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

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.

the family Paramyxoviridae, genus Morbillivirus, and is closely related to rinderpest virus, another important member of the genus Morbillivirus.Morbilliviruses are enveloped and have a single-stranded negative-sense RNA genome of approximately 16 kb (1).The clinical presentations of PPR include pyrexia, mucopurulent ocular and nasal discharges, conjunctivitis, and mucosal erosion with high morbidity (90%-100%) and mortality (up to 90%) (2,3).PPR is now considered one of the major transboundary animal diseases with frequent outbreaks being reported in many regions of the world including Europe, Asia, Morocco, and Africa (4)(5)(6)(7)(8)(9).Accordingly, PPR is listed as a reportable disease, and any occurrence or outbreak(s) of the disease must be reported to World Organization for Animal Health or WOAH (previously OIE).The WOAH and the Food and Agriculture Organization of the United Nations (FAO) developed a Global Strategy for the Control and Eradication of PPR and have set a goal to eradicate the disease by 2030, which will be the second such strategy after the successful eradication of rinderpest in 2011.
Reliable and effective inactivation of highly pathogenic viruses including PPRV is crucial before they can be transferred from higher level biocontainment such as BSL3 to a lower level of containment such as BSL2 for safe operation and manipulation, includ ing diagnosis, surveillance, and research.Incomplete inactivation of highly pathogenic viruses may compromise biosafety and potentially lead to accidental release of the virus into susceptible animal populations.
Prior to application, all virus inactivation methods must be validated to ensure that the virus is completely inactivated.One of the most trusted and useful methods to confirm virus inactivation is by virus isolation (VI) using susceptible cells lines.However, chemical agents including Triton X-100 and VLbs contain ingredients (detergents and chaotropic guanidinium salts) that are cytotoxic (11,19,27).Any live virus, if it survives after exposure to these chemical agents, cannot be recovered by VI using cell lines unless the cytotoxic ingredients of the chemical agents are either removed or diluted beyond their toxic levels.There are multiple ways toxic chemicals can be removed from samples including dialysis, ultra-filtration (Amicon filters), or using detergent-removal spin columns or hydrophobic absorbent resins (14,(28)(29)(30).However, these methods are tedious, time consuming, expensive, and often risk cross-contamination.Alternatively, a simple dilution of the samples can significantly reduce cytotoxicity and allow the cell lines to remain viable and the virus (if any) to replicate.However, dilution is not always successful for samples with low virus titers as the viruses are also equally diluted by the same factor as the active ingredients and may not be detectable by VI unless they are concentrated into a smaller volume.
In this study, Nanotrap magnetic virus particles (NMVPs), recently renamed as Nanotrap microbiome A particles (CERES Nanosciences Inc. Manassas, VA, USA), were used to capture and concentrate viruses from diluted suspensions.The NMVPs are highly porous customizable homogenous hydrogel microsphere particles/beads of about 800 nm in size that have a shell made of N-isopropylacrylamide polymers coupled with chemical affinity baits that can bind a broad range of biological compounds including proteins, nucleic acids, and virions (31,32).The NMVPs have a significant degree of flexibility and porosity in response to changes in pH and/or temperature.The NMVPs have been successfully used to capture and concentrate many enveloped viruses from a variety of samples/specimens including serum, plasma, blood, cell culture, transport media, urine, oral fluid, wastewater, and sweat (33).Some of the highly pathogenic viruses that have been successfully captured and concentrated using NMVPs include Rift Valley fever virus, human immunodeficiency virus (33), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (34), Venezuelan equine encephalitis virus (35), Zika virus, Chikungunya virus, Dengue virus (36), and influenza A and B viruses (37).Furthermore, NMVPs are neither toxic to host cells nor to the viruses.The NMVPs and the captured viruses can be directly recovered by VI using appropriate cell lines (37).
Here, we report an assessment and confirmation method of PPRV inactivation by VI on Vero cell monolayers using NMVPs after virus capture from diluted suspensions of chemically treated PPRV samples.The results were further confirmed by PPRV-specific real-time RT-PCR (RT-qPCR).

Virus strains and propagation
Two PPRV isolates, PPRV-Egypt and PPRV-Nigeria, were used in this study.Both isolates were obtained from the biorepository of the Reagents and Vaccine Services Section (RVSS) of the Foreign Animal Disease Diagnostic Laboratory (NVSL-FADDL-RVSS).For consistency, a single batch of cell culture-grown PPRV-Egypt (Log 10 10 4.5 TCID 50 /mL) or PPRV-Nigeria (Log 10 TCID 50 /mL 10 4.8 ) was used that were aliquoted into 1.5 mL cryovials and stored at −70°C.The batch cultures (PPRV-Egypt or PPRV-Nigeria) were diluted 1:1 in Eagle's Minimum Essential Medium (EMEM) supplemented with 4% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and used as the working stock (WS) for each experiment reported.Unless otherwise stated, all experiments reported herein were carried out using PPRV-Egypt isolate.The viruses were grown and propagated on Vero E6 cell monolayers also referred to as Vero cells in EMEM supplemented with 7% FBS and 20% (vol/vol) of antibiotic and antimycotic cocktail "Anti/Anti" consisting of penicillin, streptomycin, and amphotericin B (Gibco/Thermo Fisher Scientific).After inoculation, the plates were incubated at 37°C in a CO 2 incubator (5% vol/vol).

