Inactivation of Risk Group-3 Agents for Transfer to BSL-2 Laboratories
There are a variety of reasons for removing biomaterials from a BSL-3 laboratory including the further study of genetic materials and proteins. Before using these materials outside of the BSL-3, they need to be treated in a manner that renders a pathogen non-viable, nucleic acids non-infectious, or biological toxins non-toxic, while keeping the characteristics of interest intact.1 At Cornell, all BSL-3 inactivation protocols must be described within the SOP packet for individual research laboratories and verified. Verification is defined as a demonstration that the use of a particular inactivation method inactivates the material in the user’s lab according to the method’s specifications. The process of verification may require viability, infectivity, or toxicity testing. Published inactivation methods will need to be demonstrated by the lab once and the data submitted to the IBC for review. For novel inactivation methods data will need to be submitted to the IBC for review and labs must maintain records of inactivation testing each time inactivation is performed.
The recommendations below are general and have been compiled from various publications. Any method used by a laboratory must be described in the lab’s SOPs and submitted at the time of the IBC application or amendment. Verification of a lab’s SOP may be requested as part of the IBC review process. An approved inactivation protocol is required for removal from the BSL-3.
For further information on inactivation protocols and definitions, see the 6th Edition of Biosafety in Microbiological and Biomedical Laboratories.
This document is guidance only and the IBC may determine a need to review or revisit at any time.
SARS-CoV-2 and other Coronaviruses
Human blood samples positive for SARS-CoV-2 are inactivated after 30 minutes at 56°C or 60 minutes at 60°C while nasal swabs require 15 minutes at 92°C.2 SARS-CoV-1 in cell culture can be inactivated after 45 minutes at 75°C, or at least 90 minutes at 56°C and 65°C.3 Because of the similarities, it is currently assumed that inactivation of SARS-CoV-2 could be achieved with the same process.
Inactivation with heat for MERS-CoV samples requires at least 25 minutes at 56°C while increasing the temperature to 65°C for 15 minutes is more than sufficient to inactivate any sample type.4
Highly denaturing conditions destabilize the viral envelope of lipid-enveloped viruses, eliminate cellular nucleases, and maintain the structure of RNA for later analyses. Lysis solutions with guanidine salts (guanidine thiocyanate, guanidine isothiocyanate) have proven to be efficient for the inactivation of enveloped RNA viruses. Not all systems have proven equivalent. For instance, Qiagen kits that use buffers ATL (1-10% sodium dodecyl sulfate SDS) and VXL (30-50% guanidine hydrochloride, 1-10% t-Octylphenoxypolyethoxyethanol [Triton X-100]) inactivate samples of SARS-CoV-2 with viral loads as high as 106TCID50.5,6 The AVL buffer (GITC 50%-70%) from Qiagen is not sufficient to inactivate SARS-CoV-2 even when supplemented with absolute ethanol or 1% Triton X-100.6
The use of TRIzol (a phenol and guanidine isothiocyanate solution) to inactivate SARS-CoV-1 infected cells and perform RNA isolation did not result in any infectious RNA.3 The use of TRIzol LS at a ratio of 4 parts TRIzol LS to 1 part viral suspension has been shown to inactivate a broad range of viral families including Coronaviridiae at titers as high as 103 TCID50.7
Lowering the pH of a sample or viral suspension to below 3 can inactivate SARS-CoV at 25°C or 37°C.3
Fixation protocols generally assume the removal of growth medium and washing with buffer before treatment with fixatives. Both 0.037% and 0.009% final concentrations of formaldehyde applied to SARS-CoV-1 cultures were insufficient to inactivate the virus at 4°C, 25°C, and 37°C with some virus remaining after three days.3 Conversely, both 0.008% and 0.002% final concentrations of glutaraldehyde were sufficient to inactivate SARS-CoV-2 after 2 days at 25°C and 1 day at 37°C.3 Current guidance from the CDC for fixation of post-mortem samples from persons with or suspected of having COVID is 72 hours for a 5 mm thick tissue sample in 10% neutral buffered formalin.8 Fixation of tissues is highly dependent on the tissue size and composition and should be evaluated case-by-case.
