PLOS ONE RESEARCH ARTICLE Inactivation of African swine fever virus inoculated in liquid plasma by spray drying and storage for 14 days at 4˚C or 20˚C Elena Blázquez ID1,2, Joan Pujols1,3, Joaquim Segalés1,3,4, Núria Navarro1,3, Carmen Rodrı́guez2, Jesús Ródenas2, Javier Polo2,5* 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA), Barcelona, Spain, 2 APC Europe, S.L.U. Granollers, Granollers, Spain, 3 OIE Collaborating Centre for Emerging and Re-Emerging Pig Diseases in Europe, IRTA- CReSA, Barcelona, Spain, 4 Departament de Sanitat i Anatomia Animals, Universitat Autónoma de a1111111111 Barcelona (UAB), Barcelona, Spain, 5 APC LLC, Ankeny, IA, United States of America a1111111111 a1111111111 * javier.polo@apc-europe.com a1111111111 a1111111111 Abstract African swine fever virus (ASFV) is a dsDNA virus that can cause high mortality in pigs of all OPEN ACCESS ages. Spray-dried porcine plasma (SDPP) is a highly digestible ingredient used in feed because it benefits performance, gut function and immunity. The objectives were to test if Citation: Blázquez E, Pujols J, Segalés J, Navarro N, Rodrı́guez C, Ródenas J, et al. (2023) the spray-drying (SD) conditions along with post-drying storage of product for 14 days can Inactivation of African swine fever virus inoculated inactivate ASFV inoculated in liquid plasma. Fresh liquid porcine plasma was inoculated in liquid plasma by spray drying and storage for 14 with ASFV (BA71V) to a final concentration of 105.18 ±0.08 TCID50/mL of liquid plasma. Tripli- days at 4˚C or 20˚C. PLoS ONE 18(8): e0290395. cate 2-L samples of spiked plasma were SD in a lab drier set at an outlet temperature of https://doi.org/10.1371/journal.pone.0290395 80˚C or 71˚C. The final dried samples were stored at 4˚C or 20˚C for 14 d. Liquid and SD Editor: Kelli L. Barr, University of South Florida, samples were analyzed for ASFV infectivity in two mirror 24-well plaques containing VERO UNITED STATES cells monolayers. Wells were inoculated with different dilutions of SDPP dissolved 1:9 in Received: January 16, 2023 PBS. One plaque was immediately frozen at -80˚C and the other was incubated at 37˚C for Accepted: August 7, 2023 3 d. Each dilution was replicated 9 times. After incubation both plaques were analyzed for Published: August 22, 2023 ASFV by qRT-PCR. Results indicated that the SD process inactivated between 3.2 to 4.2 Copyright: © 2023 Blázquez et al. This is an open Logs ASFV TCID50/mL and 2.53 to 2.75 Logs TCID50/mL when the outlet temperature were access article distributed under the terms of the 80˚C and 71˚C respectively. All SD samples stored at 4˚C or 20˚C for 14 d were absent of Creative Commons Attribution License, which infectious ASFV. The combination of SD and post drying storage at both temperatures for permits unrestricted use, distribution, and 14 d was able to inactive >5.18 ±0.08 Log10 of ASFV inoculated in liquid porcine plasma, reproduction in any medium, provided the original author and source are credited. demonstrating that the manufacturing process for SDPP can be considered safe regarding ASFV. Data Availability Statement: All relevant data are within the paper. Funding: Funding for this study was provided by APC Europe, S.L.U., Granollers, Spain, and APC LLC, Ankeny, IA, 50021, USA. These companies Introduction manufacture animal blood products for animal Spray-dried porcine plasma (SDPP) is a highly digestible, high-protein ingredient that is consumption. The funders provided support in the widely used in feed because it benefits growth performance, feed efficiency, intestinal integrity, form of salaries for authors EB, CR, JC, LR and JPolo, but did not have any additional role in the and immune parameters [1–3]. study design, data collection and analysis, decision The manufacturing process of SDPP begins with the collection of blood from healthy ani- to publish, or preparation of the manuscript. The mals declared fit for slaughter for human consumption by the veterinary authorities. Blood is PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 1/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying specific roles of these authors are articulated in the collected into containers with anticoagulant, chilled, and centrifuged to separate plasma. Liq- ‘author contributions’ section. uid plasma is then concentrated and spray-dried at 80˚C throughout its substance to produce Competing interests: The authors have read the SDPP [3, 4]. Spray-drying involves very rapid desiccation of the liquid [5], results in a dried journal’s policy and the authors of this manuscript powder with low moisture (< 9%) and