Conceptualization of project, project administration, performing necropsies, funding acquisition, coordination of field sample transfer, writing, review, editing, J.A.R. and I.M.; preparation of original draft, data curation, sequencing and bioinformatics, C.D.M.; writing, review, editing, preparation of challenge material, J.A.R. and N.N.G.; qPCR, primary pathologist, performing necropsies, gross pathology and histological evaluation, data acquisition, oversight and analysis of molecular work (DNA extraction and PCR), writing, review, and editing, J.D.T.; performing molecular work, data acquisition and analysis, necropsy assistance, C.K.; preparation of challenge material, sample collection, data curation, review of manuscript, and editing, D.A.M.; viral titrations, sample collection, review, and editing, T.K.; review and editing of manuscript, D.B.; sample collection, sample processing, review, and editing of manuscript, D.W.M.; sample collection and processing, review, and editing, K.C.; necropsy assistance, sample processing, review, and editing, B.L.A.; sample collection, review and editing, V.B.; secondary pathologist: performed necropsies and slide review, review, and editing, J.R.; assistance in the transfer of original virus isolate from USDA-FADDL to KSU BRI, K.D. and C.J.C.; collection of field samples, coordination of field sample transfer, review, and editing, U.A.; coordination of sample transfer, review and editing, B.Z.; sequencing and bioinformatics, writing, editing, and review, J.A.S.-N. All authors have read and agreed to the published version of the manuscript.

Figure 1. ASFV-MNG19 Challenge Study Schematic. Six crossbred male pigs approximately 2-months-old were challenged with 360 HAD50 of ASFV-MNG19. Samples were collected on days −1, 1, 3, 5, and 7 DPC. Samples collected included whole blood (EDTA), OP swabs, and oral fluids collected from chewed ropes at the pen level. Body temperature and clinical scores were assessed daily. One pig (#307) was necropsied on 5 DPC as a result of complications following sample collection. Another pig (#306) was found dead on 6 DPC and subsequently necropsied. The remaining 4 pigs were humanely euthanized and necropsied at 7 DPC due to severe clinical signs.

Figure 1. ASFV-MNG19 Challenge Study Schematic. Six crossbred male pigs approximately 2-months-old were challenged with 360 HAD50 of ASFV-MNG19. Samples were collected on days −1, 1, 3, 5, and 7 DPC. Samples collected included whole blood (EDTA), OP swabs, and oral fluids collected from chewed ropes at the pen level. Body temperature and clinical scores were assessed daily. One pig (#307) was necropsied on 5 DPC as a result of complications following sample collection. Another pig (#306) was found dead on 6 DPC and subsequently necropsied. The remaining 4 pigs were humanely euthanized and necropsied at 7 DPC due to severe clinical signs.

Figure 2. Phylogenetic tree of whole-genome sequences of ASFVs constructed using maximum likelihood approach with 1000 bootstrap replicates. The blue and red dots indicate the ASFV-MNG19 original stock and inoculum used in this challenge study, respectively.

Figure 2. Phylogenetic tree of whole-genome sequences of ASFVs constructed using maximum likelihood approach with 1000 bootstrap replicates. The blue and red dots indicate the ASFV-MNG19 original stock and inoculum used in this challenge study, respectively.

Figure 3. Clinical Observations of ASFV-MNG19 Infected Pigs. Daily body temperatures (A) and cumulative clinical score (B) of the 6 pigs challenged IM with ASFV-MNG19 from 0 DPC to 7 DPC. Data indicates individual values ((A) expressed as °C, (B) expressed as an integer value). Data for individual animals are shown. The dashed line in 3A indicates 40.5 °C which is considered a fever for this study.

Figure 3. Clinical Observations of ASFV-MNG19 Infected Pigs. Daily body temperatures (A) and cumulative clinical score (B) of the 6 pigs challenged IM with ASFV-MNG19 from 0 DPC to 7 DPC. Data indicates individual values ((A) expressed as °C, (B) expressed as an integer value). Data for individual animals are shown. The dashed line in 3A indicates 40.5 °C which is considered a fever for this study.

Figure 4. Quantitative real-time PCR for ASFV DNA detected in blood, OP swabs, and oral fluids. (A) The calculated copy number (CN) of ASFV DNA extracted from EDTA blood collected on −1, 1, 3, 5, and 7 DPC using a ten-point standard curve. Datapoints for individual pigs are represented. The single data point at 6 DPC represents blood collected from pig #306, which was found dead. (B) The calculated copy number of ASFV DNA extracted from OP swabs collected on −1, 1, 3, 5, and 7 DPC using a ten-point standard curve. Datapoints for individual pigs are represented. (C) The calculated copy number of ASFV DNA extracted from oral fluids (rope chewing) collected on −1, 1, 3, and 5 DPC using a ten-point standard curve. Datapoints represent the pen level.

Figure 4. Quantitative real-time PCR for ASFV DNA detected in blood, OP swabs, and oral fluids. (A) The calculated copy number (CN) of ASFV DNA extracted from EDTA blood collected on −1, 1, 3, 5, and 7 DPC using a ten-point standard curve. Datapoints for individual pigs are represented. The single data point at 6 DPC represents blood collected from pig #306, which was found dead. (B) The calculated copy number of ASFV DNA extracted from OP swabs collected on −1, 1, 3, 5, and 7 DPC using a ten-point standard curve. Datapoints for individual pigs are represented. (C) The calculated copy number of ASFV DNA extracted from oral fluids (rope chewing) collected on −1, 1, 3, and 5 DPC using a ten-point standard curve. Datapoints represent the pen level.