Heating cultures to 100°C for at least 5 minutes in a boiling-water bath is sufficient to inactivate M. tuberculosis.9
DNA extraction methods that include a 20-minute incubation in chloroform at room temperature, a thirty-minute incubation in 70% ethanol at room temperature, or a dry water bath at 80°C for fifteen minutes have been shown to inactivate M. tuberculosis and preserve DNA integrity.10
Extraction of nucleic acids with denaturing organic solvents such as phenol or chaotropic reagents such as guanidine isothiocyanate will inactivate M. tuberculosis. Mycobacterium tuberculosis nucleic acids can be routinely extracted by lysis with 4M GITC, lysozyme treatment with bead beating followed by phenol:chloroform separation of the nucleic acids.11
Lipid extraction using chloroform:methanol (2:1 v/v) and mechanical mixing for 10 minutes or longer will inactivate tubercule baccili.11
Antimicrobial fixation of Mycobacterium tuberculosis with paraformaldehyde solution have been used for almost 2 decades. Bacteria are removed from medium and resuspended in buffer containing final concentrations 2-4% paraformaldehyde and incubated for no less than 1 hour at 4°C.12
Human Immunodeficiency Virus (HIV)
Heat inactivation of HIV at 56°C for 2 hours is a commonly accepted method.13
Current published methods for inactivation of HIV and other retroviruses include treatments with final concentrations of 0.0125% glutaraldehyde, 0.04-2% formaldehyde, at least 0.1% β-propiolactone, 0.5% paraformaldehyde, 100% ethyl ether, and 0.3% hydrogen peroxide.14 A 10-minute incubation with final concentrations of 50% ethanol or 0.3% hydrogen peroxide are sufficient.15 Using ethyl ether requires a 1-hour incubation, while the use of B-propiolactone or formaldehyde can take at least 18 hours for full inactivation at lower concentrations.16,17 At least five minutes of incubation with 0.0125% glutaraldehyde is required for inactivation. In addition to the previously listed chemical inactivators, incubation with 0.2% Triton X-100 for 1 hour at 37°C can also be used to inactivate HIV.17,18
Influenza A Virus and other Orthomyxoviruses
Influenza A virus can be inactivated by heat treatment under 56°C for 30 minutes, 65°C for 10 minutes, 70°C, 75°C, and 100°C for 1 minute.19,20
The use of 0.02% formalin for at least 18 hours at 37°C is sufficient to inactivate influenza A virus in cell culture. In addition, at least 0.2% Triton X-100 for one hour at room temperature is sufficient for inactivation.20
Similar to Coronaviridae, above, highly denaturing conditions destabilize the lipid envelope of influenza while maintaining nucleic acid for later analyses.
Chemical Fixation of Tissues
Chemical fixation of tissues with 10% formalin† from animals infected with each of the above agents is routinely accepted practice. Times for fixation will depend largely on the tissue type since diffusion rates are different for tissues of different compositions. It is recommended that the formalin used be molecular biology grade or freshly* prepared. If the formalin fixation method has been published and shown to be effective in-house validation will likely not be necessary.
†There tends to be some confusion about 10% formalin. Ten percent formalin is a 10% solution made from a stock bottle of 37-40% formaldehyde (or more precisely: a 3.7-4% solution of formaldehyde).
*Freshly prepared 10% buffered formalin solutions should be no older than 3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5.
- CDC. 2020. Biosafety in Microbiological and Biomedical Laboratories, 6th Edition. Centers for Disease Control and Prevention.
