Viral RNA detection in virus spiked PBS, plasma or Buffy coat held at room temperature up to 72 h
Ten fold dilutions of H1N1 virus in 10 μl of PBS starting with 3.55 × 106 to 3.55 × 104 EID50 of H1N1 virus in 10 μl of PBS were mixed with 130 μl of PBS, plasma, or buffy coat separately and held for different periods of time at room temperature. Nucleic acid was isolated and quantitated using the TaqMan RT-PCR assay. As shown in Figure 1, higher amounts of viral RNA were recovered from PBS relative to plasma or buffy coats, but no significant changes were found within the group of PBS, plasma, or buffy coats up to 72 h, suggesting that there is no significant H1N1 influenza RNA degradation under these conditions. We also found that viral RNA detection was greater with higher titers of virus input in buffy coats, and there was no significant change of viral RNA within 48 h of storage in buffy coats (Figure 1C).
Viral RNA recovery in Buffy coats stored at 4°C
Ten fold dilutions of H1N1 virus as described above were added to 130 μl of buffy coats and stored at room temperature, or 4 C for up to 48 h to test whether the viral RNA is degraded in buffy coats at different incubation temperatures. As shown in Figure 2, viral RNA yield was significantly increased at 4 C relative to room temperature for three different concentrations of virus incubated, although the copy number of viral RNA in buffy coats was not significantly changed all the time up to 48 h within each group of either room temperature or 4°C (Figure 1A, B or 1C).
No significant change in viral RNA yield within the group of PBS, plasma or Buffy coats at 4°C for up to 40 days
To study the recovery of viral RNA stored in buffy coats at 4°C for different periods of time, H1N1 virus at the concentrations noted above was mixed with 130 μl of buffy coats, incubated 4°C for different periods of time. After isolation of nucleic acid and viral RNA detection with TaqMan RT-PCR assay, viral RNA copy number was not significantly changed during the incubation period at 4°C for all concentrations tested (Figure 3A).
We then compared viral RNA stability after incubation in either PBS, plasma, or buffy coats in the presence of 3.55 × 106, or 3.55 × 105 EID50 of H1N1 virus at 4°C, and found a similar trend in that there was no significant change of RNA recovery within each group of PBS, plasma or buffy coats (Figure 3B & 3C), although there was gradual decline after a longer post-incubation period (Figure 3B). These data suggest that incubation time is not a major factor affecting viral RNA quantitation, using RT-PCR, and TaqMan assays to detect H1N1 viral RNA.
Virus infectivity in PBS, plasma or Buffy coats at 4°C
Given that viral RNA copy number was not significantly affected during different periods of time, and that viral RNA recovery was in the following order: PBS > plasma > buffy coats, we further determined whether virus infectivity was affected after incubation in PBS, plasma, or buffy coats. 10 μl of PBS containing 3.55 × 106, or 3.55 × 105 EID50 of H1N1 virus was incubated in PBS, plasma, or buffy coats at 4°C for different periods of time and EID50 assay was performed. We found increased loss of infectivity when virus was stored in PBS relative to buffy coats, or plasma for both virus concentrations (Figure 4A & 4B); virus infectivity was less affected by storage in plasma, although there was a gradual decline at lower virus concentration (3.55 × 105 EID50 of H1N1 virus) using the EID50 assay (Figure 4B). At higher concentrations (3.55 × 106 EID50 of H1N1 virus), virus infectivity was significantly abolished in PBS at day 10 postincubation relative to plasma and buffy coats (Figure 4A); and loss of virus infectivity was slower in buffy coats relative to PBS but higher than in plasma (Figure 3A). Although infectivity loss at lower concentrations (3.55 × 105 EID50 of H1N1 virus) in PBS, or in buffy coats was reduced to zero at day 20 postincubation, the loss of virus infectivity in buffy coats was slower than in PBS at 10 day postincubation; and virus infectivity declined only gradually when stored in plasma, but was not abolished even at 40 day post-incubation (Figure 4B). Therefore, loss of virus infectivity was highest in PBS followed by buffy coats and plasma. Virus in plasma showed no significant loss in infectivity.