Abstract
Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The results substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful method for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.
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Batéjat, C., Grassin, Q., Manuguerra, J.C., and Leclercq, I. 2020. Heat inactivation of the severe acute respiratory syndrome coronavirus 2. bioRxiv 067769.
Chan, J.F.W., Yuan, S., Kok, K.H., To, K.K.W., Chu, H., Yang, J., Xing, F., Liu, J., Yip, C.C.Y., Poon, R.W.S., et al. 2020. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet 395, 514–523.
Corman, V., Bleicker, T., Brünink, S., Drosten, C., Zambon, M., and Organization, W.H. 2020. Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR. Geneva: World Health Organization 13.
Cutts, T., Grolla, A., Jones, S., Cook, B.W.M., Qiu, X., and Theriault, S.S. 2016. Inactivation of Zaire ebolavirus variant Makona in human serum samples analyzed by enzyme-linked immunosorbent assay. J. Infect. Dis. 214, S218–S221.
Darnell, M.E., Subbarao, K., Feinstone, S.M., and Taylor, D.R. 2004. Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. J. Virol. Methods 121, 85–91.
Decrey, L., Kazama, S., and Kohn, T. 2016. Ammonia as an in situ sanitizer: influence of virus genome type on inactivation. Appl. Environ. Microbiol. 82, 4909–4920.
ECDC, European Centre for Disease Prevention and Control. 2020. COVID-19 situation update worldwide, as 5 June 2020. https://www.ecdc.europa.eu/en/geographical-distribution-2019-ncovcases
Gamarnik, A.V. and Andino, R. 1998. Switch from translation to RNA replication in a positive-stranded RNA virus. Genes Dev. 12, 2293–2304.
Herriott, R.M. 1961. Infectious nucleic acids, a new dimension in virology. Science 134, 256–260.
Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X., et al. 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506.
Kim, Y.I., Kim, S.G., Kim, S.M., Kim, E.H., Park, S.J., Yu, K.M., Chang, J.H., Kim, E.J., Lee, S., Casel, M.A.B., et al. 2020. Infection and rapid transmission of SARS-CoV-2 in ferrets. Cell Host Microbe 27, 704–709.
Knight, C.A. 1975. Action of chemical and physical agents on viruses. In Chemistry of Viruses, pp. 180–238. Springer, Berlin, Heidelberg, Germany.
Lamarre, A. and Talbot, P.J. 1989. Effect of pH and temperature on the infectivity of human coronavirus 229E. Can. J. Microbiol. 35, 972–974.
Larkin, E.P. 1977. Thermal inactivation of viruses. Army Natick Research And Development Center, Massachusetts, USA.
Leclercq, I., Batejat, C., Burguière, A.M., and Manuguerra, J.C. 2014. Heat inactivation of the middle east respiratory syndrome coronavirus. Influenza Other Respir. Viruses 8, 585–586.
Lelie, P., Reesink, H., and Lucas, C. 1987. Inactivation of 12 viruses by heating steps applied during manufacture of a hepatitis B vaccine. J. Med. Virol. 23, 297–301.
Li, D., Zhang, J., and Li, J. 2020. Primer design for quantitative realtime PCR for the emerging coronavirus SARS-CoV-2. Theranostics 10, 7150–7162.
McDonnell, G.E. 2017. Antisepsis, disinfection, and sterilization: types, action, and resistance. 2nd edn., John Wiley & Sons, New Sersey, USA.
Nuanualsuwan, S. and Cliver, D.O. 2003. Infectivity of RNA from inactivated Poliovirus. Appl. Environ. Microbiol. 69, 1629–1632.
PAHO, Pan American Health Organization. 2020a. General procedures for inactivation of potentially infectious samples with ebola virus and other highly pathogenic viral agents. https://www.paho.org/hq/dmdocuments/2014/2014-cha-proceduresinactivation-ebola.pdf
PAHO, Pan American Health Organization. 2020b. Laboratory guidelines for detection and diagnosis of the novel coronavirus (2019-nCoV) infection. https://www.paho.org/en/documents/laboratory-guidelines-detection-and-diagnosis-novel-coronavirus-2019-ncov-infection
Park, S.L., Huang, Y.J.S., Hsu, W.W., Hettenbach, S.M., Higgs, S., and Vanlandingham, D.L. 2016. Virus-specific thermostability and heat inactivation profiles of alphaviruses. J. Virol. Methods 234, 152–155.
Pratelli, A. 2008. Canine coronavirus inactivation with physical and chemical agents. Vet. J. 177, 71–79.
Reed, L.J. and Muench, H. 1938. A simple method of estimating fifty percent endpoints. Am. J. Epidemiol. 27, 493–497.
Roehrig, J.T., Hombach, J., and Barrett, A.D.T. 2008. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses. Viral Immunol. 21, 123–132.
Schlegel, A., Immelmann, A., and Kempf, C. 2001. Virus inactivation of plasma-derived proteins by pasteurization in the presence of guanidine hydrochloride. Transfusion 41, 382–389.
Siddell, S., Wege, H., and Ter Meulen, V. 1983. The biology of coronaviruses. J. Gen. Virol. 64, 761–776.
WHO, World Health Organization. 2003. First data on stability and resistance of SARS coronavirus compiled by members of WHO laboratory network. https://www.who.int/csr/sars/survival_2003_05_04/en/
WHO, World Health Organization. 2020a. 2019-nCoV outbreak is an emergency of international concern. https://www.euro.who.int/en/health-topics/health-emergencies/international-healthregulations/news/news/2020/2/2019-ncov-outbreak-is-anemergency-of-international-concern
WHO, World Health Organization. 2020b. Coronavirus. https://www.who.int/health-topics/coronavirus#tab=tab_2
WHO, World Health Organization. 2020c. WHO Director-General’s opening remarks at the media briefing on COVID-19.
Yunoki, M., Urayama, T., Yamamoto, I., Abe, S., and Ikuta, K. 2004. Heat sensitivity of a SARS-associated coronavirus introduced into plasma products. Vox Sang. 87, 302–303.
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This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942011) and conducted during the research year of Chungbuk National University in 2018 for Young Ki Choi.
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Kim, YI., Casel, M.A.B., Kim, SM. et al. Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity. J Microbiol. 58, 886–891 (2020). https://doi.org/10.1007/s12275-020-0335-6
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DOI: https://doi.org/10.1007/s12275-020-0335-6