How long does Ebola last on surfaces: answer

[Amish Country]

This is dated 2010 and may not reflect the attributes of the currently spreading strain of Ebola Zaire. I do not know if the strain has mutated significantly in this regarded since the study was done. -AC
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Journal of Applied Microbiology


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Volume 109, Issue 5
November 2010
Pages 1531?1539
The survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol
Authors

T.J. Piercy,
S.J. Smither,
J.A. Steward,
L. Eastaugh,
M.S. Lever

First published: 22 May 2010Full publication history
DOI: 10.1111/j.1365-2672.2010.04778.x
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Sophie J. Smither, Biomedical Sciences Department, Defence Science and Technology Laboratory, Room 201, Building 7a, Dstl, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK. E-mail: sjsmither@dstl.gov.uk
Abstract

Aims:  Filoviruses are associated with high morbidity and lethality rates in humans, are capable of human-to-human transmission, via infected material such as blood, and are believed to have low infectious doses for humans. Filoviruses are able to infect via the respiratory route and are lethal at very low doses in experimental animal models, but there is minimal information on how well the filoviruses survive within aerosol particles. There is also little known about how well filoviruses survive in liquids or on solid surfaces which is important in management of patients or samples that have been exposed to filoviruses.

Methods and Results:  Filoviruses were tested for their ability to survive in different liquids and on different solid substrates at different temperatures. The decay rates of filoviruses in a dynamic aerosol were also determined.

Conclusions:  Our study has shown that Lake Victoria marburgvirus (MARV) and Zaire ebolavirus (ZEBOV) can survive for long periods in different liquid media and can also be recovered from plastic and glass surfaces at low temperatures for over 3 weeks. The decay rates of ZEBOV and Reston ebolavirus (REBOV) plus MARV within a dynamic aerosol were calculated. ZEBOV and MARV had similar decay rates, whilst REBOV showed significantly better survival within an aerosol.

Significance and Impact of the Study:  Data on the survival of two ebolaviruses are presented for the first time. Extended data on the survival of MARV are presented. Data from this study extend the knowledge on the survival of filoviruses under different conditions and provide a basis with which to inform risk assessments and manage exposure to filoviruses.
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Introduction

Viral haemorrhagic fevers can be caused by a range of viral agents. The family Filoviridae consists of two genera, Ebolavirus and Marburgvirus, that contain filoviral species (Zaire ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus and Lake Victoria marburgvirus) that can cause acute and rapidly progressive haemorrhagic fever in humans. Two other Ebolavirus species, Cote d?Ivoire ebolavirus and Reston ebolavirus, cause diseases in primates but are not pathogenic in humans. Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) have been associated with a number of outbreaks with high fatality rates (up to 100%) (Feldman et al. 1993; Feldman and Klenk 1996). The average lethality for humans is c. 76% for ZEBOV and 57% for MARV (figures calculated from Bausch, et al. 2008) Both viruses are fast acting, with death often occurring within 7?10-day postinfection, although the incubation period is considered to be 2?21 days (Peters and Khan 1999; Borio et al. 2002). To date, the natural reservoir for filoviruses is not known; however reports have shown that species of African fruit bat may be the natural reservoir for the virus as antibodies to ZEBOV and MARV have been found in bat species, and human cases have been linked to bat exposure (Leroy et al. 2005; Pourrut et al. 2009; Towner et al. 2009).

Filoviruses are transmitted through contact with body fluids or tissues of humans, nonhuman primates or infected laboratory animals (Brown 1997; Mwanatambwe et al. 2001; Pinzon et al. 2004). Historically, nosocomial transmission often occurs through the reuse of incorrectly sterilized needles and syringes, or during nursing of an infected individual through contact with blood, vomit or other infected secretions (Feldman and Klenk 1996). Transmission can also occur during burials and the preparation of bodies for burial (Tukei 1996).

Filoviruses have been reported as being transmitted via the aerosol route, either experimentally or within a biocontainment facility (Jaax et al. 1995; Johnson et al. 1995; Belanov et al. 1996) and are considered to be potential biological warfare or bioterrorism agents (Borio et al. 2002; Bray 2003). In addition, there are reports that MARV was previously weaponized (Alibek and Handelman 1999). A recent review of persistence of Category A Select Agents in the environment highlighted how little basic information is known about the filoviruses (Sinclair et al. 2008). The stability of MARV within aerosols and dried on various substrates was investigated by workers in the former Soviet Union (FSU) (Belanov et al. 1996; Chepurnov et al. 1997); however survival characteristics of other filoviruses have not been investigated.

In the United Kingdom, filoviruses are classified as Advisory Committee on Dangerous Pathogens (ACDP) Hazard Group 4 pathogens [equivalent to Biosafety Level 4 (BSL-4)] and are handled under laboratory containment level 4 (CL4) to minimize the risk of infection to laboratory personnel. Filoviruses are also classed as Category A biological agents by the CDC as they have high case fatality rates and are easily disseminated and low infectious doses of filoviruses are sufficient to cause disease in animal models (Borio et al. 2002). In the field, the infrastructure to handle the pathogens is not always available, and prevention of spread is reliant on good hygiene and removal of all contaminated and infectious material. It is unknown how long the filoviruses are able to remain viable if waste and contaminated areas are not immediately sterilized. If the filoviruses were aerosolized, either accidentally or as a deliberate release, it is also unknown how long they would survive as aerosols, and therefore how many people might potentially be exposed.

Here, we provide data on the stability and viability of MARV and ZEBOV in both liquid media and on a range of solid substrates at various temperatures, over time. In addition, the stability and decay rate of MARV, ZEBOV and Reston ebolavirus (REBOV) within small-particle aerosols held within a modified version of the Goldberg drum system (Goldberg et al. 1958) was investigated.


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http://onlinelibrary.wiley.com/enhanced/doi/10.1111/j.1365-2672.2010.04778.x/

 

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Results
Survival of EBOV and MARV in liquid media over 46 days

The survival of MARV and ZEBOV in guinea pig sera and tissue culture media was assessed over a 46-day period (Fig. 1). At +4?C (Fig. 1a), virus viability reduced in titre by a maximum of 3 and 2?3 logs for MARV and ZEBOV, respectively. At room temperature (Fig. 1b), virus viability decreased more rapidly showing a 2- and 4-log reduction in virus titre, respectively, within the first 26 days. After 46 days, titres of virus were only just detectable above the detection limit of the microtitre assay in this study (10 TCID50 ml−1). There was no significant difference (P > 0?05, two-way anova) in survival of virus in either guinea pig sera or tissue culture media, however there was a significant difference between survival during storage at +4?C compared with storage at room temperature (P < 0?001, two-way anova).
Figure 1.
Figure 1.

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 Survival of Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) in liquid media at two temperatures. The survival of ZEBOV (inline image) and MARV (inline image) in tissue culture media (closed shapes) and guinea pig sera (open shapes) over 46 days at 4?C (a) and room temperature (b), was assessed by the TCID50 microtitre plate assay over time. At each time point, triplicate samples were taken; the mean count (TCID50) from triplicate samples, plus the standard error, is shown.

Survival of EBOV and MARV dried onto solid substrates over 14 days

An initial recovery experiment showed that no virus could be recovered from any substrate stored at room temperature (results not shown). All results reported are for +4?C. Neither MARV nor ZEBOV could be recovered from metal substrate at any time.

The survival of MARV and ZEBOV on plastic stored at +4?C showed a decrease in viral titre over time, with a of 3- or 4-log reductions in titres over 14 days for MARV and ZEBOV, respectively (Fig. 2b). When dried in tissue culture media onto glass and stored at +4?C, ZEBOV decreased in titre by less than a log, MARV by a log (Fig. 2a). Virus dried onto glass in guinea pig sera decreased by 3 logs (MARV) or 4 logs (ZEBOV) (Fig. 2a).
Figure 2.
Figure 2.

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 Survival of Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) dried onto different solid substrates over a 14-day period. The survival of ZEBOV (inline image) and MARV (inline image) on different substrates when dried in tissue culture media (closed shapes) or guinea pig sera (open shapes) over 14 days was assessed by the TCID50 microtitre plate assay. Samples were recovered, in triplicate, from the surfaces at various time points. (a) Survival on glass at 4?C, (b) Survival on plastic at 4?C. Each point shows the mean count (TCID50) from triplicate samples, plus the standard error.

