Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G–Deficient Candidate Vaccine Strain

Abstract

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT.

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

“…The majority of existing ILTV classification systems are based on identifying the sequence variations in representative regions of the genome that are first amplified by PCR. Sequence variation is identified in these regions by a range of methodologies including RFLP [ 9 , 10 ], Sanger sequencing [ 7 , 8 ] or fluorescently labelled TaqMan ® probes [ 14 , 40 , 41 ]. Both the number of regions being analyzed and the method of analysis will influence the precision of the classification and differentiation of ILTV isolates.…”

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

“…Deletion of gG from the ILTV genome alters the immune response to ILTV infection [32], resulting in a more protective, cell-mediated immune response [20]. This vaccine also offers the potential to discriminate between vaccinated birds and birds infected with wildtype viruses using differential PCR [33] or ELISAs [19] in DIVA (differentiation of infected and vaccinated animals) control programmes. Although a number of gene-deleted ILTV strains have been shown to have in vivo phenotypes that may make them suitable for use as vaccines [12–16, 34–41], the ΔgG ILTV vaccine strain is the most extensively investigated of these deletion mutants, providing a high level of confidence in the performance of the vaccine under different conditions.…”

Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis ofTurkey Meningoencephalitis Virus

“…The concept was further expanded for molecular differentiation between vaccine viruses and wild-type strains in the case of Bovine Herpesvirus type 1 (Schynts et al, 1999), Classical Swine Fever (Widen et al, 2014) and the Bluetongoue virus (Van Rijn et al, 2013). Only two studies on DIVA PCR of poultry viruses were published describing a real-time DIVA to differentiate the wild-type ILTV and the vaccine virus in which the glycoprotein G gene was deleted (Shil et al, 2015), and the virulent Marek's disease virus compared to the serotype 1 vaccine, strain Rispens (Baigent et al, 2016). Given the intensive and diverse vaccination in the poultry industry, the present TMEV DIVA system comprises not only a significant diagnostic asset in the diagnosis of turkeys but can also encourage the future development of additional DIVA systems of other poultry viruses.…”

“…DIVA assays were initially developed to identify differential antibodies to avian influenza viruses and vaccines (Suarez, 2012). Subsequently, DIVA assays were developed to provide differential molecular detection assays for vaccine and field viruses for Bluetongue viruses (van Rijn et al, 2013), bovine herpesvirus, type 1 (Schynts et al, 1999), classical swine fever virus (Widen et al, 2014) and the Infectious Laryngotracheitis Virus (ILTV) (Shil et al, 2015).…”