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

Abstract

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...

Randomly primed, strand-switching, MinION-based sequencing for the detection and characterization of cultured RNA viruses

“…Illumina sequencing was completed for full-length sequences of the lineage-typing regions for swine IAV and resulted in ≥99.9% pairwise identity with the full-length CDS from MinION sequencing. The high pairwise identity between the Sanger or Illumina and MinION sequences is consistent with previous results using MinION sequencing, 6,7 which demonstrated the ability to obtain accurate MinION sequences by increasing depth of coverage. 20,25 Sanger sequencing and BLASTn results for the 5’-UTR for BVDV and VP2 for EHDV matched the MinION lineage-typing region BLASTn results.…”

“…Once the viral species was identified, custom lineage-typing Centrifuge indices were also built to classify beyond the viral species and assess for the possibility of mixed infections of the same virus species, as described previously for IBV. 7 Briefly, for each main lineage per virus, one complete sequence of the lineage-typing region was selected and then combined with the respective genome of the cell line. One sequence per lineage is required because some lineages are overrepresented in GenBank and, as a result of the scoring system in Centrifuge, these overrepresented lineages would be unequally weighted.…”

Randomly primed, strand-switching MinION-based sequencing for the detection and characterization of cultured RNA viruses

“…Sanger sequencing was performed on partial lineage-typing sequences for BVDV, CDV, and EHDV and compared with the full-length lineage-typing regions obtained from MinION sequencing, resulting in 100.0% pairwise identity. The 100% pairwise identity between the Sanger and MinION sequences is consistent with previous results using MinION sequencing, 2,25 that demonstrated the ability to obtain accurate MinION sequences by increasing depth of coverage. 47,48 Sanger sequencing and BLASTn results for the 5□ UTR for BVDV and VP2 for EHDV matched the MinION lineage-typing region BLASTn results.…”

“…Once the viral species was identified, custom lineage-typing Centrifuge indices were also built to further classify beyond viral species and assess for the possibility of mixed infections of the same virus species, as previously described for IBV. 25 Briefly, for each main lineage per virus, one complete sequence of the lineage-typing region was selected and then combined with the respective genome of the cell line. Indices were constructed using the following: 20 N-terminal protease fragment ( N pro ) sequences for BVDV 26 with the bovine genome (GCF_002263795.1_ARS-UCD1.2), 13 hemagglutinin ( H ) gene sequences for CDV 27 with the African green monkey genome (GCF_000409795_Chlorocebus_sabeus_1.1), nine VP2 sequences for EHDV 28 with the bovine genome (GCF_002263795.1_ARS-UCD1.2), 32 spike 1 ( S1 ) sequences for IBV 29 with the chicken genome (GCF_000002315.4_Gallus_gallus-5.0), 18 hemagglutinin ( HA ) sequences and 11 neuraminidase ( NA ) sequences for IAV 30 with the canine genome (GCF_000002285.3_CanFam3.1) or chicken genome (GCF_000002315.6_GRCg6a), and 18 ORF5 sequences for PRRSV 31 with the swine genome (GCF_000003025.6_Sscrofa11.1) (Supplementary Table 2).…”

Special issue on applied next-generation sequencing in veterinary diagnostic laboratories

“…This issue of the Journal of Veterinary Diagnostic Investigation , the Special issue on Applied Next-Generation Sequencing , includes a series of 10 papers that represent the application of NGS to veterinary testing. These range from the most common, genome sequencing, using PCR and viral culture for enrichment, to detect and/or subtype various viral pathogens, 3,5,6,9,11 to less but increasingly common applications such as shotgun metagenomics for virus detection in clinical samples. 10 Deep amplicon sequencing is presented for targeted detection of multiple pathogens with a single NGS assay.…”