Ananlysis On The Molecular Characterizations Of Coxsackievirus A4 Associated With Hand-Foot-Mouth Disease In China Mainland

BackgroundHuman enterovirus (HEV) are noneloped single-stranded positive-sense RNA viruses belonging to the Enterovirus genus of the Picornaviridae family, which can be divided into four groups:EV-A, EV-B, EV-C and EV-D, and 119 serotypes of HEV have been reported at present. Most EV infections are asymptomatic or only cause mild symptoms, such as non-specific fever, rash, or mild upper respiratory tract symptoms (for example common cold). It also can cause a diversity of clinical diseases, including acute hemorrhagic conjunctivitis, aseptic meningitis, acute flaccid paralysis, hand, foot and mouth disease, myocarditis and neonatal septicemia. In recent years, the incidence of diseases caused by HEV infection has been showing a rising trend.The genome of HEV consists of a single-stranded positive-sense RNA of approximately 7400 nucleotides. The whole genome contains only one open reading frame (ORF) encoding the Polyprotein. This Polyprotein is composed of four structural proteins (VP1-VP4) and seven non-structural proteins. VP1 protein is the most external and immunodominant of the enterovirus capsid protein, and the main structural of recognition and combination between viruses and cell receptor. VP1 is prone to mutation under the pressure of body’s immune system. Therefore, analysis of VP1 nucleotide sequence can not only help to identify serotypes but also indicate the homologous identity and phylogenetic relationships.Hand, foot and mouth disease (HFMD) was a common infectious disease caused by a variety of enteric viruses. Since the outbreak of HFMD occurred in 2007, the number of HFMD cases increased year by year. According to recent reports, HFMD became more and more serious in mainland China and threaten children’s health and safety. Enterovirus 71 (EV71) and coxsackieviruses A16 (CA16) are the two major causative agents of HFMD. The genetic diversity and molecular evolution of other HEVs, unlike those of EV71 and CA16, has not been fully described. In recent years, other enterovirus types, including CVA4. have been associated with increasingly occurred sporadic HFMD cases and outbreak events globally. In the present study, the molecular characterization and molecular epidemiological of CVA4 associated with HFMD in mainland China were analysized.Objective:To clarify the genetic variation pattern of CVA4 associated with HFMD in mainland China from 2008-2012 through the molecular characterization and molecular epidemiological study. To enrich molecular epidemiological information of CVA4 and to further master the basic epidemic state of CVA4 circulated the Mainland China in order to provide scientific basis for the prevention and control of HFMD.Methods:1. Virus Isolation. There are 57 CVA4 that isolated from 1246 HFMD, viral encephalitis, herpangina clinical samples in Ganyu county, Jiangsu Province, in 2012. And other 19 CVA4 isolation was performed on specimens from HFMD cases in mainland China from 2008-2012.2. Gene Sequencing. Viral RNA was extracted, the VP1 and 3D coding region were amplified by RT-PCR and sequenced.3. Bioinformation Analysis. Homology analysis on VP1 sequences from CVA4 strains in this study and others submitted in GenBank were conducted by BioEdit Sequence Alignment Editor software 5.0. Analysis. Phylogenetic analysis constructed by the Neighbor-Joining method based on the alignment of the complete VP1 and 3D gene sequences of CVA4 strains in this study and the corresponding strains from GenBank were performed by Mega 5.0.Result:1. A total of 350 virus strain were identified from the clinical specimens of Ganyu County, Jiangsu Province. Of which, CVB3 was the most frequently detected type (142), followed by CVA4 (57), CVA10 (31), CVA16 (29), EV71 (24), and so on. CVA4 was the second frequently detected type (16%)./There were 57 CVA4 virus strain isolated from Ganyu County, of which the identities among the 564 of nucleotide in the 5’-VPl region were 94.0%-100%, and the identities of amino acid were 97.8%-100%. Sequence determination was proceed among the VP1 coding region while 2 isolates were chosen. Then 21 CVA4 representative strains in this study were