The diagnosis of virological etiology is critical and challenging at the beginning of the HFMD outbreak. During the Fuyang 2008 outbreak, because the clinical symptoms of HFMD showed atypical manifestations, which was different from common cutaneous lesions, local doctors misdiagnosed HFMD as pneumonia. In addition, the identification of HEV71 infection was hampered by the sudden death of 22 children within 5 days of pulmonary edema or hemorrhage, heart and lung failure. RT-PCR and sequencing were primarily performed to identify the virological etiology of the outbreak. Laboratory testing showed that 39% of the cases were confirmed as HEV71 infection. Together with clinical manifestations and epidemiological data, we concluded that HEV71 was identified as the major etiological pathogen of the Fuyang HFMD outbreak.
In the Fuyang HFMD outbreak, diseases were manifested with cutaneous lesions in mild cases and CNS symptoms in fatal or severe cases. The findings were consistent with studies of the 1997 outbreak in Malaysia and the 1998 outbreak in Taiwan, in which manifestations of HEV71 infections predominate with neurological complications in fatal or severe cases[2, 3]. Analyses of the VP1 sequence showed high homology between severe or fatal cases and mild HFMD cases and no significant nucleotide differences were found. Other groups have also studied the complete genome of HEV71 isolated from fatal and non-fatal HFMD cases and found no genetic differences that could be correlated with the severity of clinical manifestation[12, 13]. Studies on the pathogenic mechanism for HEV71 have pointed to altered innate immunity. An epidemiologic study during the same outbreak implicated treatment of initial fever of HEV71 infections with glucocorticoids, which block the innate immune response, as contributing to the high rate of severe disease. Experimental animal and clinical studies also support this finding indicating that weakened innate immunity is associated with severe disease and death from HEV71 infection[15–17]. This in combination with introduction of the new recombinant virus may help to explain the increased severity of the Fuyang outbreak.
In this study, genetic recombination event were found between the Fuyang HEV71 isolates, including both fatal cases and non-fatal cases, and CV-A16, which was similar to other studies[18, 19]. For enteroviruses and dengue viruses, genetic recombination is a well-known phenomenon and recombination could result in the emergence of viruses with altered pathogenic potentials. Except for the Fuyang HEV71 isolates, genetic recombination was also found in the HEV71 viruses, circulated in mainland of China during 1998-2008. However, it was very difficult to clarify the time of recombination and the role of the HEV71 recombinant in HFMD outbreaks remains unclear.
Previous studies showed that all known HEV71 strains could be divided into three distinct genogroups (A, B, C) and 10 subgenogroups (A, B1-5, C1-4) based on VP1 gene sequences; the subgenotype C4 could be further divided into C4a and C4b clusters. Based on the phylogenetic analyses, Fuyang isolates belonged to C4a cluster of the subgenotype C4 and showed high homology with the isolates circulating in other provinces of the mainland of China between 2007 and 2008, including those from the HFMD outbreak in Linyi city in Shandong province in 2007. The C4 subgenotype of HEV71 was initially identified in Guangdong province in 1998 and has been continuously circulating for at least 10 years, which may reflect the pattern of endemic circulation of subgenotype C4 viruses. Interestingly, C4b viruses were the predominant circulating strain in mainland of China prior to 2004. Since then, however, C4a viruses have become the predominant strains, which have been continuously circulating and causing epidemic in the mainland of China so far .
The subgenotype C4 HEV71 also circulated in neighboring countries and regions, such as, in chronological order, Taiwan, Japan, Vietnam and Thailand; other subgenogroups including B2, B4, B5, C1, C2 co-circulated with C4 between 1990 and 2008[5, 7, 22]. Interestingly, the subgenotype C4 was the only subgenotype found in China since 1998.
A total of 489,073 HFMD cases, including 126 fatal cases, were reported in China in 2008 (data source: NNDRS). Recombinant HEV71 infection may become more serious public health threat for children under the age of 5 in China, because little is known about the genetics and transmission trend of this fasting mutating virus. To tackle the increasing threat, there is an urgent need for establishing an effective HEV71 surveillance system in China and isolating more viruses, so a complete genetic baseline could be set up for the entire country.