There was no significant difference in the initial recovery of either virus suspended in either sera or tissue culture media dried onto glass or plastic (P > 0?05, two-way anova). Further statistical analysis showed that there was no significant difference in the decay rates of the two viruses across the range of substrates and liquid storage media (P > 0?05, using ancova analysis) over 14 days.
Survival of EBOV and MARV dried on to solid substrates over 50 days

The survival of MARV and ZEBOV on plastic and glass substrates was assessed when stored at +4?C, over 50 days (Fig. 3). No significant differences in the initial recovery of either virus across the two substrates and liquid media were detected (P > 0?05, two-way anova). At day 26, there were only three samples from which virus could be recovered; ZEBOV in tissue culture media on glass and MARV in tissue culture media on both glass and plastic. At day 50, the only sample from which virus could be recovered was that of ZEBOV from tissue culture media dried onto glass.
Figure 3.
Figure 3.

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 Survival of Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) dried onto different solid substrates over a 50-day period. The survival of ZEBOV (inline image) and MARV (inline image) on different substrates when dried in tissue culture media (closed shapes) or guinea pig sera (open shapes) over 50 days was assessed by the TCID50 microtitre plate assay. Samples were recovered from the surfaces, in triplicate, at various time points over a longer time period. (a) Survival on glass at 4?C, (b) Survival on plastic at 4?C. Each point shows the mean count (TCID50 ml−1) from triplicate samples, plus the standard error.
Inactivation rates of MARV, ZEBOV and REBOV within an aerosol

The stability of MARV, ZEBOV and REBOV was assessed over 90 min using a 40-l Goldberg drum. For each virus, three replicates were carried out for each time point using BA as an internal control. The average decay rate of BA spores during the filovirus runs was 1?42% min−1. This value represents the physical decay rate in the system used. Statistical analysis performed on the BA data showed no significant difference in decay rates (P > 0?05, ancova) between runs for different viruses (Fig. 4b), ensuring that any difference in decay rates of the filoviruses can be attributed to differences between the aerosol characteristics of the different virus species. All three filoviruses under investigation could be detected after 90 min in a dynamic aerosol (Fig. 4a). ZEBOV and MARV had similar decay rates and REBOV appeared to decay at a slower rate (Fig. 4a).
Figure 4.
Figure 4.

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 Aerosol decay of filoviruses and Bacillus atropheus (BA) spores in the Goldberg drum. (a) Lake Victoria marburgvirus (MARV) (inline image), Zaire ebolavirus (ZEBOV) (inline image) and Reston ebolavirus (REBOV) (inline image) were held in a 40-l Goldberg drum as a dynamic aerosol over a period of 90 min at 50?55% relative humidity and 22 ? 3?C. At various time points, samples were taken and the viral titre was determined by TCID50 assay. Each virus was run in the drum on three occasions, and nonlinear regression was performed to obtain the decay curve fitted to the equation of one-phase exponential decay. (b) BA spores were aerosolized in the drum in parallel with the filoviruses to control for internal loss and physical decay. The decay curves for BA run in triplicate with MARV (inline image), ZEBOV (inline image) and REBOV (inline image) are shown. Each point shows the mean count (TCID50 ml−1 or CFU ml−1) from triplicate runs, plus the standard error.

Data from replicate runs of each virus were fitted to the equation for one-phase exponential decay, and the total decay rates for MARV, ZEBOV and REBOV were calculated as 4?81, 4?29 and 2?72% min−1, respectively (composite of biological and physical decay). Taking into account the physical decay rates observed for BA, this suggests the biological decay rate of the filoviruses is 3?04% min−1 for MARV, 3?06% min−1 for ZEBOV and 1?55% min−1 for REBOV. Statistical analysis showed no significant difference between the decay rates of ZEBOV and MARV (P > 0?05, ancova); however there was a significant difference between the decay rates of ZEBOV and MARV compared to REBOV (P < 0?01, ancova). GraphPad Prism analysis also showed that one-phase exponential decay was the more appropriate model to fit the data for all three viruses, compared to two-phase exponential decay (P = 0?3451 to 0?5832 for one-phase decay).

From the total decay rates, the theoretical decay (as a percentage of original titre) over time was calculated (Fig. 5). The time taken for 50% of the initial virus concentration to decay (i.e. the half-life of the virus) is 14, 15 and 24 min for MARV, ZEBOV and REBOV, respectively. The time for 99% of the initial virus concentration to decay was estimated as 93 min for MARV, 104 min for ZEBOV and 162 min for REBOV.
Figure 5.
Figure 5.

