Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE The survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol T.J. Piercy, S.J. Smither, J.A. Steward, L. Eastaugh and M.S. Lever Biomedical Sciences Department, Defence Science and Technology Laboratory, Salisbury, Wiltshire, UK Keywords Abstract aerosol, decay, ebola, filovirus, marburg, survival. Aims: Filoviruses are associated with high morbidity and lethality rates in humans, are capable of human-to-human transmission, via infected material Correspondence such as blood, and are believed to have low infectious doses for humans. Sophie J. Smither, Biomedical Sciences Filoviruses are able to infect via the respiratory route and are lethal at very low Department, Defence Science and Technology doses in experimental animal models, but there is minimal information on Laboratory, Room 201, Building 7a, Dstl, how well the filoviruses survive within aerosol particles. There is also little Porton Down, Salisbury, Wiltshire SP4 0JQ, UK. E-mail: [email protected] 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 2010 ⁄ 0516: received 30 March 2010, revised filoviruses. 7 May 2010 and accepted 11 May 2010 Methods and Results: Filoviruses were tested for their ability to survive in dif- ferent liquids and on different solid substrates at different temperatures. The doi:10.1111/j.1365-2672.2010.04778.x 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 temper- atures 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 filo- viruses under different conditions and provide a basis with which to inform risk assessments and manage exposure to filoviruses. to 100%) (Feldman et al. 1993; Feldman and Klenk Introduction 1996). The average lethality for humans is c. 76% for Viral haemorrhagic fevers can be caused by a range of ZEBOV and 57% for MARV (figures calculated from viral agents. The family Filoviridae consists of two genera, Bausch, et al. 2008) Both viruses are fast acting, with Ebolavirus and Marburgvirus, that contain filoviral species death often occurring within 7–10-day postinfection, (Zaire ebolavirus, Sudan ebolavirus and Bundibugyo ebola- although the incubation period is considered to be virus and Lake Victoria marburgvirus) that can cause acute 2–21 days (Peters and Khan 1999; Borio et al. 2002). To and rapidly progressive haemorrhagic fever in humans. date, the natural reservoir for filoviruses is not known; Two other Ebolavirus species, Cote d’Ivoire ebolavirus and however reports have shown that species of African fruit Reston ebolavirus, cause diseases in primates but are not bat may be the natural reservoir for the virus as antibod- pathogenic in humans. Zaire ebolavirus (ZEBOV) and ies to ZEBOV and MARV have been found in bat species, Lake Victoria marburgvirus (MARV) have been associated and human cases have been linked to bat exposure (Leroy with a number of outbreaks with high fatality rates (up et al. 2005; Pourrut et al. 2009; Towner et al. 2009). ª 2010 British Crown copyright 2010/DSTL – published with the permission of the Controller of Her Majesty’s Stationery Office Journal of Applied Microbiology 109, 1531–1539 ª 2010 The Society for Applied Microbiology 1531 Survival of filoviruses T.J. Piercy et al. Filoviruses are transmitted through contact with body Materials and methods fluids or tissues of humans, nonhuman primates or infected laboratory animals (Brown 1997; Mwanatambwe Virus stocks and containment et al. 2001; Pinzon et al. 2004). Historically, nosocomial transmission often occurs through the reuse of incorrectly ZEBOV strain E718, MARV strain Popp and REBOV were sterilized needles and syringes, or during nursing of an obtained from the Health Protection Agency, Porton infected individual through contact with blood, vomit or Down. ZEBOV strain E718 was a clinical isolate from the other infected secretions (Feldman and Klenk 1996). 1976 outbreak associated with a case fatality rate of 88% Transmission can also occur during burials and the prep- and was one of four isolates (including ZEBOV-Mayinga) aration of bodies for burial (Tukei 1996). taken from the original outbreak. MARV-Popp Filoviruses have been reported as being transmitted (Poppinga) was also a clinical isolate from the original via the aerosol route, either experimentally or within a outbreak in Germany in 1967. The REBOV stock used in biocontainment facility (Jaax et al. 1995; Johnson et al. aerosol decay experiments was originally obtained from 1995; Belanov et al. 1996) and are considered to be Fort Detrick (USAMRIID); however the passage history of potential biological warfare or bioterrorism agents the strain is unknown and is currently being sequenced. (Borio et al. 2002; Bray 2003). In addition, there are Solid and liquid survival studies