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An alternative method of measuring aerosol survival using spiders’ webs and its use for the filoviruses
Journal of Virological Methods 177 (2011) 123–127
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
An alternative method of measuring aerosol survival using spiders’ webs and its use for the filoviruses S.J. Smither ∗ , T.J. Piercy, L. Eastaugh, J.A. Steward, M.S. Lever Biomedical Sciences Department, Dstl, Porton Down, Wiltshire, SP4 0JQ, UK
a b s t r a c t Article history: Received 11 April 2011 Received in revised form 22 June 2011 Accepted 29 June 2011 Available online 5 July 2011 Keywords: Ebola Marburg Aerosol Survival Spiders’ webs
Understanding the ability to survive in an aerosol leads to better understanding of the hazard posed by pathogenic organisms and can inform decisions related to the control and management of disease outbreaks. This basic survival information is sometimes lacking for high priority select agents such as the filoviruses which cause severe disease with high case fatality rates and can be acquired through the aerosol route. Microthreads in the form of spiders’ webs were used to capture aerosolised filoviruses, and the decay rates of Zaire ebolavirus and Marburgvirus were determined. Results were compared to data obtained using a Goldberg drum to measure survival as a dynamic aerosol. The two methods of obtaining aerostability information are compared. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
The ability of pathogenic micro-organisms to survive in aerosols is critical to disease transmission. Micro-organisms including bacteria and viruses can be aerosolised either medically, through coughing and sneezing, accidentally through aerosolisation from liquids or during laboratory manipulations, or deliberately in the case of a biowarfare or bioterrorism act. Appreciating the hazard posed by airborne infectious agents is crucial for managing and controlling the hazard; determining aerostability furthers the understanding of the epidemiology of diseases that may be acquired by the aerosol route. Studies into aerosol survival may be limited by a lack of appropriate facilities and equipment and further limited if the pathogens of interest must be handled under biological containment. Contrarily, it is often the hazard posed by high hazard-group pathogens that needs to be understood; a recent review of category A select agents showed gaps in the knowledge on aerosol survival for a number of pathogens (Sinclair et al., 2008). Understanding aerosostability also requires knowledge on the effect of external parameters as any aerosol will be affected by the environment (Tang, 2009). To model the effect of external parameters (such as weather conditions) for aerosolised dangerous pathogens, either appropriate simulants must be used, or external
Abbreviations: MARV, Lake Victoria marburgvirus; ZEBOV, zaire ebolavirus; TCID50 , 50% tissue culture infectious dose. ∗ Corresponding author at: Biomedical Sciences Department, Building 7a, Dstl, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK. Tel.: +44 0 1980 614758; fax: +44 0 1980 614307. E-mail address: [email protected] (S.J. Smither).
factors need to be reproduced safely in the laboratory. Many aerosol studies rely on filling a defined space (such as a drum or chamber; Goldberg et al., 1958) with aerosol and sampling over time. These studies are useful for determining basic information on stability within an aerosol, but the methods are not always flexible enough to alter the conditions to which the aerosol is exposed. A more flexible method of studying aerosol survival using captured aerosols is described; the method was originally proposed by Dessens (1949), commonly credited to May and Druett (1968) who first used it for studying microbiological aerosol decay. Aerosol droplets are captured on microthreads in the form of spiders’ web silk. This system has potential for studying the effect of external factors on survival as after the micro-organisms are captured on the threads, the webs can be manipulated which could allow holding the aerosol in test environments mimicking external factors, (e.g. incubators or humidity chambers to mimic different meteorological conditions), or exposing the captured aerosol to chemicals or light sources to mimic the outside environment. Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) belong to the family Filoviridae and are amongst the most dangerous pathogens known to man with case fatality rates in outbreaks often exceeding 80%. Information on survival of filoviruses in aerosols is useful to medical personnel in outbreak situations, laboratory workers who might be exposed to aerosols and policy and decision makers who might be faced with a potential attack using aerosolised filovirus. Previously a Goldberg drum has been used to study the aerosol survival of several species of the family Filoviridae (Piercy et al., 2010) and showed that, as a dynamic aerosol, ZEBOV and MARV were quite aerostable, and in the case
0166-0934/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.06.021
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S.J. Smither et al. / Journal of Virological Methods 177 (2011) 123–127
