Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 50 and 30 ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present stu...

Full description

Autores:: Rosales Munar, Alicia
Álvarez Díaz, Diego Alejandro
Laiton Donato, Katherine
Peláez Carvajal, Dioselina
Usme Ciro, José Aldemar

Tipo de recurso:: Article of journal

Fecha de publicación:: 2020

Institución:: Universidad Cooperativa de Colombia

Repositorio:: Repositorio UCC

Idioma:

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repository_id_str
dc.title.spa.fl_str_mv	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
title	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
spellingShingle	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome Extremos de genoma Virus dengue RACE-PCR Poli(A) polimerasa Genome ends Dengue virus RACE-PCR Poly(A) polymerase
title_short	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
title_full	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
title_fullStr	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
title_full_unstemmed	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
title_sort	Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome
dc.creator.fl_str_mv	Rosales Munar, Alicia Álvarez Díaz, Diego Alejandro Laiton Donato, Katherine Peláez Carvajal, Dioselina Usme Ciro, José Aldemar
dc.contributor.author.none.fl_str_mv	Rosales Munar, Alicia Álvarez Díaz, Diego Alejandro Laiton Donato, Katherine Peláez Carvajal, Dioselina Usme Ciro, José Aldemar
dc.subject.spa.fl_str_mv	Extremos de genoma Virus dengue RACE-PCR Poli(A) polimerasa
topic	Extremos de genoma Virus dengue RACE-PCR Poli(A) polimerasa Genome ends Dengue virus RACE-PCR Poly(A) polymerase
dc.subject.other.spa.fl_str_mv	Genome ends Dengue virus RACE-PCR Poly(A) polymerase
description	Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 50 and 30 ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 50 and 30 ends of dengue virus types 1 to 4. The 50 and 30 ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 50 end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 30 end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 50 and 30 ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.
publishDate	2020
dc.date.issued.none.fl_str_mv	2020-04-29
dc.date.accessioned.none.fl_str_mv	2021-01-20T23:10:15Z
dc.date.available.none.fl_str_mv	2021-01-20T23:10:15Z
dc.type.none.fl_str_mv	Artículo
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dc.identifier.issn.spa.fl_str_mv	1999-4915
dc.identifier.uri.spa.fl_str_mv	10.3390/v12050496
dc.identifier.uri.none.fl_str_mv	https://hdl.handle.net/20.500.12494/32699
dc.identifier.bibliographicCitation.spa.fl_str_mv	Rosales-Munar, A., Alvarez-Diaz, D. A., Laiton-Donato, K., Peláez-Carvajal, D., & Usme-Ciro, J. A. (2020). Efficient method for molecular characterization of the 5’ and 3' ends of the dengue virus genome. Viruses, 12(5), 496.
identifier_str_mv	1999-4915 10.3390/v12050496 Rosales-Munar, A., Alvarez-Diaz, D. A., Laiton-Donato, K., Peláez-Carvajal, D., & Usme-Ciro, J. A. (2020). Efficient method for molecular characterization of the 5’ and 3' ends of the dengue virus genome. Viruses, 12(5), 496.
url	https://hdl.handle.net/20.500.12494/32699
dc.relation.isversionof.spa.fl_str_mv	https://www.mdpi.com/1999-4915/12/5/496
dc.relation.ispartofjournal.spa.fl_str_mv	Viruses