Virus isolation and assessment of cytotoxicity using Vero cells and serial passages
Vero cells grown on 6-well microtiter plates were used for the assessment of cytotoxicity and virus isolation.Cells were grown on microtiter plates in a CO 2 incubator (above) at 1.0E+05 cells/mL until the cell density reached 70%-75% confluency, which was generally achieved in 3 days.
The chemical agents used for virus inactivation and cytotoxicity experiments included lysis/binding solution (LBs-MM) from the MagMAX Core Nucleic Acid Purification Kit (MM-C; Thermo Fisher Scientific, Waltham, MA, USA), buffer AVL (lysis buffer) plus ethanol (AVL/Ethanol) from the QIAmp Viral RNA Kit (QVR; Germantown, MD, USA), and Triton X-100 (Sigma-Aldrich, St Luis, MO, USA).The details of the experimental conditions used for cytotoxicity and virus inactivation experiments are described below under the sections "Assessment of cytotoxicity" and "Assessment of chemical inactivation of PPRV by virus isolation following virus capture using NMVPs", respectively.Briefly, following treatment with the chemical agents (cytotoxicity) or inoculation with the virus (VI), Vero cells were incubated in CO 2 incubator for 1 h to facilitate absorption and equilibration, and then the cell monolayers on 6-well plates were overlaid with EMEM (4 mL/well), supplemented with 4% FBS plus antibiotics, and incubated in CO 2 incubator (5% vol/vol) for an extended period (up to 7-9 days).
For the assessment and confirmation of cytotoxicity and virus inactivation, the Vero cells were subjected up to three serial passages.The initial treatment of Vero cells, either with the chemical agents or virus inoculation, is referred to as the "first passage" or "1P." After the first passage (1P), Vero cells were examined daily under a microscope (Evos XL Core, Invitrogen/Thermo Fisher Scientific) to monitor cytotoxicity (chemical agents) or the development of cytopathic effect or CPE (virus infection).If the cells exhibited cytotoxicity or no CPE after 1P, they were subjected up to two additional serial passages where the cell cultures (1P) were subjected to freeze-thaw cycles (freezing at −70°C for 15 min followed by thawing at RT for 15 min), and this cycle was repeated three times.The freeze-thaw cycles facilitate rupture and loosening of cells from primary vessel and release the virus particles.After freeze-thaw cycles, the content of the plates was transferred to a 1.5-mL Eppendorf tube and clarified by centrifugation at 5,000 × g at RT for 10 min; and the clarified supernatants were used to inoculate fresh Vero cells for the second passage (2P) and examined for cytotoxicity or CPE.A third passage (3P) was carried out similarly as above only if the cells exhibited cytotoxicity or there was no CPE after 2P.
The supernatants of the VI cultures after 1P, 2P, and 3P were also subjected to RNA extraction and RT-qPCR (see RNA extraction and quantitative real-time RT-PCR for verification of the VI results).

Assessment of cytotoxicity
The cytotoxicity of LBs-MM, AVL/Ethanol, and Triton X-100 was evaluated by a dilution study where the Vero cells were treated with serially diluted chemical agents in EMEM with 4% FBS plus antibiotics.For LBs-MM, a working stock of this chemical was prepared by mixing equal volumes (350 µL each) of lysis solution and binding solution (700 µL final volume) as per manufacturer's instructions and used as undiluted (0:0) and at 1:10 and 1:100 dilutions.For AVL/Ethanol, a working stock was prepared by mixing equal volume (560 µL each) of buffer AVL (lysis buffer) and 96%-100% ethanol (1,120 µL final volume) (AVL/Ethanol) as per manufacturer's instructions and used as undiluted (0:0) and at 1:10 and 1:100 dilutions.For Triton X-100 (10% vol/vol), the detergent was diluted in EMEM and used at final concentrations of 1% (vol/vol), 0.1% (vol/vol), and 0.01% (vol/vol).The chemical agents at the above dilutions were then added onto Vero cells in duplicate wells at a rate of 200 µL/well for LBs-MM or Triton X-100, and at 140 µL/well for AVL/Ethanol to evaluate cytotoxicity.The supernatants of the treated cell cultures after 1P were subjected to 2P or 3P on fresh Vero cells at the same rate (200 or 140 µL/well) if they exhibited cytotoxicity within 7 days after 1P or 2P, respectively, as described above.
The cytotoxicity was found to be neutralized at the 1:100 dilution for LBs-MM or AVL/ Ethanol and at 0.01% for Triton X-100 as described in the Results section.Accordingly, all chemically treated virus samples were diluted as such to neutralize cytotoxicity before VI.

