Genotyping of Infectious Laryngotracheitis Virus (ILTV) Isolates from Western Canadian Provinces of Alberta and British Columbia Based on Partial Open Reading Frame (ORF) a and b

Abstract

Infectious laryngotracheitis virus (ILTV) causes an acute upper respiratory disease in chickens called infectious laryngotracheitis (ILT). Live attenuated vaccines are effective in disease control; however, they have residual virulence, which makes them able to replicate, cause disease and revert to the original virulent form. Information is scarce on the molecular nature of ILTV that is linked to ILT in Canada. This study aims to determine whether isolates originating from ILT cases in Western Canada are a wild type or vaccine origin. Samples submitted for the diagnosis of ILT between 2009-2018 were obtained from Alberta (AB, n = 46) and British Columbia (BC, n = 9). For genotyping, a Sanger sequencing of open reading frame (ORF) a and b was used. A total of 27 from AB, and 5 from BC samples yielded a fragment of 1751 base pairs (bp). Three of the BC samples classified as group IV (CEO vaccine strains) and 2 as group V (CEO revertant). Of the AB samples, 22 samples clustered with group V, 3 with group VI (wild type), and 2 with group VII, VIII, and IX (wild type). Overall, 17 non-synonymous single nucleotide polymorphisms (SNPs) were detected. Further studies are underway to ascertain the virulence and transmission potential of these isolates.

Keywords: Canada; Sanger sequencing; genotyping; infectious laryngotracheitis; live attenuated vaccine.

Figure 1

Visualization of polymerase chain reaction (PCR) product run on a 1% agarose gel of PCR targeting open reading frame (ORF) a and b (USDA reference genome coordinates 22,364–23,364). The amplicon size is 1000 bp. The DNA ladder used was 1 kilo base pairs plus (kb+). Lane 1 is the DNA ladder. The lane labeled 2 is a negative control. Lane labeled as 3 is a known positive sample to an infectious laryngotracheitis virus (ILTV).

Figure 2

Alignment of the nucleotide sequences of 32 Canadian ILTV strains and 34 ILTV reference strains using MUltiple Sequence Comparison by Log-Expectation (MUSCLE) and Geneious software package. Vertical lines indicate single nucleotide polymorphism (SNP) positions. Samples are color-coded as the phylogenetic tree.

Figure 3

Alignment of the amino acid sequence of 32 Canadian ILTV strains and 36 ILTV reference strains using MUSCLE and Geneious software package. Vertical lines indicate nucleotide change positions. Samples are color-coded as the phylogenetic tree.

Figure 4

PHYML phylogenetic tree with nucleotide sequences of 66 partial ORF a and b of ILTV. The cladogram includes 34 ILTV reference strains downloaded from the GenBank and 32 ILTV Canadian strains characterized in the current study in bold (AB = 27, BC = 5). The genotype nomenclature used is based on [34]. Geneious version 10.0 was created by Biomatters. Available from http://www.geneious.com.