Molecular Evidence of Persistent Epidemic and Evolution of Subgenotype B1 Coxsackievirus A16-Associated Hand, Foot, and Mouth Disease in China

Abstract

The molecular epidemiology of CVA16 in China between 1999 and 2008 reflects a pattern of endemic cocirculation of clusters B1a and B1b within subgenotype B1 viruses. The annual evolution rate of CVA16 was estimated as approximately 0.91 ؋ 10 ؊2 substitutions per synonymous nucleotide/year and is slightly lower than that of HEV71.Coxsackievirus A16 (CVA16) and human enterovirus 71 (HEV71) are the two major causative agents of hand, foot, and mouth disease (HFMD) (12,17). In recent years, numerous large outbreaks of HEV71-associated HFMD, which were often accompanied by severe complications, including death, increased research interest in HEV71 strains (1,14,16). In contrast, little interest was paid to CVA16 strains because CVA16-associated HFMD was usually mild and benign (2, 12). Although CVA16 is genetically most closely related to HEV71, the genetic diversity and molecular evolution of CVA16, unlike those of HEV71, have not been fully described (5,7,12,13). Cocirculation of CVA16 and HEV71 has been proven to have contributed to the serious outbreaks of HFMD that have occurred in China since 2007 (17); therefore, the genetic variability and the evolution of CVA16 were determined in this study.The 42 CVA16 strains evaluated in this study were isolated from HFMD patients from different geographical locations in the Shandong, Gansu, Inner Mongolia, and Qinghai provinces of China between 2007 and 2008 (see supplemental data). To investigate the molecular epidemiology of CVA16 in mainland China, 24 additional Chinese CVA16 sequences found in Beijing and Guangdong provinces between 1999 and 2005 and 35 international CVA16 sequences (obtained from the GenBank database) were also analyzed.The complete VP1/capsid sequences of the CVA16 strains were obtained as previously described, using in-house primers that flanked the VP1 region (13, 17): CVA16-VP1-S, 5Ј-ATTGGTG CTCCCACTACAGC-3Ј (nucleotides 2335 to 2354, relative to strain CVA16/G-10), and CVA16-VP1-A, 5Ј-GCTGTCCTCCC ACACAAGAT-3Ј (nucleotides 3426 to 3445, relative to strain CVA16/G-10).A total of 66 Chinese CVA16 sequences were divided into three lineages on the basis of phylogenetic analysis (Fig. 1). A 6.5 to 8.1% nucleotide divergence was found among these three lineages, suggesting that the CVA16-associated HFMD outbreaks in China were a result of the coincident circulation of three genetically distinct viruses.To determine the molecular epidemiology of Chinese CVA16 strains associated with HFMD epidemics, a phylogenetic dendrogram was constructed with 21 Chinese CVA16 sequences (randomly selected on the basis of their genetic relationships) that circulated during the period 1999-2008 in addition to the 35 international CVA16 sequences that represented two known genotypes (A and B) (13) (Fig. 2).As in a previous study (13), all CVA16 strains could be grouped into genotypes A and B. The prototype G-10 strain differed from the other strains by 27.5 to 30.2% and clustered separately from all other CVA16 strains, including Chinese CVA16 strains, which clearly belo...

Genus-Specific Substitution Rate Variability among Picornaviruses

“…2. Published VP1 substitution rates for coxsackievirus B5 (CVB5), echovirus 9 (E9), echovirus 11 (E11), echovirus 30 (E30), HAV, and HPeV were obtained via BEAST analyses similar to those used in this study (15,23,34,(46)(47)(48); those for EV71, FMDV-A, and FMDV-O were obtained via analyses performed in TipDate (58), a precursor to BEAST (29); and the remaining rates were estimated via linear regression (3,4,44,50,66,70,71). These mean rates of enterovirus VP1 evolution range from 3.40 ϫ 10 Ϫ3 to 1.19 ϫ 10 Ϫ2 nucleotide substitutions per site per year (ns/s/y), and mean VP1 rates for nonenteroviruses range from 9.76 ϫ 10 Ϫ4 to 2.79 ϫ 10 Ϫ3 ns/s/y.…”

Molecular Analysis of Enteroviruses

“…Additionally, the nucleotide mutation (A→G) at position 485 in the 5' UTR may be one of the molecular determinants of EV-A71 attenuation of neurovirulence in cynomolgus monkeys [111]. The average evolutionary rate calculated for CV-A16 B1a and B2 was 0.91 × 10 -2 synonymous substitutions per nucleotide per year based on VP1 gene [64]. The evolutionary rate of CV-A16 is relatively slow given that the prototype CV-A16 strain was first identified in South Africa in 1951, and that all other CV-A16 strains formed a single genogroup B following approximately 60 years of evolution [64].…”

“…EV-A71 and CV-A16 are members of EV-A species, and are major and independent etiologic agents of hand-foot-mouth disease [64,65]. To understand the molecular epidemiology and evolution of EV-A71, whole-genome sequences [1] and several distinct regions of the EV-A71 genome have been employed for analysis, such as 5' UTR [19,66], 3D polymerase region [67], VP4 region [68], and VP1 region [19,49,61,69,70].…”

“…For CV-A16, the VP1 and VP4 genes are mostly used for classifying the virus [19, [77][78][79]. It has been classified on VP1 analysis into geno groups A and B, with geno group B further divided into subgenogroups B1 (including B1a, B1b and B1c), B2 [64,80] and a possible B3 [19]. Molecular analysis of VP1, combined with 5' UTR, may also enhance the reliability of molecular surveillance and the understanding of genetic evolution of CVA16 [19].…”

Human picornaviruses associated with neurological diseases and their neutralization by antibodies

“…This discrepancy should be taken into consideration when choosing vaccine strains and designing neutralizing antibodies. CV-A16 is less genetically diverse, showing a relatively slow evolution rate, and has three genotypes: A, B1 (B1a, B1b, B1c) and B2 [134]. EV-A71 can undergo intra-and intergenotype shifts that occur due to recombination events during co-circulation with different EV-A71 or CV-A16 genotypes [135].…”