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320 | Coppo et al.

protein synthesis inhibitor cycloheximide. Other works focused expressed in Escherichia coli have been used to determine the
on the ICP4 gene had also determined immediate early expres- intracellular location of some of these proteins in an attempt to
sion of this gene in cell culture (Johnson et al., 1995b). Previous identify the potential functions of some of these viral proteins
work had also categorized the UL54 ORF (ICP27) as an imme- (Ziemann et al., 1998b; Veits et al., 2003c).
diate early (α) gene (Johnson et al., 1995a), but this was not The amino acid sequences of peptides encoded by ORFs A–E
confirmed by others, as Mahmoudian et al. (2012) categorized it (which are conserved in the genus Iltovirus) share no detectable
into a subcategory in the early phase due to its almost complete similarities between each other, or with other known viral or
transcription inhibition in the presence of cycloheximide (99%) animal sequences available (Ziemann et al., 1998a). Similarly, no
and phosphonoacetic acid (90%). Table 11.1 summarizes the detectable similarities have been found for ORF F. Experiments
transcription kinetics observed for all ILTV ORFs analysed to to localize the ORF A–E products in ILTV infected cells using
date. indirect immunofluorescence and confocal microscopy revealed
At least 10 micro-RNA (miRNA) molecules encoded by ILTV that ORF C was predominantly found in the cytoplasm, but also
have been identified by deep sequencing, and transcription of all inside the host cell nucleus. Unfortunately, the specific antisera
of these has been demonstrated in cultured cells (Rachamadugu raised against the other ORF products (ORFs A, B, D and E) were
et al., 2009; Waidner et al., 2009). Two of the miRNAs (I5 and not suitable for immunofluorescence in ILTV infected cells (Veits
I6) map antisense to the ICP4 coding region in the internal et al., 2003c) and therefore the intracellular localization of these
and terminal repeats. The other miRNA (I1–I4) were mapped proteins could not be ascertained. Successful isolation of ORF
to non-coding sequences in the UL region at the terminus A–E deletion mutants expressing green fluorescence protein indi-
of the genome (Waidner et al., 2009). An additional miRNA cated that these genes are dispensable during in vitro infection of
(I7) maps to the origin of replication in the UL region (OriL) cultured cells. Furthermore, in vitro infection experiments using
(Rachamadugu et al., 2009). There appears to be variations in the deletion mutants of each of the unique ORF A–E genes indicated
relative abundance of the different miRNA that are transcribed that some functional interdependence occurred between the dif-
at different stages of the ILTV replication cycle (Rachamadugu ferent ORF products, as, for instance, the expression of the ORF
et al., 2009; Waidner et al., 2009); however, detailed studies into D peptide was reduced during infection with the ORF E deletion
the kinetics of miRNA transcription remain to be undertaken. mutant, and the expression of the ORF A peptide was increased
It has been postulated that the miRNA mapping to the major during infection with the ORF B deletion mutant (Veits et al.,
transcriptional activator ICP4 may be derived from processing 2003c). Transcription analysis of these genes indicated that they
of the putative latency associated transcripts (LATs) described all exhibited predominantly late (γ) transcription kinetics, but
earlier by Johnson et al. (1995b). Based on nucleotide sequence they all could also escape the effects of cycloheximide and/or
analyses of ICP4, Johnson et al. (1995) found a number of poten- phosphonoacetic acid in varying degrees, indicating that their
tial transcripts with splice donor and acceptor sites, a feature of transcription may be subject to more complex, yet unidentified,
LATs that map antisense to ICP4 in Varicella Zoster virus and control (Veits et al., 2003c; Mahmoudian et al., 2012). It has also
Suid alphaherpesvirus 1 (Pseudorabies virus, PRV). Later work been suggested that ORFs A–C and ORFs D–E may be under the
demonstrated that the miRNA mapping antisense to the ICP4 control of distinct types of promoter (Mahmoudian et al., 2012).
gene could interfere with the expression of ICP4 by directly Further characterization of the viral replication kinetics and cell-
cleaving the ICP4 transcript (Waidner et al., 2011). Taking into to-cell spread characteristics of the deletion mutants in different
account that ICP4 is the only immediate early (α) gene expressed cultured cell types, indicated that, although not essential for
during ILTV infection, it is possible that miRNA regulation of infection, the lack of each particular gene resulted in reduced viral
ICP4 may influence the balance between lytic and latent states. progeny and/or plaque size relative to the wild-type parent strain.
Latency associated transcripts have not been identified for ILTV, In vitro infection of cultured cells with deletion mutants lack-
but in other herpesvirus species LATs play important roles in the ing ORFs A, D or E produced smaller plaque sizes and reduced
establishment, maintenance and reactivation from latent states viral titres. In contrast, while ORF B deletion mutants produced
(Morgan et al., 2001; Kent et al., 2003). Studies examining the wild-type sized plaques but significantly reduced viral production,
transcriptome in latently ILTV-infected trigeminal ganglia could ORF C deletion mutants exhibited reduced plaque sizes but wild-
be helpful for achieving a better understanding of latency in ILTV, type like kinetics of viral replication. Despite several attempts,
which is an important feature in the epizootiology of ILT that is isolation of quintuple deletion mutants was not possible and only
still very poorly understood. triple deletion mutants were successfully isolated (with muta-
tions in ORF A, B and C), and these mutants displayed impaired
Viral proteins and their functions viral growth characteristics (Veits et al., 2003c). Further in vitro
Considerable effort has been dedicated to characterizing the and in vivo infection studies with a different ORF C deletion
properties of the peptides encoded by ILTV-specific genes, mutant derived from the virulent ILTV United States Depart-
however, still very little is known about their function. Deletion ment of Agriculture challenge strain (USDAch), confirmed that
mutants lacking each of ORFs A–E (Veits et al., 2003c; García et the lack of ORF C results in reduced plaque size but comparable
al., 2016), UL0 (Veits et al., 2003b) and UL[–1] (Nadimpalli et intra- and extra-cellular viral titre, when compared with the parent
al., 2017) have been isolated following homologous recombina- strain. When inoculated via intra-tracheal/ocular route to spe-
tion, and specific antisera raised in rabbits against fusion proteins cific pathogen free (SPF) chickens, the ORF C deletion mutant