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

Virus Production

HBV particles were produced in the HepAD38 cell line, harboring an over-length HBV genome under the control of a tetracycline-repressed promoter element (the tet-off system). The production process is described elsewhere. Briefly, HepAD38 cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin 50 µg/mL streptomycin, 400 µg/mL Geneticin, 5 µg/mL insulin, 50 µM hydrocortisone hemisuccinate and 0.3 µg/mL tetracycline for 2 weeks. After reaching cell confluency, tetracycline was removed from the medium to induce virus production. Cell culture supernatants were collected once weekly, filtered with a Millipore sterile vacuum filter (0.2 µm), and stored at 4°C.

Testing HBV Stability in the Environment

To investigate the effect of different temperatures on HBV infectivity, viral suspensions were exposed to different temperatures at indicated time points in 0.2-mL polymerase chain reaction tubes (Eppendorf, Germany), using Takara Thermal Cycler Dice Touch (Takara, Australia). After heat treatment, samples were immediately kept on ice before infection of the cells. To examine the impact of different pH values, 6 solutions with different pH values were prepared as described elsewhere. The virus was incubated with different solutions at 37°C for 10 minutes at a volume ratio of 1:4. The viral suspension was then immediately serially diluted in Dulbecco’s modified Eagle medium and then titrated in 384-well microplates.

Stability of HBV Particles at Different Temperatures

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 >106 median tissue culture infectious doses (TCID50)/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, 102 TCID50/mL). In summary, these results indicate a high environmental stability of HBV in suspension with surviving times of several weeks at room temperatures.

Impact of Human Serum and pH on HBV Stability

As HBV particles in the infected host are circulating in the presence of serum, the effect of healthy human serum (50%) on HBV stability was analyzed by incubation of viral particles at room temperature for 7 days and comparing them to HBV incubated side by side in 10% FBS. The addition of human serum did not reduce HBV infectivity over time and was comparable to findings of incubation with FBS (Figure 1C). Next, the impact of changes in the pH on viral stability was tested by incubating HBV at pH 3–8 for 10 minutes at 37°C. Afterward, the viral titer was determined by a limiting dilution assay, which demonstrated that HBV infectivity was not affected at pH 3–8 (Figure 1D).

Susceptibility of HBV to Ethanol, 1-Propanol, and 2-Propanol

Ethanol, 1-propanol, and 2-propanol are common active ingredients of commercial alcohol-based antiseptics and disinfectants used in healthcare systems. To determine the efficacy of these different alcohols on HBV infectivity, HBV was incubated for 1 minute (Figure 2A) or 5 minutes (Figure 2B) with ethanol, 1-propanol, or 2-propanol at concentrations ranging from 0% to 60%. For ethanol, a concentration of >40% was required for HBV inactivation, irrespective of the exposure time. The most effective alcohol to inactivate HBV was 1-propanol, reducing viral titers to undetectable levels at a concentration of 30% after exposure for only 1 minute (Figure 2A). In the case of 2-propanol, incubation of HBV at 40% alcohol led to a 100-fold reduction of infectivity independent of the incubation period, but to completely abrogate the infectivity of HBV particles 60% 2-propanol was necessary. The longer incubation time of 5 minutes did not result in higher inactivation levels (Figure 2B). In conclusion, HBV demonstrated the highest susceptibility to 1-propanol, while for ethanol and 2-propanol alcohol concentrations of >40% were required to inactivate HBV particles.

Virucidal Activity of Commercially Available Hand Disinfectants Against HBV

After having tested the effect of alcohols on the stability of HBV, next we evaluated 5 commercially available hand disinfectants (Sterillium Classic Pure, Manorapid Synergy, Poly-Alcohol Hand Antiseptic, Sterillium Med, and Desderman Pure) for their ability to inactivate HBV in a quantitative suspension test. Importantly, all hand disinfectants demonstrated virucidal activity at the ready-to-use concentration and inactivated HBV to undetectable levels (Figure 2C). Residual virus was only detected after treatment with the 2-propanol-based poly-alcohol hand antiseptic. Next, we evaluated whether a 1:10 dilution of the commercial antiseptics affected the virucidal activities of the products. Dilution of the alcohol-based products abrogated the inactivation properties, reaching only a 50-fold reduction for Sterillium Classic Pure and Manorapid Synergy (Figure 2D).

