Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Abstract

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.

Figure 1

Immunostaining of swine PCLS infected by swine influenza A viruses. PCLS were infected by subtypes H1N1 (H1N1/1981), H1N2 (H1N2/2000) and H3N2 (H3N2/2007) swine influenza viruses. Cryosections were prepared at 24 hpi and used for detection of infected cells, ciliated cells, and mucus-producing cells. Infected cells were stained with anti-nucleoprotein (NP) antibody; ciliated cells were stained using anti-β-tubulin antibody (red) and mucus-producing cells were stained using anti-Muc5Ac antibody (green). A, control slices showing ciliated and mucus-producing cells; B, co-staining of ciliated and virus-infected cells; C, co-staining of mucus-producing cells and virus-infected cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Figure 2

Infectivity kinetics in supernatants of PCLS infected by swine influenza A viruses. PCLS were mock-infected or infected by subtypes H1N1, H1N2, H3N2 swine influenza virus. Up to 7 dpi, infectious virus released into the supernatants of PCLS was titrated at daily intervals by endpoint dilution titration (TCID₅₀/mL; 50% tissue culture infectious dose/mL).

Figure 3

Ciliary activity of swine PCLS infected by swine influenza A viruses. PCLS were mock-infected or infected by subtypes H1N1, H1N2, H3N2 swine influenza virus. Up to seven dpi, PCLS were analyzed for ciliary activity at daily intervals.

Figure 4

Dispnoea score. Dyspnoea (score) observed in pigs after experimental infection with influenza A viruses, dpi, days post infection; statistics: asterisk (1 p < 0.05, 2 p < 0.010, 3 p < 0.005, comparison of following groups: a H1N1/1981:H1N1/2006, b H1N1/1981:H1N2/2000, c H1N1/1981:H3N2/2006, d H1N1/2006:H1N2/2000, e H1N1/2006:H3N2/2006, f H1N2/2000:H3N2/2006).

Figure 5

Rectal temperature. Rectal temperatures (°C) in pigs after experimental aerosol infection with swine influenza A viruses, dpi, days post infection; statistics: asterisk (1 p < 0.05, 2 p < 0.010, 3 p < 0.005, comparison of following groups: a H1N1/1981:H1N1/2006, b H1N1/1981:H1N2/2000, c H1N1/1981:H3N2/2006, d H1N1/2006:H1N2/2000, e H1N1/2006:H3N2/2006, f H1N2/2000:H3N2/2006).

Figure 6

Viral lung load. Viral lung load (EID₅₀/10 mg lung) in pigs 3 days after aerosol infection with swine influenza A viruses; statistics: asterisk (1 p < 0.05, 2 p < 0.010, 3 p < 0.005, comparison of following groups: a H1N1/1981:H1N1/2006, b H1N1/1981:H1N2/2000, c H1N1/1981:H3N2/2006, d H1N1/2006:H1N2/2000, e H1N1/2006:H3N2/2006, f H1N2/2000:H3N2/2006).