[{"command":"settings","settings":{"pluralDelimiter":"\u0003","suppressDeprecationErrors":true,"entitySetting":{"type":"bibcite_reference","bundle":"journal_article","mapping":{"node":{"blog":"blog","class":"classes","events":"calendar","faq":"faq","link":"links","news":"news","page":"","person":"people","presentation":"presentations","software_project":"software","software_release":"software"},"bibcite_reference":{"*":"publications"},"paragraph":{"class_material":"classes"}},"viewmode":"teaser"},"user":{"uid":0,"permissionsHash":"7f1a171f8b0b5a764cab6d1b118f6329cfc3469f3145adbaf7b7495bbf60a5ea"}},"merge":true},{"command":"add_js","selector":"body","data":[{"src":"\/files\/js\/js_DOoUrEhS-bkVvWBnZGMbXVBHnh5Ov7QOD3C6k4k3980.js?scope=footer\u0026delta=0\u0026language=en\u0026theme=texasbio_eligendi\u0026include=eJzLL44vKE3KyUxOLMnMzyvWTykqLUjM0ctHFdbLzSxO1ikrzixJ1U_OzytJrSgpTcxxK83JCctMLQcAsjEbVw"}]},{"command":"insert","method":"replaceWith","selector":"#","data":"\n  \u003Cdiv class=\u0022field field--name-field-widget-title field--type-string field--label-visually_hidden field--mode-full\u0022\u003E\n    \u003Cdiv class=\u0022field--label sr-only\u0022\u003EWidget Title\u003C\/div\u003E\n              \u003Cdiv class=\u0022field--item\u0022\u003EViral factors involved in pathogenesis \u003C\/div\u003E\n          \u003C\/div\u003E\n\n\u003Cul  id=\u0022list-of-posts\u0022 more_link_id=\u0022node-readmore\u0022 class=\u0022publications view-teaser grid-view\u0022\u003E\n \u003Cli\u003E\n\u003Carticle class=\u0022bibcite-reference bibcite bibcite--teaser\u0022\u003E\n  \n  \n  \n\n  \u003Cdiv class=\u0022bibcite__content\u0022\u003E\n    \u003Cdiv class=\u0022bibcite-citation\u0022\u003E\n      \u003Cdiv class=\u0022csl-bib-body\u0022\u003E\u003Cdiv class=\u0022csl-entry\u0022\u003ENogales, Aitor, Luis Martinez-Sobrido, and Fernando Almazan. (2024) 2024. \u201c\u003Ca href=\u0022\/lms-lab\/publications\/reverse-genetics-zika-virus-using-bacterial-artificial-chromosome\u0022 hreflang=\u0022en\u0022\u003EReverse Genetics of Zika Virus Using a Bacterial Artificial Chromosome.\u003C\/a\u003E\u201d. \u003Ci\u003EMethods in Molecular Biology (Clifton, N.J.)\u003C\/i\u003E 2733: 185-206. https:\/\/doi.org\/10.1007\/978-1-0716-3533-9_12.\u003C\/div\u003E\u003C\/div\u003E\n  \u003C\/div\u003E\n\n  \u003Cdiv class=\u0022field field--name-publishers-version field--type-link field--label-visually_hidden field--mode-teaser\u0022\u003E\n    \u003Cdiv class=\u0022field--label sr-only\u0022\u003EPublisher\u0027s Version\u003C\/div\u003E\n              \u003Cdiv class=\u0022field--item\u0022\u003E\u003Ca href=\u0022https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/38064034\u0022\u003EPublisher\u0026#039;s Version\u003C\/a\u003E\u003C\/div\u003E\n          \u003C\/div\u003E\n                \u003Cdiv class=\u0022field--label field--abstract\u0022\u003E\n      \u003Cbutton class=\u0022btn-abstract collapsed\u0022 data-toggle=\u0022collapse\u0022 data-target=\u0022#collapseAbstract\u0022 aria-expanded=\u0022false\u0022 aria-controls=\u0022collapseAbstract\u0022\u003EAbstract \u003C\/button\u003E\n    \u003C\/div\u003E\n                  \u003Cdiv class=\u0022field--item abstract--content collapse\u0022 id=\u0022collapseAbstract\u0022 aria-expanded=\u0026quot;false\u0026quot;\u003E\u003Cp\u003EZika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has become a global threat to human health. Although ZIKV has been known to circulate for decades causing mild febrile illness, the more recent ZIKV outbreaks in the Americas and the Caribbean have been associated with severe neurological disorders and congenital abnormalities. The development of ZIKV reverse