{"id":20183,"date":"2021-04-07T12:47:18","date_gmt":"2021-04-07T12:47:18","guid":{"rendered":"https:\/\/evaggelatos.com\/?p=20183"},"modified":"2021-04-08T09:15:22","modified_gmt":"2021-04-08T09:15:22","slug":"%ce%b7-%ce%bc%ce%b5%ce%b8%ce%bf%cf%84%cf%81%ce%b5%ce%be%ce%ac%cf%84%ce%b7-%ce%b1%ce%bd%ce%b1%cf%83%cf%84%ce%ad%ce%bb%ce%bb%ce%b5%ce%b9-%cf%84%ce%b7%ce%bd-%ce%b1%ce%bd%cf%84%ce%b9%ce%b3%cf%81%ce%b1","status":"publish","type":"post","link":"https:\/\/evaggelatos.com\/?p=20183","title":{"rendered":"\u0397 \u03bc\u03b5\u03b8\u03bf\u03c4\u03c1\u03b5\u03be\u03ac\u03c4\u03b7 \u03b1\u03bd\u03b1\u03c3\u03c4\u03ad\u03bb\u03bb\u03b5\u03b9 \u03c4\u03b7\u03bd \u03b1\u03bd\u03c4\u03b9\u03b3\u03c1\u03b1\u03c6\u03ae \u03c4\u03bf\u03c5 SARS\u2010CoV\u20102 virus"},"content":{"rendered":"<div class=\"article-citation\">\n<div class=\"citation\">\n<h1 class=\"citation__title\">Methotrexate inhibits SARS\u2010CoV\u20102 virus replication \u201cin vitro\u201d<\/h1>\n<div class=\"loa-wrapper loa-authors hidden-xs\">\n<div id=\"sb-1\" class=\"accordion\">\n<div class=\"accordion-tabbed\">\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a4_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Caruso%2C+Arnaldo\" data-id=\"a4\" data-db-target-for=\"a4\" aria-controls=\"a4\" aria-haspopup=\"true\">Arnaldo Caruso<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a5_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Caccuri%2C+Francesca\" data-id=\"a5\" data-db-target-for=\"a5\" aria-controls=\"a5\" aria-haspopup=\"true\">Francesca Caccuri<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a6_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Bugatti%2C+Antonella\" data-id=\"a6\" data-db-target-for=\"a6\" aria-controls=\"a6\" aria-haspopup=\"true\">Antonella Bugatti<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a7_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Zani%2C+Alberto\" data-id=\"a7\" data-db-target-for=\"a7\" aria-controls=\"a7\" aria-haspopup=\"true\">Alberto Zani<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a8_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Vanoni%2C+Marco\" data-id=\"a8\" data-db-target-for=\"a8\" aria-controls=\"a8\" aria-haspopup=\"true\">Marco Vanoni<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a9_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Bonfanti%2C+Paolo\" data-id=\"a9\" data-db-target-for=\"a9\" aria-controls=\"a9\" aria-haspopup=\"true\">Paolo Bonfanti<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a10_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Cazzaniga%2C+Marina+E\" data-id=\"a10\" data-db-target-for=\"a10\" aria-controls=\"a10\" aria-haspopup=\"true\">Marina E. Cazzaniga<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a11_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Perno%2C+Carlo+F\" data-id=\"a11\" data-db-target-for=\"a11\" aria-controls=\"a11\" aria-haspopup=\"true\">Carlo F. Perno<\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a12_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Messa%2C+Cristina\" data-id=\"a12\" data-db-target-for=\"a12\" aria-controls=\"a12\" aria-haspopup=\"true\">Cristina Messa<i class=\"icon-mail_outline\" aria-hidden=\"true\"><\/i><\/a><\/div>\n<div class=\"accordion-tabbed__tab-mobile accordion__closed\"><a id=\"a13_Ctrl\" class=\"author-name accordion-tabbed__control\" role=\"button\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/doSearch?ContribAuthorStored=Alberghina%2C+Lilia\" data-id=\"a13\" data-db-target-for=\"a13\" aria-controls=\"a13\" aria-haspopup=\"true\">Lilia Alberghina<\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n<div class=\"epub-sections\">\n<div class=\"epub-section\"><span class=\"epub-state\">First published: <\/span><span class=\"epub-date\">14 September 2020<\/span><\/div>\n<div class=\"epub-section\"><a class=\"epub-doi\" href=\"https:\/\/doi.org\/10.1002\/jmv.26512\" aria-label=\"Digital Object Identifier\">https:\/\/doi.org\/10.1002\/jmv.26512<\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n<div class=\"fixedCoolBar\">\n<nav class=\"coolBar stickybar js--fixed\" aria-label=\"Article navigation and tools\">\n<div class=\"stickybar__wrapper coolBar__wrapper clearfix\">\n<div class=\"rlist coolBar__zone\">\n<div class=\"coolBar__section\"><\/div>\n<\/div>\n<div class=\"coolBar__second rlist\">\n<div class=\"relative\"><\/div>\n<\/div>\n<\/div>\n<\/nav>\n<\/div>\n<div id=\"accessDenialslot\" class=\"accessDenialslot\"><\/div>\n<div class=\"article__body \">\n<article>\n<div class=\"abstract-group\">\n<section id=\"section-1-en\" class=\"article-section article-section__abstract\" lang=\"en\" data-lang=\"en\">\n<h2 id=\"d95669569\" class=\"article-section__header section__title main abstractlang_en main\">Abstract<\/h2>\n<div class=\"article-section__content en main\">\n<p>In early 2020 the new respiratory syndrome COVID\u201019 (caused by the zoonotic SARS\u2010CoV\u20102 virus) spread like a pandemic, starting from Wuhan, China, causing severe economic depression. Despite some advances in drug treatments of medical complications in the later stages of the disease, the pandemic&#8217;s death toll is tragic, as\u00a0no vaccine or specific antiviral treatment is currently available. By using a systems approach, we identify the host\u2010encoded pathway, which provides ribonucleotides to viral RNA synthesis, as a possible target. We show that methotrexate, an FDA\u2010approved inhibitor of purine biosynthesis, potently inhibits viral RNA replication, viral protein synthesis, and virus release. The effective antiviral methotrexate concentrations are similar to those used for established human therapies using the same drug. Methotrexate should be most effective in patients at the earliest appearance of symptoms to effectively prevent viral replication, diffusion of the infection, and possibly fatal complications.