performed by nucleotide sequencing. It showed that there was a little difference in the nucleotide and the amino acid among the 21 CVA4 strains, the homology were 94.1%～100% and 97.3%～100%, respectively.2. Homology analysis on VP1 sequences from CVA4 strains in this study and other strains, isolated in mainland China, submitted in GenBank were done:The nucleotide sequence homology between different strains was 85.5%-100%. and the amino acid sequence homolog was 97.5%-100%. Two CVA4 strains isolated in Shandong province in 1998 is genetically distinct from other provinces strains (by 88.5%-89.9% identities), and 97.5%-98.4% in amino acid sequence. Except for two strains isolated in Shandong province, the nucleotide sequence and the amino acid sequence identities between the CVA4 strains isolated from HFMD cases and that from AFP cases were 94.8%-98.4%. The nucleotide sequence and the amino acid sequence identities between the CVA4 strains isolated from HFMD mild cases and that from severe cases were 95.5%-98.8%, and found no significant difference.3. The phylogenetic trees were constructed by the complete VP1 nucleotide sequences of CVA4 from the present study and those downloaded from GenBank:The strains are clustered in five distinct genotypes, designated A, B, C, D, and E. The Mean distance between genotypes of CVA4 over nucleotide acid sequences were 14.5%-26.7%, and within genotypes were 2.0%-3.5%. All CVA4 strains isolated in mainland China between 1998 and 2012 belonged to genotype E, and can be clustered into two subgenotypes, El and E2s, and 21 strains in this study belonged to subgenotype E2.The phylogenetic trees were constructed by the VP13’420bp nucleotide sequences of CVA4 from the present study and those downloaded from GenBank:The strains are clustered in five distinct Lineages, designated A, B, C, D, and E. Two phylogenetic trees showed no difference.The 57 CVA4 strains in Ganyu Jiangsu Province belong to the subgenotype E2, it is based on VP15’ 564bp nucleotide sequences.4. Based on the genetic distance of the full-length VP1 region, we selected five strains of CVA4 reported in this study as representative strains to analyze their phylogenetic relationship with other available CVA4 strains and all the EV-A prototype strains, higher homology sequences after BLAST and EV71 C4a represent isolates in 3D region. Phylogenetic tree shows that the five CVA4 strains were most closely related to a strain isolated from Shenzhen City of Guangdong province in 2009(HQ727260) and a EV71 isolated from Guangdong province in 2009 (JF 799986), showed a relative high sequence similarity 94.6%-96%; this EV71 strain showed a relative low sequence similarity with the prototype of EV71 and subgenotype C4a represent strains (73.3%-81.7%), suggested recombination between China mainland CVA4 and this EV71 strain may be occurred in 3D non-capsid region.Conclusion:1. CVA4 is one of the main causative agents of hand foot and mouth disease (HFMD), viral encephalitis, herpangina in Ganyu, Jiangsu province in 2012. CVA4 circulated in the local large-scale, and belonged to many different viral transmission chains.2. Our study performed phylogenetic analysis of worldwide CVA4 strains based on all the available complete VP1 sequences for the first time, which indicated that they can be divided into five genotypes, designated A, B, C, D, and E. Consisting with the result, phylogenetic analysis based on 420 nucleotides of 3’VP1 region showed all the CVA4 strains can also be clustered into five lineages, lineage A to E, which further confirmed the genotyping. All the CVA4 strains isolated from mainland China belonged to genotype E, and can be clustered into two subgenotypes, E1 and E2.3. Phylogenetic analysis showed recombination may have occurred between an EV71 strain isolated from Guangdong Province in 2009 and CVA4 strains which currently were prevalent in mainland China in 3D region. Therefore, the role of 3D region as a donor in the enterovirus recombination process as well as the biological characters, transmissibility, pathogenicity of EV71 after recombination should be paid close attention.4. These results also provide important viological basic information for the diagnosis and prevention of HFMD and other diseases caused by CVA4.