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Avian Pathology ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20 Avian infectious laryngotracheitis: Virus‐host interactions in relation to prospects for eradication Trevor J. Bagust & Michael A. Johnson To cite this article: Trevor J. Bagust & Michael A. Johnson (1995) Avian infectious laryngotracheitis: Virus‐host interactions in relation to prospects for eradication, Avian Pathology, 24:3, 373-391, DOI: 10.1080/03079459508419079 To link to this article: http://dx.doi.org/10.1080/03079459508419079 Published online: 12 Nov 2007. Submit your article to this journal Article views: 939 View related articles Citing articles: 35 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Download by: [88.99.165.207] Date: 30 April 2017, At: 06:27 Avian Pathology (1995) 24, 373-391 REVIEW ARTICLE Avian infectious laryngotracheitis: virus-host interactions in relation to prospects for eradication TREVOR J. BAGUST & MICHAEL A. JOHNSON Commonwealth Scientific and Industrial Research Organisation, Animal Health Research Laboratory, Parkville, Melbourne, Victoria, 3052, Australia SUMMARY This review examines the virology, immunology and molecular biology of infectious laryngotracheitis virus (ILTV) and its interactions with the chicken, in the context of assessing the feasibility of eradication. Establishment of the latent phase during infection of the host, its central role in biological survival of ILTV and the host-viral events that are associated with reactivation of infection, are considered. In counterpoint there are several features of the biology of ILTV in its natural mode of infection which can be exploited in eradicating this pathogen from intensive poultry production sites. These include the high degree of host-specificity of ILTV, dependence on contact for spread, the short-lived infectivity outside the chicken and the stability of the genome and lack of significant antigenic variation. Further, ILTV cannot replicate productively in its main target organ, the trachea, in the face of local specific cell-mediated immunity. Genetically-engineered vaccines that are capable of generating immunity, but without the ILTV latent infections induced by conventional modified-live ILT vaccine strains, are now well into development. This paper postulates that, used in conjunction with specific site quarantine and hygiene measures, such vaccines can provide the technological tools required to eradicate ILTV from production sites, and then regionally, in developed poultry industries from around the year 2000. INTRODUCTION Whether the European-Australian acronym "ILT" or the American "LT" is used for infectious laryngotracheitisj this viral respiratory pathogen of chickens (now known also as Gallid herpesvirus 1, Roizman, 1982), has proved to be of enduring interest since being the first major avian viral disease for which an effective vaccine was developed (Hudson & Beaudette, 1932). Application of virulent ILT virus (ILTV) to the mucous membrane of the vent of young layers resulted in rapid development of immunity in the respiratory tract. This was the first method to control previously widespread and severe ILT outbreaks. In the 6 decades since the pioneering observations on ILTV were made in the Received 21 March 1995; Accepted 21 March 1995. 0307-9457/95/030373-19 © 1995 Houghton Trust Ltd 374 T. J. BAGUST & M. A. JOHNSON 1930s (see Hitchner, 1975), some hundreds of scientific papers on ILTV have been published each decade. Whilst the great majority of these have been vaccines-related, very few have been directed to examining the feasibility of eradication of ILTV on production sites or regionally. Reviewing this aspect of prevention of ILT disease now however could well prove timely for two reasons. Firstly, scientific understanding of the pathogenesis, immunology and viral ecology of ILT in both the acute and latent phases of infection has reached an advanced stage. Secondly, there are major developments occurring in molecular biological technology. These can be applied to control avian infectious diseases using a new generation of genetically-engineered vaccines and delivery systems. This paper aims, therefore, to focus on the knowledge that is currently available for assessing the feasibility of excluding ILT infection from flocks of chickens on intensive production sites. For detailed information on historical or other aspects of ILT disease and virus, other reviews which might be consulted include Jordan (1966, 1981), Hitchner (1975) or the current ILT chapter in Diseases of Poultry (Hanson & Bagust, 1991). ILT DISEASE AND VIRUS IN MODERN PERSPECTIVE Forms of disease in differing production systems Epizootic forms of ILT were often described in earlier years associated with haemorrhagic tracheitis, expectoration of blood-stained mucus and mortalities of 20% or higher. Benign forms of ILT with low morbidity and very low mortalities (0.1-2%) are the more regular features of modern ILT in the developed intensive poultry industries of Europe, the USA or Australasia (Jordan, 1966; Hanson & Bagust, 1991). In countries with modern intensive poultry industries, ILT in layer flocks is no longer recognised as a cause of significant economic losses and can be prevented or readily controlled if adequate vaccination regimens are maintained. Outbreaks on single or several sites may occasionally occur, usually originating in pullets for which vaccination has been overlooked or immunity has waned prematurely. These cases are the exception and are usually quickly rectified. For intensive broiler production systems, the high degrees of site isolation and quarantine tend to combine with the short growing periods of some 6 to 7 weeks so as to usually exclude the risk of exposure to ILT. Broilers may therefore no longer be routinely vaccinated in some