genetics approaches have allowed researchers to address key questions on the biology of ZIKV by genetically engineering infectious recombinant (r)ZIKV. This has resulted in a better understanding of the biology of ZIKV infections, including viral pathogenesis, molecular mechanisms of viral replication and transcription, or the interaction of viral and host factors, among others aspects. In addition, reverse genetics systems have facilitated the identification of anti-ZIKV compounds and the development of new prophylactic approaches to combat ZIKV infections. Different reverse genetics strategies have been implemented for the recovery of rZIKV. All these reverse genetics systems have faced and overcome multiple challenges, including the viral genome size, the toxicity of viral sequences in bacteria, etc. In this chapter we describe the generation of a ZIKV full-length complementary (c)DNA infectious clone based on the use of a bacterial artificial chromosome (BAC) and the experimental procedures for the successful recovery of rZIKV. Importantly, the protocol described in this chapter provides a powerful method for the generation of infectious clones of other flaviviruses with genomes that have stability problems during bacterial propagation.\u003C\/p\u003E\n\u003C\/div\u003E\n        \n  \u003C\/div\u003E\n\u003C\/article\u003E\n\u003C\/li\u003E\n \u003Cli\u003E\n\u003Carticle class=\u0022bibcite-reference bibcite bibcite--teaser\u0022\u003E\n  \n  \n  \n\n  \u003Cdiv class=\u0022bibcite__content\u0022\u003E\n    \u003Cdiv class=\u0022bibcite-citation\u0022\u003E\n      \u003Cdiv class=\u0022csl-bib-body\u0022\u003E\u003Cdiv class=\u0022csl-entry\u0022\u003E\u00c1vila-P\u00e9rez, Gin\u00e9s, Aitor Nogales, Ver\u00f3nica Mart\u00edn, Fernando Almazan, and Luis Martinez-Sobrido. (2018) 2018. \u201c\u003Ca href=\u0022\/lms-lab\/publications\/reverse-genetic-approaches-generation-recombinant-zika-virus\u0022 hreflang=\u0022en\u0022\u003EReverse Genetic Approaches for the Generation of Recombinant Zika Virus.\u003C\/a\u003E\u201d. \u003Ci\u003EViruses\u003C\/i\u003E 10 (11). https:\/\/doi.org\/10.3390\/v10110597.\u003C\/div\u003E\u003C\/div\u003E\n  \u003C\/div\u003E\n\n  \u003Cdiv class=\u0022field field--name-publishers-version field--type-link field--label-visually_hidden field--mode-teaser\u0022\u003E\n    \u003Cdiv class=\u0022field--label sr-only\u0022\u003EPublisher\u0027s Version\u003C\/div\u003E\n              \u003Cdiv class=\u0022field--item\u0022\u003E\u003Ca href=\u0022https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/30384426\u0022\u003EPublisher\u0026#039;s Version\u003C\/a\u003E\u003C\/div\u003E\n          \u003C\/div\u003E\n                \u003Cdiv class=\u0022field--label field--abstract\u0022\u003E\n      \u003Cbutton class=\u0022btn-abstract collapsed\u0022 data-toggle=\u0022collapse\u0022 data-target=\u0022#collapseAbstract\u0022 aria-expanded=\u0022false\u0022 aria-controls=\u0022collapseAbstract\u0022\u003EAbstract \u003C\/button\u003E\n    \u003C\/div\u003E\n                  \u003Cdiv class=\u0022field--item abstract--content collapse\u0022 id=\u0022collapseAbstract\u0022 aria-expanded=\u0026quot;false\u0026quot;\u003E\u003Cp\u003EZika virus (ZIKV) is an emergent mosquito-borne member of the \u003Ci\u003EFlaviviridae\u003C\/i\u003E family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barr\u00e9 syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.