RNA Interference Research On NP, PA Gene Of H6N2 Subtype Avian Influenza Virus

Avian influenza is the respiratory or systemic infectious disease which is induced by virus from being sticky Section A-type influenza virus, almost all the wild and domestic birds are infected. World Health Organization regulates that the disease must be reported as a national infectious diseases. In 1887, Avian influenza was first reported in domestic birds of Italy. Followed, an outbreak of epidemic throughout the world causing enormous economic losses. Between the beginning of the twentieth century and the late nineteenth century, the disease occurs mainly in Europe, South America, Southeast Asia, some countries in the region and Egypt. Avian influenza as a result of the high mutation rate, in recent years, the number and scale of Avian influenza around the world is expaning and human infection with avian influenza cases have turn up.Faced with the increasingly serious situation, the researchers are working hard to find a new method for control of influenza. The study found that RNAi is the ideal way to used in the prevention and treatment of influenza.RNAi(RNA interference) or PTGS by dsRNA homologous target mRNA resulted in degradation. first, dsRNA was cut into about 21~24 nt fragments by the Dicer enzyme(A-type RNA enzymeâ…¢), and then lead to mRNA degradation.RNAi as a new technology, with high efficiency, sequence-specific features such as identification, can specifically inhibit gene expression, from the source of virus replication Currently, RNAi has a Bright futureIn virus researchIn this paper, H6N2 avian influenza virus was used for RNA interference research and the main content included purification of H6N2 avian influenza virus and determination of virulence, NP, PA gene sequence analysis and RNA interference experiments.SPF chicken embryo were used for the proliferation of the virus.Toxicity of the virus was tested through Half of the amount of infection and Half lethal dose. the virus titer was detected by hemagglutination assayTotal RNA was extracted from purified virus and sequenced NP, PA gene coding region of virus. According to the results of NP and PA sequencing to determine the target site of siRNA interference to NP and PA genes. two pairs of siRNA to NP, PA gene was designed.Chicken embryos were transfected with chemically synthesized siRNA and viruses. the effects of siRNA interference were detected by hemagglutination test and real-time RT-PCR. The results show that siRNA designed could inhibit transcription and expression of target gene. It has varying degrees of inhibition on influenza virus in Chicken embryos. Its also have varying degrees of protection for SPF chicken embryo attacked with influenza virus.SPF chicken embryo fibroblast cells were transfected with Chemical synthesised siRNA, for detection of the specificity and the interference effects of designed siRNA by cytopathic effect, hemagglutination test and real-time RT-PCR. The results show good specificity of siRNA and no effect of inhibition on cells growth. At the same time, the designed siRNA inhibited the target gene transcription and expression, and the replication of influenza virus in chicken embryo fibroblast cells was also inhibited in varying degree.