regions. The second major ILT infection and disease situation occurs in the same countries but outside the intensive industry, and is in backyard or fancier chicken flocks, where a somewhat laissez-faire approach to ILT control can apply. The birds on these sites may be of various ages and generations, be vaccinated or not and immune or otherwise depending on natural exposure. On such sites ILTV strains of varying virulence can co-exist for generations as endemic infections, with sporadic natural spread producing immunity but little evidence of disease unless direct or indirect contact occurs with susceptible chickens elsewhere. AVIAN INFECTIOUS LARYNGOTRACHEITIS 375 However, these sites can remain as major reservoirs of ILTV and some of these strains may be moderately pathogenic. Hence, any planning for regional ILT eradication must also take such small sites into consideration. The third scenario for ILTV infection and disease remains in the village poultry of many of the world's economically-developing ("industrialising") countries. Periodic outbreaks of ILT disease among fully-susceptible village poultry in African or Asian villages can be of a severity sufficient to decimate village chicken populations (Biggs, 1982). ILT vaccines may be unavailable through constraints such as isolation of rural regions or a lack of financial resources or Government extension assistance to these areas. This permits ILTV to persist throughout these regions in its classical forms as a fearsome poultry pathogen. Resolution, even if practicable, will require an altered and integrated developmentally-based approach to upgrading poultry health in production within these countries (Bagust, 1994). Acute ILT infection: pathogenesis and transmission The target organ system for ILTV infection and disease is the respiratory tract. The epithelium of the trachea and larynx is always affected whilst other mucus membranes, respiratory sinuses, air sacs and lung tissue may periodically also become infected. This depends on the route, challenge level and sequelae of infection. Whether chickens are exposed to ILTV by nasal, oral, conjunctival or other routes, e.g., via the infraorbital sinus, the most active replication of ILT occurs within the tissues of the trachea (see Hanson & Bagust, 1991). Numerous experimental infection studies have examined the pathogenesis of ILTV infection, using chickens of different ages, routes of infection, strains of virus and various laboratory technologies for detection of the virus or its antigens. To indicate the major points, the comprehensive studies of Hitchner et al, (1977), Bagust et al, (1986) and Williams et al, (1992) can be considered together, and show the features which are described below. Involvement of respiratory tissues Active virus replication occurs only during the 1st week of infection, although low levels of ILTV can sporadically be detected up to 10 days post-infection (p.i.). Bagust et al, (1986) quantified virus replication occurring in the trachea and found titres in trachéal exúdate and tissues from days 2 to 5 of 102 to 107 plaque-forming units per gram or ml of trachéal washings or tissues. Subsequent virus replication was rarely detectable by infectivity or fluorescent antibody (FA) beyond day 7 p.i. Williams et al, (1992) recorded virus yields in trachéal swabs as a mean daily titre of 2.7 logio TCID 50 /ml between days 3 to 7, then falling to the order of 1 logio or less. Of the three major studies denoted however, only Williams et al, (1992) have reported being able to detect ILTV infectivity in the trachea other than sporadically at 8 to 10 days p.i., although antigen detection by 376 T. J. BAGUST & M. A. JOHNSON FA testing has been described over somewhat longer periods (Wilks & Kogan, 1979). Clinical signs of respiratory disease occur 6 to 12 days after natural exposure of a flock but in experimental infections, where the virus inoculum is routinely delivered directly to the trachea, between 2 to 4 days p.i. Chickens recover from primary ILT disease within 7 to 10 days or so of showing clinical signs, the appearance of these reflecting the phase of active virus replication which is occurring in the trachea. From 10 days through to 4 weeks or so after trachéal infection, whilst shedding of ILTV infectivity may have ceased, the latency phase of ILTV infection is being established (see later). Involvement of non-respiratory tissues Infection of the conjunctiva regularly occurs in both natural and experimental infections, via spread of contaminated exudates from adjacent clinically-affected birds. After local replication, the trachéal tissues are then involved. There is little evidence for the occurrence of a viraemic phase, and then only sporadically in young chickens (Bagust et al, 1986), although avian macrophages are known to be able to support the multiplication of ILTV strains in vitro (Von Bülow & Klasen, 1983; Calnek et ai, 1986). ILTV invasion of the trigeminal ganglion (TRG) was discovered to occur regularly from days 3 to 6 during the acute phase of ILT infections by Bagust et al, (1986). The exact route of infection of the TRG is not known but neural migration can be strongly inferred as this ganglion is known to provide the main sensory innervation to the tissues of the upper respiratory tract, the mouth and the eyes whilst the distal ganglia are also involved in the sensory innervation of the trachea (Bubien-Waluszewska, 1981). Williams et al, (1992), using polymerase chain reaction technology, have subsequently confirmed that the trachéal ganglion is the main site of ILTV latency (see later). Recently, ILT viruses have been detected in two samples of proximal femora that were obtained from lame broiler chickens (Jones et al, 1993). The significance of this observation is not yet clear, and further investigations are merited. Transmission