dc.relation.references.spa.fl_str_mv	Brathwaite Dick, O.; San Martín, J.L.; Montoya, R.H.; del Diego, J.; Zambrano, B.; Dayan, G.H. The History of Dengue Outbreaks in the Americas. Am. J. Trop. Med. Hyg. 2012, 87, 584–593. [CrossRef] WHO. Dengue Guidelines for Diagnosis, Treatment, Prevention and Control; World Health Organization, Ed.; WHO: Geneva, Switzerland, 2009; pp. 1–160. Bhatt, S.; Gething, P.W.; Brady, O.J.; Messina, J.P.; Farlow, A.W.; Moyes, C.L.; Drake, J.M.; Brownstein, J.S.; Hoen, A.G.; Sankoh, O.; et al. The global distribution and burden of dengue. Nature 2013, 496, 504–507. [CrossRef] Twiddy, S.S.; Farrar, J.J.; Vinh Chau, N.; Wills, B.; Gould, E.A.; Gritsun, T.; Lloyd, G.; Holmes, E.C. Phylogenetic relationships and differential selection pressures among genotypes of dengue-2 virus. Virology 2002, 298, 63–72. [CrossRef] Lindenbach, B.D.; Murray, C.L.; Thiel, H.J.; Rice, C.M. Flaviviridae. In Fields in Virology, 6th ed.; Knipe, D.M., Ed.; Williams & Wilkins Lippincott: Philadelphia, PA, USA, 2013; pp. 712–746. Clyde, K.; Kyle, J.L.; Harris, E. Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J. Virol. 2006, 80, 11418–11431. [CrossRef] Filomatori, C.V.; Lodeiro, M.F.; Alvarez, D.E.; Samsa, M.M.; Pietrasanta, L.; Gamarnik, A.V. A 50 RNA element promotes dengue virus RNA synthesis on a circular genome. Genes Dev. 2006, 20, 2238–2249. [CrossRef] Khromykh, A.A.; Meka, H.; Guyatt, K.J.; Westaway, E.G. Essential role of cyclization sequences in flavivirus RNA replication. J. Virol. 2001, 75, 6719–6728. [CrossRef] Friebe, P.; Harris, E. Interplay of RNA elements in the dengue virus 50 and 30 ends required for viral RNA replication. J. Virol. 2010, 84, 6103–6118. [CrossRef] Clyde, K.; Barrera, J.; Harris, E. The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis. Virology 2008, 379, 314–323. [CrossRef] Gebhard, L.G.; Filomatori, C.V.; Gamarnik, A.V. Functional RNA Elements in the Dengue Virus Genome. Viruses 2011, 3, 1739–1756. [CrossRef] Villordo, S.M.; Carballeda, J.M.; Filomatori, C.V.; Gamarnik, A.V. RNA Structure Duplications and Flavivirus Host Adaptation. Trends Microbiol. 2016, 24, 270–283. [CrossRef] Paranjape, S.M.; Harris, E. Y box-binding protein-1 binds to the dengue virus 30 -untranslated region and mediates antiviral effects. J. Biol. Chem. 2007, 282, 30497–30508. [CrossRef] [PubMed] Li, Z.; Yu, M.; Zhang, H.; Wang, H.Y.; Wang, L.F. Improved rapid amplification of cDNA ends (RACE) for mapping both the 50 and 30 terminal sequences of paramyxovirus genomes. J. Virol. Methods 2005, 130, 154–156. [CrossRef] [PubMed] Miller, E. Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus. Methods Mol. Biol. 2016, 1373, 169–183. [CrossRef] [PubMed] Usme, J.; Gómez, A.; Gallego, J. Molecular detection and typing of dengue virus by RT-PCR and nested PCR using degenerated oligonucleotides. Rev. Salud Uninorte 2012, 28, 1–15. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [CrossRef] [PubMed Usme-Ciro, J.A.; Mendez, J.A.; Laiton, K.D.; Paez, A. The relevance of dengue virus genotypes surveillance at country level before vaccine approval. Hum. Vaccines Immunother. 2014, 10, 2674–2678. [CrossRef] IUPAC Codes. Available online: https://www.bioinformatics.org/sms2/iupac.html (accessed on 2 April 2020). Chu, P.W.; Westaway, E.G. Replication strategy of Kunjin virus: Evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. Virology 1985, 140, 68–79. [CrossRef] Leitmeyer, K.C.; Vaughn, D.W.; Watts, D.M.; Salas, R.; Villalobos, I.; de Ramos, C.; Rico-Hesse, R. Dengue virus structural differences that correlate with pathogenesis. J. Virol. 1999, 73, 4738–4747. [CrossRef] Goo, L.; VanBlargan, L.A.; Dowd, K.A.; Diamond, M.S.; Pierson, T.C. A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis. PLoS Pathog. 2017, 13, e1006178. [CrossRef] Filomatori, C.V.; Carballeda, J.M.; Villordo, S.M.; Aguirre, S.; Pallarés, H.M.; Maestre, A.M.; Sánchez-Vargas, I.; Blair, C.D.; Fabri, C.; Morales, M.A.; et al. Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells. PLoS Pathog. 