43 Lack of cross-protection among IBV serotypes is a challenge to controlling IB 15 therefore, control of IB relies heavily on serotype-specific live attenuated vaccines. 7 IB is often complicated by secondary bacterial (e.g., Escherichia coli, Mycoplasma sp.) and viral infections (e.g., influenza A virus, Newcastle disease virus, infectious laryngotracheitis virus). 8 Clinical signs of disease are diverse and include respiratory distress, severe ocular discharge, poor body weight gain, decreased egg production, flushing (renal disease), and occasionally mortality in chickens. Infectious bronchitis (IB), which is caused by infectious bronchitis virus (IBV Avian coronavirus), is one of the most important diseases of poultry, causing severe economic losses worldwide. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer’s instructions into a 1D MinION sequencing library, and then sequenced on the MinION. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Infectious bronchitis (IB) causes significant economic losses in the global poultry industry.
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