very low water activity (aw < 0.6), which has a detri- have the following competing interests: EB, CR, JR mental effect on bacteria and virus survivability, as has been shown for multiple bacteria and and JPolo are employed by APC Europe, S.L.U. viruses of interest in swine industry [6–12]. Furthermore, some microorganisms, especially Granollers, Spain and JPolo is employed by APC LLC, Ankeny, IA, USA. APC Europe and APC LLC bacteria and enveloped viruses, are not able to survive for a prolonged time in dried materials manufactures and sells spray-dried animal plasma; like SDPP with very low water activity [3, 5, 8, 12, 13]. Therefore, a minimum post-drying however, the companies did not have any period of 14 days at 20˚C has been established as processing standards of the European Animal additional role in the study design, data collection Protein Association (EAPA) (EAPA Code of Practice, https://www.eapa.biz/quality-safety) and analysis, decision to publish, or preparation of and been adopted by most SDPP manufacturers. the manuscript. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data African swine fever virus (ASFV) is an enveloped dsDNA virus belonging to the Asfarviridae and materials. JPujols, NN and JS declared no family [14] that can cause high mortality in pigs of all ages. In 2007, a virulent ASFV strain conflict of interest. (genotype II) appeared in Georgia causing mortality rates up to 100%, and from there spread across the Caucasus region into the Russian Federation, Easter Europe, and other West Euro- pean countries (Belgium, Germany, Italy, Greece and Czech Republic). Subsequently, from 2018 onwards, ASFV further spread to China, South-East Asia, and more recently, in the Dominican Republic and Haiti, where millions of animals have succumbed to the disease [15, 16]. At global level there is no commercial vaccine available to fight against the ASF pandemic; however, recent results with recombinant live attenuated viruses provided hope for a safe and effective vaccine against ASFV as demonstrated by the launching of the first commercial vac- cine against ASFV in Vietnam. This vaccine product is based on a recombinant deletion mutant lacking the I177L gene from the Georgia 2007 ASFV isolate [15–17]. The prevalence of African Swine Fever (ASF) has increased concerns that feed ingredients could represent a risk factor for spreading ASF. Spray-drying has shown an ASFV inactivation capacity of 4.11 ± 0.20 Log10 TCID50/mL [18] resulting in the recognition of SDPP as a negligi- ble risk for transmitting the disease [19]. However, other studies are necessary to characterize the inactivation capacity of other processing steps included in the manufacturing process of SDPP, such as the post-processing storage period at 20˚C. The objective of the present study was to determine the level of ASFV inactivation in liquid plasma artificially contaminated with ASFV by the safety steps involved in commercial SDPP production including spray-drying in combination with storage for 14 days at different temperatures. Material and methods African swine fever virus ASFV strain Badajoz-71 adapted to Vero cells (ASFV-BA71-V) [20] was provided by Dr. Marı́a Luisa Salas from Centro de Biologı́a Molecular Severo Ochoa (CBMSO), Madrid, Spain. The virus was propagated in Vero cells (ATCC CCL-81) grown in DMEM (ThermoFisher, Waltham, MA, USA) supplemented with 10% FBS (BioWest, Florida, USA), 200 mM gluta- mine (ThermoFisher), penicillin 100 UI/mL (ThermoFisher), streptomycin 100 μg/mL (Ther- moFisher), and nystatin 40 UI/mL (Sigma-Aldrich, Missouri, USA). Viral stock solution was produced in this way in successive passages obtaining a final viral titer of 106.9 TCID50/mL measured by immunoperoxidase monolayer assay (IPMA). Plasma The plasma used for this experiment was obtained from pigs slaughtered in European porcine abattoir facilities from animals inspected and approved as fit for slaughter for human PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 2/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying consumption by official veterinary authorities. Blood was collected in stainless steel containers, with an anticoagulant (sodium tripolyphosphate), refrigerated and transported to the APC Europe facilities (APC-Europe S.L.U., Granollers, Spain). Plasma was separated in less than 24 hours from collection by commercial centrifugation. Eight liters of fresh plasma were UV-C irradiated at 10000 J/L for this study using the UV-C SurePure SP-1 device as previously described [21, 22] to inactivate any microorganisms before the ASFV inoculation. A 10 mL sample of