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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #358030

Research Project: Genetic and Biological Determinants of Avian Herpesviruses Pathogenicity, Transmission, and Evolution to Inform the Development of Effective Control Strategies

Location: Endemic Poultry Viral Diseases Research

Title: MinION sequencing to genotype US strains of infectious laryngotracheitis virus

item Spatz, Stephen
item GARCIA, MARICARMEN - University Of Georgia
item RIBLET, SYLVA - University Of Georgia
item Ross, Teresa
item VOLKENING, JEREMY - Base2bio
item Taylor, Tonya
item Kim, Taejoong
item Afonso, Claudio

Submitted to: Avian Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2019
Publication Date: 3/11/2019
Publication URL:
Citation: Spatz, S.J., Garcia, M., Riblet, S., Ross, T.A., Volkening, J.D., Taylor, T.L., Kim, T.N., Afonso, C.L. 2019. MinION sequencing to genotype US strains of Infectious Laryngotracheitis Virus. Avian Pathology. 48(3):255-269.

Interpretive Summary: Classification of strains of infectious laryngotracheitis has been problematic due to the large DNA genome of these viruses. Traditionally cataloging viruses is based on slight changes in the bases that comprise the virus's DNA. We have identified these changes by seqeuncing whole genomes of these viruses and used this information to group viruses into genotypes. Initially genotyping was done using DNA sequencing technology (Sanger or didoxy)that dates back to the late 1970s. We have modernized this to use the latest sequencing technology based on Oxford Nanopore technology. This technology reads the electrical resistance as the bases in the DNA molecule transverses a nanopore held within a microchip. The method detailed in the paper will allow practitioners to quickly determine the field strains circulating in a country and track its spread.

Technical Abstract: Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing.