Abstract
For diagnosis of positive-sense single-stranded RNA viruses, primers are usually raised against the sequence encoding capsid proteins, since structural proteins are more conserved. This chapter focuses on the design of primers for a group of novel viruses lacking a capsid, known as papaya Umbra-like viruses (unassigned genus) associated with Papaya Sticky Disease, which represent a threat to papaya production. Based on sequence alignments of a region encoding the RNA-dependent RNA Polymerase, universal primers to detect all the known viruses from four countries are proposed. The Forward universal primer can be used in combination with clade- and subclade-specific primers for rapid virus identification. We walk the reader through downloading sequences from nucleotide databases, doing sequence alignments and phylogenetic tree construction to identify conserved and variable regions as valid primer targets; we also show how to design and analyze the primers.
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Abbreviations
- bp:
-
Base pair
- BLAST:
-
Basic Local Alignment Search Tool
- CDS:
-
Coding sequence
- dsRNA:
-
Double-stranded RNA
- Indels:
-
Insertion–deletions
- kb:
-
Kilobases
- kcal/mol:
-
Kilocalorie per mole
- ML:
-
Maximum Likelihood
- NCBI:
-
National Center for Biotechnology Information
- NGS:
-
Next-Generation Sequencing
- NJ:
-
Neighbor-Joining
- PMeV:
-
Papaya meleira virus
- PMeV-1:
-
Papaya meleira virus 1
- PMeV-2:
-
Papaya meleira virus 2
- PMeV-Mx:
-
Papaya meleira virus-Mexican variant
- PpVQ:
-
Papaya virus Q
- PRSV-P:
-
Papaya ringspot virus type P
- PSD:
-
Papaya Sticky Disease
- PCR:
-
Polymerase chain reaction
- qPCR:
-
Quantitative PCR
- RdRP:
-
RNA-dependent RNA Polymerase
- (+) RNA:
-
Positive-sense single-stranded RNA
- RT-PCR:
-
Reverse transcription polymerase chain reaction
- T m :
-
Melting temperature
References
Dolja VV, Koonin EV (2018) Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer. Virus Res 244:36–52. https://doi.org/10.1016/j.virusres.2017.10.020
Roossinck MJ, Martin DJ, Roumagnac P (2015) Plant virus metagenomics: advances in virus discovery. Phytopathology 105:6, 716–727
Dolja VV, Krupovic M, Koonin EV (2020) Annu Rev Phytopathol 58:23–53. https://doi.org/10.1146/annurev-phyto-030320-041346
Rubio L, Galipienso L, Ferriol I (2020) Detection of plant viruses and disease management: relevance of genetic diversity and evolution. Front Plant Sci 11:1092. https://doi.org/10.3389/fpls.2020.01092
Alcala-Briseño RI, Casarrubias-Castillo K, López-Ley D et al (2020) Network analysis of the Papaya Orchard Virome from two agroecological regions of Chiapas, Mexico. mSystems 5:e00423-19. https://doi.org/10.1128/mSystems.00423-19
Garcia-Camara I, Tapia-Tussell R, Magaña-Alvarez A et al (2019) Empoasca papayae (Hemiptera: Cicadellidae)-mediated transmission of Papaya meleira virus-Mexican variant in Mexico. Plant Dis 103:2015–2023
Sá-Antunes TF, Maurastoni M, Madroñero LJ (2020) Battle of three: the curious case of papaya sticky disease. Plant Dis 104:2754–2763. https://doi.org/10.1094/PDIS-12-19-2622-FE
Campbell P (2018) New test to offer early detection of papaya sticky disease. Papaya Press. https://australianpapaya.com.au/website/wp-content/uploads/2018/05/PAPAYAPRESS-MAY.pdf
Maciel-Zambolim E, Kunieda-Alonso S, Matsuoka K, De Carvalho M, Zerbini F (2003) Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil. Plant Pathol 52:389–394
Abreu EFM, Daltro CB, Nogueira EOPL et al (2015) Sequence and genome organization of papaya meleira virus infecting papaya in Brazil. Arch Virol 160:3143–3147
Perez-Brito D, Tapia-Tussell R, Cortes-Velazquez A et al (2012) First report of papaya meleira virus (PMeV) in Mexico. Afr J Biotechnol 11:13564–13570
Abreu PMV, Piccin JG, Rodrigues SP et al (2012) Molecular diagnosis of Papaya meleira virus (PMeV) from leaf samples of Carica papaya L. using conventional and real-time RTPCR. J Virol Methods 180:11–17
Zamudio-Moreno E, Ramirez-Prado J, Moreno-Valenzuela O et al (2015) Early diagnosis of a Mexican variant of Papaya meleira virus (PMeV-Mx) by RT-PCR. Genet Mol Res 14:1145–1154
Quito-Avila DF, Alvarez RA, Ibarra MA et al (2015) Detection and partial genome sequence of a new umbra-like virus of papaya discovered in Ecuador. Eur J Plant Pathol 143:199–204
Sa Antunes TFS, Amaral RJV, Ventura JA et al (2016) The dsRNA virus papaya meleira virus and an ssRNA virus are associated with papaya sticky disease. PLoS One 11:e01552
Tapia-Tussell R, Magaña-Alvarez A, Cortes-Velazquez A et al (2015) Seed transmission of Papaya meleira virus in papaya (Carica papaya) cv. Maradol. Plant Pathol 64:272–275
Resource Coordinators NCBI (2016) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 44:D7–D19. https://doi.org/10.1093/nar/gkv1290
Zhang Z, Schwartz S, Wagner L et al (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. https://doi.org/10.1089/10665270050081478
Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20:1160–1166. https://doi.org/10.1093/bib/bbx108
Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13(1):134. https://doi.org/10.1186/1471-2105-13-134
Karsch-Mizrachi I, Takagi T, Cochrane G & International Nucleotide Sequence Database Collaboration (2018) The international nucleotide sequence database collaboration. Nucleic Acids Res 46:D48–D51. https://doi.org/10.1093/nar/gkx1097
Sievers F, Higgins DG (2021) The clustal omega multiple alignment package. In: Katoh K (ed) Multiple sequence alignment. Methods in molecular biology, vol 2231. Humana Press, New York, NY, pp 3–16. https://doi.org/10.1007/978-1-0716-1036-7_1
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340
Roshan U, Livesay DR (2006) Probalign: multiple sequence alignment using partition function posterior probabilities. Bioinformat 22:2715–2721. https://doi.org/10.1093/bioinformatics/btl472
Löytynoja A (2014) Phylogeny-aware alignment with PRANK. In: Russel D (ed) Multiple sequence alignment methods. Methods in molecular biology (Methods and protocols), vol 1079. Humana Press, Totowa, NJ, pp 155–170. https://doi.org/10.1007/978-1-62703-646-7_10
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Sokal RR, Michener CD (1958) A statistical method for evaluating systematic relationships. Univ Kans Sci Bull 38:1409–1438
Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 8:1586–1591. https://doi.org/10.1093/molbev/msm088
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. https://doi.org/10.1093/sysbio/syq010
Jukes TH, Cantor CR, Munro HN, Allison JB (1969) Evolution of protein molecules. In: Mammalian protein metabolism. Academic, New York
Acknowledgments
This work was funded by the CONACYT research grant A1-S-19850 to L.L.O.
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Ramirez-Prado, J.H., Lopez-Ochoa, L.A. (2022). Universal Primers for Detection of Novel Plant Capsid-Less Viruses: Papaya Umbra-like Viruses as Example. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 2392. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1799-1_15
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DOI: https://doi.org/10.1007/978-1-0716-1799-1_15
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