of ILTV by direct contact occurs naturally via the upper respiratory and ocular routes, mainly when infectious respiratory exudates are aerosolised or expectorated. However, there is no evidence for egg-transmission of ILTV or for shedding of ILTV on shells of eggs laid by infected hens. The chicken is the only significant primary host species for ILTV, and the existence of another reservoir host species is unknown, although some other avian species such as pheasants can sometimes be naturally infected through contact with chickens (see Hanson & Bagust, 1991). ILT latency: sites of establishment and reactivation Latency establishment circumvents the usual immune clearance mechanisms of AVIAN INFECTIOUS LARYNGOTRACHEITIS 377 the host and allows persistence of ILTV infection in host flocks even of low numbers of chickens. Integration of the viral genomic material into the host DNA occurs during latency (see later). A functional virological definition of latency requires that when putative latently-infected host tissues are placed in susceptible cell cultures, they must remain free of viral infection for several days before replication commences and infectious virus particles are produced. When organ cultures are established from the tracheas of chickens previously infected with either virulent wild-strain or vaccine-strains of ILTV, virus is not released until after some days in culture (Turner, 1972; Bagust, 1986; Hughes et al, 1989). The most detailed studies with ILT in trachéal organ cultures (TOC) systems have shown that wild-strain ILTV could be detected in 38% of chickens examined 3 to 15 months after trachéal exposure, while a similar proportion of chickens infected with an Australian ILT vaccine-strain were detected from 2 to 10 months p.i. (Bagust, 1986). Also reported in this study was the finding that the sites of establishment of latent infection were highly focal in that only 8.3% of the TOC preparations, though obtained from locations throughout entire tracheas, were positive. It may be deduced from these results, along with those of Hughes et al, (1987), that all field—and vaccine—strains of ILTV will establish latent phases of infection. Onset of the latent ("hidden") phase of ILTV infection appears to commence during the immediate post-acute phase of infection, i.e., 7 to 10 days after trachéal exposure. Latent ILTV infections are not readily demonstrable during the first few months after infection (Bagust et al, 1986; Hughes et al, 1987, 1991), and this finding probably reflects the host chicken initially exerting a high level of immune control and surveillance. From around 3 months p.i. and then throughout the life of the host chicken, reactivation of latent ILTV infection with shedding of infectious virus into the trachea will occur sporadically. Hughes et al, (1989), in a landmark study which went far towards explaining how apparently spontaneous outbreaks of ILT occur in field situations, demonstrated that rates of shedding of ILTV into the trachea were significantly increased ( P < 0.001) by the stresses of either the onset of lay or the mixing with unfamiliar birds. A number of studies have been directed towards elucidating the site(s) of ILTV latency establishment from which, after reactivation, infectious virus can eventually manifest in the trachea! epithelium. Whilst the TRG is known to be a site of latent infection for alphaherpesviridae of several other animal species (Ackermann et al, 1982; Feldman, 1991), and to be associated with nervous supply to the trachea and larynx (see Pathogenesis), it is only quite recently that firm evidence was obtained for the association of ILTV with the TRG. Two observations have strongly implicated the TRG in ILT latency—first, evidence from Australia of the involvement of the TRG regularly during the pathogenesis of acute trachéal infection with ILTV (Bagust, 1985) and then a German report (Kaleta et al, 1986) of isolation of ILTV from the TRGs of two field chickens some 15 months 378 T. J. BAGUST & M. A. JOHNSON after vaccination. Confirmation that the TRG is the major site of ILTV latency was finally obtained through polymerase chain reaction studies in the UK by Williams et al, (1992). Spreading of ILTV between production sites From the prece*ding two sections, it can be seen that the sole source of infection on a production site will be the chickens. Whilst these may have been adequately vaccinated, ILTV production will be continually occurring through reactivation of latent infections in a small proportion of these chickens. Stressing of these birds through coming into lay or mixing of flocks will cause a higher proportion of the hens in a flock to shed virus. Further, after exposure of a flock to "live" ILT vaccine, or after an outbreak, it cannot be assumed for practical purposes that active ILTV shedding has ceased until some 2 weeks after exposure or the onset of the last clinical cases in a naturally-infected flock. ILTV transfer between sites also occurs regularly by indirect contact, through infectious trachéal exudates from chickens and surface contamination of personnel, service vehicles and poultry equipment (reviewed by Hanson & Bagust, 1991). Hence risk management measures will be required in production sites which adjoin one which has undergone a recent outbreak of ILT. Whilst ILTV is known to be readily susceptible to various disinfectants and heat treatments (Hanson & Bagust, 1991), the ability of ILTV infectivity to persist in trachéal exudates and in carcasses for periods between 21 to 80 days with ambient environmental temperatures up to 20°C (see review by Jordan, 1966) appears no longer to be common knowledge. Hence high priority must be accorded to disposal of carcasses and measures to control potential predatory and other migratory animals (rodents, birds) on production sites. THE