Evaluating the stability of highly pathogenic avian influenza viruses on human skin and measuring the effectiveness of disinfectants are crucial for preventing contact disease transmission. We constructed an evaluation model using autopsy skin samples and evaluated factors that affect the stability and disinfectant effectiveness for various subtypes. The survival time of the avian influenza A(H5N1) virus on plastic surfaces was ≈26 hours and on skin surfaces ≈4.5 hours, >2.5-fold longer than other subtypes. The effectiveness of a relatively low ethanol concentration (32%–36% wt/wt) against the H5N1 subtype was substantially reduced compared with other subtypes. Moreover, recombinant viruses with the neuraminidase gene of H5N1 survived longer on plastic and skin surfaces than other recombinant viruses and were resistant to ethanol. Our results imply that the H5N1 subtype poses a higher contact transmission risk because of its higher stability and ethanol resistance, which might depend on the neuraminidase protein.
Highly pathogenic subtypes of avian influenza virus (AIV) can infect humans and cause fatal respiratory failure (1–3). Since 2003, cases of avian influenza A(H5N1) and avian influenza A(H7N9) transmission from birds to humans have been confirmed in the Middle East, West Africa, Europe, and Asia. In >50% of these cases, the outcome was fatal (4,5). Recently, subtype H5N6, H5N8, and H9N2 AIVs have been confirmed to infect humans (6,7). The H5N9 subtype has also been reported to be highly transmissible (8). Most of these cases of AIV infection have been caused by contact transmission from infected birds (9–14). Therefore, preventing contact transmission is crucial for controlling the spread of AIV infection.
Knowledge of viral stability is vital to understanding contact transmission (15,16), and several studies have assessed the stability of AIVs under various conditions (17–25). Viral stability has been reported to decrease under conditions of high temperature, high salinity, or low pH (17,19,21–25). However, because contact transmission occurs when the virus enters the human body through the skin, evaluating the stability, or survival time, of AIV on human skin and the effectiveness of disinfectants against AIV on skin are essential to assess contact-transmission risks and develop more effective infection control methods (26–29). However, clinical research in this regard is limited because of the risks involved in applying highly pathogenic AIV directly to the skin of human study participants. Therefore, the stability of AIVs and the efficacy of related disinfectants remain unknown.
Moreover, although previous studies have suggested that the stability of different AIV subtypes might vary, these differences were not clearly defined (20–22,25). Current contact transmission control methods are based on the assumption that no great differences in stability among AIV subtypes or in the effectiveness of available disinfectants against them exist (30,31). If substantial differences exist in terms of stability and disinfectant effectiveness among subtypes, then the optimal infection control methods might differ for each subtype. Therefore, developing optimal methods for controlling the transmission of each subtype requires an accurate analysis of the differences among subtypes.
An ex vivo evaluation model using skin collected from autopsy specimens has been developed that accurately and safely assesses the stability of highly pathogenic pathogens and the effectiveness of different disinfectants (26–28). In this study, we aimed to elucidate the differences in the stability of AIV subtypes and disinfectant efficacy against AIV on the surface of human skin by using this constructed model. Furthermore, we aimed to elucidate the genetic mechanisms responsible for stability differences among subtypes by using recombinant viruses.
Recombinant H5N1 viruses with the neuraminidase (NA) or hemagglutinin (HA) gene of the H5N3 subtypes (rH5N1-H5N3-NA and rH5N1-H5N3-HA), or recombinant H5N3 viruses with the NA, HA, nonstructural protein (NS), or matrix protein (M) gene of the H5N1 subtypes (rH5N3-H5N1-NA, rH5N3-H5N1-NS, rH5N3-H5N1-M, and rH5N3-H5N1-HA) were generated as target viruses by using a reverse-genetics system. We evaluated the recombinant viruses A/crow/Kyoto/53/04(H5N1) (H5N1-Ky), A/chicken/Egypt/CL6/07(H5N1) (H5N1-Eg), A/Anhui/1/23(H7N9) (H7N9), A/duck/Hong Kong/820/80(H5N3) (H5N3), A/turkey/Ontario/7732/66(H5N9) (H5N9), a clinical H3N2 strain (H3N2), A/Puerto Rico/8/1934(H1N1) (H1N1-PR8), and A/Osaka/64/2009 (H1N1-Ok-pdm).
