| Epidemics of Hand Foot and Mouth Disease (HFMD) occurred in Beijing, Shandong and Shenzhen respectively during 2009. In this paper, research in HFMD was conducted in two areas:On one hand, we focused on the etiology of HFMD in three main areas, including Shandong, Beijing and Shenzhen, where the epidemic of HFMD is more serious. To investigate etiology of HFMD comprehensively, two-step nested RT-PCR system with CODEHOP universal primers was used to screen for Enterovirus in the 347 throat swabs of HFMD patients admitted to hospital from Shandong, Beijing and Shenzhen during 2009. As for positive cases, sequencing of amplification fragments was carried out and serotypes of enteorvirus were determined through BLAST in GenBank. The result is concluded as follow:Enterovirus 71 (EV71) and Coxsackieviruses A16 (CVA16) are still the main pathogens of HFMD in China. Analysis of molecular epidemiology indicate that EV71 C4 subgenotype is still the predominant virus in China and CVA16 has been popular as other strains belonging to B1 subgenotype. Besides EV71 and CVA16, many other Enteroviruses can also cause HFMD, including Coxsackievirus A (CVA), CVA2, CVA4, CVA5, CVA6, CVA10, CVA12, Coxsackie virus B5 and Echovirus 25 (Echo25)。Predominant serotype of Enterovirus is different in three region. EV71 is predominant in Shandong and Beijing and positive rates were 58.8% and 51.4% respectively. CVA16 is predominant in Shenzheng and positive rate is 50%. Non-EV71/CVA16 in different regions have different distributions:CVB5, CVA2 and CVA5 were only detected in patients in Shandong Province; CVA4 and Echo25 were only detected in patients in Shenzhen; CVA6, CVA10, CVA12 are prevalent in Shandong and Beijing. Interestingly, Fifteen (4.3%) mixed infections of two or even three enterovirus were detected in the 347 throat swabs, most of which (13,86.7%) were cases infected by CVA16 and other enteroviruses.In our survey, CVB5, with positive rate as high as 20% and second only to EV71, become another important pathogen of HFMD in Linyi, Shandong. In addition, the number of enterovirus serotype is more than that of other places and provide the basis for recombination within or between serotypes of enterovirus. We analyzed 01/CVB5/SD/CHN/09 (CVB5/09) in a more detail. Phylogenetic tree was constructed based on 5'part of VP1 sequence, it is showed that the two evolutionary topology similar. The global epidemic CVB5 can be divided into four genotypes and CVB5/09 belong to genotype 2, together with Shandong strains in 1999 and Zhejiang strains in 2002 causing aseptic encephalitis. Complete genome sequencing of CVB5/09 showed that Compared with the Currently existing three CVB5 genome (CVB5/Funlkne,CVB5/UK,CVB5/Korea), nucleic acid and amino acid sequence identity was above 80.6% and 95%, respectively. In contrast, compared with other enteroviruses, it was under 80%.In addition, we conducted a whole genome sequencing and annotation of representatives of another three serotypes of enteovirus,01/CVA4/SHZH/CHN/09 (CVA4/09),01/CVA5/SD/CHN/09 (CVA5/09) and 01/CVA10/SD/CHN/09 (CVA10/09) and then analyzed genetic characteristics. The results showed that genome structure of the three strains in 2009 was similar to that of their own prototype. Phylogenetic analysis showed that evolution trees based on VP1 and 3D sequence were different and CVA5/09 and CVA10/09 formed an independent branch together in the latter, which gived us a tip that various serotypes of enterovirus cocirculating in one place may recombinate with each other. Then, similar analysis was conducted on strains isolated in 2009 and their own prototypes using Simplot software. It was showed that CVA5/09 and CVA10/09 may recombinate with eath other.On the other hand, the study on antiviral effect of small interfering RNA inhibiting replication of EV71, which is the major pathogen of HFMD in China, was carried out in vitro. In this study, three pairs of siRNA (Stealth-990 (VP2), Stealth-2570 (VP1) and Stealth-3020 (VP1)), targeting on VP2 and VP1 of genome repectively, were designed and synthesized by chemical method. Using modle of RD cell infected by EV71, the antiviral effect of three siRNA on EV71 was evaluated in the level of mRNA and viral replication in vitro, through determining the sequence copy number by real-time PCR and virus titer in supernatant by TCID50. The results show that three pairs of siRNA can inhibit the replication of EV71 SHZH98 and EV71 Fuyang0805 in vitro effectively and mixed preparation of effective siRNAs can also inhibit the virus replication.In recent years, numerous large outbreaks of HFMD, especially caused by EV71 infection resulting in neurological disease, occurred in China, causing the rapid increase in severe cases. HFMD has become a major public health issue in China. Understanding of etiology of HFMD and treatment of patients with EV71 infection has important social significance for prevention and control of HFMD. |
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