Optimization of virus capture from diluted samples using Nanotrap magnetic virus particles
For optimization of virus capture by NMVPs from diluted samples, 200 µL of PPRV WS was diluted 1:10 (2 mL), 1:25 (5 mL), 1:50 (10 mL), 1:100 (20 mL), and 1:200 (40 mL) in EMEM; and then each dilution was subjected to virus capture using 50, 75, 100, 150, and 200 µL of NMVPs (SKU 44202; CERES Nanosciences Inc., Manassas, VA, USA).After adding NMVPs, the contents were mixed (inverting tubes five to six times; no vertexing) and then incubated at RT for 30 min to facilitate virus capture.Next, the tubes were placed on an appropriate magnetic stand (DynaMag-2, -5, −15, or −50 magnets; Thermo Fisher Scientific) for 5 min to collect the NMVPs.The supernatants were discarded, and the clarified NMVPs were resuspended in 200 µL of EMEM; the RNA was extracted using the QVR kit and analyzed by virus-specific RT-qPCR as described below under the section "RNA extraction and quantitative real-time RT-PCR." Based on the cycle threshold (Ct) values corresponding to the virus captured by NMVPs (results not shown), it was determined that 100 µL of NMVPs (equivalent to 1.5 mg of magnetic particles) is optimal for PPRV capture from a wide range of dilutions between 1:10 (2 mL) and 1:200 (40 mL).Therefore, 100 µL of NMVPs was used to capture PPRV from diluted samples in all subsequent experiments within this study.
For the assessment of virus capture from chemically treated samples, 200 µL of PPRV WS was treated with 700 µL of LBs-MM and incubated for 2 min at RT. Next, 200 µL of the treated virus was diluted 1:100 (20 mL) in EMEM to neutralize cytotoxicity (described above under "Assessment of cytotoxicity"), and then 100 µL of NMVPs was added to capture the potential residual viruses (if any).After clarification on a magnetic stand, the supernatant, referred to as Sup-T (supernatant collected from treated PPRV), and the NMVPs (resuspended in 200 µL EMEM) were collected and saved.The controls used for validation include two positive controls (PCs) referred to as PC-V and PC-NMVPs, respectively, and one negative control (NC).Untreated PPRV WS (200 µL) diluted with 700 µL of EMEM (same volume as LBs-MM) was used as the starting material for both PCs.One 200 µL aliquot of the diluted virus was directly inoculated onto Vero cells for VI, which is referred to as PC-V; while another 200 µL aliquot of the same diluted virus was further diluted 1:100 in EMEM (20 mL; same volume dilution as was used to neutralize cytotoxicity of treated samples) and then subjected to virus capture using 100 µL of NMVPs.After clarification on a magnetic stand, the supernatant referred to as Sup-U (supernatant collected from untreated PPRV) and the NMVPs were collected and saved; the NMVPs were reconstituted in EMEM (200 µL) and inoculated onto Vero cells for VI and referred to as PC-NMVPs.To assess relative distribution and viability of the residual viruses between the two fractions, the saved NMVPs and the supernatants from both treated (Sup-T) and untreated (Sup-U) PPRV were analyzed separately by VI (up to three passages) and PPRV RT-qPCR.

Assessment of chemical inactivation of PPRV by virus isolation following virus capture using NMVPs
All virus inactivation experiments were carried out under biosafety level 3 (BSL-3) lab conditions.PPRV was treated with LBs-MM (MM-C) or AVL/Ethanol (QVR) as per manufacturer's instructions.For MM-C, 200 µL of PPRV WS was mixed with 700 µL of LBs-MM for 10 s by vortexing; and the content was incubated at RT for 2 min.For QVR, 140 µL of the PPRV WS mixed with 560 µL of AVL for 10 s by vortexing; and the content was incubated at RT for 10 min.Next, 560 µL of ethanol (96%-100%) was added, and the content (final volume 1260 µL) was mixed by vortexing for 10 s.For Triton X-100, 200 µL of the PPRV WS was mixed with 20 µL of the detergent (10% vol/vol) to make a final concentration of 1% and then incubated at RT for 10 min.
After the above treatments, 200 µL (LBs-MM or Triton X-100) or 140 µL (AVL/Ethanol) of the treated samples was diluted 1:100 (20 or 14 mL) to neutralize cytotoxicity, and the potential residual viruses were captured using NMVPs.Clarified NMVPs and the captured viruses were resuspended in 200 or 140 µL of EMEM (original starting volume of the virus) and inoculated onto fresh Vero cells for VI (1P).
Each treatment group also included appropriate positive and negative controls (PC-V, PC-NMVPs, and NC) for validation of the VI results.For PC-V used in the Triton X-100 treatments, 200 µL of PPRV WS was mixed with 20 µL of EMEM; and 200 µL of the diluted virus was inoculated onto Vero cells for VI.For PC-V used in LBs-MM treatments, 200 µL of PPRV WS was mixed with 700 µL of EMEM; and 200 µL of the diluted virus was inoculated onto Vero cells for VI.For PC-V used in AVL/Ethanol treatment, 140 µL of PPRV WS was mixed with 1,120 µL of EMEM; and 140 µL of the diluted virus was inoculated onto Vero cells for VI.For PC-NMVPs, the same virus dilutions that were used as the starting material for PC-Vs (above) were further diluted 1:100 (200 µL diluted to 20 mL for LBs-MM or Triton X-100 and 140 µL diluted to 14 mL for AVL/Ethanol), and then the viruses were captured using NMVPs (100 µL).The NMVPs and the potential captured residual viruses (if any) were clarified on a magnetic stand, resuspended in EMEM (200 or 140 µL), and inoculated onto Vero cells for VI.The Vero cells inoculated with EMEM only (200 or 140 µL per well) served as the NC.
The VI cultures after first inoculation (1P) from each treatment group were subjected to serial passages (2P and 3P) to confirm virus inactivation (no CPE), and the cell culture supernatants after each passage (1P, 2P, or 3P) corresponding to the individual treatment group were also analyzed separately by RT-qPCR for verification of the VI results.The virus was considered completely inactivated based on the following findings: the VI cultures/supernatants exhibited no CPE 7 days post-incubation after each passage (1P, 2P, or 3P), and the Ct values were undetectable; the PCs (PC-V and PC-NMVPs) were positive by VI (CPE) or RT-qPCR (detectable Cts); and the NCs were negative (no CPE or detectable Ct).A flowsheet diagram of the laboratory protocols used for virus capture (NMVPs) and chemical inactivation of PPRV is shown in Fig. 1.

