Work

Production and application of reference materials for high containment RNA viruses

Year: 2019

Type: article

Abstract: Purpose: Accurate diagnosis in an outbreak scenario is crucial for the provision of appropriate healthcare and treatments. For emerging pathogens, the assays used for the diagnosis are either absent or poorly developed. A vital tool is the provision of appropriate reference standards, which allows for harmonisation of results from laboratories worldwide. Use of killed virus will be ideal, but for high containment level pathogens (BSL4) proving inactivation may be challenging, and their distribution globally hampered by bureaucratic requirements and legislation. As an alternative we evaluated a lentiviral vector platform to package RNA of the pathogen of interest within an HIV-like particle to act as reference material in nucleic acid amplification technology (NAT)-based tests. Methods & Materials: During the Ebola outbreak in Western Africa in 2013–15, we developed a chimeric virus by incorporating Ebola virus RNA into virus-like particles for use with diagnostic kit or in house quantitative PCR and digital PCR. Ebola virus Makona strain sequences were synthesised in vitro and incorporated within a lentiviral vector plasmid. This was transfected into 293T cells together with a packaging plasmid containing HIV-1 gag and pol. The chimeric particles were harvested, purified and formulated in a universal buffer before lyophilisation. Results: The freeze-dried preparations were evaluated in an WHO-sponsored International Collaborative study in which participants were asked to test the candidate material using assays routinely used in their laboratories. The HIV-Ebola chimeric particles were able to harmonise data received from the participants and reduce the inter-laboratory variation. Based on these results, the WHO Expert committee on Biological Standardisation established the material as an International Reference Reagent. Conclusion: The lentiviral packaging system represent an enabling technology to assist the development and calibration of diagnostic kits. It is a safe alternative to handling a high containment level pathogen and obviates the issues related to bioterrorism law. Furthermore, this type of reference material can be prepared contemporaneously or in advance of any viral outbreak to assist calibration of sensitive and specific assays and their harmonisation through standardisation at the time of the emergency when they are most urgently needed.

Source: International Journal of Infectious Diseases

DOI: 10.1016/j.ijid.2018.11.314