2017, 13, e1006265. [CrossRef] Sirigulpanit, W.; Kinney, R.M.; Leardkamolkarn, V. Substitution or deletion mutations between nt 54 and 70 in the 50 non-coding region of dengue type 2 virus produce variable effects on virus viability. J. Gen. Virol. 2007, 88, 1748–1752. [CrossRef] Tajima, S.; Nukui, Y.; Takasaki, T.; Kurane, I. Characterization of the variable region in the 30 non-translated region of dengue type 1 virus. J. Gen. Virol. 2007, 88, 2214–2222. [CrossRef] [PubMed] Alvarez, D.E.; De Lella Ezcurra, A.L.; Fucito, S.; Gamarnik, A.V. Role of RNA structures present at the 30UTR of dengue virus on translation, RNA synthesis, and viral replication. Virology 2005, 339, 200–212. [CrossRef] [PubMed] Drake, J.W.; Holland, J.J. Mutation rates among RNA viruses. Proc. Natl. Acad. Sci. USA 1999, 96, 13910–13913. [CrossRef] [PubMed] Ong, S.H.; Yip, J.T.; Chen, Y.L.; Liu,W.; Harun, S.; Lystiyaningsih, E.; Heriyanto, B.; Beckett, C.G.; Mitchell,W.P.; Hibberd, M.L.; et al. Periodic re-emergence of endemic strains with strong epidemic potential-a proposed explanation for the 2004 Indonesian dengue epidemic. Infect. Genet. Evol. 2008, 8, 191–204. [CrossRef] [PubMed] Sasmono, R.T.; Wahid, I.; Trimarsanto, H.; Yohan, B.; Wahyuni, S.; Hertanto, M.; Yusuf, I.; Mubin, H.; Ganda, I.J.; Latief, R.; et al. Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia. Infect. Genet. Evol. 2015, 32, 165–177. [CrossRef] [PubMed] Christenbury, J.G.; Aw, P.P.; Ong, S.H.; Schreiber, M.J.; Chow, A.; Gubler, D.J.; Vasudevan, S.G.; Ooi, E.E.; Hibberd, M.L. A method for full genome sequencing of all four serotypes of the dengue virus. J. Virol. Methods 2010, 169, 202–206. [CrossRef] Dash, P.K.; Sharma, S.; Soni, M.; Agarwal, A.; Sahni, A.K.; Parida, M. Complete genome sequencing and evolutionary phylogeography analysis of Indian isolates of Dengue virus type 1. Virus Res. 2015, 195, 124–134. [CrossRef] Wongsurawat, T.; Jenjaroenpun, P.; Taylor, M.K.; Lee, J.; Tolardo, A.L.; Parvathareddy, J.; Kandel, S.; Wadley, T.D.; Kaewnapan, B.; Athipanyasilp, N.; et al. Rapid Sequencing of Multiple RNA Viruses in Their Native Form. Front. Microbiol. 2019, 10, 260. [CrossRef] Tan, C.C.S.; Maurer-Stroh, S.; Wan, Y.; Sessions, O.M.; de Sessions, P.F. A novel method for the capture-based purification of whole viral native RNA genomes. AMB Express 2019, 9, 45. [CrossRef]
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spelling	Rosales Munar, AliciaÁlvarez Díaz, Diego AlejandroLaiton Donato, KatherinePeláez Carvajal, DioselinaUsme Ciro, José Aldemar122021-01-20T23:10:15Z2021-01-20T23:10:15Z2020-04-291999-491510.3390/v12050496https://hdl.handle.net/20.500.12494/32699Rosales-Munar, A., Alvarez-Diaz, D. A., Laiton-Donato, K., Peláez-Carvajal, D., & Usme-Ciro, J. A. (2020). Efficient method for molecular characterization of the 5’ and 3' ends of the dengue virus genome. Viruses, 12(5), 496.Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 50 and 30 ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 50 and 30 ends of dengue virus types 1 to 4. The 50 and 30 ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 50 end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 30 end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 50 and 30 ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.https://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0000318507https://orcid.org/0000-0002-8093-0544https://scienti.minciencias.gov.co/gruplac/jsp/visualiza/visualizagr.jsp?nro=00000000008981jose.usmec@campusucc.edu.cohttps://scholar.google.com.co/citations?user=cU2KyT4AAAAJ&hl=en12Universidad Cooperativa de Colombia, Facultad de Ciencias de la Salud, Medicina, Santa MartaDr. Carlos