4 bound not only to digestive tissues, but also to gills and 470

mantle tissue sections, through a bond involving a sialic acid in α 2,3 linkage. 471

However, in digestive tissues, the interaction involved both sialic acid and an A-like 472

carbohydrate ligand. Unfortunately, we did not perform HBGA detection in the gills 473

and mantle, and have no information regarding the binding patterns of GII.4 NoV in 474

these tissues. Because of the complicated HBGA-like ligands present in oysters, 475

additional research is needed to resolve their individual roles in NoV contamination. 476

It is well known that NoVs are human enteroviruses and not oyster pathogens; 477

however, it is interesting to note that we observed antigen processing and presentation, 478

mismatch repair, base excision repair, and nucleotide excision repair KEGG pathways 479

during NoV contamination of oysters. This indicated that NoV contamination caused 480

an immune response as well as base, nucleotide excision, or mismatch in the DNA in 481

the oysters. After analyzing the processing and presentation of the antigen pathways, 482

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we observed the up-regulation of some immune- and disease-related genes in the 483

MHC I pathway (PA28, HSP 70, HSP90, CANX, BRp57, and CALR) and MHC II 484

pathway (GILT, CTSBLS, RFX, and NFY), although no antigens were presented to the 485

T cells. This phenomenon suggested that NoVs could not completely infiltrate the 486

digestive tract cells of the oysters, but remained in the transmembrane region, causing 487

a slight immune response. This may explain why the NoVs did not cause diseases in 488

the oysters. Further research is required for exploring the details of this phenomenon. 489

5. Conclusions 490

In this report, the transcriptome profiles of Pacific oyster after pollution with 491

GII.4 NoV were analyzed using a deep RNA sequencing technique. In polluted and 492

non-polluted Pacific oyster digestive tissue, DEGs were compared and their 493

associated pathways were analyzed. The bioaccumulation process of GII.4 NoV in 494

Pacific oyster was ascertained by detecting the regulation of a series of 495

glycosyltransferases in the glycosphingolipid biosynthesis: lacto and neo-lacto series 496

pathways. Consistent with this, the results of ELISA confirmed the up-regulation of 497

the type A and H1-like HBGA molecules by GII.4 NoV pollution. Thus, our findings 498

suggested that NoV bound to carbohydrates linked with glycosphingolipids, resulting 499

in its accumulation and maintenance in shellfish. In addition, although NoVs did not 500

cause diseases in the oysters, they did cause an immune response. Further studies will 501

be needed to verify this finding and to explore the mechanism behind it. 502

503

Acknowledgments 504

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We thank Prof. Li Li for providing Pacific oyster samples. We acknowledge Dr. 505

Miao Jin at the China Centers for Disease Control and Prevention for providing the 506

GII.4 Norovirus. We also thank Yaya Li and Qian Liu at Gene Denovo Co. 507

(Guangzhou, China) for their help with the images. We would like to thank Editage 508

[www.editage.cn] for English language editing. 509

510

Funding: This work was supported by the National Natural Science Fund of China 511

(grant number: 31471663) and the Qingdao Postdoctoral Application Research 512

Project. 513

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604

Supplementary data 605

606

Table S1. Primer sequences for differentially expressed genes. The selected genes 607

were identified by real-time PCR. The housekeeping gene, β-actin, was used as the 608

internal control. The data were analyzed using the cycle threshold (C(t)) method. 609

Fig. S1 Expression patterns of 16 DGEs revealed by qRT-PCR and RNA-seq. 610

611

Figure captions 612

613

Fig. 1 Statistical histograms of DEGs in four samples. X-axis represents pairs of 614

comparative samples; Y-axis represents number of differentially expressed genes; red 615

column shows up-regulated expression of different genes; green column shows 616

down-regulated expression of different genes. 617

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Fig. 2 Patterns of gene expressions, inferred by STEM analysis, across four time 618

points in the digestive tissues of NoV-infected oysters. 619

Fig. 3 GO classification of DEGs. 620

Fig. 4 Coefficient analysis of fold-change data from qRT-PCR and RNA-seq. Sixteen 621

unigenes were selected for qRT-PCR. Scatterplots were generated from the expression 622

ratios obtained from RNA-seq (X-axis) and qRT-PCR (Y-axis). 623

Fig. 5 Expression of HBGA-like molecules in contaminated and non-contaminated 624

oysters. Eight types of monoclonal antibodies against HBGA from humans were used 625

to analyze the expression of HBGA-like molecules in digestive tissue of oysters. P/N 626

values were used to evaluate the levels of these HBGA-like molecules. 627

Tables 628

Table 1 Statistical information uploaded to SRA database 629

STUDY SAMPLE EXPERIMENT RUN

PRJNA353875

(SRP093716)

