| [Background]Human enteroviruses (HEVs) belong to the genus Enterovirus, family Picornaviridae. Genus Enterovirus comprises109serotypes which are grouped into four species:A to D. HEVs evolve actively. Different serotypes possess similar pathogenicity, and different diseases can be associated with a single serotype.According to the antigenicity and pathogenicity, HEVs are previously classified into64serotypes:polioviruses (PV) type1-3, coxsackievirus A (CVA)1-22and24, CVB1-6, echovirus (Echo)1-7,9,11-21,24-27, and29-33, and newer enteroviruses68-71. In1999, the molecular typing method based on VP1sequences was introduced by Oberste et al, and a lot of serologically untypeable HEVs were identified. More than50new serotypes were identified, and with the increasing application of molecular typing method, more new HEV serotypes will be identified in the future.HEVs can cause many diseases, especially in children. HEV associated diseases is an important public health problem. Their infection is known to be generally asymptomatic, but sometimes may cause illnesses such as summer cold, acute flaccid paralysis (AFP), aseptic encephalitis and meningitis (AM), acute myocarditis, epidemic myalgia, acute hemorrhagic conjunctivitis (AHC), hand, foot, and mouth disease (HFMD), herpangina, type1diabetes, and even death. Newer HEVs are important human pathogens. Although they were identified in recent years, there are several outbreaks and epidemics of associated diseases. EV71associated HFMD outbreak or epidemics are severe public health problem. EV70is pathogen of AHC. Other associated symptoms include fever, digestive indisposition, respiratory disease, and severe disease in central nervous system such as aseptic encephalitis and meningitis. Pathogenicity of other newer HEV serotypes is under further investigation.Shandong province is one of epidemic area of HEV associated diseases with several documented outbreaks or epidemics of aseptic meningitis, HFMD, and AHC. There is no intensive research on newer HEVs in China. Only Identification of EV75,80-83,96in Yunnan province was reported. Hence, to explore the molecular epidemiology, genotype distribution, and genetic characterization will contribute significantly to the prevention and control of related diseases and the establishment of precaution mechanism.[Objectives]1. To investigate the genotype distribution of newer HEVs in Shandong province.2. To explore the biological characterization and VP1molecular evolution of newer HEVs in Shandong province.3. To study the complete genome sequence characterization and recombination of newer HEVs in Shandong province.[Methods]1. Enterovirus isolation was performed on specimens from AFP cases in Shandong province in1989-2010, HFMD cases in Shandong province in2007-2010, and aseptic meningitis cases in Linyi city in2010.2. RNA extraction, RT-PCR and sequencing of entire VP1coding region were performed for serologically untypeable isolates.3. VP1sequence alignment and molecular typing were conducted on BLAST server provided by NCBI. 4. Homologous comparison on VP1nucleotide and amino acid sequences between Shandong isolates and global isolates was performed using BioEdit7.1.3. Phylogenetic trees on VP1sequences were constructed using neighbor-joining method in Mega4.0.5. Evolutionary genetics (origin and evolution rate) of partial newer HEV serotypes (EV71, EV75, EV76, and EV90) was inferred via BEAST1.6.2, and the epidemic history in Shandong province or mainland China was re-constructed.6. Complete genome sequencing was performed on partial serotypes (EV71, EV73, EV75, EV76, EV90, EV96, and EV97). Genome alignment was conducted using BioEdit7.1.3. Recombination in the genome in Shandong newer HEV strains was analyzed using Simplot.[Results]1. Genotype distribution of newer HEVsAltogether213newer HEV strains were identified. They belonged to10serotypes:EV71(197), EV73(1), EV74(1), EV75(2), EV76(2), EV80(3), EV87(1), EV90(3), EV96(2), and EV97(1). EV71came from AFP, HFMD and AM cases, and the other9serotypes came from AFP cases.2. Molecular evolution of EV71in Shandong provinceSequencing of VP1entire coding regions were performed on59EV71strains, including13strains from AFP surveillance in1996-2010,35strains from HFMD cases in2007-2010, and11strains from AM cases in Linyi in2010. Shandong EV71strains since2003had high homologies, and they all belonged to C4a lineage of C4subgenotype, phylogenetically closely related to the mainland strains since1997. Only1EV71was isolated before2003. It was isolated from AFP cases in1996, and belonged to C2subgenotype. This is the first report of C2subgenotype EV71in mainland China.Temporal dynamics of EV71in Shandong province was observed, as was reflected via the different clusters of viruses with different isolation years. By using the Bayesian MCMC method in BEAST1.6.2software, the time of most recent common ancestor (tMRCA) of C4subgenogroup of EV71was deduced in1994, and the evolution rate was5.199×10-3nucleotides per site per year. The tMRCA of Shandong C2and C4subgenogroups was1985, implying the divergence of EV71at that time.3. VP1molecular evolution of other Shandong newer HEV serotypes(1) Global EV73strains were divided into two genogroups:A and B. Genogroup B was a major group, and its members were isolated in1964-2010, revealing the continous circulation. Shandong EV73strain belonged to genogroup B, and it had long genetic distances with other strains (mean,0.184; VP1homomogies,75.9%-84.1%).