{"id":522,"date":"2023-03-02T03:29:31","date_gmt":"2023-03-02T03:29:31","guid":{"rendered":"http:\/\/wmtc2006.com\/?p=522"},"modified":"2023-03-02T03:29:31","modified_gmt":"2023-03-02T03:29:31","slug":"the-mixtures-were-then-layered-onto-cells-in-96-well-plates","status":"publish","type":"post","link":"https:\/\/wmtc2006.com\/?p=522","title":{"rendered":"\ufeffThe mixtures were then layered onto cells in 96-well plates"},"content":{"rendered":"

\ufeffThe mixtures were then layered onto cells in 96-well plates. and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines and therapeutics. family [8]. It has five structural genes, which encode N (nucleocapsid), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large protein). The genome of VSV is simple and easy to manipulate [9]. VSV can tolerate and package foreign envelope proteins on its surface, which makes VSV an ideal tool for studying the function of heterologous glycoproteins from other viral pathogens as a form of recombinant virus [10C12]. In this report, we describe the generation and characterization of a replication-competent VSV virus that expresses SARS-CoV-2 spike (S) protein. The recombinant virus was able to infect SARS-CoV-2 susceptible cells. The infection could be neutralized using sera from COVID-19 convalescent patients and monoclonal antibodies targeting the S protein. Levels of neutralizing activity based on the replication-competent VSV system strongly correlated with levels from both pseudotype and live virus systems. Therefore, replication-competent rVSV is a promising tool to study SARS-CoV-2 entry process and immune responses to vaccines. Materials and methods Ethics statement The rVSV studies were conducted under biosafety level 2 (BSL2) conditions. Research with live SARS-CoV-2 was performed in a biosafety level 3 (BSL3) facility. Human serum samples from COVID-19 convalescent patients were obtained from the Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China. A written consent form was obtained from the patients before they participated in this study. Serum samples were incubated at 56C for 30?min to inactivate potential viruses. Cells, antibodies, and proteins Vero cells (American Type Culture Collection [ATCC], CCL-81), HEK293T (ATCC, CRL-3216), BHK-21 (ATCC, CCL-10), ST (ATCC, CRL-1746), PK15 (ATCC, CCL-33), A549 (ATCC, CCL-185), HeLa (ATCC, CCL-2), MDBK (ATCC, CCL-22), and Huh7.5 (human hepatocarcinoma cells) were maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillinCstreptomycin. All cells were incubated at 37C with 5% CO2. Cor-nuside Rabbit anti-SARS-CoV-2 S-RBD polyclonal antibody (Cat. 40150-RP01) and Cor-nuside<\/a> SARS-CoV-2 Spike RBD domain (Cat: 40592-V08B-B) were purchased from Sino Biological Inc. (Beijing, China). Construction, rescue, and characterization of FGF18<\/a> rVSV viruses The rVSV vector was designed, synthesized, and constructed as described previously [9]. The eGFP encoding sequences were added at nt62 to generate the rVSV-eGFP-G plasmid, which was used as the backbone for other plasmids. The humanized spike protein coding sequence of SARS-CoV-2 Wuhan-Hu-1 strain (GeneBank: “type”:”entrez-protein”,”attrs”:”text”:”YP_009724390.1″,”term_id”:”1796318598″,”term_text”:”YP_009724390.1″YP_009724390.1) was synthesized by Genewiz Suzhou and inserted between MluI and NotI sites into the rVSV-eGFP-G plasmid. The leader sequence of the S coding sequence was the original signal peptide. Primers used to amplify the spike gene were 5′-gtttccttgacacgcgtaccatgttcgtgttcctcgtgc-3 and 5′-gtgcagggcggccgctcaggtgtagtggagcttcacgc-3. The resulting backbone plasmid was named rVSV-eGFP-SARS-CoV-2, with the VSV glycoprotein coding sequence (3845C5380) being replaced by that of the SARS-CoV-2 spike protein. The rVSVs were rescued using a reverse genetics approach [13]. Briefly, HEK293T cells Cor-nuside were transfected with 1.6?g Cor-nuside of rVSV backbone plasmid and five supporting plasmids encoding T7 polymerase (8.1?g), N (1.286?g), P (639?ng), Cor-nuside M (169.9?ng), and L (169.9?ng) of VSV using the calcium phosphate method. Recovery of the viruses was determined by cytopathic effects and eGFP expression. Viruses in the supernatant were harvested and passaged on Vero cells to obtain virus stocks. The rVSVs were purified by one step ultracentrifugation at 35,000?rpm (SW41 rotor, Beckman, Fullerton, CA, USA) at 4C for 2?h. The pellets containing purified viral particles were resuspended with PBS. Expression of spike protein in infected cells and on purified viral particles was.<\/p>\n","protected":false},"excerpt":{"rendered":"

