Detection And Analysis Of Avian Bronchitis Virus Isolates In Shanghai And RNA Interference Effect

Avian Infectious Bronthitis virus(IBV) can cause pathological changes of respiratory tract, abdominal reproductive system lesion, and proventriculusopathogenic, nephripathogenic lesions. IBV genome was translated into six structural and non-structural proteins. Spike protein(S) which was located on the membrane of the virion was the main immuno-protein. The pro-protein of S was cleaved into two subunit, S1 and S2. S2 was targeted onto the membrane by hydrophobic bond to form the bond region and S1 was connected with S2 by non bi- bond and formed the ball region of S protein.S was the main protein for determining the tropism and sero-type of the virus, especially S1 fragment was the one of recognizing and binding to the receptor on the cells. However, S1 can only promote the infection and fused with the cell depending on the collaboration of S2 subunit. Many researches were concerned on the importance of S1 in infection, while few is about S2. In the present study, a RT-PCR method was developed for amplification S2 fragment of isolates in Shanghai in order to investigate its properties and its relationship with S1 phylogenic evolution. This may provide a basis for understanding on pathogenic mechanism of the virus.IBV is of highly mutation and share many sero-types. Few or no protection was observed among different sero-type. It is important and necessary to find a new way for prophylaxis and therapy of IBV infection.RNA interference (RNAi) is a newly developed method for antiviral therapy. It depends on the specific double-stranded RNA, which can degrade complementary target sequence of virus RNA(mRNA). Though siRNA confers transient interference of gene expression in a sequence-specific manner, they represent a previously unrecognized method that may have significant medical application on antiviral therapy, especially some of the important disease with no good method for therapy. In short period, RNAi was successful to interfering many virus' multiplication in laboratory, such as, Hepatitis B, Hepatitis C,Poliovirus, Rotavirus, Respiratory Syncytial Virus(RSV), Influenza virus, Papillomavirus, Sever Acute Respiratory Symptom virus(SARS), etc. However, no research has been conducted on the RNAi effect of avian virus infection. This study was designed to investigate RNAi effect on IBV infection in both cell line and chicken embryoned eggs, aiming to explore ways for IBV therapy.1. Isolation of IBV in Shanghai and phylogenic analysis of S2 fragmentKidney and/or trachea from the chickens suspicious of IBV infection were collected and inoculated into 9 day-old SPF chicken embryos after treatment with antibiotics. By three passages in chicken embryos, RT-PCR was conducted for nuclear acid detection. IBV were further confirmed by sequencing and electro-microscoping. Ten viruses were isolated among 12 chicken farming, which was from 13 day-old to the adults.A pair of primer was designed and synthesized for amplification of IBV S2 gene. Simulated PCR reveal that this pair of primer can amplify all the S2 gene of IBV published at GenBank. Using reference strain H52 and IBV isolate as template, RT-PCR system was established, and the amplified product was 493bp. The sensitivity was 10-50ELD50, and the specificity was 0.1ng. 34 samples suspected to be IBV infection was detected using the method, and 32 were positive. By inserting the fragment into pMD18-T vector, the plasmids were tested by PCR and sequenced, showing the isolates were the nephripathogenic strains.By using MEGA software, phylogenic analysis of IBV S2 gene was carried out. High identity was among the isolates in Shanghai district, and much more difference is seen between the isolates and the ones isolated in other places. Eight sequences of native isolates at GenBank were carried out for analysis. The S2 genes of the epidemic viruses share high identities with each other, while has much more difference with the proventriculopathogenic IBV and other virus cause respiratory symptom. Thus, IBV changed not so much in one area in certain period, but share much differenc...