High Environmental Stability of Hepatitis B Virus and Inactivation Requirements for Chemical Biocides

Than, Thoa Thi; Jo, Eunji; Todt, Daniel; Nguyen, Phuong Hong; Steinmann, Jochen; Steinmann, Eike; Windisch, Marc P

For the production of HBV particles, the human hepatoma cell line HepAD38 was used, which resulted in the generation of HBV infectious particles with titers of >10 6 median tissue culture infectious doses (TCID 50 )/mL. This virus suspension was the basis for conducting viral stability and quantitative suspension tests with biocides. Experiments were performed in the presence of PEG, which enhances binding of HBV particles but does not enable noninfectious virions to enter target cells. First, we evaluated HBV stability at different temperatures by incubation of cell culture-derived HBV particles at 4°C, 21°C, and 37°C for up to 28 days. Subsequently, infectivity was determined by inoculation of naive HepG2-NTCP cells, and HBV infection rates were determined by immunofluorescence analysis. Interestingly, no changes in infectivity were observed at 4°C, and only a minor reduction (10%) was determined after 28 days at room temperature (21°C; Figure 1A ). For samples that were stored at 37°C, the HBV infection rate was reduced in a time-dependent manner, however, reaching 50% inhibition of infection at >20 days. Next, the long-term stability of HBV was assessed by incubation of the virus at 4°C for up to 270 days. Remarkably, HBV infectivity remained stable until day 180 of incubation at 4°C, with only a minor reduction of approximately 3-fold ( Figure 1B) , and even after 270 days only a 150-fold reduction could be observed. Of note, the number of infectious particles detected at 270 days was approximately 100-fold above the detection limit of the assay (cutoff, 10 2 TCID 50 /mL). In summary, these results indicate a high environmental stability of HBV in suspension with surviving times of several weeks at room temperatures.