Determining the minimum concentration of the chemical agents for com plete inactivation of PPRV
In this experiment, the virus was treated with serially diluted chemical agents to determine their minimum concentration that would completely inactivate the virus.The PPRV WS (200 or 140 µL) was treated with LBs-MM or AVL/Ethanol at 0:0 and at 1:10 or 1:100 dilutions, and with Triton X-100 at 1%, 0.1%, and 0.01% final concentrations under similar conditions as described above.After the treatments, the samples were diluted 1:100 (in EMEM) to neutralize cytotoxicity; and the residual viruses were captured using NMVPs.Clarified NMVPs and the captured viruses were resuspended in EMEM (200 or 140 µL) and inoculated onto Vero cells for VI for (1P).Additional passages (2P or 3P) were carried out as described above if needed to validate virus inactivation.
The supernatants of the VI cultures after each passage (1P, 2P, or 3P) were also analyzed separately by RT-qPCR for verification of the VI results.

Further verification of chemical inactivation of PPRV by dilutions followed by concentration on membrane filter
For further verification of chemical inactivation of PPRV in VLbs, the PPRV WS (200 or 140 µL) was treated with undiluted (0:0) LBs-MM or AVL/Ethanol as described above.Treated samples were diluted 1:100 to neutralize cytotoxicity and then concentrated on a membrane filter (Pierce Protein Concentrators PES, 50K MWCO, 5-20 mL) by centrifuga tion (3,000 × g for 30 min at 10°C).The filtrates (flow throughs) were saved, and the retentate (concentrated virus) was washed with EMEM (20 or 14 mL) by centrifugation (same tube) to remove residual chemicals.The filtrate of the second centrifugation was discarded, and the concentrated virus (volume reduced to ~200 µL) along with the saved filtrates from the first centrifugation was inoculated onto Vero cells for VI.For PCs, appropriately diluted (1:100; 20 or 14 mL) untreated PPRV WS was similarly concentrated on membrane filters by centrifugation as above and then inoculated onto Vero cells for VI.

RNA extraction and quantitative real-time RT-PCR
The RNA extractions were carried out manually on spin columns of the QVR Extraction Kit (Qiagen) using 200 µL or 140 µL of sample volume, and the extracted RNA was eluted with 60 µL of elution buffer (Buffer AVE).The MM-C Kit was not used for RNA extraction since it also uses magnetic beads that would be competing with the NMVPs to bind the same target (viral RNA), and it could yield inconsistent results.Therefore, for consistency, all extractions were performed using the silica-based spin columns of QVR.Preferential use of spin column-based extraction kits for purification of RNA from NMVPs and the captured viruses have also been reported by others (34)(35)(36).
The PPRV-specific RT-qPCR assays were carried out on an Applied Biosystems 7500 Fast thermocycler (Thermo Fisher Scientific) according to Batten et al. (38) using the Path-ID Multiplex One-Step RT-PCR Kit (Thermo Fisher Scientific).Briefly, the RT-qPCR reaction mastermix consisted of 12.5 µL of 2× multiplex RT-PCR buffer, 2 µL of multi plex enzyme mix, 0.25 µM each of the forward and reverse primers (Integrated DNA Technology, Newark, NJ, USA), and the TaqMan probe labeled with FAM as reporter dye at the 5′-end and MGB as the quencher dye at the 3′'-end (Thermo Fisher Scientific), 5 µL of template (extracted RNA) plus the required amount of nuclease-free water to adjust the final volume to 25 µL.The thermocycling conditions for RT-qPCR included one cycle of reverse transcription at 45°C for 10 min, one cycle of denaturation at 95°C for 10 min followed by 40 cycles of amplifications with each cycle consisting of 95°C denaturation for 15 s and 60°C annealing/elongation for 1 min.

Verification of chemical inactivation using PPRV-Nigeria
The chemical inactivation protocols developed and optimized for PPRV-Egypt were further validated using PPRV-Nigeria.The PPRV-Nigeria WS (1:1; above) was treated with LBs-MM or AVL/Ethanol at 0:0, 1:10, and 1:100 dilutions and Triton X-100 at 1%, 0.1%, and 0.01% final concentrations under the similar conditions as described above for inactivation of PPRV-Egypt.After the above treatments, the samples were diluted 1:100 to neutralize cytotoxicity and subjected to virus capture using NMVPs.The clarified NMVPs and the captured viruses were resuspended in EMEM and inoculated onto Vero cells for VI.Each treatment group also included appropriate PCs (PC-V or PC-NMVPs) and NC (EMEM) for verification of the VI results.Due to slow growth of PPRV-Nigeria, the Vero cells inoculated with the virus were examined every day and up to 9 days.The VI cultures (1P) were subjected to second (2P) and third (3P) passages to confirm virus inactivation (no CPE).The supernatants of the VI cultures after each passage (1P, 2P, or 3P) were also analyzed separately by RT-qPCR for further verification of the VI results.The assessment of complete inactivation of PPRV-Nigeria was based on the similar conditions as used for PPRV-Egypt.

