| 【Objectives】1.To establish the method of EV71 immunomagnetic beads combined with routine detection,separate and detect EV71 from environment specimen,and explore the role of environment specimen in EV71 transmission.2.To investigate the genetic characteristics of EV71 separated from different clinical symptoms and obtain the potential virulence sites of EV71 related to severe cases.3.To construct the mutant infectious clone,rescue virus,determined the virulence of rescued virus by cell assay and animal model,and confirm the effect of virulence sites variation for the virulence of virus.【Methods】1.Preparation of immunomagnetic beads of EV71,establishment of the method of enrichment and isolation of EV71 from environment specimens,the complex of immunomagnetic bead-virus were detected by RT-PCR、RT-q PCR and virus isolation.The homology analysis and phylogenetic characteristics were based on the VP1 nucleotide sequences.2.Forty genomes of virus isolates from Fujian province were sequenced.Together with genome sequences of 48 viral isolates from other provinces in mainland China were classified into severe and mild viral strains according to the clinical manifestations of case.Investigating the genetic characteristics of EV71,and analysis the variation of nucleotides and amino acids between severe and mild strains.3.With site mutation method,mutant infectious clones were constructed based on Nero-pcr-XL 149-08 plasmid and rescued in RD cells.The biological characteristics of mutant rescued virus were determined by cell assays,then the rescued viruses infected the neonatal mouse to determined the virulence.Finally,the virulence change of rescued virus was further verified by transgenic mouse.【Results】1.Immunomagnetic enrichment(IME)was high sensitivity and specificity.346 specimens,including cases and environment specimens,were processed by IME,the positive rates of RT-q PCR、RT-PCR and virus isolation were 20.52%、5.78% and 9.25%,respectively,which were significantly higher than Non-IME(15.90%,3.47% and 4.05%,p<0.05).The isolation rate of EV71 from case increased from 27.45% to 43.14% through IME.The isolation rate of EV71 from household items increased from 0 to 5.29% through IME.The positive rate of toys and stationery were highest among the household items,which accounted for 52.00% and 24.00%,respectively.Among the environment specimens of kindergarten,the positive rate of RT-q PCR was 6.12%,but no EV71 can be isolated.The nucleotide homology between the case isolates and the family environment isolates was 98.0-100%.2.Forty genomes of viral isolates from Fujian province during 2003-2015 were obtained.All of viruses could be divided into two lineages of C4 a and C4 b.The viruses circulating in mainland China before 2004 were C4 b,the viruses which circulated after 2004 belong to C4 a.There were 4 mutations on EV71 genome showed significantly different between severe and mild strains.The mutation sites were located in: 5’UTR nt99-100(--→C,χ2=4.041,p<0.05)、nt576(T→C,χ2=4.346,p<0.05);VP2-144(T→S,χ2=4.433,p<0.05)、VP1-22(H→Q,χ2=4.041,p<0.05)。3.Mutant infectious clones of M1、M2、M3、M1+2、M1+3、M2+3、M1+2+3 and M4+5 were constructed successfully by site-directed mutagenesis method.All of them were successfully rescued by RD cells.The results of cell assays showed that the rescued virus M1+2+3 with a higher virulence and tolerated to temperature;the rescue virus M2 with a lower virulence and sensitive to temperature.The result of Balb/c mouse showed that the rescued virus M1+2+3 had higher mortality,the rescued virus M2 had a lower mortality.The result of SCARB2 transgenic mouse showed that rescued virus M1+2+3 had higher virulence and could cause typical clinical symptoms of HFMD and pathological changes in brain tissue.【Conclusions】1.The detection limit of RT-PCR、RT-q PCR and virus isolation can be improved by using IME;EV71 can be isolated from environment samples by using IME;the EV71 strains isolated in the environment shared high homology with the case isolates;toys and stationery play an important role in the transmission of EV71.EV71 had a higher detection rate in home environment specimens,especially in toys and stationery,so it is necessary to strengthen the effective disinfection of the family environment.2.The genotype of EV71 isolated from Fujian province is consistent with EV71 from other province,all of them belong to C4 subtype.The mutation sites between severe and mild strains were 5’UTR nt99-100(--→C)、nt576(T→C);VP2-144(T→S)、VP1-22(H→Q).3.The rescued virus with nt99-100(--→C)、nt576(T→C)and VP2-144(T→S)can enhance the virulence of virus,indicating the coordination existed among the 3 sites;and nt576(T→C)is related to the virulence of EV71 from China,but it is not only one site,it was a result of multiple sites. |
PDF Full Text Request