the 8-L batch was stored at –80˚C and later used as a control to determine ASFV antibodies (INgezim PPA COMPAC, INGENASA; Madrid, Spain) or genome by real time PCR (qRT-PCR) analysis [23]. ASFV spray-drying inactivation Three 2-L batches of UV-C irradiated liquid porcine plasma were used for the study. From each batch, 200 mL of the 2 L were collected and stored at –80˚C for later analysis. The remain- ing 1800 mL of liquid plasma per batch were inoculated with 200 mL of ASFV-BA71-V stock solution (106.9 TCID50/mL) achieving a viral theoretical titer of 105.9 TCID50/mL. The spray-drying process was conducted using a laboratory spray-drier (Büchi Mini Spray Dryer B-290, Büchi Labortechnik, Switzerland) simulating industrial process conditions. The laboratory spray-drier was set to an inlet air temperature of 200 ± 5˚C and outlet air tempera- ture set at 80 ± 1˚C or at 71 ± 1˚C as previously described [10]. Airflow through the column and the suspension flow to the nozzle was set at 45 m3 h-1 (at 20˚C) and 0.2 lh-1, respectively. The airflow through the feed nozzle adjusted to 0.7 m3h-1 (at 20˚C). Residence time was esti- mated to be 0.41 s. Before processing and collecting dried samples for analysis, the lab spray-dryer processing parameters were stabilized by running it with non-inoculated UV-C irradiated liquid porcine plasma at the target temperature for 10 min. After this time, the sample collector for the spray- dryer was replaced with a clean one and the previously collected powder was discarded. This preliminary drying time assures that the system was stabilized at the desired processing param- eters before the ASFV inoculated plasma was dried and samples for analysis were collected. Each dried inoculated sample was distributed in twenty-seven samples of 0.5 g in 0.5 cm glass tubes (inner diameter). Nine samples were obtained directly without receiving additional heat post drying. To simulate the longer retention time in commercial spray driers, an addi- tional 9 samples were placed in a water bath at 90±3˚C for 30 seconds (real temperature in the powder sample was around 70.4˚C) and another nine samples for 60 seconds (real temperature in the powder sample was around 80.7˚C). The temperature of the spray dried samples during the 30 s and 60 s post-drying heat treatment was monitored with temperature probes. In each simulation, three of these samples were analyzed directly, three samples were stored at 4˚C for 14 days and another 3 samples at 20-22˚C (room temperature) for 14 days (Fig 1). Sample analysis Spray-dried samples were reconstituted by adding 4.5 mL of PBS to 0.5 g of plasma sample (ratio SDPP 1:9 PBS). A ten-fold dilution series was done from native samples to -3 dilution. Each dilution was inoculated in 9 wells, obtaining 9 replicates of the same sample. This proce- dure was done by duplicate to obtain two identical mirror 24 wells plates. Of these two identi- cal plates, one of them was immediately frozen at -80˚C and the other one was incubated at 37˚C for 3 days. After the 3-day incubation period, the plates were frozen and thawed and the frozen plates were thawed also. Both mirror plates were analyzed for presence of ASFV by qRT-PCR [23]. From the 9 inoc- ulated wells for each dilution, 100 µL were taken from three wells and mixed to perform a PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 3/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying Fig 1. Scheme of the experiment. https://doi.org/10.1371/journal.pone.0290395.g001 single extraction from those 3 wells. In this way, from the 9 initial wells, 3 DNA extractions (INDICAL, INDICAL BIOSCIENCE, Leipzig, Germany) were performed for each dilution. Subsequently, these three extractions were analyzed by qRT-PCR to obtain three replicates of each dilution. We compared the difference between the Ct obtained in the frozen plate and the plate incubated at 37˚C, assuming that a difference of Ct in the incubated plate compared to the frozen plate implied viral replication in the Vero cell culture [24]. The reduction factor was calculated as the difference between the virus titer detected in the inoculated material at start of the experiment and the titer or absence of replication detected in the final sample after pro- cessing. Values were calculated as the reduction in Logs TCID50/mL including standard error. Standard curves were established at each outlet temperature by regressing Log10 TCID50/mL SDPP on CT results (Figs 2 and 3). Results The spray-drying process at an outlet temperature of 80˚C throughout its substance inacti- vated ASFV between 3.2±0.07 to 4.2±0.07 Logs