were conducted with reports that MARV was previously weaponized (Alibek the two most pathogenic species (ZEBOV and MARV). and Handelman 1999). A recent review of persistence of REBOV was included in the aerosol survival studies as an Category A Select Agents in the environment highlighted apathogenic species for humans but associated with anec- how little basic information is known about the filo- dotal evidence of airborne transmission. The stock of viruses (Sinclair et al. 2008). The stability of MARV Sudan ebolavirus (strain Boniface) held at Dstl Porton within aerosols and dried on various substrates was Down has been shown to have an altered phenotype (less investigated by workers in the former Soviet Union virulent in immunodeficient mice) and to have under- (FSU) (Belanov et al. 1996; Chepurnov et al. 1997); gone substantial mutation, compared to stocks held by however survival characteristics of other filoviruses have the United States (J.E. Farlow and S.M. Ibrahim, personal not been investigated. communication, manuscript in preparation). In addition, In the United Kingdom, filoviruses are classified as there was poor recovery of Sudan ebolavirus upon aero- Advisory Committee on Dangerous Pathogens (ACDP) solization (M.S. Lever, unpublished observation), and Hazard Group 4 pathogens [equivalent to Biosafety therefore Sudan ebolavirus was not used in any studies Level 4 (BSL-4)] and are handled under laboratory con- presented here. tainment level 4 (CL4) to minimize the risk of infection All work was performed within primary containment to laboratory personnel. Filoviruses are also classed as facilities housed within ACDP CL4 laboratories at Dstl, Category A biological agents by the CDC as they have Porton Down. The majority of the work was performed high case fatality rates and are easily disseminated and either within an experimental laboratory consisting of a low infectious doses of filoviruses are sufficient to cause cabinet line of interconnected microbiological safety cabi- disease in animal models (Borio et al. 2002). In the nets (Lever et al. 2008) or interconnected rigid half-suit field, the infrastructure to handle the pathogens is not isolators (for aerosol studies). always available, and prevention of spread is reliant on good hygiene and removal of all contaminated and Microtitre TCID end-point dilution assay infectious material. It is unknown how long the filovi- 50 ruses are able to remain viable if waste and contami- African Green monkey kidney cell monolayers (Vero nated areas are not immediately sterilized. If the C1008) were prepared in 96-well microtitre plates (Nunc; filoviruses were aerosolized, either accidentally or as a Thermo Fisher, Basingstoke, UK) at a concentration of deliberate release, it is also unknown how long they 2 · 104 cells per well (Vero C1008 cells obtained from would survive as aerosols, and therefore how many peo- ECACC, catalogue number 85020206; Salisbury, UK) and ple might potentially be exposed. maintained in Dulbecco’s modified Eagle’s medium, Here, we provide data on the stability and viability of DMEM, supplemented with 10% foetal calf serum (FCS), MARV and ZEBOV in both liquid media and on a range and 1% penicillin, streptomycin and l-glutamine. Plates of solid substrates at various temperatures, over time. In were incubated overnight at 37°C in a 5% CO2-humidi- addition, the stability and decay rate of MARV, ZEBOV fied atmosphere, and 80-ll maintenance media (DMEM and Reston ebolavirus (REBOV) within small-particle plus 2% FCS, 1% HEPES and 1% penicillin, streptomycin aerosols held within a modified version of the Goldberg and l-glutamine) was added to columns 2–12 of the drum system (Goldberg et al. 1958) was investigated. plate. To column 1 of the plate, 100 ll of virus test ª 2010 British Crown copyright 2010/DSTL – published with the permission of the Controller of Her Majesty’s Stationery Office 1532 Journal of Applied Microbiology 109, 1531–1539 ª 2010 The Society for Applied Microbiology T.J. Piercy et al. Survival of filoviruses sample (from survival experiments) was added, and 20 ll and glass (glass coverslips, Fisher, Loughborough, UK). was transferred across the plate (1 : 10 dilution). Control A20ll suspension of virus was then dropped onto wells (columns 11 and 12) were left in maintenance 7 ml-diameter discs of plastic, metal or glass and media, and plates were incubated at 37°C in a 5% CO2- allowed to air-dry for 30 min. Discs were stored in humidified atmosphere. At 6-day postinfection, 3 ll (final individual tubes at either +4°C or room temperature at concentration of 1Æ5%) of neutral red stain (Sigma, Gil- a relative humidity of 55 ± 5% for the duration of the lingham, UK) was added to each well, and plates were study.