Fig. 3

Virus titers in (A) the nasal turbinates collected at day 4 and (B) nose swabs collected daily until day 4, from ferrets inoculated with A/H5N1_wildtype (blue) and A/H5N1_{HAQ222L,G224S PB2 E627K} (red) throughout the 10 serial passages described in Fig. 2. Virus titers were determined by end-point titration in MDCK cells. After inoculation with A/H5N1_wildtype, virus titers in the nasal turbinates were variable but high, ranging from 1.6 × 10⁵ to 7.9 × 10⁶ TCID₅₀/gram tissue (A), with no further increase observed with repeated passage. After inoculation with A/H5N1_{HA Q222L,G224S PB2 E627K}, virus titers in nasal turbinates averaged 1.6 × 10⁴ in the first three passages, 2.5 × 10⁵ in passages four to seven, and 6.3 × 10⁵ TCID₅₀/gram tissue in the last three passages, suggestive of improved replication and virus adaptation. A similar pattern of adaptation was observed in the virus titers in the nose swabs of animals inoculated with A/H5N1_{HA Q222L,G224S PB2 E627K} (B). These titers also increased during the successive passages, with peak virus shedding of 1 × 10⁵ TCID₅₀ at 2 dpi after 10 passages. Altogether, these data indicate that A/H5N1_{HA Q222L,G224S PB2 E627K} adapted to more efficient replication in the ferret URT upon repeated passage, with evidence for such adaptation by passage number 4. In contrast, analyses of the virus titers in the nose swabs of the ferrets collected at 1 to 4 dpi throughout the 10 serial passages with A/H5N1_wildtype revealed no changes in patterns of virus shedding. Asterisks indicate that a nose wash was collected before the nose swab was taken, which may influence the virus titer that was detected.