Comparative genome analysis of Korean field strains of infectious laryngotracheitis virus

Abstract

Attenuated live infectious laryngotracheitis (ILT) virus (ILTV) vaccines have been used to prevent and control the outbreak of ILT worldwide. Recent studies using high-throughput sequencing technology have increased the number of complete genome sequences of ILTVs, enabling comparative genome analysis. Although 37 complete genome sequences of ILTV, including vaccine strains, have been reported, the complete genome sequence of any field strain of ILTV in South Korea is yet to be published. In this study, we determined and analyzed the complete genome sequences of three virulent Korean field strains of ILTV (40798/10/Ko, 0206/14/Ko, and 30678/14/Ko). Two of the Korean field strains (40798/10/Ko and 0206/14/Ko) displayed fewer non-synonymous single nucleotide polymorphisms than those of the Serva vaccine strain, indicating that these Korean field strains of ILTV most likely originated from the vaccine strain. The third ILTV strain, 307678/14/Ko, had two regions in the genome showing recombination between the Serva vaccine-like strain and the Australian A20 vaccine-like strain. Comparative genome analysis of ILTV using the Korean field strains with variable virulence can shed light on the recent trend of the emergence of virulent ILTV strains in the field. A few amino acid changes in the genome of ILTV vaccines could enhance the virulence in the vaccine strain, and natural recombination should be considered one of the major risks for the generation of revertant strains of ILTV under field conditions.

Fig 1. Complete genome alignment between the Korean field strains of ILTV and ILTV vaccine strain.

The Serva vaccine was used as a reference. The alignment was performed using MAFFT. Vertical black lines represent single-nucleotide differences, and dashes represent gaps.

Fig 2. Comparison of the size of pocks produced by the Korean field strains and vaccines of ILTV on the CAM.

The mean pock size on CAMs induced by each strain was measured 5 days post inoculation. Significant differences (P-values: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001) were determined by the Newman–Keuls multiple comparisons post-hoc test.

Fig 3. Complete genome alignment between 0206/14/Ko, 40798/10/Ko, and two attenuated Serva vaccine strains.

The Serva vaccine was used as a reference. The alignment was performed using MAFFT. Locations of non-synonymous SNPs in the CDSs between the two Korean strains and vaccine strains are indicated. Vertical black lines represent single-nucleotide differences and dashes represent gaps.

Fig 4. Phylogenetic tree analyses for the 30678/14/Ko strain of ILTV using alignments of the complete genome and sub-regions of the complete genome between ILTV strains.

Multiple genome alignment was performed using MAFFT. Maximum likelihood phylogenetic trees using the T92+G+I model were generated using alignments of the complete genome (a) and three genomic regions showing clustered SNPs in the unique long (b) and the internal repeat and unique short regions (d), which are indicated as shaded boxes in genome alignment, and the middle of the genomic region (c). Five hundred bootstrap replications were used to assess the significance of the tree topology. Bars indicate nucleotide substitutions per site.

Fig 5. Recombination analysis for the 30678/14/Ko strain of ILTV.

Bootscan analyses were performed using SimPlot with 30678/14/Ko as a query. The crossover points of predicting recombination sites are indicated on the alignment of the complete genome sequence without a terminal repeat region with a color-coded dashed line.