Distribution Of Norovirus In Shellfish And The Binding Mechanism To HBGA-like Molecules In Oysters

Noroviruses(NoVs)are the main pathogens causing non-bacterial acute gastroenteritis worldwide.Shellfish is one of the important carriers of NoVs transmission.People infected with NoVs are often associated with raw shellfish or shellfish with incomplete heating.China is a big consumer of shellfish culture.Food-borne diseases caused by NoVs contamination of shellfish can not be ignored in shellfish food safety.However,NoVs can not be cultured in vitro at present.NoVs contaminated in shellfish are often caused by low viral content,complex genotypes and interference of shellfish components,which makes it difficult to detect the content,species and distribution of NoVs in shellfish.Studies have shown that there are receptors similar to human tissue blood group antigens(HBGAs)in oysters,which can specifically bind oyster tissues,but the binding mechanism between the receptors and different types of NoVs in oysters is still unclear.Therefore,it is of great significance to establish a rapid detection and clustering method of NoVs in shellfish,to carry out HBGA-like molecules detection and distribution survey,to study the changes of HBGA-like molecules and NoVs distribution under NoVs stress,and to explore the mechanism of combining NoVs with HBGA-like molecules in oysters for accurate detection of NoVs in shellfish and early warning of consumption safety,and to ensure the sustainable and healthy development of shellfish industry in China.This paper takes NoVs as the research object,a method of rapid detection and clustering of NoVs in shellfish based on SYBR Green I were established.A method for the detection of HBGA-like molecules compounds in eight species of oysters was established,and the distribution of HBGA-like molecules compounds in oysters was preliminarily explored.The expression of HBGA-like molecules and distribution of NoVs in response to NoV challenge were studied.P-particles of six major NoVs strains were prepared and the binding properties of HBGA-like molecules compounds in oysters were analyzed.A NoVs P protein specific molecular probe was screened out.This study laid a foundation for exploring the binding mechanism of NoVs in oysters,the results are as follows:1.Establishment of the rapid detection and Gene grouping method for Noroviruses in shellfishGene sequence of six major prevalent genogroup I and genogroup II NoVs strains and international general reference NoVs were analyzed and one pair of primers in conservative region was selected to establish a rapid detection and Gene grouping method for Genogroup I and Genogroup II Noroviruses in shellfish.The actual samples were also tested and validated.The results shows that the P289/290 primers can simultaneously detect GI and GII Noroviruses at the same time,and the fluorescence quantitative detection method has a good linear relationship between virus at 10~3-10~9copies(R~2=0.993),and the Tm value of melting curve can distinguish the genogroup I and genogroup II Noroviruses significantly(p<0.05).The positive detection rate of 120shellfish samples was 5.83%,and the sequencing gene grouping result in the positive sample are consistent with those of rapid gene grouping in this paper.This method can detect and grouping of genogroup I and genogroup II Noroviruses in shellfish due to its low cost,rapid,accurate and good repeatability.2.Detection and distribution of HBGA-like molecules in OystersThe dilution concentration and second antibody concentration of monoclonal antibody in ELISA reaction were optimized.The optimal washing times,concentration of closed milk powder and time of TMB coloration were screened.The background interference was significantly reduced.An ELISA method for detecting eight kinds of HBGAs was established.Distribution and detection rate of HBGA-like molecules in240 oyster samples were analyzed by the established method.The rusults showed that there were more A,H1 and Leb HBGA-like molecules in gill tissue of oyster,and more A and H1 HBGA-like molecules in digestive tissue and mantle.The detection rate of type A HBGA-like molecules in digestive tissue,gill and mantle was 100%,93.33%and 95%,respectively.The detection rates of H1 and Leb HBGA-like molecules in gills of oysters were 65.42%and 89.58%,respectively.According to seasonal classification,type A HBGA-like molecules in gill,digestive tissue and mantle of oyster had higher detection rate(>85%)in four seasons while the high detection rate of type A HBGA-like molecules in muscle was 63.3%in spring and 68.3%in winter,respectively.Type Leb HBGA-like molecules in gills of oysters had a higher detection rate(>71.7%)in four seasons and the detection rates of type H1,Lea,Lex,Ley and Pre HBGA-like molecules in all oyster tissues were higher in spring and winter than in summer and autumn(p<0.05).Distribution of type A HBGA-like molecules in gill,digestive tissue and mantle was obvious acording to locate HBGAs in different tissue by immunohistochemical method.Type H1 HBGA-like molecules were found in gill and digestive tissue tissues of oysters,and also in mantle.In conclusion,there