Assessment of virus capture from diluted suspensions using NMVPs
In the virus capture optimization study, it was determined that 100 µL of NMVPs efficiently captures and concentrates PPRV (200 µL) from a wide range of dilutions between 1:10 (2 mL) and 1:200 (40 mL).This amount (100 µL) of NMVPs was used to capture PPRV from treated (LBs-MM) or untreated samples after 100-fold dilutions (200 µL diluted to 20 mL).After virus capture, the suspensions were clarified on a magnetic stand, and both NMVPs (reconstituted in 200 µL EMEM) and the supernatants (Sup-T or Sup-U) were saved and analyzed separately by VI and RT-qPCR.The VI cultures were subjected up to three serial passages (3P) to confirm virus inactivation.The results (Table 1) show comparable Ct values for the untreated PPRV (PC-V; 22.362; T4) and that captured by NMVPs (PC-NMVPs; 22.993; T1), while the Ct values were higher for the supernatants (Sup-U; 28.801; T1), indicating efficient virus capture.However, a low level of residual viruses remained in the supernatant that was detectable by VI after 2P (CPE), indicating the residual viruses were below the limit of detection of VI after 1P.
The results (Table 1) also showed a much higher Ct value (35.121;T2) for the NMVPs reconstituted from the treated samples (LBs-MM), which most likely correspond to the PPRV RNA released from the virus after lysis.Further analysis of the NMVPs (reconstitu ted) and the supernatants (Sup-T) from the treated samples by VI (T2, Table 1) shows no live virus detectable (no CPE) in either fraction (NMVPs or Sup-T) by VI up to 3P, and the supernatant of the VI cultures (1P, 2P or 3P) also tested negative (undetectable Ct) by RT-qPCR (not shown), indicating the absence of any live virus in the treated samples.These findings also correlate well with the results of VI on treated (LBs-MM) PPRV, which shows no detectable live virus captured by NMVPs (no CPE after 3P) (B2; Fig. 2).

Assessment of chemical inactivation of PPRV by VI using NMVPs
The assessment of chemical inactivation of PPRV was based on the combined results of VI and RT-qPCR experiments summarized in Table 2. Vero cells inoculated with untreated PPRV-Egypt developed CPE between 6 (<25%) and 7 (>50%) days post-inoculation.Accordingly, Vero cells inoculated with the virus (treated or untreated) were examined for CPE up to 7 days post-inoculation after each passage for the assessment of viral inactivation.The results show an absence of CPE on Vero cells inoculated with treated viruses captured by NMVPs at the following dilutions of the chemicals and the passages: LBs-MM at 0:0 and 1:10 dilution after 3P (B1 and B2; Fig. 2), AVL/Ethanol at undiluted (0:0) after 3P (B1; Fig. 3), and Triton X-100 at 1% and 0.1% concentrations after 3P (B1 and B2; Fig. 4).These results were further supported by RT-qPCR (Table 2) showing undetectable Ct values for the VI cultures corresponding to the above treatments.The surviving viruses were recoverable by VI after treatment with LBs-MM or AVL/Ethanol at 1:100 dilution (B3, Fig. 2; B3, Fig. 3) and Triton X-100 0.01% (B3, Fig. 4).It is interesting to note that AVL/Ethanol at 1:10 dilution was cytotoxic (A2; Fig. 3) but had no effect on PPRV as the viruses were fully recovered by VI (B2; Fig. 3).The above findings are further supported by RT-qPCR (Table 2).These results indicate PPRV is relatively more sensitive to LBs-MM (MM-C) or Triton X-100 than AVL/Ethanol (QVR).

Assessment of chemical inactivation of PPRV by dilution followed by concentration on membrane filter
For further verification of virus inactivation by the chemical agents, PPRV WS was treated with undiluted (0:0) LBs-MM or AVL/Ethanol, and then the samples were diluted (1:100) in EMEM and concentrated on 50 MWCO membrane filters by centrifugation.For positive controls, untreated PPRV was similarly diluted (1:100) in EMEM and concentrated on membrane filter by centrifugation.The viability of the concentrated viruses (treated or untreated) was assessed by VI on Vero cells.No CPE was observed on Vero cells inoculated with PPRV concentrated from treated (LBs-MM or AVL/Ethanol) samples (not shown); however, CPE was observed on cells inoculated with PPRV concentrated from untreated samples (not shown), further supporting this chemical inactivation methodol ogy.

