Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia

Canine parvovirus (CPV-2) is the causative agent of haemorrhagic gastroenteritis in canids. Three antigenic variants—CPV-2a, CPV-2b and CPV-2c—have been described, which are determined by variations at residue 426 of the VP2 capsid protein. In Colombia, the CPV-2a and CPV-2b antigenic variants have...

Full description

Autores:
Giraldo Ramírez, Sebastián
Rendón Marín, Santiago
Ruiz Sáenz, Julián
Tipo de recurso:
Article of investigation
Fecha de publicación:
2020
Institución:
Universidad Cooperativa de Colombia
Repositorio:
Repositorio UCC
Idioma:
OAI Identifier:
oai:repository.ucc.edu.co:20.500.12494/33013
Acceso en línea:
https://hdl.handle.net/20.500.12494/33013
Palabra clave:
Antigenicity
Sequencing
Virus
Rights
openAccess
License
Atribución – No comercial – Compartir igual
id COOPER2_d9561675ee1fc1bcfede1f132f0f3c93
oai_identifier_str oai:repository.ucc.edu.co:20.500.12494/33013
network_acronym_str COOPER2
network_name_str Repositorio UCC
repository_id_str
dc.title.spa.fl_str_mv Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
title Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
spellingShingle Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
Antigenicity
Sequencing
Virus
title_short Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
title_full Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
title_fullStr Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
title_full_unstemmed Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
title_sort Phylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in Colombia
dc.creator.fl_str_mv Giraldo Ramírez, Sebastián
Rendón Marín, Santiago
Ruiz Sáenz, Julián
dc.contributor.author.none.fl_str_mv Giraldo Ramírez, Sebastián
Rendón Marín, Santiago
Ruiz Sáenz, Julián
dc.subject.spa.fl_str_mv Antigenicity
Sequencing
Virus
topic Antigenicity
Sequencing
Virus
description Canine parvovirus (CPV-2) is the causative agent of haemorrhagic gastroenteritis in canids. Three antigenic variants—CPV-2a, CPV-2b and CPV-2c—have been described, which are determined by variations at residue 426 of the VP2 capsid protein. In Colombia, the CPV-2a and CPV-2b antigenic variants have previously been reported through partial VP2 sequencing. Mutations at residues Asn428Asp and Ala514Ser of variant CPV-2a were detected, implying the appearance of a possible new CPV-2a variant in Colombia. The purpose of the present study was to characterise the full VP2 capsid protein in samples from Antioquia, Colombia. We conducted a cross-sectional study with 56 stool samples from dogs showing clinical symptoms of parvoviral disease. Following DNA extraction from the samples, VP2 amplification was performed using PCR and positive samples were sequenced. Sequence and phylogenetic analyses were performed by comparison with the VP2 gene sequences of the different CPV-2 worldwide. VP2 was amplified in 51.8% of the analysed samples. Sequencing and sequence alignment showed that 93.1% of the amplified samples belonged to the new CPV-2a antigenic variant previously. Analysing the amino acid sequences revealed that all CPV-2a contain Ala297Asn mutations, which are related to the South America I clade, and the Ala514Ser mutation, which allows characterization as a new CPV-2a sub-variant. The Colombian CPV-2b variant presented Phe267Tyr, Tyr324Ile and Thr440Ala, which are related to the Asia-I clade variants. The CPV-2c was not detected in the samples. In conclusion, two antigenic CPV-2 variants of two geographically distant origins are circulating in Colombia. It is crucial to continue characterising CPV-2 to elucidate the molecular dynamics of the virus and to detect new CPV-2 variants that could be becoming highly prevalent in the region.
