Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. 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. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. 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. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. 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. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of infectious bronchitis 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. While serotyping by definition requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. 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 mixed isolates. This paper describes a MinION-based AmpSeq method that genetically typed IBV from clinical samples, including samples with multiple isolates. Total RNA was extracted from fifteen tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples.AmpSeq accurately detected and genotyped both IBV lineages in three of five samples containing two IBV lineages. Additionally, one sample contained three IBV lineages, and AmpSeq accurately detected two of the three. Strain identification, including detection of different strains from the same lineage, was also possible with this AmpSeq method. The results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.Infectious bronchitis virus is an enveloped, pleomorphic gammacoronavirus with an unsegmented, single-stranded, positive-sense, 26-27.8 Kb, RNA genome that encodes the nonstructural polyprotein,1a and 1b, and several structural proteins: spike (S), envelop (E), membrane (M) and nucleocapsid (N). 38,41 In addition, two accessory genes, expressing 3a, 3b and 5a and 5b, respectively, have also been described. 6,14,38 The S protein is highly glycosylated and post-translational cleavage leads to two subunits: S1 and S2. 10,48 Besides acting as the viral attachment protein, the S1 protein is a major target of neutralizing antibodies. 7 As with many attachment proteins that are targets of virus-neutralizing antibodies, the S1 subunit is highly diverse with almost 50% of the amino acids differing among IBV serotypes. 2,21,38 Such variation leads to important biological differences between IBV serotypes and the emergence of novel variants. More than 60 serotypes of IBV have been reported, but the most common serotypes of All rights reserved. No reuse allowed without permission.Massachusetts. 16 This genetic diversity leads to emergence of new serotypes and a lack of complete cross-serotype protection by vaccines. 29 The correlation of IBV genotypes and serotypes of IBV has been reported 45 ; therefore, accurate genotypic identification o...

In collaboration with the American College of Veterinary Pathologists