| Objective: To explore the distribution of human enterovirus(HEV)serotype an d genomic characteristics of Coxsackieviruses B5(CVB5)in environmental Sur veillance in Shanghai from 2013 to 2015.Methods: The sampling point was respectively established in Minghang and Jiading District.One sewage sample was collected at each sampling point every month from2013 to 2015.The specimens were concentrated by anionic membrane absorption,eluted with beef extract solution,and used to inoculate RD,Hep-2,and L20 B cell lines.RT-PCR and Sanger sequencing were performed to identify the positive specimens.Homology analysis of VP1 sequences was conducted by Bio Edit(version7.0.0).Phylogenetic analysis performed by the neighbor-joining method based on the alignment of VP1 gene sequences by MEGA(version 6.0).The dominant serotypes and the pattern of changes of HEV were analyzed,combining with HEV data of environmental,acute flaccid paralysis(AFP)case and healthy children surveillance.The whole genome sequence of was sequenced by RT-PCR.MEGA and Simplo were used to analyze the homology and similarity.Results: A total of 336 HEV were isolated from sewage samples during the study period,including 261 non-polio enterovirus(NPEV)and 75 poliovirus(PV)strains.Major dominant serotypes of NPEV were CVB5、ECHO6、ECHO7、ECHO11、CVB3、ECHO3.CVB5 had the largest number of isolates.The C6 and C8 subtypes of CVB5 were circulated in Shanghai.There were great nucleotide differences between ECHO6 which had a rapid evolution.Multiple branches of ECHO6 exited in Shanghai.ECHO7,ECHO11,CVB3,ECHO3 kept with obvious regional clustering and had low speed of nucleotide and amino acid mutation.Compared with the prototype strain Faulkner,the genomic sequences of two CVB5 isolates from environmental surveillance showed that the nucleotide homology and acid homology was respectively 79.5%～80.6% and 96%～96.6%.HJ330-MH2013 had the highest similarity with the ECHO3/HNWY-01 strain in 5 ’NTR and VP4 region.A CVB5 isolated from environment surveillance had a substitution of serine(S)to asparagines(N)at amino acid 95 in the VP1 region.Conclusion: PV strains were identified as vaccine strains in environmental surveillance from 2013 to 2015 in Shanghai.The dominant serotypes of HEV were CVB5,ECHO6,ECHO7,ECHO11,CVB3 and ECHO3.Different serotypes had different circulation patterns.CVB5 isolate may exist recombination.A substitution of serine(S)to asparagines(N)at amino acid 95 in the VP1 region may be a major virulence determinant for CVB5. |
