Arch Virol (2009) 154:115119 DOI 10.

1007/s00705-008-0264-x

BRIEF REPORT

Testing thermal resistance of viruses

Andreas Sauerbrei P. Wutzler

Received: 22 August 2008 / Accepted: 3 November 2008 / Published online: 28 November 2008 Springer-Verlag 2008

Abstract Representative viral strains recommended for virucidal testing of biocides in human medicine were used for testing viral resistance to dry heat using the new Keredusy hot instrument. The results demonstrate that poliovirus type 1 could be inactivated by treatment at 75C for 1 h. For inactivation of adenovirus type 5, 2 h at 85C was needed. The infectivity of polyomavirus SV40 could only be inuenced signicantly by a temperature of 95C over a period of 1 h, whereas vaccinia virus and bovine viral diarrhea virus needed a time interval of 2 h at 95C. The infectivity of bovine parvovirus could not be inuenced signicantly by exposure to 95C for 2 h. In conclusion, human viruses and their surrogates for testing biocides may have a considerable thermal resistance that makes them difcult to be inactivated only by dry heat.

Up to 10% of all infections acquired in the hospital are caused by viruses [17]. In many paediatric settings, viruses account for more than 30% of the cases of hospitalacquired infections [13]. Therefore, it is important to provide information on the possibilities of viral inactivation. Infectivity of viruses can be destroyed effectively by the use of biocides, eg. aldehydes, chlorine-based and peroxygen compounds. By raising temperatures, the virucidal efcacy of biocides can be increased. In practice, not only chemical or thermo-chemical disinfection methods but also thermal disinfection and sterilization procedures are
A. Sauerbrei (&) P. Wutzler Institute of Virology and Antiviral Therapy, Friedrich-Schiller University of Jena, Hans-Knoll-Strasse 2, 07745 Jena, Germany e-mail: andreas.sauerbrei@med.uni-jena.de

recommended. Therefore, detailed information on the inactivation of human viruses by dry heat is required. To date, most data on the thermal resistance of viruses were collected under moist heat conditions. Heating sterilized albumin preparations at 60C for 10 h has been shown historically to yield a hepatitis virus-free, efcacious product [10]. Whereas infectivity of coxsackievirus B3 has been reported to be destroyed by moist heat at 55C for 15 min [6], hepatitis A virus could only be inactivated completely by a temperature of 100C for 2 min [5]. Animal parvoviruses have been reported to be the most heat-resistant viruses against moist heat, depending on the environment in which the viruses were suspended [12]. Thus, bovine parvovirus (BPV) strain Haden was recommended for use in the evaluation of thermo-chemical and thermal disinfection procedures in view of virucidal effectiveness [3]. Using BPV, it has been reported that low moisture may stabilize viruses against heat [4]. In conclusion, viruses may be more resistant to dry heat than to moist heat. In this paper, data on the resistance of different viruses to dry heat using the new Keredusy hot instrument are reported. The following representative viral strains recommended for virucidal testing of biocides in human medicine [1] were used as test viruses: (1) poliovirus type 1 vaccine strain LSc-ab (species Poliovirus, genus Enterovirus, family Picornaviridae), (2) adenovirus type 5 strain adenoid 75 (species Human adenovirus C, genus Mastadenovirus, family Adenoviridae), (3) vaccinia virus strain Elstree (species Vacciniavirus, genus Orthopoxvirus, family Poxviridae), (4) bovine viral diarrhea virus (BVDV) strain NADL (species Bovine viral diarrhea virus, genus Pestivirus, family Flaviviridae) as a surrogate for hepatitis C virus, (5) polyomavirus SV40 strain 777 (species Simian virus 40, genus Polyomavirus, family Polyomaviridae), and

