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Viral infectivity is maintained by an RNA protection buffer
Journal of Virological Methods 128 (2005) 189–191
Short communication
Viral infectivity is maintained by an RNA protection buffer Christine Uhlenhaut ∗ , Maren Kracht Center for Biological Safety, Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany Received 10 February 2005; received in revised form 26 April 2005; accepted 3 May 2005 Available online 4 June 2005
Abstract RNA stabilization buffers are valuable additives which are used widely. Although their effect on RNA protection has been well assessed, the impact on viral infectivity was unsettled. The potential stabilizing or inactivating effects of RNAlater (Qiagen) on the infectivity of different relevant and model viruses including HIV were assessed. Our results show that RNAlater not only protects RNA as published by others but also stabilizes virus infectivity of enveloped and non-enveloped model viruses for a considerable period of time on a relatively high level (storage at room temperature for 50 days yielded infectious titers >100 TCID50 ). © 2005 Elsevier B.V. All rights reserved. Keywords: Virus safety; RNA stabilization; RNAlater
RNA stabilization and protection during transportation and storage of samples is a prerequisite, e.g., for reliable gene expression analysis. Not only ubiquitous RNases and easy hydrolysis of RNA but also short half-lives of mRNA subpopulations pose significant problems. The need for stabilization of RNA is met currently by different RNA protection systems. These efficient buffers are used widely for sampling, storing and for stable transportation of diagnostic material, viruses or various other potentially infectious materials such as whole blood, PBMCs, tissue or feces. RNAlater (Qiagen) is a high salt solution (25 mM sodium citrate, 10 mM EDTA, 70 g ammonium sulphate/100 ml solution, pH 5.2) and was developed as a non-toxic storage reagent for tissue (cellular RNA) but can also be used for RNA protection in lymphocytes or fecal samples (Lee et al., 2002; Whittier et al., 2004). Most investigations focus on the detection of genome equivalents of viruses rather than on viral infectivity (Lee et al., 2002). The effect of RNAlater (Qiagen) on the infectivity of several
Abbreviations: D-MEM, Dulbecco’s modified Eagle’s medium; HIV, human immunodeficiency virus; MEV, Maus Elberfeld virus; PBS, phosphate buffered saline; TCID50 , tissue culture infectious dose 50 per mL; VSV, vesicular stomatitis virus ∗ Corresponding author. Tel.: +49 18887542558; fax: +49 18887542605. E-mail address: [email protected] (C. Uhlenhaut). 0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.05.002
viruses was evaluated. A non-enveloped virus, Encephalomyocarditis virus (EMCV) strain Maus Elberfeld virus (MEV), genus Cardiovirus, family Picornaviridae, was used as a model for hepatitis A virus, Rhino-, and Enteroviruses. Vesicular stomatitis virus (VSV), genus Vesiculovirus, family Rhabdoviridae, was used as general model for enveloped viruses, especially as a model for Lyssavirus. Because of its fatal pathogenic potential for humans and its common incidence in clinical samples, human immunodeficiency virus 1 (HIV-1) was used. Two different HIV-1 subtype B strains were tested in parallel (HTLV IIIb and NL4.3). Aliquots (0.5 ml) of virus stocks in cryotubes that had been stored at −70 ◦ C were thawed and mixed immediately with an equal volume of either phosphate buffered saline (PBS) or RNAlater. The samples were incubated at room temperature on a desk in a laboratory that had only artificial light during working hours. The sample stored for 72 h was prepared first, the one incubated for 48 h on the second day and the one incubated for 24 h on day three. Viruses were pelleted by ultracentrifugation (2 h, 70,000 × g, 4 ◦ C). The pellet was resuspended in 1 ml growth medium. MEV was titrated on HEp2 cells and VSV on BHK21 cells, using D-MEM supplemented with 5% FCS as growth medium. Eight parallel cultures in 96-well plates were inoculated with 0.1 ml of a dilution (dilution steps
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C. Uhlenhaut, M. Kracht / Journal of Virological Methods 128 (2005) 189–191
Table 1 Relevant and model viruses Virus
MEV VSV
Treatment with
Incubation for 24 h
48 h
72 h
50 days
PBS RNAlater
3.2 × 107
2.4 × 107
1.8 × 107
3.2 × 107 1.7 × 107
1.1 × 102 5.7 × 104
PBS RNAlater
1.8 × 108 2.4 × 107
1.8 × 106 1.3 × 107
5.6 × 102 1.3 × 107
>101 4.3 × 102
3.2 × 107
Table 2 HIV Virus
HIV NL4.3 HTLV IIIb
Treatment with
Incubation for (followed by 4 days at 4 ◦ C) 24 h