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 Theoretical survival of filoviruses in an aerosol over time. Based on the decay rates of the three filovirus obtained from the Goldberg Drum analysis, the theoretical survival curves of Lake Victoria marburgvirus (?), Zaire ebolavirus (????) and Reston ebolavirus (???) were plotted as a percentage of initial virus titre over time. The starting amount for each virus was 100%, and the amount of virus remaining, as a percentage, over time is shown.
Discussion

This study has demonstrated that filoviruses are able to survive and remain infectious for cell culture, for extended periods when suspended within liquid media and dried onto surfaces. In addition, decay rates of a range of filoviruses, within small-particle aerosols, have been calculated, and these rates suggest that filoviruses are able to survive and remain infectious for cell culture for at least 90 min.

Recovery of virus from liquid media (tissue culture media and sera) was significantly higher in samples stored at +4?C compared to room temperature. MARV and ZEBOV, dried onto solid substrates, were recovered in high titres from both plastic and glass surfaces. It has also been shown that low titres of virus could be recovered from samples suspended in tissue culture media and dried onto both plastic and glass until day 26, but only virus dried onto glass substrate was recovered by day 50, when stored at +4?C. The only significant differences that could be detected across the range of conditions were attributable to the suspending liquid dried onto glass, where virus in tissue culture media could be recovered for significantly longer than virus dried in guinea pig sera.

Data from researchers of the FSU indicated that MARV in human blood was able to survive on steel, glass and cotton wool for at least 6 days (Belanov et al. 1996). Results from our study extend these findings to include survival data for EBOV as well as MARV and also include data for the survival of filoviruses on plastic and glass substrates over a longer period of time.

There was no survival of MARV on metal surfaces in our study, contrary to the results recorded by scientists of the FSU (Belanov et al. 1996). The two studies were performed at different relative humidities, and this may have influenced the survival rates. In addition no details of the steel used in the study conducted in the FSU were provided, and therefore no details of any possible virucidal components within the metal were available. The lack of recovery of virus from metal substrates may be attributed to several factors; previous work (Sommer et al. 1999; Pawar et al. 2005) has shown the binding of micro-organisms to metal surfaces to be poor because of the high surface energy, high electronegative and hydrophilic properties of metal. Other research has also shown that positively charged metallic ions such as copper and silver have a high bactericidal activity (Friedman and Dugan 1968; Bitton and Freihofer 1977; Slawson et al. 1990) and hence by extrapolation may also have a high virucidal activity. Short survival times for several haemorrhagic fever viruses on metal surfaces have been observed; on aluminium discs, the time taken for the initial virus titre to decay by 90% for hantavirus 76?118, Sicilian virus Sabin and Crimean?Congo haemorrhagic fever virus was between 1?08 and 1?45 h (Sinclair et al. 2008).

Viruses display a range of survival times within the environment. Variola virus, the causative agent of smallpox, for example, can remain infectious in dust and on tissue for up to 1 year, whereas influenza virus has a low level of survival and only retains its infectivity on surfaces for a matter of days (Harper 1961). Data generated in this study suggested that both EBOV and MARV could be recovered from contaminated substrates for at least 50 days. No comparable data have been published by other groups; however in a recent study undertaken to assess the risk of transmission of Sudan ebolavirus from bodily fluids and fomites, virus was detected by PCR in a number of samples. No data were recorded however, as to the length of time virus was able to survive in these samples (Bausch et al. 2007).

Data on the survival of filoviruses on substrates and in liquid media presented in this study should be set within the context of the infectivity of the viruses. It has been reported that the infectious dose of filoviruses for mice and nonhuman primates is low (e.g. 400 PFU, Johnson et al. 1995; 1 PFU, Bray et al. 1999). Studies with the wild-type ZEBOV and MARV strains used in these survival studies in a susceptible mouse model demonstrated 100% mortality with <10 TCID50 of either virus by the aerosol or intraperitoneal challenge route (M.S. Lever, personal communication). These low infectious dose reports suggest that if the initial viral titre is high, infectious quantities of viable virus could be recovered from samples stored at +4?C for periods of up to 46 days in liquid media, and from samples dried onto glass at both 26 and 50 days. This demonstrates the need for good control measures when handling and disposing of clinical samples that may be contaminated with filoviruses.