IMMUNOBIOLOGY OF ILTV Host.responses following infection A variety of responses are generated by the immune system following infection by ILTV (reviewed by Jordan, 1981; Hanson & Bagust, 1991). Best known are virus-neutralising antibodies which become detectable in the serum within 5 to 7 days of trachéal exposure, peak around 21 days, and then wane over the next several months to low levels at which they can persist for a year or more. Mucosal antibodies (IgG and IgA) which are capable of binding to ILTV antigen and of low levels of virus-neutralising and ELISA antibody activity become detectable in trachéal secretions and washings from approximately 7 days p.i. (Bagust, 1986; York et al, 1989) and plateau at days 10 to 28. York et al, (1989) observed the numbers of IgA- and IgG-synthesising cells in the trachea to be substantially increased by days 3 through to 7 p.i. Cell-mediated immune (CMI) responses to ILTV infection have generally only been studied indirectly (e.g., using bursectomised chickens) because of the complexity of requirements in conducting CMI experimentation, but DTH AVIAN INFECTIOUS LARYNGOTRACHEITIS 379 responses to ILTV have been detected (York & Fahey, 1990). The duration of CMI responses can only be intuited from observations that levels of ILT vaccineinduced protection are highest for 15 to 20 weeks (see Hanson & Bagust, 1991), after which vaccine breakdowns in the field become more common. ILT immune protection Primary ILT vaccination confers protection against challenge which is partial by 3 to 4 days post-exposure, and complete from 1 week (see reviews by Hitchner, 1975; Jordan, 1981). High levels of protection then ensue for 15 to 20 weeks post-vaccination, with variable degrees of protection occurring within a flock over the next year. Revaccination with infectious ("live") vaccines may or may not assist in maintaining protection levels (Jordan, 1981), as the infectivity of the new vaccine virus may well just be inactivated at the portal of entry to the host chicken. Numerous laboratory and field studies have independently confirmed that immune protection to ILTV challenge is neither indicated by nor conferrable through the presence of serum antibody. Transfer of immunity to ILTV challenge, using spleen cells and peripheral blood leukocytes obtained from congenie immune donors, has been reported by Fahey et al., (1984). However, the onset of ILT immunity in the recipient chickens could not be demonstrated until some 7 days after transfer of the immune cells, and possible reasons for this delay in onset require to be examined. Studies by Fahey & York (1990) using vaccinated-bursectomised chickens have demonstrated that mucosal antibody is not essential in preventing the replication of virus in vaccinated chickens. These authors concluded that the effector mechanism of protection from ILT is the local cell-mediated immune response in the trachea. Effector mechanism(s) of cytotoxic lymphocytes or lymphokines released by activated T-cells, or both, have been surmised. Of most importance however within the present context of assessing the feasibility of eradication of ILTV has been the decisive demonstration in two studies (York et al., 1989; Fahey & York, 1990), that when chickens are challenged with virulent ILTV 28 to 30 days after vaccination, viral replication in the trachéal epithelium is completely prevented. Both the effector mechanism and duration of this protection post-vaccination urgently need to be resolved. The latent phase of an ILTV infection may be induced as well as maintained by immune-mediated mechanisms. It is known that ILTV latency is established in the face of development of detectable host immune responses 1 to 2 weeks after trachéal exposure. Latency establishment is the biological survival mechanism which permits ILTV to evade host immune surveillance and clearance by the host. As immunity begins to wane, or effectiveness is reduced due to stress factors (Hughes et al., 1989), reactivation to produce infectious ILTV can occur. For ILTV, in contrast to latent infections by the alphaherpesviridae of other domestic animals, experimental treatments using regimens of immunosuppressive drugs such as corticosteroids or cyclophosphamide (Bagust, 1986; Hughes et al, 1989) 380 T. J. BAGUST & M. A. JOHNSON have not been shown to induce reactivation of ILTV infectivity in chickens known to be latently infected. MOLECULAR BIOLOGY OF ILTV The viral genome and antigens ILTV is a member of the alphaherpesviridae, with a genome consisting of a linear 155 kilobase double-stranded molecule, consisting of unique long (UO and unique short (Us) segments flanked by inverted repeats (IRs) (Leib et al, 1987; Johnson et al, 1991). From random DNA sequencing of ILTV DNA, Griffin (1989) was able to identify 21 genes. Of these, 20 were identified by comparison to varicella-zoster virus (VZV) and 19 by comparison to herpes simplex virus type 1 (HSV-1); only 12 genes were found by comparison with the gammaherpesvirus Epstein-Barr virus (EBV). Sequence data of the thymidine kinase gene and the upstream overlapping genes of ILTV also provided evidence that homology exists at the DNA level between ILTV and other alphaherpesviruses (Griffin & Boursnell, 1990; Keeler et al, 1991). A number of ILTV genes which have HSV homologues have now been sequenced including glycoprotein B (gB) (Kongsuwan et al, 1991; Griffin, 1991; Poulsen et al, 1991), glycoprotein C (gC) (Kingsley et al, 1994), glycoprotein D (gD) (Johnson et al, 1995a), ICP4 (Johnson et al, 1995b), ICP27, glycoprotein K (gK) and DNA helicase complex and a gene with homology to glycoprotein X (gX) of pseudorabies virus (PRV) (Kongsuwan et al, 1993a). All of these genes were located in colinear positions with respect to the prototype genome HSV-1. The level of amino acid identity of these ILTV genes compared