publishDate 2020
dc.date.issued.none.fl_str_mv 2020-04
dc.date.accessioned.none.fl_str_mv 2021-01-28T22:13:51Z
dc.date.available.none.fl_str_mv 2021-01-28T22:13:51Z
dc.type.none.fl_str_mv Artículos Científicos
dc.type.coar.none.fl_str_mv http://purl.org/coar/resource_type/c_2df8fbb1
dc.type.coarversion.none.fl_str_mv http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.driver.none.fl_str_mv info:eu-repo/semantics/article
dc.type.version.none.fl_str_mv info:eu-repo/semantics/publishedVersion
format http://purl.org/coar/resource_type/c_2df8fbb1
status_str publishedVersion
dc.identifier.issn.spa.fl_str_mv 1999-4915
dc.identifier.uri.spa.fl_str_mv doi.org/10.3390/v12050500
dc.identifier.uri.none.fl_str_mv https://hdl.handle.net/20.500.12494/33013
dc.identifier.bibliographicCitation.spa.fl_str_mv Giraldo-Ramirez, Sebastián; Rendon-Marin, Santiago; Ruiz-Saenz, Julián. 2020. "Phylogenetic, Evolutionary and Structural Analysis of Canine Parvovirus (CPV-2) Antigenic Variants Circulating in Colombia" Viruses 12, no. 5: 500. https://doi.org/10.3390/v12050500
identifier_str_mv 1999-4915
doi.org/10.3390/v12050500
Giraldo-Ramirez, Sebastián; Rendon-Marin, Santiago; Ruiz-Saenz, Julián. 2020. "Phylogenetic, Evolutionary and Structural Analysis of Canine Parvovirus (CPV-2) Antigenic Variants Circulating in Colombia" Viruses 12, no. 5: 500. https://doi.org/10.3390/v12050500
url https://hdl.handle.net/20.500.12494/33013
dc.relation.isversionof.spa.fl_str_mv https://www.mdpi.com/1999-4915/12/5/500
dc.relation.ispartofjournal.spa.fl_str_mv Viruses
dc.relation.references.spa.fl_str_mv Goddard, A.; Leisewitz, A.L. Canine parvovirus. Vet. Clin. N. Am. Small Anim. Pract. 2010, 40, 1041–1053.
Cotmore, S.F.; Agbandje-McKenna, M.; Chiorini, J.A.; Mukha, D.V.; Pintel, D.J.; Qiu, J.; Soderlund-Venermo, M.; Tattersall, P.; Tijssen, P.; Gatherer, D.; et al. The family Parvoviridae. Arch. Virol. 2014, 159, 1239–1247
Reed, A.P.; Jones, E.V.; Miller, T.J. Nucleotide sequence and genome organization of canine parvovirus. J. Virol. 1988, 62, 266–276.
Hueffer, K.; Parker, J.S.; Weichert, W.S.; Geisel, R.E.; Sgro, J.Y.; Parrish, C.R. The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor. J. Virol. 2003, 77, 1718–1726
Parker, J.S.; Parrish, C.R. Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking. J. Virol. 2000, 74, 1919–1930.
Miranda, C.; Thompson, G. Canine parvovirus: The worldwide occurrence of antigenic variants. J. Gen. Virol. 2016, 97, 2043–2057.
Chang, S.F.; Sgro, J.Y.; Parrish, C.R. Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties. J. Virol. 1992, 66, 6858–6867
Truyen, U.; Evermann, J.F.; Vieler, E.; Parrish, C.R. Evolution of canine parvovirus involved loss and gain of feline host range. Virology 1996, 215, 186–189.
Parrish, C.R. Host range relationships and the evolution of canine parvovirus. Vet. Microbiol. 1999, 69, 29–40
Parrish, C.R.; Aquadro, C.F.; Strassheim, M.L.; Evermann, J.F.; Sgro, J.Y.; Mohammed, H.O. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J. Virol. 1991, 65, 6544–6552.
Tsao, J.; Chapman, M.S.; Agbandje, M.; Keller, W.; Smith, K.; Wu, H.; Luo, M.; Smith, T.J.; Rossmann, M.G.; Compans, R.W.; et al. The three-dimensional structure of canine parvovirus and its functional implications. Science 1991, 251, 1456–1464.
Buonavoglia, C.; Martella, V.; Pratelli, A.; Tempesta, M.; Cavalli, A.; Buonavoglia, D.; Bozzo, G.; Elia, G.; Decaro, N.; Carmichael, L. Evidence for evolution of canine parvovirus type 2 in Italy. J. Gen. Virol. 2001, 82 Pt 12, 3021–3025.
Decaro, N.; Buonavoglia, C. Canine parvovirus—A review of epidemiological and diagnostic aspects, with emphasis on type 2c. Vet. Microbiol. 2012, 155, 1–12
Duque-Garcia, Y.; Echeverri-Zuluaga, M.; Trejos-Suarez, J.; Ruiz-Saenz, J. Prevalence and molecular epidemiology of Canine parvovirus 2 in diarrheic dogs in Colombia, South America: A possible new CPV-2a is emerging? Vet. Microbiol. 2017, 201, 56–61.
Zhou, P.; Zeng, W.; Zhang, X.; Li, S. The genetic evolution of canine parvovirus—A new perspective. PLoS ONE 2017, 12, e0175035
Grecco, S.; Iraola, G.; Decaro, N.; Alfieri, A.; Alfieri, A.; Gallo Calderon, M.; da Silva, A.P.; Name, D.; Aldaz, J.; Calleros, L.; et al. Inter- and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America. Virus Evol. 2018, 4, vey011.
