Development and application of high-resolution melting analysis for the classification of infectious laryngotracheitis virus strains and detection of recombinant progeny

Omid Fakhri,Sang-Won Lee,Amir H Noormohammadi,Carol A Hartley,Glenn F Browning,Joanne M Devlin

doi:10.1007/s00705-018-4086-1

Abstract

Live attenuated vaccines against infectious laryngotracheitis virus (ILTV) are widely used in the poultry industry to control disease and help prevent economic losses. Molecular epidemiological studies of currently circulating strains of ILTV within poultry flocks in Australia have demonstrated the presence of highly virulent viruses generated by genomic recombination events between vaccine strains. In this study, high-resolution melting (HRM) analysis was used to develop a tool to classify ILTV isolates and to investigate ILTV recombination. The assay was applied to plaque-purified progeny viruses generated after co-infection of chicken embryo kidney (CEK) monolayers with the A20 and Serva ILT vaccine strains and also to viruses isolated from field samples. The results showed that the HRM analysis is a suitable tool for the classification of ILTV isolates and can be used to detect recombination between ILTV vaccine strains in vitro. This method can be used to classify a broad range of ILTV strains to facilitate the classification and genotyping of ILTV and help to further understand recombination in these viruses.

Highlights

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV, species Gallid alphaherpesvirus 1), has an impact on the poultry industry worldwide [1]
Intraspecific genomic recombination between vaccine strains of ILTV has been reported to occur in the field [18] and has been demonstrated for field strains under laboratory conditions [14, 18,19,20]
We aimed to develop a reliable cost- and timeefficient method that can be applied to the classification and genotyping of ILTV strains and the identification of ILTV recombinant viruses

Summary

Introduction

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV, species Gallid alphaherpesvirus 1), has an impact on the poultry industry worldwide [1]. All of the ILTV strains form a single serotype [5], yet have shown a notable level of genotypic variation in different studies (0.1–0.8% nucleotide sequence difference) [6]. A number of different methods have been published to classify these viruses by genotype [7,8,9,10], including polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) [9,10,11,12,13], targeted sequencing [7, 8], and strainspecific fluorescent probe hydrolysis [14]. Multiple regions of the ~150-kbp genome are analyzed in order to more precisely differentiate genotypes. These methods have been used to classify ILT viruses to facilitate epidemiological studies and clinical diagnosis [15]

Objectives

Methods

Results

Conclusion