Isolation and Molecular Characterization of Avian Infectious Bronchitis Virus Based on S1 Gene and Effect of TLR Ligands on Virus Replication and Cytokine Expression in Embryonated Chicken Eggs
Abstract
Infectious bronchitis (IB) is an acute, highly contagious and economically important disease of
chickens. In the present study, isolation of IBV virus and sequencing of S1 (partial) gene was carried
out. An in ovo model was also employed to study the antiviral activity of TLR ligands (Pam3CSK4,
LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing
of TLR ligands may reduce viral load of IBV in Specific Pathogen Free (SPF) embryonated chicken
eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in CAM of
ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high,
intermediate and low) of TLR-2 (Pam3CSK4),TLR-4 (LPS) and TLR-21 (CpG ODN). In addition, to
know the timing of TLR ligand treatment, six time interval were analyzedviz36, 24 and 12 h prior to
infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post
IBV infection. For study of relative expression of immunestimulatory gene (IFN-α, IFN-β, IFN-γ, IL-
1β, iNOS and OAS) in CAM, TLR ligands were given through intra allantoic route and CAM were
collected at 4, 8 and 16 h post treatment. To estimate the viral load in kindeys and trachea of embryos,
18 day old ECEs were treated with optimum dose of TLR ligands before IBV infection. Out of 105
samples examined, four samples/isolates demonstrated 99-100% sequence homology with the vaccine
strain 4/91.The intermediate dose of all the three TLR ligands significantly reduced virus titers and
used for further study. However, the LPS reduced virus titer pre- and post-IBV infection but
Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands
induced interferon, proinflammatory cytokine and other antiviral genes in CAM. These TLR ligands
significantly reduced the viral load in kidneys and trachea of ECEs. The present study points toward
novel opportunities for rational design of LPS as immunostimulatory agent in chickens with special
reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance
resistance against viral infection in neonatal chicken and that these TLR ligands may contribute
towards the development of more effective and safer vaccines including in ovo vaccine. Further studies
are required to confirm the dose and time of treatment with different TLR ligands in SPF as well as
commercial birds
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