Epidemiology And Spatiotemporal Dynamics Of Norovirus Associated With Sporadic Acute Gastroenteritis During 2013-2018, Zhoushan Islands,China

ObjectivesTo understand the norovirus genotypes distribution,the dynamic changes of dominant epidemic strains and epidemiological characteristics in the sporadic acute gastroenteritis patients in Zhoushan Island from 2013 to 2018.To understand the genetic characteristics,evolution events and spatiotemporal dynamics of GII.P16-GII.2 and GII.P17-GII.17 norovirus in Zhoushan Island.Methods1849 fecal swabs and stool samples and case questionnaires were collected from intestinal clinic of Zhoushan Hospital from 2013 to 2018,which met the criteria of diagnosis of viral diarrhea.Norovirus was detected by fluorescence quantitative RT-PCR and the epidemiological characteristics of norovirus were analyzed.Norovirus positive samples were amplified by RT-PCR method and the positive products were sequenced and genotyped.The phylogenetic tree was constructed using MEGA software.The epidemiological characteristics of norovirus,genotype distribution and dynamic changes of dominant epidemic strains in sporadic acute gastroenteritis patients in the Zhoushan Islands were analyzed at the molecular level.The VP1 region of GII.17 and GII.2 and RdRp region of GII.P16 norovirus in Zhoushan Island were amplified by RT-PCR method.The positive products were sequenced and ML tree was constructed using RAxML.MEGA software was used to measure the genetic distance between groups.Simplot was used to identify recombination events.Finally,the Bayesian Markov chain-Monte Carlo?MCMC?method and the BSSVS model in the BEAST.v.1.82 were used to analyze the molecular evolution of GII.P16-GII.2 and GII.P17-GII.17 in Zhoushan Island and to reconstruct Phylogeographic tree.ResultsA total of 1849 samples of acute gastroenteritis were collected from Zhoushan Hospital between January 2013 to August 2018.The average annual detected rate of norovirus was 7.24%.The positive rate showed an increasing trend by year(?²_trend=22.72,P<0.05).The ratio of male to female was 1.65:1,and the highest detection rate was found in 15-40 age group?11.00%?.The main incidence peak occurred in December-April?18.62%?.134 samples subsequently identified as human norovirus?HuNoV?positive.120 samples was belong to GII norovirus?89.55%?and 14?10.55%?was GI norovirus.Phylogenetic analysis of partial RdRp and VP1 gene regions identified 10 genotypes of the GII genogroup and 6 genotypes of the GI genogroup The common three genotypes were GII.P17-GII.17?38.75%?,GII.Pe-GII.4_Sydney₂012?18.03%?,GII.P16-GII.2?17.12%?.The distribution of different genotypic age groups was statistically different??₌²17.34,P=0.008<0.05?.GII.Pe-GII.4_Sydney₂012 was the main epidemic strain in 2013-2014 and then GII.P17-GII.17 was epidemic in 2015-2016.In 2017-2018,GII.P16-GII.2 and GII.P17-GII.17 were alternately prevalent.The results of Simplot analysis showed that the GII.P16-GII.2 norovirus in Zhoushan Island was recombined from the GII.P16RdRp region and the GII.2VP1region.The recombination site was located at the 5052-5053bp of the ORF1-ORF2junction region,and GII.P16 could be divided into three clusters.Zhoushan island GII.P16 belongs to GII.P16c cluster.The evolutionary analysis of GII.P16 norovirus showed that its evolution rate was 2.64×10^-3(2.17-3.08×10^-3)substitution/site/year.It was deduced that the most recent common ancestor of GII.P16 type norovirus appeared in 1983?1978-1989?.The new GII.P16c cluster emerged in2016-2017 was diverged from 2010-2012 Japanese GII.P16-GII.2 and was appeared in 2012-2013.GII.2 norovirus can be divided into four clusters and Zhoushan Islands sequences belongs to GII.2d cluster.The phylogenetic results of GII.2norovirus showed that its evolutionary rate was 3.36×10^-3(2.66-4.04×10^-3)substitution/site/year.The most recent common ancestor of the GII.2 virus appeared in 1967?1957-1975?.The new GII.2d cluster emerged in 2016-2017 was diverged from 2010-2012 Japanese GII.P16-GII.2 and was appeared in 2012-2013.The BSSVS model showed that the re-emerging GII.P16-GII.2 strains were first introduced into Hong Kong from Japan and then Hong Kong became the center of GII.P16-GII.2 norovirus outbreak in China,which mainly spread to coastal cities.GII.P16-GII.2 norovirus in Zhoushan Island was most likely to be introduced from Jiangsu Province in 2016.The results of ML tree showed that the GII.P17-GII.17 norovirus could be divided into four clusters,and sequences isolated from Zhoushan Islands was belonged to cluster D.The evolution analysis showed that the evolution rate of 31GII.P17-GII.17 in Zhoushan Islands was 1.14×10^-3(0.62-1.73×10^-3)substitution/site/year,and the most recent common ancestor appeared in 1997?1987-2009?.A new variant of GII.P17-GII.17 norovirus emerged in 2014-2015 appeared in 2011?2009-2012?.The phylogenetic results of the GII.17 norovirus showed that Hong Kong was the epicenters of the new variant GII.P17-GII.17 norovirus outbreak.The results of BSSVS model speculated that GII.P17-GII.17 norovirus in Zhoushan Islands was most likely introduced by Hong Kong and Japan.ConclusionsThe average annual norovirus infection rate associated with sporadic acute gastroenteritis during 2013-2018 in the Zhoushan Islands was 7.24%but showed an increasing trend.We have detected ten GII norovirus genotype and six GI norovirus genotyp.The norovirus in Zhoushan Islands is highly diverse and Zhoushan Islands are susceptible to epidemics from new emerging strains which main introduced by Hong Kong.Continuous surveillance to monitor genotypes and the emergence of new strains for development of prevention and control strategies is necessary.