Determination of the minimum protective dose of a glycoprotein-G-deficient infectious laryngotracheitis virus vaccine delivered via eye-drop to week-old chickens.

Infectious laryngotracheitis (ILT) is an upper respiratory tract disease of chickens that is caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. This disease causes significant economic loses in poultry industries worldwide. Despite widespread use of commercial live attenuated vaccines, many poultry industries continue to experience outbreaks of disease caused by ILTV. Efforts to improve the control of this disease have resulted in the generation of new vaccine candidates, including ILTV mutants deficient in virulence factors. A glycoprotein G deletion mutant vaccine strain of ILTV (ΔgG ILTV), recently licenced as Vaxsafe ILT (Bioproperties Pty Ltd), has been extensively characterised in vitro and in vivo, but the minimum effective dose required to protect inoculated animals has not been determined. This study performed a vaccination and challenge experiment to determine the minimum dose of ΔgG ILTV that, when delivered by eye-drop to seven-day-old specific pathogen-free chickens, would protect the birds from a robust challenge with a virulent field strain of virus (class 9 ILTV). A dose of 10(3.8) plaque forming units was the lowest dose capable of providing a high level of protection against challenge, as measured by clinical signs of disease, tracheal pathology and virus replication after challenge. This study has shown that the ΔgG ILTV vaccine strain is capable of inducing a high level of protection against a virulent field virus at a commercially feasible dose. These results lay the foundations upon which a commercial vaccine can be developed, thereby offering the potential to provide producers with another important tool to help control ILTV

Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host

<div><p>To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following <i>in vivo</i> co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.</p></div

Summary of recombinant and parental virus progeny detected in co-inoculated SPF chickens.

<p>Summary of recombinant and parental virus progeny detected in co-inoculated SPF chickens.</p

Single nucleotide polymorphisms (SNPs) selected within the CSW and V1-99 genomes.

<p>Single nucleotide polymorphisms (SNPs) selected within the CSW and V1-99 genomes.</p

Single nucleotide polymorphism (SNP) genotyping assay.

<p><b>A)</b> Schematic representation of the distribution of the TaqMan probes along the unique long (UL), unique short (US), internal and terminal repeat (IR–TR) regions of the genome of CSW-1 ILTV (grey) and V1-99 ILTV (black). The SNPs targeted are indicated with arrows and were located in the UL46, UL36, UL8, UL0, ICP4 and US3 genes <b>B)</b> Scatter plot results from SNP genotyping assays applied to three 10-fold dilutions of positive control starting from 1x10⁵ genome copy numbers per reaction (pure CSW-1 (<i>y</i> axis as ■) or V1-99 (<i>x</i> axis as ●) ILTV DNA), negative controls (no template and DNA negative extractions as ◆) and mixed controls (CSW-1 and V1-99 ILTV genome copy numbers in ratios of 10:1, 1:1 and 1:10 as ▲) obtained from the SNP genotyping assay platform within the Stratagene Mx3000P QPCR 4.1v software with measurements of fluorescence for Orange 560 (<i>x</i>-axis) and FAM (<i>y</i>-axis) dyes labeling V1-99 or CSW-1, respectively.</p

Oligonucleotide probes and primers used to target single nucleotide polymorphisms (SNPS) within the CSW-1 and V1-99 ILTV genomes

<p>Oligonucleotide probes and primers used to target single nucleotide polymorphisms (SNPS) within the CSW-1 and V1-99 ILTV genomes</p

Progeny viruses detected four days after co-inoculation of SPF chickens with CSW-1 and V1-99 ILTV.

<p>Results from Taqman SNP genotying assays are shown for 87 viruses isolated and plaque purified from five SPF chickens (A to E) co-inoculated with CSW-1 and V1-99 ILTV. Each row corresponds to an isolated virus, with each SNP represented as CSW-1 origin (grey) or V1-99 (black). Each recombination pattern was given a unique genotype code (last column).</p

Growth and entry kinetic for the parent viruses, CSW-1 and V1-99 ILTV.

<p><b>A)</b> Entry kinetics of CSW-1 and V1-99. LMH cells were infected with virus, after which they were overlaid with methylcellulose medium and incubated at 37°C. Entry at different time points was calculated by comparing the number of plaques formed after inoculation as percentage of plaques formed after an inoculation period of 60 minutes. This experiment was performed 3 times independently. Mean results are shown. Error bars represent the standard deviation. <b>B)</b> Growth kinetics of CSW-1 and V1-99. LMH cells were inoculated in triplicate at a multiplicity of infection (MOI) of 0.002. At 24 hours intervals virus genome concentration in the cell-free supernatant was determined by qPCR. Error bars represent standard deviations and asterisks indicate values that were significantly different (<i>p</i> < 0.05 Student t test).</p