Validation of Inactivation Methods for Arenaviruses

Olschewski, Silke; Thielebein, Anke; Hoffmann, Chris; Blake, Olivia; Müller, Jonas; Bockholt, Sabrina; Pallasch, Elisa; Hinzmann, Julia; Wurr, Stephanie; Neddersen, Neele; Rieger, Toni; Günther, Stephan; Oestereich, Lisa

doi:10.3390/v13060968

Cited by 6 publications

(11 citation statements)

References 31 publications

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“…Moreover, some chemicals are difficult to remove with this technique [ 23 , 24 ]. In light of this difficulty, we used a recently described in vitro protocol where the use of Amicon ® Ultra-15 100K Centrifugal Filter Devices allows both virus sample concentration and the simultaneous removal of cytotoxic substances via ultrafiltration [ 15 ]. Importantly, this method ensured a high virus recovery rate in a non-toxic, small sample volume that could easily be added to fresh cells in order to test the remaining infectivity of the samples.…”

Section: Discussionmentioning

confidence: 99%

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Inactivation Methods for Experimental Nipah Virus Infection

Widerspick

Vázquez

Niemetz

et al. 2022

Viruses

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Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe disease in humans and livestock. Due to its high pathogenicity in humans and the lack of available vaccines and therapeutics, NiV needs to be handled in biosafety level 4 (BSL-4) laboratories. Safe inactivation of samples containing NiV is thus necessary to allow further processing in lower containment areas. To date, there is only limited information available on NiV inactivation methods validated by BSL-4 facilities that can be used as a reference. Here, we compare some of the most common inactivation methods in order to evaluate their efficacy at inactivating NiV in infected cells, supernatants and organs. Thus, several physical and chemical inactivation methods, and combinations thereof, were assessed. Viral replication was monitored for 3 weeks and NiV presence was assessed by RT-qPCR, plaque assay and indirect immunofluorescence. A total of nineteen methods were shown to reduce NiV infectious particles in cells, supernatants and organs to undetectable levels. Therefore, we provide a list of methods for the safe and efficient inactivation of NiV.

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Section: Discussionmentioning

confidence: 99%

“…After incubation with the inactivation reagents, samples were then washed with 200 mL of PBS using Amicon ® Ultra-15 100K Centrifugal Filter Devices (Millipore, Burlington, MA, USA), as described by Olschewski et al (2021) [ 15 ]. This method has been shown to efficiently remove the inactivating agents while concentrating the sample.…”

Section: Methodsmentioning

confidence: 99%

Inactivation Methods for Experimental Nipah Virus Infection

Widerspick

Vázquez

Niemetz

et al. 2022

Viruses

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“…Each inactivation study comes with its own challenges. Chemical inactivation often requires the removal of cytotoxic reagents that interfere with testing the viability of inactivated viruses in cell culture [ 8 , 9 , 10 , 11 , 12 , 13 ]. Physical inactivation, such as gamma irradiation and heat, relies on highly specific parameters, including the sample volume, number of cells or viral particles, protein content, temperature, and inactivation time [ 5 , 14 , 15 , 16 ].…”

Section: Resultsmentioning

confidence: 99%

“…Heat treatment at ≥95 °C, usually in the presence of SDS, is commonly used to inactivate enveloped negative sense RNA viruses to prepare cell lysates for downstream analyses such as Western blot [ 10 , 12 , 13 ]. We demonstrate that a 10-min incubation at a sample temperature of 99+ °C (block temperature setting: 120 °C) is sufficient to inactivate 1 mL of 2.4 × 10 7 EBOV-infected cells ( Figure 6 , sample 5).…”

Section: Resultsmentioning

confidence: 99%

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses

Olejnik,

Hume,

Ross

et al. 2023

Pathogens

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The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.

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“…Aldehyde or solvent fixation is used for microscopy samples while chemical inactivation is frequently employed prior to nucleic acid or protein extractions. The used buffers classically contain compounds such as sodium dodecyl sulfate (SDS) or guanidine thiocyanate [ 1 ], which denature the sample and do not keep cells intact. Since they perturb the native protein complexes, they are not suitable for native mass spectrometry (nMS) or protein immunoprecipitation assays without biocontainment measures.…”

Section: Introductionmentioning

confidence: 99%

A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells

et al. 2023

Self Cite

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Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.

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Validation of Inactivation Methods for Arenaviruses

Cited by 6 publications

References 31 publications

Inactivation Methods for Experimental Nipah Virus Infection

Inactivation Methods for Experimental Nipah Virus Infection

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses

A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells

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