Evaluation of henipavirus chemical inactivation methods for the safe removal of samples from the high-containment PC4 laboratory

https://doi.org/10.1016/j.jviromet.2021.114287Get rights and content

Highlights

  • Fixation methods for safe removal of samples from high containment laboratories.

  • 10 % neutral buffered formalin inactivates Nipah virus within tissues ≤48 h.

  • 4 % paraformaldehyde inactivates henipavirus-infected cell monolayers ≤15 min.

Abstract

Henipaviruses, Hendra (HeV) and Nipah (NiV), are highly pathogenic zoonotic agents that pose a serious health risk to human life, and as such are restricted to physical containment 4 (PC4) laboratories. For further analysis of virus-infected biological specimens, it is necessary to ensure absolute inactivation of any infectious virus present before removal from the PC4 laboratory. To evaluate the inactivation of HeV and NiV within infected samples, two chemical inactivation methods were assessed. Henipavirus-infected cell monolayers treated with 4 % paraformaldehyde (PFA) showed the complete inactivation of infectious virus, with an inactivation period of 15 min resulting in more than 8-log decrease in infectious titre. NiV-infected tissue samples treated with 10 % neutral-buffered formalin (NBF) showed a complete reduction of infectious virus in 7/8 ferret organs incubated for 24 h, with the remaining tissue demonstrating complete virus inactivation after 48 h. The chemical inactivation methods described herein evaluated two simple methods of henipavirus inactivation, resulting in the complete inactivation of infectious virus – an essential requirement for the safe removal and handling of biological samples from the PC4 laboratory.

Introduction

The henipavirus genus comprises of five species: Cedar virus, Ghanian bat virus, Mojiang virus, Hendra virus (HeV), and Nipah virus (NiV) (Rima et al., 2019) Belonging to the Paramyxoviridae family, henipaviruses are non-segmented, enveloped, negative-sense RNA viruses, with HeV and NiV capable of highly pathogenic zoonotic transmission (Wang et al., 2001). Serological evidence and real time reverse transcription PCR (RT-PCR) has identified the host reservoir of henipaviruses as the fruit bat of the genus Pteropus, with spill over events occurring sporadically and infecting mainly horses, pigs and humans (Mackenzie et al., 2003; Chua et al., 2002). Given the serious health risk to human health, high fatality rate, and a lack of vaccine or therapeutics for human use, these pathogens are limited to handling at physical containment level 4 (PC4) – the highest level of biological safety in the world (Middleton et al., 2014).

First identified in 1994 in Brisbane, Australia, HeV was determined to cause fatal acute respiratory disease and encephalitis in horses, with horse-to-human transmission resulting from close contact with infected horses (Halpin et al., 2000; Murray et al., 1995). In 1999, Nipah virus (NiVMal) was isolated in peninsular Malaysia as the etiological agent of an outbreak with similar disease characteristics, where 265 human cases resulted in 105 deaths. During this outbreak, NiV was amplifying within the pig population, with pig to human transmission occurring (Harcourt et al., 2000). In 2001, Nipah (NiVBan) outbreaks in Bangladesh caused febrile encephalitis and severe respiratory symptoms similar to the Malaysia outbreak. Furthermore, these outbreaks recorded the first human-to-human NiV transmission events. Since the first NiVBan outbreak, NiV-positive cases have been recorded almost annually in the India-Bangladesh region (Hsu et al., 2004; Gurley et al., 2007).

To perform further analyses on biological specimens infected with these viruses, it may be necessary to remove samples from the PC4 laboratory. For this to be possible it is paramount that all infectious virus in the sample be reliably inactivated prior to removal for safe handling at lower physical containment levels. Although there are virus inactivation methods published for other notifiable viruses such as Ebola (Haddock et al., 2016; Alfson and Griffiths, 2018), avian influenza (De Benedictis et al., 2007) and rabies virus (Wu et al., 2017), there are currently no published validation studies available for the chemical inactivation of henipaviruses for the safe removal of animal tissue and cell monolayers from the PC4 laboratory. Use of validated methods of inactivation may allow for removal of samples from containment without the need to test every sample for inactivation, based on policies of individual Institutional Biosafety Committees and relevant Government Regulators.

In this study we validated two chemical inactivation methods for the safe removal and handling of henipavirus-infected biospecimens from the PC4 laboratory. To evaluate the effectiveness of virus inactivation, 10 % neutral-buffered formalin (NBF) and 4 % paraformaldehyde (PFA) were used to treat animal tissue and infected cell monolayers, respectively. During infection of Vero cells, both HeV and NiV reliably produce a characteristic cytopathic effect (CPE), also known as syncytia (Crameri et al., 2002), therefore blind cell passaging and 50 % tissue culture infectious dose (TCID50) assays were used to visually confirm any presence of infectious virus and quantify virus titres of infected samples. Real-time RT-PCR used to demonstrate the presence of virus in unfixed animal tissues. NiV infection within tissues assayed was also supported with histopathogical analysis. Both methods described herein were shown to completely inactivate infectious virus present, therefore validating their use for treating biospecimens for removal and safe handling at lower biosafety levels.

Section snippets

Virus and cells

Vero cells (ATCC) were maintained in cell culture medium containing Minimum Essential Media (MEM; Gibco, USA), 1 % Antibiotic-Antimycotic (Gibco, USA), and 5 % heat-inactivated foetal bovine serum (FBS; Gibco, USA), and incubated in 5 % CO2 at 37 °C. To assess virus inactivation of NiV-infected tissue, fresh ferret samples were opportunistically obtained from control ferrets belonging to another NiV challenge study in which these tissues were surplus to needs.

The henipavirus stocks used for

Results

The effectiveness of 4 % PFA and 10 % NBF to completely inactivate infectious virus within a biological sample was evaluated. Virus isolations through blind passaging of supernatants/cell suspensions, TCID50 assays and RT-PCR assays were used to determine if these methods were able to inactivate virus whilst also determining the infectious virus titre present within a sample.

Discussion

We assessed the required period to inactivate henipaviruses within biological samples using two commonly used fixation chemicals. Both fixatives were selected for their ability to inactivate virus and to maintain the structural integrity of cell structures within biological samples as is necessary for downstream analyses such as histopathology and microscopy.

In this study we observed a complete reduction of infectious virus when NiV-infected tissues (no greater than 1 cm in diameter) were fixed

Author contributions

SJE: conceptualisation, methodology, validation, formal analysis, investigation, resources, writing – original draft, supervision; SC: investigation and writing – review & editing; WWS: methodology, formal analysis, writing – review & editing; SJ: investigation and writing – review & editing; BR: investigation; GAM: conceptualisation, methodology, formal analysis, resources, writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors acknowledge the capabilities of the Australian Centre for Disease Preparedness (grid.413322.5) in undertaking this research, including infrastructure funded by the National Collaborative Research Infrastructure Strategy.

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