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.

Plaque assays, RT-PCR, and Sanger sequencing of a variable regions of the HN and polymerase genes and variable non-coding regions were used to obtain isolates with each of the high-frequency variant genotypes (i.e. nt sites 8, 053, 8,073, 8,265, and 11,925) . Also included in the analysis was the leader region, including nt sites 20 and 24 which have been associated with virulence in mice (Fujii et al. 2002) but were not included in the Illumina sequence analysis. Analysis of the plaque genotypes showed that nt 20 of the leader region was mutated from G to A in 27% of the clones, with most of the mutations occurring later in passage. Seventeen percent of clones from P1 had an A present (10/58), whereas by P10 an A was detected in 48% of clones (13/27). After plaque selection and genotyping, twelve clones with sequence that represented high-frequency variants were used to construct populations of single and mixed genotypes for testing in mice (Table 2, Supplementary Fig. S5 ). Eight groups of mice were infected with homogeneous or heterogeneous mixtures of clones (six mixtures were assayed). The egg-passaged seed stock used to infect mice for P1 served as a 'high diversity' control because it contains a full mutant spectrum versus the other inocula that contained one to six clones. Virulence of each inoculum was gauged using weight loss and lung histopathology scores. Infection with the high-diversity seed stock displayed the greatest virulence as compared with the clone mixtures (Table 2, Supplementary Fig. S5 ). An inoculum representing a homogenous preparation of one of the two major genotypes present in the seed stock also had higher virulence as compared with the other clone preparations, although the difference statistically significant for only two of the other inoculums. This clone contained the wild type genotype with an Asp residue in the polymerase gene that was selected for during serial passage (N1124D).