{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:28:27Z","timestamp":1785432507319,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T00:00:00Z","timestamp":1777420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Ministero della Salute","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/00789fa95","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>West Nile virus (WNV) diagnosis relies on nucleic acid amplifications, but these techniques do not discriminate between infectious and non-infectious viral particles. This limitation can be bypassed by using a genome-binding dye (PMAxx) that is unable to cross membranes and can only bind to the genomes of non-intact (i.e., non-infectious) viral particles. This study evaluated a workflow combining PMAxx treatment with digital PCR to improve the molecular discrimination of intact WNV particles. Fifty-five samples (35 plasma, 20 urine) from 41 patients with WNV fever (WNF) or WNV neuroinvasive disease (WNND) were analyzed. Samples were tested with\/without PMAxx treatment. Overall, PMAxx treatment resulted in a significant reduction in detectable viral RNA (median reduction: 1.0 Log copies\/mL; p &lt; 0.0001), indicating that a substantial fraction of RNA detected by standard methods originated from non-infectious particles. This reduction was more visible in urine (1.8 Log copies\/mL) than in plasma (0.4 Log copies\/mL), suggesting a higher proportion of degraded viral particles or free RNA in urine. Stratification by clinical presentation showed significant reductions in both WNF and WNND patients, with no significant differences between groups. This approach may represent a valuable adjunct for improving diagnostic interpretation and epidemiological assessment of WNV infection, particularly in matrices characterized by prolonged RNA persistence.<\/jats:p>","DOI":"10.3390\/ijms27094004","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T08:03:47Z","timestamp":1777536227000},"page":"4004","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Molecular Differentiation of Intact West Nile Virus Using a PMAxx\u2122-Enabled Digital PCR Workflow"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0955-2074","authenticated-orcid":false,"given":"Giuseppe","family":"Sberna","sequence":"first","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0640-8667","authenticated-orcid":false,"given":"Francesca","family":"Colavita","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cosmina","family":"Mija","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabiano","family":"Brillo","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1952-5117","authenticated-orcid":false,"given":"Fabrizio","family":"Carletti","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-9748-5340","authenticated-orcid":false,"given":"Silvia","family":"Cammisa","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Flavia","family":"Smoquina","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7489-5271","authenticated-orcid":false,"given":"Fabrizio","family":"Maggi","sequence":"additional","affiliation":[{"name":"National Institute for Infectious Diseases Lazzaro Spallanzani\u2014IRCCS, 00149 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1001\/jama.2013.8042","article-title":"West Nile virus: Review of the literature","volume":"310","author":"Petersen","year":"2013","journal-title":"JAMA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S1474-4422(07)70030-3","article-title":"West Nile virus","volume":"6","author":"Kramer","year":"2007","journal-title":"Lancet Neurol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"606","DOI":"10.3390\/v6020606","article-title":"Clinical manifestations and outcomes of West Nile virus infection","volume":"6","author":"Sejvar","year":"2014","journal-title":"Viruses"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2500634","DOI":"10.2807\/1560-7917.ES.2025.30.35.2500634","article-title":"Outbreak of autochthonous West Nile virus infection in Lazio region, Italy, July to August 2025: Preliminary investigation","volume":"30","author":"Mussetto","year":"2025","journal-title":"Euro Surveill."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4066","DOI":"10.1128\/JCM.38.11.4066-4071.2000","article-title":"Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan RT-PCR assay","volume":"38","author":"Lanciotti","year":"2000","journal-title":"J. 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