| Hand, foot and mouth disease is one of the statutory infectious diseases of class C in our country. It has become an important infectious disease with the highest morbidity and mortality among class C infectious diseases and it threats the health of infants and public. Enterovirus 71, coxasckievirus A and coxasckievirus B have been reported to be pathogens of hand, foot and mouth disease(HFMD) while EV71 and CVA16 are the two most popular genotypes. Besides herpes and fever observed in hand, Foot and mouth, aseptic meningitis, brainstem encephalitis, neurogenic pulmonary edema, myocarditis, acute flaccid paralysis may either occurs. Especially severe symptoms are easy to develop rapidly and result in death. Infants are susceptible to EV71 virus, especially the children under the age of six. At present, there is no specific clinical medicine against EV71 virus except symptomatic treatment. Lack of animal model infected with EV71 makes it urgent to find drugs and antiviral targets against EV71.Objective Suckling mice model infected with EV71 was established to provide information for the establishment of EV71 mice model and technical support for the antiviral drugs screening and activity evaluation;This study is to find host mi RNAs associated with EV71 virus infection to provide not only a deeper understanding of the host antiviral defense mechanisms, but also lay foundation to look for gene medicine and target gene anti-EV71 by bioinformatics; To study the inhibitory action and mechanism of FNC in RD and Vero cells infected with EV71 to provide lead compound or candidate drugs for prevent or treatment against EV71.Methods 1) Mice model infected with EV71 was generated after eight consecutive passages of the parental EV71 strain in mice. Virus virulence was observed by the determination of RNA copy number of different mouse-adapted EV71 strains and survival rate of mice infected with different mouse-adapted EV71 strains. In order to search for mutations, the full genetic sequencing of mouse-adapted EV71 strain(P8-1J-V) was compared with the original virus. EV71(H) was propagated in Vero cells and the procedure was repeated for 3 passages. Mice model infected with EV71(H) of the third generation was established by intraperitoneally injection of the virus.2) In this study, host mi RNAs closely related to the expression of EV71 virus was predicted and researched firstly by bioinformatics. In order to study the inhibitory effect of mi RNA inhibitor against EV71, q RT-PCR, CPE, plaque, immunofluorescence and western blot were evaluated. 3) Antiviral drugs were screened and selected by CCK8. The cytotoxicity, CPE inhibition, virus titers and VP1 expression of FNC were determined. FNC was added to Vero cells infected with EV71 to study its direct virucidal effect, function on viral life cycle and effect on activity of 3C protein. Virus was passaged for 14 times consecutively and FNC was given to inhibit the virus at the same time. FNC could restrain the 3C protein activity by 3C protein activity detection experiments. Drug resistance experiment was identified by analyzing FNC-resistant EV71 virus. Results 1) Mice model infected with EV71(Br Cr) was established successfully and virus virulence was strengthened. The mortality rates of 7-day-old ICR and BALB/c suckling mice infected with mouse-adapted EV71 strain(P8-1J-V) reached 100%. Bases 1819:C'T, 2428:C'G, 2731: A'G, 5259:G'A mutations were found through the whole genome sequencing. Mice model infected with EV71(H) was established and mortality rate of the 12-day-old ICR suckling mice infected EV71(H) virus was more than 70% no matter virus was diluted five times or not. 2) hsa-mi R-146 a, hsa-mi R-155 and hsa-mi R-210 were associated with EV71 infection. EV71 infection increased the levels of hsa-mi R-146a、hsa-mi R-155 and hsa-mi R-210. The expression of hsa-mi R-146 a, hsa-mi R-155 and hsa-mi R-210 could be inhibited after being treated with corresponding mi RNA inhibitors, while the virus titers, viral copy and expression of VP1 protein decreased. 3) It was found that FNC had the ability to inhibit EV71 and it could inhibit the cytopathy of cells. IC50 of FNC in RD and Vero cells infected with EV71 were(88.40±17.99),(66.07±5.27),(33.76±4.65) and(10.16±2.46),(12.08±2.07),(6.40±2.32) n M respectively. FNC could inhibit virus titers in cell culture supernatant and VP1 expression in cells. It was found that FNC had no direct virucidal effect on EV71. FNC(0.33 g/m L) had weak inhibitory effect of 3C protease activity, but it could significantly inhibit the replication of RD cells infected with EV71. The antiviral effect of FNC was no significantly decrease in RD cells infected with the passage 14 FNC-resistant virus. Conclusions Mice models infected with EV71(Br Cr and H) were established; hsa-mi R-146 a, hsa-mi R-155 and hsa-mi R-210 have negative feedback regulation in the process of EV71 virus replication; FNC had notable anti-EV71 activity and it may inhibit virus replication to achieve its inhibitive effection. |
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