<\/p>\n<\/div>\n<\/section>\n<section id=\"section-1-en\" class=\"article-section article-section__abstract\" lang=\"en\" data-lang=\"en\">\n<h2 id=\"d95669570\" class=\"article-section__header highlights abstractlang_en highlights\">Highlights<\/h2>\n<div class=\"article-section__content en highlights\">\n<p>&nbsp;<\/p>\n<ul class=\"unordered-list\">\n<li>The COVID\u201019 pandemic caused by the SARS\u2010CoV\u20102 virus is a global public health threat causing over 800000 deaths, millions of infected people, and severe economic depression.<\/li>\n<li>No vaccine or specific antiviral treatments are currently available.<\/li>\n<li>By inhibiting the host\u2010encoded pathway, which provides ribonucleotides to viral RNA synthesis, <strong>the FDA\u2010approved drug methotrexate efficiently blocks SARS\u2010CoV\u20102 replication.<\/strong><\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<\/div>\n<\/section>\n<\/div>\n<div class=\"pb-dropzone\" data-pb-dropzone=\"below-abstract-group\"><\/div>\n<section class=\"article-section article-section__full\">\n<section id=\"jmv26512-sec-0010\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0010-title\" class=\"article-section__title section__title section1\">1 INTRODUCTION<\/h2>\n<p>Due to the massive diffusion of the COVID\u201019 pandemic, the present worldwide health emergency, with over 845,000 deaths and millions of infected people, is severely disrupting social and economic activities. If no efficient treatment or specific vaccine is soon\u00a0available worldwide, the occurrence of a severe global recession, which would bring significant social and economic turmoil, is feared in\u00a0the future.<\/p>\n<p>COVID\u201019 is an infectious respiratory disease caused by SARS\u2010CoV\u20102, a new zoonotic virus that undergoes mutational variability.<sup><a id=\"jmv26512-bib-0001R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0001\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 1 - Nature Med\">1<\/a><\/sup> Inhalation of virus\u2010containing respiratory droplets and airborne transmission represents the most relevant infective routes.<sup><a id=\"jmv26512-bib-0002R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0002\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 2 - Proc Natl Acad Sci USA\">2<\/a><\/sup> The vast majority of infected people are asymptomatic or present mild health problems. Asymptomatic and symptomatic patients may present a comparable viral load, and both may be infective.<sup><a id=\"jmv26512-bib-0003R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0003\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 3 - Cell Death Dis\">3<\/a><\/sup> The median incubation period (from infection to symptom onset) is 5.1 days. Over 97% of symptomatic patients develop signs of disease within 11.5 days.<sup><a id=\"jmv26512-bib-0004R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0004\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 4\">4<\/a><\/sup><\/p>\n<p>If the invading SARS\u2010CoV\u20102 virus elicits a strong protective immune response, the patient shows non\u2010severe symptoms. If the immune system fails to eliminate the virus, the patient develops viral pneumonia symptoms, with dyspnea, which may lead to\u00a0severe and potentially lethal complications.<sup><a id=\"jmv26512-bib-0005R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0005\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 5 - JAMA\">5<\/a>&#8211;<a id=\"jmv26512-bib-0007R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0007\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 7 - Lancet\">7<\/a><\/sup> This more dangerous stage is characterized by a dysfunctional immune response that leads to\u00a0a cytokine storm, which predisposes to hemostatic abnormalities.<sup><a id=\"jmv26512-bib-0005R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0005\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 5 - JAMA\">5<\/a>&#8211;<a id=\"jmv26512-bib-0007R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0007\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 7 - Lancet\">7<\/a><\/sup> High levels of interleukin\u20106 (IL\u20106), together with significant levels of SARS\u2010CoV\u20102 RNA, are an index of a high lethality risk. In the most severe cases, acute respiratory distress syndrome (ARDS), often complicated by pulmonary thromboembolism, occurs finally, leading to death.