Figure 5. Viral titers in blood and OP swabs. (A) The log transformed viral titers (TCID50/mL) of ASFV-MNG19 in EDTA blood collected on −1, 1, 3, 5, and 7 DPC. All samples were run in triplicate. The datapoints represent the calculated titers using the Spearman–Karber method. The dashed line represents the limit of detection for the assay. Datapoints for individual pigs are represented. The single data point at 6 DPC represents samples collected from pig #306 that was found dead. (B) The log transformed viral titers (TCID50/mL) of ASFV-MNG19 in OP swabs collected on −1, 1, 3, 5, and 7 DPC. All samples were run in triplicate. The datapoints represent the calculated titers using the Spearman–Karber method. The dashed line represents the limit of detection for the assay. Datapoints for individual pigs are represented.

Figure 5. Viral titers in blood and OP swabs. (A) The log transformed viral titers (TCID50/mL) of ASFV-MNG19 in EDTA blood collected on −1, 1, 3, 5, and 7 DPC. All samples were run in triplicate. The datapoints represent the calculated titers using the Spearman–Karber method. The dashed line represents the limit of detection for the assay. Datapoints for individual pigs are represented. The single data point at 6 DPC represents samples collected from pig #306 that was found dead. (B) The log transformed viral titers (TCID50/mL) of ASFV-MNG19 in OP swabs collected on −1, 1, 3, 5, and 7 DPC. All samples were run in triplicate. The datapoints represent the calculated titers using the Spearman–Karber method. The dashed line represents the limit of detection for the assay. Datapoints for individual pigs are represented.

Figure 6. Gross pathology lesions following ASFV-MNG19 infection. (A) Spleen: severe splenomegaly; (B) Lymph nodes: hemorrhage (from left to right: renal lymph node, gastrohepatic lymph node, and cranial mediastinal lymph node); (C) Lungs: pulmonary edema with congestion, consolidation (yellow arrow), and ecchymosis; (D) Kidney: retroperitoneal serous effusion indicated by the yellow star, petechial hemorrhages of renal cortex indicated by the yellow arrow.

Figure 6. Gross pathology lesions following ASFV-MNG19 infection. (A) Spleen: severe splenomegaly; (B) Lymph nodes: hemorrhage (from left to right: renal lymph node, gastrohepatic lymph node, and cranial mediastinal lymph node); (C) Lungs: pulmonary edema with congestion, consolidation (yellow arrow), and ecchymosis; (D) Kidney: retroperitoneal serous effusion indicated by the yellow star, petechial hemorrhages of renal cortex indicated by the yellow arrow.

Figure 7. Histological lesions following ASFV-MNG19 infection. (A) Spleen: Marked lympholysis and collapse of periarterial sheaths and fibrin thrombi (asterisk) in marginal zone (100×). (B) Renal lymph node: Marked lympholysis and necrosis (asterisk and insert) with loss of cortical and sinusoidal lymphocytes accompanied by marked filling and expansion of the subcapsular and medullary sinuses with hemorrhage and fibrin (100× and 400× insert). (C) Lung: Marked expansion of the alveolar spaces by congestion, fibrin thrombi (asterisk) and necrotic cellular debris, eosinophilic proteinaceous material; edema, fibrin and occasional macrophages fill the alveolar spaces (200×). (D) Kidney: Multifocal congestion and fibrin thrombosis (insert-asterisk) of the interstitial capillaries accompanied by hemorrhage and coagulative necrosis and dystrophic mineralization (arrow) of tubules (200×, 400×-insert).

Figure 7. Histological lesions following ASFV-MNG19 infection. (A) Spleen: Marked lympholysis and collapse of periarterial sheaths and fibrin thrombi (asterisk) in marginal zone (100×). (B) Renal lymph node: Marked lympholysis and necrosis (asterisk and insert) with loss of cortical and sinusoidal lymphocytes accompanied by marked filling and expansion of the subcapsular and medullary sinuses with hemorrhage and fibrin (100× and 400× insert). (C) Lung: Marked expansion of the alveolar spaces by congestion, fibrin thrombi (asterisk) and necrotic cellular debris, eosinophilic proteinaceous material; edema, fibrin and occasional macrophages fill the alveolar spaces (200×). (D) Kidney: Multifocal congestion and fibrin thrombosis (insert-asterisk) of the interstitial capillaries accompanied by hemorrhage and coagulative necrosis and dystrophic mineralization (arrow) of tubules (200×, 400×-insert).

Table 1. The ten sequences with the highest similarity to ASFV-MNG19.

Table 1. The ten sequences with the highest similarity to ASFV-MNG19.

AccessionNamePercent IdentityMK128995.1China/2018/AnhuiXCGQ99.99MN172368.1ASFV/pig/China/CAS19-01/201999.99MK333180.1Pig/HLJ/201899.99ON075797.1Korea/YC1/201999.98MW306191.1ASFV/Primorsky 19/WB-672399.99MW396979.1ASFV/Timor-Leste/2019/199.98MN715134.1ASFV_HU_201899.98OL692744.1IND/AR/SD-61/202099.98OL692743.1IND/AS/SD-02/202099.98MW049116.1ASFV Korea/pig/Yeoncheon1/201999.98