Dataset Information

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 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.

SUBMITTER: Butt SL

PROVIDER: S-EPMC7201198 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Butt Salman L SL Erwood Eric C EC Zhang Jian J Sellers Holly S HS Young Kelsey K Lahmers Kevin K KK Stanton James B JB

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20200305 2

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 <i>S1</i> subunit of the spike gene is cur ...[more]

PMID: 32133932

Similar Datasets

Project description:Infectious bronchitis virus (IBV) is a worldwide prevalent RNA virus that causes highly contagious and economically devastating disease in chicken. The virus exists in many different genetic forms which made the disease control very difficult. The present study describes the development and validation of TaqMan probe-based real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) targeting the S1 coding region of S gene characteristic for the GII-1 lineage (formerly the D1466-like variant) of IBV. These strains are quite different from other European IBV belonging to different lineages of the GI genotype. The developed method was 30-fold more sensitive than used so far for standard nested RT-PCR with detection limit of 56 RNA copies per reaction. The specificity of the assay was also evaluated with a panel of different poultry pathogens. Repeatability and reproducibility of the method was very high with coefficients of variation lower than 4%. One hundred and twenty-seven IBV-positive samples were tested by this method and GII-1 strains were detected in four of them (3·15%) which indicate a decrease in the GII-1 IBV prevalence in Poland. The assay was proven to be a valuable tool for rapid diagnosis of GII-1 lineage of IBV strains and moreover it enabled the monitoring of viral loads which can be used to assess disease progression. SIGNIFICANCE AND IMPACT OF THE STUDY:This study reports a TaqMan probe-based real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for rapid and accurate identification of GII-1 lineage (formerly D1466-like variant) of infectious bronchitis virus (IBV). The assay revealed to be more sensitive than standard nested RT-PCR assay, previously used for this purpose. The developed assay has been tested on numerous field samples and revealed 3·15% prevalence of this lineage of IBV in Polish chicken population. Moreover, this new assay enables the assessment of viral load measurement which might be useful for epidemiology and pathogenesis studies.

Project description:Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns, and characteristics of the Mass-type IBV strains.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets