Vet. Res. 38 (2007) 261–279 261 c INRA, EDP Sciences, 2007 DOI: 10.1051/vetres:200657 Review article

Molecular biology of avian infectious laryngotracheitis

Walter Fa*, Jutta Va, Dorothee Ha, Harald Gb,JensP.Tb, Thomas C. Ma

a Institute of Molecular Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Boddenblick 5A, 17493 Greifswald – Insel Riems, Germany b Institute of Infectology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Boddenblick 5A, 17493 Greifswald – Insel Riems, Germany

(Received 6 April 2006; accepted 19 October 2006)

Abstract – Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an econom- ically important disease, which results in delayed growth, reduced egg production, and also frequently in death of the animals. After acute infection of the upper respiratory tract, the virus can establish latency in the central nervous system, and subsequent reactivations can lead to infec- tion of naive . For prevention of ILT, conventionally attenuated live vaccines are available. However, these vaccine strains are genetically not characterized, and reversions to a virulent phe- notype occur. Although molecular analyses of ILTV are hampered by the lack of an optimal cell culture system, the complete nucleotide sequence of the ILTV genome has recently been eluci- dated, and several ILTV recombinants lacking nonessential, but virulence determining genes have been constructed. Animal trials indicated that genetically engineered stable gene deletion mutants are safe alternatives to the current vaccine strains. Furthermore, since live ILTV vaccines are suit- able for fast and inexpensive mass administration, they are promising as vectors for immunogenic proteins of other chicken pathogens. Thus, immunization with ILTV recombinants expressing avian influenza virus hemagglutinin was shown to protect chickens against ILT and fowl plague. Using monospecific antisera and monoclonal antibodies several virion proteins of ILTV have been iden- tified and characterized. Since they include immunogenic envelope glycoproteins, these results can contribute to the improvement of virus diagnostics, and to the development of marker vaccines.

infectious laryngotracheitis virus / DNA sequence / monoclonal antibodies / recombinant live- virus vaccines / viral vectors

Table of contents 1. Infectious laryngotracheitis...... 262 2. Virion structure, replication and morphogenesis ...... 262 3. Genome structure, gene content, and phylogenetic aspects...... 265 4. Identification of viral proteins and diagnostic tools ...... 268 5. Genetically engineered ILTV mutants, and other novel approaches for vaccine development ...... 271 6. ILTV as a vector for antigens of other chicken pathogens ...... 273 7. Conclusion...... 273

* Corresponding author: walter.fuchs@fli.bund.de

Article available at http://www.edpsciences.org/vetres or http://dx.doi.org/10.1051/vetres:200657 262 W. Fuchs et al.

1. INFECTIOUS of cell-mediated immune responses [22]. LARYNGOTRACHEITIS Nevertheless, ILTV-specific antibodies can be used for serological diagnosis by virus Infectious laryngotracheitis (ILT) is a neutralization, agar-gel immunodiffusion, worldwide occurring respiratory disease of indirect immunofluorescence, and enzyme chickens, which is caused by a virus des- linked immuno sorbent assays (ELISA) ignated as ILTV or gallid herpesvirus 1 [42, 43, 50, 68]. The ELISA technique has (GaHV-1). A detailed review on ILT has also been used for detection of viral pro- recently been published [39]. Therefore, teins in tracheal exudates [98]. However, clinical and immunological aspects will more recently protein detection by im- only be briefly mentioned in the present re- munoperoxidase tests, and DNA detection port. ILT was first described in the USA in by conventional polymerase chain reaction 1925 [62], and shortly thereafter its viral (PCR) or real-time PCR have become pre- aetiology was demonstrated [6]. The sever- ferred methods of virus diagnosis [1, 16, ity of clinical symptoms depends on the 38, 54, 81, 96]. In combination with analy- virulence of a particular strain or isolate, ses of restriction fragment polymorphisms and mortality rates can vary between 0 and and DNA sequencing the PCR technique more than 70%. Symptoms of milder forms also facilitates differentiation of virus iso- of ILT are nasal discharge, conjunctivi- lates for epidemiological and phylogenetic tis and reduced egg production, whereas studies [16, 54]. severe forms are additionally character- For prevention of ILT, live-virus vacci- ized by gasping, coughing, expectoration nation has been in use for many decades. of bloody mucus, and marked dyspnoea Early after discovery of the disease, chick- (Fig. 1D) which may lead to suffocation. ens were immunized by cloacal application Pathomorphological alterations can be ob- of virulent virus [9]. Later, virulent field served in conjunctiva, larynx, trachea, and strains of ILTV were attenuated by passage sometimes also in the lungs (Fig. 2E) and in embryonated chicken eggs or cell cul- air sacs. The incubation period ranges from tures [29,77]. The obtained vaccine strains ca. 6 to 12 days after natural infection can be administered individually by eye through ocular or respiratory routes, and drop, but are also suitable for mass appli- from 2 to 4 days after experimental in- cation via aerosol or drinking water [39]. tratracheal infection. After an acute phase Most of these vaccines are efficacious, but of ca. 7 days, ILTV can establish life- many of them possess considerable resid- long latent infections in the central nervous ual virulence, which can further increase system, in particular within the trigeminal during animal passages [37,58]. Thus, sev- ganglion [4,96]. Sporadic reactivations are eral outbreaks of ILT have been attributed mostly inapparent, but lead to productive to revertant vaccine strains [3, 36]. replication in the respiratory tract and virus shedding, which can result in infection of susceptible contact animals [44]. 2. VIRION STRUCTURE, ILTV infection of chickens frequently REPLICATION AND induces production of virus-neutralizing MORPHOGENESIS antibodies [42], but antibody titers corre- late only poorly with protective immunity Electron microscopy revealed that ILTV [8, 84]. Furthermore, bursectomized ani- particles exhibit the typical morphology of mals, although unable to produce ILTV- herpesvirions (Figs. 2A, 2B, 3), consisting specific antibodies, were protected against of a DNA-containing core within an icosa- reinfection, indicating a dominant role hedral capsid which is surrounded by a Molecular biology of ILTV 263

Figure 1. Safety and efficacy of live-virus vaccination with gJ-negative ILTV. (A) Eight-week old chickens were infected by intratracheal administration of 2 × 104 plaque forming units (PFU) of ILTV-∆gJ or wild-type ILTV (ILTV-WT). (B) After four weeks, immunized and naïve (NI) animals were challenged with 105 PFU of ILTV. From days 1 to 10 after each infection the animals were daily classified as healthy (0), ill (1), severely ill (2), or dead (3). The cumulative clinical scores for each group were plotted and mortality rates (%) are indicated. The pictures show a protected chicken (C) and a control animal suffering from severe dyspnea (D) at day 4 after challenge. For more details see reference [26]. (A colour version of this figure is available at www.edpsciences.org/vetres.) 264 W. Fuchs et al.