of MARV, more stable than previously reported (Belanov et al., 1996). ZEBOV and MARV are hazard group 4 pathogens that must be handled under maximum containment (Biosafety Level 4, BSL-4 or Advisory Committee for Dangerous Pathogens, ACDP-Level 4). Limited facilities capable of performing aerosol studies with such pathogens exist. Database searches suggest the microthread technique has not been used with viruses that are highly pathogenic in humans. It was determined if the microthread technique could be used to estimate the aerosol survival of ZEBOV and MARV as captured aerosols. There are existing data on the decay rates of ZEBOV and MARV as dynamic aerosols, so comparisons of the methods of determining aerosol survival could be made, and confidence in the results observed using the Goldberg drum could be obtained. The microthread process is as follows: Orb web spiders, Zygiella x-notata of size 5–15 mm were used for production of webs (Fig. 1). Webs were spun over frames using a web winder calibrated for 50 turns. Prior to use webs were sterilised in a hot air oven at 60 ◦ C for 1 h. Auxiliary kit used in experiments included web holders and containers for washing the webs. The final piece of equipment, the ‘sow’ connects to the Henderson apparatus and holds the webs for exposure to an aerosol (Fig. 1). Sterilised webs are inserted into the holders and placed within the sow. Different size and capacity sows can be used. The position of webs within the sow was numbered to allow random selection of webs at different time-points. Aerosol is generated from a liquid suspension using a Collison nebuliser and the aerosol is conditioned in the contained Henderson apparatus (Henderson, 1952, Druett, 1969) and then exposed to the webs held in the sow. After webs have been sprayed they can be retained in the sow or removed for exposure to alternative environmental conditions. Recovery of micro-organisms from webs is through washing the webs in an appropriate wash/collection fluid contained within vessels designed to fit the web frames. Enumeration is then performed using the wash solution. Sows and web frames are lightweight and can be manipulated easily within high containment. ZEBOV strain E718 and MARV strain Popp (described previously in Piercy et al., 2010) were grown in Monkey kidney C1008 Vero cells and harvested at day 9. Viral titre was enumerated and 4 ml stocks were stored at −80 ◦ C. The spray solution was harvested virus in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 2% fetal calf serum (FCS), and 1% penicillin, streptomycin and l-glutamine (all Sigma). The wash solution was DMEM with 2% FCS and l-glutamine. Penicillin and streptomycin (1%) were added after samples were removed for bacterial enumeration. Bacillus atropheus (BG) was sprayed alongside ZEBOV or MARV by addition of 1 ml BG spores at 1 × 107 cfu ml−1 to 4 ml virus at between 1 × 106 and 1 × 108 TCID50 ml−1 . Virus/bacterial suspension was sprayed for 2 min over the webs within the sow. The aerosol stream was maintained at 50–55% relative humidity and 22 ◦ C ± 3 ◦ C. After spraying, the webs were held in the sow i.e. maintained in the dark under ambient conditions. At time t = 0, 5, 15, 30, 45 and 60 min, 3 webs (determined randomly across the sow) were removed and washed in 3 ml wash solution until all visible web strands had been removed from the web frame into solution. A 100 l sample was removed from the wash samples for BG enumeration prior to the addition of Penicillin and Streptomycin and downstream virological quantification. For BG quantification, 100 l sample was serially diluted 1:10 in PBS to a dilution of 10−3 , 100 l of each dilution was plated out onto Nutrient agar in duplicate and incubated at room temperature for 3 days for BG quantification. The 50% Tissue Culture Infectious dose (TCID50 ) assay was used for virus enumeration and has been described previously (Piercy
et al., 2010). Briefly, Vero C1008 cell monolayers are infected with virus sample (wash solution from the microthreads) and serially diluted across a 96-well plate, a column of 8 wells per dilution. After 6 days of incubation, cells are stained with neutral red (1.5%) for 24 h and fixed with 10% formal saline and the presence of cytopathic effect is scored for each well. The TCID50 value is calculated by the method of Reed and Muench (1938). The limit of the assay (under conditions described) is 3.72 TCID50 ml−1 . Five replicate runs were performed for each virus, and, for all experiments, the same Henderson apparatus and Collison nebuliser were used to produce and condition the aerosol particles (Henderson, 1952). This allowed consistent and defined aerosolisation and delivery onto the microthreads. Data was analysed in Microsoft Excel and Prism v4. In Prism, an F-test was used to determine if slopes were different at the 95% confidence level. Data using the microthreads was compared with data previously obtained using the Goldberg drum which measured stability as a dynamic aerosol. Aerosol decay in the Goldberg drum was calculated by filling a stainless steel drum (40 l) with aerosolised virus and BG. The drum was filled for 2 min and mixed for 5 min prior to sampling at various time points by withdrawing