Effects of Alkaline Solutions on the Structure and Function of Influenza A Virus

Abstract

Influenza A virus (IAV) infection contributes to high annual morbidity and mortality, thus necessitating measures aimed at protecting against the disease. Alcohol-based disinfectants are commonly used to inactivate IAV, but they have several undesirable properties. In search of other means which would inactivate IAV, we focused on the effect of alkaline solutions on IAV. We found the viral infectivity remarkably decreased with treatment of an alkaline solution at pH 12.0 for 1 min, where destruction of the viral spikes was observed using an electron microscope. A more detailed examination revealed that the infectivity of IAV was remarkedly reduced by brief treatment with the alkaline solution at pH 11.75 or above, most likely due to the degradation of viral hemagglutinin protein. These results show that at a high pH, the haemagglutinin protein is degraded, resulting in very rapid inactivation of IAV.

Keywords: alkaline condition; hemagglutinin; hydrolysis; influenza A virus.

Figure 1

MDCK cells were infected with IAV, which had been treated with alkaline solutions for 1 min and incubated for 72 h. Then, the viral infectivity was evaluated by a plaque assay and the results of quadruple samples were presented as mean ± standard deviation.

Figure 2

IAVs were treated with alkaline solution and subjected to SDS-PAGE under non-reducing conditions followed by Western blot under non-reducing conditions, as described in the Materials and Methods. Data are representative of three independent experiments. The HA protein was detected by a rabbit monoclonal antibody specific for the HA protein (A) and the NP protein was detected by a rabbit monoclonal antibody specific for the nucleoprotein protein (B).

Figure 3

IAV particles treated with alkaline solutions were negatively stained and images were obtained using transmission electron microscopy. Yellow indicates viral spikes. Data are representative of two independent experiments.

Figure 4

MDCK cells were infected with IAV which had been treated with more detailed alkaline solutions for 1 min (n = 4) and incubated for 72 h, and the viral infectivity was evaluated by a plaque assay. The results are presented as mean ± standard deviation. * p < 0.01.

Figure 5

IAVs treated with alkaline solutions for 1 min were subjected to serial two-fold dilution in PBS, mixed with guinea pig erythrocytes in a round-bottom 96-well plate, and incubated at 4 °C for 120 min. The graphical results are representative of three independent experiments.

Figure 6

IAVs were treated with alkaline solutions and subjected to SDS-PAGE under non-reducing (A,C) or reducing (B) conditions followed by Western blot, as described in the Materials and Methods. Data are representative of three independent experiments. The HA protein was detected by a rabbit monoclonal antibody specific for the HA protein (A,B) and the NP protein was detected by a rabbit monoclonal antibody specific for the nucleoprotein protein (C).

Figure 7

IAVs were treated with alkaline solutions and subjected to SDS-PAGE under non-reducing conditions followed by silver staining, as described in the Section 2.