48 h
72 h
PBS RNAlater
Not done Not done
7.8 × 102 2.3 × 102
4.5 × 102 4.5 × 102
PBS RNAlater
7.0 × 103 4.1 × 103
4.1 × 103 2.3 × 103
1.2 × 104 2.3 × 103
1:3) as described elsewhere (Scheidler et al., 1998). Virus stocks and RNAlater were subjected to ultracentrifugation and processed like the virus-containing samples. HIV was titrated in duplicate in 24-well microtiter plates on CEMx174 cells grown in RPMI 1640 supplemented with 10% FCS; virus titers were calculated according to Spearman and K¨arber (1974, in Scheidler et al., 1998). Virus suspensions were mixed with RNAlater or with PBS and incubated for different time periods (Table 1) and the virus titer was determined. Virus stocks were subjected to ultracentrifugation, resuspended in growth medium and the titer was determined in parallel (MEV 1.9 × 106 TCID50 ; VSV 3.0 × 107 TCID50 ). The experiments with incubation periods of up to 3 days were repeated once; experiments with incubation periods of 50 days were performed in triplicate. Two strains of HIV-1 were investigated (NL4.3, HTLV IIIb; Table 2). Viruses were mixed with either PBS or RNAlater and incubated as described above for 24, 48 and 72 h, respectively. At the end of the incubation, virus suspensions were stored for 4 days at 4 ◦ C to mimic storage of clinical samples, e.g., over the weekend. The titer of the virus stock for HIV NL4.3 was 6.3 × 104 and for HTLV IIIb 6.3 × 104 . Experiments with HIV were carried out once. Although RNAlater stabilizes RNA in biological samples, it was unexpected that virus infectivity was preserved in virus suspensions stored at room temperature for a prolonged time. The effect of RNAlater was most prominent in the short-time incubation of VSV. Whereas no loss of infectivity was observed in presence of RNAlater during the incubation period of 72 h, infectivity titers decreased after 48 h by a factor of 102 and after 72 h by a factor of 104 . No infectious virus could be detected in the PBS sample after 50 days, whereas infectious virus was still present in the sample stored in presence of RNAlater (4.3 × 102 TCID50 ).
No significant difference in MEV titers could be observed in the first 72 h of incubation with and without RNAlater (pvalue = 0.3842); however, a significant difference was seen in the samples stored for 50 days at room temperature (pvalue = 0.0330). A titer of approximately 102 TCID50 was detected in the sample mixed with PBS, whereas the titer in the sample with RNAlater was approximately 500 times higher. The titer of HIV was reduced by a factor of 10–50 in both samples compared to the original virus stocks, but showed no further decrease on day two and three. The stability of HIV, especially in presence of plasma or serum, was described previously (Moudgil and Daar, 1993). Biological samples with RNAlater should be considered as potentially infectious even after prolonged storage or transportation time at ambient temperature, because virus infectivity of non-enveloped as well as of enveloped viruses is stabilized, and appropriate precautions should be taken when handling this material. However, this stabilizing effect is also advantageous and can be exploited when infectious biological material has to be transported under conditions when a cooling chain cannot be maintained. Virus samples transported under these conditions are stabilized and subsequent cultivation might be possible.
Acknowledgements Beatrice Hahn and Georg Pauli suggested the importance of the performed analysis. Georg Pauli provided substantial support and experience for experiment design. We thank Fabian H. Leendertz and Heinz Ellerbrok for discussion and critical reading of the manuscript. Sybille Somogyi kindly provided NL4.3. Special thanks to Ursula Erikli for copy-editing. This work was funded by the Robert Koch-Institute, Center for Biological Safety (ZBS 1), Berlin, Germany.
C. Uhlenhaut, M. Kracht / Journal of Virological Methods 128 (2005) 189–191
References Lee, D.H., Li, L., Andrus, L., Prince, A.M., 2002. Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT. Transfusion 42, 409–413. Moudgil, T., Daar, E.S., 1993. Infectious decay of human immunodeficiency virus type 1 in plasma. J. Infect. Dis. 167, 210–212.
191
Scheidler, A., Rokos, K., Reuter, T., Ebermann, R., Pauli, G., 1998. Inactivation of viruses by -propiolactone in human cryo poor plasma and IgG concentrates. Biologicals 26, 135–144. Whittier, C.A., Horne, W., Slenning, B., Loomis, M., Stoskopf, M.K., 2004. Comparison of storage methods for reverse transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples. J. Virol. Methods 116, 11–17.
