3.2 UL21.5 mRNA in plasma is mostly packaged in virions
We tested UL21.5 mRNA both in purified virus preparations and in plasma
of infected patients before or after RNAse digestion (RNAse protection
assay) to verify whether it is free-floating in plasma (and therefore
undetectable after digestion) or packaged in virions (and therefore
resistant to RNAse digestion). Plasma amples with UL21.5 mRNA levels
above the quantification limit (n=28) were included in this analysis. To
verify the efficacy of the RNA digestion treatment, different
concentrations of rhinovirus RNA solution (from 109 to
104 copies/ml) were treated with the same protocol
that was subsequently applied to the plasma samples, resulting in
complete degradation of the RNA at all the concentrations tested (Figure
2A).
After RNAs digestion of purified virus preparations of AD169 and VR1814,
UL21.5mRNA was still detectable with a reduction of 0 and 0.3
Log10, respectively (Figure 2B).
Similarly, after RNAse digestion of the plasma, UL21.5 mRNA was still
detectable in all samples, with a median reduction of 0.1 (IQ range
0-0.3) Log10 (Figure 2C). This indicates that the UL21.5
mRNA detected in plasma is mainly packaged in virions. As control, in
the first 14 samples tested 106copies/ml of rhinovirus
RNA were added to plasma. After RNAse treatment, rhinovirus RNA was
undetectable in all samples.