Partial Gene Sequence Analysis And Immunogenicity Analysis Of Infectious Laryngotracheitis Virus Isolates

AILT (Avian Infectious Laryngotracheitis) is an acute and contact Upper respiratory infections Caused by infectious laryngotracheitis virus. Characterized by dyspnea, cough and cough up exudate containing blood.Larynx, trachea mucosa swelling, bleeding and erosion during necropsy can be found in the necropsy process. In the early stages of the disease,affected cells can form nuclear inclusions. After the disease was first reported in 1925 in the United States, has been all over the world. The disease spread fast, high mortality, incidence and prevalence of many regions in China, endangering the development of the poultry industry. AILT is first infectious diseases through effective vaccine immunization for prevention and control. Commonly used in the prevention and control of ILT vaccine is live attenuated vaccine, but the vaccine can cause latent infections and in the communication process between flocks and flocks, the virus virulence atavistic, poor security, easy to cause disease so there is a big application restrictions. This experiment mainly on three chicken infectious laryngotracheitis virus isolates of some genes and immunogenicity is analyzed, so as to ILTV vaccine research provide some reference.First of all, the isolation and preliminary identification of three suspected chicken infectious laryngotracheitis virus Inoculate SPF chicken embryos by chicken embryos villi allantois membrane (CAM) inoculation and observe its culture characteristics, collect villi allantois membrane and allantoic fluid, extraction of genomic DNA.Then, four pairs of primers were designed according to the conserved sequences of ILTV gB、gC、gD and gG genes published in GenBank to amplify specific fragments for sequencing.Sequence analysis showed that the nucleotide sequence and published in GenBank sequence similarity were over 99%, there is a high homology.Second immunogenicity analysis about three strains of the virus, the strains infection of villi allantois membrane after freezing and thawing and allantoic fluid mixing the laryngeal inoculation test on chicken. Meanwhile ILTV commodity attenuated vaccine immune experiment chicken through some eye drops of nose, and set the immune control chicken group. After vaccination,7 d,14 d and 21 d separation blood lymphocytes isolated for RNA extraction and reverse transcription, using relative quantitative PCR (select β-actin gene as an internal, using SYBR Green dye method) to detect IL-2, IL-6 expression level changes. Establish standard curve and analysis of the melting curve. And 21 days after vaccination, infection with virulent ILTV all test chickens at 28d,35d blood to detect changes in the expression of IL-2, IL-6’s. The results show CT values of each gene detection range around 14-33, have good linear relation, R2 were greater than 0.990, the melting curve analysis showed that the product for the specific unimodal. And these three strains of the virus IL-2, IL-6 expression levels compared to the control group, commercialization attenuated vaccine group were significantly different (P<0.05). Explain JS1, JS2, JS3 ILTV virus isolates resistant to ILTV virulent attacks as attenuated vaccines, effectiveness even better than the merchandise attenuated vaccine to protect chickens resistant to virulent attacks.