samples from the drum for 1 min using an all glass impinger sampling at 4 l min−1 . Enumeration of virus and BG in the impinger was using the same assays as described above. Three replicates runs were performed in the drum for each virus. The method is described further in Piercy et al. (2010). Validation experiments (not shown) confirmed even deposition of aerosol particles across the length of the sow. Positions in the sow were numbered so webs could be removed in a random order. Decay of MARV, ZEBOV and BG is shown (Fig. 2). For each, results show 5 replicates of web runs, and 3 replicates of drum runs (data from Piercy et al., 2010). The capacity of the drum, compared to the current sow, enabled an extra time-point to be taken at 90 min. There was slightly more variation between runs with the microthread method which is why more replicates were performed. The efficiency of washing of threads, and therefore of sampling was not determined directly. However, web strands could be visualised so it was possible to observe when all webs had been removed from a frame, and the inclusion of BG as a control showed consistent collection efficiency. Using the microthread technique the filoviruses showed decay over time, but could still be recovered from a captured aerosol after one hour. Using the microthread technique, BG spores showed no decay over time. Previously, using the Goldberg drum (Piercy et al., 2010) BG decayed at a rate of ∼1.5% per minute and others have made similar observations of BG decay using the dynamic aerosol system. The decay of BG in the drum is likely due to the spores impacting on the side and falling out of the aerosol. The physical decay in the drum system means any decay rates determined will be a combination of physical and biological decay. Using the microthread system, there appears to be minimal or no decay of BG on the webs, (top panel Fig. 2). This observation suggests there is no physical decay associated with the web system, which is advantageous compared to the drum system, as it means results are not complicated, and observations are solely down to biological decay. The TCID50 assay was used to measure virus amounts over time as TCID50 per ml of collection fluid (wash solution). This assay measures viable virus particles and had been used in the past, so for consistency in comparison with other data produced in the laboratory, and confidence of measuring viable filovirus, the assay was preferred to PCR methods. From these preliminary microthread aerosol studies, and the decay equation Yt = Y0 e−kt fitted to the average count over time, the decay of MARV and ZEBOV were estimated at 3.15% min−1 and 3.43% min−1 , respectively. From our drum experiments the total decay rates were estimated to be 4.81% and 4.29% min−1 for MARV and ZEBOV, respectively. These drum
S.J. Smither et al. / Journal of Virological Methods 177 (2011) 123–127
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Fig. 1. The microthread process for analysing aerosol decay. Spiders (A and B) are used to wind webs over frames (C and D). (D) shows a frame post spray with visible coating on web threads. Auxiliary equipment used includes the Sow (arrow in E) which holds the frames for exposure, and the Collison nebuliser (circled in E) which produces the aerosol spray that is conditioned in the Henderson apparatus. Close up of web holder (F) and glass vessels for washing webs (G) also shown. All photos taken by S.J. Smither and M.S. Lever. Photos C–E were taken during experiments with MARV, and show the apparatus in action within a BSL-4 laboratory using a rigid half suit isolator for primary containment.
data are a sum of biological and physical decay. If physical decay associated with the drum system is approximately 1.5% min−1, then the biological decay of the filoviruses in the drum can be estimated as 3.31% min−1 (MARV) and 2.79% min−1 (ZEBOV). Therefore the decay rates from the two systems appear similar and suggest an aerosol decay rate for the filoviruses of ∼3% min−1 . F-test analysis on the data from web and drum runs suggested there was no statistically significant difference between the gradient of the slopes for both MARV and ZEBOV using either method of measuring aerosol decay. Linear regression of the BG decay from the web and drum sprays indicated there was a statistically significant difference between the two methods for BG (P = 0.000467). These initial results suggest that both the drum and the microthread techniques are useful methods for evaluating aerosol decay. Under similar conditions (ambient temperature and humidity and enclosed in the dark) they produced similar results so either method can be used in future studies. The web system offers a number of advantages of the drum system in terms of flexibility and ease of use, particularly at high containment. The microthread system is more flexible than the drum system as exposed webs can be manipulated in a number of ways. Having demonstrated the two systems produce similar results, it increases confidence in the baseline decay of MARV and ZEBOV. Prior to the drum data, no decay rate for any ebolavirus had been published, and the published data for MARV reported a decay rate of 11.5% min−1 (Belanov et al., 1996).