\ufeffThe mixtures were then layered onto cells in 96-well plates. and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[9],"tags":[],"class_list":["post-522","post","type-post","status-publish","format-standard","hentry","category-hydrolases"],"yoast_head":"\n\ufeffThe mixtures were then layered onto cells in 96-well plates - Discovery and characterization of Histamine-2 Receptor Antagonists<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/wmtc2006.com\/?p=522\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"\ufeffThe mixtures were then layered onto cells in 96-well plates - Discovery and characterization of Histamine-2 Receptor Antagonists\" \/>\n<meta property=\"og:description\" content=\"\ufeffThe mixtures were then layered onto cells in 96-well plates. and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines [...]\" \/>\n<meta property=\"og:url\" content=\"https:\/\/wmtc2006.com\/?p=522\" \/>\n<meta property=\"og:site_name\" content=\"Discovery and characterization of Histamine-2 Receptor Antagonists\" \/>\n<meta property=\"article:published_time\" content=\"2023-03-02T03:29:31+00:00\" \/>\n<meta name=\"author\" content=\"wpadmin\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"wpadmin\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"3 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"Article\",\"@id\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522#article\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522\"},\"author\":{\"name\":\"wpadmin\",\"@id\":\"http:\\\/\\\/wmtc2006.com\\\/#\\\/schema\\\/person\\\/5a1d916d8ed9c89b34ac45c96f81e776\"},\"headline\":\"\ufeffThe mixtures were then layered onto cells in 96-well plates\",\"datePublished\":\"2023-03-02T03:29:31+00:00\",\"mainEntityOfPage\":{\"@id\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522\"},\"wordCount\":609,\"articleSection\":[\"Hydrolases\"],\"inLanguage\":\"en-US\"},{\"@type\":\"WebPage\",\"@id\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522\",\"url\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522\",\"name\":\"\ufeffThe mixtures were then layered onto cells in 96-well plates - Discovery and characterization of Histamine-2 Receptor Antagonists\",\"isPartOf\":{\"@id\":\"http:\\\/\\\/wmtc2006.com\\\/#website\"},\"datePublished\":\"2023-03-02T03:29:31+00:00\",\"author\":{\"@id\":\"http:\\\/\\\/wmtc2006.com\\\/#\\\/schema\\\/person\\\/5a1d916d8ed9c89b34ac45c96f81e776\"},\"breadcrumb\":{\"@id\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522#breadcrumb\"},\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\\\/\\\/wmtc2006.com\\\/?p=522\"]}]},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\\\/\\\/wmtc2006.com\\\/?p=522#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Home\",\"item\":\"http:\\\/\\\/wmtc2006.com\\\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"\ufeffThe mixtures were then layered onto cells in 96-well plates\"}]},{\"@type\":\"WebSite\",\"@id\":\"http:\\\/\\\/wmtc2006.com\\\/#website\",\"url\":\"http:\\\/\\\/wmtc2006.com\\\/\",\"name\":\"Discovery and characterization of Histamine-2 Receptor Antagonists\",\"description\":\"Just another WordPress site\",\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"http:\\\/\\\/wmtc2006.com\\\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"en-US\"},{\"@type\":\"Person\",\"@id\":\"http:\\\/\\\/wmtc2006.com\\\/#\\\/schema\\\/person\\\/5a1d916d8ed9c89b34ac45c96f81e776\",\"name\":\"wpadmin\",\"image\":{\"@type\":\"ImageObject\",\"inLanguage\":\"en-US\",\"@id\":\"https:\\\/\\\/secure.gravatar.com\\\/avatar\\\/e57206ca88c051844b618224f501afdd8e9328b238000fd2f37bf30c03bcff14?s=96&d=mm&r=g\",\"url\":\"https:\\\/\\\/secure.gravatar.com\\\/avatar\\\/e57206ca88c051844b618224f501afdd8e9328b238000fd2f37bf30c03bcff14?s=96&d=mm&r=g\",\"contentUrl\":\"https:\\\/\\\/secure.gravatar.com\\\/avatar\\\/e57206ca88c051844b618224f501afdd8e9328b238000fd2f37bf30c03bcff14?s=96&d=mm&r=g\",\"caption\":\"wpadmin\"},\"sameAs\":[\"http:\\\/\\\/wmtc2006.com\"],\"url\":\"https:\\\/\\\/wmtc2006.com\\\/?author=1\"}]}<\/script>\n<!