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(6) bovine parvovirus (BPV) strain Haden (species Bovine parvovirus, genus Bocavirus, family Parvoviridae). Table 1 summarizes cell cultures and media used for viral growth and titration. All media were supplemented with 510% fetal calf serum (FCS), 100 U/mL penicillin, and 100 lg/ mL streptomycin sulfate. For viral propagation, the media were used without FCS. With the exception of KOP-R and Klu-R cells, which were incubated in a humid atmosphere containing 1% CO2, cells were cultured at 5% CO2 and 37C. For poliovirus, adenovirus, vaccinia virus, polyomavirus and BPV, the supernatants of virus-infected cell cultures were used for subsequent experiments. For the preparation of BVDV, virus-infected cells were disrupted by ultrasonic treatment, and both the supernatant of sonicated cell suspension and the supernatant of virus-infected cell culture were mixed, and viruses were concentrated by ultracentrifugation. All viruses were used at concentrations of 105.51010.2 tissue culture infective dose 50% (TCID50)/ mL. Viral stocks were stored at -80C. Carrier tests were carried out using a modied method described previously [14]. Stainless steel disks (EN 10088-1), 20 mm in diameter with a thickness of 1.21.5 mm and a plane surface (quality 2 B according EN 10088-2), were used as viral carriers. The metal disks were cleaned by placing in 5% Decon (Decon Laboratories, Hove, UK) for 60 min, rinsed with sterile distilled water for 20 s, treated with 70% (v/v) ethanol for 15 min and air dried. At the centre of each disk carrier, 100 lL of virus suspension was deposited. The disks were dried under a laminar ow box for 2 h and stored at room temperature for up to 5 h. For thermal inactivation of viruses, the Keredusy hot instrument (Medizin&Service GmbH, Chemnitz, Germany) was used. This instrument provided dry airow of dened temperature. Within a hose system, the airow was led to a plastic box containing a plastic carrier of the metal disks. In the plastic carrier, three metal disks could be placed and xed one on top of another with vertical distance of about

5 mm to allow contact of airow with the inoculated surface of each metal carrier. The temperature in the box containing carriers was controlled by using a temperature sensor and a digital thermometer. The different virus strains dried on stainless steel carriers were exposed to temperatures between 40 2 and 95 2C for 1 and 2 h. The treated disks were placed in the wells of 12-well cell culture plates, and 1 mL ice-cold cell culture medium plus glass beads was added. After vortexing for 1 min, suspensions were serially diluted ten-fold with ice-cold cell culture medium, and 200 lL of each was seeded into micro-plate wells with cell culture for measuring the TCID50. For each dilution, 8 wells were inoculated. In parallel, untreated inoculated disks served as control. Three inoculated steel disks were treated for one experiment, and all experiments were performed twice. For statistical analysis, the reduction values of viral titres expressed as log10 and the 95% condence intervals were calculated [1]. According to virucidal testing of chemical biocides, the criterion used for assessment of signicant reduction in viral titre was a C4 log10 reduction of virus concentration, corresponding to 99.99% inactivation. One-hour exposure to dry heat at a temperature of 40C did not inuence the infectivity of any of the virus strains included in this study (Table 2). After exposure to higher temperatures, the most sensitive virus was poliovirus, whose infectivity in cell culture was reduced signicantly at 7595C after 1 h. The polyomavirus SV40 was inactivated signicantly when it was exposed to 95C for 1 h. Other viral strains, including adenovirus type 5, vaccinia virus, BVDV and BPV, could not be inactivated signicantly by 1-h exposition to temperatures between 75 and 95C. Thermal inactivation was more effective when the viral strains were exposed to temperatures between 75 and 95C for 2 h (Table 3). The results conrmed that poliovirus was the most sensitive virus that was inactivated signicantly at a temperature of 75C for 2 h. The

Table 1 Viral strains, cell cultures and media used for this study Viral strains Poliovirus type 1 vaccine strain LSc-ab Adenovirus type 5 strain adenoid 75 Vaccinia virus strain Elstree Bovine viral diarrhea virus (BVDV) strain NADL Polyomavirus SV40 strain 777 Bovine parvovirus (BPV) strain Haden Cell cultures African green monkey cells (BGM) Carcinomic human alveolar basal epithelial cells (A549) African green monkey cells (BGM) Bovine pharyngeal cells (KOP-R) African green monkey kidney broblasts (CV-1) Fetal calf lung cells (Klu-R) Media Earls modied Eagles medium (EMEM) Dulbeccos modied Eagles medium (DMEM) Dulbeccos modied Eagles medium (DMEM) Minimal essential medium ? Hanks solution Dulbeccos modied Eagles medium (DMEM) Minimal essential medium ? Hanks solution