The findings with the drum, and now verified with the web method suggest a much lower decay rate which suggested the hazard posed by aerosolised filoviruses may have been underestimated. Future studies using the microthread system will look at the effect of different environmental parameters on aerosol survival; particularly investigating the effect of temperature, humidity or exposure to (simulated) sunlight to mimic conditions of a release in the environment. These studies with altered parameters would be harder to perform in a drum or chamber as it is technically challenging to expose a dynamic aerosol to consistent altered environmental conditions. Aerosols captured on microthreads however, can be more easily manipulated as the aerosol can be moved around. The microthread system can be more easily used to mimic realistic outdoor conditions in which an aerosol release might occur which would lead to more relevant models for studying epidemiology or the effect of a release of virus. This system has also allowed for the study of ‘open air factor’ (OAF) where substances in the air can affect aerostability (Druett and May, 1968; May et al., 1969; Hood, 1971). Previously, the microthread technology has been used infrequently with viruses and there seems to have been no previous microthread experiments with highly virulent human pathogenic viruses of concern. It has been demonstrated that the method can be used at high containment and therefore could also be applied to a wide range of other viruses of interest where the handling pre-
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cautions are less stringent. Primarily in the past the microthread system has been used to study the relationship of OAF on viruses. Newcastle Disease virus exposed to open air on microthreads gave similar results to studies with virus in a “freebourne aerosol” (Hugh-Jones et al., 1973). The microthread method was also used to look at the survival of foot-and-mouth disease virus; no virus decay was observed on webs held in the sow, but decay was seen on webs exposed to external conditions (Donaldson and Ferris, 1975). The decay rate of Semliki Forest on microthreads was greater than achieved in the Goldberg drum or other closed containers (0.1% vs ∼2%; Benbough and Hood, 1971). A direct comparison of decay rates obtained using the two different methods has been provided. It has been observed that, for initial experiments with the filoviruses, similar results are obtained with either method. This suggests either technique could be used for future work, the choice likely to be dependent on which method is more suitable for the studies being performed. Experiments have shown that the Collison nebuliser used to generate the aerosol particles in this study produces particles of a average size of 4 m (Thomas et al., 2008). In the future it is hoped to connect apparatus capable of producing large particle aerosols to the microthread system to determine if larger particles (average size 12 m) show different decay rates. It is unlikely large particle aerosols could be maintained in a dynamic aerosol system, demonstrating another advantage of the microthread system. It is also hoped to develop methods of preserving the captured aerosol particles on the microthreads so the captured aerosol particles can be visualised and enumerated. The microthread system initially appears to have given more variable results between replicate runs than the drum system which may make it harder to observe subtle differences in decay rates. The power of the experiments however, can be increased if the number of runs and replicates is increased. The drum system is limited by the capacity of the drum and the number of samples that can be removed (which may also introduce a dilution factor) and the conditions the aerosol can be exposed to are also limited. The microthread system is limited by the size of the sow and the number of webs it holds but sows are smaller and easier to manipulate so more suited to work in high containment. Larger sows, or multiple sows connected in parallel could increase the capacity of the experiment and therefore the number of samples that can be collected. In conclusion, the microthread method of using spiders’ webs to capture aerosols is useful for measuring aerosol decay. The microthread method has been demonstrated with two filovirus species and gained consistent results comparable to those obtained using a dynamic aerosol system. The microthread method is flexible for use at high containment and would allow the study of the effect of different parameters on persistence within an aerosol. Acknowledgements The authors gratefully acknowledge S. Eley for technical discussions and helpful advice, and T. Hawkyard and S Bailey for recommendations in using the spiders’ web methodology. References Fig. 2. Aerosol decay of BG, MARV and ZEBOV using the Goldberg drum or Microthread technique. Filoviruses MARV (middle) or ZEBOV (bottom) were aerosolised along with BG spores (top). Aerosols were either maintained as a dynamic aerosol (circles, solid line) in a Goldberg drum, or captured on spiders’ webs (diamonds, dashed line). Samples were collected over time and enumerated for virus and bacteria. Drum data is from previous studies (Piercy et al., 2010). Drum data is from three replicates, web data is from five replicates. Error bars are standard error of the mean. Best fit lines were used for F-test analysis in Prism 4. Virus is measured as TCID50 per ml collection fluid.
Belanov, E.F., Muntianov, V.P., Kriuk, V.D., Sokolov, A.V., Bormotov, N.I., P’Iankov, O.V., Sergeev, A.N., 1996. Survival of Marburg virus infectivity on contaminated surfaces and in aerosols. Vopr. Virusol. 41, 32–34 (translation). Benbough, J.E., Hood, A.M., 1971. Viricidal activity of open air. J. Hyg. (London) 69, 619–626. Dessens, H., 1949. The use of spiders’ threads in the study of condensation nuclei. Q. J. R. Met. Soc. 75, 23–26. Donaldson, A.I., Ferris, N.P., 1975. The survival of foot-and-mouth disease virus in open air conditions. J. Hyg. (London) 74, 409–416.
S.J. Smither et al. / Journal of Virological Methods 177 (2011) 123–127 Druett, H.A., 1969. A mobile form of the Henderson apparatus. J. Hyg. (London) 67, 437–448. Druett, H.A., May, K.R., 1968. Unstable germicidal pollutant in rural air. Nature 220, 395–396. Goldberg, L.J., Watkins, H.M., Boerke, E.E., Chatigny, M.A., 1958. The use of a rotating drum for the study of aerosols over extended periods of time. Am. J. Hyg. 68, 85–93. Henderson, D.W., 1952. An apparatus for the study of airborne infection. J. Hyg. 50, 53–68. Hood, A.M., 1971. An indoor system for the study of biological aerosols in open air conditions. J. Hyg. (London) 69, 607–617. Hugh-Jones, M., Allan, W.H., Dark, F.A., Harper, G.J., 1973. The evidence for the airbourne spread of newcastle disease. J. Hyg. (London) 71, 325–339. May, K.R., Druett, H.A., 1968. A microthread technique for studying the viability of microbes in a simulated airborne state. J. Gen. Microbiol. 51, 353–366.
