Inhibition Of Gene Expression And Replication Of Major Hand, Foot, And Mouth Disease Pathogens By RNA Interference

Hand, foot, and mouth disease (HFMD) is a common illness which mainly affects young children (< 5 years of age), resulting in the appearance of vesicular rashes on hands, feet, and buccal mucosa. HFMD usually spontaneously resolves, but severe meningitis, encephalitis, myocarditis and even death can supervene. HFMD can be caused by many causative agents which belong to the family Picornaviridae under the genus Enterovirus, including Coxsackievirus A16, A4, A5, A9, A10, B2, B5, Enterovirus 71 and partial Echoviruses. Of these, human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are two major causative agents of HFMD. EV71 and CA16 infections manifesting as HFMD and herpangina are clinically indistinguishable, but EV71 infection is more frequently associated with aseptic meningitis, encephalitis, poliomyelitis-like paralysis and serious neurological complications and fatalities. CA16-caused HFMD was considered to be mild and harmless due to its relatively benign symptoms. Recent studies showed that CA16 infection is not always a benign infection:it can be associated with serious myocarditis, pericarditis and fatal shock.RNA interference (RNAi) is an evolutionarily conserved mechanism of sequence-specific post-transcriptional gene silence triggered by double-stranded RNA. This process is via a two-step mechanism. Firstly, long dsRNAs are cleaved by a host ribonuclease-III like enzyme (named dicer) into small interfering RNA (siRNAs) of 21-23 nt. These siRNAs are associated with a multiprotein complex known as RNA-induced silencing complex (RISC) and ultimately target homologous mRNA for degradation based on complementary base pairing. In mammalian cells, introduction of 21-23nt siRNAs exhibit an RNAi effect without inducing a nonspecific interferon response. Many studies have shown that RNAi could be used for suppressing gene expression when delivered into mammalian cells in vitro because of the specificity and efficiency of RNAi machinery. Recently, various studies demonstrated that RNAi can also be used for clearing mammalian cells from viral infection, such as influenza A virus, influenza B virus, human immunodeficiency virus type 1 (HIV-1), hepatitis B virus, and coxsackievirus B3. There has been considerable interest in the development of siRNA as a possible treatment for HFMD.In this study, initially established and analyzed the entire nucleotide sequence of CA16 strain Shzh05-1, EV71 strain Shzh-98 and EV71 strain Fuyang-0805. The CA16 strain Shzh05-1 was obtained from the Department of Microbiology at Shenzhen Center for Diease Control and Prevention (Shzhen, People’s Republic of China) and progagated in Vero cells. EV71 strain Fuyang-0805 was isolated from pharyngeal swab of a child in the city of Fuyang with a clinical diagnosis of HFMD and propagated in cultured Rhabdomyosarcoma (RD) cells. The complete genome sequence of the above two strains were established by overlap sequenceing and submitted to GenBank (GenBank accession no. EU262658 and FJ439769).For CA16 virus, this study is the first report of siRNAs interfering with CA16 reproduction in cell culture experiments. We designed 30 siRNAs targeting conserved regions between Shzh05-1, Tainan/5079/98, and Shzh00-1 (because of scarce genetics data, the only two Asia strains providing complete nucleotide sequences in NCBI are Tainan/5079/98, and Shzh00-1) and confirmed 13 out of 30 siRNAs could potently inhibit the replication of CA16 in cultured cells. For further preventing escape mutants, we cotransfected these 13 siRNAs targeting different regions of CA16 RNA into cultured cells and compared their inhibitory effect with single transfected siRNAs. The results show that siRNAs transfected by this method could also effectively inhibit the propagation of CA16 though without enhanced inhibitory effect.For EV71 virus, this study is also the first report of siRNAs inhibiting replication of multiple EV 71 China strains in cell culture experiments. To limit escape mutants, all of the siRNAs targets designed in this study were conserved among EV 71 China strains. The three siRNAs targeting conserved regions between Shzh-98 and other strains in China could potently inhibit the replication of the EV 71 strain Shzh-98 in cultured (RD) cells. For further preventing escape mutants, the three siRNAs were mixed of equal quantity (MIX) and then transfected into cells. Co-transfection of these three siRNAs targeting different conserved regions of the EV 71 genome could also effectively inhibit the propagation of the EV 71 strain Shzh-98. The inhibitory effect of MIX was tested in one epidemic strain (Fuyang-0805) isolated from a child in the city of Fuyang with a clinical diagnosis of HFMD in 2008. The MIX of the three siRNAs could also effectively limit the propagation of clinical isolated strain Fuyang-0805. Nucleotide sequence analyses of the three siRNAs indicated that the mixture may have a much broader antiviral effect in other EV 71 strains besides China strains because of the extremely conserved targets of the three siRNAs among EV 71 strains circulating throughout AsiaIn conclusion, this rapid identification of active siRNAs could be very useful for combating the sudden emergence of HFMD.