8cd45aba9810f4fec40f8bf377c02f10 pmc_10819582.pdf 90cf0dd3fc579702dbcecb099134c662d92b0fee pmc_10819582.pdf 3dc99e1404196d6c030696edd29b200a9a58e68b84fe431d51156cbd0d2518b4 pmc_10819582.pdf Title: Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model Subject: Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture. Keywords: porcine epidemic diarrhea virus (PEDV); strain; cell passaged; S-INDEL; non-S INDEL; piglet Author: Carlos López-Figueroa, Esmeralda Cano, Núria Navarro, Mónica Pérez-Maíllo, Joan Pujols, José I. Núñez, Júlia Vergara-Alert and Joaquim Segalés Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Dec 27 10:46:34 2023 CET ModDate: Wed Dec 27 11:43:04 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3245654 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DIMMOC+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 GFKJOY+VnURWPalladioL Type 1 Custom yes yes yes 16 0 SGKJAS+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 CLIIXX+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 WCSHDP+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 HOWKDX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 48 0 ECTHDD+VnURWPalladioL-Italic Type 1 Custom yes yes yes 68 0 TSMJFB+CMSY10 Type 1 Builtin yes yes yes 73 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 78 0 DNFNBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 102 0 DNFNCA+PalatinoLinotype CID TrueType Identity-H yes yes yes 105 0 DNFNCB+PalatinoLinotype TrueType WinAnsi yes yes no 111 0 DNFNCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 114 0 DNFNNP+PalatinoLinotype TrueType MacRoman yes yes no 117 0 DNFNBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 129 0 DNFNCA+PalatinoLinotype CID TrueType Identity-H yes yes yes 132 0 DNFNCB+PalatinoLinotype TrueType WinAnsi yes yes no 138 0 DNFNCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 141 0 DNFNNP+PalatinoLinotype TrueType MacRoman yes yes no 144 0 DNFNBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 159 0 DNFNCA+PalatinoLinotype CID TrueType Identity-H yes yes yes 162 0 DNFNCB+PalatinoLinotype TrueType WinAnsi yes yes no 168 0 DNFNCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 171 0 DNFNNP+PalatinoLinotype TrueType MacRoman yes yes no 174 0 DNFNBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 188 0 DNFNCA+PalatinoLinotype CID TrueType Identity-H yes yes yes 191 0 DNFNCB+PalatinoLinotype TrueType WinAnsi yes yes no 197 0 DNFNCC+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 200 0 DNFNCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 206 0 DNFNNP+PalatinoLinotype TrueType MacRoman yes yes no 209 0 DNFNBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 227 0 DNFNCA+PalatinoLinotype CID TrueType Identity-H yes yes yes 230 0 DNFNCB+PalatinoLinotype TrueType WinAnsi yes yes no 236 0 DNFNCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 239 0 DNFNNP+PalatinoLinotype TrueType MacRoman yes yes no 242 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-03-08 02:36:24 CET RepresentationInformation: pmc_10819582.