Inactivation Methods for Experimental Nipah Virus Infection

Abstract

Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe disease in humans and livestock. Due to its high pathogenicity in humans and the lack of available vaccines and therapeutics, NiV needs to be handled in biosafety level 4 (BSL-4) laboratories. Safe inactivation of samples containing NiV is thus necessary to allow further processing in lower containment areas. To date, there is only limited information available on NiV inactivation methods validated by BSL-4 facilities that can be used as a reference. Here, we compare some of the most common inactivation methods in order to evaluate their efficacy at inactivating NiV in infected cells, supernatants and organs. Thus, several physical and chemical inactivation methods, and combinations thereof, were assessed. Viral replication was monitored for 3 weeks and NiV presence was assessed by RT-qPCR, plaque assay and indirect immunofluorescence. A total of nineteen methods were shown to reduce NiV infectious particles in cells, supernatants and organs to undetectable levels. Therefore, we provide a list of methods for the safe and efficient inactivation of NiV.

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses

“…All samples showed the expected results, with only the positive control (NiV + DMEM) and NiV challenge samples showing CPE and staining for NiV ( Figure 4 , samples 2 and 3). The NiV-infected cells lysed in TRIzol showed complete inactivation ( Figure 4 , sample 5), consistent with previous results [ 12 ].…”

“…We performed similar testing of the ability of 10% formalin and 4% PFA to inactivate EBOV- and NiV-infected cells and 4% PFA to inactivate LASV-infected cells, which also showed complete inactivation at both 30 min and 6 h, consistent with previous reports ( Table 2 ) [ 10 , 12 , 19 , 23 , 24 , 25 ]. Based on the formalin and PFA inactivation results, we developed an agent- and procedure-specific SOP.…”

“…Each inactivation study comes with its own challenges. Chemical inactivation often requires the removal of cytotoxic reagents that interfere with testing the viability of inactivated viruses in cell culture [ 8 , 9 , 10 , 11 , 12 , 13 ]. Physical inactivation, such as gamma irradiation and heat, relies on highly specific parameters, including the sample volume, number of cells or viral particles, protein content, temperature, and inactivation time [ 5 , 14 , 15 , 16 ].…”

“…The use of size exclusion columns is only suitable for testing small numbers of cells, as the columns easily clog, even with moderate cell numbers. The approach we chose was to wash the fixed cells, which retains virus while being able to remove the fixative, prior to adding the sample to fresh cells, similar to previous reports [ 12 , 20 ]. Here, we present example data, using 10% neutral-buffered formalin to inactivate cells infected with LASV.…”

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers

“…This shows that the specific conditions of inactivation must be carefully determined, including virus species, cell numbers/infectious viral particles, lysis buffer volume, and incubation times. Besides RLT, TRIzol is also commonly used for virus inactivation in high containment settings [ 10 , 17 , 19 , 23 , 24 , 25 , 26 , 27 ], yet neither of these buffers is recommended for use with low cell numbers unlike TCL, which can be used even at the single cell level [ 1 , 2 ]. Our analysis showed limited virus loss for both EBOV and SARS-CoV-2 due to purification over the Amicon columns to remove cytotoxic components ( Figure S2 ), similar to previously described SARS-CoV-2 yields using these columns [ 28 ].…”