(2) Shandong EV74strain,05293, had a mean genetic distance of0.194with other strains in GenBank, suggesting it had no close relationship with others. Because of the little genetic data of EV74available in GenBank, and the high divergence among global strains, the classification of genogroup could not be performed.(3) Shandong EV75strains,97102and97209, both came from AFP cases in1997. Global EV75strains segregated into two genogroups, A and B. Genogroup B contained5members, all from AFP cases in mainland China (Shandong and Yunnan). The mean genetic distance between Shandong and Yunnan strains was0.128, and the parameter between Shandong strains and other global strains was0.247. Shandong EV75strains had100%nucleotide homology with each other, and71.0%-77.8%homologies with other strains. The tMRCA of global EV75was dated back to1956with the evolution rate of1.647×10-2nucleotides per site per year.(4) Shandong EV76strains,04360and05048, had high VP1nucleotide homology (99.7%) with each other, and they belonged to the same transmission chain. They were isolated from AFP cases in Wenshang county and Hedong district, respectively, suggesting the long distance transmission between the two areas at that time. Shandong strains had86.0%VP1nt similarity with proto FRA91-10369,86.9%with Yunnan strain171-99, and80.7%-94.7%with other strains.(5) Shandong EV80strains,97249,98323and04HZ+1, had76.4%-81.7%VP1nt similarities with proto CA67-10387. A high similarity was observed between98323and04HZ+1, reflecting close relationship, while97249had relative low VP1 homologies with the other two Shandong strains (80.3%-82.0%).(6) Only one EV87strain was isolated in Shandong province, and this is the first report of isolation of EV87in China. The nucleotide and amino acid identities with proto BAN01-10396were80.3%and97.0%, respectively.(7) Three EV90strains,01336,01421and03446, were isolated in Shandong province. This is the first report of EV90in China. Shandong strains belonged to genogroup B, and had96.7%-98.0%VP1nucleotide similarities among themselves, reflecting close relationship. Shandong strains had90.8%-91.4%VP1nucleotide similarities with proto BAN99-10399, and77.7%-92.3%with other strains.(8) Two EV96strains,05517and09228C1, were isolated in Shandong province. They had81.6%VP1nucleotide similarities with each other, reflecting remote relationship. They had78.1%-81.0%nucleotide similarities with proto BAN00-10488, and77.6%-87.9%with other strains. They formed a distinct cluster in the phylogenetic tree.(9) Only1EV97strain was isolated in Shandong province, and this is the first report of EV97in China. It had remote genetic relationship with other6EV97strains available in GenBank. A deletion of18nt in3'end of VP1coding region was observed.4. Complete genome and recombination analysis of Shandong newer HEVsThe complete genome sequence analysis of EV71, EV73, EV75, EV76, EV90, EV96and EV97revealed the evidence of recombination in non-structural region of Shandong newer HEV. In this region, Shandong EV71strains had higher similarities with CVA14proto G14strain and CVA16proto G10strain than with EV71proto BrCr; EV73and EV75Shandong strains had highest similarities with domestic Echo30strain Zhejiang/17/03/CSF; EV76Shandong strain had highest similarities with EV89and EV90proto strains; EV90Shandong strain01421had highest similarity with Finland strain F950027; EV97Shandong strain99188had highest similarities with Echo9strain DM and Echo30strain Zhejiang/17/03/CSF.Complete genome sequence analysis of EV90, EV96and EV97revealed deletions of nucleotide in capsid protein coding region, and the number of deletion was3or18nucleotides, separately, resulting in no frame shift. The position of deletion was the hot region of length variance in different serotypes, such as VP3or3' end of VP1region. This implyed the nucleotide insertion or deletion might happen before the serotype alteration caused by accumulation of point mutation.[Conclusions and suggestions]1. In this study, several newer HEV serotypes were identified from HEV associated diseases. This is the first report of EV87, EV90, EV97and C2subgenotype of EV71in mainland China.Altogether213newer HEV strains of10serotypes were identified, as were EV71, EV73, EV74, EV75, EV76, EV80, EV87, EV90, EV96, and EV97. EV71came from AFP, HFMD and AM cases, and the other9serotypes came from AFP cases.2. Compared to other serotypes, EV71was the most common newer HEV serotypes, and it had resulted in the HFMD epidemic in mainland China since2007.Our work demonstrated EV71was the causative agent of HFMD outbreak in Linyi in2007, and revealed the existence of other subgenotypes of EV71in mainland China before the broad epidemic of C4subgenotype. More retrospective study will provide a broad view of circulation of other subgenotypes in mainland China. Also, EV71was an important causative agent of AM in Shandong province.3. Except EV71, other newer serotypes had low isolation rate, suggesting they had not circulated extensively in China yet.4. Great nucleotide divergence was observed between Shandong newer HEVs and other strains available in GenBank, suggesting a remote relationship. Nucleotide insertions and deletions participate in the evolution of HEV capsid protein region.5. Evidence of recombination was revealed in the complete genome analysis of Shandong newer HEV strains. More genome sequence data is needed to identify the exact donator.6. On account of the fact that newer HEV strains were frequently isolated in surveillance cases, we recommend the surveillance and molecular epidemiology study on newer HEVs should be strengthened in the future.
|
PDF Full Text Request