Faz, M.; Martinez, J.S.; Gomez, L.B.; Quijano-Hernandez, I.; Fajardo, R.; Del Angel-Caraza, J. Origin and genetic diversity of canine parvovirus 2c circulating in Mexico. Arch. Virol. 2019, 164, 371–3
Ikeda, Y.; Mochizuki, M.; Naito, R.; Nakamura, K.; Miyazawa, T.; Mikami, T.; Takahashi, E. Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats. Virology 2000, 278, 13–19.
Xiao, C.; Rossmann, M.G. Interpretation of electron density with stereographic roadmap projections. J. Struct. Biol. 2007, 158, 182
dc.rights.license.none.fl_str_mv Atribución – No comercial – Compartir igual
dc.rights.accessrights.none.fl_str_mv info:eu-repo/semantics/openAccess
dc.rights.coar.none.fl_str_mv http://purl.org/coar/access_right/c_abf2
rights_invalid_str_mv Atribución – No comercial – Compartir igual
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.extent.spa.fl_str_mv 500
dc.coverage.temporal.spa.fl_str_mv 12(5)
dc.publisher.spa.fl_str_mv MDPI
Universidad Cooperativa de Colombia, Facultad de Ciencias de la Salud, Medicina Veterinaría y Zootecnia, Bucaramanga
dc.publisher.program.spa.fl_str_mv Medicina veterinaria y zootecnia
dc.publisher.place.spa.fl_str_mv Bucaramanga
institution Universidad Cooperativa de Colombia
bitstream.url.fl_str_mv https://repository.ucc.edu.co/bitstreams/2b22bccc-677d-46ed-8727-a8345fcefc85/download
https://repository.ucc.edu.co/bitstreams/996b4248-8b1a-478a-8d0b-11a3c549544b/download
https://repository.ucc.edu.co/bitstreams/3b7357ba-c328-4296-bcf5-2df5515db590/download
https://repository.ucc.edu.co/bitstreams/d51051fa-1516-480f-8cb6-7d99f3830f4c/download
https://repository.ucc.edu.co/bitstreams/d101ee50-82b9-49a0-8fdd-af6cf9285d49/download
https://repository.ucc.edu.co/bitstreams/489a83a5-7dc6-47d6-943c-76c3062a6c16/download
https://repository.ucc.edu.co/bitstreams/97460ace-37a5-4afa-bf66-eb3f6eb46805/download
bitstream.checksum.fl_str_mv f10c8e08d949b00d8bc6b71c83c4e2ec
316b2d4e7b775914d676f8504b59de56
3bce4f7ab09dfc588f126e1e36e98a45
1e7e99a51d9298f546cbda87d64727c8
3a13c16585e5c6c06edf85fd140fa4ac
6ffaab7af82de08bddfc49bb38d6a462
5a15ba390abf175ca1664971cd9b330f
bitstream.checksumAlgorithm.fl_str_mv MD5
MD5
MD5
MD5
MD5
MD5
MD5
repository.name.fl_str_mv Repositorio Institucional Universidad Cooperativa de Colombia
repository.mail.fl_str_mv bdigital@metabiblioteca.com
_version_ 1811565394648891392
