Exploration of the neurovirulence of enterovirus 71 with mouse-adapted strains

• Main Authors: Ya-Fang Wang, 王雅芳
• Other Authors: Chun-Keung Yu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/53742531854580087413

博士 === 國立成功大學 === 基礎醫學研究所 === 95 === Enterovirus 71 (EV71), a single positive strand RNA virus that belongs to Picornaviridae, has caused significant morbidity and mortality worldwide since it was first described in 1969 in the United States. EV71 infections usually cause epidemic HFMD and occasionally have been associated with aseptic meningitis, encephalitis and polio-like illness. The outbreak of EV71 in Taiwan in 1998 killed 78 children. It has been suggested that EV71 may become the most important neurotropic enterovirus after the eradication of poliovirus. One of the major barriers to study the pathogenesis of EV71 and vaccine development is the lack of a suitable animal model. In an attempt to establish a murine model of EV71 infection, we found that mouse-adapted EV71 strains, which were isolated from the brain of ICR neonatal mice infected intraperitoneally at the age of day 1, were more virulent than the parental strain. Furthermore, MP4 (the fourth passage of mouse-adapted EV71 strain), in contrast to its parental strain EV71/4643, could orally infect and replicate in the central nervous system of neonatal mice, indicating an increase in neuroinvasiveness of the virus after adaptation. In vitro mouse-adapted EV71 strains exhibited a more rapid growth rate, bigger plaque size, lower temperature-sensitivity and more resistant to heat treatment at 45�aC and acidic condition than parental strain 4643. Oral inoculation of MP4 into 7-day-old mice caused encephalitis, flaccid paralysis and death of mice at 7-9 days post infection. Pathological examination revealed inflammatory cell infiltration and neuron apoptosis in the brain and spinal cord, massive necrosis in the limb muscles and severe lymphocytes depletion in the lymphoid organs. After oral inoculation, there was an early and transient virus replication in the intestines, whereas the spinal cord, brain and muscle became the sites of viral replication during the late phase of the infection. Protection against EV71 challenge was demonstrated following administration of hyper-immune serum after inoculation of the virus. Nucleotide (nt) sequence analysis of the mouse-adapted EV71 strains genome revealed there were thirty-one nucleotide changes after four in vivo passages, resulting in five amino acid (aa) substitutions. Interestingly, MP1, the first passage of the virus, had 29 nt and 4 aa changes with increased resistant phenotypes (rct marker, thermostability and acid resistance) but not in vivo toxicity. Five additional nt changes and three aa substitutions (1 in VP1, E710Q, and 2 in 2A, V964M and A1009V) were occurred in the transition of MP1 to MP2. MP2 and MP3, the second and third passages of parental strain 4643 in vivo, were strongly acid resistance and more thermostable and lethal to 1-day-old mice than the 4643 with a LD50 of 1 log-fold decrease. MP4, the virus strain after the forth passage of 4643, was highly toxic to 1-day-old mice (2 log-fold decrease in LD50) and acquired the ability to invade the central nervous system after oral delivery. The MP4 had a total of 31 nt and 5 aa different than those of 4643, and only one aa different between MP1 and MP4 which located in the VP1 region (E710Q). Studies with the luciferase reporter plasmid demonstrated that the IRES activity of MP4 was higher than parental strain 4643 in both RD cells and SK-N-SH cells. Furthermore, the full-length infectious cDNA of 4643 and MP4 were constructed and used to generate the chimeric viruses. The 4M-1, a cDNA clone after replacing the 5’ terminal 964 nucleotides of MP4 genome with the corresponding region of the 4643 strain, produced MP4-like plaques in RD cells and showed neurovirulence similar to that of MP4 in neonatal mice, but had a kinetic of replication in RD cells similar to 4643. We concluded that, unlike poliovirus, the 5’ UTR of EV71 may be important for viral replication and that its function alone is not sufficient for large plaque phenotype and mouse virulence. We believe that both mouse-adapted EV71 strains and murine model will provide excellent frameworks for examining the pathogenesis of EV71, verifying the virulence determinants of EV71, and developing vaccines and anti-EV71 drugs.