Molecular Epidemiological Study On Norovirus In Environmental Sewage In Guangzhou

ObjectiveThrough high-throughput sequencing technology combined with bioinformatics analysis,the distribution and dynamic changes of genotypes in Norovirus GI and GII groups were elucidated in environmental wastewater from 2013 to 2018 in Guangzhou.In addition,the epidemiological data of Norovirus epidemic in Guangdong and phylogenetic analysis were combined to explore the role of Norovirus monitoring of environmental sewage in epidemic warning and prevention.MethodsThe Guangzhou Liede Wastewater Treatment Plant was selected as the sampling point,and the sewage samples from January 2013 to August 2018 were collected by the timed sampling method,and 4 samples were collected in the middle of each month.After the sample is subjected to virus concentration and enrichment,the viral nucleic acid is extracted.Firstly,the real-time fluorescent PCR method was used to detect the overall situation of Norovirus GI and GII groups in environmental sewage.Then,viral nucleic acids per month were mixed into one with the ratio of 1:1:1:1,and amplified by nested PCR.The PCR product was sequenced by high-throughput sequencing after identified and purified.The DNASTAR software was used for homology analysis to calculate nucleotide and amino acid homology between the sequences.The major epidemic variants of GII.4,GII.17 and GII.2 downloaded from GenBank were used as reference sequences to construct a phylogenetic tree together with the sequence in the sewage.Results 1.Overall detection of GI and GII groups Norovirus in sewageFrom January 2013 to August 2018,a total of 272 samples of sewage were collected.The detection rate of NoV GII group（90.07%）was higher than that of GI group（65.44%）.The difference was statistically significant（P<0.001）;165 of them were sewage.At the same time,the GI group and the GII group NoV were detected.From the perspective of the year distribution,the detection rate of NoV in the GII group in the sewage was higher than that in the GI group in 2013-2015,and the difference in the detection rate of NoV in the GI and GII groups was not statistically significant in 2016-2018.From the distribution of the month,the total detection rate in December was the highest,accounting for 95.00%,mainly in winter and spring.2.Detection of genotypes of GI group in sewageA total of 10 different Norovirus GI genotypes were detected in the sewage.The main genotypes were GI.8,GI.2,GI.3 and GI.5,accounting for 26.16%,25.42%,11.60% and 10%,respectively.The remaining proportions of GI.9,GI.4,GI.1,GI.6,GI.7 and GI.12 are all less than 10%.From 2013 to 2016,GI.8 was the main-genotype,and from 2017,GI.2 was the most detected genotype,accounting for 40%.3.Detection of genotypes of GII group in sewageA total of 17 different Norovirus GII genotypes were detected in the sewage.The dominant genotypes were GII.4（39.17%）,GII.17（35.43%）,and GII.2（13.74%）.In addition,GII.1,GII.3,GII.5,GII.6,GII.7,GII.8,GII.9,GII.10,GII.12,GII.13,GII.14,GII.16,GII.20 and GII.21 were also detected in the sewage,all of which are <5%.Among them,GII.4 genotype was detected in a certain proportion during the whole research period,especially in 2013.In 2014,the GII.17 genotype of detection rate increased significantly.Until the first half of 2016,GII.17 has been in a dominant position.However,from October 2016,the total detection ratio of GII.2 genotype exceeds that of GII.17 and GII.4.4.Homology analysis and phylogenetic analysis of GII.4 genotypeIn 2013-2018,the nucleotide homology between the GII.4 was 91.10%-100.00%,and the amino acid homology was 98.50%-100.00%.Phylogenetic analysis showed that the sewage was mainly GII.4_Sydney₂012 mutant and the nucleotide and amino acid homology to the reference sequence were 98.9%-100% and 99.1%-100%.The remaining few sequences were GII.4_NewOrleans₂009 variants.5.Homology analysis and phylogenetic analysis of GII.17 genotypeIn the sewage from January 2013 to October 2014,the nucleotide homology of GII.17 was 85.10%-100.00%,and the amino acid homology was 90.50%-100.00%.It can be divided into three major evolutionary branches: Kawasaki₂014 variant,Kawasaki₃23 variant and HongKong₂015 variant.The Kawasaki₂014 variant contains all sequences from September and October 2014.6.Homology analysis and phylogenetic analysis of GII.2 genotypeIn the sewage from January 2013 to October 2016,the nucleotide homology of GII.2 type was 82.80%-100.00%,and the amino acid homology was 83.10%-100.00%.Phylogenetic analysis showed that the 2016 sequence and the 2013-2015 period were located on different evolutionary branches.Most of the sequences in 2016 were the new GII.2 variants popular in Guangdong from 2016 to 2017.The other two evolutionary branches are the GII.2 strains that were prevalent in Japan in 2002-2006 and 2012-2015.Conclusions 1.The detection rate of NoV in the GI and GII groups in the environmental sewage was high,and the type was diverse,suggesting that it was at a higher epidemic intensity in the covered population,and many different types of NoV existed in the population.2.A certain proportion of GII.4_Sydney₂012 mutants are detected every year in the sewage,indicating that this genotype has been repeatedly exploded and circulated in the population after being replaced by new genotypes,suggesting that we still need to replace the currently substituted GII.4 type.Be vigilant and prevent the risk of its prevalence.3.The increase of GII.17_Kawasaki₂014 and GII.2_GuangDong₂016-2017 mutants in sewage is earlier than the Norovirus outbreak time in the population,suggesting that environmental wastewater monitoring can provide a basis for the prediction and warning of Norovirus outbreaks.