Our main observation was that human influenza viruses can survive and maintain their infectiousness for several days when they are deposited on banknotes. The duration of viral infectiousness was related to both the concentration of the inoculum and the presence of a beneficial microenvironment. When high-concentration inocula were mixed with respiratory mucus, the infectiousness of influenza A (H3N2) and influenza B viruses increased in an unexpected way by up to 17 days. The concentrations of virus needed to achieve this prolonged survival were around 8.9 × 105
/ml. This concentration is within the range of concentrations found in clinical specimens since at the peak of symptoms during naturally acquired influenza A, the median virus titers in nasopharyngeal secretions can reach 6.3 × 104
). This protective role of respiratory secretions for the survival of a virus is in agreement with previous studies performed in the 1940s in which Parker et al. showed that there was increased viral stability in the presence of human mucus (17
). However, despite the fact that the virus could be cultivated over a long sampling period, the concentration of infectious virus diluted in mucus and deposited on banknotes dropped quite rapidly (approximately 102
-fold after 2 h and 105
-fold after 2 weeks) (data not shown).
The clinical relevance of these observations was confirmed by using the nasopharyngeal secretions collected from children with influenzalike illnesses which had been screened in our routine laboratory during the ongoing season. Respiratory secretions from these influenza A (H3N2) virus-positive cases were directly deposited on banknotes. The virus survived for at least 24 h in 50% of the cases and for 48 h or more in more than one-third of the cases. These findings are similar to our experimental findings and confirm that the viral load in naturally infected individuals is high enough to significantly contaminate the environment. Unfortunately, we are unable to provide quantitative data for this part of the study. It should also be kept in mind that children harbor a higher viral load, which suggests that environmental contamination might be more frequent in this population.
A study of influenza virus stability in the environment needs to take many different factors into consideration, which must be identified and evaluated. A striking example in our study was the protective effect of respiratory mucus on influenza viruses. Other factors that have been recognized and whose effects on virus infectivity have been evaluated include the type of surface (nonporous versus porous) (2
), the type of virus used, viral concentration, temperature, humidity (19
), and the light and UV conditions (7
), as well as the pH. These factors were not considered in the present experiments. Furthermore, given that many environmental studies were performed several decades ago, differences in methodology, such as the detection methods (egg culture, cell culture, in vivo inoculation) should also be borne in mind. Thus, in agreement with previous results (2
), we found that influenza A viruses can survive on inert and nonporous surfaces for days or even weeks. On porous surfaces, such as paper or tissue, the survival rate appeared to be shorter and limited to 12 and 8 h for influenza A and B viruses, respectively. Although banknotes could be considered to be an inhospitable surface for any biological agent, we learned that the main raw material for the fabrication of Swiss banknotes is cotton which is covered by a resin (kinegram). This resin represents a nonporous surface, which we found to exhibit no significant pH variation (data not shown). Whether similar results would be obtained with banknotes from other countries and with different characteristics needs to be studied.
Survival in the environment is, however, not sufficient to sustain transmission and represents only the first requirement and the first step before possible self-inoculation via fingers. The nasal route for establishing influenza virus infection is known to be effective in both humans and animals (8
). Some observations suggest that this fact needs to be considered, and a recent review even concluded that contaminated fomites play a predominant role in influenza virus transmission between humans (4
). Moreover, contamination of hands after contact with an influenza virus-contaminated surface has been demonstrated. Indeed, infectious influenza virus was isolated from hands after contact with a porous surface contaminated for 15 min, as well as after contact with a nonporous surface contaminated for 24 h (2
). Subsequent hand-to-hand transmission was also demonstrated, and competent influenza virus was recoverable for at least 5 min from fingers after brief contact with the previously experimentally contaminated hand of a patient infected with influenza virus.
Similarly, nasal inoculation with other respiratory viruses via contaminated fingers is also known to be effective, particularly for rhinovirus (10
). Our observations demonstrate that even in the absence of mucus, HRV2 and HRV37 survived for 2 and 5 days, respectively, on banknotes. Rhinovirus is known to survive on fomites (10
), and previous studies showed that HRV14 and HRV37 could survive for 14 and 24 h on nonporous surfaces (12
). This suggests that our findings might also be relevant for other respiratory viruses.
From the perspective of a possible influenza A (H5N1) pandemic, specific biological properties of avian strains have to be considered. In humans, the virus is excreted at high concentrations (1.8 × 104
to 9.8 × 104
copies per ml) in both respiratory secretions and feces (5
). Avian influenza A viruses that are excreted at very high levels in bird feces were able to survive for more than 60 days in distilled water at 28°C and for 91 days at 4°C (23
). All these data suggest that environmental contamination, not only from respiratory secretions but also from feces, might be more frequent than expected in the event of an H5N1 pandemic. The severe acute respiratory syndrome experience has also shown that emerging enveloped RNA viruses can develop versatile biological properties that enable them to be transmissible in an efficient manner via environmental contamination (15
An interesting observation in our study was that a constant level of viral RNA could be detected for more than 10 days. It should be noted that a real-time PCR assay that targets small stretches of nucleic acid was used and was still positive, while the larger genomic RNA could be fragmented. These results suggest that real-time PCR can be considered an appropriate detection tool for environmental screening.
We showed that infectious virus can survive for several days on banknotes. This requires a relatively large inoculum and the presence of a protective matrix, such as respiratory mucus. Pandemic events depend on the presence of sufficient quantities of virus with pandemic properties, as well as suitable vehicles for its transmission, including environmental vectors, such as banknotes. The results of our study show that influenza virus stability is not the sole determining factor in a pandemic. As hundreds of billions of banknotes are probably exchanged every day worldwide, infection from hands contaminated with virus picked up from virus-contaminated banknotes cannot be totally ignored. Given the unexpected stability of influenza virus in this nonbiological environment, our current understanding of the conditions favoring influenza virus survival needs to be revised, particularly in the context of pandemic preparedness.