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May, K.R., Druett, H.A., Packman, L.P., 1969. Toxicity of open air to a variety of microorganisms. Nature 221, 1146–1147. Piercy, T.J., Smither, S.J., Steward, J.A., Eastaugh, L., Lever, M.S., 2010. The survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol. J. Appl. Micobiol. 109, 1531–1539. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497. Sinclair, R., Boone, S.A., Greenberg, D., Keim, P., Gerba, C.P., 2008. Persistence of category A select agents in the environment. Appl. Environ. Microbiol. 74, 555–563. Tang, J.W., 2009. The effect of environmental parameters on the survival of airborne infectious agents. J. R. Soc. Interface 6, S737–S746. Thomas, R.J., Webber, D., Sellors, W., Collinge, A., Frost, A., Stagg, A.J., Bailey, S.C., Jayasekera, P.N., Taylor, R.R., Eley, S., Titball, R.W., 2008. Characterization and deposition of respirable large- and small-particle bioaerosols. Appl. Environ. Microbiol. 74 (October), 6437–6443.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
An alternative method of measuring aerosol survival using spiders’ webs and its use for the filoviruses S.J. Smither ∗ , T.J. Piercy, L. Eastaugh, J.A. Steward, M.S. Lever Biomedical Sciences Department, Dstl, Porton Down, Wiltshire, SP4 0JQ, UK
a b s t r a c t Article history: Received 11 April 2011 Received in revised form 22 June 2011 Accepted 29 June 2011 Available online 5 July 2011 Keywords: Ebola Marburg Aerosol Survival Spiders’ webs
Understanding the ability to survive in an aerosol leads to better understanding of the hazard posed by pathogenic organisms and can inform decisions related to the control and management of disease outbreaks. This basic survival information is sometimes lacking for high priority select agents such as the filoviruses which cause severe disease with high case fatality rates and can be acquired through the aerosol route. Microthreads in the form of spiders’ webs were used to capture aerosolised filoviruses, and the decay rates of Zaire ebolavirus and Marburgvirus were determined. Results were compared to data obtained using a Goldberg drum to measure survival as a dynamic aerosol. The two methods of obtaining aerostability information are compared. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
The ability of pathogenic micro-organisms to survive in aerosols is critical to disease transmission. Micro-organisms including bacteria and viruses can be aerosolised either medically, through coughing and sneezing, accidentally through aerosolisation from liquids or during laboratory manipulations, or deliberately in the case of a biowarfare or bioterrorism act. Appreciating the hazard posed by airborne infectious agents is crucial for managing and controlling the hazard; determining aerostability furthers the understanding of the epidemiology of diseases that may be acquired by the aerosol route. Studies into aerosol survival may be limited by a lack of appropriate facilities and equipment and further limited if the pathogens of interest must be handled under biological containment. Contrarily, it is often the hazard posed by high hazard-group pathogens that needs to be understood; a recent review of category A select agents showed gaps in the knowledge on aerosol survival for a number of pathogens (Sinclair et al., 2008). Understanding aerosostability also requires knowledge on the effect of external parameters as any aerosol will be affected by the environment (Tang, 2009). To model the effect of external parameters (such as weather conditions) for aerosolised dangerous pathogens, either appropriate simulants must be used, or external
Abbreviations: MARV, Lake Victoria marburgvirus; ZEBOV, zaire ebolavirus; TCID50 , 50% tissue culture infectious dose. ∗ Corresponding author at: Biomedical Sciences Department, Building 7a, Dstl, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK. Tel.: +44 0 1980 614758; fax: +44 0 1980 614307. E-mail address: [email protected] (S.J. Smither).
