Human Rhinovirus (HRV) is a major pathogeny associated with the common cold and is responsible for induction or exacerbation of asthma [1]. In recent years, studies have reported that HRV infection in early childhood markedly increased risk of wheezing and developing asthma [2-4]. The magnitude of this association appears larger than that of respiratory syncytial virus (RSV), the classic respiratory pathogen of infancy [5,6]. HRV infections could increase the severity of infection in patients with immunodeficiency diseases and abnormal immune function. HRV is highly transmissible, leading to high infection rates in the respiratory tract particularly in younger children and older men. This infection not only causes severe morbidity and mortality but also results in economic and social burden. The high prevalence of HRV infections and high association between HRV and asthma, severe upper respiratory tract infection, and bacterial super-infection underscores a need to prevent HRV infection and reduce its prevalence globally. However, HRV is highly heterogeneous and have high mutation rates, thus making it challenging to develop effective vaccines against these evolving viruses. Therefore, there is a need to identify effective measure for the immediate containment of the infection and prevention of potential future outbreaks. In addition to good sanitation and hygienic practices, effective disinfection strategies are also necessary. Understanding the potential effects of chemical and physical treatments on inactivation of HRV is significant for the implementation of proper public intervention measures. This study was designed to evaluate the effectiveness of various physical conditions (temperature, ultraviolet light (UV)) and chemical agents (sodium hypochlorite, PAA, Virkon-S, ethanol) against HRV.

Cell culture and HRV virus

HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS, glutamine, and antibiotics as described previously [7]. HRV86 was propagated in HeLa cells with 2% serum for maintenance and infection. 2 × 10^-6.59/ml TCID₅₀ HRV86 was used in this study.

Infectivity and inactivation of heat

To determine the effect of heat on infectivity, HRV86 viruses firstly were exposed to various temperatures. After 100 μl of viral stock solutions containing 1 × TCID₅₀, 10 × TCID₅₀, 100 × TCID₅₀ were incubated Room temperature, 37°C for 6 h, 12 h, 18 h, 24 h, 36 h, 48 h and 72 h. Treated virus was inoculated into HeLa cells respectively. For thermal inactivation, 100 μl of viral stock solutions containing 1 × TCID₅₀, 10 × TCID₅₀, 100 × TCID₅₀, 1000 × TCID₅₀ were respectively exposed at 60°C for 5 min, 10 min, 15 min, 30 min, 1 h and 2 h. The heat-inactivated HRV inoculated into HeLa cells. Then cytopathic effect (CPE) in HeLa cells was observed for 24 h after passaging three times.

UV inactivation

A 100 μl virus stock solution containing 1 × TCID₅₀, 10 × TCID₅₀, 100 × TCID₅₀, or 1000 × TCID₅₀ was exposed to UV light (distance of 30 cm and wavelength of 250-270 nm) in a biosafety cabinet (Thermo Company) at room temperature for 5, 10, 15, 20, 30, 45, 60, and 120 min, respectively. UV treated virus was inoculated into HeLa cells. CPE was observed as above described.

Disinfectants inactivation

Commercial disinfectants, including sodium hypochlorite (Beijing Yasite LTD. China), peracetic acid (Xilong Chemical Co., Ltd. China), glutaraldehyde (Xilong Chemical Co., Ltd. China), Virkon-S (Antec™ International, UK), ethanol (Dezhou ANNJET disinfection products Co. Ltd. China) were assessed for their effectiveness in the inactivating HRV86. Briefly, all disinfectants were diluted with distilled water following the manufacturers’ protocol. A 100 μl virus stock solution containing 100 × TCID₅₀ was absorbed into filter and exposed with above disinfectants, respectively for 2, 5, 10, 20 or 30 min at room temperature. Then filter were washed with 1 ml PBS for three times. Treated virus was inoculated into HeLa cells and CPE was observed as described.

Cytotoxicity

The cytotoxic effects of HRV after exposure to various disinfectants were assessed as previously described [8] except for the replacement of the virus by PBS.

