0e25ad24d1be82987aa79b80255fd6eb pmc_33371460.pdf 961709ba24e4002fea4d0a1417f8c317c0d8ec28 pmc_33371460.pdf b6070eb0f86ad884d4e9006809911367d4e0811f89c19dea0964ffa9c5ac520d pmc_33371460.pdf Title: Live Attenuated African Swine Fever Viruses as Ideal Tools to Dissect the Mechanisms Involved in Cross-Protection Subject: African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas. Keywords: African swine fever virus; live attenuate vaccine; cross-protection Author: Elisabeth Lopez, Juanita van Heerden, Laia Bosch-Camós, Francesc Accensi, Maria Jesus Navas, Paula López-Monteagudo, Jordi Argilaguet, Carmina Gallardo, Sonia Pina-Pedrero, Maria Luisa Salas, Jeremy Salt and Fernando Rodriguez Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Mon Dec 21 15:58:05 2020 CET ModDate: Mon Dec 21 15:58:05 2020 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 11 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1447222 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- XFTBXK+URWPalladioL-Ital Type 1 Custom yes yes no 77 0 XIXXJP+URWPalladioL-Bold Type 1 Custom yes yes no 78 0 SOZKOC+VnURWPalladioL-Bold Type 1 Custom yes yes no 79 0 LXUYTR+URWPalladioL-Roma Type 1 Custom yes yes no 80 0 VJAUGW+VnURWPalladioL Type 1 Custom yes yes no 81 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 82 0 MHAHCD+Rpxr Type 1 Builtin yes yes no 83 0 HOWKDX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 88 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 105 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 106 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 107 0 ICYUJJ+Pxsy Type 1 Builtin yes yes no 166 0 WPPLLU+EURM10 Type 1 Builtin yes yes no 178 0 KCAOOE+PalatinoLinotype CID TrueType Identity-H yes yes yes 196 0 KCAOPF+PalatinoLinotype TrueType WinAnsi yes yes no 197 0 KCAPIE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 198 0 KCAPIG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 199 0 KCBAIB+TimesNewRoman CID TrueType Identity-H yes yes yes 200 0 KCAOOE+PalatinoLinotype CID TrueType Identity-H yes yes yes 247 0 KCAOPF+PalatinoLinotype TrueType WinAnsi yes yes no 248 0 KCAPIE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 249 0 KCAPIG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 250 0 KCBAIB+TimesNewRoman CID TrueType Identity-H yes yes yes 251 0 BCDEEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 331 0 BCDNEE+Calibri CID TrueType Identity-H yes yes yes 332 0 BCDFEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 333 0 BCDGEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 334 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 335 0 TimesNewRomanPSMT TrueType WinAnsi no no no 336 0 BCDKEE+Calibri-Bold TrueType WinAnsi yes yes no 351 0 Times New Roman,Bold TrueType WinAnsi no no no 364 0 Symbol CID TrueType Identity-H yes no yes 375 0 VSHNAE+Rpcxr Type 1 Builtin yes yes no 460 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 03:47:41 CEST RepresentationInformation: pmc_33371460.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 11:43:40 CEST Size: 1447222 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 FreeObjects: 2 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: ぉのぴひはつふっぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸲ Item: Title: きちぴづひどちぬびぜ〴ぜ〰ちのつぜ〴ぜ〰きづぴとはつびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮ㄩ‾㸊⡜㌷㙜㌷㝜〰ざどひふびづびぜ〴ぜ〰ちのつぜ〴ぜ〰ぃづぬぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㈩‾㸊⡜㌷㙜㌷㝜〰ぁのどねちぬぜ〴ぜ〰しづぬてちひづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぅへばづひどねづのぴちぬぜ〴ぜ〰ぁばばひはちっとぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべぴどっちぬぜ〴ぜ〰げづちつはふぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸱ Children: Item: Title: ざどひふびづびぜ〴ぜ〰ちのつぜ〴ぜ〰ぃづぬぬびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㈩‾㸊⡜㌷㙜㌷㝜〰ぁのどねちぬぜ〴ぜ〰しづぬてちひづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぅへばづひどねづのぴちぬぜ〴ぜ〰ぁばばひはちっとぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべぴどっちぬぜ〴ぜ〰げづちつはふぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸱ Item: Title: ぁのどねちぬぜ〴ぜ〰しづぬてちひづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぅへばづひどねづのぴちぬぜ〴ぜ〰ぁばばひはちっとぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべぴどっちぬぜ〴ぜ〰げづちつはふぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸱ Item: Title: ぅへばづひどねづのぴちぬぜ〴ぜ〰ぁばばひはちっとぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぁのちぬべぴどっちぬぜ〴ぜ〰げづちつはふぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸱ Item: Title: ぁのちぬべぴどっちぬぜ〴ぜ〰げづちつはふぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸱ Children: Item: Title: ぁこうざぜ〴ぜ〰けふちのぴどてどっちぴどはのぜ〴ぜ〰ぢべぜ〴ぜ〰げづちぬ〭ごどねづぜ〴ぜ〰ぐぃげぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸲ Item: Title: ぁのぴどぢはつべぜ〴ぜ〰いづぴづっぴどはのぜ〴ぜ〰ぢべぜ〴ぜ〰ぅがぉこぁぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸳ Item: Title: ごぜ〴ぜ〰ぃづぬぬ〭こばづっどてどっぜ〴ぜ〰ぉねねふのづぜ〴ぜ〰げづびばはのびづぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸲⸴⸴ Item: Title: ぉのてづっぴどはの〭ぉのとどぢどぴどはのぜ〴ぜ〰ぁびびちべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰こぴちぴどびぴどっちぬぜ〴ぜ〰ぁのちぬべびどびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: こぴちぴどびぴどっちぬぜ〴ぜ〰ぁのちぬべびどびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: げづびふぬぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡦楧畲攮捡灴楯渮㌩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: げづてづひづのっづび Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LXUYTR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: LXUYTR+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VJAUGW+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: VJAUGW+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: WPPLLU+EURM10 FontSubset: true FirstChar: 13 LastChar: 22 FontDescriptor: FontName: WPPLLU+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: MHAHCD+Rpxr FontSubset: true FirstChar: 11 LastChar: 47 FontDescriptor: FontName: MHAHCD+Rpxr Flags: Symbolic FontBBox: -40, -283, 904, 728 FontFile: true Font: BaseFont: ICYUJJ+Pxsy FontSubset: true FirstChar: 2 LastChar: 48 FontDescriptor: FontName: ICYUJJ+Pxsy Flags: Symbolic FontBBox: -21, -994, 1292, 912 FontFile: true Font: BaseFont: VSHNAE+Rpcxr FontSubset: true FirstChar: 169 LastChar: 169 FontDescriptor: FontName: VSHNAE+Rpcxr Flags: Symbolic FontBBox: -212, -286, 1344, 973 FontFile: true Font: BaseFont: XFTBXK+URWPalladioL-Ital FontSubset: true FirstChar: 44 LastChar: 121 FontDescriptor: FontName: XFTBXK+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XIXXJP+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: XIXXJP+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SOZKOC+VnURWPalladioL-Bold FontSubset: true FirstChar: 226 LastChar: 226 FontDescriptor: FontName: SOZKOC+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 75, 757, 188, 793 Annotation: Subtype: Link Rect: 474, 757, 519, 793 Annotation: Subtype: Link Rect: 155, 641, 166, 655 Annotation: Subtype: Link Rect: 269, 641, 280, 655 Annotation: Subtype: Link Rect: 380, 628, 391, 641 Annotation: Subtype: Link Rect: 326, 614, 337, 628 Annotation: Subtype: Link Rect: 464, 614, 475, 628 Annotation: Subtype: Link Rect: 466, 428, 518, 447 Annotation: Subtype: Link Rect: 166, 38, 233, 50 Annotation: Subtype: Link Rect: 405, 38, 519, 50 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 443, 696, 450, 705 ActionDest: 406 Annotation: Subtype: Link Rect: 201, 655, 208, 665 ActionDest: 406 Annotation: Subtype: Link Rect: 208, 655, 215, 665 ActionDest: 406 Annotation: Subtype: Link Rect: 465, 574, 472, 584 ActionDest: 406 Annotation: Subtype: Link Rect: 438, 561, 445, 570 ActionDest: 406 Annotation: Subtype: Link Rect: 258, 534, 265, 543 ActionDest: 406 Annotation: Subtype: Link Rect: 