<sup><a id=\"jmv26512-bib-0008R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0008\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 8 - Lancet\">8<\/a>, <a id=\"jmv26512-bib-0009R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0009\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 9 - Cell Death Differ\">9<\/a><\/sup> Extension of the viral infection to extrapulmonary districts<sup><a id=\"jmv26512-bib-0010R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0010\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 10 - Nat Med\">10<\/a><\/sup> may have a role in the diffused post\u2010COVID syndrome.<sup><a id=\"jmv26512-bib-0011R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0011\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 11 - JAMA\">11<\/a><\/sup><\/p>\n<p>Patients with mild symptoms receive mostly symptomatic treatments.<sup><a id=\"jmv26512-bib-0006R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0006\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 6 - Nat Rev Immunol\">6<\/a><\/sup> Patients in the severe stage receive anti\u2010inflammatory drugs, including low doses of steroids or drugs acting on the IL\u20106 axis.<sup><a id=\"jmv26512-bib-0012R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0012\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 12 - Int J Mol Sci\">12<\/a><\/sup>\u00a0As\u00a0the COVID\u201019 pandemic would be substantially constrained by a drug blocking the reproduction of viral particles, several studies are ongoing<sup><a id=\"jmv26512-bib-0012R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0012\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 12 - Int J Mol Sci\">12<\/a><\/sup> to reposition available drugs looking for their ability to interfere with essential viral functions.<\/p>\n<p>The novelty of the present report is provided by considering, in a system biology approach, not only the events of viral entrance and reproduction into lung type II pneumocytes but also their necessary collaboration with the human host cellular pathways, among which the one providing nucleotides required for the synthesis of viral RNA. <strong>The synthesis of purines (a component of nucleotides) is inhibited by methotrexate (4\u2010amino\u201010\u2010methyl folic acid, MTX),<sup><a id=\"jmv26512-bib-0013R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0013\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 13 - Int J Mol Sci\">13<\/a><\/sup> an FDA approved drug, used to treat several cancer types and, at lower doses, autoimmune diseases.<\/strong> Herein, we present the first round of experiments &#8220;in vitro,&#8221; which clearly show that MTX blocks, with high efficiency, SARS\u2010CoV\u20102 replication in the post\u2010entry phase.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0020\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0020-title\" class=\"article-section__title section__title section1\">2 MATERIALS AND METHODS<\/h2>\n<section id=\"jmv26512-sec-0030\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0030-title\" class=\"article-section__sub-title section2\">2.1 Cells<\/h3>\n<p>African green monkey kidney Vero E6 cell line was obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM; Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo\u2010Fisher Scientific) at 37\u00b0C in a humidified atmosphere of 5% CO<sub>2<\/sub>.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0040\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0040-title\" class=\"article-section__sub-title section2\">2.2 Virus<\/h3>\n<p>We successfully isolated SARS\u2010CoV\u20102 in Vero E6 cells from the nasopharyngeal swab sample of a COVID\u201019 patient. The identity of the strain was verified in Vero E6 cells by real\u2010time polymerase chain reaction (PCR) and by metagenomic sequencing, from which the reads mapped to nCoV\u20102019 (genomic data are available at EBI under study accession n. PRJEB38101). We propagated the clinical isolate in Vero E6 cells and determined the viral titer by a standard plaque assay. We performed infection experiments in a biosafety level\u20103 (BLS\u20103) laboratory at a multiplicity of infection (MOI) of 0.05 and 1.0.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0050\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0050-title\" class=\"article-section__sub-title section2\">2.3 Efficacy study of MTX<\/h3>\n<p>Vero E6 cells were seeded at a density of 5\u2009\u00d7\u200910<sup>4<\/sup> cells\/well in a 24\u2010well plate and treated with different doses of MTX (Sigma\u2010Aldrich). The efficacy of MTX was determined by CellTiter\u2010Glo (Promega) that measures the ATP levels and direct counting of the viable cell number after trypan\u2010blue staining.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0060\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0060-title\" class=\"article-section__sub-title section2\">2.4 Evaluation of antiviral efficacy of methotrexate<\/h3>\n<p>Vero E6 cells were infected for one h with the SARS\u2010CoV\u20102 isolate at an MOI of 0.05. Infection was carried out in DMEM medium without FBS. Then, after virus removal and washing with warm phosphate\u2010buffered saline (we cultured cells in a medium containing 2% FBS in the presence or the absence of MTX at the concentration of 25, 2.5, or 0.25\u2009\u03bcM. At 48\u2009h postinfection, both cells and supernatants were collected for further viral genome quantification analysis.