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Testing thermal resistance of viruses Table 2 Reduction in viral titres after exposure of model viruses to temperatures of 40, 75, 85 and 95C for 1 h Model virus Viral titre (log10 95% condence interval) 40 2C Control Poliovirus type 1 Adenovirus type 5 75 2C Titre reduction Control 85 2C Titre reduction Control 95 2C Titre reduction Control

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Titre reduction

7.4 0.28 0.7 0.13 9.4 0.25 0.5 0.15

7.7 0.22 4.3 0.13 9.4 0.30 0.7 0.16 7.1 0.25 0.3 0.15 5.2 0.44 0.9 0.17 8.6 0.26 0.8 0.17 4.5 0 0 0.29

6.7 0.33 4.8* 9.4 0.30 1.2 0.17 6.8 0.37 1.6 0.15 5.4 0.28 1.5 0.20 7.9 0.24 2.7 0.19 4.6 0.41 0.1 0.23

7.1 0.26 5.6* 9.4 0.28 3.7 0.15 6.7 0.26 2.1 0.15 5.9 0.25 2.8 0.17 8.6 0.25 5.2 0.12 5.6 0.41 0.8 0.20

Vaccinia virus 6.9 0.31 0 0.18 Bovine viral diarrhea virus 5.6 0.38 0.2 0.17 Polyomavirus SV40 Bovine parvovirus 5.9 0.29 0.1 0.13 4.6 0.41 0 0.20

The values are means of the results of two single experiments. Values in bold type represent signicant reduction in viral titre * 95% condence interval could not be calculated

Table 3 Reduction in viral titres after exposure of model viruses to temperatures of 75, 85 or 95C for 2 h Model virus Viral titre (log10 95% condence interval) 75 2C Control Poliovirus type 1 Adenovirus type 5 Vaccinia virus Bovine viral diarrhea virus Polyomavirus SV40 Bovine parvovirus 7.0 0.27 8.4 0.29 7.0 0.30 5.4 0.21 7.1 0.29 5.4 0.50 Titre reduction 4.3* 1.9 0.17 0.6 0.15 1.0 0.13 2.3 0.16 0.1 0.29 85 2C Control ND 8.0 0.31 6.6 0.22 5.4 0.22 7.3 0.29 5.6 0.41 Titre reduction ND 5.5* 1.6 0.14 1.7 0.14 3.4 0.17 0.6 0.28 95 2C Control ND 8.0 0.24 7.1 0.21 5.2 0.23 7.9 0.30 4.9 0.25 Titre reduction ND 6.2* 4.3* 4.0* 5.1* 0.9 0.14

The values are means of the results of two single experiments. Values in bold type represent signicant reduction in viral titre * 95% condence interval could not be calculated; ND not done

infectivity of adenovirus type 5 was reduced signicantly when the virus was treated with dry heat at 85 and 95C. Strains of vaccinia virus, BVDV und polyomavirus SV40 could only be inactivated by a temperature of 95C for 2 h. The BPV strain Haden had the highest resistance to dry heat. The infectivity of this viral strain could only be inuenced slightly by treatment with temperatures up to 95C over an interval of 2 h. It has been accepted generally that viruses can show considerable thermal resistance after being dried on surfaces and exposed to dry heat [16]. However, detailed information on resistance of viruses belonging to different viral families is not available. To date, ndings on viral resistance to dry heat have only been provided in few studies using single viruses such as the BPV strain Haden prepared in lyophilisates [4, 11]. This virus was found to be very resistant up to 100C [4]. To our knowledge, the present study provides, for the rst time, data on the resistance of representative lipophilic and hydrophilic viruses against dry heat. The carrier test used simulated practical clinical conditions comparable with drying of virus-containing blood or secretions, even though

