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PPRV belongs to the family Paramyxoviridae in the order Mononegavirales, which also includes Rhabdoviridae, Nyamaviridae, Bornaviridae, Filoviridae, and Pneumoviridae (Amarasinghe et al. 2018). These viruses share a common feature in reverse genetics—their RNA is not an infectious unit before they are packaged by nucleoproteins and transcribed by polymerase and other required co-factors. The history of successful reverse genetics for these virus families dates from the first rescue of an RV (Schnell et al. 1994). The possibility of virus engineering by nucleotide insertion or deletion at will has revolutionized our molecular understanding of these viruses. In the following sections we will present an overview of recent innovations in design and new strategies that can serve as references for improving or establishing PPRV reverse genetic systems.
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The success of reverse genetics for non-segmented negative-sense RNA viruses is influenced by several factors, including the intact full-length cDNA of the virus to be rescued and its correct 5' and 3'-ends. T7 RNA polymerase activity tends to initiate from error-free templates at both 5' and 3'-ends of an RNA transcript. The so-called leader and trailer regions play a critical role in RNA transcription and virus replication (Yunus et al. 1999; Hanley et al. 2010) and thus these regions must be kept intact. In all reverse genetics systems analyzed thus far, mutations in both the leader and trailer sequences have shown a negative influence on RNA transcription and virus replication (Peeples and Collins 2000; Hanley et al. 2010). To avoid extraneous nucleotides from inserting in the 5' and 3'-ends of the RNA template during in vitro transcription, several methods have been described for yielding target RNAs with precise and defined ends (Pleiss et al. 1998; Helm et al. 1999; Kao et al. 1999; Avis et al. 2012). It is now believed that the possible heterologous 5' and 3'-ends during run-off transcription with T7 RNA polymerase can be controlled by self-cleaving trans and cis-acting ribozymes. Thus a hammerhead ribozyme (HHRZ) at the 5'-end and HDVRZ at the 3'-end have been introduced and are widely used with high efficiency (Been and Wickham 1997; Wichlacz et al. 2004; Avis et al. 2012; Szafraniec et al. 2012; Meyer and Masquida 2014). However, there is no common standard design applicable for all viruses and thus each system must be adapted and optimized for each particular virus. For example, insertion of HHRZ between the T7 promoter and start codon of the minigenome significantly improved rescue efficiency of the RV minigenome. This approach increased rescue efficiency by 100-fold for a full-length RV in combination with HDVRZ flanking the 3'-end of the antigenome (Ghanem et al. 2012). Surprisingly, the same approach showed poor performance with MV or Borna disease virus (BDV; Martin et al. 2006). Moreover, HHRZ sequences are obtained from different families of endonucleolytic ribozymes and may possess variations in cleavage efficiency among different sequences (Hammann et al. 2012).
With continued improvements in molecular biology, reverse genetics technology has progressed within the last two decades. In traditional reverse genetics, T7 RNA polymerase was widely used for negative-sense RNA viruses for which the majority of RNA transcription is accomplished in the cytoplasm (Edenborough and Marsh 2014). To improve the reverse genetics of these viruses, other alternative systems were developed, such as replacement of the T7 promoter by the human cytomegalovirus promoter (CMV), which is directly recognized by eukaryotic RNA polymerase (Inoue et al. 2003; Martin et al. 2006; Wang et al. 2015; Liu et al. 2017a). Following the same approach, Hu et al. (2012) developed a CMV promoter-based system and successfully rescued PPRV from full-length PPRV cDNA for the first time. In previous attempts, a similar approach was applied to improve RV recovery (Inoue et al. 2003). Although the T7 promoterbased system has been widely used to rescue negativesense RNA viruses, this system requires the use of an exogenous T7 RNA polymerase—usually from the vaccinia virus (vTF7-3)—which interferes with the viability of transfected cells (Nakatsu et al. 2006). This problem was overcome by adding vaccinia virus replication inhibitors, such as cytosine arabinoside and rifampicin, which increased the viability of transfected cells (Kato et al. 1996). Additionally, using mutant vaccinia virus (MVAT7) that grows in avian but not mammalian cells also avoided the vaccinia-induced cytotoxicity (Sutter et al. 1995; Wyatt et al. 1995). Efforts to develop helper virusfree systems with transgenic cell lines expressing T7 RNA polymerase have also been successful (Martin et al. 2006; Zheng et al. 2009; Li et al. 2011). Furthermore, the use of T7 RNA polymerase-expressing plasmids prior to or during co-transfection with the full-length antigenome and helper plasmids for generating infectious viruses has also been reported (Lowen et al. 2004; Witko et al. 2006; Freiberg et al. 2008; Jiang et al. 2009). Detailed information on the different systems used to rescue negative-sense RNA viruses in traditional reverse genetics have been extensively reviewed elsewhere (Radecke and Billeter 1997; Huemer et al. 2000; Edenborough and Marsh 2014).