S0(SRS1813038) SRX2366913 SRR5043896

S12(SRS1813040) SRX2366915 SRR5043898

S24(SRS1813039) SRX2366914 SRR5043897

S48(SRS1813041) SRX2366916 SRR5043899

630

631

632

633

634

635

636

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637

Table 2 Base statistical analysis before and after filter 638

Sample

Before Filter After Filter

Raw Data

(bp) Q20 N GC

Clean Data

(bp) Q20 N GC

S0 2913443250 93.85% 0.00% 43.64% 2875896500 94.31% 0.00% 43.62%

S12 6175145000 96.44% 0.00% 44.48% 6134197500 96.68% 0.00% 44.47%

S24 8281903000 93.73% 0.00% 43.58% 8161837250 94.25% 0.00% 43.55%

S48 4014234500 93.70% 0.00% 43.65% 3959743250 94.19% 0.00% 43.62%

639

640

641

642

643

644

645

646

647

648

649

650

651

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652

Table 3 Mapping information for the clean data 653

Sample Total reads Unmapped

reads

Unique mapped

reads

Multiple mapped

reads

Mapping

ratio

S0 19453802 6883805 12076003 493994 64.61%

S12 41670526 14174186 26315512 1180828 65.99%

S24 53755694 18507410 33778232 1470052 65.57%

S48 27281172 9716274 16845392 719506 64.38%

654

655

656

657

658

659

660

661

662

663

664

665

666

667

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668

Table 4 Ten KEGG pathways with the highest representation of DEGs 669

Pathway

No. of DEGs with pathway annotation

Pathway

ID All profiles

(% of 3991)

Profile 7

(% of 1390)

Profile 14

(% of 1222)

Profile 15

(% of 688)

Profile 8

(% of 713)

Profile 5

(% of 726)

Profile 6

(% of 727)

DNA replication 34 (0.85%) 1 (0.07%) 28 (2.29%) 2 (0.29%) 0 (0.00%) 0 (0.00%) 2 (0.28%) ko03030

Antigen

processing and

presentation

33 (0.83%) 4 (0.29%) 3 (0.25%) 1 (0.15%) 9 (1.26%) 1 (0.14%) 0 (0.00%) ko04612

Cell cycle 77 (1.93%) 10 (0.72%) 29 (2.37%) 10 (1.45%) 8 (1.12%) 1 (0.14%) 2 (0.28%) ko04110

Mineral

absorption 40 (1%) 4 (0.29%) 0 (0.00%) 1 (1.15%) 7 (0.98%) 3 (0.41%) 1 (0.14%) ko04978

Glycosphingolip

id biosynthesis -

lacto and

neolacto series

33 (0.83%) 5 (0.36%) 3 (0.25%) 3 (0.44%) 2 (0.28%) 5 (0.69%) 2 (0.28%) ko00601

Mismatch repair 22 (0.55%) 2 (0.14%) 9 (0.74%) 3 (0.44%) 2 (0.28%) 1 (0.14%) 4 (0.55%) ko03430

Progesterone-me

diated oocyte

maturation

43 (1.08%) 7 (0.50%) 10 (0.28%) 6 (0.87%) 6 (0.84%) 1 (0.14%) 2 (0.28%) ko04914

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Base excision

repair 31 (0.78%) 6 (0.43%) 13 (1.06%) 2 (0.29%) 0 (0.00%) 0 (0.00%) 3 (0.41%) ko03410

Nucleotide

excision repair 30 (0.75%) 4 (0.29%) 16 (1.31%) 4 (0.58%) 1 (0.14%) 0 (0.00%) 3 (0.41%) ko03420

Protein digestion

and absorption 53 (1.33%) 2 (0.14%) 3 (0.25%) 3 (0.44%) 2 (0.28%) 7 (0.96%) 2 (0.28%) ko04974

670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

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686

Table 5 Major DEGs involved in glycosphingolipid biosynthesis: lacto and neolacto 687

series pathways. 688

Gene ID

（（（（Locus tag）））） Homologous function

Fold

change

in 12 h

Fold

change

in 24 h

Fold

change

in 48 h

Profile

Gene

bank

number

CGI_10024598 Beta-1,3-galactosyltransferase 1

（B3GALT1） −3.83 −3.21 −1.21 5 JH816189

CGI_10027819 Beta-1,3-galactosyltransferase 5

（B3GALT5） −1.75 1.26 0.64 7 JH818873

CGI_10002434 Beta-1,4-N-acetylgalactosaminyltransf

-erase bre-4（bre-4） −0.68 −1.44 0.17 5 JH816103

CGI_10028074 Beta-1,4-N-acetylgalactosaminyltransf

-erase bre-4（bre-4） −0.84 −1.06 -1.19 4 JH819182

CGI_10023468 Beta-1,3-galactosyl-O-glycosyl-glycop

-rotein

Beta-1,6-N-acetylglucosaminyltransfer

-ase 3（GCNT3））））

−1.48 −1.89 -1.3 4 JH819075

CGI_10026669 Beta-1,3-galactosyl-O-glycosyl-glycop

-rotein

Beta-1,6-N-acetylglucosaminyltransfer

-ase 3（GCNT3））））

−0.81 −1.74 -0.31 2 JH816980

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CGI_10028006 UDP-GlcNAc:betaGal

beta-1,3-N-acetylglucosaminyltransfer

-ase 4(B3GNT4)

−1.17 2.57 2.73 15 JH823233

XLOC_023979 Galactoside

2-alpha-L-fucosyltransferase 2 0.43 2.66 3.08 15

New gene

CGI_10017887 Galactoside

2-alpha-L-fucosyltransferase 2

(FUT 2)

0.32 1.68 1.25 15 JH816140

XLOC_023978 Galactoside

2-alpha-L-fucosyltransferase 2 −0.63 1.58 −0.97 14

New gene

689

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dat

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cor=0.835p−value<2.2e-16

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Highlights

1. RNA-seq proved to be an effective method to explore the expression of genes

associated with viral infection.

2. 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.

3. Analysis of differently expressed unigenes related to glycosphingolipid

biosynthesis-lacto and neo lacto series help to reveal the molecular mechanism of

oysters concentrated and accumulated NoVs, and to provide a theoretical basis on

control the virus transmission and ensure the safety of shellfish consumption.