Assessment and verification of chemical inactivation of peste des petits ruminants virus by virus isolation following virus capture using Nanotrap magnetic virus particles

Abstract

ABSTRACT This study reports development and optimization of a new method for the assessment and verification of the inactivation of peste des petits ruminants virus (PPRV) by chemical agents, including Triton X-100 and commercially available viral lysis buffers. Virus inactivation was confirmed by virus isolation (VI) on Vero cells following capture of the potential residual viruses from treated samples using Nanotrap magnetic virus particles (NMVPs). Since chemical agents are cytotoxic, treated PPRV samples could not be used directly for VI on Vero cell monolayers; instead, they were diluted in Eagle’s Minimum Essential Medium (EMEM) to neutralize cytotoxicity and then subjected to virus capture using NMVPs. The NMVPs and the captured viruses were then clarified on a magnetic stand, reconstituted in EMEM, and inoculated onto Vero cells that were examined for cytopathic effect (CPE). No CPE was observed on cells inoculated with treated viruses captured by NMVPs; but CPE was observed on cells inoculated with untreated viruses, including those captured by NMVPs. For further verification, the supernatants of the VI cultures (treated or untreated) were subjected to RNA extraction and PPRV-specific real-time RT-PCR (RT-qPCR). The cycle threshold values were undetectable for the supernatants of VI cultures inoculated with NMVPs reconstituted from treated PPRV but detectable for the supernatants of VI cultures inoculated with untreated PPRV or the NMVPs reconstituted from untreated PPRV, indicating complete inactivation of PPRV. This new method of verification of virus inactivation using NMVPs can be applied to other high impact viruses of agricultural or public health importance. IMPORTANCE Research including diagnosis on highly contagious viruses at the molecular level such as PCR and next-generation sequencing requires complete inactivation of the virus to ensure biosafety and biosecurity so that any accidental release of the virus does not compromise the safety of the susceptible population and the environment. In this work, peste des petits ruminants virus (PPRV) was inactivated with chemical agents, and the virus inactivation was confirmed by virus isolation (VI) using Vero cells. Since the chemical agents are cytotoxic, inactivated virus (PPRV) was diluted 1:100 to neutralize cytotoxicity, and the residual viruses (if any) were captured using Nanotrap magnetic virus particles (NMVPs). The NMVPs and the captured viruses were subjected to VI. No CPE was observed, indicating complete inactivation, and the results were further supported by real-time RT-PCR. This new protocol to verify virus inactivation can be applicable to other viruses.

Keywords

• PPRV
• Triton X-100
• lysis buffer
• virus inactivation
• Nanotrap magnetic virus particles
• VI