Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus

Abstract

Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR-restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 10(2) or 10(3) plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 10(4) PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain.

Fig 1. Kinetics of virus growth (A) and plaque development (B) of class 2 (open circles) and class 9 (closed circles) ILTV in LMH cells.

(A) LMH cells were inoculated at multiplicity of infection of 0.001 and inocula removed at one hour post-inoculation. Triplicate wells were harvested at 24 hour intervals after infection and virus genome concentrations in the cell-free supernatant determined using qPCR. Error bars indicate standard deviations and asterisks indicate time points at which the mean concentrations of the two viruses were significantly different (P<0.05; Student’s t-test). (B) LMH cells were inoculated with virus, after which they were overlaid with methyl cellulose medium and incubated at 37°C. Nineteen to 26 individual plaques were photographed at each time point, and plaque area measured using Image J, calibrated using a stage micrometer. Plaques induced by each of the viruses differed significantly in mean area at each time point (P < 0.004; Student’s t-test).

Fig 2. Comparison of titres of class 2 and class 9 ILTV in CAM extracts and allantoic fluid.

Virus was inoculated onto the CAMs of embryonated hen eggs (5 x 10³ PFU for each virus), which were then incubated for 6 days. Mean virus titres are shown. Error bars indicate standard deviations.

Fig 3. Entry kinetics of class 2 (open circles) and class 9 (closed circles) ILTV into LMH cells.

Entry at different time points was calculated by comparing the number of plaques formed after inoculation for that period of time to the number of plaques formed after an inoculation period of 60 minutes. The means and standard deviations (error bars) from 3 independent experiments are shown.

Fig 4. Virus shedding in class 2 ILTV (left hand panels) and class 9 ILTV (right hand panels) infected birds as detected by tracheal (A, B, E, F) and conjunctival (C, D, G, H) swabbing.

Swabs were collected from directly inoculated (A-D, solid line) or in-contact (E-H, broken line) birds inoculated with 10² (circles), 10³ (squares) or 10⁴ (triangles) PFU of virus or sterile media (x). Genome concentrations were determined using an ILTV-UL15-specific qPCR. Mean log₁₀ viral genome concentrations for each group are shown, with error bars indicating standard deviations.