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Characterization of VP1 sequence of Coxsackievirus A16 isolates by Bayesian evolutionary method
© The Author(s). 2016
Received: 31 March 2016
Accepted: 29 June 2016
Published: 28 July 2016
Coxsackievirus A16 (CV-A16), a major etiopathologic cause of pediatric hand, foot, and mouth disease (HFMD) worldwide, has been reported to have caused several fatalities. Revealing the evolutionary and epidemiologic dynamics of CV-A16 across time and space is central to understanding its outbreak potential.
In this study, we isolated six CV-A16 strains in China’s Jilin province and construct a maximum clade credibility (MCC) tree for CV-A16 VP1 gene by the Bayesian Markov Chain Monte Carlo method using 708 strains from GenBank with epidemiological information. The evolution characteristics of CV-A16 VP1 gene was also analysed dynamicly through Bayesian skyline plot.
All CV-A16 strains identified could be classified into five major genogroups, denoted by GI–GV. GIV and GV have co-circulated in China since 2007, and the CV-A16 epidemic strain isolated in the Jilin province, China, can be classified as GIV-3. The CV-A16 genogroups circulating recently in China have the same ancestor since 2007. The genetic diversity of the CV-A16 VP1 gene shows a continuous increase since the mid-1990s, with sharp increases in genetic diversity in 1997 and 2007 and reached peak in 2007. Very low genetic diversity existed after 2010. The CV-A16 VP1 gene evolutionary rate was 6.656E-3 substitutions per site per year.
We predicted the dynamic phylogenetic trends, which indicate outbreak trends of CV-A16, and provide theoretical foundations for clinical prevention and treatment of HFMD which caused by a CV-A16.
In the pediatric population, hand, foot, and mouth disease (HFMD) is a common self-limiting condition caused by various serotypes of the enterovirus A species that is typically characterized by fever, pharyngalgia, malaise, erythema and herpetic lesions on hands and feet, as well as exanthema on oral mucosa and tongue . A minority of patients develop severe neurologic complications such as acute flaccid paralysis, encephalitis, pulmonary edema, and myocarditis, with fatal outcomes in some severely afflicted patients [2, 3]. Over the last few decades, HFMD has been a very common pediatric infection in the Asia–Pacific region, with sporadic outbreaks reported in Europe and North America. In mainland China, HFMD has been listed as a notifiable disease since 2008 [4–6].
Coxsackievirus A16 (CV-A16) and enterovirus 71 (EV-A71) were major pathogens of HFMD in the past few decades, with EV-A71 more frequently associated with neurologic diseases . Vaccine trials for the first EV-A71 vaccine have reached phase 3 clinical testing in China, with an estimated vaccine protection rate of 90 % against clinical EV-A71 infection-associated HFMD and 80.4 % against other EV-A71-associated diseases . The other major pathogen of HFMD, CV-A16 has had epidemic presence in China and abroad for many years, but generating much lower social concern compared to EV71.
The first CV-A16 was identified in 1951 in South Africa, although there were no reports of an HFMD epidemic at the time . New Zealand reported the first case of HFMD in the world in 1957 . The relationship between CV-A16 and HFMD was confirmed by Atsop in 1959, and officially coined the term “Hand, Foot and Mouth Disease” based on clinical symptoms . Although it is the first-identified HFMD virus, there is no specific antiviral treatment for CV-A16, which thus deserves more research attention. Recent years have seen extensive research efforts toward phylogenetic analysis of the HFMD pathogen based on bioinformatics in order to understand correlations between virus genogroup changes and disease epidemic trends [8, 11, 12]. At present, most extant phylogenetic trees are constructed based on neighbor-joining (NJ) distance, maximum parsimony (MP), and maximum likelihood (ML) [8, 13]. In reality, however, the difference between sequences does not completely represent the evolutionary distance, with large potential for errors . The posterior probability is derived based on the Markov chain by Bayesian statistics, which allow researchers to use prior knowledge for guiding the construction of phylogenetic trees and to infer the maximum posteriori probability for estimating the most likely phylogenetic tree . Moreover, the Bayesian method uses the posterior probability to visually represent phylogenetic relationships, thereby eliminating the need for bootstrapping [15, 16], and is widely used to construct the phylogenetic trees of swine-origin influenza A (H1N1) virus, measles viruses (MV), and EV-A71 and for accurate judgments of the relationship between genetic diversity (g) and epidemics of associated diseases [16–18].
This study was undertaken to investigate evolutionary and epidemiological dynamics of HFMD, with particular focus on CV-A16 genetic history and dynamics, within and between countries where this disease is endemic to facilitate prediction of its emergence in new locations as well as to provide the basis for an effective public health response framework. Bayesian analyses were performed to construct maximum clade credibility (MCC) tree for all sequenced and downloaded sequences, and global population dynamics of CV-A16 over the previous 30 years were reconstructed to examine temporal trends in genetic diversity and association with major epidemics.
