Inactivation of murine norovirus by chemical biocides on stainless steel

Human noroviruses (NoVs) are members of the genus Norovirus and belong to the family Caliciviridae possessing a single-stranded genome without an envelope. NoV is responsible for more than 80% of nonbacterial gastroenteritis in Europe and the United States [1, 2]. The most common routes of norovirus transmission are ingestion of contaminated food or water, direct and indirect person-to-person contact and aerosolization of viral particles.

In young children, a long-term shedding for more than one month was observed in 22.6% of those with adequate follow-up [3]. This study in a children's hospital also revealed that 59% of all NoV infections were hospital-acquired. A recent publication confirmed the extensive contamination of environmental surfaces resulting in a prolonged norovirus outbreak [4]. These findings highlight the role of widespread environmental contamination in hospitals and other medical settings. Effective disinfection of surfaces and healthcare equipment is crucial for the prevention of virus transmission. Consequently, inactivation studies simulating practical conditions with different chemical biocides are important to develop surface disinfectants effective against human NoVs.

Due to the lack of a cell culture system for important nosocomial pathogens such as human NoVs, it is necessary to use a surrogate virus which can easily be cultivated in cell culture. In the past, the most suitable surrogate for human NoVs when studying the virucidal activity of chemical disinfectants in suspension and on carriers was the feline calicivirus (FCV) [5‐7]. Within the family Caliciviridae, FCV belongs to the genus Vesivirus and it is known to be a respiratory pathogen. Therefore, survival and inactivation characteristics may differ from those of human NoVs, which are transmitted by the fecal-oral route.

Recently, propagation of the murine norovirus (MNV) in dendritic cells and macrophages was achieved [8]. Like human NoV, MNV is a member of the genus Norovirus and passes through the gastrointestinal tract. Because of this, MNV seems to be a more suitable surrogate for human NoV than FCV [9, 10].

This study presents the results of activities of different biocides against the MNV dried on stainless steel to simulate practical conditions. These data should facilitate our understanding of inactivation of human NoV by surface disinfectants.

Norovirus infections spread rapidly particularly in senior residences, hospitals, hotels, schools and even cruise ships. Apparently, contaminated surfaces may play an important role in the spread of human NoV [12‐15]. In a rehabilitation centre, the protracted course of norovirus-induced gastroenteritis was also connected to an environmental contamination [16]. Therefore, measures for the prevention and control of NoV transmission should include surface disinfection with products highly effective against this viral pathogen.

Many surface cleaners in the household are based on 0.5% hypochlorite. It was shown by reverse transcriptase polymerase chain reaction (RT-PCR) for human NoVs that the cleaning of fecally contaminated surfaces with a cloth soaked in detergents or treatment with 0.5% hypochlorite alone failed to eliminate human NoVs. The virus could still be detected on up to 28% of the surfaces by RT-PCR. Only wiping the surface with a cloth soaked in detergents followed by applying a combined hypochlorite/detergent treatment was successful to achieve a sufficient efficacy against human NoVs [17].

Since there is only limited knowledge about human NoV inactivation on surfaces, it was the aim of this study to evaluate the virucidal properties of some often-used chemical ingredients of surface disinfectants in a standardized test procedure. The importance of carrier-based methods to test efficacy of disinfectants against vertebrate viruses has been stressed [18].

Meanwhile, carrier test methods including a ring trial launched under the initiative of the Organisation for Economic Cooperation and Development have been published [19‐21]. In our study the protocol used followed the draft of the European Committee for Standardization TC 216 with 0.03% BSA and 0.3% BSA plus 0.3% sheep erythrocytes as interfering substances without mechanical action [22].

The absence of a cell culture assay for human NoV makes it necessary to introduce a reliable surrogate virus when studying the environmental persistence and efficacy of chemical disinfectants. Because FCV can be propagated in cell culture, it has been extensively studied in inactivation studies in the past [5, 23]. In contrast to FCV, MNV is transmitted by the fecal-oral route, thus making this virus a promising surrogate for human NoVs. Consequently, stability and inactivation by alcohol-based hand rubs and commercial products used in veterinary medicine for surface disinfection have been successfully tested with MNV in suspension and carrier tests [24‐26].

In this study, MNV showed a remarkable stability while drying. The difference in the virus titre before and after drying on the carrier did not exceed 0.35 log₁₀ (data not shown in table). In contrast, the poliovirus titre decreased by about 3 log₁₀ during the drying process [27].

In general, non-enveloped viruses can persist for approximately up to two months on inanimate surfaces [28]. For example, reovirus was able to survive on polyvinyl chloride carriers for a period of 30 days dried in organic matrix [29].

