Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, is normally mild but can have life-threatening manifestations. It can be caused by enteroviruses, particularly Coxsackieviruses and human enterovirus 71 (HEV71) with highly variable clinical manifestations. In the spring of 2008, a large, unprecedented HFMD outbreak in Fuyang city of Anhui province in the central part of southeastern China resulted in a high aggregation of fatal cases. In this study, epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. Of the 6,049 cases reported between 1 March and 9 May of 2008, 3023 (50%) were hospitalized, 353 (5.8%) were severe and 22 (0.36%) were fatal. HEV71 was confirmed as the etiological pathogen of the outbreak. Phylogenetic analyses of entire VP1 capsid protein sequence of 45 Fuyang HEV71 isolates showed that they belong to C4a cluster of the C4 subgenotype. In addition, genetic recombinations were found in the 3D region (RNA-dependent RNA polymerase, a major component of the viral replication complex of the genome) between the Fuyang HEV71 strain and Coxsackievirus A16 (CV-A16), resulting in a recombination virus. In conclusion, an emerging recombinant HEV71 was responsible for the HFMD outbreak in Fuyang City of China, 2008.

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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...

Emerging epidemics of hand-foot-and-mouth disease (HFMD) associated with enterovirus 71 (EV71) has become a serious concern in mainland China. It caused 126 and 353 fatalities in 2008 and 2009, respectively. The epidemiologic and pathogenic data of the outbreak collected from national laboratory network and notifiable disease surveillance system. To understand the virological evolution of this emerging outbreak, 326 VP1 gene sequences of EV71 detected in China from 1987 to 2009 were collected for genetic analyses. Evidence from both traditional and molecular epidemiology confirmed that the recent HFMD outbreak was an emerging one caused by EV71 of subgenotype C4. This emerging HFMD outbreak is associated with EV71 of subgenotype C4, circulating persistently in mainland China since 1998, but not attributed to the importation of new genotype. Originating from 1992, subgenotype C4 has been the predominant genotype since 1998 in mainland China, with an evolutionary rate of 4.6∼4.8×10−3 nucleotide substitutions/site/year. The phylogenetic analysis revealed that the majority of the virus during this epidemic was the most recent descendant of subgenotype C4 (clade C4a). It suggests that the evolution might be one of the potential reasons for this native virus to cause the emerging outbreak in China. However, strong negative selective pressure on VP1 protein of EV71 suggested that immune escape might not be the evolving strategy of EV71, predicting a light future for vaccine development. Nonetheless, long-term antigenic and genetic surveillance is still necessary for further understanding.

BackgroundLarge nationwide outbreaks of hand, foot, and mouth disease (HFMD) occurred in China from 2008; most of the cases were in children under 5 years. This study aims to identify the situation of natural human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) infections in children before 2008 in China.ResultsRetrospective seroepidemiologic studies of HEV71 and CVA16 were performed with 900 serum samples collected from children ≤5 years of age in 2005. The samples were collected from 6 different geographical areas (Anhui, Guangdong, Hunan, Xinjiang, Yunnan, and Heilongjiang provinces) in mainland China. Of the 900 samples, 288 were positive for HEV71; the total positive rate was 32.0% and the geometric mean titer (GMT) was 1:8.5. Guangdong (43.7% and 1:10.8), Xinjiang (45.4% and 1:11.1), and Yunnan (43.4% and 1:12.0) provinces had relatively high rates of infection, while Heilongjiang province (8.1% and 1:4.9) had the lowest rate of infection. On the other hand, 390 samples were positive for CVA16; the total positive rate was 43.4% and the GMT was 1:9.5. Anhui (62.2% and 1:16.0) and Hunan (61.1% and 1:23.1) had relatively high rates, while Heilongjiang (8.0% and 1:4.6) had the lowest rate. Although there is a geographical difference in HEV71 and CVA16 infections, low neutralizing antibody positive rate and titer of both viruses were found in all 6 provinces.ConclusionsThis report confirmed that HEV71 and CVA16 had wildly circulated in a couple provinces in China before the large-scale outbreaks from 2008. This finding also suggests that public health measures to control the spread of HEV71 and CVA16 should be devised according to the different regional characteristics.