TCID50/mL. When the outlet temperature was reduced to 71˚C, ASFV inactivation was reduced to 2.53±0.12 to 2.75±0.04 Logs TCID50/mL (Table 1). The ASFV inoculated spray-dried samples obtained from both outlet temperatures with dif- ferent residence times (0 to 60 seconds) when stored either at 4˚C or 20˚C for 14 days were completely inactivated (Table 1). Our data suggested that both storage temperatures after the spray-drying process inactivated at least an additional 2.65±0.08 Log of ASFV. PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 4/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying Fig 2. Standard curve of ASFV Log TCID50/mL vs Ct for samples spray-dried at 80˚C. https://doi.org/10.1371/journal.pone.0290395.g002 Discussion Spray-dried plasma (SDP) is a functional protein source that significantly improves daily gain, feed intake, production efficiency and reduces post weaning lag [2, 25–29]. The manufacturing process of SDP involves several safety features as veterinary inspection at abattoir, pooling of plasma, spray-drying process and storage [3, 4]. Veterinary inspection is crucial to ensure that blood from only healthy animals slaughtered for human consumption is the exclusive source of raw material to be used for the manufacturing of blood products. However, veterinarian inspections might not find subclinical illnesses or early viremia/bacteremia, therefore, imple- menting, understanding, and validating the biosafety measures are therefore crucial for the manufacturing process of SDP. Several viruses of concern to the swine industry, including the Porcine respiratory and reproductive syndrome virus (PRRSV), Pseudorabies virus (PRV), and Porcine epidemic diar- rhea virus (PEDV), have been shown to be effectively inactivated by spray-drying [9–12], as well as bacteria such as Escherichia coli or Salmonella enterica [7, 8]. The phenomena involved in spray-drying inactivation of these pathogens rely on the rapid desiccation [5], cytoplasmic membrane damage [30, 31], genetic material destruction, and inactivation of other proteins, including enzymes [31]. Under commercial conditions, during the spray-drying process, SDP is exposed to a minimum of 80˚C throughout substance [3], a temperature that is recognized as effective to inactivate pathogens such as ASFV, CSFV, Swine vesicular disease virus and Foot and mouth disease virus (FMDV) in cooked meat products (“Council Directive 2002/99/EC of 16 December 2002). In the present study we tested the commercial outlet air temperature conditions of 80˚C and 71˚C. Under these conditions we PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 5/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying Fig 3. Standard curve of ASFV Log TCID50/mL vs Ct for samples spray-dried at 71˚C. https://doi.org/10.1371/journal.pone.0290395.g003 observed higher ASFV inactivation at 80˚C (range between 3.23–4.20 Log reduction Value (LRV)) than 71˚C (range 2.53±0.12–2.75±0.04 LRV), results that agrees with publications indi- cating that pathogen inactivation during spray-drying is related to the inlet and outlet air tem- peratures, especially the outlet temperature [3, 5, 32]. These results also agree with the code of practice that established 80˚C outlet temperature as the recommended drying temperature for commercial blood products intended for use in swine feedstuff (EAPA, 2022). In addition, the results confirm that the higher residence time of commercial driers (30 to 60 seconds) increases the inactivation of ASFV inoculated in liquid plasma compared to the residence time of laboratory driers (<1 sec), as demonstrated in previous publications that show higher resi- dence time inside spray driers may increase the inactivation of microorganisms [5, 32]. Table 1. African swine fever virus (ASFV) inactivation during spray-drying at different outlet temperatures and residence times and further storage at either 4˚C or 20˚C for 14 d. Initial ASFV inoculation in liquid plasma was 105.18 ±0.08 TCID50/mL. Conditions SDPP (TCID50/mL) LRVs during SD Storage of SDPP (4˚C or 20˚C) for 14days (TCID50/mL) (TCID50/mL) SD at 80˚C; No post treatment 1.94±0.07 3.23±0.07 Negative SD at 80˚C; RT 30 sec at 80˚C 1.35±0.06 3.82±0.07 Negative SD at 80˚C; RT 60 sec at 80˚C 0.97±0.07 4.20±0.07 Negative SD at 71˚C; No post treatment 2.59±0.14 2.58±0.14 Negative SD at 71˚C; RT 30 sec at 80˚C 2.64±0.12 2.53±0.12 Negative SD at 71˚C; RT 60 sec at 80˚C 2.42±0.04 2.75±0.04 Negative SD = spray-drying; RT = residence time; SDPP = spray-dried porcine plasma; LRVs = Log10 reduction values https://doi.org/10.1371/journal.pone.0290395.