Development and testing of a method for validating chemical inactivation of ebola virus

Kendra J. Alfson; Anthony Griffiths

doi:10.3390/v10030126

Development and testing of a method for validating chemical inactivation of ebola virus

Kendra J. Alfson, Anthony Griffiths

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

Abstract

Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV (Zaire ebolavirus) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (Zaire ebolavirus). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.

Original language	English (US)
Article number	126
Journal	Viruses
Volume	10
Issue number	3
DOIs	https://doi.org/10.3390/v10030126
State	Published - Mar 13 2018
Externally published	Yes

Keywords

Chemical inactivation
Cytopathic effect
Ebola virus
Limit of detection
Viability testing

ASJC Scopus subject areas

Infectious Diseases
Virology

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10.3390/v10030126

Cite this

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title = "Development and testing of a method for validating chemical inactivation of ebola virus",

abstract = "Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV (Zaire ebolavirus) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (Zaire ebolavirus). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.",

keywords = "Chemical inactivation, Cytopathic effect, Ebola virus, Limit of detection, Viability testing",

author = "Alfson, {Kendra J.} and Anthony Griffiths",

note = "Funding Information: Acknowledgments: This research was internally funded. This work was conducted in facilities constructed with support from the Research Facilities Improvement Program (Grant Number C06 RR012087) from the NCRR. Heinz Feldmann kindly provided the recombinant Ebola virus containing GFP. The authors would also like to thank Yenny Goez-Gazi, Matthew Stautzenberger and Ricardo Carrion Jr. for assistance in obtaining the liver tissue used and Chris Reed (USAMRIID) for his advice on TEM grid preparation. Funding Information: This research was internally funded. This work was conducted in facilities constructed with support from the Research Facilities Improvement Program (Grant Number C06 RR012087) from the NCRR. Heinz Feldmann kindly provided the recombinant Ebola virus containing GFP. The authors would also like to thank Yenny Goez-Gazi, Matthew Stautzenberger and Ricardo Carrion Jr. for assistance in obtaining the liver tissue used and Chris Reed (USAMRIID) for his advice on TEM grid preparation. Publisher Copyright: {\textcopyright} 2018 by the authors. Licensee MDPI, Basel, Switzerland.",

year = "2018",

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doi = "10.3390/v10030126",

language = "English (US)",

volume = "10",

journal = "Viruses",

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AU - Alfson, Kendra J.

AU - Griffiths, Anthony

N1 - Funding Information: Acknowledgments: This research was internally funded. This work was conducted in facilities constructed with support from the Research Facilities Improvement Program (Grant Number C06 RR012087) from the NCRR. Heinz Feldmann kindly provided the recombinant Ebola virus containing GFP. The authors would also like to thank Yenny Goez-Gazi, Matthew Stautzenberger and Ricardo Carrion Jr. for assistance in obtaining the liver tissue used and Chris Reed (USAMRIID) for his advice on TEM grid preparation. Funding Information: This research was internally funded. This work was conducted in facilities constructed with support from the Research Facilities Improvement Program (Grant Number C06 RR012087) from the NCRR. Heinz Feldmann kindly provided the recombinant Ebola virus containing GFP. The authors would also like to thank Yenny Goez-Gazi, Matthew Stautzenberger and Ricardo Carrion Jr. for assistance in obtaining the liver tissue used and Chris Reed (USAMRIID) for his advice on TEM grid preparation. Publisher Copyright: © 2018 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2018/3/13

Y1 - 2018/3/13

N2 - Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV (Zaire ebolavirus) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (Zaire ebolavirus). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.

AB - Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV (Zaire ebolavirus) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (Zaire ebolavirus). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.

KW - Chemical inactivation

KW - Cytopathic effect

KW - Ebola virus

KW - Limit of detection

KW - Viability testing

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ER -

Development and testing of a method for validating chemical inactivation of ebola virus

Abstract

Keywords

ASJC Scopus subject areas

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