Heat inactivation

The infectivity of HRV86 after exposure to various environmental temperatures (RT (25 ± 2°C), 37°C, or 60°C) was determined. The effects of temperature on HRV inactivation were time-and titersdependent. RT impact greatly on HRV virus infectivity of 1 × TCID₅₀. Although HRV was able to retained activation at RT for 24 h, its infectivity was decreased from 18 h when used at 1 × TCID₅₀. And, the virus was able to retained activation at RT for 48 h at both 10 × TCID₅₀ and 100 × TCID₅₀ titers (Table 1). However, we found that the validation of HRV was impaired severally at 37°C. Complete loss of viral infectivity at both 1 × TCID₅₀ titers and validation of HRV only maintained for 24 h in 100 × TCID₅₀ titers (1/4) (Table 1). Complete loss of viral infectivity was observed at 36 h in the 100 × TCID₅₀ titers when kept at 37°C. However, complete loss of viral infectivity were observed both 1 × TCID₅₀, 10 × TCID₅₀ titers and 100 × TCID₅₀ titers at 37°C after exposed for 6 h or longer.

Table 1: Effect of temperatures on infectivity of HRV86.

To find appropriately heat inactivation of viruses, exposure time was shorted within 2 h and virus titers were also increased to 1000 × TCID₅₀ (Table 2). It was observed that HRV86 was completely inactivated in the 1 × TCID₅₀ or 10 × TCID₅₀ titers when exposed at 60°C for 5 min or longer (Table 2). However, 100 × TCID₅₀ or 1000 × TCID50 titers remain partial vitality when exposed at 60°C for 5 min, in which 2 of 4 wells and 1 of 4 wells CPE was respectively observed at 5 min in the 100 × TCID₅₀ or 1000×TCID₅₀ titers (Table 2). However, no CPE was observed when HRV was treated over 10 min at 60°C (Table 2). These results showed that HRV was completely inactivated at 60°C after exposure for 10 min.

Table 2: Heat inactivation of HRV86 with 60°C.

UV inactivation

To examine the effects of continuous UV irradiation on HRV infectivity, 100 μl aliquots of HRV stock (1 × TCID₅₀, 10 × TCID₅₀, 100 × TCID₅₀, or 1000 × TCID₅₀) were exposed to UV light irradiation at different time points. After 1 × TCID₅₀, 10 × TCID₅₀, 100 × TCID₅₀, or 1000 × TCID₅₀ of HRV were treated with UV radiation for 5, 10, 15, 20, 30, 45, 60, and 120 min, infectivity of HRV decreased in a timedependent manner. When 100 μl of 1 × TCID₅₀ HRV86 were exposed under UV radiation for 10 min, 2 of 4 wells appeared CPE (Table 3). HRV was completely inactivated by UV radiation for 15 min at the same titer. When 100 μl of 10 × TCID₅₀ HRV were exposed under UV radiation for 15 min or 20 min, 2 of 4 wells and 1 of 4 wells appeared CPE (Table 3), respectively. Additionally, at this titer, exposure of up to or over 30 min, HRV infectivity on cells lost thoroughly (Table 3). However, HRV titers at 100 × TCID₅₀ or 1000 × TCID₅₀ were completely inactivated by UV radiation after exposure for 45 min or longer (Table 3).

Table 3: Inactivation of HRV86 with UV irradiation.

Disinfectants inactivation

To investigate the resistance of the HRV viruses against various disinfectants, 100 μl of stock solutions containing 100 × TCID₅₀ HRV virus titers were treated with sodium hypochlorite, glutaraldehyde, peracetic acid, Virkon-S, or 75% ethanol at room temperature.

Inactivation by sodium hypochlorite

Sodium hypochlorite is a common disinfectant. To determine the effects of sodium hypochlorite on the inactivation of HRV, aliquots of HRV stock were treated with 0.1 g/L, 0.2 g/L, 0.25 g/L, or 0.5 g/L of sodium hypochlorite for 2, 10, 20, or 30 min, respectively. Exposure of HRV to 0.1 g/L sodium hypochlorite beyond 10 min completely inactivated HRV (Table 4). Over a concentration of 0.2 g/L, sodium hypochlorite sufficiently inactivated HRV by 2 min (Table 4). These findings suggest that sodium hypochlorite at 0.2 g/L is suitable for use against the most etiologic agents of common cold. Based on these results, treatment of contaminated surfaces with sodium hypochlorite may reduce infection of contaminated surfaces and thereby reduce the risk of laboratory transmission during outbreaks.

