Survey of enterovirus infections from hand, foot and mouth disease outbreak in china, 2009

Background In China, a rapid expansion of Hand, foot, and mouth disease (HFMD) outbreaks has occurred since 2004 and HFMD has become an important issue for China. However, people are still only concerned with human enterovirus 71(HEV-71) and coxsackie virus A16 (CV-A16). Much of what is known about the other enterovirus infections relies on fractional evidence and old epidemic data, with little knowledge concerning their distribution. To alert potential threatens of the other enteroviruses, our study genetically characterized specimens from different regions of China and yielded novel information concerning the circulating and phylogenetic characteristics of enteroviral strains from HFMD cases. Findings A total of 301 clinical throat swabs were randomly obtained from patients suffering from HFMD from the southern, northern and central regions of China during outbreaks in 2009. 266 of 301 (88.4%) HFMD cases were found positive for HEV and seven genotypes, HEV-71, CV-A16, -B5, -A4, -A6, -A10, and -A12, were detected. Conclusions The HFMD pathogen compositions in the different regions of China were significantly different. HFMD epidemics might persist for a long time in China due to the multiple pathogen compositions, the enteroviral characteristic of recombination and co-infection, the ever-increasing travel and migration and the deficiency of effective vaccine. Our study deserves the attention on HFMD control and vaccine development.

In recent years, the prevalence of HFMD in the Asia-Pacific region, especially in Southeast Asia, has greatly increased. In China, a rapid expansion of HFMD outbreaks has occurred since 2004 [4][5][6][7] Semi-nested RT-PCR on the 5' partial region of VP1 with sense (GCIATGYTIGGIACICAYRT; CCAGCACT-GACAGCAGYNGARAYNGG) and antisense (CICCIG-GIGGIAYRWACAT; TACTGGACCACCTGGNGG-NAYRWACAT) primers was used to type HEV directly from specimens as previously described [8]. Amplified DNA was purified using a commercial procedure (QIAquick Gel Extraction Kit (Cat. No 28704), Qiagen, Valencia, CA) and then sequenced on an ABI3730 automated sequencer (Applied Biosciences, Foster City, CA) using Big Dye reagents (version 3.0). Using the MEGA4 software, partial sequences were aligned with the sequences retrieved from GenBank by using the neighbor-joining method.
All the identified HEV-71 strains belonged to subgenogroup C4 (Figure 1a). High homologous (95.1%-100.0%) was noticed between the isolated stains from three different regions, but lower homologous showed between the new isolated stains and C4 previous strains isolated in 2001 and 2003. Notably, C4 is predominantly responsible for almost all HEV-71 infections in more than 10 years in Mainland China [9]. In contrast, the analysis of recent and previous HEV71 isolates in the Western Pacific Region showed that several subgenogroups, B1, B2, B3, B4, C1, C2, C3 and C4, were cocirculating in Australia, Malaysia, Singapore, Taiwan and Japan, respectively [3,10]. It seemed that the HEV71 strains evolved independently in Mainland China.
All CV-A16 strains evaluated in this study grouped with cluster B1a and B1b with 87.8-100% identity (Figure 1b), consistent with Chinese CV-A16 strains isolated between 1999 and 2008, which were also found in Taiwan, Malaysia, Thailand, Australia, Vietnam, and Saudi Arabia [11]. It indicated that the Chinese CV-A16 strains coevolved and co-circulated with those from surrounding countries. Interestingly, this was very different with Chinese HEV71 (the genetic evolution of Chinese HEV71 didn't share with neighboring countries), although both of them were genetically closely related and shared the similar transmission mode.
CV-B5 was present in 5.  [GenBank: GQ214173 and GU947787] strains were more closely related to those in our study than the earlier Shandong strains and the other Japanese, German and American strains. We found only four sequences that could be compared with our three sequenced CV-A12 isolates, and the 2003 Japanese strains showed the closest relationship with our new CV-A12 strains.

Discussion
The In our study, we detected and analyzed four other HEVs (CV-A4, -A6, -A10, and -A12) that were simultaneously coexisting in China. Although some HFMD outbreaks and sporadic cases have been associated with them, eg., CV-A6 had been identified as the etiologic agent of HFMD outbreak in Finland [2], they only were detected from few HFMD patients in our study. Therefore, their relationship with HFMD may not be identified in China in 2009. However, the coexisting of multiple enteroviruses and the viral ability of co-infection and recombination suggested that, except HEV-71 and CV-A16, other enteroviruses also had the possibility to cause HFMD epidemic in China. It deserves our attention on HFMD control and vaccine development. HFMD epidemics might persist for a long time in China due to the multiple pathogen compositions, the enteroviral characteristic of recombination and co-infection, the ever-increasing travel and migration and the deficiency of effective vaccine.