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 6/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying In previous studies spray-drying at 80˚C for 60 s, was able to inactivate 4.11 Log TCID50/ mL of ASFV [15]. In the present study, we wanted to characterize the loss of viability that occurs naturally in the storage step of SDPP manufacturing process, comparing two different storage temperatures. Post-drying storage for 14 days at 4˚C or 21±2˚C resulted in complete inactivation of ASFV. The additional 1.95±0.08 Log10 TCID50/mL inactivation at 80˚C or 2.65 ±0.08 Log10 TCID50/mL at 71˚C partially agrees with results obtained by Fischer et al., 2021 [24], who achieved a complete inactivation of 5.7 HAD/ml after two weeks of storage at room temperature, but limited inactivation was achieved for SDPP spiked with ASFV and stored at 4˚C. However, we observed a 2.65±0.08 Log10 TCID50/mL loss of viability when SDPP was stored at 4˚C. The differing results between our study and theirs may be that in our study we inoculated ASFV into the liquid plasma before drying, whereas they spiked ASFV on previ- ously dried porcine plasma. Therefore, the initial viral titer after spray drying in our study was lower than the initial viral titer of the spiked SDPP used in the study by Fischer et al. (2021). In addition, we started the storage period just after spray-drying of the liquid plasma without time for the virus to recover from the heat and desiccation effects associated with the spray- drying process. In contrast, the current results are consistent with the inactivation of 2 Log10 TCID50 observed by Dee et al., 2018 [33] over 30 days at room temperature. Furthermore, in a previous study [18] it was demonstrated that the minimum infectious dose in feed mixed with ASFV contaminated liquid porcine plasma was higher than 5.0 Log10 TCID50/pig. Considering this minimum infective dose of ASFV in the feed, the proven inacti- vation capacity of the spray-drying process (> 4 log TCID50/mL) and the subsequent inactiva- tion during post-drying storage (inactivation between 1.95 to 5.7 Log10 TCID50/mL) within two weeks at room temperature, the manufacturing process for SDPP has a high inactivation capacity against ASFV. The overall results demonstrated that spray-dry process at 80˚C or 71˚C and the subsequent storage at room temperature for 14 days inactivates >5.18 ±0.08 Log10 TCID50/mL of ASFV result in a safe feed ingredient for use in swine feed. Acknowledgments The authors want to acknowledge the protocol review and suggestions received from the tech- nical members of the European Animal Protein Association (EAPA), especially from Lourens Heres (Sonac/Darling Ingredients, The Netherlands) and Isabelle Kalmar (Veos N.V., Belgium). Author Contributions Conceptualization: Elena Blázquez, Joan Pujols, Joaquim Segalés, Carmen Rodrı́guez, Jesús Ródenas, Javier Polo. Data curation: Elena Blázquez, Joan Pujols, Joaquim Segalés, Jesús Ródenas, Javier Polo. Formal analysis: Elena Blázquez, Joan Pujols, Joaquim Segalés, Jesús Ródenas, Javier Polo. Funding acquisition: Joan Pujols, Joaquim Segalés, Carmen Rodrı́guez, Javier Polo. Investigation: Elena Blázquez, Joaquim Segalés, Jesús Ródenas, Javier Polo. Methodology: Elena Blázquez, Núria Navarro, Jesús Ródenas. Project administration: Elena Blázquez, Joan Pujols, Joaquim Segalés, Javier Polo. Resources: Elena Blázquez, Joan Pujols, Javier Polo. Software: Elena Blázquez. PLOS ONE | https://doi.org/10.1371/journal.pone.0290395 August 22, 2023 7/9 PLOS ONE ASFV inactivation in liquid plasma by Spray-drying Supervision: Joan Pujols, Joaquim Segalés, Carmen Rodrı́guez, Javier Polo. Validation: Elena Blázquez, Joan Pujols, Joaquim Segalés, Javier Polo. Visualization: Elena Blázquez, Javier Polo. Writing – original draft: Elena Blázquez, Joaquim Segalés, Javier Polo. Writing – review & editing: Elena Blázquez, Joaquim Segalés, Carmen Rodrı́guez, Jesús Ródenas, Javier Polo. References 1. Coffey RD, Cromwell GL. Spray-Dried Animal Plasma in Diets for Weanling Pigs. Pigs News Informa- tion. 2001; 22: 39–48. 2. Torrallardona D. Spray dried animal plasma as an alternative to antibiotics in weanling pigs. Asian-Aus- tralas J Anim Sci. 2010; 23: 131–148. https://doi.org/10.5713/ajas.2010.70630 3. Pérez-Bosque A, Polo J, Torrallardona D. Spray dried plasma as an alternative to antibiotics in piglet feeds, mode of action and biosafety. Porcine Health Manag. 2016; 2: 1–10. https://doi.org/10.1186/ s40813-016-0034-1 PMID: 28405442 4. Blázquez E, Rodrı́guez C, Ródenas J, Segalés J, Pujols J, Polo J. 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