spelling Giraldo Ramírez, SebastiánRendón Marín, SantiagoRuiz Sáenz, Julián12(5)2021-01-28T22:13:51Z2021-01-28T22:13:51Z2020-041999-4915doi.org/10.3390/v12050500https://hdl.handle.net/20.500.12494/33013Giraldo-Ramirez, Sebastián; Rendon-Marin, Santiago; Ruiz-Saenz, Julián. 2020. "Phylogenetic, Evolutionary and Structural Analysis of Canine Parvovirus (CPV-2) Antigenic Variants Circulating in Colombia" Viruses 12, no. 5: 500. https://doi.org/10.3390/v12050500Canine parvovirus (CPV-2) is the causative agent of haemorrhagic gastroenteritis in canids. Three antigenic variants—CPV-2a, CPV-2b and CPV-2c—have been described, which are determined by variations at residue 426 of the VP2 capsid protein. In Colombia, the CPV-2a and CPV-2b antigenic variants have previously been reported through partial VP2 sequencing. Mutations at residues Asn428Asp and Ala514Ser of variant CPV-2a were detected, implying the appearance of a possible new CPV-2a variant in Colombia. The purpose of the present study was to characterise the full VP2 capsid protein in samples from Antioquia, Colombia. We conducted a cross-sectional study with 56 stool samples from dogs showing clinical symptoms of parvoviral disease. Following DNA extraction from the samples, VP2 amplification was performed using PCR and positive samples were sequenced. Sequence and phylogenetic analyses were performed by comparison with the VP2 gene sequences of the different CPV-2 worldwide. VP2 was amplified in 51.8% of the analysed samples. Sequencing and sequence alignment showed that 93.1% of the amplified samples belonged to the new CPV-2a antigenic variant previously. Analysing the amino acid sequences revealed that all CPV-2a contain Ala297Asn mutations, which are related to the South America I clade, and the Ala514Ser mutation, which allows characterization as a new CPV-2a sub-variant. The Colombian CPV-2b variant presented Phe267Tyr, Tyr324Ile and Thr440Ala, which are related to the Asia-I clade variants. The CPV-2c was not detected in the samples. In conclusion, two antigenic CPV-2 variants of two geographically distant origins are circulating in Colombia. It is crucial to continue characterising CPV-2 to elucidate the molecular dynamics of the virus and to detect new CPV-2 variants that could be becoming highly prevalent in the region.http://scienti.colciencias.gov.co:8081/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000153095https://orcid.org/0000-0002-1447-1458https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000000695julian.ruizs@campusucc.edu.cohttps://scholar.google.com/citations?user=o3Y7mZwAAAAJ&hl=es500MDPIUniversidad Cooperativa de Colombia, Facultad de Ciencias de la Salud, Medicina Veterinaría y Zootecnia, BucaramangaMedicina veterinaria y zootecniaBucaramangahttps://www.mdpi.com/1999-4915/12/5/500VirusesGoddard, A.; Leisewitz, A.L. Canine parvovirus. Vet. Clin. N. Am. Small Anim. Pract. 2010, 40, 1041–1053.Cotmore, S.F.; Agbandje-McKenna, M.; Chiorini, J.A.; Mukha, D.V.; Pintel, D.J.; Qiu, J.; Soderlund-Venermo, M.; Tattersall, P.; Tijssen, P.; Gatherer, D.; et al. The family Parvoviridae. Arch. Virol. 