There have been increasing reports of successful negative-sense RNA virus rescue using cellular inherent RNA polymerase Ⅰ (Pol Ⅰ) (Murakami et al. 2008; Suphaphiphat et al. 2010) and RNA polymerase Ⅱ (Pol Ⅱ)-driven systems under the control of a CMV promoter (Li et al. 2011; Wang et al. 2015). It was initially thought that the possibility of BDV rescue with RNA Pol Ⅰ and RNA Pol Ⅱ was due to its unique genetics and biological features of being the only member of Mononegavirales to exhibit nuclear replication (de la Torre 2002; Perez et al. 2003; Lipkin et al. 2011). A CMV promoter-driven RNA Pol Ⅱ system was first thought to function better with nuclear-replicating viruses; however, this hypothesis does not correlate with the recent rescue of other non-nuclear replicating viruses, such as PPRV and Newcastle disease virus (NDV), using the same promoter (Hu et al. 2012; Wang et al. 2015; Liu et al. 2017a). Other exceptions to this hypothesis include influenza viruses, MV, and Ebola virus. These cytoplasmic-based RNA transcription viruses have been rescued by cellular RNA Pol Ⅰ or RNA Pol Ⅱ (Edenborough and Marsh 2014). Despite these findings, further investigations are still needed on the utility of Pol Ⅱ for virus rescue of other negative-sense RNA viruses due to the possible splicing or polyadenylation of Pol Ⅱ transcripts (Martin et al. 2006).
The above examples leave room for hypothesizing alternative methods that can be explored for virus rescue of other non-nuclear transcription viruses including PPRV. Of note, the Pol Ⅰ and Pol Ⅱ-driven systems—under the control of a CMV promoter—can avoid helper virus-induced cytopathic effects (CPE) after cell transfection, which may be confused with the CPE induced by the rescued virus. Indeed, the CMV promoter was successfully used to rescue NDV, although it was shown to be less efficient in low virulent strains (Liu et al. 2017a). This low efficiency in rescuing low virulent strains was previously linked with system complexity involving several different-sized plasmids and the poor capacity of low virulent strains to be rescued, as described in the past for segmented influenza viruses (Neumann et al. 2005). In this regard, considering that all available methods for PPRV rescue employ the PPRV Nigeria75/1 strain (an attenuated vaccine strain) as template, we propose that a comparison of rescue efficiency of PPRV Nigeria75/1 strain with other virulent PPRV strains is necessary to assess possible limitations of virus rescue, which may be linked with low virulence of PPRV Nigeria75/1. However, due to biosecurity concerns, such comparative studies may require specialized laboratories that are licensed to handle live virulent PPRV.
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The Paramyxoviridae family includes enveloped viruses with linear non-segmented negative-sense RNAs that are approximately 15.5 kb in length. Their active polymerase is usually a complex of at least two components and the initiation of the viral cycle is a complex of virion-associated RdRp that generates a functional RNP complex. Although molecular techniques such as PCR, gene cloning, and the use of endonuclease restriction enzymes have become routine, generation of an intact, error-free, and stable clone of more than 15 kb is not always easy. The PPRV genome (15, 948 nts) is one of the longest paramyxoviruses after the recently described novel Feline morbillivirus (16, 050 nts; Woo et al. 2012; Marcacci et al. 2016). Such large genome sizes are relatively difficult for generating error-free, fulllength cDNA using conventional cloning techniques. To overcome these potential sequence errors, a synthetic approach was applied to generate error-free cDNA in a PPRV rescue assay (Muniraju et al. 2015). However, this approach is not 100% error-free in cases where wild-type strains are to be rescued due to possible errors that may exist in the sequences available in GenBank. These limitations linked to genome size and the ability to generate stable fulllength cDNA plasmids were previously reported during an attempt at establishing a one plasmid-based system to rescue NDV. The 33 kb pMG-725/GFP-NPL plasmid was unstable due to its size and was lost during passaging in new bacterial culture medium (Liu et al. 2017b).
