Page 328 - Avian Virology: Current Research and Future Trends

Infectious Laryngotracheitis Virus | 319

ORFs (Ziemann et al., 1998a; Veits et al., 2003c). Two additional (Loncoman et al., 2018a,b), even if virus replication is unchanged
ILTV-specific genes, UL0 and UL[–1] are encoded in the UL (Loncoman et al., 2018b). Under field conditions, full genome
region of the genome. They are adjacent to each other and located sequencing, followed by bioinformatics analysis of genome
upstream to the conserved UL1 ORF (glycoprotein L, gL) at the sequences remains a powerful approach to detecting and describ-
far right end of the UL region of the ILTV genome (Fuchs and ing recombination in field isolates of ILTV (Lee et al., 2013;
Mettenleiter, 1996; Ziemann et al., 1998b) (Fig. 11.1). These Agnew-Crumpton et al., 2016). The frequency of recombination
genes appear to have emerged due to an ancient duplication in ILTV appears to be high in comparison to some other herpes-
event, as they share a significant level of identity of their predicted viruses, including Marek’s disease virus, in which recombination
amino acid sequence (Ziemann et al., 1998b). ORF F has been has also recently been detected (Loncoman et al., 2017b). This
located downstream to the UL56 ORF at the left end of the UL high frequency of recombination has implications for vaccine
region (Johnson et al., 1997) and confirmed by whole genome safety, as vaccine viruses have the potential to recombine with field
sequence analysis (Lee et al., 2011a). strains, or other vaccine viruses, to generate ILTV recombinants
with higher levels of virulence (S.W. Lee et al., 2012; Loncoman
ILTV phylogeny and recombination et al., 2017b). Recombination should also be considered when
The need to distinguish between virulent and avirulent strains inferring phylogenetic relationships between ILTV isolates, as
of ILTV compelled many of the early studies that sought to dif- ignoring recombination present in any sequences used to generate
ferentiate ILTV strains and to examine the genetic relationships phylogenetic trees can result in inaccurate descriptions of phylo-
between isolates. Many of the early studies used restriction genetic relationships between viruses. Instead recombinants can
enzyme digestion of whole genomic DNA to differentiate between be identified and removed from such analyses, or only genome
ILTV isolates (Kotiw et al., 1982; Andreasen et al., 1990) and also sequence regions unbroken by recombination events can be used.
southern blotting hybridization techniques (Kotiw et al., 1986). Alternatively, ‘recombination aware’ phylogenetic analyses can be
Later, polymerase chain reaction (PCR) was used to amplify performed (Martin et al., 2015).
a selection of genes, followed by restriction enzyme digestion
and analysis of fragments (PCR restriction fragment length Viral gene transcription and micro-RNA
polymorphism, PCR-RFLP). The genes targeted in these PCR- Transcription in ILTV, as in other herpesviruses, follows a cascade
RFLP systems, and in similar systems that utilized DNA sequence pattern, where genes are classified into immediate early genes (α),
analysis of targeted genome regions, have recently been reviewed early genes (β) and late genes (γ1 and γ2) depending on their
(Menendez et al., 2014). Typically, the target genes used in these temporal transcription and their dependence on previous viral
PCR-RFLP systems varied between countries or regions. Thus, protein synthesis or viral DNA replication (Prideaux et al., 1992;
these systems facilitated analyses of phylogenetic relationships Mahmoudian et al., 2012). Each group of genes is normally asso-
between viruses within the same country or geographical region ciated with distinct functions in viral replication, where α-genes
but made analyses of relationships between strains across differ- are mostly transcription factors that are expressed independently
ent regions more difficult (see Strain Identification section). More of de novo protein synthesis, β-genes are typically involved in viral
recently, full genome sequencing of vaccine and field isolates of DNA metabolism and replication, and γ-genes are involved in
ILTV have enabled a more comprehensive examination of ILTV capsid assembly and morphogenesis and are normally completely
phylogeny, worldwide (Lee et al., 2011a,b; Chandra et al., 2012; or partially dependent on viral DNA replication (Prideaux et al.,
Spatz et al., 2012; García et al., 2013b; Kong et al., 2013; Piccirillo 1992; Mahmoudian et al., 2012). Initial work using metabolic
et al., 2016). radio-labelling characterized the expression of ILTV genes in
Full genome sequencing and analysis of ILTV isolates led to cell culture, and identified 16 polypeptides encoded by the ILTV
the detection of ILTV recombination. Recombination between genome, which were expressed in a cascade pattern, with different
different strains of ILTV was first reported in 2012 (S.W. Lee et al., peptides falling into each of the categories mentioned. Unfortu-
2012). This report demonstrated natural (field) recombination nately, the lack of more specific reagents targeting ILTV products
between two vaccine strains of ILTV to generate, new, virulent prevented the identification of the peptides observed (Prideaux
viruses in Australian poultry flocks (S.W. Lee et al., 2012). Since et al., 1992). Later work, using reverse transcription and PCR to
ILTV recombination was first identified, subsequent studies have determine the transcription kinetics of 74 ORFs encoded in the
indicated that recombination is an important process in ILTV ILTV genome, categorized genes into more highly resolved tran-
evolution and genome diversification and have demonstrated scription categories: immediate early; early I, II and II; early/late
frequent ILTV recombination under field conditions (Lee et al., I and II; and late I, II and II (Mahmoudian et al., 2012). With the
2013; Agnew-Crumpton et al., 2016) and also under experimen- exception of ORF F and UL56, which have not been investigated,
tal conditions (Loncoman et al., 2017a). Under experimental all the ILTV ORFs are transcribed and expressed during in vitro
conditions, single nucleotide polymorphism (SNP) genotyping ILTV infection in cultured cells (Ziemann et al., 1998a,b; Veits et
assays have been developed to study ILTV recombination and al., 2003b,c; Mahmoudian et al., 2012; Nadimpalli et al., 2017).
have shown that up to 75% of progeny viruses isolated after Mahmoudian et al. (2012) found only one ORF falling into the
high-dose co-infection of chickens are recombinants (Loncoman immediate early (α) gene category, ICP4, which was consistent
et al., 2017a). Fewer recombinant viruses are detected after co- with results from Prideaux et al. (1992) who found that only one
inoculation of vaccinated chickens, compared with naïve chickens 200 kDa polypeptide was expressed under the influence of the