Table 4: Inactivation of HRV86 by Sodium hypochlorite.

Virkon inactivation

Virkon-S was next used at 1 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L for 5, 10, 20 and 30 min to assess its effects on HRV infectivity. At 1 g/L, Virkon-S led to partial inactivation of HRV, as CPE appeared in only 1 of 4 wells after 30 min (Table 5). Similar results were observed when used at 5 g/L for 5 min (Table 5).

Table 5: Inactivation of HRV86 by Virkon-S.

However, prolong treatment time for over 10 min, HRV infectivity on cells was complete lost (Table 5). Similar results were also observed when used at 10 g/L or more and exposed for 5 min or longer.

Glutaraldehyde inactivation

Glutaraldehyde is popularly used in hospitals, which is able to effectively eliminate many types of bacteria, fungi, and bacillus. To further understand the effects of this disinfectant on HRV infectivity, 100 × TCID₅₀ of HRV86 was treated with 10 g/L, 20 g/L, 50 g/L, or 100 g/L glutaraldehyde for 5, 10, 20, or 30 min, respectively. In all experiments, no residual infectivity was observed until the third passage. These results indicated that effective viral inactivation was achieved, and that glutaraldehyde is an effective disinfectant to inactivate HRV.

Peracetc acid inactivation

According to the US Center of Disease Control (CDC) “Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008” [9], peracetic acid (PAA) is described as a very effective virucide and sporocide. To understand the extent in which PAA inactivates HRV, aliquots of HRV viral stocks were treated with 3 g/L, 1.5 g/L, 0.75 g/L and 0.375 g/L diluted PPA and exposed for 2, 10, 20, or 30 min. These results showed that 100 × TCID₅₀ of the infectivity of HRV was completely loss when treated with 3 g/L diluted PAA for 2 min or longer (Table 6). However, it took 30 min for 1.5 g/L, 0.75 g/L and 0.375 g/L diluted PAA to completely inactivate HRV (Table 6). Thus, these results showed that 3g/L diluted PAA could effectively disinfectant HRV.

Table 6: Inactivation of HRV86 by Peracetic acid.

75% Alcohol inactivation

Because 75% alcohol is one of the most common disinfectant used in hospitals, households, schools, and other public areas, we next investigated the virucidal activities of 75% alcohol in HRV from 45 s to 30 min. As shown in Table 7, 75% alcohol did not inactivate HRV after treating for 45 s. There was some inhibition of HRV infectivity by 75% alcohol after exposure for 1 or 2 min (Table 7). However, complete inactivation of HRV was observed when treated for 5 min or longer.

Table 7: Inactivation of HRV86 by 75% alcohol.

Heat treatment is one of widely used viral inactivation methods against viruses [10]. Heat-inactivated virus exhibits denatured viral proteins and dissociated viral particles into noninfectious viral subunits [11]. Many viruses, such as avian influenza A (H7N9), HCV, and EBOV have been shown to be sensitive to heat treatment [12-14]. In this study, an analysis was conducted on the inactivation of HRV at various environmental temperatures. Our results showed that HRV are very sensitive to temperature. HRV survival at RT were dependent on virus titers and exposure time. HRV infectivity reduced gradually upon longer exposure time and at lower viral titers (Table 1). Of note, the stability of HRV was severely impaired at 37°C (Table 1). It is possible that HRV virus may have a different structure when incubated at 37 °C and at room temperature just like Dengue virus [15]. Our results suggest that survival of HRV are very fragile in the environment. In this current study, exposure for 5 min at 60°C was sufficient to eliminate the infectivity of HRV86 in all 4 viral titers (Table 2). Therefore, disinfection and inactivation of daily necessities in the cold season for 5 min at 60°C could eliminate HRV infectivity and effectively prevent colds caused by rhinovirus.

