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Abstract
Respiratory viral diseases are common in commercial poultry, and major diseases such as infectious bronchitis (IBV), Newcastle disease (NDV), infectious laryngotracheitis (ILTV), and avian metapneumovirus (AMPV) can severely affect the performance of poultry, causing high costs to the industry resulting in significant economic loss and even trade restraints and embargos on poultry products. Constant surveillance and identification of the etiologic agentwithin an infected poultry flock are crucial, so appropriate countermeasures can be promptly implemented. To aid accurate and rapid diagnosis of respiratory viruses in the field, a panel consisting of ten sets of TaqMan-based quantitative RT-PCR assays that shared the same thermocycling conditions with fast run times was developed and the potential of introducing serotyping ELISA for IBV was examined. Internal positive controls (IPC) were incorporated in five of TaqMan-based quantitative RT-PCR assays, and the analytical sensitivity and specificity of all tests were evaluated rigorously using synthetic DNA standards that wereidentical with the target sequence and by using clinical and biological tissue specimens. All developed IBV screening and serotyping qRT-PCR assays achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of 10 target copies per reaction, amplification efficiencies ranging between 90%-115%. NDV, ILTV, AMPV-A, AMPV-B qRTPCR assays also achieved the same dynamic range and limit of detection, with amplification efficiencies ranging between 86.8%-98.2%. Further validation of specificity using clinical andbiological specimens was also successful and met all the initial requirements. In a separate, but complementary, study, the potential of VSV particles expressing IBV spike proteins as serotyping ELISA antigens was examined. Genes of the IBV spike protein were incorporated into the outer shell of the viral envelope of VSV and the functionality of these VSV particles bearing different IBV spikes (Mass, Ark) as serotyping ELISA antigens was studied. This proof of concept study proved some potential of the IBV spike pseudotyped VSV particles as serotyping antigens. The primary focus of this whole study was the development and provision of a board panel of diagnostic assays and practical concepts that could aid in identifying respiratory viral etiologic agents that are distributed worldwide.