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To determine any possible effect the loss of N40 may have had on virus replication, its plaque phenotype was analysed. Confluent monolayers of MDCK cells were infected with the two N40 deletion viruses. Cells were overlayed with a mixture of Avicel and DMEM supplemented with N-acetyl trypsin. At 24 or 72 hpi, cells were fixed and stained either with crystal violet or by immunostaining and plaque size was compared to those of WT infected cells. As can be seen in Figure 6.4, plaques seemed slightly smaller 24 hpi, however no difference was observed in plaque size at day 3 post infection. rWSN WT rWSN ΔN40 rWSN ΔF2/ΔN40 Figure 6.4: Plaque phenotype of viruses with a N40 deletion: Confluent monolayers of MDCK cells were infected with recombinant viruses. Cells were overlayed with 1.2% Avicel-DMEM and incubated at 37°C for 3 days (top panel) or 24 h (bottom panel). Mean

values ± SEM for ∼30

plaques/recombinant virus (3 dpi) are plotted.

To evaluate this further, viral growth was determined under multi-step conditions or single-step conditions. Confluent MDCK cells were infected with low or high MOI (0.001 or 3, respectively) and supernatants were collected at the indicated time points to measure viral growth. Beside MDCK cells, viral growth in a multi-step cycle was also determined in A549 cells (Figure 6.5(B)). As shown in Figure 6.5, the loss of N40 had a minor effect on viral replication in MDCK cells; in A549 cells no difference in growth was observed. This finding was independent of PB1-F2 expression and the

(A)

(B)

Figure 6.5: Multi-step growth curve of viruses with a N40 deletion: Triplicate

MDCK cell (A) or A549 cell (B) monolayers were infected with the recombinant

WSN viruses WT, ∆N40 and ∆F2/∆N40 at an MOI of 0.001. Media was

supplemented with N-acetyl trypsin. Supernatants were harvested at the indicated time points post infection and virus titres were determined by plaque assay. Mean

values±SEM of each set of triplicates are plotted.

Wise et al. reported that viral growth in a multi-step cycle was unaffected by the loss of N40, similar to the results seen in this study. However they found a delay of viral growth under single cycle conditions (Wise et al., 2009). To evaluate this for the influenza A virus strain A/WSN/33, confluent monolayers of MDCK cells were infected with either the WT or one of the

(A) (C) NS1 PB1 (B) α-tubulin NS1 PB1 N40 4 8 12 4 8 12 4 8 12 MOCK MOCK rWSN WT rWSN ΔN40 _ΔF2/ΔN40rWSN hpi

Figure 6.6: Single-step growth curve of viruses with a N40 deletion: (A) Triplicate MDCK cell monolayers were infected with the recombinant WSN viruses

WT,∆N40 and∆F2/∆N40 at an MOI of 3. Supernatant was harvested at indicated

time points post infection and virus titres were determined by plaque assay. Mean

values ±SEM of each set of triplicates are plotted. (B) Synthesis of viral protein

was analysed by Western blot at indicated time points. 4 - 12% gradient Bis-Tris

gels were used to detect PB1, N40 and NS1. α-tubulin was used as an internal

mutant viruses at an MOI of 3 and supernatants were harvested at the indicated time points (Figure 6.6 (A)). Both N40 deficient viruses were attenuated under these conditions. Following this, viral protein synthesis was determined by western blot analysis. Although accumulation of NS1 was similar in cells infected with either virus, PB1 expression levels were decreased at later time points in cells infected with N40 deficient viruses compared to cells infected with the WT virus (Figure 6.6 (B and C)).

Based on the results described in Chapter 3 and the results in this Chapter, a mixed infection assay was used to further evaluate possible effects of N40 and PB1-F2 on viral growth. When cells were infected at a high MOI, viruses overexpressing N40 in a PB1-F2 deficient background (∆AUG) showed similar growth to WT viruses. On the other hand, viruses deficient for N40 showed an attenuated phenotype. The opposite result was found when infecting at a low MOI: ∆AUG viruses were attenuated, viruses deficient for N40 showed a similar growth phenotype to WT virus. The potential of the presence of high levels of N40 in combination with the availability of PB1-F2 in the same cell was examined by infecting MDCK cells with both viruses simultaneously at an MOI of 3. Viral infections follow a Poisson distribution and ∼80% of cells are expected to be infected with more than one virus particle at an MOI of 3 (Knipe et al., 2001). Cells were also infected with either of the two viruses at an MOI of 3 or 6 and with WT virus (MOI 6). The results are shown in Figure 6.7. As described before, cells overexpressing N40 showed a small plaque phenotype, whereas the plaque size of ∆N40 viruses was comparable to WT viruses. Infection of cells with a mixture of ∆AUG and ∆N40 viruses caused a mixed plaque phenotype, although the majority of plaques were similar to those seen for WT and∆N40 viruses (Figure 6.7(A)).

All viral infections gave rise to high levels of released virus and viral proteins were detected at all time points examined (Figure 6.7(B and C)). Whereas PB1-F2 was absent in cells infected with ∆AUG viruses and N40 was absent in cells infected with∆N40 viruses, PB1-F2 and N40 were detected in cells infected with a mixture of rWSN∆AUG and rWSN∆N40.

Regarding viral growth, findings could be summarised as follows:

(1) Under conditions of high MOI,∆AUG viruses grew similar or even better then WT viruses, whereas∆N40 viruses showed an attenuation.

Figure 6.7: Comparison ofin vitrophenotypes of WT,∆AUG,∆N40 and mixed infections: Triplicates of MDCK cell monolayers were infected with rWSN WT,

rWSN ∆AUG, rWSN ∆N40 or a mixture of ∆AUG and ∆N40. (A) Supernatants

were harvested at 8 hpi and plaque phenotype was determined.(B)At the indicated

time points, cells were lysed and accumulation of viral proteins was determined by western blot analysis. 4-12% gradient Bis-Tris gels were used to detect PB1, N40

and NS1. 16% Tricine-SDS-PAGE was used to detect PB1-F2. α-tubulin was used

as an internal loading control. (C) Supernatants were harvested at the indicated

time points and viral growth was measured by plaque assay. Mean values±SEM

(2) Only minor differences were observed comparing infections with MOI of 3 and MOI of 6. The differences in the percentage of infected cells for both MOI are thought to be small, as MOI of 3 led to 95%, MOI of 6 to about 99%, infected cells (Knipe et al., 2001).

(3) Viral growth in a mixed infection was more similar to growth seen for

∆N40 virus rather than∆AUG virus infections.

6.4

Effect of loss of N40 on virulence in mice