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Previously, we generated a TK&amp;gE-deleted PRV (PRV<jats:sup>\u0394<\/jats:sup><jats:sup>TK&amp;gE\u2212AH02<\/jats:sup>) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4\u2009~\u20095 week-old PRV antibody negative piglets and provide rapid and 100\u2009% protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRV<jats:sup>TK&amp;gE\u2212AH02<\/jats:sup> may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)\u0394TK\/gE and PRV-S(UL35-36)\u0394TK\/gE compared to that of PRV-S(UL46-27)\u0394TK\/gE at 6\u00a0h post infection (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.05). Moreover, at 12\u00a0h post infection, cells infected with PRV-S(UL11-10)\u0394TK\/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)\u0394TK\/gE (<jats:italic>P<\/jats:italic>\u2009=\u20090.097) and PRV-S(UL46-27)\u0394TK\/gE (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRV<jats:sup>\u0394<\/jats:sup><jats:sup>TK&amp;gE\u2212AH02<\/jats:sup>).<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12917-021-02887-w","type":"journal-article","created":{"date-parts":[[2021,5,12]],"date-time":"2021-05-12T17:03:24Z","timestamp":1620839004000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Identification of four insertion sites for foreign genes in a pseudorabies virus vector"],"prefix":"10.1186","volume":"17","author":[{"given":"Chuanjian","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiqi","family":"Guo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rongli","family":"Guo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saisai","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yating","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengwei","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhisheng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yamei","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jichun","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,5,12]]},"reference":[{"issue":"1","key":"2887_CR1","first-page":"99","volume":"31","author":"TC Mettenleiter","year":"2000","unstructured":"Mettenleiter TC: Aujeszky\u2019s disease (pseudorabies) virus: the virus and molecular pathogenesis\u2013state of the art, June 1999. 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