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0070\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0070-title\" class=\"article-section__sub-title section2\">2.5 Viral RNA extraction and quantitative real\u2010time reverse\u2010transcription PCR (qRT\u2010PCR)<\/h3>\n<p>RNA was extracted from clarified cell culture supernatants (16,000<i>g<\/i>\u2009\u00d7\u200910\u2009min) and infected cells using a QIAamp Viral RNA Mini Kit and RNeasy Plus mini kit (Qiagen), respectively, according to the manufacturer&#8217;s instructions.<\/p>\n<p>RNA was eluted in 30\u2009\u03bcl of RNase\u2010free water and stored at \u201380\u00b0C till use. The qRT\u2010PCR was carried\u2010out following previously described procedures with minor modifications.<sup><a id=\"jmv26512-bib-0014R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0014\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 14 - Cell Res\">14<\/a><\/sup> Briefly, reverse transcription and amplification of the S gene were performed using the one\u2010step QuantiFast Sybr Green RT\u2010PCR mix (Qiagen) as follows: 50\u00b0C for 10\u2009min, 95\u00b0C for 5\u2009min; 95\u00b0C for 10\u2009s, 60\u00b0C for 30\u2009s\u00a0(40 cycles) (primers: RBD\u2010qF1: 5\u2019\u2010CAATGGTTTAACAGGCACAGG\u20103\u2019 and RBD\u2010qR1: 5\u2019\u2010CTCAAGTGTCTGTGGATCACG\u20103). A standard curve was generated by determination of copy numbers derived from serial dilutions (10<sup>3<\/sup>\u201310<sup>9<\/sup> copies) of a pGEM T\u2010easy vector (Promega) containing the receptor\u2010binding domain of the S gene (primers: RBD\u2010F: 5\u2019\u2010GCTGGATCCCCTAATATTACAAACTTGTGCC\u20103\u2019; RBD\u2010R: 5\u2019\u2010TGCCTCGAGCTCAAGTGTCTGTGGATCAC\u20103\u2019). Each quantification was run in triplicate.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0080\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0080-title\" class=\"article-section__sub-title section2\">2.6 Western blot analysis<\/h3>\n<p>Protein samples (30\u2009\u00b5g) obtained from lysis in RIPA buffer (Cell Signaling Technology, Danvers) of Vero E6 infected cells were separated on 10% sodium dodecyl sulphate\u2010polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride\u00a0membranes (Millipore, Sigma). After being blocked with 3% bovine serum albumin in tris\u00a0buffered\u00a0saline buffer containing 0.05% Tween 20, the blot was probed with a human serum (1:1000 dilution) containing IgG to the SARS\u2010CoV\u20102 nucleoprotein (NP) and with mouse anti\u2010human GAPDH monoclonal antibody (G\u20109;\u00a0Santa Cruz Biotechnology). The antigen\u2010antibody complexes were detected using peroxidase\u2010conjugated goat anti\u2010human or goat antimouse IgG (Sigma) and revealed using the enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology).<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0090\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0090-title\" class=\"article-section__sub-title section2\">2.7 Statistical analysis<\/h3>\n<p>Data were analyzed for statistical significance using the one\u2010way analysis of variance, and the Bonferroni post test was used to compare data. Differences were considered significant when <i>p<\/i>\u2009&lt;\u2009.05. Statistical tests were performed using GraphPad Prism 8.<\/p>\n<\/section>\n<\/section>\n<section id=\"jmv26512-sec-0100\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0100-title\" class=\"article-section__title section__title section1\">3 RESULTS<\/h2>\n<section id=\"jmv26512-sec-0110\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0110-title\" class=\"article-section__sub-title section2\">3.1 Effects of methotrexate on Vero E6 cells growth and metabolism<\/h3>\n<p>At first, we assayed the effect of various concentrations of MTX on cell proliferation of Vero E6 cells, an established model system for SARS\u2010CoV\u20102 isolation and replication. After 48\u2009h of culture, 0.015\u2009\u00b5M MTX has a negligible effect on the extent of growth, assayed as the viable cell number detected by the Trypan Blue dye exclusion test. At this very low concentration, MTX inhibition may be competitively displaced by the intracellular substrate of DHFR. MTX concentrations ranging from 0.06 to 2.5\u2009\u00b5M have a potent inhibitory effect (around 45%) that becomes substantially stronger (85%) at MTX 25\u2009\u00b5M (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0001\">1A<\/a>). MTX\u2010treated cells display a normal surface\u2010adherent phenotype (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0001\">1B<\/a>).