Page 65 - Avian Virology: Current Research and Future Trends

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have substitution rates of 1.6 × 10 (Beaty and Lee, 2016). The Reverse genetics
high genetic stability seen in NDV is also observed for other Reverse genetics is an approach to study the functions of a gene
members of the family Paramyxoviridae (Pomeroy et al., 2008), or a specific gene sequence by analysing its phenotypic effects.
which appears to be a property of this family. Although the rea- Reverse genetics is a powerful tool not only to study the structure
sons for genetic stability of NDV is not known, both structural and function of viral genes and their proteins, but also to engineer
and host selection pressures probably limit change in the genome. live vaccines and vaccine vectors. However, unlike DNA viruses,
NDV follows the ‘rule of six’ of the family Paramyxoviridae, in the genome of RNA viruses cannot be directly manipulated.
which the genome must be an exact multiple of six nt in length Therefore, initiation of these studies for an RNA virus requires
for efficient replication (Kolakofsky et al., 1998). The rule of six establishment of a method to produce infectious viruses from
restricts replication of genomes those are not multiple of six due cloned complementary DNA (cDNA), allowing manipula-
to insertions, deletions or recombination. The genome change of tion of the genome at the DNA level. Because the genome of a
NDV is also restricted by hexameric phasing, which is the periodic negative-strand RNA virus is not infectious alone (as opposed
manner the N protein interacts with the genome. Each group of to a positive-strand RNA virus), the viral proteins necessary for
six nt is bound by the same N molecule, the position of a given nt a first round of transcription must be present inside the cell for
within that group corresponds to phase 1 to 6 (Lamb and Parks, production of infectious virus. The basic methodology to pro-
2013). Phase is an important feature for viral transcription, it duce a recombinant non-segmented negative-strand RNA virus
might restrict synonymous mutations that disrupt the conserved (NS-NSV), such as NDV, has been to create an artificial viral rep-
phasing patterns (Beaty and Lee, 2016). lication cycle inside a cell. In all NS-NSV, the N-RNA is only the
Recombination, which is an important mechanism for genetic functional template for transcription and replication. Therefore,
diversity in many viruses, rarely occurs in negative-sense RNA N-RNA, which is the viral genomic or antigenomic RNA encap-
viruses (Chare et al., 2003). A possible reason for the low rate or sidated with N protein, must be created de novo from cDNAs.
absence of recombination is that both genomic and antigenomic To initiate replication, the N-RNA needs the viral polymerase
RNAs are never free in the cytoplasm for interaction. They are (P and L proteins). Therefore, the rescue of recombinant NDV
always tightly encapsidated with the N protein. The polymerase is requires co-transfection with four separate plasmids, one contain-
always bound to the nucleocapsid, which makes it difficult for the ing the NDV antigenome (positive-strand) and the other three
polymerase to switch templates during RNA replication. Another containing N, P, and L genes (Fig. 2.8). Inside the cell, positive-
reason for the low rate of recombination is the ‘rule of six’, which strand RNA is synthesized from the plasmid containing NDV
dictates that recombinants will only be viable when the total antigenome, which is then encapsidated with N protein. Once
number of nt is a multiple of six (Calain and Roux, 1993). the positive-strand N-RNA is formed inside the cell, the P and
Many attempts have been made using temperature sensitive L proteins synthesize the negative-strand viral genome, thereby
mutants to identify NDV recombinants in vitro, but all have been initiating the infection cycle. In NS-NSV, the antigenomic
unsuccessful (Granoff, 1959a,b; Dahlberg and Simon, 1969a,b). (positive-strand) not the genomic (negative-strand) RNA is used
Phylogenetic analysis of F and HN genes of NDV strains iso- for N-RNA formation because of the concern that simultaneous
lated over a period of 50 years have shown existence of distinct expression of naked negative-strand genomic RNA and positive-
lineages (Sakaguchi et al., 1989; Toyoda et al., 1989). It was strand N, P, and L mRNAs would result in hybridization and
concluded that the different lineages evolved by accumulation generation of double-strand RNA, leading to unsuccessful recov-
of point mutations and no gene exchange by recombination had ery of the virus. Therefore, all NDV recovery systems have used
occurred. A separate analysis of the M gene among NDV strains antigenomic RNA to form RNP.
also showed no evidence of recombination (Seal et al., 2000). The plasmids used in the NDV recovery systems are under the
However, there have been reports showing indirect evidence control of a T7 RNA polymerase promoter. Therefore, the host
of natural recombination in NDV (Han et al., 2008a; Qin et al., cell must express the bacteriophage T7 RNA polymerase. This
2008; Chong et al., 2010; Zhang et al., 2010). These studies were is typically accomplished either by co-infection of the cell with
performed by phylogenetic and recombination analysis of a single a recombinant vaccinia virus expressing the T7 polymerase (e.g.
gene or full NDV genome. These analyses occasionally identified vTF7-3 or MVA-T7), co-transfection of cells with a separate plas-
a virus with sequence discontinuity suggestive of a recombina- mid expressing T7 polymerase or use of a cell line that expresses
tion breakpoint. The putative parents for the recombinant were T7 polymerase (Buchholz et al., 1999).
identified from the sequence database. Some of the previously Construction of a rescue system first requires analysis of the
reported recombinants upon resequencing showed no evidence complete genome sequence of the NDV strain to identify the
of recombination, suggesting recombination of NDV is not as naturally occurring unique restriction enzyme (RE) sites. In
common as has been reported (Han and Worobey, 2011; Song cases where the convenient unique RE sites are not available the
et al., 2011). Although the occurrence of recombination in NDV nt sequences (preferably in the 3′ non-coding region of a gene)
cannot be completely ruled out, the evidence based on phyloge- are modified to create unique RE sites. The total genome length
netic analysis should be considered speculative. It will require in is adjusted to maintain the rule of six. The cDNA fragments are
vitro and in vivo recombination experiments with known parents amplified from the genomic RNA by RT-PCR using specific
to unequivocally demonstrate recombination in NDV. primers and a high-fidelity polymerase. Although, sequential