PMID- 35980204 OWN - NLM STAT- MEDLINE DCOM- 20221028 LR - 20240205 IS - 2165-0497 (Electronic) IS - 2165-0497 (Linking) VI - 10 IP - 5 DP - 2022 Oct 26 TI - Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus. PG - e0237922 LID - 10.1128/spectrum.02379-22 [doi] LID - e02379-22 AB - Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 biology and the impact of antiviral therapeutics is a robust method to detect the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2-expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals is limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively) or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bioreporter-expressing rSARS-CoV-2 for the study of SARS-CoV-2 in vitro and in vivo. IMPORTANCE Despite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use in specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualized SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS). FAU - Chiem, Kevin AU - Chiem K AD - Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA. FAU - Park, Jun-Gyu AU - Park JG AD - Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA. FAU - Morales Vasquez, Desarey AU - Morales Vasquez D AD - Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA. FAU - Plemper, Richard K AU - Plemper RK AD - Center for Translational Antiviral Research, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA. FAU - Torrelles, Jordi B AU - Torrelles JB AD - Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA. FAU - Kobie, James J AU - Kobie JJ AD - Department of Medicine, Division of Infectious Diseases, University of Alabama at Birmingham, Birmingham, Alabama, USA. FAU - Walter, Mark R AU - Walter MR AD - Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA. FAU - Ye, Chengjin AU - Ye C AUID- ORCID: 0000-0002-1934-9494 AD - Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA. FAU - Martinez-Sobrido, Luis AU - Martinez-Sobrido L AUID- ORCID: 0000-0001-7084-0804 AD - Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA. LA - eng GR - R01 AI141607/AI/NIAID NIH HHS/United States GR - R01 AI141222/AI/NIAID NIH HHS/United States GR - R01 AI145332/AI/NIAID NIH HHS/United States GR - R01 AI142985/AI/NIAID NIH HHS/United States GR - R43 AI165089/AI/NIAID NIH HHS/United States GR - R01 AI161175/AI/NIAID NIH HHS/United States GR - R01 AI161363/AI/NIAID NIH HHS/United States GR - 75N93021C00014/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20220818 PL - United States TA - Microbiol Spectr JT - Microbiology spectrum JID - 101634614 RN - EC 3.4.17.23 (Angiotensin-Converting Enzyme 2) RN - 0 (Antiviral Agents) RN - EC 1.13.12.- (Luciferases) RN - 0 (Antibodies, Neutralizing) SB - IM MH - Cricetinae MH - Mice MH - Animals MH - Humans MH - *COVID-19 MH - Angiotensin-Converting Enzyme 2/genetics MH - SARS-CoV-2/genetics MH - Virus Replication MH - Antiviral Agents/pharmacology MH - Luciferases/genetics/pharmacology MH - Antibodies, Neutralizing MH - Mice, Transgenic PMC - PMC9603146 OTO - NOTNLM OT - SARS-CoV-2 OT - coronavirus OT - fluorescent OT - infection OT - luciferase OT - recombinant OT - reporter OT - transmission COIS- The authors declare a conflict of interest. C.Y. and L.M.-S. are co-inventors on a patent that includes claims related to reverse genetics approaches to generate recombinant SARS-CoV-2. EDAT- 2022/08/19 06:00 MHDA- 2022/10/29 06:00 PMCR- 2022/08/18 CRDT- 2022/08/18 09:04 PHST- 2022/08/19 06:00 [pubmed] PHST- 2022/10/29 06:00 [medline] PHST- 2022/08/18 09:04 [entrez] PHST- 2022/08/18 00:00 [pmc-release] AID - 02379-22 [pii] AID - spectrum.02379-22 [pii] AID - 10.1128/spectrum.02379-22 [doi] PST - ppublish SO - Microbiol Spectr. 2022 Oct 26;10(5):e0237922. doi: 10.1128/spectrum.02379-22. Epub 2022 Aug 18.