Verification of chemical inactivation using PPRV-Nigeria
The chemical inactivation protocols developed and optimized for PPRV-Egypt were applied on PPRV-Nigeria for further verification.Vero cells inoculated with untreated PPRV-Nigeria developed CPE between 7 (<25%) and 9 (>50%) days post-inoculation.Accordingly, Vero cells inoculated with the virus (treated or untreated) were examined for CPE up to 9 days post-inoculation for the assessment of viral inactivation.Examination of the images of Vero cells indicates the viruses were completely inactivated (no CPE after 3P) by LBs-MM at undiluted (0:0; not shown) and at 1:10 dilution (A1; Fig. 5); by undiluted (0:0) AVL/Ethanol (A2; Fig. 5) and by Triton X-100 at 1% (not shown) and 0.1% concentrations (A3; Fig. 5).The virus survived when exposed to 1:100 dilution of LBs-MM (B1; Fig. 5) or AVL/Ethanol (not shown) and 0.01% of Triton X-100 (B3; Fig. 5).PPRV was shown to be partially inactivated by AVL/Ethanol at 1:10 dilution (no CPE after 1P; not shown) but fully recovered (developed CPE) after 2P (B2; Fig. 5), and these results were further supported by RT-qPCR (not shown).These findings correlate well with the findings of the PPRV-Egypt studies, indicating consistency in the virus inactivation protocol applicable to both isolates of PPRV.

