Pathogenicity Of Infectious Bronchitis Virus Guangxi Isolates And Development Of Real-Time RT-PCR Assays For Chicken Cytokines

Infectious bronchitis (IB) is a highly contagious, acute disease of chickens caused by infectious bronchitis virus (IBV), it’s caused huge losses to the global poultry industry. Today, many researches just concentrate on the molecular biology of IBV, it is also necessary to study the pathogenicity, tissue tropism and dynamic changes of virus concentration in vivo after infection of local isolates. The wide application of live and inactivated virus vaccines can’t prevent and control IB completely, it’s a hotspot of research to develop safe and efficient new vaccines. Cytokines, as important immunomodulators, their gene expression are often used to evaluate the effectiveness of new vaccines.Firstly, a real-time quantitative PCR assay with high specificity, sensitivity and reproducibility was developed for the detection of IBV. The amplification efficiency was101.5%and the coefficient of variation of repeated trials was0.69%～2.51%, the minimum detectable concentration was down to7.27×101copies/μLSecondly,4IBV Guangxi representative isolates GX-YL11072(LX-type), GX-GL11079(LX-type), GX-NN1201(HN08-type) and GX-NN09032(new type), which were isolated in recent years were chosen to infect chicks, which were healthy and non-immunized and25birds for each virus were used, with105TOC-ID50/0.2ml of the virus through eyes and nasals drop. Every The signswere observed daily and the trachea, lungs, kidneys, oviducts and cecal tonsil of birds at day post infection (DPI)1,3,7,14,21and28were sampled for the quantification of IBV. The results showed that all the four isolates can cause coughing, sneezing and other respiratory symptoms with latent period of1-4d, the morbidity and mortality was30%-100%and0%-10%. GX-GL11079caused the most severe clinical symptoms and the enlargement of kidneys, deposition of urate in the kidneys; yellow thick mucus was observed in the trachea of chicks challenged with GX-NN09032.Virus quantification of most visceral organs peaked at3DPI in groups infected with GX-NN1201, GX-YL11072, GX-GL11079, while the highest titres appeared at7DPI in the group infected with GX-NN09032; higher IBV titres were detected in group infected with GX-GL11079than those in other three groups; trachea had the highest virus titres and could be detected until21DPI, while in the cecal tonsil, IBV were still detectable at28DPI in all groups. The results showed that these four IBV Guangxi isolates have a wide range of tissue tropism in chicken.Thirdly, the real-time RT-PCR assays for the relative quantification of IFN-γ, IL-4, IL-10and IL-12were developed. The amplification efficiency was98.8%-103.3%with high specificity and reproducibility. The cytokine genes expressions in the spleens of chicks were detected after immunized with attenuated vaccine strain H120. The results showed that IFN-y and IL-12genes expression peaked at7DPI (P<0.01), while IL-4and IL-10genes expression reached the peak at DPI14(P<0.01) with9.3times and3.1times of those of the control group respectively, no significant difference of all the4cytokine genes expression with control group was observed at DPI21.The results of the study demonstrated that a specific and sensitive real-time RT-PCR assay for the detection of IBV was developed.All the4IBV Guangxi representative isolates were pathogenic for chicks, while isolate GX-GL11079had the strongest pathogenicity, and had a wide range of tissue tropism with the trachea was the major target organ.The developed real-time RT-PCR assays for the relative quantification of IFN-y, IL-4, IL-10and IL-12can be used to monitor the cytokines response in vivo.