\u003C\/p\u003E\n\u003C\/div\u003E\n        \n  \u003C\/div\u003E\n\u003C\/article\u003E\n\u003C\/li\u003E\n \u003Cli\u003E\n\u003Carticle class=\u0022bibcite-reference bibcite bibcite--teaser\u0022\u003E\n  \n  \n  \n\n  \u003Cdiv class=\u0022bibcite__content\u0022\u003E\n    \u003Cdiv class=\u0022bibcite-citation\u0022\u003E\n      \u003Cdiv class=\u0022csl-bib-body\u0022\u003E\u003Cdiv class=\u0022csl-entry\u0022\u003E\u00c1vila-P\u00e9rez, Gin\u00e9s, Jun-Gyu Park, Aitor Nogales, Fernando Almazan, and Luis Martinez-Sobrido. (2019) 2019. \u201c\u003Ca href=\u0022\/lms-lab\/publications\/rescue-recombinant-zika-virus-bacterial-artificial-chromosome-cdna-clone\u0022 hreflang=\u0022en\u0022\u003ERescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome CDNA Clone.\u003C\/a\u003E\u201d. \u003Ci\u003EJournal of Visualized Experiments : JoVE\u003C\/i\u003E, no. 148. https:\/\/doi.org\/10.3791\/59537.\u003C\/div\u003E\u003C\/div\u003E\n  \u003C\/div\u003E\n\n  \u003Cdiv class=\u0022field field--name-publishers-version field--type-link field--label-visually_hidden field--mode-teaser\u0022\u003E\n    \u003Cdiv class=\u0022field--label sr-only\u0022\u003EPublisher\u0027s Version\u003C\/div\u003E\n              \u003Cdiv class=\u0022field--item\u0022\u003E\u003Ca href=\u0022https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/31282877\u0022\u003EPublisher\u0026#039;s Version\u003C\/a\u003E\u003C\/div\u003E\n          \u003C\/div\u003E\n                \u003Cdiv class=\u0022field--label field--abstract\u0022\u003E\n      \u003Cbutton class=\u0022btn-abstract collapsed\u0022 data-toggle=\u0022collapse\u0022 data-target=\u0022#collapseAbstract\u0022 aria-expanded=\u0022false\u0022 aria-controls=\u0022collapseAbstract\u0022\u003EAbstract \u003C\/button\u003E\n    \u003C\/div\u003E\n                  \u003Cdiv class=\u0022field--item abstract--content collapse\u0022 id=\u0022collapseAbstract\u0022 aria-expanded=\u0026quot;false\u0026quot;\u003E\u003Cp\u003EThe association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and\/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and\/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial\u00a0propagation.\u003C\/p\u003E\n\u003C\/div\u003E\n        \n  \u003C\/div\u003E\n\u003C\/article\u003E\n\u003C\/li\u003E\n\u003C\/ul\u003E\n  \u003Cnav role=\u0022navigation\u0022 aria-labelledby=\u0022pagination-for-flavivirus-viral-factors-involved-in-pathogenesis-\u0022 id=pager-heading\u003E\n    \u003Ch3 id=\u0022pagination-for-flavivirus-viral-factors-involved-in-pathogenesis-\u0022 class=\u0022visually-hidden\u0022\u003Epagination for flavivirus viral factors involved in pathogenesis \u003C\/h3\u003E\n    \u003Cul class=\u0022js-pager__items pager-mini\u0022\u003E\n            \u003Cli class=\u0022current\u0022\u003E\n        \u003Cspan aria-live=\u0022polite\u0022\u003E\n            \u003Cspan class=\u0022visually-hidden\u0022\u003EFlavivirus - viral factors involved in pathogenesis \u003C\/span\u003E\n            1 of 2\n          \u003C\/span\u003E      \u003C\/li\u003E\n              \u003Cli\u003E\n          \u003Ca href=\u0022\/lms-lab\/refresh-widget-content\/2920?page=1\u0026amp;selector=list-of-posts\u0026amp;pagerid=pager-heading\u0026amp;moreid=node-readmore\u0022 class=\u0022use-ajax next\u0022 rel=\u0022next\u0022\u003E\u003Cspan aria-hidden=\u0022true\u0022\u003E\u203a\u203a\u003C\/span\u003E\u003Cspan class=\u0022visually-hidden\u0022\u003ENext page\u003C\/span\u003E\u003C\/a\u003E\n        \u003C\/li\u003E\n          \u003C\/ul\u003E\n  \u003C\/nav\u003E\n\n\u003Cdiv class=\u0022node-readmore\u0022 id=node-readmore\u003E\u003C\/div\u003E\n","settings":null},{"command":"insert","method":"replaceWith","selector":"#","data":"","settings":null},{"command":"insert","method":"replaceWith","selector":"#","data":"","settings":null},{"command":"insert","method":"replaceWith","selector":".field--name-field-widget-title","data":"","settings":null}]