Effect of Toll Like Receptor Ligands on Innate Immunity against Newcastle Disease Virus in Embryonated Chicken Eggs

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Date
2019
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LUVAS
Abstract
Newcastle Disease virus (NDV) continues to cause serious economic losses in global chicken production. The objectives of the present study were to develop a rapid and efficient method for determining pathotype of NDV from field specimens by using Reverse Transcription polymerase chain reaction (RT-PCR) and in silico restriction endonuclease enzymes analysis (REA) and to employ the in ovo model to study the antiviral activities of Toll like receptor TLR ligands. The field samples were investigated for identification, differentiation and pathotyping of NDV by amplification of partial F gene of 356 base pairs (bp) using RT-PCR followed by pathotyping by in silico REA using HhaI and BglI enzymes. To differentiate virulent and avirulent NDV, the RT-PCR product of F gene in size of 356 (bp) was analyzed by HhaI and BglI restriction enzymes. Out of one hundred twenty field samples examined, nineteen were found positive for NDV and among these nineteen positive samples fourteen samples were lentogenic, two samples were mesogenic and one sample was velogenic NDV.NDV was isolated from one of the sample using SPF ECE after two blind passages, confirmed as NDV by RT-PCR, followed by sequence analysis and HI test. To determine the antiviral response, TLR ligands (Poly I/C, MPLA and CpG-ODN) were given in ovo at the appropriate dose and time, and their effect on reduction of NDV titres in SPF ECE was observed. Moreover, the mechanisms involved in the TLR-mediated antiviral responses in the chorioallantoic membrane (CAM) were also investigated. SPF ECE (9-10 day old) were treated with TLR3, 4 and 21 ligands using different doses and times pre- and post- NDV infection. Six time intervals were analysed viz. three (36 h, 24 h and 12 h) pre-infection, at the time of infection, two (12 h and 24 h) post NDV infection. It was observed that treatment of SPF ECE with TLR ligands reduced NDV replication as determined by qPCR. Further comparative expression study of immune-stimulatory genes like interferon (IFN)-γ IFN-α, IFN-β, IL-1β, iNOS and OAS in the CAM, was done in treated SPF ECEs at different time interval viz. 4h, 8h and 16h post treatment with different TLR ligands The timing and dosage of TLR ligands administration had significant impacts on the outcome of the treated eggs. The present study demonstrated that the in ovo route may be employed to determine the antiviral characteristics of TLR ligands against NDV. These results suggest that treatment with the intermediate dose of MPLA (2 μg/SPF ECE), CpG-ODN 1826 (4 μg/SPF ECE), and Poly I/C (100 μg/SPF ECE) significantly reduced the virus titre as post treatment mortality of embryo was found to be low. Treatment with MPLA significantly decreased the virus titre at each time interval except 36 h prior to infection while poly I/C reduced the virus titre significantly in early treatment, and CpG-ODN was most effective when used 12 h prior to treatment. The cytokine expression study showed significant varied fold change in the titre of IFN genes (IFN-γ IFN-α, IFN-β) as well as IFN stimulating genes (IL-1β, iNOS, OAS) which may have important role in decreasing virus titre after treatment with TLR ligands.
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