A. SariolMedicinaSanta Martahttps://www.mdpi.com/1999-4915/12/5/496VirusesBrathwaite Dick, O.; San Martín, J.L.; Montoya, R.H.; del Diego, J.; Zambrano, B.; Dayan, G.H. The History of Dengue Outbreaks in the Americas. Am. J. Trop. Med. Hyg. 2012, 87, 584–593. [CrossRef]WHO. Dengue Guidelines for Diagnosis, Treatment, Prevention and Control; World Health Organization, Ed.; WHO: Geneva, Switzerland, 2009; pp. 1–160.Bhatt, S.; Gething, P.W.; Brady, O.J.; Messina, J.P.; Farlow, A.W.; Moyes, C.L.; Drake, J.M.; Brownstein, J.S.; Hoen, A.G.; Sankoh, O.; et al. The global distribution and burden of dengue. Nature 2013, 496, 504–507. [CrossRef]Twiddy, S.S.; Farrar, J.J.; Vinh Chau, N.; Wills, B.; Gould, E.A.; Gritsun, T.; Lloyd, G.; Holmes, E.C. Phylogenetic relationships and differential selection pressures among genotypes of dengue-2 virus. Virology 2002, 298, 63–72. [CrossRef]Lindenbach, B.D.; Murray, C.L.; Thiel, H.J.; Rice, C.M. Flaviviridae. In Fields in Virology, 6th ed.; Knipe, D.M., Ed.; Williams & Wilkins Lippincott: Philadelphia, PA, USA, 2013; pp. 712–746.Clyde, K.; Kyle, J.L.; Harris, E. Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J. Virol. 2006, 80, 11418–11431. [CrossRef]Filomatori, C.V.; Lodeiro, M.F.; Alvarez, D.E.; Samsa, M.M.; Pietrasanta, L.; Gamarnik, A.V. A 50 RNA element promotes dengue virus RNA synthesis on a circular genome. Genes Dev. 2006, 20, 2238–2249. [CrossRef]Khromykh, A.A.; Meka, H.; Guyatt, K.J.; Westaway, E.G. Essential role of cyclization sequences in flavivirus RNA replication. J. Virol. 2001, 75, 6719–6728. [CrossRef]Friebe, P.; Harris, E. Interplay of RNA elements in the dengue virus 50 and 30 ends required for viral RNA replication. J. Virol. 2010, 84, 6103–6118. [CrossRef]Clyde, K.; Barrera, J.; Harris, E. The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis. Virology 2008, 379, 314–323. [CrossRef]Gebhard, L.G.; Filomatori, C.V.; Gamarnik, A.V. Functional RNA Elements in the Dengue Virus Genome. Viruses 2011, 3, 1739–1756. [CrossRef]Villordo, S.M.; Carballeda, J.M.; Filomatori, C.V.; Gamarnik, A.V. RNA Structure Duplications and Flavivirus Host Adaptation. Trends Microbiol. 2016, 24, 270–283. [CrossRef]Paranjape, S.M.; Harris, E. Y box-binding protein-1 binds to the dengue virus 30 -untranslated region and mediates antiviral effects. J. Biol. Chem. 2007, 282, 30497–30508. [CrossRef] [PubMed]Li, Z.; Yu, M.; Zhang, H.; Wang, H.Y.; Wang, L.F. Improved rapid amplification of cDNA ends (RACE) for mapping both the 50 and 30 terminal sequences of paramyxovirus genomes. J. Virol. Methods 2005, 130, 154–156. [CrossRef] [PubMed]Miller, E. Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus. Methods Mol. Biol. 2016, 1373, 169–183. [CrossRef] [PubMed]Usme, J.; Gómez, A.; Gallego, J. Molecular detection and typing of dengue virus by RT-PCR and nested PCR using degenerated oligonucleotides. Rev. Salud Uninorte 2012, 28, 1–15.Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [CrossRef] [PubMedUsme-Ciro, J.A.; Mendez, J.A.; Laiton, K.D.; Paez, A. The relevance of dengue virus genotypes surveillance at country level before vaccine approval. Hum. Vaccines Immunother. 2014, 10, 2674–2678. [CrossRef]IUPAC Codes. Available online: https://www.bioinformatics.org/sms2/iupac.html (accessed on 2 April 2020).Chu, P.W.; Westaway, E.G. Replication strategy of Kunjin virus: Evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. Virology 1985, 140, 68–79. [CrossRef]Leitmeyer, K.C.; Vaughn, D.W.; Watts, D.M.; Salas, R.; Villalobos, I.; de Ramos, C.; Rico-Hesse, R. Dengue virus structural differences that correlate with pathogenesis. J. Virol. 