Circulating Coxsackievirus A16 Identified as Recombinant Type A Human Enterovirus, China

To determine the relationship of coxsackievirus A16 (CA16) to prototype CA16-G10, we conducted a phylogenetic analysis of circulating CA16 strains in China. Complex recombinant forms of CA16-related viruses involving multiple human enteroviruses, subgroup A (CA4, CA16, and enterovirus 71), are prevalent among patients with hand, foot, and mouth disease.

the parental strain of circulating CA16 strains. As a result, we found that current CA16 strains in China, although still related to CA16-G10, are recombinant HEV-A.
Because the shzh00-1, shzh05-1, and GZ08 sequences clustered within the HEV-A group and were determined to be CA16 on the basis of the VP1 region (data not shown), we examined these 3 sequences for evidence of recombination. Shzh00-1 was chosen as representative because it was isolated earlier. It has 7,410 nt in its genome, including the 5′ untranslated region (UTR) (1-745), structural protein (746-3331), and nonstructural proteins P2 (3332-5065) and P3 (5066-7327), with the rest of its genome as 3′ UTR (7328-7410). Bootscanning with a sliding window of 500 nt, overlapping by 20 nt, was performed with the SimPlot program (version 3.5.1) (14) to investigate the possibility of recombination within the shzh00-1 sequence. Various HEV-A sequences were used as reference sequences. The results indicated that the 5′ UTR of the shzh00-1 sequence had relatively high similarity to CA4 ( Figure 1, panel B). The P1 (VP4, VP2, VP3, and VP1) region was more similar to that of CA16-G10. However, part of the P2/P3 region of the shzh00-1 sequence (2C, 3A, 3B, and 3C) had relatively high similarity to EV71A but not to CA16-G10 ( Figure 1, panel B). Similar results were obtained for the shzh05-1 and GZ08 sequences (data not shown). Thus, shzh00-1, shzh05-1, and GZ08 are recombinant type A HEVs that contain CA4, CA16, and EV71.
To examine the circulation status of recombinant CA16 in China, we characterized 24 additional CA16-related sequences from HFMD patients from central (Hangzhou, Zhejiang Province) and northeastern (Changchun, Jilin Province) China (Table). The break point around position 3555 interested us most because it not only roughly separated the P1 region from P2/P3 but also divided the open reading frame (746-7327) into CA16-like and non-CA16-like fragments. Thus, we selected this region spanning the recombination break point between the CA16 sequence and the downstream sequence.
Bootscanning of these new sequences showed that the sequences from Changchun and Hangzhou have a recombination pattern similar to that of shzh00-1 (   was shown for bootscanning analysis with human enterovirus A (HEV-A) sequences and shzh00-1 as references. The results suggested changchun104 was similar to shzh00-1. The red vertical line indicates position 3,555, which corresponded to shzh00-1. B) hangzhou212, hangzhou023, and all Changchun sequences clustered with shzh00-1, shzh05-1, and GZ08, with a very high bootstrap value (100%). Another Hangzhou sequence, hangzhou114, was most likely related to CA4.
indicates Hangzhou sequences; indicates Changchun sequences, and indicates shzh00-1, shzh05-1, and GZ08. Scale bar indicates nucleotide substitutions per site. C) Map of the People's Republic of China indicating the provinces where shzh00-1like CA16 sequences were characterized.
analysis indicated that 23 of the Changchun and Hangzhou sequences formed a strong cluster with shzh00-1, shzh05-1, and GZ08 ( Figure 2, panel B). Notably, 1 sequence clustered with the CA4 reference sequence, indicating that CA4-related viruses are also circulating among HFMD patients in China. These data suggest that shzh00-1 and these Changchun and Hangzhou sequences are likely derived from a common ancestor. New sequences identifi ed in this study have been submitted to GenBank under accession nos. HQ450559-HQ450582.

Conclusions
As many as 40% of cases of HFMD in China have been attributed to CA16 infection on the basis of partial viral genome determination (11). In the current study, we demonstrated that circulating CA16 viruses in China are actually complex recombinant viruses involving multiple type A HEVs, including CA4, CA16, and EV71 ( Figure 1). The 5′ UTR region (207-647 bp) of these viruses had the highest similarity to CA4. Most of the P1 region resembled that of the prototype CA16-G10 strain. The nonstructural protein domains (P2 and P3) had a 1.5-kb fragment (4,406-5,854 bp) that was most similar to EV71A. Several regions of shzh00-1 remain unclassifi ed, including part of 2A, 2B, part of 2C, and 3D.
Over the past 30 years, numerous large outbreaks of CA16-associated HFMD, along with outbreaks caused by EV71, have been reported in China. However, full molecular characterization of circulating CA16 viruses in China had not been conducted. The current study provides evidence that circulating recombinant forms of the CA16-related viruses are prevalent among HFMD patients throughout China (Figure 2, panel C). The origin, including the place and date, of the current recombinant CA16 viruses is not clear. The exact parental viral strains involved in the generation of CA16 recombinant viruses are also unknown. Some of the parental virus strains of China CA16 could have become extinct or are yet to be discovered. Notably, CA4 was detected in 1 of our samples from Zhejiang Province (central China) and has also been reported in Gansu Province (northwestern China) (15). The identifi cation of CA16-related HFMD, based mainly on VP1 sequences, has been widely reported in many parts of China, including Guangdong, Fujian, Jiangsu, and Inner Mongolia Provinces, and Beijing and Shanghai. Unfortunately, the sequences of other parts of these viral genomes, especially the regions spanning the recombination site, have not been determined. Further detailed characterization of HEV sequences from HFMD patients is still needed, but the information obtained thus far has implications for addressing the future emergence of new pathogenic HEVs and for vaccine development to manage the increasing prevalence of HFMD.