to homologues of other members of the alphaherpesviridae would indicate that ILTV is an ancient member of this family of viruses. The study of the protective antigens of ILTV has focused on the glycoproteins, as these molecules are responsible for stimulating both humoral and cell-mediated responses to other herpesviruses (Spear, 1985). The characterization of ILTV glycoproteins is still in an early stage, although a number have now been characterized by sequencing including gB, gC, gD, gX, gK (see above references) and a unique glycoprotein, gp60 (Kongsuwan et al, 1993b, 1995). York et al, (1987) showed that chicken antisera to ILT vaccine strain (SA-2) and to a virulent isolate immunoprecipitated five major viral glycoproteins of 205, 160, 115, 90 and 60 Kilodaltons (kDa), respectively. Additional glycoprotein bands were recognized by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus infected cells. Further work using monoclonal antibodies defined the five major glycoproteins into two groups; the 205 complex (205, 160, 115 and 90 kDa glycoproteins) and the 60 kDa glycoproteins (York et al, 1990). Monoclonal antibodies which reacted with the 205 kDa complex were used to screen a Agtl 1 expression library to identify and characterize glycoprotein B (Kongsuwan et al, 1991). This approach was also used to characterize the 60 kDa glycoprotein AVIAN INFECTIOUS LARYNGOTRACHEITIS •• 3 8 1 (gp60); Kongsuwan etal, 1993b, 1995). This glycoprotein, which maps in the U s region, has no homologue with other alphaherpesviruses and is therefore unique to ILTV. Although gp60 is one of the primary proteins recognized by sera from ILTV-infected chickens, its precise role in immunity or on the pathogenesis of ILTV has not been determined. The depletion of gp60 from total glycoprotein preparations using monoclonal antibody did not reduce the efficacy of preparations as a vaccine (York, 1989) and infected birds have been found to produce both humoral and cell-mediated responses to gp60 (York & Fahey, 1990). Finally, a glycoprotein with homology to PRV gX, a member of the gG family of glycoproteins, has also been characterized. PRV gX is not incorporated into PRV virions (Ben-Porat & Kaplan, 1970), is the predominant protein in the culture medium of PRV-infected cells (Rea et al, 1985) and is immunogenic. ILTV gX, like PRV gX, is secreted into tissue culture medium and hence is not a structural component of the virion, whilst being immunogenic (Kongsuwan et al, 1993a). Attempts at vaccination with GST-gX fusion protein, followed by challenge, showed that birds were not protected from clinical disease nor from replication of the challenge virus in the trachea (C. Prideaux and K. Kongsuwan, personal communication 1994). Mechanisms of latency and reactivation in host cells Latency is a property of all herpesviruses, and this mechanism is marked by two key features, being (1) the maintenance of multiple copies of circular extrachromosomal viral DNA in nuclei of one or two unique cell types, and (2) limited transcription from these viral genomes. At some time after the establishment of latency, conditions may be presented which trigger small numbers of latently infected cells to enter into the productive stage of infection, and infectious virus re-emerges. This infection may be clinically distinct from the primary infections, with no apparent lesions. However, in some cases it can produce recrudescence of clinical disease. Most of the knowledge surrounding latency mechanisms has been derived from studies on HSV and EBV, and excellent current reviews are available, e.g., Wagner (1994). Advances in knowledge have centred around detailed investigations on the genes involved in latency, latency associated transcripts (LATs), the cells involved in latency and application of genetic engineering techniques. There have been a number of studies with HSV in animal models which have shown that the establishment of latency in the cells of a target tissue by the original viral strain, precluded colonization of the same tissue by another strain (Klein et al, 1978; Centifanto-Fitzgerald et al, 1982; Meignier et al, 1983; Thomas et al, 1985). Other studies however have indicated that different strains can establish latency in the same tissue of the host animal (Lewis et al, 1984; Whetstone & Miller, 1989). A general premise of vaccination strategies used has been that attenuated strains should reduce the incidence of establishment of latency by wild type (WT) virus. Vaccination regimes in Australia show for ILTV that widespread use of tissue-culture adapted strains can reduce the incidence of 382 T. J. BAGUST & M. A. JOHNSON virulent outbreaks, presumably by a combination of immunity and early colonisation by the SA-2 strain inhibiting latency establishment by other ILT field viruses. The most recent studies on PRV infection suggest that there is indeed a significant inverse correlation between the level of latency established by attenuated strains and the level of latency established by a superinfecting WT virus (Schang et al., 1994). This study indicates that vaccination with attenuated virus, using routes of inoculation that can maximize the establishment of latency, might significantly decrease latency establishment by WT strains. Further, genetically engineered viruses that have undergone deletions for the genes of both glycoprotein I (gl) and thymidine kinase (TK) are impaired from establishing latency because of an inability to target the virus to the tissues of the central nervous system, a function of gl, and impaired replication in neurones, a function of TK. Therefore, attenuated strains which are gl + may be better in precolonization of neurones of the TG and consequently have a greater capacity to interfere with WT latency. ILT VACCINES AND THE NEW TECHNOLOGIES Conventional ILT vaccines All of the current