1999, 73, 4738–4747. [CrossRef]Goo, L.; VanBlargan, L.A.; Dowd, K.A.; Diamond, M.S.; Pierson, T.C. A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis. PLoS Pathog. 2017, 13, e1006178. [CrossRef]Filomatori, C.V.; Carballeda, J.M.; Villordo, S.M.; Aguirre, S.; Pallarés, H.M.; Maestre, A.M.; Sánchez-Vargas, I.; Blair, C.D.; Fabri, C.; Morales, M.A.; et al. Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells. PLoS Pathog. 2017, 13, e1006265. [CrossRef]Sirigulpanit, W.; Kinney, R.M.; Leardkamolkarn, V. Substitution or deletion mutations between nt 54 and 70 in the 50 non-coding region of dengue type 2 virus produce variable effects on virus viability. J. Gen. Virol. 2007, 88, 1748–1752. [CrossRef]Tajima, S.; Nukui, Y.; Takasaki, T.; Kurane, I. Characterization of the variable region in the 30 non-translated region of dengue type 1 virus. J. Gen. Virol. 2007, 88, 2214–2222. [CrossRef] [PubMed]Alvarez, D.E.; De Lella Ezcurra, A.L.; Fucito, S.; Gamarnik, A.V. Role of RNA structures present at the 30UTR of dengue virus on translation, RNA synthesis, and viral replication. Virology 2005, 339, 200–212. [CrossRef] [PubMed]Drake, J.W.; Holland, J.J. Mutation rates among RNA viruses. Proc. Natl. Acad. Sci. USA 1999, 96, 13910–13913. [CrossRef] [PubMed]Ong, S.H.; Yip, J.T.; Chen, Y.L.; Liu,W.; Harun, S.; Lystiyaningsih, E.; Heriyanto, B.; Beckett, C.G.; Mitchell,W.P.; Hibberd, M.L.; et al. Periodic re-emergence of endemic strains with strong epidemic potential-a proposed explanation for the 2004 Indonesian dengue epidemic. Infect. Genet. Evol. 2008, 8, 191–204. [CrossRef] [PubMed]Sasmono, R.T.; Wahid, I.; Trimarsanto, H.; Yohan, B.; Wahyuni, S.; Hertanto, M.; Yusuf, I.; Mubin, H.; Ganda, I.J.; Latief, R.; et al. Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia. Infect. Genet. Evol. 2015, 32, 165–177. [CrossRef] [PubMed]Christenbury, J.G.; Aw, P.P.; Ong, S.H.; Schreiber, M.J.; Chow, A.; Gubler, D.J.; Vasudevan, S.G.; Ooi, E.E.; Hibberd, M.L. A method for full genome sequencing of all four serotypes of the dengue virus. J. Virol. Methods 2010, 169, 202–206. [CrossRef]Dash, P.K.; Sharma, S.; Soni, M.; Agarwal, A.; Sahni, A.K.; Parida, M. Complete genome sequencing and evolutionary phylogeography analysis of Indian isolates of Dengue virus type 1. Virus Res. 2015, 195, 124–134. [CrossRef]Wongsurawat, T.; Jenjaroenpun, P.; Taylor, M.K.; Lee, J.; Tolardo, A.L.; Parvathareddy, J.; Kandel, S.; Wadley, T.D.; Kaewnapan, B.; Athipanyasilp, N.; et al. Rapid Sequencing of Multiple RNA Viruses in Their Native Form. Front. Microbiol. 2019, 10, 260. [CrossRef]Tan, C.C.S.; Maurer-Stroh, S.; Wan, Y.; Sessions, O.M.; de Sessions, P.F. A novel method for the capture-based purification of whole viral native RNA genomes. AMB Express 2019, 9, 45. [CrossRef]Extremos de genomaVirus dengueRACE-PCRPoli(A) polimerasaGenome endsDengue virusRACE-PCRPoly(A) polymeraseEfficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus GenomeArtículohttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1http://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionAtribucióninfo:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2PublicationLICENSElicense.txtlicense.txttext/plain; charset=utf-84334https://repository.ucc.edu.co/bitstreams/0df08498-84b6-4a2a-934b-aa6786a80d9d/download3bce4f7ab09dfc588f126e1e36e98a45MD52ORIGINAL28. Efficient Method for Molecular Characterization of the 5 and 3 Ends of the Dengue Virus Genome. Rosales-Munar et al 2020.pdf28. Efficient Method for Molecular Characterization of the 5 and 3 Ends of the Dengue Virus Genome. 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Efficient Method for Molecular Characterization of the 5’ and 3’ Ends of the Dengue Virus Genome

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