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-03-01 02:38:11 CET Size: 3245654 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 413 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Viral Inoculum Destination: subsection.2.1 Item: Title: Sequencing and Phylogenetic Analysis of the Inocula Destination: subsection.2.2 Item: Title: Experimental Design Destination: subsection.2.3 Item: Title: Nucleic Acid Extraction and RT-PCR/PCR for Detection of Viral Pathogens Destination: subsection.2.4 Item: Title: ELISA Tests Destination: subsection.2.5 Item: Title: PEDV Genome Sequencing Destination: subsection.2.6 Item: Title: Pathological Analyses and Immunohistochemistry Destination: subsection.2.7 Item: Title: Statistical Analyses Destination: subsection.2.8 Item: Title: Results Destination: section.3 Children: Item: Title: Both CALAF Inocula Had Similar Genomes and Showed More Variation in Their Sequences When Compared to the PEDV-USA One Destination: subsection.3.1 Item: Title: Clinical Outcome Varies Depending on the PEDV Isolate Destination: subsection.3.2 Item: Title: PEDV Antigen Was Present in the Jejunum and Ileum, but Only the PEDV-USA and CALAF-HOMOG Isolates Caused Intestinal Damage Destination: subsection.3.3 Item: Title: All PEDV Isolates Replicated in the Jejunum and Were Shed in Feces at 48 hpi with Different Viral Loads Depending on the Isolate Destination: subsection.3.4 Item: Title: Sequences of Inocula and Viruses Recovered from Intestinal Contents at 48 hpi Were Highly Conserved Destination: subsection.3.5 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model Author: Carlos López-Figueroa, Esmeralda Cano, Núria Navarro, Mónica Pérez-Maíllo, Joan Pujols, José I. Núñez, Júlia Vergara-Alert and Joaquim Segalés Subject: Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture. Keywords: porcine epidemic diarrhea virus (PEDV); strain; cell passaged; S-INDEL; non-S INDEL; piglet Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Dec 27 10:46:34 CET 2023 ModDate: Wed Dec 27 11:43:04 CET 2023 ID: 0xd9f71c2e1c9ec2978d6717085a8f7d58, 0xd9f71c2e1c9ec2978d6717085a8f7d58 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: GFKJOY+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: GFKJOY+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: WCSHDP+URWPalladioL-Ital FontSubset: true FirstChar: 38 LastChar: 121 FontDescriptor: FontName: WCSHDP+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ECTHDD+VnURWPalladioL-Italic FontSubset: true FirstChar: 160 LastChar: 225 FontDescriptor: FontName: ECTHDD+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: SGKJAS+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: SGKJAS+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: TSMJFB+CMSY10 FontSubset: true FirstChar: 2 LastChar: 48 FontDescriptor: FontName: TSMJFB+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: DIMMOC+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 243 FontDescriptor: FontName: DIMMOC+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: CLIIXX+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: CLIIXX+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 47, 279, 152, 288 Annotation: Subtype: Link Rect: 34, 269, 68, 276 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 125, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 149, 87 Annotation: Subtype: Link Rect: 34, 756, 147, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 108, 610, 119, 624 Annotation: Subtype: Link Rect: 228, 610, 239, 624 Annotation: Subtype: Link Rect: 362, 610, 373, 624 Annotation: Subtype: Link Rect: 104, 17, 233, 28 Annotation: Subtype: Link Rect: 411, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 400, 87, 419 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 202, 731, 209, 740 ActionDest: 254 Annotation: Subtype: Link Rect: 431, 706, 438, 715 ActionDest: 254 Annotation: Subtype: Link Rect: 469, 693, 476, 702 ActionDest: 254 Annotation: Subtype: Link Rect: 277, 668, 284, 677 ActionDest: 254 Annotation: Subtype: Link Rect: 541, 655, 548, 665 ActionDest: 254 Annotation: Subtype: Link Rect: 549, 655, 556, 665 ActionDest: 254 Annotation: Subtype: Link Rect: 446, 605, 453, 614 ActionDest: 254 Annotation: Subtype: Link Rect: 454, 605, 461, 614 ActionDest: 254 Annotation: Subtype: Link Rect: 464, 568, 471, 577 ActionDest: 254 Annotation: Subtype: Link Rect: 288, 530, 295, 539 ActionDest: 254 Annotation: Subtype: Link Rect: 343, 480, 350, 489 ActionDest: 254 Annotation: Subtype: Link Rect: 336, 442, 348, 451 ActionDest: 254 Annotation: Subtype: Link Rect: 349, 442, 361, 451 ActionDest: 254 Annotation: Subtype: Link Rect: 218, 354, 230, 363 ActionDest: 254 Annotation: Subtype: Link Rect: 392, 329, 399, 338 ActionDest: 254 Annotation: Subtype: Link Rect: 256, 291, 268, 301 ActionDest: 254 Annotation: Subtype: Link Rect: 435, 241, 447, 250 ActionDest: 254 Annotation: Subtype: Link Rect: 334, 204, 341, 213 ActionDest: 254 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 492, 702, 504, 711 ActionDest: 254 Annotation: Subtype: Link Rect: 259, 589, 271, 598 ActionDest: 254 Annotation: Subtype: Link Rect: 474, 551, 486, 560 ActionDest: 254 Annotation: Subtype: Link Rect: 373, 199, 385, 208 ActionDest: 254 Annotation: Subtype: Link Rect: 200, 86, 212, 96 ActionDest: 254 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 213, 427, 225, 436 ActionDest: 254 Annotation: Subtype: Link Rect: 204, 364, 216, 373 ActionDest: 254 Annotation: Subtype: Link Rect: 518, 97, 532, 109 ActionDest: 65 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 544, 567, 556, 576 ActionDest: 254 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 475, 465, 487, 474 ActionDest: 254 Annotation: Subtype: Link Rect: 266, 390, 278, 399 ActionDest: 257 Annotation: Subtype: Link Rect: 278, 390, 290, 399 ActionDest: 257 Annotation: Subtype: Link Rect: 342, 274, 349, 286 ActionDest: 92 Annotation: Subtype: Link Rect: 534, 237, 541, 249 ActionDest: 92 Annotation: Subtype: Link Rect: 511, 224, 518, 236 ActionDest: 92 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 267, 355, 274, 367 ActionDest: 92 Annotation: Subtype: Link Rect: 540, 343, 547, 355 ActionDest: 92 Annotation: Subtype: Link Rect: 283, 293, 290, 305 ActionDest: 92 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 382, 605, 389, 617 ActionDest: 149 Annotation: Subtype: Link Rect: 367, 165, 374, 177 ActionDest: 149 Annotation: Subtype: Link Rect: 354, 115, 361, 127 ActionDest: 149 Annotation: Subtype: Link Rect: 512, 90, 519, 102 ActionDest: 178 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 527, 665, 534, 677 ActionDest: 178 Annotation: Subtype: Link Rect: 197, 653, 204, 665 ActionDest: 178 Annotation: Subtype: Link Rect: 405, 628, 412, 640 ActionDest: 178 Annotation: Subtype: Link Rect: 308, 105, 315, 117 ActionDest: 213 Annotation: Subtype: Link Rect: 453, 105, 460, 117 ActionDest: 213 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 542, 403, 549, 415 ActionDest: 213 Annotation: Subtype: Link Rect: 217, 118, 229, 127 ActionDest: 257 Annotation: Subtype: Link Rect: 232, 118, 244, 127 ActionDest: 257 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 501, 718, 508, 727 ActionDest: 254 Annotation: Subtype: Link Rect: 508, 718, 520, 727 ActionDest: 254 Annotation: Subtype: Link Rect: 273, 555, 285, 564 ActionDest: 257 Annotation: Subtype: Link Rect: 286, 555, 298, 564 ActionDest: 257 Annotation: Subtype: Link Rect: 435, 442, 447, 451 ActionDest: 254 Annotation: Subtype: Link Rect: 447, 442, 459, 451 ActionDest: 257 Annotation: Subtype: Link Rect: 462, 442, 474, 451 ActionDest: 257 Annotation: Subtype: Link Rect: 548, 417, 555, 426 ActionDest: 