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328 | Coppo et al.

and single deletion mutants did not reach this same conclusion. median embryo infective dose (EID ) per bird. Birds typically
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The single gI and gE deletion mutants were capable of replicating develop clinical signs and lesions between 3 and 6 days after
in cell culture to similar titres of those reached by the wild-type inoculation, which are accompanied by viral shedding that can be
parent or rescuant viruses, but produced significantly smaller detected by PCR or culture using specimens collected from the
sized plaques, thus confirming its role in cell-to-cell-spread. The upper respiratory tract (trachea, conjunctiva, palatine cleft) by
absence of gI did not affect the expression of gE, and vice-versa, swabbing (in live animals) or scraping off of the mucosal surfaces
but the absence of both gI and gE had a significant impact on viral upon post-mortem examination (Fahey et al., 1983; Bagust et
replication and plaque size. It has been argued that the replica- al., 1986; Guy et al., 1990; Fuchs et al., 2000; Kirkpatrick et al.,
tion defect of the first double deletion mutant created by Devlin 2006a; Oldoni et al., 2009). A recent study has shown that the
et al. (2006a) may have been the result of inadvertent changes in route of inoculation determines which sites of the upper respira-
the nucleotide sequences of adjacent genes, especially US6 (gD), tory tract are infected after inoculation with vaccine or virulent
which has been found to be indispensable for replication (Pav- field strain. An interesting observation from this study was that,
lova et al., 2013). However, analysis of the nucleotide sequences in contrast with what was observed in other infected tissues,
upstream to the gI/gE deletion revealed no disruptions in the microscopic lesions were not apparent in the Harderian gland,
sequence of US6 in the deletion mutant (Devlin et al., 2006a), despite the positive detection of viral antigen and genomes in this
which suggests that replication defects in this mutant may have tissue, which suggests that Harderian gland may be an important
originated through changes outside US6. None of the gE or gI site for virus uptake (Beltrán et al., 2017). During experimental
deletion recombinants has been investigated in vivo. acute infection, ILTV not only infects the upper respiratory tract
Like gD, the protein encoded by US9 has also been categorized but also undergoes systemic distribution and has been detected
as a late gene (Mahmoudian et al., 2012; Pavlova et al., 2013) and by viral isolation in liver and lungs between 5 and 7 days post-
expressed as two major forms with apparent masses of 37 and infection (pi) (Bagust et al., 1986), and thymus between 5 and
25 kDa that are not modified by N-glycosylation (Pavlova et al., 9 days pi (Oldoni et al., 2009). Using quantitative PCR (qPCR),
2013). Only trace amounts of this protein are incorporated into ILTV has also been detected in tissues collected from experi-
virions and indirect immunofluorescence localized this protein in mentally inoculated chickens including trigeminal ganglia (4–5
the cytoplasm of infected cells. An ILTV deletion mutant lacking days pi), thymus (5–9 days pi), caecal tonsils (4–5 days pi) and
the US9 ORF was isolated and the absence of this gene did not sporadically in the cloaca (Oldoni et al., 2009). Later studies
appear to affect the replication or cell-to-cell spread of the recom- have found ILTV in almost every organ in the chicken includ-
binant (Pavlova et al., 2013). ing brain, lungs, heart, glandular stomach, spleen, duodenum,
pancreas, small and large intestine, caecum, kidney, and bursa of
Fabricius, as early as 1 day pi and as late as 28 days pi (Wang et al.,
Pathogenesis and immunity 2013; Zhao et al., 2013), sometimes also causing lesions (Wang
ILTV infects epithelial cells of the respiratory tract, where it et al., 2013). Infectious laryngotracheitis virus has also being
produces a lytic cycle of infection (Calnek et al., 1986). Systemic detected by PCR and nested qPCR in feather shafts (Davidson
infections have been described after experimental inoculations, et al., 2009; Davidson et al., 2016). Although viraemia could not
where the virus has been detected in tissues outside the respira- be detected by viral isolation in cell culture (Bagust et al., 1986),
tory tract (Bagust et al., 1986; Oldoni et al., 2009; Wang et al., the identification of ILTV in extra-respiratory tissues indicates
2013). During acute infection ILTV can infect sensory neurones that ILTV is systemically distributed. The application of more
of the peripheral nervous system (Bagust et al., 1986), where the sensitive molecular detection methods, such as qPCR on blood
virus establishes a latent infection. Until now only the trachea samples to detect viraemia, have not been reported in the litera-
and trigeminal ganglia have been identified as sites of viral latency ture. It has been hypothesized that monocytes/macrophages may
(Bagust, 1986; Williams et al., 1992). Reactivation of latent infec- serve as vehicles for systemic viral spread (Oldoni et al., 2009),
tion has been described (Hughes et al., 1989, 1991b; Coppo et as these cells, but not lymphocytes, are permissive to infection
al., 2012). There is an age-associated susceptibility to ILTV. As in vitro (Chang et al., 1977; Calnek et al., 1986; Loudovaris et al.,
birds become more resistant to infection as they grow, they can 1991a,b). However, recent experimental work conducted in our
withstand infection with increasing doses of ILTV at older ages laboratories (unpublished) has failed to consistently detect ILTV
(Fahey et al., 1983). genomes in peripheral blood mononuclear cells (PBMCs) from
acutely infected chickens, suggesting that ILTV transport may
Acute infection occur in a different compartment.
Several chicken experimental infection models of ILT have been Infection of the trachea with ILTV is normally accompanied
developed for the study of acute lytic infections, but not for latent with a reduction of the tracheal luminal diameter, in most cases
infections. Thus, the features of acute ILTV infection are much due to diphtheritic lesions in the upper and mid-trachea, com-
better understood than those associated with latent infection and monly with caseous plugs in the larynx and upper trachea. Such
reactivation. Acute experimental infection models utilize inocula- obstruction in combination with an already rigid structure (chick-
tions via ocular and/or intra-tracheal route with varying doses of ens have complete cartilaginous tracheal rings) is responsible for
viral inoculum, typically ranging from 10 to 10 plaque forming the respiratory distress observed in diseased chickens (Linares et
2
5
units (PFU), median tissue culture infective dose (TCID ) or al., 1994; Bagust et al., 2000) (see ‘Clinical features, diagnosis and
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