- Shi J, Wen Z, Zhong G, et al. Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS–coronavirus 2. Science. April 2020. doi:10.1126/science.abb7015
- Darnell MER, Subbarao K, Feinstone SM, Taylor DR. Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. J Virol Methods. 2004;121(1):85-91. doi:10.1016/j.jviromet.2004.06.006
- Leclercq I, Batéjat C, Burguière AM, Manuguerra JC. Heat inactivation of the Middle East respiratory syndrome coronavirus. Influenza Other Respi Viruses. 2014;8(5):585-586. doi:10.1111/irv.12261
- Pastorino B, Touret F, Gilles M, de Lamballerie X, Charrel R. Evaluation of heating and chemical protocols for inactivating SARS-CoV-2. bioRxiv. 2020;(iii):0-8. doi:10.1101/2020.04.11.036855
- Pastorino B, Touret F, Gilles M, de Lamballerie X, Charrel RN. Heat inactivation of different types of SARS-CoV-2 samples: What protocols for biosafety, molecular detection and serological diagnostics? Viruses. 2020;12. doi:10.3390/v12070735.
- Kochel TJ, Kocher GA, Ksiazek TG, Burans JP. Evaluation of TRIzol LS inactivation of viruses. Appl Biosaf. 2017;22(2):52-55. doi:10.1177/1535676017713739
- CDC. 2020. Collection and Submission of Postmortem Specimens from Deceased Persons with Confirmed or Suspected COVID-19. https://www.cdc.gov/coronavirus/2019-ncov/hcp/guidance-postmortem-specimens.html
- Bemer-Melchior P, Drugeon HB. Inactivation of Mycobacterium tuberculosis for DNA typing analysis. J Clin Microbiol. 1999;37(7):2350-2351. doi:10.1128/jcm.37.7.2350-2351.1999
- Billard-Pomares T, Bleibtreu A, Walewski V, et al. Proposition of a safe Mycobacterium tuberculosis complex denaturation method that does not compromise the integrity of DNA for whole-genome sequencing. Tuberculosis. 2019;117:62-64. doi:10.1016/j.tube.2019.06.003
- Mycobacteria Protocols. Parish T, Brown AC, editors. Totowa, NJ: Humana Press; 2009. doi:10.1007/978-1-59745-207-6
- Schwebach JR, Jacobs WR, Casadevall A. Sterilization of Mycobacterium tuberculosis Erdman Samples by Antimicrobial Fixation in a Biosafety Level 3 Laboratory. J Clin Microbiol. 2001;39: 769–771. doi:10.1128/JCM.39.2.769-771.2001
- Rossio JL, Esser MT, Suryanarayana K, et al. Inactivation of Human Immunodeficiency Virus Type 1 Infectivity with Preservation of Conformational and Functional Integrity of Virion Surface Proteins. J Virol. 1998;72(10):7992-8001. doi:10.1128/jvi.72.10.7992-8001.1998
- Programs NRC (US) and I of M (US) P on NE and BD. Inactivation and Disinfection of HIV: A Summary. 1994. https://www.ncbi.nlm.nih.gov/books/NBK236636/. Accessed November 23, 2020.
- Martin LS, McDougal JS, Loskoski SL. Disinfection and Inactivation of the Human T Lymphotropic Virus Type III/Lymphadenopathy-Associated Virus. J Infect Dis. 1985;152(2):400-403.
- Quinnan G, Wells M, Wittek A, et al. Inactivation of human T-cell lymphotropic virus, type III by heat, chemicals, and irradiation. Transfusion. 1986;26(5):481-483. doi:10.1046/j.1537-2995.1986.26587020131.x
- Spire B, Montagnier L, Barré-Sinoussi F, Chermann JC. Inactivation of lymphadenopathy associated virus by chemical disinfectants. Lancet. 1984;324(8408):899-901. doi:10.1016/S0140-6736(84)90657-3
- Ukkonen P, Korpela J, Suni J, Hedman K. Inactivation of human immunodeficiency virus in serum specimens as a safety measure for diagnostic immunoassays. Eur J Clin Microbiol Infect Dis. 1988;7(4):518-523. doi:10.1007/BF01962603
- Zou S, Guo J, Gao R, et al. Inactivation of the novel avian influenza A (H7N9) virus under physical conditions or chemical agents treatment. Virol J. 2013;10(1):289. doi:10.1186/1743-422X-10-289
- Jonges M, Liu WM, Van Der Vries E, et al. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J Clin Microbiol. 2010;48(3):928-940. doi:10.1128/JCM.02045-09
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