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The Experimental Research of RNAi Technology Inhibiting Respiratory Syncytial Virus

Author: LiNa
Tutor: ZhouYanMing;CuiYuXia
School: Zunyi Medical College,
Course: Pediatrics
Keywords: Respiratory syncytial virus Small interfering RNA Antiviral activity Airway inflammation Mice
CLC: R725.6
Type: Master's thesis
Year: 2011
Downloads: 8
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Abstract


Respiratory syncytial virus (RSV)is one of the most important lower repiratory pathogens in infants and young children,especially causing pneumonia and bron chiolitis. It is one of main reasons that caused the abby to be in hospital.There is no reliable vaccine or antiviral drug against RSV at present. RNA interference (RNAi) is the procss of depleting RNA targets by the use of short double-stranded homologous mRNA,and has been widely used anti-tumor, anti-viral therapy.Objective:1. To explore the inflammation and immune status of respiratory syncytial virus (RSV)-infected BALB/c mice.2.To study the effect of siRNA(small interference RNA,siRNA)7816 on inhibiting RSV in BALB/c miceMethods:1.Balb/c mice were randomly divided into two groups(n=6), and BALB/c mice were infected with RSV via intranasal instillation of RSV suspension, Airway inflammation and immunology was revealed by Pulmonary histopathology, the levels of IFN-γ, IL-12 and IL-10 in bronchoalveolar lavage fluid (BALF) detected by ELISA and white blood cell counts in BALF counted by microscope.2.Balb/c mice were randomly divided into six groups(n=6),and then infected mice were treated at 24 hours and 48 hours post inoculation with various dose specific siRNA7816 targeting RSV-M2 gene. The mouse were killed on day 3 post-infection (PID3), and had their lung tissue removed in super clean bench. The distribution of fluorescein-labeled siRNA7816 was then evaluated in the frozen section of the lung tissue. Pulmonary viral titers of the Lung tissue were assayed by plaque forming experiment and plaque forming inhibition rate of each treatment group were calculated. Pulmonary RSV-M2 expression was analyzed by RT-PCR. Pulmonary viral antigen was detected by immuno-histochemisty.Airway inflammation was revealed by Pulmonary histopathology, the levels of IFN-γ，IL-12 and IL-10 in bronchoalveolar lavage fluid (BALF) detected by ELISA and white blood cell counts in BALF counted by microscope.Result:1. Following RSV infection, histopathology showed an inflammatory reaction in the lung tissue of BALB/c mice. A significant increase in leukocytes count was observed in BALF (P<0.05). especially lymphocytes (P<0.05). The levels of IFN-y, IL-12 and IL-10 were also increased (P<0.05).2.Following RSV infection, virus could be isolated from the mice lung tissue, and viral titers and the number of the plaques were increased; RT-PCR showed an elevated RSV-M2 mRNA level; and immunohisto-chemistry revealed an increased mean optical density of RSV antigen in the lung tissue.siRNA administered intranasally were well distributed in the lung tissue of BALB/c mice. When treating the BALB/c mice with 0.5nM、1.0nM and 2.0nM siRNA7816, plaque forming inhibitory rates were 55.48%、75.74%、84.96% respectively, and RSV-M2 mRNA level decreased by 28.98%、54.40%、76.79%respectively, and mean optical denity of RSV antigen declined by 13.43%、22.37%和28.93% respectively.Obvious alleviation of the pathological damage to the lung was observed following siRNA7816 medication. The leukocytes count, the percentage of lymphocytes and the levels of IFN-γ, IL-12 and IL-10 in BALF showed were decreased.The differences were significant as compared with the infection control group (P<0.05). Comparised between the treatment groups,2.0 nMsiRNA therapeutic dose had the better effect.The difference between the negative control group and the infection control group had no significance (P>0.05).Conclusion:1.Intranasally administration of RSV to the BALB/c mice resulted in replication of the virus.2.After RSV infection, causing the Lung tissue and airway inflammation in BALB/c mice. siRNA7816 could suppress inflammation reaction.3. siRNA7816 effectively inhibited RSV replication in the lung tissue.4.siRNA7816 induced specific inhibition of the virus replication.

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