vaccines for prevention and control of outbreaks of ILT are based upon modified-live (ML) strains of this virus. These possess varying degrees of residual virulence, naturally or through attenuation, and are grown using chicken embryos or tissue culture. Various vaccination routes have been developed during the past 60 years (see Hanson & Bagust, 1991). From the initial cloacal route, the application of ILT vaccines has evolved through feather follicle abrasion, then intranasal or sub-conjunctival instillation, and currently via the drinking water or using aerosols with selected strains. The value of using ML ILT vaccines to prevent or control disease is beyond question. It has to be borne in mind however that ML vaccines regularly retain inherent pathogenicity (requiring careful control of the dosage and conditions of administration), and are capable of spreading from vaccinated to non-vaccinated birds to cause serious problems (e.g., Clarke et al, 1980). The levels of virulence of ML ILT vaccine strains are liable to be increased by bird-bird or flock-flock passages after vaccination, and this may result in reversion to virulence (Guy et al, 1991). Life-long latent ILTV infections, with sporadic reactivation and shedding of virus (Bagust 1985, 1986; Hughes et al., 1991) are a further complication with the use of ML ILT vaccines. Successful application of recombinant DNA technology to ILTV vaccines, especially in the areas of safety and of delivery, would therefore be a very significant advance. Strategies for developing recombinant vaccines and vector systems Increasing knowledge of the antigens of ILTV makes it possible to use genetic engineering to produce sub-unit vaccines, to construct virus vectors expressing ILT antigen(s), or to reconstruct ILTV itself as a viral vector. AVIAN INFECTIOUS LARYNGOTRACHEITIS 383 There is now good evidence that vaccination with herpesvirus glycoproteins can induce protective immunity (Ghiasi et al, 1994). For example, HSV gB has been demonstrated, both with temperature sensitive mutants (Glorioso et al, 1984) and immunopurified proteins (Chan et ai, 1985), to evoke circulating antibody and cell-mediated responses which can protect mice against lethal challenge. Further, HSV gB expressed either by recombinant vaccinia virus vector (Cantin et al, 1987), adenovirus vector (McDermott et al, 1989), or baculovirus vector (Ghiasi et al, 1994), has protected mice against lethal challenge. For ILT, a subunit vaccine containing only glycoproteins of the 205 kDa complex protected 100% of chickens against clinical disease and also against viral replication (York & Fahey, 1991). Since this 205 kDa complex contains ILTV gB (Kongsuwan et al, 1991), this glycoprotein could prove to be a major protective immunogen of ILTV and therefore a prime candidate for inclusion in subunit or recombinant vaccines. These might be any of fowlpox virus-ILT gB, fowl adenovirus-ILT gB, Marek's disease virus-ILT gB, or herpesvirus of turkeysILT gB viral vectors. Such an approach might also be extended to produce multi-valent recombinant viral vectors by including other glycoproteins that can induce a protective response such as gD and gC. It would then be possible to vaccinate flocks even though these chickens have not experienced ILTV infection. Another approach is to engineer ILTV as a vector to include other antigens whose expression is controlled by ILT promoters. The prime site for insertion is in the ILT gX (gG) gene. Since PRV gX can be deleted from the herpesvirus genome without reducing its ability to kill mice (Thomsen et al, 1987a), and foreign genes can be inserted into gX-deleted genomes (Thomsen et al, 1987b; Mettenleiter et al, 1990; van Zijl et al, 1991), it is most likely that this site in ILTV can also be used to construct viral vectors expressing foreign antigens. It has, however, been difficult to produce recombinant ILTVs. The recent development of a continuous cell line that can support ILTV replication (Scholz et al, 1993) may well assist in future. Other recent reports of the construction of ILTV recombinants have included Guo et al, (1994), who inserted the jS-gal marker gene into an open reading frame and has claimed the isolation of a recombinant. No evidence has been given to date however of virus isolation or characteristics of the recombinant, although inspection of the sequence showed that the marker gene to be inserted into ILTV ICP4. Another group (Okamura et al, 1994) has reported successfully inserting the /?-gal marker gene into the TK gene and have isolated a stable recombinant. This would seem to have been made possible by the use of a strain of ILTV which is capable of propagation in chicken embryo fibroblasts, thus allowing successful transfection/recombination experiments. It should be noted however that one of the disadvantages attending the use of ILTV as a vector is that vaccination is unlikely to completely prevent latency. Further, any such recombinant virus will have to be constructed so that rearrangements in vivo cannot occur, i.e., with wild type virus in the field. This will require the precise deletion of non-essential genes as has been demonstrated for 384 T. J. BAGUST & M. A. JOHNSON pseudorabies virus vaccines. Nevertheless, even that highly successful vaccine appears unable to prevent some Aujeszky's disease virus infection within swine herds (Stegeman et al, 1994). More positively, an ILTV TK~, gX~ recombinant could provide a most promising tool for eradication. Alternative future approaches may involve not using ILTV at all in the field, but rather to rely upon other vectors such as fowl adenovirus or fowlpox to deliver the major ILTV neutralising antigen (gB). However, other glycoprotein genes might also be required and it is yet not known what effect this type of regime might have on preventing latency. Any validation protocols for future putative ILT vaccines should therefore include systematic studies on this aspect. ASSESSING PROSPECTS FOR ERADICATION OF ILT The feasibility of site eradication? The following features of the biology and ecology of ILTV in both virus-host and virus-site relationships are major indicators of the potential eradicability of ILTV. 1. ILT virus is not egg-transmitted: hence the infection status of the parent flocks is not transmitted to progeny chickens whether placed on the same or other sites. 