Using Next Generation Sequencing To Analyze The Genetic Diversity,Genotype Distribution And Molecular Phylogenetics Of Noroviruses In Sewage

Background and objectiveNorovirus(Norovirus,NoV)is responsible for about 20%of acute gastroenteritis(Acute gastroenteritis,AGE)cases worldwide.The epidemic trend of NoV has changed from a GⅡ.4-dominated epidemic to a multivariate epidemic,which poses a challenge to the current hospital-based clinical case surveillance system.Our previous research suggests that environmental sewage can reflect the dynamic distribution and epidemic characteristics of NoV in the population.Based on the high-throughput sequencing method established in the previous stage to determine the abundance of NoV genotypes in sewage,this study is to clarify the genetic diversity and dynamic genotype distribution of NoV in sewage in Guangdong Province in 2019,to analyze the epidemic and phylogenetic characteristics of the epidemic strains in sewage,which further identify the emergence of novel variant and reflect the molecular epidemiological characteristics of NoV in the population.It is to explore the role of environmental sewage surveillance in the early warning and prevention and control of the NoV epidemics after comparing with the molecular epidemiological data of the NoV-associated AGE outbreak in Guangdong Province.Methods1.Collecting environmental sewage samples annually in Guangzhou,Foshan,Jiangmen,Dongguan and Zhuhai City,and the samples were performed virus concentration by electronegative membrane adsorption-ultrasonic elution method and then conducted RT-qPCR to analyze the overall detection of NoV GⅠ and GⅡ in the sewage.2.Semi-nested PCR was to amplify the VP1 fragments of GⅠ and GⅡ,which were then performed high-throughput sequencing to analyze the genetic diversity and dynamic distribution characteristics of GⅠ and GⅡ genotypes in sewage.3.The sequenced NoV fragments were subjected to OTU cluster analysis,and then the GⅡ.4,GⅡ.2,and GⅡ.17 fragments were performed phylogenetic analysis.Results1.In 2019,the overall detection rate of NoV in the GⅠ and GⅡ genogroups were 88.33%(53/60)and 85.00%(51/60),respectively.The detection rates of GⅠ and GⅡ in winter and spring were higher than those in summer and autumn.2.For GⅠ genogroup,a total of 9 genotypes were detected,among which GⅠ.1 and GⅠ.3,followed by GⅠ.5,were the main epidemic genotypes in sewage in five cities.For GⅡ genogroup,a total of 17 GⅡ genotypes were detected,among which GⅡ.4,GⅡ.2,and GⅡ.13 were the dominant genotypes,followed by GⅡ.17,in sewage in Jiangmen City,and GⅡ.A,GⅡ.2 and GⅡ.17 were the main epidemic genotypes in remaining 4 cities.Except for Dongguan City,the proportion of GⅡ.4 in the other 4 cities began to increase at the month of Aug,and became the dominant epidemic genotype in the period from August to December.This suggests that the NoV of sewage in 5 cities in Guangdong Province in 2019 showed genetic diversity,and regional and temporal distribution differences.3.The number of GⅡ.2,GⅡ.17 and GⅡ.4 associated AGE cases and the proportion of them in sewage samples showed similar dynamic trend,especially the proportion of GⅡ.4 in sewage began to increase at the month of Aug,and the number of outbreaks caused by GⅡ.4 also start to elevate at the month of Sep.4.By constructing a phylogenetic tree based on the partial VP1 fragment of the GⅡ.4 variants,and the maximum sequence similarity(mean+2SD)was used to determine the sequence similarity threshold of the OTU cluster,that is 98.93%.5.Based on the OTU clustering method,a total of 131 GⅡ.2 sequences were to construct phylogenetic tree which divided into 2 clusters and the main cluster of it consisted of 96.94%of these sequences and the GⅡ.2[P16]variant prevalent in 2016-2019.Totally 111 GⅡ.17 sequences were generated and constructed phylogenetic tree that is divided into 3 clusters.The major cluster is formed by 63.06%of the sequences and Kawasaki 2014 variants in 2014-2015 epidemics.A total of 198 GⅡ.4 sequences were generated and constructed phylogenetic tree which was divided into 3 clusters.The major cluster included 59.09%of these GⅡ.4 sequences and GⅡ.4 Sydney 2012[P31]dominated in 2018-2019.Conclusions1.It is more sensitively to detect potential new variants hat caused the NoV-associated outbreak through environmental sewage surveillance.For example,the new GⅡ.4 variant that caused the epidemic was detected in environmental sewage one month in advance.2.The OTU clustering method can easily calculate the proportion of NoV genotypes and variants in sewage,and can simplify the process of phylogenetic analysis.3.Environmental sewage surveillance based on high-throughput sequencing can detect NoV genotypes that are difficultly detectable in clinical samples,which is used to optimize current clinical surveillance and can more fully reflect the genetic diversity of NoV in the population,such as the dominant genotypes including GⅡ.2,GⅡ.17,and GⅡ.4 have different variants prevailing in the population at the same period.