| [Background]Human enterovirus (HEV) is single-stranded RNA viruses belonging to the genus Enterovirus, family Picornaviridae, order Picornavirales. The viral genome contains a5’-and3’-untranslated regions (UTR) which contain necessary structure of the viral RNA replication and translation, moreover, a polyadenylation (PolyA) tail is essential for the3’UTR. The coding region is divided into structural protein coding region (PI) and non-structural protein coding regions (P2and P3), while the P1encoding four capsid proteins, VP1to VP4. The VP1protein, which locates at the outermost, contains the major antigenic sites, and its coding region is the main target for the genotyping and molecular epidemiological studies.According to the divergence of the VP1nucleotide sequence, HEV are divided into four groups, HEV-A to HEV-D. Currently, HEV-B group, containing59serotypes, is the largest group in the four groups. It is known that HEV-B not only contains a large number of serotypes, but with a wide rang of transmission, which can lead to a variety of outbreaks. In addition, HEV has a rapid genomic variation, and new serotypes are emerging. All of these have caused great concern of attention, and HEV-B group is considered as one of the current hot.The complexity of the genetic variation has been known to occur in the HEV genome for almost fifty years. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. The emergence of cVDPVs by recombination reveals a fascinating mechanism of variability in HEV, and analysis of this phenomenon should provide us with new insight into HEV evolution.The evolutionary mechanism of HEV genome includes both point mutation and genetic recombination. Nucleotide insertions, deletions and substitutions are the main forms of point mutations in HEV genome, and the recombination are known to be homologous and non-homologous recombination. According to the reports, genetic recombination was obtained not only within the prototype strains but also the circulating enteroviruses. In addition, the recombination can be hunt between the same serotype, but may also exist between different serotypes. In the majority of cases, recombination within VP1capsid protein seems to be rather an exception than a rule, although the VP1gene is the focus of HEV evolutionary genetic research. It has been shown recombination was more prone to occur in the non-structural genome region, and in a cell-free system that3D polymerase alone is sufficient for recombination. The occurrence of HEV recombinanation will be obtained by comparing the different characteristics of VP1and3D phylogenetic trees, and further analysis can be carried out for the gene sources of recombination. As HEV are locating and spreading in an area, what are the characteristics of the VP1evolutionary genetics and the population dynamics, and what is the recombination rate among different HEV serotypes? Further researchs need to be carried out to reveal these questions.[Objectives]1. To establish databases of VP1and3D complete sequences of predominant HEV-B serotypes (CVA9, CVB3, CVB5, Echo6, Echo7, Echoll, Echo14, Echo19, Echo25and Echo30) isolated in Shandong province, and to describe the genetic characterization.2. To analyze the evolutionary genetics characteristics of the predominant serotypes, such as the gene origin, evolution rate, epidemic history reconstruction.3. To investigate the occurrence of genetic recombination of the predominant serotypes, and to clarify the recombication pattern of cocirculating strains in Shandong province and the frequency of the recombination. [Methods]1. Virus Isolation. Enterovirus isolation was performed on specimens from AFP and AM cases in Shandong province in1989-2010. Two cell lines, human rhabdomyosarcoma (RD) cell and human epidermoid carcinoma (Hep-2) cell, were used for virus proliferation.2. Gene Sequencing. Viral RNA was extracted, the VP1and3D coding region were amplified by RT-PCR and positive products were sequenced.3. Serotype Identification. The VP1sequence was compared with sequences available in GenBank using BLAST, obtained online from NCBI.4. Bioinformation Analysis. The phylogenetic trees of VP1and3D were generated by Mega4.0. Recombinant analysis was carried out by comparing the VP1and3D phylogenetic trees. Evolutionary genetics of predominant HEV serotypes was inferred via BEAST1.6.2, and the epidemic history in Shandong province was reconstructed.[Results]1. Predominant HEV-B Serotypes Identification. In the previous study, molecular serotyping methods were used for the serotypes identification in Shandong province. In this study, the work was continued to identify the HEV serotypes, and a total of1010Shandong HEV strains (includes58HEV serotypes) were isolated from AFP and AM cases between1989and2010. CVA9, CVB3, CVB5, Echo6, Echo7, Echo11, Echo14, Echo19, Echo25and Echo30were acconted for78.4%(792/1010) of all identified serotypes, were the predominant serotypes.2. Recombinant Analysis of Predominant HEV-B Serotypes in Shandong Province. Totally,458Shandong strains were used for the phylogenetic analysis. A frequent recombinant phenomenon was observed in non-structural gene among the10predominant HEV-B serotypes, and13clusters were divided among the predominant HEV-B Shandong serotypes.(1) In the3D phylogenetic tree, serotypes belonging to different clusters were not the same, and the same serotypes of Shandong strains were divided into different clusters. Each cluster contained about two to nine serotypes, and The predominant clusters were such as1,2,3,9,12,13.(2) The isolation time of different clusters were different. The longest time span of the cluster was about21years, and the predominant isolation time of different clusters were all about15(14.8±4.6) years.3. Evolutionary Origin of Predominant HEV-B Strains in Shandong Province. The most recent common ancestor of the10predominant serotypes can be traced back between1885and1923, and the mean evolution rate of VP1sequence was1.007×10-3～9.856×10-3substitutions per site per year. CVA9, Echo6, Echo7and Echo25showed the same ancestor with strains isolated in another province of China and foreign countries. CVB3, Echo11, Echo14, Echol9and Echo30presented the same ancestor with strains in another province of China.[Conclusions]1. CVA9, CVB3, CVB5, Echo6, Echo7, Echoll, Echo14, Echo19, Echo25and Echo30are the predominant HEV-B serotypes in Shandong province. For a22-year surveillance, the isolates of CVA9, CVB3, CVB5, Echo6, Echo7, Echo11, Echo14, Echo19, Echo25and Echo30strains in Shandong province are larger than other HEV serotypes, and are the predominant HEV-B serotypes.2. Recombination is the evolutionary characteristic of non-structural coding region among HEV. The phylogenetic trees based on3D region show that each predominant serotype will cluster in one genogroup with other HEV-B serotypes. Altogether,13different clusters can be divided in the3D phylogenetic tree. Each serotype can be divided into several clusters and the predominant epidemic time of different transmission chains are not all the same. Our results suggest that recombination is a significant and relatively frequent mode in the evolution of HEV genomes.3. Variation in the VP1sequences of dominant HEV-B Shandong strains is extensive, and different transmission chains are co-circulating in Shandong. Epidemic history reconstruction indicating that the ancestors of the dominant HEV-B Shandong strains can be traced back to97years ago, and different evolution rates for different transmission chains can be observed in the molecular clock phylogenetic tree. These results suggest the constant growth population size and a strict clock model were not suitable for the evolutionary analysis of predominant HEV-B Shandong strains. |
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