2. ILT infection is essentially confined to chickens: the chicken is the primary host species, and through latency establishment, is also the reservoir host. 3. Levels of ILTV infectivity are usually low: titres of infectivity in excess of very low levels will only occur in the first 7 days or so after trachéal infection of a chicken whether exposed through vaccination or by natural infection. 4. Spread of ILTV infection between sites can be strongly circumscribed by industry precautions: relatively simple site quarantine and hygiene procedures will prevent ILTV from entering intensive broiler production sites. 5. ILTV infectivity is readily inactivated outside the host chicken by disinfectants and by low levels of heat. Carry-over between successive production batches in a house can therefore be prevented by adequate shed cleanup. 6. Immunity will absolutely protect against ILTV challenge: for 4 to 5 months post-vaccination, replication of challenge ILTV can be prevented at the trachea! surface. Denial of the trachéal epithelium as a substrate for viral replication will prevent latency establishment by ILTV. 7. ILTV strains are antigenically homogenous: the DNA genome is stable, and antigenic variation is not a problem in ILT vaccines as cross-neutralization and cross-protection occurs between all known strains of ILTV. 8. Immunity to ILTV is cell-mediated: maternal antibody to ILTV does not interfere with the vaccination of very young chickens, or even in ovo. AVIAN INFECTIOUS LARYNGOTRACHEITIS 385 When these features are considered separately, and together, the prospects for systematic site and then regional eradication of ILTV can be seen to be most positive providing that the (vaccine-based) technological tools required can be developed. These will be recombinant vaccines which contain the ILTV immunogens that can induce protection through immune responses in the trachea. Some practical considerations For an eradication approach to ILT the benefit-costs ratio and the likely time scale for introduction are both major issues once feasibility is accepted. For benefit-cost considerations, were developments needed through recombinant-DNA technology to be focused solely on ILT, the costs at all levels in a developed poultry industry would outweigh the gains from initial eradication. However, there are several other major avian viral pathogens (infectious bursal disease virus, Marek's disease virus, infectious bronchitis virus, Newcastle disease virus) for which recombinant vector vaccines are projected to be available around year 2000. ILT recombinant vaccines should also now be included. This would allow the costs of development to be amortised, with particular value for ILTV because of the numerous biological and ecological features (see previous section) that render it specially susceptible to eradication measures. While awaiting new-generation vaccines however, any region likely to be designated for ILT eradication could most usefully be systematically swamped with ML ILT vaccines on all production sites whether large, small or fancier-type. Broiler production sites which do not vaccinate now however could be excluded, provided that their strict quarantine and hygiene measures (Hanson & Bagust, 1991; Bagust, 1992) were maintained. Government-industry cooperation will be needed for success of such eradication programmes. Laudable results obtained from earlier attempts at exclusion of ILT from some regions of the USA (e.g., Mallison et al., 1981) should not be lost sight of. Whilst the eradication of ILT from Australia, a sub-continent bounded by seas, has long been a vision for the poultry industry (Hart, 1992), its realisation must await the commercial availability of ILT vaccines that have been engineered for this task. IN CONCLUSION Demand and need will inevitably increase over the next decade for avian vaccines which can not only prevent diseases, but can also facilitate exclusion or eradication of endemic pathogens from production sites. The ILT vaccines which have been developed and used to date have generally served the developed poultry industries well. In the final analysis however, they operate through controlled exposure to infection, and they perpetuate persistence of ILTV on sites through latent infections. Advancing technology is now presenting opportunities to develop alternative strategies as we have indicated here. ILT control through site and then regional eradication would be a further 386 T. J. BAGUST & M. A. JOHNSON major development in avian health. Interestingly, it is only the recombinant-DNA ILT vaccines which will have the potential to provide the solutions needed to meet the deep apprehensions of poultry producers concerning ILT control. Explicitly, if vaccination is stopped, with ILTVs then being excluded from production sites only by quarantine and hygiene, what concrete measures can be taken to provide cover for these flocks should ILTV exposure occur? Whilst conventional ML ILT vaccines cannot provide immune cover in the absence of active infection (and thence latency, etc.), recombinant DNA vector-delivered vaccines do have the clear potential to provide the immunologically-based tools needed for such a task. Historically, there have been relatively few significant avian pathogens for which the eradication option can realistically be planned. For ILTV , however, research and development, as well as preliminary strategic planning in health by developed poultry industries, can realistically be directed towards such an approach now. ACKNOWLEDGEMENTS We would wish to honour the work of numerous scientists, but this review is particularly dedicated for their pioneering research studies on ILTV to Professors S. Hitchner and L. Hanson (USA), Professor B. Sinkovic and Dr L. Hart (Australia) and to Dr F. Jordan and his colleagues at Liverpool University, UK. 