factors need to be reproduced safely in the laboratory. Many aerosol studies rely on filling a defined space (such as a drum or chamber; Goldberg et al., 1958) with aerosol and sampling over time. These studies are useful for determining basic information on stability within an aerosol, but the methods are not always flexible enough to alter the conditions to which the aerosol is exposed. A more flexible method of studying aerosol survival using captured aerosols is described; the method was originally proposed by Dessens (1949), commonly credited to May and Druett (1968) who first used it for studying microbiological aerosol decay. Aerosol droplets are captured on microthreads in the form of spiders’ web silk. This system has potential for studying the effect of external factors on survival as after the micro-organisms are captured on the threads, the webs can be manipulated which could allow holding the aerosol in test environments mimicking external factors, (e.g. incubators or humidity chambers to mimic different meteorological conditions), or exposing the captured aerosol to chemicals or light sources to mimic the outside environment. Zaire ebolavirus (ZEBOV) and Lake Victoria marburgvirus (MARV) belong to the family Filoviridae and are amongst the most dangerous pathogens known to man with case fatality rates in outbreaks often exceeding 80%. Information on survival of filoviruses in aerosols is useful to medical personnel in outbreak situations, laboratory workers who might be exposed to aerosols and policy and decision makers who might be faced with a potential attack using aerosolised filovirus. Previously a Goldberg drum has been used to study the aerosol survival of several species of the family Filoviridae (Piercy et al., 2010) and showed that, as a dynamic aerosol, ZEBOV and MARV were quite aerostable, and in the case
0166-0934/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.06.021
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of MARV, more stable than previously reported (Belanov et al., 1996). ZEBOV and MARV are hazard group 4 pathogens that must be handled under maximum containment (Biosafety Level 4, BSL-4 or Advisory Committee for Dangerous Pathogens, ACDP-Level 4). Limited facilities capable of performing aerosol studies with such pathogens exist. Database searches suggest the microthread technique has not been used with viruses that are highly pathogenic in humans. It was determined if the microthread technique could be used to estimate the aerosol survival of ZEBOV and MARV as captured aerosols. There are existing data on the decay rates of ZEBOV and MARV as dynamic aerosols, so comparisons of the methods of determining aerosol survival could be made, and confidence in the results observed using the Goldberg drum could be obtained. The microthread process is as follows: Orb web spiders, Zygiella x-notata of size 5–15 mm were used for production of webs (Fig. 1). Webs were spun over frames using a web winder calibrated for 50 turns. Prior to use webs were sterilised in a hot air oven at 60 ◦ C for 1 h. Auxiliary kit used in experiments included web holders and containers for washing the webs. The final piece of equipment, the ‘sow’ connects to the Henderson apparatus and holds the webs for exposure to an aerosol (Fig. 1). Sterilised webs are inserted into the holders and placed within the sow. Different size and capacity sows can be used. The position of webs within the sow was numbered to allow random selection of webs at different time-points. Aerosol is generated from a liquid suspension using a Collison nebuliser and the aerosol is conditioned in the contained Henderson apparatus (Henderson, 1952, Druett, 1969) and then exposed to the webs held in the sow. After webs have been sprayed they can be retained in the sow or removed for exposure to alternative environmental conditions. Recovery of micro-organisms from webs is through washing the webs in an appropriate wash/collection fluid contained within vessels designed to fit the web frames. Enumeration is then performed using the wash solution. Sows and web frames are lightweight and can be manipulated easily within high containment. ZEBOV strain E718 and MARV strain Popp (described previously in Piercy et al., 2010) were grown in Monkey kidney C1008 Vero cells and harvested at day 9. Viral titre was enumerated and 4 ml stocks were stored at −80 ◦ C. The spray solution was harvested virus in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 2% fetal calf serum (FCS), and 1% penicillin, streptomycin and l-glutamine (all Sigma). The wash solution was DMEM with 2% FCS and l-glutamine. Penicillin and streptomycin (1%) were added after samples were removed for bacterial enumeration. Bacillus atropheus (BG) was sprayed alongside ZEBOV or MARV by addition of 1 ml BG spores at 1 × 107 cfu ml−1 to 4 ml virus at between 1 × 106 and 1 × 108 TCID50 ml−1 . Virus/bacterial suspension was sprayed for 2 min over the webs within the sow. The aerosol stream was maintained at 50–55% relative humidity and 22 ◦ C ± 3 ◦ C. After spraying, the webs were held in the sow i.e. maintained in the dark under ambient conditions. At time t = 0, 5, 15, 30, 45 and 60 min, 3 webs (determined randomly across the sow) were removed and washed in 3 ml wash solution until all visible web strands had been removed from the web frame into solution. A 100 l sample was removed from the wash samples for BG enumeration prior to the addition of Penicillin and Streptomycin and downstream virological quantification. For BG quantification, 100 l sample was serially diluted 1:10 in PBS to a dilution of 10−3 , 100 l of each dilution was plated out onto Nutrient agar in duplicate and incubated at room temperature for 3 days for BG quantification. The 50% Tissue Culture Infectious dose (TCID50 ) assay was used for virus enumeration and has been described previously (Piercy