DISCUSSION
Proper handling of materials containing highly pathogenic viruses is extremely important for laboratory biosafety and biosecurity.For research and diagnostic purposes, it is often necessary to transfer inactivated virus-containing materials from endemic to non-endemic regions within a country or from a high-level biocontainment (BSL3 or BSL4) lab to a low-level containment (BSL2) lab.Complete inactivation of the viruses is, therefore, critical prior to carrying out such transfers/operations.Examples of such operations include the 2014 Ebola virus (EBOV) outbreak in Africa and, most recently, the 2019 pandemic of SARS-CoV-2.In both cases, infectious specimens/materials from disease outbreak sites had to be transported to laboratories to perform critical opera tions including research and diagnosis.Accordingly, both EBOV and SARS-CoV-2 have been targeted for the development of reliable virus inactivation protocols.It has been reported that EBOV and SARS-CoV-2 can be successfully inactivated by chemical methods using Triton X-100 or the VLb of several commercial nucleic acid extraction kits including MagMAX and QVR (12,(14)(15)(16)(17)(18)(19)(20).
There is no report on PPRV inactivation using any of the chemical agents as described above.In this study, we developed and evaluated a virus inactivation protocol using Triton X-100 and the VLb of two commercial nucleic acids extraction kits, MM-C and QVR; and the virus inactivation was verified and confirmed by using NMVPs (virus capture) followed by VI and RT-qPCR.One of the advantages of using Triton X-100 or VLbs is that the integrity of the viral genome remains mostly intact, which allows minimal interference in the downstream applications such as PCR and NGS.
Triton X-100 is a mild non-ionic detergent and a surfactant that can break/disrupt protein-lipid and lipid-lipid associations of the membrane of enveloped viruses, which is thought to be one of the mechanisms contributing to loss of viability and virus inactivation (26,(39)(40)(41).The VLbs of the commercial extraction kits also carry detergents such as Triton X-100 (some of the commercial kits) in addition to chaotropic agents  such as guanidium thiocyanate, which primarily act as protein denaturants inactivating nucleases.The chaotropic agents can also break non-covalent interactions of biomo lecules (e.g., hydrogen bonds, dipole-dipole interactions, hydrophobic interactions) resulting in disruption of membranes and loss of viability/infectivity of viruses while eliminating cellular nucleases (26).
Treatment with chemical agents such as Triton X-100 or VLb does not always guarantee complete inactivation of the viruses, which can be a major concern with respect to biosafety and biosecurity since it could potentially lead to the release of live viruses into the environment and exposure to susceptible hosts.Unreliable and incomplete virus inactivation using commercial VLbs have been reported (14)(15)(16)18); and there have been many cases of laboratory-acquired infections due to either incomplete inactivation or mishandling of infectious agents (42)(43)(44)(45).To avoid such occurrences, virus inactivation protocols must be thoroughly validated.One of the gold standard methods for verification of virus inactivation is VI using susceptible cell lines; however, due to cytotoxicity of the chemicals such as detergents and/or chaotropic salts (11,19,21,30), it is difficult to test viability of the residual viruses in treated samples by VI.Therefore, for successful implementation of VI, the toxic chemicals must either be removed or diluted to non-toxic levels before cell culture.Dilution of chemically treated viruses in appropriate diluents (PBS or viral growth medium such as EMEM) followed by concentration by centrifugation on membrane filters prior to VI has been reported (29,30).In this study, the toxic chemicals in the treated samples were successfully neutralized by dilutions in EMEM, and the potential residual viruses (if any) were captured and concentrated using NMVPs.The NMVPs and the captured viruses were then clarified on a magnetic stand and inoculated onto Vero cells for VI to assess virus inactivation.
The cytotoxicity of the chemical agents was evaluated by serial dilutions followed by exposure to Vero cells.The microscopic examination of Vero cells exposed to chemical agents revealed cytotoxicity that gradually declined through dilutions or serial passages on Vero cells (Table 2; Fig. 2 to 4).Vero cells exhibited no cytotoxicity when exposed to LBs-MM or AVL/Ethanol at a 1:100 dilution and Triton X-100 at 0.01% vol/vol (A3; Fig. 2  to 4).Neutralization of cytotoxicity at 0.01% Triton X-100 and at 1:100 or higher dilutions of VLbs of MagMAX or QVR was also reported by others (11,18).Therefore, for the assessment of virus inactivation, all chemically treated PPRV samples were appropriately diluted (1:100 or 0.01%) prior to VI.
The efficacy of virus capture by NMVPs from diluted suspensions of PPRV was evaluated separately by RT-qPCR and VI.Based on the Ct values of the viruses captured by NMVPs, it was determined that 100 µL of NMVPs is optimum to capture PPRV from a wide range of dilutions (1:10 to 1:200).Efficient virus capture from diluted suspensions using 100 µL of NMVPs has also been reported for multiple enveloped viruses including SARS-CoV-2 and influenza A and B (34,46).Comparable Ct values between the PPRV RNA extracted from the untreated virus (T4; Table 1) and that extracted from the NMVPs after virus (untreated) capture (PC-NMVPs; T1; Table 1) indicate efficient virus recovery.A low level of residual viruses was still detectable (Ct 28.801; T1.Table 1) in the supernatants (Sup-U) that were recoverable by VI after 2P (T1; Table 1).The higher Ct values (35.121) corresponding to the NMVPs recovered from the treated (LBs-MM) samples (T2; Table 1) most likely contributed by PPRV RNA released from the virus after lysis.The absence of any live virus in the treated samples was further confirmed by VI which shows neither NMVPs (reconstituted) nor the supernatants (Sup-T) from the treated samples developed any CPE in VI cultures up to 3P (T2, Table 1), and these results (absence of any virus particles) were further supported by undetectable Ct (RT-qPCR) in the supernatants of the VI cultures (not shown).Assuming a 100% amplification efficiency of RT-qPCR and that the Ct values increased by ~3 units (3.33 to be exact) at every 10-fold dilution of the template (RNA) (link: Agilent Genomics : Tools -Bio Calculators), the amount of the viruses captured by NMVPs is estimated to be 100-fold higher than the amount of the residual viruses in the supernatant.Efficient virus capture (nearly 100%) by NMVPs from diluted suspensions have also been reported for several enveloped viruses (33,35,36).
The above results show NMVPs are not toxic to either host cells (Vero) or the virus (PPRV), and the captured viruses remained viable and recoverable by VI, which has also been reported by others on other viruses (33,37,46).
The chemical inactivation of PPRV reported in this study was verified using two strains of PPRV (Egypt and Nigeria), and both were shown to be completely inactivated by undiluted (0:0) LBs-MM or AVL/Ethanol and 1% Triton X-100 (Fig. 2 to 5; Table 2).Complete inactivation of PPRV by undiluted (0:0) LBs-MM or AVL/Ethanol was further supported by dilutions, followed by concentration of the treated virus on membrane filters by centrifugation.The concentrated virus recovered from the treated samples failed to develop CPE on Vero cells, which is consistent with the results of virus (treated) capture using NMVPs.These findings reaffirmed the efficacy of virus inactivation by these chemicals that have been previously reported on several highly pathogenic and zoonotic viruses (13,14,16,19,21,29).PPRV (Egypt or Nigeria) was also shown to be completely inactivated by the chemical agents at further dilutions including LBs-MM at 1:10 (B2; Fig. 2) and Triton X-100 at 0.1% (B2; Fig. 4), but they survived at 1:100 of LBs-MM, 1:10 and 1:100 of AVL/Ethanol, and at 0.01% of Triton X-100 (Fig. 2 to 5; Table 2).Triton X-100 alone at 0.1% was also shown to strongly reduce the infectivity of West Nile Virus and EBOV (4-6 logs), but a complete inactivation of the virus could not be achieved (11).Studies on EBOV show the virus was partially inactivated by AVL (undiluted) or 0.1% Triton X-100 but completely inactivated by AVL plus 0.1% (vol/vol) Triton X-100 (14) or AVL plus ethanol (15).AVL plus ethanol was also shown to completely inactivate PPRV (this study).One of the interesting findings of the dilution studies was that AVL/Ethanol at 1:10 is toxic to Vero cells (A2; Fig. 3), but it only partially inactivated PPRV as the residual viruses were successfully recovered by VI (2P) after virus capture (B2, Fig. 3; B2, Fig. 4).These results suggest PPRV is more susceptible to LBs-MM or Triton X-100 than AVL/Ethanol.Some of the differences between the PPRV inactivation (this study) and the inactivation of other viruses could be due to the relative tolerance of the individual viruses to the chemical agents and/or the initial concentration of the viruses being used for virus inactivation.In this regard, a complete inactivation of virus may occur at lower concentrations (this study) but not at higher titers/concentrations (11).
The assessment of the combined results of VI and RT-qPCR as described above reveals that PPRV can be completely inactivated by either of the three chemical agents alone: LBs-MM, AVL/EtOH, or Triton X-100.This assessment reaffirms safe handling, operations, and transfer of chemically inactivated PPRV samples from high-level biocontainment (BSL3 or BSL4) to a low-level containment (BSL2) without compromising biosafety and biosecurity, and it also minimizes the risk associated with any accidental release of the virus into the environment or susceptible hosts.
In a separate study (not shown), it was found that the genomic integrity of chemically inactivated PPRV remained mostly intact (personal communication, Zaheer Ahmed), further supporting the advantages of the chemical inactivation methods used in this study.
This is the first report on the use of NMVPs for verification of virus inactivation combined with VI.NMVPs have been shown to capture viruses, while still retaining their ability to detect the analyte of interest (33), and this capacity of the NMVPs has been further exploited to improve diagnostic sensitivity of the viruses in dilu ted samples/specimens (33,34,46).The new virus inactivation verification method developed in this study can be safely used in laboratories working on PPRV.The chemistry of NMVPs favors capture of enveloped viruses (33,37,46) including PPRV as demonstrated in this study.Furthermore, this study provides an effective working protocol to dilute the active ingredients of the virus-inactivating chemical agents from treated samples and to capture and concentrate the residual viruses by using NMVPs after VI.This assessment and verification method of virus inactivation using NMVPs can be applied to other high impact viruses of agricultural or public health importance.