-- \/ Yoast SEO plugin. -->","yoast_head_json":{"title":"\ufeffThe mixtures were then layered onto cells in 96-well plates - Discovery and characterization of Histamine-2 Receptor Antagonists","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"https:\/\/wmtc2006.com\/?p=522","og_locale":"en_US","og_type":"article","og_title":"\ufeffThe mixtures were then layered onto cells in 96-well plates - Discovery and characterization of Histamine-2 Receptor Antagonists","og_description":"\ufeffThe mixtures were then layered onto cells in 96-well plates. and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines [...]","og_url":"https:\/\/wmtc2006.com\/?p=522","og_site_name":"Discovery and characterization of Histamine-2 Receptor Antagonists","article_published_time":"2023-03-02T03:29:31+00:00","author":"wpadmin","twitter_card":"summary_large_image","twitter_misc":{"Written by":"wpadmin","Est. reading time":"3 minutes"},"schema":{"@context":"https:\/\/schema.org","@graph":[{"@type":"Article","@id":"https:\/\/wmtc2006.com\/?p=522#article","isPartOf":{"@id":"https:\/\/wmtc2006.com\/?p=522"},"author":{"name":"wpadmin","@id":"http:\/\/wmtc2006.com\/#\/schema\/person\/5a1d916d8ed9c89b34ac45c96f81e776"},"headline":"\ufeffThe mixtures were then layered onto cells in 96-well plates","datePublished":"2023-03-02T03:29:31+00:00","mainEntityOfPage":{"@id":"https:\/\/wmtc2006.com\/?p=522"},"wordCount":609,"articleSection":["Hydrolases"],"inLanguage":"en-US"},{"@type":"WebPage","@id":"https:\/\/wmtc2006.com\/?p=522","url":"https:\/\/wmtc2006.com\/?p=522","name":"\ufeffThe mixtures were then layered onto cells in 96-well plates - Discovery and characterization of Histamine-2 Receptor Antagonists","isPartOf":{"@id":"http:\/\/wmtc2006.com\/#website"},"datePublished":"2023-03-02T03:29:31+00:00","author":{"@id":"http:\/\/wmtc2006.com\/#\/schema\/person\/5a1d916d8ed9c89b34ac45c96f81e776"},"breadcrumb":{"@id":"https:\/\/wmtc2006.com\/?p=522#breadcrumb"},"inLanguage":"en-US","potentialAction":[{"@type":"ReadAction","target":["https:\/\/wmtc2006.com\/?p=522"]}]},{"@type":"BreadcrumbList","@id":"https:\/\/wmtc2006.com\/?p=522#breadcrumb","itemListElement":[{"@type":"ListItem","position":1,"name":"Home","item":"http:\/\/wmtc2006.com\/"},{"@type":"ListItem","position":2,"name":"\ufeffThe mixtures were then layered onto cells in 96-well plates"}]},{"@type":"WebSite","@id":"http:\/\/wmtc2006.com\/#website","url":"http:\/\/wmtc2006.com\/","name":"Discovery and characterization of Histamine-2 Receptor Antagonists","description":"Just another WordPress site","potentialAction":[{"@type":"SearchAction","target":{"@type":"EntryPoint","urlTemplate":"http:\/\/wmtc2006.com\/?s={search_term_string}"},"query-input":{"@type":"PropertyValueSpecification","valueRequired":true,"valueName":"search_term_string"}}],"inLanguage":"en-US"},{"@type":"Person","@id":"http:\/\/wmtc2006.com\/#\/schema\/person\/5a1d916d8ed9c89b34ac45c96f81e776","name":"wpadmin","image":{"@type":"ImageObject","inLanguage":"en-US","@id":"https:\/\/secure.gravatar.com\/avatar\/e57206ca88c051844b618224f501afdd8e9328b238000fd2f37bf30c03bcff14?s=96&d=mm&r=g","url":"https:\/\/secure.gravatar.com\/avatar\/e57206ca88c051844b618224f501afdd8e9328b238000fd2f37bf30c03bcff14?s=96&d=mm&r=g","contentUrl":"https:\/\/secure.gravatar.com\/avatar\/e57206ca88c051844b618224f501afdd8e9328b238000fd2f37bf30c03bcff14?s=96&d=mm&r=g","caption":"wpadmin"},"sameAs":["http:\/\/wmtc2006.com"],"url":"https:\/\/wmtc2006.com\/?author=1"}]}},"_links":{"self":[{"href":"https:\/\/wmtc2006.com\/index.php?rest_route=\/wp\/v2\/posts\/522","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/wmtc2006.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/wmtc2006.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/wmtc2006.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/wmtc2006.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=522"}],"version-history":[{"count":1,"href":"https:\/\/wmtc2006.com\/index.php?rest_route=\/wp\/v2\/posts\/522\/revisions"}],"predecessor-version":[{"id":523,"href":"https:\/\/wmtc2006.com\/index.php?rest_route=\/wp\/v2\/posts\/522\/revisions\/523"}],"wp:attachment":[{"href":"https:\/\/wmtc2006.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=522"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/wmtc2006.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=522"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/wmtc2006.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=522"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}