2014, 159, 1239–1247Reed, A.P.; Jones, E.V.; Miller, T.J. Nucleotide sequence and genome organization of canine parvovirus. J. Virol. 1988, 62, 266–276.Hueffer, K.; Parker, J.S.; Weichert, W.S.; Geisel, R.E.; Sgro, J.Y.; Parrish, C.R. The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor. J. Virol. 2003, 77, 1718–1726Parker, J.S.; Parrish, C.R. Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking. J. Virol. 2000, 74, 1919–1930.Miranda, C.; Thompson, G. Canine parvovirus: The worldwide occurrence of antigenic variants. J. Gen. Virol. 2016, 97, 2043–2057.Chang, S.F.; Sgro, J.Y.; Parrish, C.R. Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties. J. Virol. 1992, 66, 6858–6867Truyen, U.; Evermann, J.F.; Vieler, E.; Parrish, C.R. Evolution of canine parvovirus involved loss and gain of feline host range. Virology 1996, 215, 186–189.Parrish, C.R. Host range relationships and the evolution of canine parvovirus. Vet. Microbiol. 1999, 69, 29–40Parrish, C.R.; Aquadro, C.F.; Strassheim, M.L.; Evermann, J.F.; Sgro, J.Y.; Mohammed, H.O. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J. Virol. 1991, 65, 6544–6552.Tsao, J.; Chapman, M.S.; Agbandje, M.; Keller, W.; Smith, K.; Wu, H.; Luo, M.; Smith, T.J.; Rossmann, M.G.; Compans, R.W.; et al. The three-dimensional structure of canine parvovirus and its functional implications. Science 1991, 251, 1456–1464.Buonavoglia, C.; Martella, V.; Pratelli, A.; Tempesta, M.; Cavalli, A.; Buonavoglia, D.; Bozzo, G.; Elia, G.; Decaro, N.; Carmichael, L. Evidence for evolution of canine parvovirus type 2 in Italy. J. Gen. Virol. 2001, 82 Pt 12, 3021–3025.Decaro, N.; Buonavoglia, C. Canine parvovirus—A review of epidemiological and diagnostic aspects, with emphasis on type 2c. Vet. Microbiol. 2012, 155, 1–12Duque-Garcia, Y.; Echeverri-Zuluaga, M.; Trejos-Suarez, J.; Ruiz-Saenz, J. Prevalence and molecular epidemiology of Canine parvovirus 2 in diarrheic dogs in Colombia, South America: A possible new CPV-2a is emerging? Vet. Microbiol. 2017, 201, 56–61.Zhou, P.; Zeng, W.; Zhang, X.; Li, S. The genetic evolution of canine parvovirus—A new perspective. PLoS ONE 2017, 12, e0175035Grecco, S.; Iraola, G.; Decaro, N.; Alfieri, A.; Alfieri, A.; Gallo Calderon, M.; da Silva, A.P.; Name, D.; Aldaz, J.; Calleros, L.; et al. Inter- and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America. Virus Evol. 2018, 4, vey011.Faz, M.; Martinez, J.S.; Gomez, L.B.; Quijano-Hernandez, I.; Fajardo, R.; Del Angel-Caraza, J. Origin and genetic diversity of canine parvovirus 2c circulating in Mexico. Arch. Virol. 2019, 164, 371–3Ikeda, Y.; Mochizuki, M.; Naito, R.; Nakamura, K.; Miyazawa, T.; Mikami, T.; Takahashi, E. Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats. Virology 2000, 278, 13–19.Xiao, C.; Rossmann, M.G. Interpretation of electron density with stereographic roadmap projections. J. Struct. Biol. 2007, 158, 182AntigenicitySequencingVirusPhylogenetic, evolutionary and structural analysis of Canine Parvovirus (CPV-2) antigenic variants circulating in ColombiaArtículos Científicoshttp://purl.org/coar/resource_type/c_2df8fbb1http://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionAtribución – No comercial – Compartir igualinfo:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2PublicationORIGINAL2021_phylogenetic_evolutionary_structural.pdf2021_phylogenetic_evolutionary_structural.pdfapplication/pdf1460441https://repository.ucc.edu.co/bitstreams/2b22bccc-677d-46ed-8727-a8345fcefc85/downloadf10c8e08d949b00d8bc6b71c83c4e2ecMD512021_phylogenetic_evolutionary_structural-FormatoLicenciaUso.pdf2021_phylogenetic_evolutionary_structural-FormatoLicenciaUso.pdfapplication/pdf207981https://repository.ucc.edu.co/bitstreams/996b4248-8b1a-478a-8d0b-11a3c549544b/download316b2d4e7b775914d676f8504b59de56MD53LICENSElicense.txtlicense.txttext/plain; charset=utf-84334https://repository.ucc.edu.co/bitstreams/3b7357ba-c328-4296-bcf5-2df5515db590/download3bce4f7ab09dfc588f126e1e36e98a45MD54THUMBNAIL2021_phylogenetic_evolutionary_structural.pdf.jpg2021_phylogenetic_evolutionary_structural.pdf.jpgGenerated Thumbnailimage/jpeg5481https://repository.ucc.edu.co/bitstreams/d51051fa-1516-480f-8cb6-7d99f3830f4c/download1e7e99a51d9298f546cbda87d64727c8MD552021_phylogenetic_evolutionary_structural-FormatoLicenciaUso.pdf.jpg2021_phylogenetic_evolutionary_structural-FormatoLicenciaUso.pdf.jpgGenerated Thumbnailimage/jpeg5467https://repository.ucc.edu.co/bitstreams/d101ee50-82b9-49a0-8fdd-af6cf9285d49/download3a13c16585e5c6c06edf85fd140fa4acMD56TEXT2021_phylogenetic_evolutionary_structural.pdf.txt2021_phylogenetic_evolutionary_structural.pdf.txtExtracted texttext/plain58376https://repository.ucc.edu.co/bitstreams/489a83a5-7dc6-47d6-943c-76c3062a6c16/download6ffaab7af82de08bddfc49bb38d6a462MD572021_phylogenetic_evolutionary_structural-FormatoLicenciaUso.pdf.txt2021_phylogenetic_evolutionary_structural-FormatoLicenciaUso.pdf.txtExtracted texttext/plain5935https://repository.ucc.edu.co/bitstreams/97460ace-37a5-4afa-bf66-eb3f6eb46805/download5a15ba390abf175ca1664971cd9b330fMD5820.500.12494/33013oai:repository.ucc.edu.co:20.500.12494/330132024-08-09 12:27:32.196open.accesshttps://repository.ucc.edu.coRepositorio Institucional Universidad Cooperativa de