Alternative methods for generating long template cDNAs, such as the faster and more economic methods used in clone screening (Guo et al. 2007), should thus be considered. Recently, fast screening of the clones after transformation was shown to be more advantageous compared with conventional restriction and PCR methods (Liu et al. 2017b). Similar innovations in facilitating long DNA cloning, such as enzyme-free cloning and the recently modified enzyme-free cloning protocols, are continuously being developed (Tillett and Neilan 1999; Blanusa et al. 2010; Matsumoto and Itoh 2011). A new strategy for rapid generation of complete cDNA clones of negative-sense RNA and recombinant viruses (Nolden et al. 2016) is based on direct cloning of cDNA copies of a complete virus genome into reverse genetics vectors through a technique called "linear-to-linear RedE/T" recombination. This convenient technique has been long used to manipulate molecules such as yeast, bacteria, P1-derived artificial chromosome vectors, and Escherichia coli chromosomes (Zhang et al. 1998). Techniques associated with this method have been shown to be appropriate for direct cloning of long DNA sequences (Fu et al. 2012; Wang et al. 2016) and may constitute alternative methods of reducing the usual conflicts between restriction sites on plasmid vectors and gene inserts. In addition, these alternative methods reduce errors when compared with longterm manipulations of genome sequences under conventional techniques.
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In virology, virus isolation is the core of any advancement in research. Isolation of a virus usually requires sensitive cells that allow viral growth and replication. Moreover, virus rescue from cDNA requires highly sensitive and permissive cell lines to allow effective replication and propagation of a rescued virus. In laboratory settings, several viruses of grazing animals, such as PPRV, sheep and goat pox virus (SPV), and Orf virus, usually exhibit poor growth in vitro and show difficulty adapting to commonly used cell lines or animal models (personal communication). Consequently, compared with other morbilliviruses, isolation of a field PPRV strain can be difficult due to the lack of sensitive cell lines or inadequate conditions of transportation and stocking of samples (Bhuiyan et al. 2014), especially in poor endemic countries. In this section, we will explore the available options for PPRV growth and replication in different cell lines to aid appropriate cell line choice for virus rescue assays.
Conventional cell lines that exhibit high performance in growth and propagation of PPRV are rarely available (Fakri et al. 2016). Fortunately, there is an increasing number of reports of engineered cell lines expressing known receptors such as SLAM and nectin-4 for other morbilliviruses that have been shown to be or may be more sensitive to supporting PPRV growth and replication (Hsu et al. 2001; Adombi et al. 2011; Muhlebach et al. 2011; Noyce and Richardson 2012; Noyce et al. 2013). In addition, a lymph node suspension cell line derived from cow showed higher sensitivity to PPRV in comparison with adherent Vero cells. The high titer of PPRV in the cowderived lymph node cell line was linked to the fact that lymphoid cells are major targets of different morbilliviruses (Mofrad et al. 2016). In a comparative study of potential permissive cell lines for PPRV growth and propagation, both BHK-21A and HEK 293T cells were able to produce PPRV titers (Silva et al. 2008). Research results have led to different opinions on the growth and replication of PPRV in various cell lines (Seki et al. 2003; Emikpe et al. 2009; Sannat et al. 2014; Muniraju et al. 2015; Fakri et al. 2016; Latif et al. 2018). Therefore, it is critical to select a highly sensitive cell line during PPRV rescue assay; engineered cell lines that exhibit high sensitivity for PPRV growth and replication are listed in Table 1.
Table 1. Samples of engineered sensitive cell lines for PPRV growth and replication.