Sendai virus intra-host population dynamics and host immunocompetence influence viral virulence during in vivo passage

Peña, José; Chen-Harris, Haiyin; Allen, Jonathan E.; Hwang, Mona; Elsheikh, Maher; Mabery, Shalini; Bielefeldt-Ohmann, Helle; Zemla, Adam T.; Bowen, Richard A.; Borucki, Monica K.

Although SeV primers were designed to be as sensitive to variant SeV genomes as possible, amplification of some regions of viral genome proved to be extremely difficult for many of the samples, with selenium deficient diet passage 1 samples showing the poorest amplification results. Many different combinations of primers were used in order to amplify as much of the viral genome as possible (Supplementary Table S1 ). To determine if the poor PCR amplification results were due to low numbers of viral genomes present in each sample, viral titers were determined by both plaque assay and Taqman PCR. The average number of positive PCR results per region (best possible score is two primers per region) was compared with the viral titer (PFU/ ml) and to the C t (cycle threshold) average of two replicates. The average C t had a negative correlation with positive PCR results (r ¼ À0.97), however there was no correlation between PCR outcome and viral titer as determined by plaque assay (r ¼ À0.35). Number of viral genomes per PCR reaction was estimated using standard curve derived from control SeV plasmid dilutions and values ranged from $800 genomes (sample SD4.P10, PCR score of 0) per PCR reaction to 25 million genomes (FD2.P10, PCR score of 2). These data indicate that lack of PCR amplification most likely resulted from a low number of total viral genomes, present in the sample (both infectious and non-infectious).