Stability of Influenza Virus on Plastic
Within ten hours, all influenza virus subtypes—aside from H5N1—were totally dormant. The H5N1 subtype strains that were tested, however, H5N1-Ky and H5N1-Eg, remained contagious on the plastic surface for ten hours before going completely inactive for twenty-four hours. Furthermore, at the majority of time points, the titers of H5N1-Ky and H5N1-Eg that remained on the plastic surface were significantly higher than those of the other subtypes (, panel A)
Next, for the virus samples that were still on the surface, we computed the half-lives and survival times of the virus titers. All subtypes had survival times of approximately 8–10 hours, with the exception of the H5N1 subtype. For instance, the H5N3 subtype had a 10-year survival rate. 01 (95% CI 8. 35–11. 91) hours. In contrast, the survival time of H5N1-Ky was 26. 35 (95% CI 23. 84–29. 01) hours and survival time of H5N1-Eg was 26. 30 (95% CI 23. 64–29. both considerably longer than those for other subtypes (;, panel A) at 14) hours. Furthermore, compared to other subtypes, the half-lives of the H5N1-Ky and H5N1-Eg strains were more than twice as long ( ;, panel B)
| Subtype | Median survival time (95% CI), h† | Median half-life (95% CI), h‡ | ||
|---|---|---|---|---|
| 4 (log10 FFU) | 3 (log10 FFU) | 2 (log10 FFU) | ||
| H5N1-Ky | 26.35 (23.84–29.01) | 1.28 (1.15–1.43) | 1.71 (1.54–1.91) | 2.56 (2.30–2.86) |
| H5N1-Eg | 26.30 (23.64–29.14) | 1.27 (1.13–1.43) | 1.69 (1.51–1.90) | 2.54 (2.27–2.85) |
| H7N9 | 7.97 (6.82–9.27) | 0.40 (0.34–0.49) | 0.54 (0.45–0.65) | 0.81 (0.67–0.98) |
| H5N3 | 10.01 (8.35–11.91) | 0.52 (0.42–0.65) | 0.70 (0.57–0.87) | 1.05 (0.85–1.30) |
| H5N9 | 8.88 (7.67–10.23) | 0.46 (0.39–0.55) | 0.61 (0.51–0.73) | 0.92 (0.77–1.09) |
| H3N2 | 9.28 (7.94–10.79) | 0.48 (0.40–0.58) | 0.64 (0.54–0.77) | 0.96 (0.80–1.16) |
| H1N1-PR8 | 9.70 (8.29–11.30) | 0.51 (0.42–0.61) | 0.68 (0.56–0.82) | 1.01 (0.85–1.22) |
| H1N1-Ok-pdm | 8.78 (7.60–10.10) | 0.45 (0.38–0.54) | 0.60 (0.51–0.72) | 0.91 (0.76–1.08) |
Constructing a Model to Evaluate Virus Stability and Disinfectant Effectiveness
Human skin was taken from forensic autopsy specimens that were acquired from Kyoto Prefectural University of Medicine’s (Kyoto, Japan) Department of Forensic Medicine. Samples of abdominal skin ranging in age from 20 to 70 years were cut into squares roughly 4 cm by 8 cm in size. Autopsy specimens with significant burn or drowning injuries to the skin were not included (26, 32). According to reports, collected skin can be used for grafting even 24 hours after death, and after 14 days in culture, the skin maintains its physiologic function reasonably well 36 hours after excision (33–35). Thus, to preserve the physiologic function of the epidermis, skin specimens were obtained at ≈1 day after death for this study. We created an ex vivo model using the skin autopsy specimens to assess the stability of various viruses on human skin and the potency of various disinfectants against viruses on skin. PBS was used to wash the skin after the panniculus adiposus was removed, and the skin was then placed in a culture insert (Corning Inc.). , https://www. corning. com) on a membrane with a pore size of 8. 0 µm. The culture inserts were placed in six-well plates containing 1. 0 mL of Dulbecco modified Eagle’s medium (DMEM) (Sigma-Aldrich, https://www. sigmaaldrich. com) (26,27).
Stability of Influenza Virus on Human Skin Surface
All subtypes (except H5N1) were completely inactive within 1. 5 hours. However, even after one hour, the H5N1-Ky and H5N1-Eg stains were still present on the skin. 5 hours but were completely inactive within 3 hours. Furthermore, compared to other subtypes, the titers of H5N1-Ky and H5N1-Eg that were still present on the skin were significantly higher (, panel B)
All subtypes (apart from H5N1) had survival times of approximately two hours. For instance, the H5N3 subtype had a two-year survival time. 10 (95% CI 1. 94–2. 26) hours. In contrast, the survival time of H5N1-Ky was 4. 66 (95% CI 4. 21–5. 13) hours and survival time of H5N1-Eg was 4. 54 (95% CI 4. 16–4. 97) hours, both considerably longer than the other subtypes under investigation (;, panel C) Moreover, the half-lives of H5N1-Ky and H5N1-Eg were more than twice as long as those of other subtypes, and they displayed the same tendency as the survival time ( ;, panel D)
| Subtype | Median survival time (95% CI), h† | Median half-life (95% CI), h‡ | ||
|---|---|---|---|---|
| 4 (log10 FFU) | 3 (log10 FFU) | 2 (log10 FFU) | ||
| H5N1-Ky | 4.66 (4.21–5.13) | 0.20 (0.18–0.23) | 0.27 (0.24–0.30) | 0.40 (0.36–0.45) |
| H5N1-Eg | 4.54 (4.14–4.97) | 0.20 (0.18–0.22) | 0.26 (0.24–0.29) | 0.40 (0.36–0.44) |
| H7N9 | 1.96 (1.84–2.08) | 0.08 (0.08–0.09) | 0.11 (0.11–0.12) | 0.17 (0.16–0.18) |
| H5N3 | 2.10 (1.94–2.26) | 0.09 (0.08–0.10) | 0.12 (0.11–0.13) | 0.18 (0.17–0.20) |
| H5N9 | 2.03 (1.89–2.17) | 0.09 (0.08–0.09) | 0.12 (0.11–0.13) | 0.18 (0.16–0.19) |
| H3N2 | 2.03 (1.89–2.17) | 0.09 (0.08–0.10) | 0.12 (0.11–0.13) | 0.18 (0.16–0.19) |
| H1N1-PR8 | 1.97 (1.83–2.12) | 0.08 (0.08–0.09) | 0.11 (0.10–0.12) | 0.17 (0.15–0.18) |
| H1N1-Ok-pdm | 2.10 (1.93–2.27) | 0.09 (0.08–0.10) | 0.12 (0.11–0.13) | 0.18 (0.17–0.20) |
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