FIG 4
FIG 4 Cytotoxicity or CPE on Vero cells after treatments with Triton X-100 or inoculation with NMVPs reconstituted from virus (PPRV-Egypt) samples after treatment with Triton X-100.A1, A2, and A3 represent Vero cells treated with Triton X-100 at final concentrations of 1%, 0.1%, and 0.01%, respectively; B1, B2, and B3 represent cells inoculated with NMVPs reconstituted from virus samples after treatment with Triton X-100 at final concentrations of 1%, 0.1%, and 0.01%, respectively; B4 (PC-NMVPs) represents Vero cells inoculated with NMVPs reconstituted from diluted (1:100) suspensions of untreated virus; A4 (NC) represents Vero cells inoculated with EMEM only.Images of the Vero cells were taken 7 days post-incubation after the number of passages (P) as shown.Arrows point to the NMVPs observed on some monolayers.

a
T1 (PC-NMVPs): 200 μL of PPRV WS (PPRV-Egypt) was diluted with 700 μL of EMEM and 200 μL of the diluted virus further diluted 1:100 in EMEM (20 μL) and subjected to virus capture using 100 μL of NMVPs.The NMVPs and the supernatants (Sup-U) were clarified on a magnetic stand and analyzed separately by RT-qPCR and VI.T2: 200 μL of PPRV WS was treated with 700 μL of LBs-MM and 200 μL of the treated virus further diluted 1:100 in EMEM (20 μL) and subjected to virus capture using 100 μL of NMVPs.The NMVPs and the supernatants (Sup-T) were clarified on a magnetic stand and analyzed separately by RT-qPCR and VI.T3 (PC-V): 200 μL of PPRV WS was diluted with 700 μL of EMEM and 200 μL of the diluted virus inoculated onto Vero cells for VI.T4: 200 μL of PPRV WS was diluted with 700 μL of EMEM, and 200 μL of the diluted virus was extracted and analyzed by RT-qPCR.NC: 200 μL of EMEM inoculated onto Vero cells, and the cell cultures were analyzed separately by VI and RT-qPCR.b Y/N, YES/NO.

c
PC-V: 200-or 140 μl of WS (PPRV-Egypt) proportionately diluted with EMEM (same volume as the chemical agents used for virus inactivation) and 200-or 140 μl of diluted virus inoculated onto Vero cells and subjected up to 3 serial passages (1P, 2P and 3P) and examined for CPE.The supernatant of the VI cultures after each passage were also analyzed separately by RT-qPCR to confirm the results of VI. d NC: Vero cells inoculated with EMEM (200-or 140 μl) only and the supernatants of the cell cultures were analyzed separately by VI and RT-qPCR.e nd, not determined.f Y/N, Yes/No.

TABLE 1
Assessment of virus capture from diluted suspensions of PPRV by NMVPs a

TABLE 2
Assessment of chemical inactivation of PPRV by VI and RT-qPCR after virus capture using NMVPs a Virus inactivation: 200-or 140 μl of WS (PPRV-Egypt) treated with serially diluted LBs-MM, AVL/Ethanol or Triton X-100 (shown above) under the conditions as described in materials and methods.Next, the treated samples were diluted 1:100 (EMEM) and subjected to virus capture using NMVPs; the clarified NMVPs and the captured viruses were resuspended in EMEM (200-or 140 μl) and inoculated onto Vero cells for VI.The VI cultures corresponding to each treatment group were inoculated onto fresh Vero cells and subjected up to 3 serial passages (1P, 2P and 3P) and examined for CPE as described in materials and methods.The supernatants of the VI cultures after each passage (1P, 2P or 3P) were also analyzed separately by RT-qPCR to confirm the results of VI.Each experiment was repeated at least twice and the average results including SD (Ct values) are shown.b PC-NMVPs: 200-or 140 μl of WS (PPRV-Egypt) diluted 100x (20 ml or 14 ml) in EMEM and then subjected to virus capture using NMVPs; the clarified NMVPs and the captured viruses were resuspended in EMEM (200-or 140 μl) and inoculated onto Vero cells for VI and the VI cultures were subjected up to 3 serial passages (1P, 2P and 3P) and examined for CPE.The supernatant of the VI cultures after each passage (1P, 2P or 3P) were also analyzed separately by RT-qPCR to confirm the results of VI. a