SCAD (Avian Diseases) II

S OF PAPERS 239 101 Genome sequencing and comparative analysis of avian coronavirus. M. W. Jackwood,* E. T. McKinley, J. E. Philips, and S. W. Thor, University of Georgia, Athens. Genome sequencing and comparative genomics can be used to examine past host and tissue tropism shifts to aid in identification of new variant viruses, to identify genetic differences between different pathotypes, and to elucidate the evolution and emergence of avian coronavirus variants. The avian coronaviruses are enveloped gama-coronaviruses that contain a single stranded positive sense RNA genome and are capable of causing disease in chickens, turkeys, pheasants and a variety of other avian species. The most widely studied virus is infectious bronchitis virus (IBV), which causes a highly contagious upper-respiratory tract disease in chickens. Coronaviruses have an enormous capacity for genetic change. Full genome analysis of this virus has revealed recombination events that lead to the emergence of a new enteric disease in turkeys (turkey coronavirus). Sequencing has also identified areas with high mutations within the RNA-dependent RNA-polymerase gene complex, rather than within spike where most of the changes in the genome were thought to occur. Finally, genome sequencing and analysis has lead to the discovery of an enormous amount of recombination within the IBV genome. Recombination events in coronaviruses along with mutations are a method used to develop genetic diversity, which when under selection pressures leads to evolution of the virus. 102 Simultaneous detection and identification of four major serotypes of infectious bronchitis virus using a microsphere-based assay. H. J. Roh*SC and M. W. Jackwood, University of Georgia, Athens. Infectious bronchitis virus (IBV) causes huge economic losses in commercial chickens worldwide. Economic losses are due to poor growth rate, decreased egg production and mortality. Vaccination with attenuated live virus is used to control the disease but due to multiple serotypes and antigenic variants of the virus that do not give cross protection, it is extremely important to choose the right vaccine type to achieve protection. The conventional diagnostic method for detecting and typing IBV is virus isolation followed by the virus neutralization test in embryonated eggs. A common molecular diagnostic test used for detecting and typing the virus is reverse transcriptase-polymerase chain reaction (RT-PCR) followed by nucleotide sequencing. In an effort to improve on those diagnostic tests, a microsphere-based multiplex assay was developed for simultaneous detection and identification of the 4 most common IBV serotypes diagnosed in the USA; Arkansas (Ark), Connecticut (Conn), Delaware (DE) and Massachusetts (Mass). Biotinylated primers were used to amplify the hypervariable region of the spike glycoprotein of IBV. Using serotype-specific probes for each of the 4 serotypes hybridized to microspheres with different spectral addresses, we performed a hybridization reaction with the biotin labeled amplicons. A fluorescent tag bound to the biotin labeled amplicons and the spectral addresses of the microspheres were measured in the Bio-Plex suspension Array System (Bio-Rad, Hercules, CA). Specificity, sensitivity and cross-reactivity were measured. In our preliminary data, specificity of 4 serotype-specific probes showed very low cross reactivity between the serotypes. A multiplexed microsphere assay using all 4 probes detected each serotype specifically. The limit of detection for Ark, Mass, Conn, and DE was 2.47ng, 1.34ng, 1.07ng and 2.14ng respectively. These results demonstrated the possibility that this newly developed microsphere-based assay can be used for rapid and simultaneous detection of different IBV serotypes, although validation of the assay with clinical samples needs to be conducted.