et al., 2010). Briefly, Vero C1008 cell monolayers are infected with virus sample (wash solution from the microthreads) and serially diluted across a 96-well plate, a column of 8 wells per dilution. After 6 days of incubation, cells are stained with neutral red (1.5%) for 24 h and fixed with 10% formal saline and the presence of cytopathic effect is scored for each well. The TCID50 value is calculated by the method of Reed and Muench (1938). The limit of the assay (under conditions described) is 3.72 TCID50 ml−1 . Five replicate runs were performed for each virus, and, for all experiments, the same Henderson apparatus and Collison nebuliser were used to produce and condition the aerosol particles (Henderson, 1952). This allowed consistent and defined aerosolisation and delivery onto the microthreads. Data was analysed in Microsoft Excel and Prism v4. In Prism, an F-test was used to determine if slopes were different at the 95% confidence level. Data using the microthreads was compared with data previously obtained using the Goldberg drum which measured stability as a dynamic aerosol. Aerosol decay in the Goldberg drum was calculated by filling a stainless steel drum (40 l) with aerosolised virus and BG. The drum was filled for 2 min and mixed for 5 min prior to sampling at various time points by withdrawing samples from the drum for 1 min using an all glass impinger sampling at 4 l min−1 . Enumeration of virus and BG in the impinger was using the same assays as described above. Three replicates runs were performed in the drum for each virus. The method is described further in Piercy et al. (2010). Validation experiments (not shown) confirmed even deposition of aerosol particles across the length of the sow. Positions in the sow were numbered so webs could be removed in a random order. Decay of MARV, ZEBOV and BG is shown (Fig. 2). For each, results show 5 replicates of web runs, and 3 replicates of drum runs (data from Piercy et al., 2010). The capacity of the drum, compared to the current sow, enabled an extra time-point to be taken at 90 min. There was slightly more variation between runs with the microthread method which is why more replicates were performed. The efficiency of washing of threads, and therefore of sampling was not determined directly. However, web strands could be visualised so it was possible to observe when all webs had been removed from a frame, and the inclusion of BG as a control showed consistent collection efficiency. Using the microthread technique the filoviruses showed decay over time, but could still be recovered from a captured aerosol after one hour. Using the microthread technique, BG spores showed no decay over time. Previously, using the Goldberg drum (Piercy et al., 2010) BG decayed at a rate of ∼1.5% per minute and others have made similar observations of BG decay using the dynamic aerosol system. The decay of BG in the drum is likely due to the spores impacting on the side and falling out of the aerosol. The physical decay in the drum system means any decay rates determined will be a combination of physical and biological decay. Using the microthread system, there appears to be minimal or no decay of BG on the webs, (top panel Fig. 2). This observation suggests there is no physical decay associated with the web system, which is advantageous compared to the drum system, as it means results are not complicated, and observations are solely down to biological decay. The TCID50 assay was used to measure virus amounts over time as TCID50 per ml of collection fluid (wash solution). This assay measures viable virus particles and had been used in the past, so for consistency in comparison with other data produced in the laboratory, and confidence of measuring viable filovirus, the assay was preferred to PCR methods. From these preliminary microthread aerosol studies, and the decay equation Yt = Y0 e−kt fitted to the average count over time, the decay of MARV and ZEBOV were estimated at 3.15% min−1 and 3.43% min−1 , respectively. From our drum experiments the total decay rates were estimated to be 4.81% and 4.29% min−1 for MARV and ZEBOV, respectively. These drum
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Fig. 1. The microthread process for analysing aerosol decay. Spiders (A and B) are used to wind webs over frames (C and D). (D) shows a frame post spray with visible coating on web threads. Auxiliary equipment used includes the Sow (arrow in E) which holds the frames for exposure, and the Collison nebuliser (circled in E) which produces the aerosol spray that is conditioned in the Henderson apparatus. Close up of web holder (F) and glass vessels for washing webs (G) also shown. All photos taken by S.J. Smither and M.S. Lever. Photos C–E were taken during experiments with MARV, and show the apparatus in action within a BSL-4 laboratory using a rigid half suit isolator for primary containment.
data are a sum of biological and physical decay. If physical decay associated with the drum system is approximately 1.5% min−1, then the biological decay of the filoviruses in the drum can be estimated as 3.31% min−1 (MARV) and 2.79% min−1 (ZEBOV). Therefore the decay rates from the two systems appear similar and suggest an aerosol decay rate for the filoviruses of ∼3% min−1 . F-test analysis on the data from web and drum runs suggested there was no statistically significant difference between the gradient of the slopes for both MARV and ZEBOV using either method of measuring aerosol decay. Linear regression of the BG decay from the web and drum sprays indicated there was a statistically significant difference between the two methods for BG (P = 0.000467). These initial results suggest that both the drum and the microthread techniques are useful methods for evaluating aerosol decay. Under similar conditions (ambient temperature and humidity and enclosed in the dark) they produced similar results so either method can be used in future studies. The web system offers a number of advantages of the drum system in terms of flexibility and ease of use, particularly at high containment. The microthread system is more flexible than the drum system as exposed webs can be manipulated in a number of ways. Having demonstrated the two systems produce similar results, it increases confidence in the baseline decay of MARV and ZEBOV. Prior to the drum data, no decay rate for any ebolavirus had been published, and the published data for MARV reported a decay rate of 11.5% min−1 (Belanov et al., 1996).