Data produced in this study have shown that MARV, ZEBOV and REBOV have total decay rates in small particle aerosols of 4?81, 4?29 and 2?72% min−1, respectively. There was a significant difference between the decay rates of ZEBOV and MARV when compared to REBOV (P < 0?01, ancova), but no significant difference between the decay rates of ZEBOV and MARV (P > 0?05, ancova). To our knowledge, this is the first time the decay rates of the genera Ebolavirus have been reported.

Many factors affect the inactivation rate of viruses in aerosols, such as relative humidity, the suspending fluid from which the virus is sprayed and the system used to measure aerosol decay. It is challenging to therefore compare directly the inactivation rates determined in this study with published data generated under different conditions for other viruses. However, in this study the average decay rate of the bacterial tracer (used to distinguish between physical and viability losses within the Goldberg Drum) was 1?42% min−1, which was comparable to the 1?5% reported elsewhere (Ehrlich and Miller 1971; Larson et al. 1980). Inactivation rates for filoviruses within aerosols determined in this study were much lower than values for MARV in aerosols reported by the FSU scientists (Belanov et al. 1996), but comparable with values reported for other lipid-containing viruses, known to be transmissible via the airborne route. Such viruses include, Venezuelan equine encephalomyelitis virus (VEEV) (1?3?2?99% min−1), influenza A virus (1?9% min−1) and vaccinia virus (0?34% min−1) (Harper 1961). Other quoted decay rates recorded for viruses at a similar relative humidity (50%) and temperature (20?25?C) as was used in this study include Japanese encephalitis virus (3?3% min−1) (Larson et al. 1980) and yellow fever virus (7?04% min−1) (Mayhew and Hahon 1970).

The time taken for initial viral titres to decrease by 90 or 99% has been reviewed for an arenavirus and an alphavirus (Lassa and VEEV), and two flaviviruses (Japanese encephalitis virus and Saint Louis encephalitis virus). The times varied greatly and were dependant on the humidity and temperature, from 0?7 of an hour to over 2000 h (Sinclair et al. 2008) with corresponding inactivation coefficients of between 0?02 and 1?41. Inactivation coefficients from this study for the filoviruses of 0?027 to 0?049 fall within the range observed for other haemorrhagic fever viruses; however it would be expected that decay constants and survival times would vary dependent on humidity and temperature, as has been observed for other enveloped viruses.

Data generated by FSU scientists found that the decay rate of MARV (strain Popp) was 11?5% min−1 (Belanov et al. 1996), which was much higher than values suggested by this study. Data from the two studies, however, cannot be compared directly because of differences in the equipment and conditions used. The work carried out by the FSU researchers used human saliva as a suspension fluid, and no mixing of the aerosol after generation was undertaken. Such factors may in part have contributed to the increased rate of decay. In addition, differences in enumeration methods (guinea pig lethal doses, compared to tissue culture infectious doses) make direct comparisons difficult.

The infectious dose of filoviruses, via the aerosol route, in nonhuman primates (reviewed in Leffel and Reed 2004) and in susceptible mice (M.S. Lever, personal communication), is very low. Such data, obtained from experimental animal models, combined with the aerosol decay rates determined in this study, would suggest that filovirus, at infectious levels, may remain a potential aerosol threat for at least one and a half hours. Epidemiological evidence, however, would suggest that during outbreaks, filoviruses are rarely transmitted by the airborne route. The lower decay rate observed for REBOV in our study may support observations made during the original outbreak in captive primates and subsequent studies where airborne transmission may be involved in the spread of filoviruses (Jaax et al. 1995; Johnson et al. 1995 and Jahrling et al. 1996). The ZEBOV and MARV used in the studies are from human clinical isolates, whereas REBOV is nonpathogenic in humans. This might suggest that the factors that cause a decrease in virulence in humans also contribute to an increase in aerostability. In the future, sequence analysis, protein structural information and characterization of protein expression from the filoviruses after aerosolization may yield further insight into the survival characteristics of the viruses.

This study has shown that human pathogenic filoviruses may survive in an aerosol in the dark to detectable levels for at least 1?5 h. If filoviruses were deliberately (Borio et al. 2002; Leffel and Reed 2004), or accidentally aerosolized during normal laboratory or clinical practices (Dimmick et al. 1973; Bennett and Parks 2006), they may pose a significant threat to humans, as they are able to remain infectious over a significant period of time. The results presented in this study are able to provide basic survival data on which hazard management, risk assessments, decontamination and control measures can be implemented to help prevent infection and transmission of disease.