<\/p>\n<section class=\"article-section__inline-figure\">\n<figure id=\"jmv26512-fig-0001\" class=\"figure\"><a href=\"https:\/\/onlinelibrary.wiley.com\/cms\/asset\/e2495cf6-20c1-4b72-b23a-fda2cc70a393\/jmv26512-fig-0001-m.jpg\" target=\"_blank\" rel=\"noopener\"> <picture><source srcset=\"\/cms\/asset\/e2495cf6-20c1-4b72-b23a-fda2cc70a393\/jmv26512-fig-0001-m.jpg\" media=\"(min-width: 1650px)\" \/><img decoding=\"async\" class=\"figure__image\" title=\"image\" src=\"https:\/\/onlinelibrary.wiley.com\/cms\/asset\/c36bf417-3486-4fe8-bfe9-e9724b1698d7\/jmv26512-fig-0001-m.png\" alt=\"image\" data-lg-src=\"\/cms\/asset\/e2495cf6-20c1-4b72-b23a-fda2cc70a393\/jmv26512-fig-0001-m.jpg\" \/><\/picture><\/a><figcaption class=\"figure__caption\">\n<div class=\"figure__caption__header\">\n<p><strong class=\"figure__title\">Figure 1<\/strong><\/p>\n<div class=\"figure-extra\"><a class=\"open-figure-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#\">Open in figure viewer<\/a><a class=\"ppt-figure-link\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/downloadFigures?id=jmv26512-fig-0001&amp;doi=10.1002%2Fjmv.26512\"><i class=\"icon-Icon_Download\" aria-hidden=\"true\"><\/i>PowerPoint<\/a><\/div>\n<\/div>\n<div class=\"figure__caption figure__caption-text\">Determination of antimetabolic and proliferative efficacy of MTX. (A) Inhibition of cell growth after 48\u2009h of culture in the presence of MTX at different concentrations (25, 2.5, 0.25, 0.125, 0.06, 0.03, and 0.015\u2009\u03bcM). (B) 10\u00d7 bright\u2010field images of Vero E6 cells after incubation for 48\u2009h at 37\u00b0C with the indicated MTX concentrations with an initial cell density of 5\u2009\u00d7\u200910<sup>4<\/sup> cells\/well. (C) measurement of ATP by CellTiter\u2010Glo as a luminescent readout of cell viability. Red dots in (A) and (C) refer to cells not treated with MTX<\/div>\n<\/figcaption><\/figure>\n<\/section>\n<p>MTX severely inhibits the accumulation of cellular ATP (assayed with CellTiter\u2010Glo). For an extensive range of MTX concentrations, intracellular ATP levels&#8217; inhibition remains reasonably constant, ranging from 73% inhibition at MTX 0.06\u2009\u00b5M to 82% for 25\u2009\u00b5M (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0001\">1C<\/a>).<\/p>\n<p>Taken together, these data indicate that at concentrations between 0.06 and 2.5\u2009\u00b5M, the proliferative ability of MTX\u2010treated Vero E6 cells correlates with down\u2010modulation of their metabolic activity, with negligible effects on cell morphology and minor cytotoxicity.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0120\" class=\"article-section__sub-content\">\n<h3 id=\"jmv26512-sec-0120-title\" class=\"article-section__sub-title section2\">3.2 MTX potently inhibits SARS\u2010CoV\u20102 replication<\/h3>\n<p>We infected Vero E6 cells at either low MOI (MOI\u2009=\u20090.05) or high MOI (MOI\u2009=\u20091.0). Quantification of released RNA after 48\u2009h indicates that viral production is very similar at both MOIs (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0002\">2A<\/a>). Therefore, in the following experiments, we use an MOI of 0.05.<\/p>\n<section class=\"article-section__inline-figure\">\n<figure id=\"jmv26512-fig-0002\" class=\"figure\"><a href=\"https:\/\/onlinelibrary.wiley.com\/cms\/asset\/1b4c08ee-8b39-4790-8390-bc2f7c0237a7\/jmv26512-fig-0002-m.jpg\" target=\"_blank\" rel=\"noopener\"> <picture><source srcset=\"\/cms\/asset\/1b4c08ee-8b39-4790-8390-bc2f7c0237a7\/jmv26512-fig-0002-m.jpg\" media=\"(min-width: 1650px)\" \/><img decoding=\"async\" class=\"figure__image\" title=\"image\" src=\"https:\/\/onlinelibrary.wiley.com\/cms\/asset\/23af8414-56ce-4aea-97c1-e121fe87ec04\/jmv26512-fig-0002-m.png\" alt=\"image\" data-lg-src=\"\/cms\/asset\/1b4c08ee-8b39-4790-8390-bc2f7c0237a7\/jmv26512-fig-0002-m.jpg\" \/><\/picture><\/a><figcaption class=\"figure__caption\">\n<div class=\"figure__caption__header\">\n<p><strong class=\"figure__title\">Figure 2<\/strong><\/p>\n<div class=\"figure-extra\"><a class=\"open-figure-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#\">Open in figure viewer<\/a><a class=\"ppt-figure-link\" href=\"https:\/\/onlinelibrary.wiley.com\/action\/downloadFigures?id=jmv26512-fig-0002&amp;doi=10.1002%2Fjmv.26512\"><i class=\"icon-Icon_Download\" aria-hidden=\"true\"><\/i>PowerPoint<\/a><\/div>\n<\/div>\n<div class=\"figure__caption figure__caption-text\">Antiviral activity of MTX. (A) Vero E6 cells were infected with SARS\u2010CoV\u20102 at a multiplicity of infection (MOI) of 0.05 or 1.0 for 1\u2009h at 37\u00b0C and then washed and cultured for 48\u2009h. Viral yield was quantitated in the cell supernatant by quantitative real\u2010time reverse\u2010transcription PCR (qRT\u2010PCR). At least three independent replicates were performed. Data are representative of two independent experiments with similar results. In (B)\u2013(E),<strong> Vero E6 cells were infected with SARS\u2010CoV\u20102 at an MOI of 0.05 in the absence or in the presence of different doses of MTX.<\/strong> (B) Cells were imaged with an optical microscope to detect typical SARS\u2010CoV\u20102\u2010induced cytolytic effects. (C) Viral yield was quantitated in the cell supernatant by qRT\u2010PCR. <strong>At least three independent replicates were performed. Data are representative of two independent experiments with similar results. (***<i>p<\/i>\u2009&lt;\u2009.001)<\/strong>. (D) Quantitation of SARS\u2010CoV\u20102 genomes at the intracellular level by qRT\u2010PCR. At least three independent replicates were performed. Data are representative of two independent experiments with similar results. (**<i>p<\/i>\u2009&lt;\u2009.01; ***<i>p<\/i>\u2009&lt;\u2009.001). (E) Nucleoprotein (NP) expression in infected cells was analyzed by Western blot (left panel). Densitometric analysis of Western blot results is shown in the right panel. Bars represent mean % inhibition of NP expression in cells treated with MTX at different concentrations, normalized to control SARS\u2010CoV\u20102\u2010infected cells<\/div>\n<\/figcaption><\/figure>\n<\/section>\n<p>Next, we assessed whether MTX could affect the replication of the SARS\u2010CoV\u20102 virus.