Colombiabdigital@metabiblioteca.comVU5JVkVSU0lEQUQgQ09PUEVSQVRJVkEgREUgQ09MT01CSUEKUkVQT1NJVE9SSU9TIElOU1RJVFVDSU9OQUxFUwpMSUNFTkNJQSBERSBVU08KClBvciBtZWRpbyBkZWwgcHJlc2VudGUgZG9jdW1lbnRvLCBlbCBBdXRvcihlcyksIG1heW9yIChlcykgZGUgZWRhZCwgcXVpZW4gZW4gYWRlbGFudGUgc2UgZGVub21pbmFyw6EgZWwgQVVUT1IsIGNvbmZpZXJlIGEgbGEgVU5JVkVSU0lEQUQgQ09PUEVSQVRJVkEgREUgQ09MT01CSUEsIGNvbiBOSVQuIDg2MC0wMjk5MjQtNywgdW5hIExJQ0VOQ0lBIERFIFVTTyBkZSBvYnJhLCBiYWpvIGxhcyBzaWd1aWVudGVzIGNvbmRpY2lvbmVzLgoKQ0zDgVVTVUxBUwoKUFJJTUVSQS4gT2JqZXRvLiBFTCBBVVRPUiBwb3IgZXN0ZSBhY3RvIGF1dG9yaXphIGxhIHV0aWxpemFjacOzbiBkZSBsYSBvYnJhLCBkZSBjb25mb3JtaWRhZCBjb24gbG8gZXN0aXB1bGFkbyBhIGNvbnRpbnVhY2nDs246IAoKKGEpIFBhcmEgZWZlY3RvcyBkZSBsYSBwcmVzZW50ZSBsaWNlbmNpYSBzZSBhdXRvcml6YSBsYSByZXByb2R1Y2Npw7NuIGRlIGxhIG9icmEgYW50ZXJpb3JtZW50ZSBjaXRhZGEsIGxhIGN1YWwgc2UgYWxvamFyw6EgZW4gZm9ybWF0byBkaWdpdGFsIGVuIGxhcyBwbGF0YWZvcm1hcyBvIHJlcG9zaXRvcmlvcyBhZG1pbmlzdHJhZG9zIHBvciBsYSBVTklWRVJTSURBRCBvIGVuIG90cm8gdGlwbyBkZSByZXBvc2l0b3Jpb3MgZXh0ZXJub3MgbyBww6FnaW5hcyB3ZWIgZXNjb2dpZG9zIHBvciBsYSBVTklWRVJTSURBRCwgcGFyYSBmaW5lcyBkZSBkaWZ1c2nDs24geSBkaXZ1bGdhY2nDs24uIEFkaWNpb25hbG1lbnRlLCBzZSBhdXRvcml6YSBhIHF1ZSBsb3MgdXN1YXJpb3MgaW50ZXJub3MgeSBleHRlcm5vcyBkZSBkaWNoYXMgcGxhdGFmb3JtYXMgbyByZXBvc2l0b3Jpb3MgcmVwcm9kdXpjYW4gbyBkZXNjYXJndWVuIGxhIG9icmEsIHNpbiDDoW5pbW8gZGUgbHVjcm8sIHBhcmEgZmluZXMgcHJpdmFkb3MsIGVkdWNhdGl2b3MgbyBhY2Fkw6ltaWNvczsgc2llbXByZSB5IGN1YW5kbyBubyBzZSB2aW9sZW4gYWN1ZXJkb3MgY29uIGVkaXRvcmVzLCBwZXJpb2RvcyBkZSBlbWJhcmdvIG8gYWN1ZXJkb3MgZGUgY29uZmlkZW5jaWFsaWRhZCBxdWUgYXBsaXF1ZW4uCgooYikgU2UgYXV0b3JpemEgbGEgY29tdW5pY2FjacOzbiBww7pibGljYSB5IGxhIHB1ZXN0YSBhIGRpc3Bvc2ljacOzbiBkZSBsYSBvYnJhIG1lbmNpb25hZGEsIGVuIGFjY2VzbyBhYmllcnRvLCBwYXJhIHN1IHV0aWxpemFjacOzbiBlbiBsYXMgcGxhdGFmb3JtYXMgbyByZXBvc2l0b3Jpb3MgYWRtaW5pc3RyYWRvcyBwb3IgbGEgVU5JVkVSU0lEQUQuCgooYykgTG8gYW50ZXJpb3IgZXN0YXLDoSBzdWpldG8gYSBsYXMgZGVmaW5pY2lvbmVzIGNvbnRlbmlkYXMgZW4gbGEgRGVjaXNpw7NuIEFuZGluYSAzNTEgZGUgMTk5MyB5IGxhIExleSAyMyBkZSAxOTgyLgoKClNFR1VOREEuIE9yaWdpbmFsaWRhZCB5IHJlY2xhbWFjaW9uZXMuIEVsIEFVVE9SIGRlY2xhcmEgcXVlIGxhIE9CUkEgZXMgb3JpZ2luYWwgeSBxdWUgZXMgZGUgc3UgY3JlYWNpw7NuIGV4Y2x1c2l2YSwgbm8gZXhpc3RpZW5kbyBpbXBlZGltZW50byBkZSBjdWFscXVpZXIgbmF0dXJhbGV6YSAoZW1iYXJnb3MsIHVzbyBkZSBtYXRlcmlhbCBwcm90ZWdpZG8gcG9yIGRlcmVjaG9zIGRlIGF1dG9yKSBwYXJhIGxhIGNvbmNlc2nDs24gZGUgbG9zIGRlcmVjaG9zIHByZXZpc3RvcyBlbiBlc3RlIGFjdWVyZG8uIEVsIEFVVE9SIHJlc3BvbmRlcsOhIHBvciBjdWFscXVpZXIgYWNjacOzbiBkZSByZWl2aW5kaWNhY2nDs24sIHBsYWdpbyB1IG90cmEgY2xhc2UgZGUgcmVjbGFtYWNpw7NuIHF1ZSBhbCByZXNwZWN0byBwdWRpZXJhIHNvYnJldmVuaXIuCgpURVJDRVJBLiBDb250cmFwcmVzdGFjacOzbi4gRWwgQVVUT1IgYXV0b3JpemEgYSBxdWUgc3Ugb2JyYSBzZWEgdXRpbGl6YWRhIGRlIGNvbmZvcm1pZGFkIGNvbiBsYSBjbMOhdXN1bGEgUFJJTUVSQSBkZSBmb3JtYSBncmF0dWl0YSwgZXMgZGVjaXIsIHF1ZSBsYSB1dGlsaXphY2nDs24gZGUgbGEgbWlzbWEgbm8gZ2VuZXJhIG5pbmfDum4gcGFnbyBvIHJlZ2Fsw61hcyBlbiBmYXZvciBkZSBlc3RlLgoKQ1VBUlRBLiBUaXR1bGFyaWRhZCBkZSBkZXJlY2hvcy4gRWwgcHJlc2VudGUgY29udHJhdG8gbm8gdHJhbnNmaWVyZSBsYSB0aXR1bGFyaWRhZCBkZSBsb3MgZGVyZWNob3MgcGF0cmltb25pYWxlcyBzb2JyZSBsYXMgb2JyYXMgYW50ZXJpb3JtZW50ZSBtZW5jaW9uYWRhcyBhIGxhIFVOSVZFUlNJREFELiDDmm5pY2FtZW50ZSBoYWNlIHJlbGFjacOzbiBhIHVuYSBsaWNlbmNpYSBubyBleGNsdXNpdmEgZW4gbG9zIHTDqXJtaW5vcyB5IGNvbmRpY2lvbmVzIGFudGVyaW9ybWVudGUgcGFjdGFkb3MuCgpRVUlOVEEuIENyw6lkaXRvcy4gTGEgVU5JVkVSU0lEQUQgc2UgY29tcHJvbWV0ZSBhIGRhciBhbCBBVVRPUiwgZWwgcmVjb25vY2ltaWVudG8gZGVudHJvIGNhZGEgZm9ybWEgZGUgdXRpbGl6YWNpw7NuIGVuIGxhIG9icmEuIExvcyBjcsOpZGl0b3MgZGViZW4gZmlndXJhciBlbiBjYWRhIHVubyBkZSBsb3MgZm9ybWF0b3MgbyByZWdpc3Ryb3MgZGUgcHVibGljYWNpw7NuLiBObyBjb25zdGl0dWlyw6EgdW5hIHZpb2xhY2nDs24gYSBsb3MgZGVyZWNob3MgbW9yYWxlcyBkZWwgYXV0b3IgbGEgbm8gcmVwcm9kdWNjacOzbiwgY29tdW5pY2FjacOzbiBvIGRlbcOhcyB1dGlsaXphY2lvbmVzIGRlIGxhIG9icmEuIExhIHV0aWxpemFjacOzbiBvIG5vIGRlIGxhIG9icmEsIGFzw60gY29tbyBzdSBmb3JtYSBkZSB1dGlsaXphY2nDs24gc2Vyw6EgZmFjdWx0YWQgZXhjbHVzaXZhIGRlIGxhIFVOSVZFUlNJREFELgogClNFWFRBLiBEdXJhY2nDs24geSB0ZXJyaXRvcmlvLiBMYSBwcmVzZW50ZSBsaWNlbmNpYSBkZSB1c28gcXVlIHNlIG90b3JnYSBhIGZhdm9yIGRlIGxhIFVOSVZFUlNJREFEIHRlbmRyw6EgdW5hIGR1cmFjacOzbiBlcXVpdmFsZW50ZSBhbCB0w6lybWlubyBkZSBwcm90ZWNjacOzbiBsZWdhbCBkZSBsYSBvYnJhIHkgcGFyYSB0b2RvcyBsb3MgcGHDrXNlcyBkZWwgbXVuZG8uCgpTw4lQVElNQS4gVXNvIGRlIENyZWF0aXZlIENvbW1vbnMuIEVsIEFVVE9SIGF1dG9yaXphcsOhIGxhIGRpZnVzacOzbiBkZSBzdSBjb250ZW5pZG8gYmFqbyB1bmEgbGljZW5jaWEgQ3JlYXRpdmUgQ29tbW9ucyBhdHJpYnVjacOzbiA0LjAgaW50ZXJuYWNpb25hbCwgcXVlIGRlYmVyw6EgaW5jbHVpcnNlIGVuIGVsIGNvbnRlbmlkby4gCgpPQ1RBVkEuIERlcmVjaG8gZGUgZXhjbHVzacOzbi4gQ2FkYSBhdXRvciBwdWVkZSBpbmRpY2FyIGVuIGVsIG1vbWVudG8gZGUgZGVww7NzaXRvIGRlbCBjb250ZW5pZG8gcXVlIGVsIHRleHRvIGNvbXBsZXRvIGRlIGxhIHByb2R1Y2Npw7NuIGFjYWTDqW1pY2EgbyBjaWVudMOtZmljYSBubyBlc3RlIGNvbiBhY2Nlc28gYWJpZXJ0byBlbiBlbCBSZXBvc2l0b3JpbyBJbnN0aXR1Y2lvbmFsIHBvciBtb3Rpdm9zIGRlIGNvbmZpZGVuY2lhbGlkYWQsIHBvcnF1ZSBzZSBlbmN1ZW50cmUgZW4gdsOtYXMgZGUgb2J0ZW5lciB1biBkZXJlY2hvIGRlIHByb3BpZWRhZCBpbmR1c3RyaWFsIG8gZXhpc3RpciBhY3VlcmRvcyBwcmV2aW9zIGNvbiB0ZXJjZXJvcyAoZWRpdG9yaWFsZXMsIHJldmlzdGFzIGNpZW50w61maWNhcywgb3RyYXMgaW5zdGl0dWNpb25lcykuIEVsIGF1dG9yIHNlIGNvbXByb21ldGUgYSBkZXBvc2l0YXIgbG9zIG1ldGFkYXRvcyBlIGluZm9ybWFyIGVsIHRpZW1wbyBkZSBlbWJhcmdvIGR1cmFudGUgZWwgY3VhbCBlbCB0ZXh0byBjb21wbGV0byB0ZW5kcsOhIGFjY2VzbyByZXN0cmluZ2lkby4gCgpOT1ZFTkEuIEVsIEFVVE9SIGFsIGFjZXB0YXIgZXN0YSBsaWNlbmNpYSBhZHVjZSBxdWUgZXN0YSBwcm9kdWNjacOzbiBzZSBkZXNhcnJvbGzDsyBlbiBlbCBwZXJpb2RvIGVuIHF1ZSBzZSBlbmN1ZW50cmEgY29uIHbDrW5jdWxvcyBjb24gTGEgVW5pdmVyc2lkYWQuCgpEw4lDSU1BLiBOb3JtYXMgYXBsaWNhYmxlcy4gUGFyYSBsYSBpbnRlcnByZXRhY2nDs24geSBjdW1wbGltaWVudG8gZGVsIHByZXNlbnRlIGFjdWVyZG8gbGFzIHBhcnRlcyBzZSBzb21ldGVuIGEgbGEgRGVjaXNpw7NuIEFuZGluYSAzNTEgZGUgMTk5MywgbGEgTGV5IDIzIGRlIDE5ODIgeSBkZW3DoXMgbm9ybWFzIGFwbGljYWJsZXMgZGUgQ29sb21iaWEuIEFkZW3DoXMsIGEgbGFzIG5vcm1hcyBJbnN0aXR1Y2lvbmFsZXMgcXVlIGFwbGlxdWVuLgoKTGEgcHJlc2VudGUgbGljZW5jaWEgc2UgYXV0b3JpemEgZW4gbGEgZmVjaGEgZGUgcHVibGljYWNpw7NuIGVuIGxvcyByZXBvc2l0b3Jpb3MgaW5zdGl0dWNpb25hbGVzLgo=