Environmental survival and microbicide inactivation
Manfred H. Wolff,1 Syed A. Sattar,2 Olusola Adegbunrin2 and Jason Tetro2
1 Faculty of Bioscience, Institute of Microbiology and Virology, Stockumer Str. 10, D-58453 Witten, Germany
2 Centre for Research on Environmental Microbiology (CREM), Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
Since their first isolation from chickens in 1937 , coronaviruses have proven to be significant pathogens of many types of wild as well as economically important domesticated animals.Though coronaviruses were first identified as human respiratory pathogens in 1965 , only recently, with their established link with the severe acute respiratory syndrome (SARS), has there been a sudden upsurge of interest in this group of viruses.
Taxonomically, these enveloped, positive-sense RNA viruses  belong in the genus Coronavirus of the family Coronaviridae in the order Nidovirales . To date, the genus contains some 14 members. Birds and mammals are the known hosts with a wide variety of species affected. In mammals, coronaviruses have been isolated from pigs, cattle, mice, rats, dogs, horses, cats, and humans , and in birds mainly from chickens  and turkeys .
Coronaviruses 229E and OC43 are recognized respiratory pathogens of humans. The causative agent of SARS (SARS-CoV), which has now been fully characterized , awaits its formal inclusion in the genus. Genomic studies show SARS-CoV to be unique as it contains elements of both mammalian and avian ancestry  and the effect of this recombination has been
disastrous for humans. In the first recorded outbreak in 2003, the virus caused 8,461 clinical cases and 804 recorded deaths globally . Fortunately, and in spite of its seemingly high mutation rates , the spread of the virus was effectively controlled, mainly through general public health
measures and basic infection control practices. Nevertheless, the SARS incident has had a significant impact on human health and the global economy  and thus highlighted the need to better understand the modes and vehicles for its spread and proper means to interrupt its environmental transmission.
Pathogenesis and shedding of infectious virus
Depending on the type of coronavirus and the animal host species, virus shedding can occur from the respiratory and/or gastrointestinal tract  and the two main portals of virus entry into susceptible animals are the nose and the mouth . In humans, coronaviruses normally cause the common cold, SARS being a notable exception because it causes severe pneumonia as well as acute gastroenteritis. The risk of environmental contamination with the SARS-CoV is thus much higher. The evidence available thus far from outbreak investigations is strongly suggestive of droplet transmission  and to a much limited degree by aerosols . It is not known if the SARS-CoV can also be acquired through the mouth or the conjunctivae, nor do we know if inhaled virus deposited in the throat can be directly translocated to the gut.
In SARS, massive spiking of the immunological response leads to
hypoxia and eventual respiratory distress syndrome . Infection of the gastrointestinal tract causes acute diarrhea and may also result in peritonitis and necrotizing colitis . The simultaneous involvement of the respiratory as well as the gastrointestinal tracts in SARS  is highly reminiscent of coronavirus infections in cattle  and other animals.
Based on the pig model, the infectious period for coronaviruses is
approximately 6 days post-infection [12, 18] with a peak at day 4 post-infection. This holds true for bovine [17, 19] and human infections [20, 21] as well. In pigs, virus release from the nose peaks on the fourth day of infection with yields of > 5.5 log10 TCID50/g of nasal discharge, with the air around such animals containing nearly 2 log10 of infectious virus/m3 . Similarly, infectious virus has been detected in the nasal discharge and feces of SARS patients .
Environmental survival and spread of coronaviruses
In general, a given pathogen must remain viable outside the host to allow for environmental spread, and the combined effect of many biotic and abiotic factors determines how long such viability can be retained. While environmental survival of coronaviruses has been studied to some degree under experimental conditions [22, 23] including SARS (Tab. 1), we know much less about the types and relative significance of vehicles in the in-nature spread of coronaviruses, in particular those that can infect humans. For example, infectious virus has been recovered from both droplets and air in the vicinity of pigs experimentally infected with the porcine respiratory coronavirus , but the potential, if any, of droplets and aerosols in the spread of the virus to susceptible animals in the vicinity is unknown.
The following is a summary of the available information on the environmental survival and spread of coronaviruses.
Environmental survival and microbicide inactivation of coronaviruses
The effect of relative humidity (RH) and air temperature has been studied on the airborne survival of experimentally aerosolized human coronavirus 229E . As is true for enveloped viruses in general, 229E survived better at 30-50% RH than at 80% RH when the air temperature was about 20°C. Under these conditions, the half-lives of the virus at 30%, 50% and 80% RH were 27, 67 and 3 hours, respectively. Lowering the air temperature to
6°C increased the half-lives of the virus at 30% and 50% RH to 34 and 103 hours, respectively. But the lower air temperature produced the most dramatic effect on virus survival at 80% RH and changed its half-life from 3 to
over 86 hours.
