Attenuated viral strains are usually obtained by multiple passages in specific cell lines or by cold-adaptation or by genetic manipulation in recombinant viruses. Attenuation of viruses may be the result of a single nucleotide change in a critical gene product and hence has the possibility of reverting to the wild type (WT) phenotype due to genetic instability. The ability to induce interferon synthesis in the host and the sensitivity to the antiviral activity of interferon are characteristic features for a given virus and hence may be used to characterize WT and attenuated strains that are derived from the parent. Such information on RSV is lacking and in this project, our goal is to define these characters of WT and three temperature sensitive mutants of RSV strain, A2. Human lung cells, A549 and MRC-5 cell lines are infected with the wild type (WT) RSV strain A2 and at different time points post infection, samples were collected to analyze for the presence of different IFNs by (a) antiviral activity and (b) by IFN-specific ELISA methods. Initial studies with RSV A2 indicated that very little antiviral activity was stimulated and released in the WT RSV infected cell supernatants. These observations are confirmed by more specific ELISA methods. Studies on attenuated vaccine starins indicated that most attenuated vaccine strain produced most antiviral activity compared to its wild type parent strain. Further studies are also planned to understand the molecular basis for this apparent lack of IFN induction by RSV infection. Similarly, the effects of various IFNs on RSV A2 replication is currently investigated in both MRC-5 and A549 cells. Cells were treated with IFNs a, b and g, at 0 - 10,000 u/ml conc. for 20-24 h prior to infection with RSV A2 and the sensitivity to IFN is evaluated in terms of virus yield, RNA replication and viral protein synthesis. As a control virus with known sensitivity to IFN, human Parainfluenza Virus type 3 (hPIV3) is used and similar parameters are evaluated. Our experimental results indicate that while hPIV3 is acutely sensitive to IFNs a and b (> 3-4 log10 reduction in virus yield), RSV A2 is highly resistant (~0.5-1.5 log10 reduction in virus yield) to the antiviral activities of various IFNs. This was also further confirmed in cell lines stabley expressing human MxA protein, an IFN-inducible protein associated with antiviral activity against certain viruses. While hPIV3's growth was found to be sensitive, RSV A2 seem to be normal in these cell lines. These studies indicate that wild type RSV A2 is resistant to IFNs and might have developed mechanisms that counter act the antiviral activities of IFNs. Further studies with attenuated, mutant viruses are underway to determine whether they exhibit similar IFN-resistant phenotype.