Short communication
Viral infectivity is maintained by an RNA protection buffer Christine Uhlenhaut ∗ , Maren Kracht Center for Biological Safety, Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany Received 10 February 2005; received in revised form 26 April 2005; accepted 3 May 2005 Available online 4 June 2005
Abstract RNA stabilization buffers are valuable additives which are used widely. Although their effect on RNA protection has been well assessed, the impact on viral infectivity was unsettled. The potential stabilizing or inactivating effects of RNAlater (Qiagen) on the infectivity of different relevant and model viruses including HIV were assessed. Our results show that RNAlater not only protects RNA as published by others but also stabilizes virus infectivity of enveloped and non-enveloped model viruses for a considerable period of time on a relatively high level (storage at room temperature for 50 days yielded infectious titers >100 TCID50 ). © 2005 Elsevier B.V. All rights reserved. Keywords: Virus safety; RNA stabilization; RNAlater
RNA stabilization and protection during transportation and storage of samples is a prerequisite, e.g., for reliable gene expression analysis. Not only ubiquitous RNases and easy hydrolysis of RNA but also short half-lives of mRNA subpopulations pose significant problems. The need for stabilization of RNA is met currently by different RNA protection systems. These efficient buffers are used widely for sampling, storing and for stable transportation of diagnostic material, viruses or various other potentially infectious materials such as whole blood, PBMCs, tissue or feces. RNAlater (Qiagen) is a high salt solution (25 mM sodium citrate, 10 mM EDTA, 70 g ammonium sulphate/100 ml solution, pH 5.2) and was developed as a non-toxic storage reagent for tissue (cellular RNA) but can also be used for RNA protection in lymphocytes or fecal samples (Lee et al., 2002; Whittier et al., 2004). Most investigations focus on the detection of genome equivalents of viruses rather than on viral infectivity (Lee et al., 2002). The effect of RNAlater (Qiagen) on the infectivity of several
Abbreviations: D-MEM, Dulbecco’s modified Eagle’s medium; HIV, human immunodeficiency virus; MEV, Maus Elberfeld virus; PBS, phosphate buffered saline; TCID50 , tissue culture infectious dose 50 per mL; VSV, vesicular stomatitis virus ∗ Corresponding author. Tel.: +49 18887542558; fax: +49 18887542605. E-mail address: [email protected] (C. Uhlenhaut). 0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.05.002
viruses was evaluated. A non-enveloped virus, Encephalomyocarditis virus (EMCV) strain Maus Elberfeld virus (MEV), genus Cardiovirus, family Picornaviridae, was used as a model for hepatitis A virus, Rhino-, and Enteroviruses. Vesicular stomatitis virus (VSV), genus Vesiculovirus, family Rhabdoviridae, was used as general model for enveloped viruses, especially as a model for Lyssavirus. Because of its fatal pathogenic potential for humans and its common incidence in clinical samples, human immunodeficiency virus 1 (HIV-1) was used. Two different HIV-1 subtype B strains were tested in parallel (HTLV IIIb and NL4.3). Aliquots (0.5 ml) of virus stocks in cryotubes that had been stored at −70 ◦ C were thawed and mixed immediately with an equal volume of either phosphate buffered saline (PBS) or RNAlater. The samples were incubated at room temperature on a desk in a laboratory that had only artificial light during working hours. The sample stored for 72 h was prepared first, the one incubated for 48 h on the second day and the one incubated for 24 h on day three. Viruses were pelleted by ultracentrifugation (2 h, 70,000 × g, 4 ◦ C). The pellet was resuspended in 1 ml growth medium. MEV was titrated on HEp2 cells and VSV on BHK21 cells, using D-MEM supplemented with 5% FCS as growth medium. Eight parallel cultures in 96-well plates were inoculated with 0.1 ml of a dilution (dilution steps
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C. Uhlenhaut, M. Kracht / Journal of Virological Methods 128 (2005) 189–191
Table 1 Relevant and model viruses Virus
MEV VSV
Treatment with
Incubation for 24 h
48 h
72 h
50 days
PBS RNAlater
3.2 × 107
2.4 × 107
1.8 × 107
3.2 × 107 1.7 × 107
1.1 × 102 5.7 × 104
PBS RNAlater
1.8 × 108 2.4 × 107
1.8 × 106 1.3 × 107
5.6 × 102 1.3 × 107
>101 4.3 × 102
3.2 × 107
Table 2 HIV Virus
HIV NL4.3 HTLV IIIb
Treatment with
Incubation for (followed by 4 days at 4 ◦ C) 24 h
48 h
72 h
PBS RNAlater
Not done Not done
7.8 × 102 2.3 × 102
4.5 × 102 4.5 × 102
