Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Salman L. Butt; Eric C. Erwood; Jian Zhang; Holly S. Sellers; Kelsey Young; Kevin K. Lahmers; James B. Stanton

The proper diagnosis of IBV as the cause of clinical respiratory disease is contingent on virus typing and differentiating live vaccine viruses from field strains. 11 Previously, it was reported that IBV genotypes correlate well with the serotypes of IBV 45 ; therefore, accurate genotypic identification of IBV will be useful to diagnose vaccine and variant viruses in clinical samples. The rapid pan-IBV and serotype specific RT-qPCR assays 4,23,36 have been used for serotype identification; however, positive results from RT-qPCR is insufficient to determine the IBV genotype and obtain isolate-level resolution of IBV; thus, sequence analysis of IBV-S1 gene is required. Previously, use of partial S1 gene sequences (450 bp) to type IBV has been described. 29 However, increasing the length of sequenced S1 gene (~1620 bp) results in more data to be used for genotyping as more of the hypervariable region is covered. 17, 27, 45 For example, the AmpSeq protocol was able to differentiate two genetically very similar (99.5% at S1 gene of IBV genome) but serotypically different GIV-L1 isolates, DE072 and GA98, within a single sample. Thus, the detection of highly diverse IBV genotypes, via the S1 subunit, with a single and rapid sequencing protocol is desirable and the ability to detect multiple isolates in a single sample would improve IBV detection in clinical samples. All rights reserved. No reuse allowed without permission.