The available epidemiological evidence strongly suggests that SARS
spreads through droplets  and such spread is much easier to control than that through aerosols. However, the pattern of spread of SARS-CoV in at least two instances is highly suggestive of airborne spread.
A cluster of 329 SARS cases was recorded in one apartment complex in Hong Kong with the majority of them occurring on several floors in one wing . This pattern of spread is highly suggestive of virus dissemination by air, and the aerosolization of the virus was speculated to have occurred from malfunctioning sewers in the building . Rodent pests in the building have been hypothesized as possible amplifiers and disseminators of the
virus ; the virus, most likely acquired from infected residents, may have multiplied in the rodents, being released in their excreta and then possibly becoming airborne. While hantaviruses, for example, can infect humans from aerosolized rodent excreta , the role of air in the transmission of SARS-CoV in this outbreak remains speculative at this stage. However, a more detailed analysis of the outbreak of SARS at that apartment complex
suggests that the virus released by the patients themselves may have spread through air .
Limited airborne spread of SARS may have occurred on board commercial aircraft. In one such instance, a symptomatic index case infected at least 22 of 120 (18.3%) passengers and crew during a three-hour flight . Some of those infected were seated over 2 m (90 inches) away from the index case, a distance much longer than the 0.9 m (36 inches) generally believed to be the limit for droplet transmission. In such retrospective investigations it is virtually impossible to rule out the role of other possible
means of virus spread. Also, the inside of an aircraft combines features which may be more conducive to airborne spread of pathogens.
A recent report from Canada suggests that oxygen delivery masks with open vents could promote the dispersal of respiratory pathogens such as SARS-CoV through their enhanced release in mists of exhaled pulmonary gases . The exhaled moist air ejected from such oxygen masks is
believed to carry pathogen-laden droplets over longer distances and possibly contribute to an increased risk of spread of respiratory infections in nosocomial settings.Additional investigations are needed to first prove that
viruses such SARS-CoV can retain their infectivity better in the warm, moisture-laden air exhaled from oxygen masks. The findings on the influence of RH and air temperature on the airborne survival of coronavirus 229E would tend to suggest otherwise .
Sewage and biosolids
SARS-CoV can survive for up to 96 hours in body fluids such as sputum, feces and serum, but is less stable in urine . The fecal excretion of SARS-CoV generated much concern on the safety of handling and spreading municipal solids on lands . There are no reports of the recovery of infectious SARS-CoV from raw sewage or sludge and if the behavior of other enveloped viruses such as HIV is an indication, it would be highly unlikely that the virus can survive in such wastes long enough to pose any risks to human health. In view of this, it is even less likely that the virus
would survive the conventional methods of sewage and biosolids treatment. Therefore, any suggestion of health risk from handling of municipal wastes or their proper disposal would be difficult to justify .
Environmental survival and microbicide inactivation of coronaviruses
Since safety and ethical considerations would not permit the experimental contamination of the hands of human subjects with SARS-CoV, we have used 229E as a surrogate to study the potential of coronaviruses to survive on hands. Preliminary findings from such experiments indicate that nearly 45% of infectious virus remains viable on the hands of adult subjects after 1 hour. This is in contrast to other enveloped respiratory viruses (e.g. parainfluenzavirus) which become essentially undetectable in about 10 minutes on human skin .
Food and water
We are not aware of any published information on the survival of coronaviruses in food. Recent studies have shown SARS-CoV to survive in water to a very limited degree . There is no evidence to suggest the spread of coronaviruses through food or water.
SARS-CoV  and 229E and OC43  can retain their infectivity for several hours on porous and non-porous environmental surfaces. While such survival is better than that of other enveloped human pathogens , there is no evidence to suggest that environmental surfaces play any direct or indirect role in the spread of coronaviruses.
Activity of microbicides against coronaviruses
Because of their enveloped nature coronaviruses are more susceptible to microbicides than non-enveloped viruses. In a comparative study, an animal
parvovirus (non-enveloped) required 20- to 500-fold higher concentrations of the tested microbicides than were needed to inactivate an animal coronavirus .
Even before the advent of SARS considerable evaluation of the activity of microbicides against this virus group had been carried out. The following is a summary of this information.
Virucidal activity of several microbicides was tested against the mouse hepatitis virus (MHV) and the canine coronavirus (CCV), Kilham rat virus and canine parvovirus . Both coronaviruses were readily inactivated by ethanol, isopropanol, benzalkonium chloride, iodophor, sodium hypochlorite, sodium chlorite, cresol soap and formaldehyde whereas the two parvoviruses proved to be considerably more resistant.
