Sequence Analysis Of Complete Genomes Of Enterovirus71and Study On Candidate Virulence Positions Of VP1Region

Enterovirus71(EV71), a member in the family of Picornaviridae, can cause hand, foot and mouth disease (HFMD). In addition, EV71can also infect the respiratory system, cardiovascular system and central nervous system, and then cause encephalitis, myocarditis, pulmonary edema, flaccid paralysis and other symptoms. In recent years, the prevalence of EV71is rising in Asia.Objective1. To amplify sequences and screen of neurovirulence candidate positions of complete genomes of enterovirus71.2. To search for potential virulence positions from VP1gene, and compare cytopathic effect caused by mutation.Methods1. Nine pairs of primers were designed to amplify complete genomes of3SC-EV71strains (SDLY96, SDLY107. SDLY153). Complete genomes of3SC-EV71strains,3MC-EV71strains (SDLY1、SDLY11、SDLY48, isolated in our laboratory) and25strains of explicit clinical symptoms selected in GenBank were aligned.2. VP1sequences of23EV71strains were amplified and sequenced, and sequence analyses were performed by BioEdit7.09and Mega4software.3. Full VP1sequences of strains SDLY11and SDLY107were amplified and sequenced, and expression vector pcDNA3.1(+)/11VP1and pcDNA3.1(+)/107VP1 were constructed. VP1sequences of3SC-EV71strains (SDLY96, SDLY107, SDLY153) and3MC-EV71strains (SDLY1, SDLY11, SDLY48) were aligned, and P98, P145and P289were screened as potential virulence positions. Primers were designed according to the potential virulence positions. Recombinant PCR was used for constructing3clone mutants, in which there was only one amino acid mutated (107VP1/K98E,107VP1/E145G,107VP1/A289T). These5expression vectors were transfected into RD cells. Giemsa staining, indirect immunofluorescence, Western blot and lactate dehydrogenase (LDH) cytotoxicity assay were performed to examine cytopathic effect (CPE).4. SDLY107strains which isolated from death cases were selected to construct full-length cDNA plasmid. Due to failure of connection, full-length cDNA plasmid pCDNA3.1(+)/SDLY107was synthetized. pCDNA3.1(+)/SDLY107was linearized and transcription in vitro to prepare RNA transcript. RNA transcript was transfected into RD cells.Results1. Complete genomes of3SC-EV71strains were amplified. Complete genomes of3SC-EV71strains,3MC-EV71strains and25strains of explicit clinical symptoms selected in GenBank were aligned. ValP814/IleP814, ValP1148/IleP1148and AlaP1728/Cys P1728/ValP1728were screened as significant positions.2. Complete VP1sequences of23EV71strains were amplified. The relationship between23strains and the reference strains of genotype A or B were relatively farther. The nucleotide and the amino acid identity of the23strains with the reference strains of group C4a were92.8%～93.5%and98.3%～99.3%, significantly higher than other subtypes.3. pcDNA3.1(+)/11VP1,pcDNA3.1(+)/107VP1,107VP1/K98E,107VP1/E145G and107VP1/A289T were constructed. These5expression vectors were transfected into RD cells. After Giemsa staining, it showed that5group of cells had different degrees of cytopathic effect. It showed that all the5group of cells could generate green fluorescence when examined by indirect immunofluorescence and all the5group of cells could expresse VP1protien (34KD) when examined by Western blot. LDH cytotoxicity assay showed that pcDNA3.1(+)/107VP1,107VP1/K98E and107VP1/A289T induced the maxmum cytopathic effect, pcDNA3.1(+)/HVP1induced the minimum cytopathic effect, and107VP1/E145G induced cytopathic effect lied between the maximum and minimum values.4. pCDNA3.1(+)/SDLY107was linearized and transcripted in vitro to prepare RNA transcript. RNA transcript was transfected into RD cells. There was no obvious cytopathic effect, and RT-PCR identification had no corresponding fragment.Conclusion1. Three positions were screened as candidate positions associated with neurovirulence or genotype, they were ValP814/IleP814in VP1, Valp1148/IleP1148in3A and AlaP1728/CysP1728/ValP1728in3C.2. According to VP1region, the23EV71strains belonged to group C4a of subgenotype C4.3. The145th amino acid protein of VP1(the710th amino acid sequence of the complete genome) was the potential virulence position, and mutation (E�'G) could caused reduction of cytopathic effect.