<strong> Vero E6 cells were infected with a primary SARS\u2010CoV\u20102 strain isolated in Brescia, Italy, and one h later they were cultured in the absence or the presence of MTX at the concentrations of 25, 2.5, or 0.25\u2009\u00b5M. MTX efficiently inhibits viral replication<\/strong> (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0002\">2B\u2010E<\/a>). All MTX doses abolish the SARS\u2010CoV\u20102 cytolytic effects on Vero E6 cells, with only limited cytopathic effects detectable at the lowest micromolar dose of MTX used (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0002\">2B<\/a>). Quantification of viral RNA copy number in the cell culture supernatants confirms the potent inhibitory effect of MTX on viral particles&#8217; production, <strong>with a reduction of nearly 70\u2010fold at 25\u2009\u00b5M and about 20\u2010fold at 0.25\u2009\u00b5M, as compared with untreated infected cells<\/strong> (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0002\">2C<\/a>). qRT\u2010PCR quantification of intracellular SARS\u2010CoV\u20102 RNA in SARS\u2010CoV\u20102\u2010infected cells shows dose\u2010dependent inhibition of intracellular SARS\u2010CoV\u20102 genome expression, ranging from 40% to 70% at the tested MTX concentrations (from 0.25 to 25\u2009\u00b5M) compared with untreated cells (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0002\">2D<\/a>). Western blot with patient antisera<sup><a id=\"jmv26512-bib-0015R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0015\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 15\">15<\/a><\/sup> recognizing the virus NP\u00a0confirms MTX&#8217;s efficacy on virus replication. MTX induces a significant (approximate 60%) inhibition of NP accumulation (Figure\u00a0<a href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-fig-0002\">2E<\/a>). As\u00a0there are many copies of NP for each RNA molecule in the virion, the detected constant immunoreactive band, observed at all MTX concentrations, may mainly reflect the viral protein present at the beginning of the treatment, masking the robust dose\u2010dependent inhibition observed in viral RNA assays experiments.<\/p>\n<\/section>\n<\/section>\n<section id=\"jmv26512-sec-0130\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0130-title\" class=\"article-section__title section__title section1\">4 DISCUSSION<\/h2>\n<p>Our results indicate that MTX efficiently inhibits viral replication at the post\u2010entry stages of the SARS\u2010CoV\u20102 infection. Its dose\u2010dependent antiviral activity is extremely potent. Consistent with data reported for Zika Virus infection<sup><a id=\"jmv26512-bib-0016R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0016\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 16 - Viruses\">16<\/a><\/sup> and the established mechanism of action of MTX,<sup><a id=\"jmv26512-bib-0013R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0013\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 13 - Int J Mol Sci\">13<\/a><\/sup> these pieces of data indicate that <span style=\"font-size: 14pt;\"><strong>the cellular purine biosynthetic pathway is a reliable target to inhibit SARS\u2010CoV\u20102 replication. Accordingly, MTX should be most effective in patients at the earliest appearance of symptoms to prevent the synthesis of new viral RNA and, therefore, the formation of virus particles able to extend the infection to contiguous lung alveolar epithelium and extrapulmonary sites<sup><a id=\"jmv26512-bib-0010R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0010\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 10 - Nat Med\">10<\/a><\/sup> and to spread the infection to other people through virus\u2010containing droplet emission.<\/strong><\/span> As\u00a0viral RNA has been detected in non\u2010survivors until the point of death, a correlation between virus persistence and poor disease outcome has been proposed.<sup><a id=\"jmv26512-bib-0017R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0017\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 17 - Lancet\">17<\/a><\/sup><\/p>\n<p>In the first 24\u2009h following different routes of administration of<strong> 15\u2009mg MTX to rheumatoid arthritis patients, the drug&#8217;s plasma concentration remains between 0.1 and 1\u2009\u03bcM,<sup><a id=\"jmv26512-bib-0018R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0018\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 18 - Br J Clin Pharmacol\">18<\/a><\/sup> that is, within the effective range reported in our study (0.25\u20132.5\u2009\u03bcM).