Proliferation And Transcriptome Analysis Of Human Norovirus Genotype Ⅱ.4 In Human Intestinal Enteroids

Human Norovirus(HuNoV)is the main pathogen of acute gastroenteritis in the world.HuNoV has been circulated in worldwide since it was firstly identified at 1972.Epidemiology of HuNoV has been conducted for many years and achieved a lot of results,however,the pathogenesis of HuNoV and host antiviral immunity against HuNoV have been delayed.The main reason is lacking suitable cluture system or animal research model,at hence,developing novel culture system for HuNoV proliferation will provide chance for pathogenesis of HuNoV and virus-host interaction.The three-dimensional culture system could overcome the limitation of traditional cell monolayer culture,let the cell growth at three-dimensional and differentitation,mimic the structure and microenvironment of host at largest degree.Currently,air-liquid interface(ALI)culture,rotation culture based cassette and organoids culture are the main three-dimensional culture methods.Organoids culture is mainly using the primary cells from its tissue to re-construct host tissue structure through regulate the culture medium or addition the essentials.This platform could be used for culturing HuNoV and human rhinovirus species C.In order to culture HuNoV and understand the interaction between HuNoV and enteroids,human enteroids three-dimensional culture system was constructed firstly and the replication ablitilities of different types of HuNoV were assessed.Finally,the interaction between HuNoV GⅡ.4 and human enteroids was determined through transciptome method.The research text as follows.Part Ⅰ:Construction of human enteroids three-dimensional culture systemIn this part,we first got the clinical jujunum tissues from hospital,then we separated crypt of jujunum and seeded on the Matrigel.Several regulation essestials such as Wnt-3A、R-Spondin、Noggin、EGF were added into the culture medium to induce the formation of human enteroids and passage culture.The proliferation of crypt of jujunum,formation of human enteroids,cell types of differentitated cells were identified by morphology,immunofluorescence and electrical microscopy.The results showed the cells of human enteroids were very similar to human jujunum at structure and morphology.The cells of human enteroids had polarities.The apical villus of human enteroids towarded to lument,the cell junction was integrity,the classical goblet cells were observed in human enteroids and the vesicle bodies were gathered at top of nuclear in goblet cells.The vesicles which containing hormone were gathered at the bottom of nuclear in secretory cells.The cell monolayer was obtained from digestion of human enteroids by trypsin and cultered in differentitation culture medium by removing Wnt-3A and R-Spondin.The goblet cells were randomly distributed in the cells and the cell types were increased with the culture time prolonged,such as goblet cells and enterocyte appeared.Part Ⅱ:Proliferation and transcriptome analysis in human enteroids infected with HuNoV GⅡ.4In this part,we selected GⅡ.4,GⅡ.3,and GⅡ.17 variants of Norovirus from children and adults,and inoculated them into human jejunum monolayer cells and found GⅡ.4.In the intestinal tissues,there is a stable amplification,and the amplification factor gradually increases with time,reaching a peak at 72 hours after inoculation.The pediatric patient-derived Norovirus GⅡ.4 variant strain was replicated in the intestinal tissue.The viral genome copies of HuNoV GⅡ.4 in layer cells was significantly higher than that of norovirus GⅡ.4 strains derived from adult patients,with a increase of 19-fold.Norovirus GⅡ.3 and GⅡ.17 variants derived from children have a lower proliferative efficiency in jejunum-like monolayer cells than GⅡ.4 variants,but exist in the bile replacement glycyrrhetine chenodeoxycholic acid(GCDCA).Under the conditions,the fold expansion after infected with GⅡ.17 variant was increased to 11.53 fold compared to 1 hour after infection.In the transcriptome study,we used high-throughput sequencing method to perform transcriptome sequencing and analysis of small intestine tissues at indicated time points after infected with HuNoV GⅡ.4 to obtain significant differentially expressed genes.We then selected specimens from different jejunal tissue donors to infect the same pediatric patient-originated norovirus GⅡ.4 variant strains,and used the established Real Time PCR assay for HuNoV GⅡ.4 to detect viral infections.We performed mRNA level sequencing of enteric cells at 0 h before infected with HuNoV GII.4 and at 1 h and 72 h post-infection.We sequenced two samples of human intestine tissue after HuNoV GII.4 infectionTaking 0 h as the control group,5376 differentially expressed genes were found by differential expression analysis,of which 49%were up-regulated and most were associated with immunity,such as interferon(IFN)and IFN-stimulated genes such as RIG-I,MDA5,and IRF4.IFN III and IFNR.However,about 51%genes were down-regulated,mainly related to growth and metabolism-related genes,such as insulin-like growth factor,ubiquitin-conjugating enzymes.The significant differentially expressed genes had a significant effect on the function of intestinal cells in small intestinal tissue by HuNoV GII.4 infection according to GO biological function enrichment analysis.According to the KEGG database search,the differentially expressed genes were enriched in more than 45 signaling pathways,such as apoptosis,cell cycle,cell structure,fatty acid metabolism,and signaling pathways related to immune responses.The transcriptional levels of type Ⅲ interferon(IFN-λ1/-2/-3)were significantly evaluated after HuNoV GII.4 infection,however,the differential expression of type Ⅰ and type Ⅱ interferon genes were not found.The changes of RIG-I and MDA5 reached peak level at 72 h psot infected with HuNoV GII.4.Additional,the level of tumor necrosis-encoding gene(TNF)was also up-regulated,while the other apoptosis-associated genes were down-regulated.Among these down-regulated genes,genes of p53 signaling pathways such as CCNE1/2,CDK1/2,TP73,RRM2,APAF1,PPM1D,and PMAIP1 were slight or significant down-regulated after infected with HuNoV GII.4.At the same time,apoptotic peptidase activating factor 1(Apaf-1),an important regulatory factor in cell mitochondrial apoptosis signaling pathway which can mediate programmed cell death,was significantly down-regulated.In addition,the host anti-apoptotic member BCL-2 was up-regulated.These results indicated that HuNoV inhibited apoptosis through down-regulated apoptosis-associated genes and up-regulated genes to promote HuNoV replication and proliferation in human enteroids.In summary,we constructed the human enteroids culture platform and successful proliferated HuNoV GII.4 in this platform.Determination the changes of trancripitional profiles of human enteroids infected with HuNoV GII.4 and indicated IFN-λ maybe play an important role in innate immunity against HuNoV GII.4.Celluar metabolism and apoptosis were regulated to promote the proliferation of HuNoV GII.4.These results will provide the scientific evidence for the pathogenesis of HuNoV and host innate immunity against HuNoV.