The relative resistance of 229E to microbicides was compared to that of coxsackievirus B3, adenovirus type 5, and parainfluenzavirus type 3 using the second tier of a quantitative carrier test (QCT-2) . Stainless steel disks were used as carriers and each one received 10 µl of the test virus, suspended in either faeces or mucin and the inoculum allowed to dry for 1 h
under ambient conditions. After 1 min exposure to 20 µl of the disinfectant, the virus from the disks was immediately eluted and plaque assayed. The efficacy criterion of a ≥ 3 log10 reduction in virus infectivity was used. As expected, the coronavirus proved to be generally less resistant to microbicides than the two non-enveloped viruses.
In a more recent study, Wood and Payne  used a suspension test to assess the activity of chloroxylenol, benzalkonium chloride and cetrimide/chlorhexidine against three types of enveloped viruses (herpesvirus type 1, HIV-1 and a human coronavirus). The coronavirus was found to be generally more resistant than the other two enveloped viruses tested.While
this observation is of interest, the findings have limited practical significance because the testing was based on a suspension test which presents the test microbicide with a weaker challenge than a carrier test protocol. A summary of all findings can be found in Table 2.
Most alcohols used in disinfection are ethanol and isopropyl alcohol, both usually at a concentration of 70% [32, 34]. Without soil load, the transmissible gastroenteritis virus (TGEV) was reduced 4.5 log10 by ethanol in the suspension test over 5 minutes . Similarly, using QCT-2 and organic load, ethanol reduced 229E  by at least 3 log10 over the course of 5 minutes.
Chlorine and other halides
In tests using varying concentrations of sodium hypochlorite, 1000 ppm was effective against TGEV , bovine coronavirus  and 229E  irrespective of test conditions. Chloramine T (C7H7SO2NNaCl) had similar results against bovine coronavirus and 229E. Povidone-iodine at a concentration of 1% reduced 229E by >3 log10 with the QCT-2 test.
Formaldehyde at 4% is used as an overall disinfecting and sterilizing solution albeit its use as a general disinfectant is not recommended. It is effective
against TGEV, reducing it over 5 log10 in under 5 minutes in suspension. At 2%, glutaraldehyde can inactivate 229E over 3 log10  in the QCT-2 test.
Environmental survival and microbicide inactivation of coronaviruses
SL, soil load; QCT-2, quantitative carrier test – Tier 2; TGEV, transmissible gastroenteritis virus.
Quaternary ammonium compounds
A quaternary ammonium disinfectant requires the presence of an ammonium ion and at least one of its hydrogen atoms substituted by an organic radical. As a result, the list of quaternary ammonium compounds is rather extensive. This type of disinfectant is usually cationic and affects a
Environmental survival and microbicide inactivation of coronaviruses
cell by adhering to its negatively charged membrane and disrupting the ionic potential of the cell. In the case of viruses, this adherence can disrupt the envelope, rendering the virus noninfectious. The quaternary ammonium compounds are best represented by chlorhexidine gluconate, which is widely used in health care institutions and in consumer disinfectants and
antiseptics. In both suspension and QCT-2 tests, chlorhexidine gluconate faired well against TGEV  and 229E [33, 34]. Concentrations ranging from 500 to 15,000 ppm were effective in reducing the virus by at least 3 log10.
The phenolic compounds have a long history as their ancestry dates back to the use of coal tar soaps, whose active ingredients were creosol-based. Although some disinfectants continue to use the same historical active ingredient, creosol, most have developed over time to become more active against various microbes. For example, o-phenylphenol at 200 ppm is highly ineffective against most viruses, including 229E in the QCT-2 test . However, the addition of either a detergent, such as sodium lauryl sulphate, or ethanol, proves highly effective against the virus over 3 log10 reduction is seen after 5 minutes. In the same way, many phenolic
compounds rely on the helper effect of other chemicals to work effectively.
Survival and inactivation of the SARS coronavirus
The World Health Organization Laboratory Network has summarized the
available data on SARS-CoV survival and inactivation , which are redrawn for this chapter as Table 3. Tables 4 and 5 summarize more recent studies on the inactivation of 229E by environmental surface disinfectants and antiseptics, respectively.
Despite the enhanced awareness of the potential of coronaviruses as animal and human pathogens, our understanding of their environmental survival and the exact means of their spread remains weak. Such information would be essential to design and implement more rational approaches to prevention and control of outbreaks of coronaviral infections. This is particularly relevant for the recently discovered SARS-CoV. However, the limited data available indicate that coronaviruses as a group are more stable in
the environment that other enveloped viruses.
The fingerpad method (ASTM 1838) was used. Ten µl of virus in soil load was placed on each fingerpad & dried. The dried inoculum was exposed to 1 ml of control or test solution for 30 seconds. The fingerpads were eluted & eluates plaque assayed.
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