<\/strong> As\u00a0the metronomic, multiple administration of low MTX doses<sup><a id=\"jmv26512-bib-0019R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0019\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 19 - J Clin Oncol\">19<\/a><\/sup> may improve drug absorption, the effective doses reported here are compatible with the therapeutically effective (and well\u2010tolerated) MTX dosage employed in clinical use. These experiments could provide the basis for designing antiviral treatments that are compatible with patient assistance at home, monitored by family doctors under COVID hospitals&#8217; supervision.<\/p>\n<p>In rheumatoid arthritis and other inflammatory syndromes, the molecular target of MTX is not DHFR.<sup><a id=\"jmv26512-bib-0013R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0013\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 13 - Int J Mol Sci\">13<\/a><\/sup> MTX decreases the levels of interleukin 6 (IL\u20106) and soluble IL\u20102 receptor, the reduction in cytokine levels being paralleled by an improvement in clinical indices.<sup><a id=\"jmv26512-bib-0020R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0020\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 20 - J Rheumatol\">20<\/a><\/sup> Therefore, in COVID\u201019 patients in a more advanced stage, the treatment with MTX is expected to decrease virion production and down\u2010regulate the IL\u20106 pathway, a strategy currently in use in different clinical trials.<sup><a id=\"jmv26512-bib-0012R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0012\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 12 - Int J Mol Sci\">12<\/a><\/sup><\/p>\n<\/section>\n<section id=\"jmv26512-sec-0140\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0140-title\" class=\"article-section__title section__title section1\">5 CONCLUSIONS<\/h2>\n<p>We show that MTX exerts a dose\u2010dependent, potent inhibition of SARS\u2010CoV\u20102 replication in model cell lines. As\u00a0it targets an enzyme essential for viral replication, MTX could be used as a first\u2010line intervention even against heavily mutated SARS\u2010CoV\u20102 variants or emerging epidemics caused by novel RNA viruses. <strong>The potential ability of MTX to down\u2010regulate the cytokine storm typical of later stages of the disease could contribute to making\u00a0oral MTX a new and effective antiviral treatment for the COVID\u201019 pandemic. MTX may also be useful in patients suffering from the post\u2010COVID syndrome,<sup><a id=\"jmv26512-bib-0011R\" class=\"bibLink tab-link\" href=\"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.26512#jmv26512-bib-0011\" data-tab=\"pane-pcw-references\" aria-label=\"Reference 11 - JAMA\">11<\/a><\/sup> in which systemic permanence and diffusion of the virus play\u00a0a role.<\/strong><\/p>\n<\/section>\n<section id=\"jmv26512-sec-0150\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0150-title\" class=\"article-section__title section__title section1\">CONFLICT OF INTERESTS<\/h2>\n<p>The authors declare that there is no conflict of interest.<\/p>\n<\/section>\n<section id=\"jmv26512-sec-0160\" class=\"article-section__content\">\n<h2 id=\"jmv26512-sec-0160-title\" class=\"article-section__title section__title section1\">AUTHOR CONTRIBUTIONS<\/h2>\n<p>Lilia Alberghina conceived the idea that led\u00a0to the systems identification of MTX as a potential anti\u2010COVID drug; Arnaldo Caruso planned the in vitro experiments. Francesca Caccuri isolated, expanded, and titrated the primary SARS\u2010CoV\u20102 isolate obtained in Brescia, Italy. Francesca Caccuri and Alberto Zani performed experiments on the antiviral activity of MTX by quantitating viral genome expression in cells and in cell culture supernatants. Antonella Bugatti performed experiments on the metabolic activity of MTX\u2010treated or MTX\u2010untreated cells and on the SARS\u2010CoV\u20102 NP antigen expression in cells by Western blot; Paolo Bonfanti, Carlo F.\u00a0Perno, and Marina E.\u00a0Cazzaniga evaluated clinical perspectives; Arnaldo Caruso and Francesca Caccuri analyzed in vitro data; Arnaldo Caruso, Marco Vanoni, and Lilia Alberghina collected data and wrote the manuscript; Cristina Messa and Carlo F. Perno assembled, coordinated and guided the multidisciplinary team; all the authors discussed, reviewed and approved the manuscript.<\/p>\n<p>\u03a0\u0397\u0393\u0397:<a title=\"Journal of Medical Virology homepage\" href=\"https:\/\/onlinelibrary.wiley.com\/journal\/10969071\">Journal of Medical Virology<\/a><\/p>\n<div class=\"pageBody article-page-tablet body-text\" data-widget-def=\"pageBody\" data-widget-id=\"72100436-7a82-49fc-933b-c6c9d8c42914\">\n<p>&nbsp;<\/p>\n<div class=\"page-body pagefulltext\">\n<div data-pb-dropzone=\"main\">\n<section data-widget-def=\"ux3-layout-widget\" data-widget-id=\"0f7bccba-8328-4dbd-a1fa-e84c526dd9ab\">\n<div class=\"container\">\n<div class=\"row\">\n<div class=\" col-md-12\">\n<div>\n<article