are different types of HBGA-like molecules in different oyster tissues,and the distribution of HBGA-like molecules is polymorphic and influenced by seasons.3.The expression of HBGA-like molecules and distribution of NoVs in response to NoV challengeOyster was exposed to GI.3/GII.4 NoVs in laboratory.After a certain period of biological accumulation,the expression of HBGAs-like in different oyster tissues was detected.The results showed that the expression of type A HBGA-like molecules increased significantly in oysters after GII.4 NoVs challege,and the expression of type H1 HBGA-like molecules in gill and digestive tissue increased significantly after GI.3or GII.4 or their mixture contaminated by oysters.Simultaneous detection of NoVs in Oyster tissues by real-time RT-PCR,The results showed that GI.3 NoVs were detected in gill,digestive tissue,mantle and closure muscles of oysters,but mainly distributed in the digestive tissue(1.50±0.17)×10~7 copies,with the least in the closure muscles,and even two results were below the detection limit.GII.4 NoV was also detected in gill,digestive tissue,mantle and obturator muscle of oyster,mainly distributed in gill and digestive tissue(8.31±0.56)×10~6 copies and(1.77±0.62)×10~6 copies,respectively.GI.3 NoV was mainly distributed in digestive tissue(7.26±1.44)×10~6 copies when oysters were exposed to mixed NoVs,while GII.4 NoV in gill and digestive tissue(4.48±0.65)×10~6 copies and(1.11±0.13)×10~6 copies,respectively.No NoVs were detected in seawater 48 hours after artificial contamination of oysters.In conclusion,it can be preliminarily inferred that the selective enrichment of NoVs in oysters by exposing NoVs to oysters,and the binding of NoVs with oyster HBGAs promotes the expression of some HBGAs-like compounds in oysters.This study laid a foundation for the study of the binding and regulation of NoVs with oyster HBGAs.4.Accumulation and Distribution of GII.4 Norovirus Artificially Contaminated in Pacific OysterIn order to study the accumulation and distribution of norovirus in oysters,Oysters were bred in certain conditions exposure to GII.4 NoV in laboratory.The content and distribution of GII.4 NoV in oyster tissues were detected by real-time quantitative RT-PCR and immunohistochemistry.The real-time quantitative RT-PCR results showed that the amount of GII.4 NoV in oyster mantle was stable at(8.75±0.36)×10~4 to (9.12±0.49)×10~4copies,while the content of GII.4 NoV in oyster gills was firstly increases and then decreases until achieves stability,the highest content is (1.15±0.15)×10~6copies at 6 hours.The content of GII.4 NoV in oyster digestive tissues was continuously increased until achieves stability at 12 hours which content is (1.06±0.14)×10~6copies GII.4 NoV mainly accumulated in oyster digestive tissues 24hours later.The content of GII.4 NoV have no significant change after Purification(P>0.05).The immunohistochemistry result showed that GII.4 NoV was widely distributed in the mantle edge,gill wall and inner wall of the digestive tissues.The change trend of GII.4 NoV distributed in oyster tissues with time was in accordance with real-time quantitative RT-PCR result.5.Preparation and in vitro binding properties of P particles of different types of NorovirusSix main popular NoVs PGEX-4T-1-P expressing bacteria were used to express the P protein labeled with GST protein.The twenty-four dimers GI NoVs P particles(32.6KDa)andGII NoVs P particles(38KDa)were successfully prepared after IPTG induced,ultrasonicated cell disruption,agarose gel purified,GST ligand removed.Binding experiment of HBGAs with prepared P particles in vitro.The results showed that different NoVs P particles had strong binding with different synthetic oligosaccharides H1 and had diversity of binding with other types.Different NoVs P particles bind to O+secretory saliva,all types of NoVs P particles do not bind to non-secretory saliva(O-),and other types have diverse binding.GI NoVs(57397 strains,54108 strains,55063 strains)P particles and oyster digestive tract tissue extract have obvious advantages in binding ability,whlie GII NoVs(57395 strains,F278 strains,L20strains)NoVs P particles have strong binding ability with gill,digestive tissue and mantle of oyster.These results were consistent with the accumulation and distribution of NoVs in oysters and indirectly proved that there are HBGAs-like binding substances with NoVs in oysters.This result may provide a reference for the study of the binding mechanism between NoVs and oyster HBGAs.6.Screening and Application of Noirovirus P Protein Specific Molecular ProbesFive groups of specific probes were screened for the preparation of NoVs P protein by fluorescent aptamer technique.After 16 rounds of cyclic screening,A1-3 sequence was screened as the specific sequence for the detection of NoVs P protein.After optimization,the optimal probe time of P protein fluorescent aptamer in A1-3 sequence reaction was 120 min.The sequence was highly specific and sensitive.After 10truncation treatments,the truncated sequence A1-3-10 with stronger characteristics and lower cost was screened out.This is a new thought and method for NoVs detection.