Figure 1.
rPan/99 wt and var viruses show similar growth phenotypes in MDCK cells and guinea pigs.
A) MDCK cells were infected at an MOI of 0.001 PFU/cell with the indicated viruses. For rPan/99wt-HIS virus, n = 6 dishes; for rPan/99var2-HA virus, n = 3 dishes. B) Groups of three guinea pigs were inoculated intranasally with 1000 PFU of the indicated virus. Virus titers in nasal washings are plotted vs. day post-infection. Average values +/− standard deviations are shown.
Figure 2.
Identification of wild-type and variant virus gene segments by high resolution melt analysis.
Examples of the Difference RFU (relative fluorescence units) curves generated by the Precision Melt Analysis software are shown for each vRNA segment. Curves colored red clustered with the rPan/99wt control and curves colored green clustered with the rPan/99var control.
Figure 3.
Reassortment in cultured cells is efficient under unbiased conditions.
The results are shown of HRM genotyping analysis of 121 plaque isolates obtained from each of two culture dishes of MDCK cells co-infected at high MOI (10 PFU/cell). In A, 108/121 (89.3%) had reassortant genotypes and in B, 106/121 (87.6%) had reassortant genotypes. This experiment was done in the presence of trypsin. Green coloring indicates a segment derived from rPan/99var virus; red coloring indicates a segment derived from Pan/99wt virus; white indicates a segment that was untyped. The right-most column in each chart shows the overall genotype: green for var, red for wt and blue for reassortant.
Figure 4.
Frequency of reassortment increases exponentially with frequency of co-infection.
MDCK cells were infected simultaneously at the indicated MOIs with rPAN/99wt and rPan/99var viruses carrying His and HA tags, respectively. At 12 h post-infection, cells were collected for flow cytometric analysis to determine co-infection frequency and cell culture medium was collected for genotyping of released virus. To prevent multiple cycles of replication, infections were performed in the absence of trypsin. Average values (n = 2 cell culture dishes) +/− standard deviation are shown. The coefficient of determination (R2) value indicates how well the exponential trend line fits the data.
Table 1.
The frequency of reassortment in vivo is dependent on inoculum dose1.
Figure 5.
Super-infection delayed by up to 12 h allowed robust co-infection and by up to 8 h allowed robust reassortment in cell culture.
MDCK cells were infected at MOI 10 PFU/cell with rPan/99var2-HA virus at 0 h and then super-infected at MOI 10 PFU/cell with rPan/99wt-HIS virus after the indicated time interval. Clonal isolates from supernatant collected 12 h after the wt virus infection were obtained and genotyped to determine the % reassortment. Flow cytometry was performed on the harvested cells to determine % co-infection. To prevent multiple cycles of replication, infections were performed in the absence of trypsin. Average values (n = 2 cell culture dishes) +/− standard deviations are shown.
Figure 6.
Infections separated by less than 18 h led to robust reassortment in vivo.
Groups of three guinea pigs were infected with 1000 PFU rPan/99var virus and, either at the same time (0 h group), or after the indicated time interval, infected with 1000 PFU rPan/99wt virus. Plaque isolates derived from nasal washings collected 48 h after wt virus infection were genotyped by HRM analysis. The average +/− standard deviation of the percentage of isolates with reassortant genotypes is shown.
Table 2.
Super-infection up to 12 h after primary infection leads to robust reassortment in vivo.
Figure 7.
Viral spread in cell culture leads to increased frequency of co-infected cells when super-infection is delayed by 8 or 12 hours.
MDCK cells were infected in the presence of trypsin at an MOI of 0.01 PFU/cell with each virus, simultaneously (0 h), or first with the var virus and then with the wt virus at the times indicated. Cell monolayers were collected and processed for flow cytometry 12 h after the wt virus infection. The average (n = 3) percentage of infected cells that were co-infected is plotted. Error bars indicate standard deviation.