title: Propidium monoazide (PMA) and ethidium bromide monoazide (EMA) improve DNA array and high-throughput sequencing of porcine reproductive and respiratory syndrome virus identification.
abstract: Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.
authors.affiliation: Groupe de recherche sur les maladies infectieuses du porc (GREMIP), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada; Swine and Poultry Infectious Diseases Research Center (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Bellehumeur
authors.affiliation: Institut de biologie intégrative et des systèmes (IBIS), Université Laval, Québec, QC, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Boyle
authors.affiliation: Swine and Poultry Infectious Diseases Research Center (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada; Institut de biologie intégrative et des systèmes (IBIS), Université Laval, Québec, QC, Canada; Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, QC, Canada; Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Québec, QC, Canada.
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Charette
authors.affiliation: Groupe de recherche sur les maladies infectieuses du porc (GREMIP), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada; Swine and Poultry Infectious Diseases Research Center (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada.
authors.forename: Josée
authors.initials: J
authors.lastname: Harel
authors.affiliation: Swine and Poultry Infectious Diseases Research Center (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada; Canadian Food Inspection Agency, Saint-Hyacinthe, QC, Canada.
authors.forename: Yvan
authors.initials: Y
authors.lastname: L'Homme
authors.affiliation: Swine and Poultry Infectious Diseases Research Center (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada; National Research Council Canada, Montréal, QC, Canada.
authors.forename: Luke
authors.initials: L
authors.lastname: Masson
authors.affiliation: Groupe de recherche sur les maladies infectieuses du porc (GREMIP), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada; Swine and Poultry Infectious Diseases Research Center (CRIPA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada. Electronic address: carl.a.gagnon@umontreal.ca.
authors.forename: Carl A
authors.initials: CA
authors.lastname: Gagnon
mesh_terms: D000818:Animals; D001386:Azides / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D008168:Lung / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis*; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology; D013552:Swine
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium; D004996:Ethidium
keywords: DNA array; EMA; Ethidium bromide monoazide; High-throughput sequencing; PMA; PRRSV; Porcine reproductive and respiratory syndrome virus; Propidium monoazide; Virus identification