Inactivation of Viable Surrogates for the Select Agents Virulent Newcastle Disease Virus and Highly Pathogenic Avian Influenza Virus Using Either Commercial Lysis Buffer or Heat

Abstract

Introduction: Federal Select Agent Program regulations require laboratories to document a validated procedure for inactivating select agents prior to movement outside registered space. Avian influenza viruses and virulent Newcastle disease virus (vNDV) are cultured in chicken amnio-allantoic fluid (AAF), but the efficacy of commercial lysis buffers to inactivate viruses in protein-rich media has not been documented.

Objectives: We assesses the efficacy of MagMAX™ lysis buffer for inactivating highly pathogenic avian influenza virus (HPAIV) and vNDV in chicken AAF and confirm the inactivation of avian influenza in serum using heat.

Methods: Low pathogenic avian influenza virus (LPAIV) and avian paramyxovirus subtype-1 (APMV-1) were incubated with lysis buffer and tested for viability. Known viable LPAIV and APMV-1 RNA was extracted from AAF using MagMAX™-96 AI/ND Viral RNA Isolation kit, and the eluate was tested for remaining infectious agent. Finally, inactivation of LPAIV in serum was examined over 3 combinations of temperature and incubation time.

Results: MagMAX™ lysis buffer inactivated both LPAIV and APMV-1 in AAF when incubated for 30 minutes at room temperature. The full extraction process eliminated viable virus from the final RNA eluate. LPAIV in serum heated to 70°C for 30 minutes was rendered noninfectious.

Conclusion: The ability of a diagnostic laboratory to move samples from one space to another is critical to maintaining biosecurity as well as efficient laboratory workflow. Our study demonstrates a method to ensure the inactivation of viable avian influenza and avian paramyxoviruses in AAF, RNA eluate, and viable avian influenza virus in sera.

Keywords: BSL-3; Federal Select Agent Program; biological select agents and toxins; biorisk management; infectious agent; pathogen.

Figure 1.

Illustrated workflow for MagMAX™ lysis buffer inactivation of APMV-1 and H5N2 LPAIV in AAF. (a) Workflow for initial trial (APMV-1 only) using variable incubation times at room temperature, prior to dilution with VTM and inoculation into 8-day old embryonated chicken eggs. (b) Workflow for all subsequent trials (APMV-1 and H5N2 LPAIV) adding 1 minute of agitation on an orbital shaker, followed by 30 minutes of incubation at room temperature, prior to dilution with VTM and inoculation into 8-day old embryonated chicken eggs. AAF, amnio-allantoic fluid; APMV-1, avian paramyxovirus subtype-1; LPAIV, low pathogenic avian influenza; VTM, viral transport media.

Figure 2.

Illustrated workflow for egg inoculation in MagMAX™ lysis buffer inactivation trials (avian paramyxovirus subtype-1 and H5N2 low pathogenic avian influenza).