Risk Assessment In Shellfish-borne Infections Of Hepatitis E Virus And It's Construction And Immunological Evaluation Of Lactic Acid Bacteria Display System

Hepatitis E virus(HEV)is a zoonotic pathogen,may threaten the food safety and public health security because of its widely across species epidemiological characteristics.A risk assessment in shellfish-borne HEV infection was executed in this study.Shellfish,as an ideal bio-indicator,we first focus on the establishment of molecular biology methods for surveillance of HEV contaminated in them around the coastal waters.Then combining with the basic theory from international codex alimentarius commission(CAC),a provisional risk assessment model for food-borne HEV infection through shellfish in our country was extrapolated.This reference model would be useful for the surveillance,prevention and control of the food-borne HEV.In addition,a live recombinant HEV genetic engineering vehicle lactobacillus was constructed in this study.The specific research results are as follows:1.The establishment of the HEV molecular detection method in shellfish.First,the positive quality control for HEV molecular detection was prepared by RNA armor technologies.The molecular detection HEV method in shellfish was then developed and established basis on the product of positive quality control,and other methods including HEV enrichment from shellfish and various RT-PCR detection were also evaluated and analyzed.Analysis results show that:(1)the HEV-like particles can be used as general genotype 1-4 HEV standard quality control in RT-PCR detection;(2)the virus recovery reached up to 5-14%in our improved shellfish enrichment method for HEV;(3)the developed nRT-PCR and RT-LAMP method for specific genotype 4 HEV detection showed as good specificity and sensitivity as the international reference qRT-PCR method,and their detection limit was from 2.5×102 to 101 copies/?L.2.Surveillance of HEV contaminated in shellfish and genetic evolutionary analysis.Results showed that(1)HEV pollution rate in offshore waters was 17.5%per kilogram shellfish,and seven contaminated area were confirmed;(2)three species of A.granosa,S.Subcrenata and R.philippinarum was all detected positive by the qRT-PCR verification,and the level of HEV contaminated in them were from 6.23x102 to 6.36×103 gene copies within a significant statistical difference(p<0.05);(3)13 isolates of HEV gene sequences were all belong to genotype 4HEV according to analysis of genetic evolution using Mega6.0 software.Those HEVs were very close to pig sourced HEV with falling into G4b,G4d subtypes and an unknown group.3.Risk assessment in shellfish-borne HEV infection.After collected and analyzed the survey data from the HEV contamination situation in shellfish and the related community dietary,a provisional quantitative risk assessment adopting the CAC microbial risk assessment method for the shellfish-borne HEV infection in the area was executed comprehensively.The risk assessment results showed that the average occurrence probability infected by HEV due to consumption of shellfish among the residents living around Huang-Bohai area were 0.000300055(about three over one million);the extrapolated average infection rate of HEV was about 0.4%per one million people and the number of infected people were 49 722 and 755 722 among ages 0 to 14 and abovel4 years old residents,respectively.Uncertainty analysis was also shared in this study,which would be useful for the reference of HEV risk assessment in shellfish or other related animal food.4.The construction and immunological evaluation of lactobacillus display system for capsid protein expression of HEV.(1)Western blotting and IFA identification results showed that the constructed recombinant plasmid pNZ8149-inaQN-ORF2 was capable of expressing a low level of HEV ORF2 capsid protein and presenting to the host bacterium L.lactis NZ3900 surface with the aid of INP membrane anchor protein.(2)ELISA detection results showed that the specific mucosa antibody IgA,secreted in immunized mice gastrointestinal,respiratory and urinary tract was effectively up-regulated compared with the control bacteria group,specific anti-ORF2 IgA in the digestive and respiratory tract can maintain at a high level from 35th to 63rd day after the first boost day,while in the genitourinary tract can only maintain to 49th day after the first boost day(P<0.05,P<0.01).(3)the mice serum specific antibody IgG ELISA detection results showed that the oral immune recombinant bacteria L.lactis NZ3900 can effectively stimulate mice systemic immune response against ORF2 antigen.Compared with the control bacteria,specific anti-ORF2 antibody IgG titre can maintain a high level in 21st to 63rd day after the first boost day.(4)the mice spleen T lymphocyte proliferation of CCK-8 test results showed that the experimental group of spleen T lymphocyte proliferation was significantly higher than that of control group from 35th to 63rd day after the first boost day(p<0.05,p<0.01).(5)ELISA test results showed that IL-4 secreted by immuned mice spleen T lymphocytes were significantly higher than that of control group on the 49th day after the first boost day(P<0.01),while the secretion level of IFN-? had no difference between experiment and control group.