Analysis of supernatants of infected Calu-3 cells grown under ALI conditions after

Calu-3 cells, a permanent human cell line with features of the bronchiolar epithelium when grown under air-liquid interface conditions (ALI), was infected with the avian viruses, which were previously used for the infection of chicken and turkey TOCs.

The influence of several passages in Calu-3 cells was monitored and analysis of cell culture supernatants was performed.

For the infection experiments, Calu-3 cells were seeded on filter inserts and cultured as described in 3.1.4. Infection was performed as described in 3.3.6 with an moi of 0.1 for the different avian H9N2 viruses. Supernatants were afterwards analyzed for the amount of virus at different time points post infection by immunoplaque assay.

Passaging of the parental virus on Calu-3 cells revealed that during passaging there was a shift in the time point, when the highest viral titer was achieved (figure 17.A). In the first passage, the highest viral titer was observed 48 h p.i., whereas in the second passage the highest titer was seen 36 h p.i.. The viral titer of the parental virus reached 4.5x10⁷ ffu/ml in the first passage. In the second passage the viral titer reached 6.3x10⁷ ffu/ml. The highest viral titer was achieved in the third passage 36 h p.i. with 1.04x10⁸ ffu/ml. During passaging only a 2.3-fold increase in viral titer was observed. The most striking feature was the 12 h accelerated replication.

Infection of Calu-3 cells with the H9N2 virus passaged four times in chicken TOCs (H9N2 ck) revealed that during passaging there also was a shift in the time point, when the highest viral titer was achieved (figure 17.B), similar to the shift with the parental virus (figure 17.A). In the first passage, the highest viral titer was observed 48 h p.i., whereas in the second passage the highest titer was seen 36 h p.i.. The viral titer of the chicken TOC-adapted virus reached 6.4x10⁷ ffu/ml in the first passage. In the second passage the viral titer reached 6.1x10⁷ ffu/ml. The highest viral titer was achieved in the fourth passage 36 h p.i. with 7.9x10⁷ ffu/ml. During passaging only a 1.4-fold increase in viral titer was observed. The most striking feature therefore was the 12 h accelerated replication. This was the same characteristic that was observed with the parental virus.

Figure 17. Effect of five passages on the infection of Calu-3 cells by different avian H9N2 viruses. Shown are the viral titers of the different passages of Calu-3 cells infected by an m.o.i. of 0.1 of A) egg-grown A/chicken/SaudiArabia/CP7/1998 (H9N2 p), B) the H9N2 virus passaged four times in chicken TOCs (H9N2 ck) or C) the H9N2 virus passaged four times in turkey TOCs (H9N2 tk). ffu:

focus-forming units; p.i.: post infection

Infection experiments of Calu-3 cells with the H9N2 virus passaged four times in turkey TOCs (H9N2 tk) revealed that during passaging there also was a shift in the time point, when the highest viral titer was achieved (figure 17.C), similar to the parental and the chicken TOC-adapted virus (figure 17.A and 17.B). In the first passage, the highest viral titer was observed 48 h p.i., whereas in the second passage the highest titer was seen 36 h p.i.. The viral titer of the turkey TOC-adapted virus reached 5.4x10⁷ ffu/ml in the first passage. In the second passage the viral titer reached 4.5x10⁷ ffu/ml. The highest viral titer was achieved in the fourth passage 36 h p.i. with 8.6x10⁷ ffu/ml. During passaging only a 1.6-fold increase in viral titer was observed. The most striking feature therefore was the 12 h accelerated replication. This was the same characteristic that was observed with the parental and the chicken TOC-adapted virus. The highest increase in viral titer that was observed during passaging was seen with the parental virus (figure 17.A).

For a better representation of the decisive results of the titration values of the different passages they were as well presented in a linear diagram. The focus was thereby laid on the values that seemed to have played the most important role during the adaptation process, the time points around 36 h and 48 h p.i..

This type of presentation illustrates that in all three investigated viruses, the highest viral titer was reached much earlier, namely 12 hours earlier than without prior adaptation to Calu-3 cells (figure 18). Furthermore, it was apparent that the parental virus showed the highest viral titer after three passages on Calu-3 cells (figure 18.D), whereas the chicken and turkey TOC-adapted viruses display the highest viral titer after four passages on Calu-3 cells (figure 18.E and 18.F). Passaging had the biggest favorable influence on the parental virus, since a 2.3-fold increase in viral titer was observed (figure 18.D). The chicken and turkey TOC-adapted viruses only showed a 1.4- (figure 18.E), respectively 1.6-fold (figure 18.F) increase in viral titer. Additionally, the results showed that continued passaging of the viruses on Calu-3 cells does not automatically lead to a further increase in viral titer. At some point during passaging the maximal viral titer was reached. For the parental virus the highest titer was reached after three passages (figure 18.D), whereas the highest viral titer for the chicken and turkey TOC-adapted viruses was reached after the fourth passage (figure 18.E and 18.F).

Figure 18. Effect of five passages on the infection by the different avian H9N2 viruses determined by virus titration (linear illustration). Shown are the viral titers of the different passages of Calu-3 cells infected by an m.o.i. of 0.1 of D) egg-grown A/chicken/SaudiArabia/CP7/1998 (H9N2 p), E) the H9N2 virus passaged four times in chicken TOCs (H9N2 ck) or F) the H9N2 virus passaged four times in turkey TOCs (H9N2 tk). Error bars are shown in figure 17.

4.2.5 Analysis of Calu-3 supernatants after infection with different avian