class=\"hide_CASA\" data-figures=\"\/action\/ajaxShowFigures?widgetId=5cf4c79f-0ae9-4dc5-96ce-77f62de7ada9&amp;ajax=true&amp;doi=10.1002%2Fjmv.26512&amp;pbContext=%3Bpage%3Astring%3AArticle%2FChapter+View%3Bctype%3Astring%3AJournal+Content%3Bwebsite%3Awebsite%3Apericles%3Barticle%3Aarticle%3Adoi%5C%3A10.1002%2Fjmv.26512%3Bissue%3Aissue%3Adoi%5C%3A10.1002%2Fjmv.v93.3%3Bjournal%3Ajournal%3A10969071%3Bwgroup%3Astring%3APublication+Websites%3BpageGroup%3Astring%3APublication+Pages%3BsubPage%3Astring%3AFull+Text%3BrequestedJournal%3Ajournal%3A10969071&amp;accordionHeadingWrapper=h2\" data-references=\"\/action\/ajaxShowEnhancedAbstract?widgetId=5cf4c79f-0ae9-4dc5-96ce-77f62de7ada9&amp;ajax=true&amp;doi=10.1002%2Fjmv.26512&amp;pbContext=%3Bpage%3Astring%3AArticle%2FChapter+View%3Bctype%3Astring%3AJournal+Content%3Bwebsite%3Awebsite%3Apericles%3Barticle%3Aarticle%3Adoi%5C%3A10.1002%2Fjmv.26512%3Bissue%3Aissue%3Adoi%5C%3A10.1002%2Fjmv.v93.3%3Bjournal%3Ajournal%3A10969071%3Bwgroup%3Astring%3APublication+Websites%3BpageGroup%3Astring%3APublication+Pages%3BsubPage%3Astring%3AFull+Text%3BrequestedJournal%3Ajournal%3A10969071&amp;accordionHeadingWrapper=h2\" data-related=\"\/action\/ajaxShowRecommended?widgetId=5cf4c79f-0ae9-4dc5-96ce-77f62de7ada9&amp;ajax=true&amp;doi=10.1002%2Fjmv.26512&amp;pbContext=%3Bpage%3Astring%3AArticle%2FChapter+View%3Bctype%3Astring%3AJournal+Content%3Bwebsite%3Awebsite%3Apericles%3Barticle%3Aarticle%3Adoi%5C%3A10.1002%2Fjmv.26512%3Bissue%3Aissue%3Adoi%5C%3A10.1002%2Fjmv.v93.3%3Bjournal%3Ajournal%3A10969071%3Bwgroup%3Astring%3APublication+Websites%3BpageGroup%3Astring%3APublication+Pages%3BsubPage%3Astring%3AFull+Text%3BrequestedJournal%3Ajournal%3A10969071&amp;accordionHeadingWrapper=h2&amp;showSubjects=true&amp;taxonomiesCodes=enter+your+taxonomy+codes+here&amp;displayCitedByLink=true&amp;displayAlmetricDropzone=true&amp;taxonomyUri=publication-features&amp;topicUri=hide-issue-metadata\" data-details=\"\/action\/ajaxShowPubInfo?widgetId=5cf4c79f-0ae9-4dc5-96ce-77f62de7ada9&amp;ajax=true&amp;doi=10.1002%2Fjmv.26512&amp;pbContext=%3Bpage%3Astring%3AArticle%2FChapter+View%3Bctype%3Astring%3AJournal+Content%3Bwebsite%3Awebsite%3Apericles%3Barticle%3Aarticle%3Adoi%5C%3A10.1002%2Fjmv.26512%3Bissue%3Aissue%3Adoi%5C%3A10.1002%2Fjmv.v93.3%3Bjournal%3Ajournal%3A10969071%3Bwgroup%3Astring%3APublication+Websites%3BpageGroup%3Astring%3APublication+Pages%3BsubPage%3Astring%3AFull+Text%3BrequestedJournal%3Ajournal%3A10969071&amp;accordionHeadingWrapper=h2&amp;showSubjects=true&amp;taxonomiesCodes=enter+your+taxonomy+codes+here&amp;displayCitedByLink=true&amp;displayAlmetricDropzone=true&amp;taxonomyUri=publication-features&amp;topicUri=hide-issue-metadata\" data-mathjax=\"https:\/\/cdnjs.cloudflare.com\/ajax\/libs\/mathjax\/2.7.5\/MathJax.js?config=TeX-AMS-MML_HTMLorMML\">\n<div class=\"row article-row\">\n<div id=\"article__content\" class=\"col-sm-12 col-md-8 col-lg-8 article__content article-row-left\" tabindex=\"-1\">\n<div class=\"article-citation\">\n<div class=\"citation\">\n<div class=\"pb-dropzone\" data-pb-dropzone=\"publicaitonContent-series-title\"><a class=\"citation--logo\" title=\"Journal of Medical Virology homepage\" href=\"https:\/\/onlinelibrary.wiley.com\/journal\/10969071\"><img decoding=\"async\" id=\"journal-banner-image\" class=\"journal-banner-image\" src=\"https:\/\/onlinelibrary.wiley.com\/pb-assets\/journal-banners\/10969071-1527767726743.jpg\" alt=\"Journal of Medical Virology\" \/><\/a><\/div>\n<div class=\"doi-access-container clearfix\"><\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/article>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/section>\n<\/div>\n<\/div>\n<p>&nbsp;<\/p>\n<\/div>\n<\/section>\n<\/section>\n<\/article>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Methotrexate inhibits SARS\u2010CoV\u20102 virus replication \u201cin vitro\u201d Arnaldo Caruso Francesca Caccuri Antonella Bugatti Alberto Zani Marco Vanoni Paolo Bonfanti Marina E. Cazzaniga Carlo F. Perno Cristina Messa Lilia Alberghina First published: 14 September 2020 https:\/\/doi.org\/10.1002\/jmv.26512 Abstract In early 2020 the new respiratory syndrome COVID\u201019 (caused by the zoonotic SARS\u2010CoV\u20102 virus) spread like a pandemic, starting &hellip; <\/p>\n<p><a class=\"more-link btn\" href=\"https:\/\/evaggelatos.com\/?p=20183\">\u03a3\u03c5\u03bd\u03ad\u03c7\u03b5\u03b9\u03b1 \u03b1\u03bd\u03ac\u03b3\u03bd\u03c9\u03c3\u03b7\u03c2<\/a><\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"class_list":["post-20183","post","type-post","status-publish","format-standard","hentry","category-uncategorized","item-wrap"],"_links":{"self":[{"href":"https:\/\/evaggelatos.com\/index.php?rest_route=\/wp\/v2\/posts\/20183","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/evaggelatos.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/evaggelatos.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/evaggelatos.com\/index.php?rest_route=\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/evaggelatos.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=20183"}],"version-history":[{"count":4,"href":"https:\/\/evaggelatos.com\/index.php?rest_route=\/wp\/v2\/posts\/20183\/revisions"}],"predecessor-version":[{"id":20211,"href":"https:\/\/evaggelatos.com\/index.php?rest_route=\/wp\/v2\/posts\/20183\/revisions\/20211"}],"wp:attachment":[{"href":"https:\/\/evaggelatos.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=20183"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/evaggelatos.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=20183"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/evaggelatos.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=20183"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}