Development and use of a quantitative real-time PCR assay for rhinovirus serotype-16

This thesis describes the development of a quantitative real-time PCR (qPCR) plasmid standard curve for the measurement of human rhinovirus serotype 16 (HRV16) mRNA. The necessity of developing such an assay was to be able to absolutely quantitate viral strand numbers in primary cultures of human airway epithelium infected with HRV16.;Previously we have shown that squamous cultures of human airway epithelium consistently produced 100 to 1000 fold more virus than mucociliary cultures. We reasoned that this difference could be due to differences in binding, internalization or replication. Levels of ICAM-1, the viral cell surface receptor, were measured by Western blotting and were found to be only four-times higher in squamous than mucociliary cultures. This suggested that differences in binding capacity were not the main reason for the differences in viral production between phenotypes. However to test this hypothesis, we needed to determine the concentration dependence of viral binding. The most sensitive and quantitative approach seemed to be to develop a real-time quantitative PCR assay for viral RNA.;The standard curve for viral RNA was obtained using the plasmid pR16.11, which contains the full-length genome of HRV16. The curve showed a high degree of linearity over a range of 101 to 106 plasmids with r2 ≥ 0.9989. Amplification efficiency of the qPCR reaction was greater than 97.6 percent. After validation of the assay we set forth to measure the differences in viral binding between the two airway epithelial phenotypes.;Using our new qPCR method we confirmed that the squamous phenotype produced approximately 100-times as much virus as the mucociliary. Concentration dependence of viral uptake after a one-hour exposure was then measured over a three-log range of HRV16 at half-log dilutions using on cultures of both phenotypes. Uptakes were very similar for both phenotypes. We were not able to detect a saturating component of viral binding.;In conclusion, we describe a method for generating a plasmid standard curve to measure numbers of HRV16 viral stands. Our results indicate the differences in viral production between squamous and mucociliary phenotypes are not due to differences in initial uptake of virus.