The toxic effect of an Enterovirus 71 mutant in mice

• Main Authors: Chun-Ting Chou, 周春婷
• Other Authors: Chun-Keung Yu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/95236807312581711621

碩士 === 國立成功大學 === 微生物暨免疫學研究所 === 91 === Enterovirus 71(EV71), a single positive strand RNA virus, belongs to Picornaviridae. EV71 infection manifests most frequently as the childhood exanthem known as hand-foot-and-mouth disease (HFMD), as well as a number of severe diseases, including encephalitis, aseptic meningitis, pulmonary edema and poliomyelitis paralysis. We have demonstrated that a mouse-adapted enterovirus 71 strain, EV71/4643/MP4 (MP4), but not the parental strain EV71/4643 (4643), could orally infect and induce neurological diseases in 7-day-old ICR mice with a mortality rate of 50~80%. Therefore, the purpose of the present study was to define the virulence of EV71/MP4 strain. MP4 was found to be more virulent than 4643 in 1-day-old mice with an increased mortality and decreased survival time (LD50: 102 pfu/mouse and 104 pfu/mouse, respectively). MP4 had a more rapid growth rate and exhibited a larger plaque size than 4643 in SK-N-SH (human neuroblastoma), CaCo-2 (human colorectal adenocarcinoma), and RD (human rhabdomyosarcoma) cells. Comparison of nucleotide and amino acid sequence revealed 99% identity. Nucleotide (nt) sequence analysis revealed that there were six point mutations on the 5'-untranslated region (UTR) of the other EV71. Comparison of nucleotide sequence of internal ribosome entry site (IRES) with 4643 and MP4, there were three point mutation: nt：307(C to T), nt：498 (A to G), and nt：577(C to T). We found both 4643 and MP4 induced SK-N-SH cells undergo apoptosis at early stage, but induced Caco-2 cells apoptosis at late stage. Our results also indicated MP4 was more virulent than 4643 at 12 hour postinfection. To further compare in vivo virulence of EV71/4643 and EV71/MP4, We orally infected 7-day-old ICR mice with 4643 or MP4. There was no virus detected in 4643-infected mice. However, virus could be isolated from the brain, spinal cord and skeletal muscle of MP4-infectd mice at 3 and 7 day postinfection. These results indicted that MP4, but not 4643 could orally infect 7-day-old ICR mice. Because our results indicated MP4 has more replication rate and neurovirulence than 4643 in vitro and in vivo, we assume that the mutation sites on the IRES of MP4 may play an important role in viral virulence. RAN interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells. We have designed six EV71-specific siRNA-1 to —6, and demonstrated that siRNA-2、siRNA-3 and the combination of siRNA-2 and -3 could effectively protect SK-N-SH cells against 4643 infection. Pre-treatment of SK-N-SH cells with the siRNAs markedly reduced the EV71-induced cytopathic effect and increased cell viability at 24 hours after infection. Thus, the specific intracellular antiviral resistant elicited by siRNA may provide a therapeutic strategy against evterovirus 71.