Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge

Krister Blodörn, 2011, Master of Science in Veterinary Medicine, Swedish University of Agricultural Sciences (SLU).
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(A) Amplification chart showing threshold and (B) standard curve for the TLR4 standard dilution series in the bovine TLR4 mRNA qPCR assay.
(A) Amplification chart showing threshold and (B) standard curve for the TLR4 standard dilution series in the bovine TLR4 mRNA qPCR assay.
The Bovine respiratory syncytial virus (BRSV) causes bronchiolitis and interstitial pneumonia, predominantly in calves, and is a major cause of bovine respiratory disease worldwide. In humans, BRSV is paralleled by the closely related Human respiratory syncytial virus (HRSV), an important cause of respiratory disease, most severe in infants. The clinical signs and pathology during RSV infection is caused, not only by the direct effects of viral replication, but also by the response of the host immune system. The immunopathology of RSV has long obfuscated our understanding of the disease, and development of effective treatment and vaccines will be very difficult until greater knowledge is gained. One of the components of the immune system that has come into focus in RSV research the last few years, is the Toll-like receptor 4 (TLR4). The TLR4 receptor is well known as the receptor that binds lipopolysaccaride (LPS), and initiates the host response to bacterial infection. Recently, it has been shown that the fusion protein of RSV also interacts with, and up-regulates the expression of, the TLR4 receptor. Whether this has a predominantly protective effect, as would be expected from an immune response, or if it is mainly detrimental to the host, remains to be determined. The objective of this work was to develop an assay for quantification of TLR4 mRNA in clinical samples and to determine if TLR4 mRNA in bronchoalveolar lavage (BAL) cell samples could be used as a marker of protection during experimental BRSV infection. Two one-step quantitative real-time PCR systems were developed and optimized in this work. One for detection of TLR4 mRNA, and the other for detection of the housekeeping gene product 28S rRNA. The assays showed good efficiency as well as intra- and inter-assay reproducibility. Furthermore, BAL cell samples collected during an experimental vaccinal challenge were used to evaluate the level of TLR4 mRNA expression in relation to detected BRSV RNA. When the results were analyzed, it appeared that TLR4 mRNA quantification can not be used as a marker of protection against BRSV infection after previous vaccination.