PBS RNAlater
7.0 × 103 4.1 × 103
4.1 × 103 2.3 × 103
1.2 × 104 2.3 × 103
1:3) as described elsewhere (Scheidler et al., 1998). Virus stocks and RNAlater were subjected to ultracentrifugation and processed like the virus-containing samples. HIV was titrated in duplicate in 24-well microtiter plates on CEMx174 cells grown in RPMI 1640 supplemented with 10% FCS; virus titers were calculated according to Spearman and K¨arber (1974, in Scheidler et al., 1998). Virus suspensions were mixed with RNAlater or with PBS and incubated for different time periods (Table 1) and the virus titer was determined. Virus stocks were subjected to ultracentrifugation, resuspended in growth medium and the titer was determined in parallel (MEV 1.9 × 106 TCID50 ; VSV 3.0 × 107 TCID50 ). The experiments with incubation periods of up to 3 days were repeated once; experiments with incubation periods of 50 days were performed in triplicate. Two strains of HIV-1 were investigated (NL4.3, HTLV IIIb; Table 2). Viruses were mixed with either PBS or RNAlater and incubated as described above for 24, 48 and 72 h, respectively. At the end of the incubation, virus suspensions were stored for 4 days at 4 ◦ C to mimic storage of clinical samples, e.g., over the weekend. The titer of the virus stock for HIV NL4.3 was 6.3 × 104 and for HTLV IIIb 6.3 × 104 . Experiments with HIV were carried out once. Although RNAlater stabilizes RNA in biological samples, it was unexpected that virus infectivity was preserved in virus suspensions stored at room temperature for a prolonged time. The effect of RNAlater was most prominent in the short-time incubation of VSV. Whereas no loss of infectivity was observed in presence of RNAlater during the incubation period of 72 h, infectivity titers decreased after 48 h by a factor of 102 and after 72 h by a factor of 104 . No infectious virus could be detected in the PBS sample after 50 days, whereas infectious virus was still present in the sample stored in presence of RNAlater (4.3 × 102 TCID50 ).
No significant difference in MEV titers could be observed in the first 72 h of incubation with and without RNAlater (pvalue = 0.3842); however, a significant difference was seen in the samples stored for 50 days at room temperature (pvalue = 0.0330). A titer of approximately 102 TCID50 was detected in the sample mixed with PBS, whereas the titer in the sample with RNAlater was approximately 500 times higher. The titer of HIV was reduced by a factor of 10–50 in both samples compared to the original virus stocks, but showed no further decrease on day two and three. The stability of HIV, especially in presence of plasma or serum, was described previously (Moudgil and Daar, 1993). Biological samples with RNAlater should be considered as potentially infectious even after prolonged storage or transportation time at ambient temperature, because virus infectivity of non-enveloped as well as of enveloped viruses is stabilized, and appropriate precautions should be taken when handling this material. However, this stabilizing effect is also advantageous and can be exploited when infectious biological material has to be transported under conditions when a cooling chain cannot be maintained. Virus samples transported under these conditions are stabilized and subsequent cultivation might be possible.
Acknowledgements Beatrice Hahn and Georg Pauli suggested the importance of the performed analysis. Georg Pauli provided substantial support and experience for experiment design. We thank Fabian H. Leendertz and Heinz Ellerbrok for discussion and critical reading of the manuscript. Sybille Somogyi kindly provided NL4.3. Special thanks to Ursula Erikli for copy-editing. This work was funded by the Robert Koch-Institute, Center for Biological Safety (ZBS 1), Berlin, Germany.
C. Uhlenhaut, M. Kracht / Journal of Virological Methods 128 (2005) 189–191
References Lee, D.H., Li, L., Andrus, L., Prince, A.M., 2002. Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT. Transfusion 42, 409–413. Moudgil, T., Daar, E.S., 1993. Infectious decay of human immunodeficiency virus type 1 in plasma. J. Infect. Dis. 167, 210–212.
191
Scheidler, A., Rokos, K., Reuter, T., Ebermann, R., Pauli, G., 1998. Inactivation of viruses by -propiolactone in human cryo poor plasma and IgG concentrates. Biologicals 26, 135–144. Whittier, C.A., Horne, W., Slenning, B., Loomis, M., Stoskopf, M.K., 2004. Comparison of storage methods for reverse transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples. J. Virol. Methods 116, 11–17.