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Newcastle Disease Virus | 59

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Figure 2.8 A schematic representation of the method used in reverse genetics to generate infectious Newcastle disease virus (NDV) from
a full-length cDNA clone. The plasmid containing the full-length cDNA of NDV genome will be transfected with the support plasmids (N, P
and L) to cells. Prior to the transfection, the cells can be infected with a recombinant vaccinia virus expressing T7 polymerase. Alternatively,
cells that constitutively express T7 polymerase can be used. Transcription from the T7 promoter (T7) will generate the correct 5′ end of the
antigenomic (+) RNA. The self-cleaving hepatitis delta virus ribozyme (HDR) sequence will generate the correct 3′ end of the antigenomic (+)
RNA. The (+) RNA synthesized by the T7 polymerase will be encapsidated by the recombinant N protein and associated with recombinant
L and P proteins to form the ribonucleoprotein (RNP) complex. The (+) RNP will be subsequently copied to form (–) RNP. The (–) RNP will be
used for transcription and replication. The newly synthesized (–) RNP will be packaged into NDV virion for budding out.

assembly of the cDNA fragments into a transcription vector is the Thirdly, poxviruses encode large number of modifying enzymes,
most popular method (Peeters et al., 1999; Römer-Oberdörfer et which may affect NDV genes. Lastly, MVA shuts down host cell
al., 1999; Krishnamurthy et al., 2000), ligation-independent clon- protein synthesis, which may cause inefficient rescue. Therefore,
ing using an In-Fusion® PCR (Clontech, USA) system has also an alternative rescue system that uses RNA polymerase II under
been used (Hu, H. et al., 2011; Li et al., 2012; Zhao et al., 2013). the control of the cytomegalovirus (CMV) promoter has been
To allow initiation of transcription, the T7 RNA polymerase pro- developed for NS-NSV (Bridgen, 2013). This system has been
moter sequence is added to the 5′-end of the antigenome cDNA. shown to be effective in recovery of a higher-titre NDV (Li et
An 84-nt hepatitis delta virus (HDV) autocleaving ribozyme al., 2011; Zhang et al., 2013; Wang et al., 2015; Chellappa et al.,
sequence is placed directly after the 3′-end of the cDNA to gener- 2017).
ate the precise 3′-end of the antigenome after transfection from Conventional rescue of NDV requires transfection of four
the plasmid. The HDV ribozyme sequence is then followed by a plasmids into the same cell and this method has been used suc-
T7 terminator sequence. Viral N, P, and L ORFs, flanked by the cessfully to recover NDV in most laboratories. However, to
T7 promoter and T7 terminator sequences are separately cloned improve the efficiency of rescue, a system using two plasmids
into a plasmid vector. Infection with MVA and transfection with has been developed (Liu et al., 2017a) and it was found that this
plasmids of mammalian cells, such as HEp-2 or BHK-21 cells, are system performs better than other rescue systems (Liu et al.,
standard procedures routinely used in most laboratories (Ayllon 2017b).
et al., 2013). Since the efficiency of producing infectious NDV Development of reverse genetics techniques for NDV is now
in the transfected cells is very low, the rescue protocol usually a well-established procedure that can be achieved in a standard
requires one or two amplification steps in embryonated chicken molecular biology laboratory. Reverse genetics systems have been
eggs. developed for different pathotypes of NDV strains: (1) lento-
Although the T7-expression system is widely used to rescue genic strains LaSota (Peeters et al., 1999; Römer-Oberdörfer et
NDV, it is not ideal for vaccine development for several reasons. al., 1999; Huang et al., 2001) and B1 (Nakaya et al., 2001); (2)
First, this method requires a stringent purification step to remove mesogenic strains Beaudette C (BC) (Krishnamurthy et al., 2000;
the T7-expressing helper virus. Secondly, in a poxvirus system Paldurai et al., 2014a) and Anhinga (Estevez et al., 2007); and (3)
there is higher chances of recombination between plasmids. velogenic strains Herts 33 (de Leeuw et al., 2005), ZJ1 (Liu et al.,