254 Annotation: Subtype: Link Rect: 292, 329, 304, 338 ActionDest: 257 Annotation: Subtype: Link Rect: 290, 304, 297, 313 ActionDest: 254 Annotation: Subtype: Link Rect: 298, 304, 310, 313 ActionDest: 254 Annotation: Subtype: Link Rect: 376, 254, 388, 263 ActionDest: 257 Annotation: Subtype: Link Rect: 342, 191, 349, 200 ActionDest: 254 Annotation: Subtype: Link Rect: 349, 191, 356, 200 ActionDest: 254 Annotation: Subtype: Link Rect: 357, 191, 369, 200 ActionDest: 254 Annotation: Subtype: Link Rect: 544, 153, 556, 162 ActionDest: 257 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 288, 744, 300, 752 ActionDest: 254 Annotation: Subtype: Link Rect: 445, 643, 457, 652 ActionDest: 254 Annotation: Subtype: Link Rect: 286, 605, 293, 614 ActionDest: 254 Annotation: Subtype: Link Rect: 507, 379, 514, 388 ActionDest: 254 Annotation: Subtype: Link Rect: 526, 277, 560, 288 Annotation: Subtype: Link Rect: 165, 265, 352, 276 Annotation: Subtype: Link Rect: 357, 232, 368, 240 ActionDest: 257 Annotation: Subtype: Link Rect: 371, 232, 382, 240 ActionDest: 257 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 95, 518, 133, 528 Annotation: Subtype: Link Rect: 267, 494, 305, 505 Annotation: Subtype: Link Rect: 232, 471, 270, 482 Annotation: Subtype: Link Rect: 218, 436, 255, 447 Annotation: Subtype: Link Rect: 221, 414, 259, 424 Annotation: Subtype: Link Rect: 95, 391, 133, 401 Annotation: Subtype: Link Rect: 520, 367, 557, 378 Annotation: Subtype: Link Rect: 161, 345, 198, 355 Annotation: Subtype: Link Rect: 416, 321, 453, 332 Annotation: Subtype: Link Rect: 459, 321, 495, 332 Annotation: Subtype: Link Rect: 368, 287, 405, 298 Annotation: Subtype: Link Rect: 411, 287, 447, 298 Annotation: Subtype: Link Rect: 207, 264, 244, 275 Annotation: Subtype: Link Rect: 250, 264, 286, 275 Annotation: Subtype: Link Rect: 222, 229, 259, 240 Annotation: Subtype: Link Rect: 266, 229, 302, 240 Annotation: Subtype: Link Rect: 100, 195, 137, 206 Annotation: Subtype: Link Rect: 143, 195, 179, 206 Annotation: Subtype: Link Rect: 117, 172, 155, 183 Annotation: Subtype: Link Rect: 161, 172, 197, 183 Annotation: Subtype: Link Rect: 131, 149, 168, 160 Annotation: Subtype: Link Rect: 175, 149, 211, 160 Annotation: Subtype: Link Rect: 157, 115, 194, 125 Annotation: Subtype: Link Rect: 200, 115, 236, 125 Annotation: Subtype: Link Rect: 437, 91, 475, 102 Annotation: Subtype: Link Rect: 212, 68, 250, 79 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 59, 719, 96, 729 Annotation: Subtype: Link Rect: 86, 684, 124, 694 Annotation: Subtype: Link Rect: 509, 660, 546, 671 Annotation: Subtype: Link Rect: 59, 638, 96, 648 Annotation: Subtype: Link Rect: 333, 614, 370, 625 Annotation: Subtype: Link Rect: 237, 591, 275, 602 Annotation: Subtype: Link Rect: 426, 568, 464, 579 Annotation: Subtype: Link Rect: 417, 545, 455, 556 Annotation: Subtype: Link Rect: 296, 522, 334, 533 Annotation: Subtype: Link Rect: 282, 499, 320, 510 Annotation: Subtype: Link Rect: 502, 442, 540, 453 Annotation: Subtype: Link Rect: 257, 408, 295, 418 Annotation: Subtype: Link Rect: 301, 408, 337, 418 Annotation: Subtype: Link Rect: 112, 374, 150, 384 Annotation: Subtype: Link Rect: 399, 350, 436, 361 Annotation: Subtype: Link Rect: 386, 327, 424, 338 Annotation: Subtype: Link Rect: 380, 292, 417, 303 Annotation: Subtype: Link Rect: 515, 269, 553, 280 Checksum: 05fda8c1 Type: CRC32 Checksum: 8cd45aba9810f4fec40f8bf377c02f10 Type: MD5 Checksum: 90cf0dd3fc579702dbcecb099134c662d92b0fee Type: SHA-1 Checksum: 3dc99e1404196d6c030696edd29b200a9a58e68b84fe431d51156cbd0d2518b4 Type: SHA-256