Rapid typing of infectious laryngotracheitis virus directly from tracheal tissues based on next-generation sequencing

Abstract

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.