A total of 52 specimens, as well as information on patient demographics, clinical symptoms, and complications were obtained from 2012 to 2013 at Disease Control and Prevention Center of Changchun, China. Stool specimens were processed as described previously for subsequent RNA extraction . Specimens of other types were used directly for viral RNA extraction. The strains used for sequencing were amplified and isolated in RD (rhabdomyosarcoma) cells as previously described .
RNA extraction, RT-PCR, and sequencing
Viral RNA was extracted using QIAamp Viral RNA MiniKit (QIAGEN, USA). TaKaRa RNA PCRTM Kit (AMV) was used to do the amplification fragment according to previous reports. The real-time PCR (RT-PCR) was firstly performed to detect the presence of the common (universal) sequence of enterovirus (EV-F, EV-R), and the specific sequences of EV-A71 (EV71S-F, EV71S-R) and CV-A16 (CV-A16S-F, CV-A16S-R). Then using primers (CV-A16VP1-F and CV-A16VP1-R) amplified CV-A16 VP1 whole sequence. Primers and reaction conditions was shown in Additional file 1: Table S1. All results of RT-PCR products were analyzed by 1 % agarose gel electrophoresis . A part of CV-A16 VP1 sequences have been submitted to GenBank (Accession no. KT000389-KT000394).
Sequence collection and phylogenetic analyses
For phylogenetic analysis, a total of 759 CV-A16 VP1 gene sequences before June 2013 were downloaded from GenBank. We retrieved 706 sequences which known collection dates and isolate country for analysis. The accession numbers and specific information of the sequences was listed in Additional file 2: Table S2. These nucleotide sequences were isolated mainly from large HFMD outbreaks and sporadic cases that occurred globally over 1981–2013. Combined with the six sequences isolated from our laboratory, a total of 708 sequences were used in phylogenetic analysis.
Alignment processing and recombination detection
The complete VP1 sequence alignment of the CV-A16 strains was conducted with the Clustal W program in MEGA 6.0. Excess sequence was cut off, and FASTA format that can be used in the BEAST 1.8.2 was exported. Then we use the SEAL (sequence simulation and alignment evaluation software, http://tree.bio.ed.ac.uk/software/seal/) software to edit the nucleotide sequence. RDP3 Restructuring Package was used to detect the recombination of all CV-A16 sequence . Then DAMBE was used for the saturation monitoring, if ISS < ISS.c and p = 0.0000 (extremely significant), then these sequences were unsaturated and suitable for construction of phylogenetic tree . Finally we calculated the best alternative model With JModeltest . The calculation was done after the selection of all the four kinds of patterns and then the statistical of AIC value. The smaller of the AIC value, the better fitting for the model with the data. Then, usually we choose the model with smallest AIC value for the construction of phylogenetic trees.
Bayesian Markov Chain Monte Carlo evolutionary analysis
Bayesian Markov chain Monte Carlo (MCMC) methods were used to construct a maximum clade credibility tree (MCC) using BEASTv1.8.2 (http://beast-mcmc.googlecode.com/files/BEASTv1.8.2.tgz). Tracer v1.6 (http://beast.bio.ed.ac.uk/Tracer) was used to output analysis of sampling data, and then the Tree Annotator program was employed to output the results of MCC tree model. In the end the MCC molecular evolutionary tree graph was illustrated with FigTree1.3 (http://tree.bio.ed.ac.uk/). At the same time, Bayesian skyline plot analyses was used to reconstruct the population history of CV-A16 by measuring the dynamics of VP1 gene genetic diversity over time with 160 typical CV-A16 VP1 (Additional file 3: Figure S1).
JModeltest result revealed that HKY was the best substitution model, and the molecular clock model chosen the Relaxed Clock: Uncorrelated Log-normal. Using the Bayesian Markov Chain Monte Carlo framework, 80 million steps were run, sampling every 8000 and removing 10 % as burn-in. Convergence was assessed using Tracer (v1.6), and effective sample size (ESS) values above 200 were accepted.
Phylogenetic analysis of CV-A16
Global distribution of genogroups of CV-A16
Isolated time and location
1951 South Africa
2008 China: Fuyang
2010 China: Ningbo
1995 Japan: Yamagata
1995, 1997 Japan: Yamagata
1998, 2005 Taiwan
2011 Japan: Yamagata
2001, 2003 Arabia;
2000, 2002–2003 Japan: Toyama
2011 Japan: Yamagata
1997–2003, 2005–2007 Malaysia
2000–2004, 2006, 2008 Japan: Yamagata
2008 South Korea
2002 Japan Toyama
2005, 2007 Malaysia
2008–2010 Japan: Yamagata
2007 Japan: Toyama
Origin and distribution of CV-A16 from Japan, Malaysia, and China
Genetic diversity analysis with Bayesian skyline plot
Codon substitution and evolution rates of CV-A16 VP1 gene
Estimates of the relative substitution rates for the core gene of all three codon positions
95 % HPD lower
95 % HPD upper
Effective sample size (ESS)
This study establishes the phylogenetic relationship, genomic diversity, and the evolutionary rate of CV-A16 for the first time using the Bayesian Markov chain method, providing new sights into the relationship of evolutionary history of virus population and disease periodicity. We reconstructed the epidemic history of CV-A16 and found that the CV-A16 virus, prevalent between 1980 and 2013, is a pathogen that originated around mid-twentieth century.
