Recombinant porcine reproductive and respiratory syndrome virus expressing luciferase genes provide a new indication of viral propagation in both permissive and target cells Fei Gao a,b , Zehui Qu a , Liwei Li a , Lingxue Yu a , Yifeng Jiang a,b , Yanjun Zhou a , Shen Yang a , Hao Zheng a , Qinfeng Huang a , Wu Tong a,b , Guangzhi Tong a,b, ⁎ a Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China b Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China abstract article info Article history: Received 10 November 2015 Received in revised form 25 April 2016 Accepted 30 May 2016 Available online xxxx Porcine reproductive and respiratory syndrome virus (PRRSV) has a condensed single-stranded positive-sense RNA genome that contains several overlapping regions. The transcription regulatory sequence (TRS) is the im- portant cis-acting element participating in PRRSV discontinuous transcription process. Based on reverse genetic system of type 2 highly pathogenic PRRSV cell-passage attenuated strain pHuN4-F112, firefly luciferase or Renilla luciferase genes were inserted between ORF1b and ORF2. An extra TRS6 was embedded behind the foreign lucif- erase genes. pA-Fluc and pA-Rluc were constructed and successfully rescued in MARC-145 cells. The phenotyp- ical characteristics of the progeny virus were indistinguishable from those of vHuN4-F112 and were genetically stable for at least 25 cell passages. Mutant virus-infected cells were lysed at different time points to assess lucif- erase activities and measure foreign gene expression levels. The results showed identical variations in the lucif- erase activities of the recombinants in MARC-145 cells, indicating that they were suitable for monitoring viral propagation in PRRSV-permissive cell cultures. They were also used to infect pulmonary alveolar macrophages, which yielded similar variations in luciferase activities. Therefore, vA-Fluc and vA-Rluc present powerful new tools to monitor PRRSV propagation in both passaged and target cells. © 2016 Elsevier Ltd. All rights reserved. Keywords: Firefly luciferase Renilla luciferase Viral propagation Relative activity 1. Introduction Nidoviruses are the causative agents of a variety of infectious dis- eases, both in humans and livestock (Cavanagh, 1997; Drosten et al., 2003; Pasternak et al., 2006; Snijder, 2001; Snijder and Meulenberg, 1998), which include porcine reproductive and respiratory syndrome virus (PRRSV) (Cavanagh, 1997; Meulenberg, 2000; Meulenberg et al., 1993), a member of the family Arteriviridae, together with equine arter- itis virus (EAV), simian hemorrhagic fever virus, and lactate dehydroge- nase-elevating virus (Pasternak et al., 2006; Snijder, 2001; Snijder and Meulenberg, 1998). Since 2006, outbreaks of highly pathogenic PRRSV, the causative agent of PRRS, have overwhelmed the swine industry in China, resulting in tremendous economic losses (Brar et al., 2015; Leng et al., 2014; Lu et al., 2015; Wang et al., 2015a; Wang et al., 2015b). The PRRSV genome, which is ~ 15 kb in length, has a 5′-cap and 3′- poly (A) tail, and consists of at least 10 open reading frames (ORFs), flanked by the 5′-untranslated region (UTR) and the 3′-UTR. The ORFs include ORF1a, ORF1b, ORF2a, ORF2, ORF3, ORF4, ORF5a, ORF5, ORF6, and ORF7 (Johnson et al., 2011; Oh and Lee, 2012; Robinson et al., 2013; Sun et al., 2013; Sun et al., 2015). PRRSV has re- stricted cell tropism, as the primary target cells of PRRSV are pulmo- nary alveolar macrophages (PAMs). Monkey embryonic kidney epithelial cells (MARC-145) are a PRRSV-permissive cell line for nor- mal usage (Tian et al., 2012). Like other nidoviruses, PRRSV adopts quite distinct discontinuous transcription mechanisms, as compared with other single-stranded, positive-sense RNA viruses, to generate a 3′ co-terminal nested set of subgenomic mRNAs (sg mRNA) in order to express its structural proteins (Pasternak et al., 2006). PRRSV sg mRNAs also contain a common 5′-leader sequence, which is located at the 5′-end of the genome. During the process of discontinuous RNA transcription, the transcription regulatory sequence (TRS) plays a critical role by orchestrating base-paring interactions of the conserved hexanucleotide motif [5′-UCAAC(U/C)-3′] between the TRS body and TRS leader sequences during synthesis of minus-strand RNA (Pei et al., 2009; Sawicki and Sawicki, 1995; Zheng et al., 2010). The conserved motif and secondary structure formed by itself and its flanking sequence are necessary for sg mRNA transcription (Zheng et al., 2010; Zheng et al., 2014). Research in Veterinary Science 107 (2016) 132–140 ⁎ Corresponding author at: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai 200241, China. E-mail address: gztong@shvri.ac.cn (G. Tong). http://dx.doi.org/10.1016/j.rvsc.2016.05.014 0034-5288/© 2016 Elsevier Ltd. 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