UV light irradiation is another commonly used physical method for viral inactivation. UV irradiation prevents viral replication and inactivates virus through formation of pyrimidine dimers in the viral genome [12,14,16-18]. This study demonstrated that the efficiency of UV irradiation to completely inactivate HRV was dependent on exposure time and virus titer (Table 3). Higher exposure time to UV irradiation was required to inactivate HRV at higher titers (Table 3). All 4 HRV titers were completely inactivated upon UV irradiation for 45 min. Therefore, UV irradiation is a highly effective method for inactivating HRV.

The efficacy of several other commonly used disinfectants was also evaluated. Because PPA can inactivate virus through oxidizing S-H groups and N-H of viral proteins of the envelope and the capsid [19], this agent has been used worldwide during food processing and animal breeding, as well as in the beverage industry [9]. In this study, the virucidal activity of PAA against HRV occurred when using concentrations as low as 3 g/L for 2 min, demonstrating that HRV is more sensitive to PPA than Polio virus (0.2% PAA solution an exposure time of 5 min) [19]. However, it took about 30 min for 0.75 g/L diluted PPA or higher to inactivate HRV.

Sodium hypochlorite is one of the most widely used disinfectants. These agents has been successfully used during many infectious disease outbreaks including SARS, EBOV, and influenza H1N1 to decontaminate hospital instruments, infectious materials, and personal protective equipment [20-22] for health care professionals. In the current West African EVD outbreak, a 0.5% chlorine solution has been recommended to disinfect surfaces contaminated with EBOV, and is now recommended for disinfection by the World Health Organization [23]. Our results showed that HRV, compared with other known viruses, was more sensitive to sodium hypochlorite. We found that 0.2 g/L sodium hypochlorite could effectively inactivate HRV within 2 min of exposure. Therefore, sodium hypochlorite is effective in decontaminating surfaces in hospitals, as well as for using among health care professional for proper hand hygiene.

Glutaraldehyde is a chemical cross-linking reagent that is mostly used for tissue fixation for electron microscopy. Although its mechanism of action remain unclear, successful inactivation of many viruses with including hepatitis B virus, human immunodeficiency virus (HIV), hepatitis C virus, and SARS, coronavirus have been reported using glutaraldehyde. [14,16,24,25]. In this study, we demonstrated that 20 min of exposure to glutaraldehyde (10 g/L) could inactivate HRV infectivity on cells. Thus, 25 g/L glutaraldehyde could be used to effectively inactivate HRV.

Virkon-S has been previously reported to be effective for inactivating avian influenza A (H7N9) virus [12], enterovirus, and decreasing transmission of foot and mouth diseases [26,27]. When exposed to 10 g/L Virkon-S for 10 min, titer of feline herpesvirus was significantly reduced [28]. We showed that Virkon-S could completely inactivate HRV infection, consistent with the results of previous studies which used 5 g/L Virkon-S for 10 min or 10 g/L Virkon-S for 5 min or longer. Virkon-S was previously found to be effective against virus, however, the concentrations tested in this study were far higher than those previously employed [29,30]. Combined with previous studies, we think that Virkon-S could have effects against a broadspectrum of viruses.

Although the Food and Drug Administration suggests ethanol concentrations should range from 60% to 95% [31], a previous study showed virucidal activity of ethanol concentrations which ranged between 58% to 75% [32]. In this study, 75% ethanol was used, as it was the concentration most commonly used in daily life, and at hospitals and laboratories. The results showed that 75% ethanol at 5 minutes effectively inactivated HRV. Additionally, we speculate that paper towel or cotton containing 75% ethanol was less effective in inactivating HRV.

Overall, our results provide effective methods for inactivating HRV, which could be implemented to control a HRV outbreak, reduced risk of transmission of HRV, decontaminate infected environments, or as preventative measures to improve public hygiene.

This study was funded by China Mega-Project for Infectious Disease (2012ZX10004401, 2012ZX10004215 and 2013ZX10004805), Special foundation for Health Industry Research Subject (201302006) and the SKLID Development Grant (2011SKLID104).