Toys may serve as important fomites for the transmission of viral diseases among children, including lipid-enveloped respiratory viruses such as influenza that spread through hand-to- mucous-membrane contact. Sampling of shared toys in day cares1 shows evidence of viral contamination; nucleic acids from respiratory viruses have been isolated from toys in day cares2 and doctors’ offices,3 and influenza nucleic acids have been isolated from toys in homes.4 Toys in common play areas in healthcare settings have been implicated as vehicles for outbreaks of viral illness.2 One challenge of understanding the potential role of toys in viral transmission is that many studies of viral contamination of toys are cross-sectional and based on detection of viral nucleic acids, making it difficult to determine whether infectious viruses are present on toys and how long they persist. Without knowing how enveloped viruses survive on fomites like toys, it is difficult to assess the potential risk of infection and effectively design control measures such as disinfection. Therefore, the purpose of this study is to determine the inactivation of an enveloped virus surrogate, bacteriophage Φ6, on the surface of a nonporous children’s toy at temperature and relative humidity (RH) levels typical of indoor environments.
Virus was propagated in host Pseudomonas syringae using the soft agar propagation method (bacteriophage and host were kindly provided by Dr. Leonard Mindich, University of Medicine and Dentistry, New Jersey). Thirty milliliter of host bacterial culture was grown for 24 hours with shaking (100 rpm, 25°C). Virus stock (2 mL) was added and incubated with shaking for another 24 hours. This virus culture (0.5 mL) and fresh host culture (0.5 mL) were added to 30 mL of soft agar (0.7% agar), dispensed into tryptic soy bottom agar plates and incubated at 25°C for 24 hours. The top layer was then harvested, pooled, purified by centrifugation (5900 × g, 30 minutes and 4°C) and stored as stock in 20% glycerol–tryptic soy broth at −80°C.
A nonporous flexible plastic children’s toy (a squeaking frog) was disinfected with 70% ethanol and cut into 1 cm2 coupons. Virus stock was diluted in phosphate-buffered saline to target a concentration of 107 plaque-forming units (PFU) in 10 μL. 10 μL was placed on toy coupons. Time 0 carriers were sampled immediately. For the other time points, coupons were placed into controlled humidity environments at 22°C at either 40% (±2%) or 60% (±2%) RH, created by placing saturated salt solutions in sealed glass containers. For sampling, coupons were placed in tubes using sterile forceps. Eluent (5 mL of 1.5% beef extract, pH 7.5) was added into each tube and agitated on a shaker at 100 rpm for 20 minutes. Eluent was assayed using the double-agar layer plaque assay on tryptic soy agar and incubated at 22°C for 24 hours. Virus survival at each time point was expressed as log10(Nt/N0), where Nt is the virus concentration in PFU on the coupon at time t, and N0 is the initial virus concentration in PFU on the coupon at time 0. Data were analyzed with Excel 2011 (Microsoft Corp.) and GraphPad Prism 5 (GraphPad, San Diego, CA).
Over 24 hours, there was an approximately 2log10 (99%) reduction in the number of infectious viruses at 60% RH, with approximately 1log10 reduction in the first 8 hours. At 40% RH, there was a more rapid decline, with 3.2log10 reduction at 2 hours and 6.8log10 at 10 hours (Fig. 1). Measurements at 40% humidity were carried out until the detection limit of 6.8log10 was reached at 10 hours.
To determine if the slopes of the inactivation curves were significantly different, data were fitted using the Weibull model, which has been previously applied to describe the effects of humidity on virus survival.5 The Weibull model is log10(Nt/N0) = −btn. Using this model, the slope n was determined for each humidity level (60%, n =0.47; 40% RH, n = 1.08). R2 values indicated good model fit (60% R2 = 0.86, 60% = 0.93). Slopes at 40 and 60% RH were significantly different (P = 0.04).
The results of this study indicate that a lipid-enveloped virus can survive on the surface of a nonporous children’s toy for hours at indoor temperature and RH levels, and the RH level affects how rapid the inactivation is. Inactivation was significantly faster at 40% RH, a level typical of indoor environments, compared with 60%, which may be high for typical indoor settings. Even at the lower RH, it may take up to 2 hours to achieve 99.9% reduction of infectious virus. This is similar to findings that suggest inactivation of influenza on nonporous fomites may be fairly rapid at low RH.6 Even at normal indoor RH, viruses deposited on toy surfaces at high levels may survive long enough to result in fomite-to-hand exposures. Bacteriophage Φ6 has been used previously as a surrogate to study the survival of influenza and other enveloped viruses;7 the structural similarity to lipid-enveloped human viruses, ease of assay and ability to produce high titer to follow inactivation over long periods of time make this phage a promising potential candidate for understanding several aspects of viral survival and transmission. Toys have been previously implicated as vehicles of outbreaks,2,8 and previous investigations have found viral nucleic acids from enveloped viruses on toys in day cares, hospital nurseries, doctors’ offices and homes.3,4,9,10 This suggests the possibility of exposure, but the significance is somewhat unclear because the presence of nucleic acid does not necessarily indicate the presence of infectious virus.9 Our results show that at 40% RH, typical of indoor environments, a period of a few hours may be sufficient for exposure, and higher indoor RH may promote even longer term survival of enveloped viruses on fomites, suggesting that control of indoor RH levels can create environments less favorable for viruses on surfaces. Periodic disinfection of toys in a pediatric healthcare setting has been evaluated as an infection control measure;10 if toys are shared in an environment where children carry respiratory illness, frequent cleaning and disinfection to interrupt viral transmission may be desirable. In indoor environments where children share toys, their role as fomites for the potential spread of enveloped respiratory viruses should be assessed and appropriate infection control measures applied.
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