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RESUME Laryngotrachéite infectieuse aviaire: interactions virus-hôte en relation avec les possibilités d'éradication Cet article de synthèse traite de la virologie, de l'immunologie et de la biologie moléculaire du virus de la laryngotrachéite infectieuse (ILTV) et de ses interactions avec le poulet dans le but 390 T. J. BAGUST & M. A. JOHNSON d'envisager la faisabilité de son eradication. L'établissement d'une phase de latence pendant l'infection de l'hôte, son rôle primordial dans la survie biologique de l'ILTV et les relations virus-hôte qui sont associées avec la réactivation de l'infection sont étudiés. Plusieurs aspects de la biologie de l'ILTV dans son mode naturel d'infection peuvent être utilisés pour éradiquer cet agent pathogène des fermes de production intensive de volailles, à savoir le haut degré de spécificité de l'hôte pour l'ILTV, la nécessité de contact pour sa dissémination, son infectivité courte en dehors du poulet, la stabilité du génome et l'absence de variation antigénique significative. De plus, l'ILTV ne peut se répliquer abondamment dans le principal organe cible, la trachée, lorsqu'il existe une immunité à médiation cellulaire spécifique locale. Des vaccins préparés par génie génétique, capables de générer une immunité sans les infections latentes de l'ILTV induites par des souches vaccinales de l'ILTV vivantes modifiées conventionnelles, sont en cours de développement. Cet article démontre qu'en conjonction avec des mesures de quarantine spécifiques à la ferme d'élevage et des mesures hygiéniques, de tels vaccins peuvent être des outils technologiques permettant l'éradication, à l'approche de l'an 2000, de l'ILTV des sites de production et par la suite, des zones géographiques où est pratiquée l'industrie de la volaille. ZUSAMMENFASSUNG Aviäre infektiöse Laryngotracheitis: Virus-Wirt-Interaktionen in bezug auf Aussichten für die Tilgung Die vorliegende Übersichtsarbeit untersucht die Virologie, Immunologie und Molekularbiologie des Virus der infektiösen Laryngotracheitis (ILTV) und seine Interaktionen mit dem Huhn im Zusammenhang mit einer Beurteilung der Möglichkeit der Tilgung. Die Etablierung der Latenzphase während der Infektion des Wirtes, deren zentrale Rolle beim biologischen Überleben des ILTV und die Wirt-Virus-Ereignisse, die mit der Reaktivierung der Infektion verbunden sind, werden betrachtet. Auf der anderen Seite gibt es verschiedene Charakteristika der Biologie von ILTV bei seiner natürlichen Infektionsweise, die beim Tilgen dieses Erregers von Plätzen mit intensiver Geflügelproduktion ausgenutzt werden können. Diese umfassen die hochgradige Wirtsspezifität von ILTV, die Abhängigkeit von Kontakt für die Ausbreitung, die kurzlebige Infektiosität außerhalb des Huhnes und die Stabilität des Genoms mit dem Ausbleiben einer signifikanten Antigenvariation. Ferner kann sich ILTV angesichts einer lokalen spezifischen zeilvermittelten Immunität in seinem hauptsächlichen Zielorgan, der Trachea, nicht produktiv replizieren. Die Entwicklung gentechnologisch hergestellter Vakzinen, die fähig sind, eine Immunität zu bewirken, aber ohne die latenten ILTV-Infektionen, die durch die konventionellen modifizierten ILT-Vakzinestämme verursacht werden, ist jetzt gut im Gange. Die vorliegende Arbeit postuliert, daß mit solchen Vakzinen, wenn sie in Verbindung mit Quarantäne- und Hygienemaßnahmen angewendet werden, das technologische Instrument geliefert wird, das erforderlich ist, um ILTV aus Produktionsstandorten zu tilgen und dann regional, in der entwickelten Geflügelwirtschaft etwa ab dem Jahr 2000. RESUMEN Laringotraqueitis infecciosa aviar: interacciones hospedador-virus en relación a la posibilidad de erradicar la enfermedad Esta revisión abarca la virología, inmunología y biología molecular del virus de la laringotraqueitis infecciosa (ILTV) y sus interacciones con la gallina en el contexto de observar las AVIAN INFECTIOUS LARYNGOTRACHEITIS 391 posibilidades de erradicar. Se considerarán el establecieminto de la fase latente durante la infección del hospedador, su papel central en la supervivencia biológica del ILTV y los eventos virus-hospedador asociados con la reactivación de la infección. En contrapartida, hay varias aspectos de la biología de la infección natural que pueden ser explotados en la erradicación de este agente de las zonas de producción avícola intensiva. Estas incluyen un grado elevado de hospedador-especificidad de ILTV, dependencia del contacto para su diseminación, infectividad reducida fuera del pollo, estabilidad del genoma de ILTV y la ausencia de una variación antigénica significativa. Además ILTV no puede replicarse productivamente en su órgano proncipal, la tráquea, en presencia de una inmunidad celular local específica. Las vacunas de ingienería genética en vías de desarrollo son capaces de generar inmunidad sin producir infecciones latentes por ILTV al contrario de lo que sucede con las vacumas modificadas convencionales. Este artículo postula que las vacunas deingieneria genética, al ser empleadas en conjunción con medidas específicas de cuarentena y medidas higiénicas, pueden proporcionar las herramientas necesarias para erradicar ILTV de las áreas de producción avícola y posteriormente regionalmente en industrias avícolas avanzadas alrededor del año 2000.