353, 507, 360, 516 ActionDest: 406 Annotation: Subtype: Link Rect: 103, 453, 110, 462 ActionDest: 406 Annotation: Subtype: Link Rect: 113, 453, 125, 462 ActionDest: 406 Annotation: Subtype: Link Rect: 153, 412, 165, 421 ActionDest: 406 Annotation: Subtype: Link Rect: 277, 385, 289, 394 ActionDest: 406 Annotation: Subtype: Link Rect: 110, 345, 122, 354 ActionDest: 435 Annotation: Subtype: Link Rect: 168, 318, 180, 327 ActionDest: 435 Annotation: Subtype: Link Rect: 204, 304, 216, 313 ActionDest: 435 Annotation: Subtype: Link Rect: 421, 277, 433, 286 ActionDest: 435 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 167, 659, 179, 668 ActionDest: 435 Annotation: Subtype: Link Rect: 374, 646, 386, 655 ActionDest: 435 Annotation: Subtype: Link Rect: 100, 578, 112, 587 ActionDest: 435 Annotation: Subtype: Link Rect: 305, 358, 317, 367 ActionDest: 435 Annotation: Subtype: Link Rect: 167, 344, 179, 353 ActionDest: 435 Annotation: Subtype: Link Rect: 94, 331, 106, 340 ActionDest: 435 Annotation: Subtype: Link Rect: 492, 168, 503, 178 ActionDest: 435 Annotation: Subtype: Link Rect: 504, 169, 515, 178 ActionDest: 435 Annotation: Subtype: Link Rect: 503, 101, 515, 110 ActionDest: 435 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 299, 709, 311, 719 ActionDest: 435 Annotation: Subtype: Link Rect: 397, 160, 409, 170 ActionDest: 435 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 371, 577, 378, 589 ActionDest: 188 Annotation: Subtype: Link Rect: 144, 203, 155, 211 ActionDest: 435 Annotation: Subtype: Link Rect: 193, 164, 200, 176 ActionDest: 188 Annotation: Subtype: Link Rect: 363, 83, 370, 95 ActionDest: 188 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 330, 713, 337, 725 ActionDest: 188 Annotation: Subtype: Link Rect: 171, 702, 183, 711 ActionDest: 435 Annotation: Subtype: Link Rect: 274, 686, 282, 698 ActionDest: 188 Annotation: Subtype: Link Rect: 307, 659, 314, 671 ActionDest: 188 Annotation: Subtype: Link Rect: 220, 632, 227, 644 ActionDest: 237 Annotation: Subtype: Link Rect: 503, 634, 515, 644 ActionDest: 435 Annotation: Subtype: Link Rect: 376, 591, 383, 603 ActionDest: 237 Annotation: Subtype: Link Rect: 398, 580, 410, 590 ActionDest: 435 Annotation: Subtype: Link Rect: 453, 567, 465, 576 ActionDest: 435 Annotation: Subtype: Link Rect: 332, 524, 339, 535 ActionDest: 237 Annotation: Subtype: Link Rect: 176, 510, 182, 522 ActionDest: 237 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 111, 726, 118, 738 ActionDest: 237 Annotation: Subtype: Link Rect: 502, 726, 509, 738 ActionDest: 237 Annotation: Subtype: Link Rect: 368, 686, 375, 698 ActionDest: 237 Annotation: Subtype: Link Rect: 447, 659, 454, 671 ActionDest: 237 Annotation: Subtype: Link Rect: 419, 648, 431, 657 ActionDest: 435 Annotation: Subtype: Link Rect: 279, 621, 291, 630 ActionDest: 435 Annotation: Subtype: Link Rect: 291, 621, 303, 630 ActionDest: 435 Annotation: Subtype: Link Rect: 252, 607, 264, 617 ActionDest: 435 Annotation: Subtype: Link Rect: 435, 537, 442, 549 ActionDest: 324 Annotation: Subtype: Link Rect: 108, 510, 115, 522 ActionDest: 324 Annotation: Subtype: Link Rect: 108, 443, 115, 454 ActionDest: 324 Annotation: Subtype: Link Rect: 302, 432, 314, 441 ActionDest: 435 Annotation: Subtype: Link Rect: 292, 378, 304, 387 ActionDest: 435 Annotation: Subtype: Link Rect: 416, 351, 428, 360 ActionDest: 435 Annotation: Subtype: Link Rect: 503, 324, 515, 333 ActionDest: 435 Annotation: Subtype: Link Rect: 283, 297, 295, 306 ActionDest: 435 Annotation: Subtype: Link Rect: 296, 297, 308, 306 ActionDest: 435 Annotation: Subtype: Link Rect: 311, 297, 323, 306 ActionDest: 455 Annotation: Subtype: Link Rect: 380, 283, 392, 292 ActionDest: 435 Annotation: Subtype: Link Rect: 423, 242, 435, 252 ActionDest: 455 Annotation: Subtype: Link Rect: 436, 242, 448, 252 ActionDest: 455 Annotation: Subtype: Link Rect: 501, 172, 508, 184 ActionDest: 324 Annotation: Subtype: Link Rect: 409, 121, 421, 130 ActionDest: 435 Annotation: Subtype: Link Rect: 245, 80, 257, 90 ActionDest: 435 Annotation: Subtype: Link Rect: 258, 80, 270, 90 ActionDest: 435 Annotation: Subtype: Link Rect: 270, 80, 282, 90 ActionDest: 435 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 196, 330, 208, 339 ActionDest: 455 Annotation: Subtype: Link Rect: 208, 330, 220, 339 ActionDest: 455 Annotation: Subtype: Link Rect: 370, 276, 381, 285 ActionDest: 455 Annotation: Subtype: Link Rect: 257, 235, 268, 244 ActionDest: 435 Annotation: Subtype: Link Rect: 117, 168, 129, 177 ActionDest: 455 Annotation: Subtype: Link Rect: 360, 168, 367, 177 ActionDest: 406 Annotation: Subtype: Link Rect: 367, 168, 379, 177 ActionDest: 435 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 389, 472, 426, 482 Annotation: Subtype: Link Rect: 432, 472, 468, 482 Annotation: Subtype: Link Rect: 330, 407, 368, 418 Annotation: Subtype: Link Rect: 233, 381, 270, 392 Annotation: Subtype: Link Rect: 276, 381, 312, 392 Annotation: Subtype: Link Rect: 100, 343, 137, 353 Annotation: Subtype: Link Rect: 143, 343, 179, 353 Annotation: Subtype: Link Rect: 127, 317, 177, 327 Annotation: Subtype: Link Rect: 335, 291, 373, 302 Annotation: Subtype: Link Rect: 242, 253, 279, 263 Annotation: Subtype: Link Rect: 144, 201, 181, 211 Annotation: Subtype: Link Rect: 244, 162, 281, 173 Annotation: Subtype: Link Rect: 258, 124, 295, 134 Annotation: Subtype: Link Rect: 479, 84, 516, 95 Annotation: Subtype: Link Rect: 100, 72, 136, 82 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 341, 688, 378, 699 Annotation: Subtype: Link Rect: 384, 688, 420, 699 Annotation: Subtype: Link Rect: 407, 663, 443, 673 Annotation: Subtype: Link Rect: 325, 624, 363, 635 Annotation: Subtype: Link Rect: 258, 586, 295, 596 Annotation: Subtype: Link Rect: 136, 547, 174, 557 Annotation: Subtype: Link Rect: 247, 508, 284, 519 Annotation: Subtype: Link Rect: 243, 470, 281, 480 Annotation: Subtype: Link Rect: 441, 430, 479, 441 Annotation: Subtype: Link Rect: 341, 391, 379, 402 Annotation: Subtype: Link Rect: 426, 366, 462, 376 Annotation: Subtype: Link Rect: 438, 327, 475, 338 Annotation: Subtype: Link Rect: 481, 327, 516, 338 Annotation: Subtype: Link Rect: 223, 289, 260, 299 Annotation: Subtype: Link Rect: 267, 289, 303, 299 Annotation: Subtype: Link Rect: 473, 262, 510, 273 Annotation: Subtype: Link Rect: 449, 236, 487, 247 Annotation: Subtype: Link Rect: 477, 211, 514, 222 Annotation: Subtype: Link Rect: 456, 172, 493, 183 Annotation: Subtype: Link Rect: 478, 146, 515, 157 Annotation: Subtype: Link Rect: 450, 107, 487, 118 Annotation: Subtype: Link Rect: 382, 69, 420, 80 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 480, 727, 516, 738 Annotation: Subtype: Link Rect: 100, 715, 135, 725 Annotation: Subtype: Link Rect: 478, 675, 516, 686 Annotation: Subtype: Link Rect: 100, 663, 136, 673 Annotation: Subtype: Link Rect: 261, 624, 299, 635 Annotation: Subtype: Link Rect: 305, 624, 341, 635 Annotation: Subtype: Link Rect: 286, 598, 324, 609 Annotation: Subtype: Link Rect: 330, 598, 366, 609 Annotation: Subtype: Link Rect: 356, 572, 394, 583 Annotation: Subtype: Link Rect: 328, 546, 365, 557 Annotation: Subtype: Link Rect: 371, 546, 407, 557 Annotation: Subtype: Link Rect: 213, 508, 251, 519 Annotation: Subtype: Link Rect: 181, 470, 219, 480 Annotation: Subtype: Link Rect: 420, 430, 457, 441 Annotation: Subtype: Link Rect: 76, 358, 166, 389 Annotation: Subtype: Link Rect: 234, 353, 407, 363 Checksum: a63901e3 Type: CRC32 Checksum: 0e25ad24d1be82987aa79b80255fd6eb Type: MD5 Checksum: 961709ba24e4002fea4d0a1417f8c317c0d8ec28 Type: SHA-1