Study On Acute Gastroenteritis Virus Infection Risk And Response Of Gut Microbiota Based On Co-Surveillance Of Five Viruses

Objective:To establish five co-surveillance method of acute gastroenteritis virus to survey the acute gastroenteritis virus in food borne vectors（feces and shellfish）;Based on the results of viruses detection,the risk of single acute gastroenteritis virus infection was quantitatively assessed;According to five co-surveillance results,shellfish were classified for food safety risk;Study the response of gut microbiota to different acute gastroenteritis virus infection,and explore the value of gut microbiota in acute gastroenteritis virus risk assessment.Methods:1.The recombinant plasmid of acute gastroenteritis virus was constructed to make the standard curve,and the multiplex RT qPCR co-surveillance method of Norovirus GⅠ（Nov GⅠ）,Norovirus GⅡ（Nov GⅡ）,Rotavirus（RoV）,Human enteric adenovirus（HAdV）and Hepatitis A virus（HAV）were established.The performance of detection was analyzed from the aspects of specificity,sensitivity and stability;2.The epidemic and pollution status of acute gastroenteritis virus in foodborne vectors（feces and shellfish）were investigated and analyzed by the established co-surveillance method;3.Based on the co-surveillance results of acute gastroenteritis virus,Monte Carlo simulation was used to evaluate the quantitative risk of acute gastroenteritis virus infection;Establish a risk classification matrix to classify the safety risk of eating shellfish in different seasons（spring,summer,autumn and winter）and different age groups（0-4 years old,5-18 years old,18-65 years old and>65 years old）;4.Fecal samples were sequenced by high-throughput.The gut microbiota of different hosts infected with acute gastroenteritis virus were analyzed by one-way ANOVA rank sum test,Nonmetric Multidimensional Scaling,Linear discriminant analysis Effect Size and Pearson correlation analysis α Diversity,β Diversity and species composition and correlation of relative abundance.Results:1.The cross-validation of NoV GⅠ,NoV GⅡ,HAV,RoV and HAdV primers/probes have no non-specific results;The minimum detection limits for NoV GⅠ,NoV GⅡ,HAV and RoV were 10 copies/μL,and the minimum detection limit for HAdV was 102 copies/μL;The coefficient of variation of the Ct value was less than 2%for three times repetitions.2.In the stool samples of 289 patients with non-bacterial acute gastroenteritis,the detection rate of acute gastroenteritis virus was 23.53%,of which the positive rate of Nov GⅠ,Nov GⅡ,RoV and HAdV was 0.69%,12.80%,5.19%and 7.61%,respectively;Among 103 commercial shellfish samples,the detection rate of acute gastroenteritis virus was 37.86%,of which the positive rate of Nov GⅠ,Nov GⅡ,HAV,RoV and HAdV was 10.68%,9.71%,7.77%,14.56%and 2.91%,respectively;3.The average probability of acute gastroenteritis caused by five acute gastroenteritis viruses in shellfish per meal is as follows:Nov GⅠ is 0.97%,Nov GⅡ is 1.02%,HAV is 5.41%,RoV is 5.93%and HAdV is 4.97%.The risk of consumption of clams is ClassⅠ,that of razor clams,blood clams,clams and oysters is Class Ⅱ,and that of mussels is ClassⅢ;The risk level of shellfish consumption in summer and autumn is Class Ⅰ,that in winter is Class Ⅱ,and that in spring is Class Ⅲ.Except that the risk level of shellfish consumption among people aged 19-64 is Class Ⅱ,the other age groups are Class Ⅰ;4.The β diversity of gut microbiota of NoV infected host was significantly different from that of the host infected by the two viruses;Compared with other gut microbiota species,there were significant differences in the abundance of Bifidobacterium in RoV infected host gut microbiota,Prevotella₉ in without virus host gut microbiota,Bacteroides,Flavobacterium and Asticcacaulis in HAdV infected host gut microbiota,which may be biomarker strains;There was a negative correlation between the species abundance of the gut microbiota of the host without virus and that of the host infected by the two viruses.Conclusion:1.The co-surveillance method of five acute gastroenteritis virus have good detection performance and can be used for large-scale and rapid detection;2.The main viruses detected in fecal samples of non-bacterial acute gastroenteritis were Nov GⅡ,HAdV and RoV,and the main viruses detected in commercial shellfish samples were RoV,Nov GⅠ and NoV GⅡ;3.Shellfish Consumption has a certain probability of causing viral acute gastroenteritis.Mussel consumption,spring shellfish consumption and shellfish consumption of people aged 19-64 had the highest risk level in their respective groups;4.The gut microbiota responds to the infection of different acute gastroenteritis viruses.Multiple virus infections cause greater damage to the homeostasis of gut microbiota,which has potential value for the risk assessment of acute gastroenteritis virus.