Bayesian derivation and the maximum likelihood method have similar characteristics in that both have excellent statistical characteristics. However, one difference is that the Bayesian method can use posterior probability, which is derived from the Markov chain to optimize criterion . A very accurate posterior probability can be obtained using the Bayesian MCMC method due to the rigorous control criterion of every link. Against this background, we established the phylogenies of the CV-A16 gene based on Bayesian derivation combined with the Markov chain model method. Compared with the phylogenetic analysis reported in the previous study  using the Neighbor-joiningmethod, the CV-A16 causing the HFMD outbreak in Yamagata, Japan, in 1995 can be independently classified under genogroup GIII using the Bayesian method (Fig. 1, Table 1). There were swift but sporadic occurrences of HFMD in Japan in various years such as 1984, 1988, and 1991 . However, the nucleotide sequence of CV-A16 were relatively stable in this period, reflecting that the epidemic disease that occurred every several years was determined by the cumulative proportion of unvaccinated children and not by the viral antigen’s evolution . Four years late, in 1995, an HFMD outbreak was reported in Japan, which was different from the past episodes; CV-A16 became the main pathogen that replaced EV71 [32, 33]. This suggested that a new CV-A16 genogroup emerged different from former CV-A16 epidemic strains. From the results of our Bayesian skyline plot (Fig. 6), we can also survey the great change in the genetic diversity of CV-A16 in 1995. Therefore, the MCC we reconstructed on the basis of spatiotemporal divergence and genetic diversity is consistent with the trends of epidemic disease. It is very interesting that genogroup GI disappeared for almost 60 years and then it is detected again in 2010 both in our and previous reports . We do not know the reasons for such large-scale changes for genogroup GI, but they may be associated with the G-10’s weakly pathogenicity which didn’t cause enough attention and lead to a lack of continuity monitor data.
From the Bayesian skyline plots (Fig. 6), we can see that every sharp change of genetic diversity resulted in a large-scale HFMD outbreak. To some extent, the increase of genetic diversity corresponding to this characteristic since mid-1990 was a marker for the emergence of a new CV-A16 genogroup. The data set also indicated agreement between genetic diversity dynamics and emergent genogroups, which reflect the earlier HFMD outbreaks. Since 2007, the genetic diversity of CV-A16 stabilized and slightly decreased, and no novel genogroup emergence was reported [12, 13, 33]. However, there were some HFMD outbreaks caused by CV-A16 in various provinces of China that may be attributable to the cumulative proportion of unvaccinated children and increased detection intensity. Since 2010, genetic diversity dynamics tended to be gentler, CVA6 replaced CVA16 become the second pathogene in Shenzhen and Guangdong, China [34, 35].
CV-A16 has long been the main pathogen of HFMD, seriously threatening human health [8, 11, 12, 33]. The reports of some deaths caused by CV-A16 infection [2, 36, 37], suggesting that more attention should be paid to the detection and prevention of CV-A16. From the data we obtained, we predicted the dynamic phylogenetic trends, which indicate outbreak trends of CV-A16, and provide theoretical foundations for clinical prevention and treatment of HFMD which caused by a CV-A16. The relatively stable nucleotide sequence will provide a great opportunity to develop a vaccine for this disease. So the development and administration of its vaccine should be accelerated.
This work was supported in part by grants from National Natural Science Foundation of China (#81271897, #81611130074), foundation of Jilin Province Science and Technology Department (#20140414048GH), the Norman Bethune Program of Jilin University (#2012219) and Research funds from the Jilin Key Laboratory of Biomedical Materials and Jilin University - Xinjiang Medical University joint research project. We would like to extend our special thanks to the two anonymous reviewers for their helpful comment on our manuscript.
National Natural Science Foundation of China (#81271897, #81611130074), Foundation of Jilin Province Science and Technology Department (#20140414048GH), The Norman Bethune Program of Jilin University (#2012219) and Research funds from the Jilin Key Laboratory of Biomedical Materials and Jilin University - Xinjiang Medical University joint research project.
FL conceived the study. GZ designed the experiments, analyzed the data and wrote the manuscript.GW guided the experiment process. XZ and CW contributed in data collection. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
This work was approved by the Ethics Committee of Jilin University, to ensure the consent of all research objects.
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