MNV can persist in various environmental conditions. After 40 days at -20°C and 4°C only a < 2 log₁₀ reduction was observed [30]. This study also revealed that MNV survived better in a stool suspension than on the surface of gauze or diaper material.

Recently, the stability of MNV mixed with artificial feces on stainless steel coupons was studied [25]. In this study, only a minimal loss of infectivity was measured at pH 2, whereas FCV was rather unstable at pH values lower than 3.

In this study we show that PAA and GDA were able to reduce the titre of MNV under clean conditions about more than 4 log₁₀ within 5 minutes of exposure time. When testing PAA a concentration of 0.1% (1000 ppm) and when testing GDA a concentration of 0.25% (2500 ppm) was needed to inactivate 99.99% of the viruses. These results were confirmed by the ability of two commercial products based upon GDA and PAA to inactivate MNV [26].

In tests with other viruses simulating practical conditions, 2.0% GDA was able to inactivate HIV, rotavirus and HAV on carriers within one minute [31‐33]. Additionally, reovirus embedded in an artificial test soil was inactivated by GDA within one minute, whereas a concentration of 0.1% took 12 minutes to inactivate this virus completely [29].

Generally, besides PAA and GDA, chlorine-based products are often recommended to inactivate non-enveloped viruses on contaminated inanimate surfaces. Testing nonporous and porous surfaces, 20 to 200 ppm of hypochlorous acid solution reduced the titres of human NoV and MNV by 3 log₁₀ within a contact time of 10 minutes [34].

In further experiments we analysed the virucidal properties of ethanol, 1-propanol and 2-propanol against MNV in the quantitative carrier test under clean conditions with concentrations from 10 to 60% (v/v). Since results from the suspension test with the MNV where ethanol in a concentration of 60% (v/v) to 80% (v/v) was more effective than 1-propanol followed by 2-propanol (unpublished), we expected ethanol to be the most effective virucidal agent, followed by 1- and 2-propanol. Surprisingly, 1-propanol was the most effective alcohol to inactivate MNV on the carrier under clean conditions. Interestingly, a concentration of only 30% (v/v) 1-propanol was sufficient to inactivate 99.99% of the virus. In comparison, the two other alcohols were completely ineffective at this concentration. Ethanol achieved a similar effectiveness only at 50–55% (v/v), while 2-propanol at a concentration of 60% (v/v) did not reach a 4 log₁₀ reduction. These results are in contrast to data with FCV. Here, 2-propanol inactivating 99% within one minute at 40 to 60% was found to be more effective than ethanol (6).

Approaching practical conditions, the soil load was increased from 0.03% BSA (clean conditions) to 0.3% BSA plus 0.3% sheep erythrocytes (dirty conditions). Previous results of suspension tests or carrier assays had shown that a higher organic load resulted in a decreased effectiveness of the biocide [35‐37]. Interestingly, our results could not confirm these findings. There was no difference in log₁₀ reduction detectable between clean and dirty conditions. All three alcohols tested showed similar reduction factors at 40% (v/v) and 60% (v/v) with 0.03% BSA as well as with 0.3% BSA plus 0.3% erythrocytes. At the tested concentration of 40% (v/v) 1-propanol was highly active against MNV after an exposure time of 5 minutes by reaching a reduction of 6 log₁₀, whereas ethanol and 2-propanol could not inactivate the virus sufficiently. The effectiveness of ethanol and 2-propanol could be enhanced by increasing the concentration of alcohol. At an alcohol concentration of 60% (v/v) ethanol reached an efficacy comparable to 1-propanol, whereas 2-propanol was not capable of reducing the virus titre by more than 3 log₁₀ at the same concentration.

In summary, our data indicate that 1-propanol was the most effective alcohol to inactivate MNV on stainless steel discs within 5 minutes of contact time. Independent of the amount of soil load, even a concentration of 30% (v/v) 1-propanol had the capability to decrease the virus titre by ≥ 4 log₁₀ within 5 minutes.

In further studies, it would be interesting to compare the inactivating properties of these biocides against MNV on other carriers often used in hospitals and other medical settings.

To interrupt the transmission of human NoV it is necessary to use highly effective surface disinfectants tested under practical conditions. Our results show that MNV as a surrogate for human NoV is inactivated under clean conditions by PAA and GDA as well as by the two alcohols ethanol and 1-propanol on stainless steel discs after 5 minutes of exposure time, whereas 2-propanol only achieved insufficient effectiveness. The effective concentrations of the chemical biocides examined provide an informative basis when evaluating these substances as possible active ingredients of surface disinfectants with short contact times.