additional proteins were not added to the viral suspensions. The experiments were possible thanks to the availability of the new Keredusy hot instrument, which has been shown to be useful for reliable testing of resistance of viruses to dry heat. The ndings of this study demonstrated that the hydrophilic poliovirus, which lacks an envelope, belongs to the most heat-sensitive viruses that could be inactivated by temperatures of C75C for 1 h. Nevertheless, this study conrms data described by Shiomi et al. [15], who reported that heating at 50C for 30 min drastically decreases the infectivity of poliovirus. These ndings are in contrast to the high resistance of this virus against chemical biocides [18]. The infectivity of adenovirus type 5, a non-enveloped virus with capsomeric lipophilicity, was reduced signicantly by dry heat at C85C for 2 h. A comparable thermal resistance was demonstrated for the less lipophilic enveloped vaccinia virus, the more lipophilic enveloped BVDV and the less hydrophilic polyomavirus SV40, which lacks an envelope. These viruses could only be inactivated within 2 h at 95C with exception of the polyomavirus SV40, whose infectivity could also be

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inuenced signicantly by 95C over a period of 1 h. As conrmed by this study, BPV has the highest thermal resistance to dry heat. Even at temperatures of 95C over a period of 2 h, the infectivity of BPV, classied as hydrophilic virus without envelope, could only be reduced by approximately 1 log10. In comparison, Brauninger et al. [4] found a similar titre reduction after exposure to dry heat of 100C for 30 min using BPV lyophilisates in 20% human albumin. The viral inactivation was highly dependent on the residual moisture of the lyophilisate. Since BPV shows thermal resistance comparable to hepatitis B virus, this virus has been proposed as surrogate to verify the efcacy of thermal disinfection techniques against hepatitis B virus [4]. Surprisingly, the thermal resistance of poliovirus and BPV, both characterized as non-enveloped viruses with high hydrophilicity, is very different. These results show that the classication of viruses according to their assembly and lipophilicity or hydrophilicity is useful to characterize sensitivity to chemical biocides, but is not helpful for characterization of thermal resistance. In addition, the ndings suggest that the chemical and physical inactivation of viruses takes place at different points of action. First of all, these results suggest that the presence or absence of a viral envelope most likely has no inuence on the thermal resistance of viruses, in contrast to their sensitivity to chemical biocides. Studies on different parvoviruses have revealed that the genomic DNA release from intact capsids seems to be a common feature accompanying thermal viral inactivation [8]. In this context, electron microscopic examination has demonstrated that poliovirus particles were converted to empty particles devoid of RNA during thermal inactivation [15]. Since infectious coxsackievirus B3 was undetectable after thermal treatment, while the viral genome was still detected, it has been suggested that the loss of viral infectivity may be mostly due to cleavage or conformation changes in the viral capsid without signicant destruction of the genome [6]. As several studies have shown, signicant differences in thermal resistance may be detected within viral families. While animal parvoviruses largely withstand pasteurization of albumin, parvovirus B19 is inactivated rapidly [2]. Furthermore hepatitis A virus has to be considered much more heat-resistant than other members of the picornavirus family such as poliovirus and coxsackievirus [5, 6]. Recently, viral strain-specic differences in heat resistance have been reported for foot-and-mouth disease virus [7]. In addition, mutations in the capsid region may contribute to the development of heat-resistant viral mutants, which have been reported for poliovirus type 1 [15] and reovirus [9]. In summary and conclusion, the Keredusy hot instrument is very useful for testing thermal resistance of viruses.

Human viruses and their surrogates may have a considerable thermal resistance when they are dried on surfaces. This makes them difcult to be inactivated only by dry heat. Therefore, the present ndings have to be considered in recommendations for viral heat inactivation.
Acknowledgments Medizin&Service GmbH (Chemnitz, Germany) supported this work by providing the Keredusy hot instrument.

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