Virucidal Efficacy of WHO-Recommended Formulations Against HBV

The WHO’s proposed guidelines on hand hygiene in healthcare recommend the use of 2 alcohol-based hand rubs (formulation I and formulation II) for surgical and hygiene hand disinfection in healthcare settings and to reduce the transmission of pathogens by hands. Formulation I is based on ethanol, whereas formulation II consists of 2-propanol. To determine the efficacy of WHO formulations I and II against HBV, we incubated viral particles for 30 seconds with both formulations at final concentrations ranging from 0% to 80%. As depicted in Supplementary Figure 1A, viral titers of 107 TCID50/mL in the control were reduced to undetectable levels at concentrations of >40% for WHO formulation I, whereas at a concentration of 40% WHO formulation II yielded a significant reduction in infectivity (Supplementary Figure 1B). Finally, we analyzed the susceptibility of HBV to these WHO formulations in comparison to that of other enveloped viruses, which we investigated previously. This comparative analysis of inactivation response curves revealed that HBV has greater stability than hepatitis C virus (HCV), influenza A(H1N1) virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and modified vaccinia virus Ankara (MVA; Supplementary Figure 1C and 1D), which is the chosen test virus for all enveloped viruses in the European Guideline for testing chemical disinfectants and antiseptics in human medicine.

Discussion

In this study, we analyzed the stability of the HBV by using a recently developed cell culture system fully susceptible and permissive to HBV infection and replication with high titer virus stocks. The high environmental stability of HBV observed in this study was also previously observed in an in vivo model system. Bond et al demonstrated in 1981 that HBV-positive human plasma, which was dried for 1 week and inoculated in chimpanzees, resulted in an active infection. This infection system is based on human hepatoma cells and viruses generated in vitro, which might differ from primary human hepatocytes and patient-derived viral particles in some aspects. However, given the recent progress that has been made in HBV research using this system, cell culture–derived HBV particles are considered the best tool to address the questions described here. However, other time points and long-term storage of plasma ex vivo were not investigated. For HCV, another blood-borne virus that belongs to the family of Flaviviridae, the environmental stability was much lower, with a half-life of 6 hours at 37°C and 11 days at 4°C.

Regarding the inactivation profiles for HBV with different alcohols, 1-propanol could be identified as the most effective alcohol in a quantitative suspension test, in line what has been described for HCV. The concentrations of the tested alcohols required for virucidal activity were slightly higher than those required for the often-used DHBV, but side-by-side comparisons using the experimental procedure have not been conducted. Importantly, 5 different alcohol-based, commercially available hand disinfectants were able to inactivate HBV after exposure for 30 seconds in a suspension test. Dilution of these products abrogated the susceptibility of HBV, but under practical conditions hand antiseptics are not diluted, precluding a recommendation for a specific product. Two WHO-recommended formulations that have been proposed as alcohol-based hand rubs to reduce the transmission of pathogens were able to completely inactivate HBV. Importantly, in comparison to the enveloped viruses EBOV, HCV, influenza A(H1N1) virus, MERS-CoV, and MVA, HBV demonstrated the highest stability. The degree of susceptibility of the different viruses to the WHO formulation likely depends on the specific surface properties of the lipophilic envelope of the respective virus. HBV subviral particles display a fluid bilayer membrane and were shown to have a strong resistance to freeze drying. However, further investigations are required in the future, ideally with serum-derived HBV, to analyze this phenomenon in more detail.

In summary, this first systematic investigation of the environmental stability of HBV and its susceptibility to different alcohols and hand antiseptics has important implications for the management of nosocomial and occupational exposures to HBV. We propose that very strict compliance with established hygienic guidelines should be mandatory to avoid nosocomial and occupational HBV infections, even if materials have been contaminated with HBV for several weeks.