The findings with the drum, and now verified with the web method suggest a much lower decay rate which suggested the hazard posed by aerosolised filoviruses may have been underestimated. Future studies using the microthread system will look at the effect of different environmental parameters on aerosol survival; particularly investigating the effect of temperature, humidity or exposure to (simulated) sunlight to mimic conditions of a release in the environment. These studies with altered parameters would be harder to perform in a drum or chamber as it is technically challenging to expose a dynamic aerosol to consistent altered environmental conditions. Aerosols captured on microthreads however, can be more easily manipulated as the aerosol can be moved around. The microthread system can be more easily used to mimic realistic outdoor conditions in which an aerosol release might occur which would lead to more relevant models for studying epidemiology or the effect of a release of virus. This system has also allowed for the study of ‘open air factor’ (OAF) where substances in the air can affect aerostability (Druett and May, 1968; May et al., 1969; Hood, 1971). Previously, the microthread technology has been used infrequently with viruses and there seems to have been no previous microthread experiments with highly virulent human pathogenic viruses of concern. It has been demonstrated that the method can be used at high containment and therefore could also be applied to a wide range of other viruses of interest where the handling pre-
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cautions are less stringent. Primarily in the past the microthread system has been used to study the relationship of OAF on viruses. Newcastle Disease virus exposed to open air on microthreads gave similar results to studies with virus in a “freebourne aerosol” (Hugh-Jones et al., 1973). The microthread method was also used to look at the survival of foot-and-mouth disease virus; no virus decay was observed on webs held in the sow, but decay was seen on webs exposed to external conditions (Donaldson and Ferris, 1975). The decay rate of Semliki Forest on microthreads was greater than achieved in the Goldberg drum or other closed containers (0.1% vs ∼2%; Benbough and Hood, 1971). A direct comparison of decay rates obtained using the two different methods has been provided. It has been observed that, for initial experiments with the filoviruses, similar results are obtained with either method. This suggests either technique could be used for future work, the choice likely to be dependent on which method is more suitable for the studies being performed. Experiments have shown that the Collison nebuliser used to generate the aerosol particles in this study produces particles of a average size of 4 m (Thomas et al., 2008). In the future it is hoped to connect apparatus capable of producing large particle aerosols to the microthread system to determine if larger particles (average size 12 m) show different decay rates. It is unlikely large particle aerosols could be maintained in a dynamic aerosol system, demonstrating another advantage of the microthread system. It is also hoped to develop methods of preserving the captured aerosol particles on the microthreads so the captured aerosol particles can be visualised and enumerated. The microthread system initially appears to have given more variable results between replicate runs than the drum system which may make it harder to observe subtle differences in decay rates. The power of the experiments however, can be increased if the number of runs and replicates is increased. The drum system is limited by the capacity of the drum and the number of samples that can be removed (which may also introduce a dilution factor) and the conditions the aerosol can be exposed to are also limited. The microthread system is limited by the size of the sow and the number of webs it holds but sows are smaller and easier to manipulate so more suited to work in high containment. Larger sows, or multiple sows connected in parallel could increase the capacity of the experiment and therefore the number of samples that can be collected. In conclusion, the microthread method of using spiders’ webs to capture aerosols is useful for measuring aerosol decay. The microthread method has been demonstrated with two filovirus species and gained consistent results comparable to those obtained using a dynamic aerosol system. The microthread method is flexible for use at high containment and would allow the study of the effect of different parameters on persistence within an aerosol. Acknowledgements The authors gratefully acknowledge S. Eley for technical discussions and helpful advice, and T. Hawkyard and S Bailey for recommendations in using the spiders’ web methodology. References Fig. 2. Aerosol decay of BG, MARV and ZEBOV using the Goldberg drum or Microthread technique. Filoviruses MARV (middle) or ZEBOV (bottom) were aerosolised along with BG spores (top). Aerosols were either maintained as a dynamic aerosol (circles, solid line) in a Goldberg drum, or captured on spiders’ webs (diamonds, dashed line). Samples were collected over time and enumerated for virus and bacteria. Drum data is from previous studies (Piercy et al., 2010). Drum data is from three replicates, web data is from five replicates. Error bars are standard error of the mean. Best fit lines were used for F-test analysis in Prism 4. Virus is measured as TCID50 per ml collection fluid.
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