Isolation and sequencing of field isolates of Avian infectious bronchitis virus in Iraq
Ruya Nabih Mohammed Atta and Dr. Aida Bara Allawe
In this study, sixty samples (Trachea and lung) were collected from different areas in Iraq. The age of chicks ranged (14-25) days. Most samples were collected from broilers. Real-Time reverse transcriptase polymerase chain reaction (RT-PCR) technique was conducted for collected samples to detect Infectious Bronchitis (IB) virus by amplification of matrix gene. Four samples out of sixty were positive by using Real-Time RT-PCR technique. Four samples were from (Karbala and Al-Taji farms). After preparation of positive samples, these samples were grown in chicken embryos by allantoic method inoculation for three passages, and then the three passages of four samples were tested by using Conventional RT-PCR technique by amplification of nucleoprotein (NP) gene which was positive for the presence of IB virus. The product of amplification of the four isolates (locally isolated) was sent by Iraqi Biotechnology Company to Korea (NICEM Company/Seol national University, South Korea) for sequencing. As a result of sequencing of Nucleoprotein gene, the phylogenic tree analysis revealed that the Iraqi isolates were similar to IBV found in Poland KYO47602.1 with idenditity 95% and to Western Africa FN430414.1 with idenditity 92%. Third passage of allantoic fluid was propagated in chicken embryo fibroblast cell culture for three passages. The cytopathic effects (CPE) were noticed by propagation of two isolates (the first isolate from Karbala farms) and other isolate from (Al-Taji farms) while the other two isolates did not induce clear CPE. The CPEs were rounded cells with vacuoles and degeneration of infected cells compared with non-infected cells. The second and the third passages of the two isolates were titrated in chicken embryo fibroblast cell culture to calculate TCID50 using Reed and Muench method. The titer of the first isolated virus was 103 in second passages and then increased to 104 in the third passage, while the titer of the second isolated virus was 102 in the second passage and then increased to 102.5 in the third passage.