{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:00:31Z","timestamp":1785416431676,"version":"3.56.0"},"posted":{"date-parts":[[2025,7,15]]},"group-title":"Microbiology","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T00:00:00Z","timestamp":1752537600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100002590","name":"American Lung Association","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100002590","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100008688","name":"Center for Research and Advanced Studies of the National Polytechnic Institute","doi-asserted-by":"crossref","award":["75N93021C00014"],"award-info":[{"award-number":["75N93021C00014"]}],"id":[{"id":"10.13039\/501100008688","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIH","award":["5R01AI145332"],"award-info":[{"award-number":["5R01AI145332"]}]},{"DOI":"10.13039\/100012403","name":"Texas Biomedical Research Institute","doi-asserted-by":"crossref","award":["Texas Biomed Forum Award"],"award-info":[{"award-number":["Texas Biomed Forum Award"]}],"id":[{"id":"10.13039\/100012403","id-type":"DOI","asserted-by":"crossref"}]},{"award":["Texas Biomed Douglass Award"],"award-info":[{"award-number":["Texas Biomed Douglass Award"]}],"id":[{"id":"https:\/\/ror.org\/00wbskb04","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Influenza A viruses (IAV) infect a wide range of mammal and bird species and are responsible for seasonal outbreaks and occasional pandemics. Studying IAV requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV expressing fluorescent proteins have allowed monitoring viral infection in cultured cells and\n                  <jats:italic>ex vivo<\/jats:italic>\n                  in the organs of infected animals. However, fluorescent-expressing IAV are often attenuated and are not suited for the imaging of infected animals using\n                  <jats:italic>in vivo<\/jats:italic>\n                  imaging systems (IVIS). To overcome this limitation, we generated a recombinant A\/California\/04\/2009 H1N1 (pH1N1) expressing nanoluciferase (Nluc) from the non-structural (NS) viral segment (pH1N1-Nluc) that replicates efficiently\n                  <jats:italic>in vitro<\/jats:italic>\n                  , with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to identify neutralizing antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection\n                  <jats:italic>in vivo<\/jats:italic>\n                  and\n                  <jats:italic>ex vivo<\/jats:italic>\n                  following infection of mice with viral titers comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A\/Puerto Rico\/8\/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAV represent a valuable tool for\n                  <jats:italic>in vitro<\/jats:italic>\n                  and\n                  <jats:italic>in vivo<\/jats:italic>\n                  studies, including the identification of antivirals and\/or neutralizing antibodies, and to assess protective efficacy of vaccines.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    Despite the availability of recombinant influenza A viruses (IAV) expressing fluorescent reporter genes to track viral infections\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>ex vivo<\/jats:italic>\n                    , these viruses are often attenuated and do not represent the best option for imaging entire animals using\n                    <jats:italic>in vivo<\/jats:italic>\n                    imaging systems (IVIS). To solve this limitation, we generated recombinant influenza pandemic A\/California\/04\/2009 H1N1 expressing nanoluciferase (pH1N1-Nluc) from the viral non-structural (NS) segment and demonstrate how expression of Nluc does not affect viral replication\n                    <jats:italic>in vitro<\/jats:italic>\n                    or viral pathogenesis\n                    <jats:italic>in vivo.<\/jats:italic>\n                    Importantly, we demonstrate the feasibility of detecting pH1N1-Nluc infection\n                    <jats:italic>in vivo<\/jats:italic>\n                    using IVIS. We also validate the flexibility of this approach by generating an influenza A\/Puerto Rico\/8\/1934 H1N1 (PR8-Nluc). Our results support the feasibility of using these recombinant IAVs expressing Nluc from the NS segment for\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>in vivo<\/jats:italic>\n                    studies, including the identification of neutralizing antibodies and\/or antivirals, and to assess protective efficacy of vaccines.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.07.15.664884","type":"posted-content","created":{"date-parts":[[2025,7,16]],"date-time":"2025-07-16T03:55:15Z","timestamp":1752638115000},"source":"Crossref","is-referenced-by-count":0,"title":["Bioluminescent reporter influenza A viruses to track viral infections"],"prefix":"10.64898","author":[{"given":"Ramya S.","family":"Barre","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed","family":"Mostafa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Chiem","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca L.","family":"Pearl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9754-5765","authenticated-orcid":false,"given":"Roy N.","family":"Platt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasija","family":"Cupic","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0191-0204","authenticated-orcid":false,"given":"Timothy J. C.","family":"Anderson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulla G.","family":"Knaus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Randy A.","family":"Albrecht","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adolfo","family":"Garc\u00eda-Sastre","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8069-0272","authenticated-orcid":false,"given":"James J.","family":"Kobie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aitor","family":"Nogales","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7084-0804","authenticated-orcid":false,"given":"Luis","family":"Martinez-Sobrido","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025071713012848000_2025.07.15.664884v1.1","doi-asserted-by":"publisher","DOI":"10.3390\/v10090497"},{"key":"2025071713012848000_2025.07.15.664884v1.2","doi-asserted-by":"publisher","DOI":"10.1111\/irv.12470"},{"key":"2025071713012848000_2025.07.15.664884v1.3","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/j.pedhc.2010.05.001","article-title":"2009 H1N1 Influenza Pandemic","volume":"24","year":"2010","journal-title":"Journal of Pediatric Health Care"},{"key":"2025071713012848000_2025.07.15.664884v1.4","doi-asserted-by":"publisher","DOI":"10.4103\/0256-4947.59365"},{"key":"2025071713012848000_2025.07.15.664884v1.5","doi-asserted-by":"crossref","unstructured":"Bu L , Chen B , Xing L , Cai X , Liang S , Zhang L , Wang X , Song W . 2022. 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