Figure 2. Reactivity of monoclonal antibodies against ILTV glycoproteins gC (A, C) and gJ (B, D, E). For immunoelectron microscopy (A, B) MAb binding to purified ILT virions was detected by gold-tagged secondary antibodies. Virus particles were stained with phosphotungstic acid. For in- direct immunofluorescence tests (C, D) chicken embryo kidney cells were fixed 24 h after infection with methanol and acetone (1:1). MAb binding was detected with fluorescein-conjugated secondary antibodies, and chromatin was counterstained with propidium iodide. Immunohistochemical stain- ing of a lung section from an ILTV-infected chicken (E) was performed by the ABC method. A gJ-specific MAb detected viral antigen in the cytoplasm of several syncytia (arrows). Bars repre- sent 150 nm (A, B), 100 µm (C, D), and 50 µm (E). (A colour version of this figure is available at www.edpsciences.org/vetres.) Molecular biology of ILTV 265 proteinaceous tegument layer and an outer and ICP4 revealed similar transactivating envelope with incorporated viral glycopro- functions [25, 41]. teins [17,75,94]. Whereas all capsids have Ultrastructural analyses of ILTV- diameters of ca. 100 nm, particle sizes vary infected cells showed the typical steps between 200 and 350 nm, since ILTV tends of herpesvirus morphogenesis (Fig. 3) to incorporate huge amounts of tegument [30, 35, 67]. In the nucleus, capsids proteins [30]. are formed and filled with viral DNA (Figs. 3A, 3C). Then, the nucleocap- Like other avian herpesviruses, ILTV sids are transported into the cytoplasm exhibits a very narrow host range both in by subsequent envelopment and de- vivo and in vitro. Besides domestic chick- envelopment at the inner and outer leaflets ens, pheasants represent the only natural of the nuclear membrane (Figs. 3B, 3C). hosts [39]. Furthermore, the virus could The cytoplasmic capsids associate with be isolated from peafowl [15], and experi- electron dense tegument, and are then mental infections of turkeys have also been re-enveloped by a second budding event described [97]. In vitro, ILTV can be prop- in the trans-Golgi region (Figs. 3D, 3E), agated on the chorioallantoic membrane of followed by release of mature particles embryonated chicken eggs [10], and in pri- by exocytosis (Figs. 3F, 3G). Uncommon mary kidney and liver cells of chickens or features frequently observed in ILTV- chicken embryos [12, 63]. Up to now, only infected cells are the formation of tubular one permanent cell line (LMH) derived structures of unknown function, and of from a chemically induced chicken liver large cytoplasmic vacuoles containing tumor was shown to permit replication of numerous virions [35]. Furthermore, ILTV [51, 78, 80]. However, the resulting besides few complete virions, many light virus titers were significantly lower than in (L) particles are formed, which consist of primary cells. After several hours, ILTV in- tegument and envelope, but do not contain fection of permissive chicken cells induces nucleocapsids (Fig. 3D) [30]. Presumably, formation of syncytia and nuclear inclu- these erroneous structures are in part sion bodies [73], and subsequent lysis of responsible for the low titers of ILTV in the cells leads to plaque formation after 3 tissue culture, which barely exceed one to 5 days. infectious unit per cell. The replication cycle of ILTV has only been poorly investigated up to now, but ap- pears to be similar to that of other alphaher- 3. GENOME STRUCTURE, pesviruses like type 1 GENE CONTENT, AND (HSV-1) [74]. One-step growth analyses in PHYLOGENETIC ASPECTS primary chicken kidney cells revealed that first infectious progeny are formed Restriction analyses revealed that the 8 to 12 h after infection, and reach a max- linear double-stranded DNA genome of imum after 24 to 30 h [25]. Metabolic la- ILTV consists of two unique regions (UL, beling of proteins in ILTV-infected cells in US), and of inverted repeat sequences combination with inhibitors of translation (IR, TR) flanking the US-region (Fig. 4) or DNA replication indicated a cascade- [45, 60]. Similar structures, which al- like regulation of viral gene expression low formation of two genome isomers as is typical for herpesviruses [72], and with differently oriented US-regions, have transfection experiments with expression also been found for many other alpha- plasmids for the ILTV homologues of the herpesviruses, and designated as type D major HSV-1 transcription activators VP16 herpesvirus genomes [75]. First sequence 266 W. Fuchs et al.

Figure 3. Electron microscopy of ILTV morphogenesis. Chicken hepatoma (LMH) cells were fixed and embedded 18 h after infection, and ultrathin sections were stained with uranyl acetate and lead salts. The micrographs show nuclear egress by primary envelopment of capsids at the nuclear membrane (B, C), and formation of mature and capsid-less particles by assembly of tegument and secondary envelopment in the trans-Golgi region of the cytoplasm (D, E). Virions are released from cells by exocytosis (F, G). Bars represent 1 µm (A), 500 nm (D), 300 nm (E, F, G), or 150 nm (B, C). Molecular biology of ILTV 267

Figure 4. Map of the double-stranded DNA genome of ILTV, which is ca. 150 kbp in size and consists of long and short unique regions (UL,US), and of inverted repeats (IR, TR) flanking the US- region. The IR- and TR-regions contain variable copy numbers of direct repeat elements (vertical lines). Compared to most other alphaherpesvirus genomes, a part of the UL region is inverted (bor- dered by vertical arrows). Origins of DNA replication (ORIL,ORIS) and selected genes (horizontal arrows), including those of conserved glycoproteins (gB to gN) are indicated. Enlarged sections show two clusters of Iltovirus-specific genes (ORF A – E, UL0, UL(-1)). Other specific features of ILTV are the translocation of UL47 to the US region, and the absence of an UL16 homologue (highlighted). data of ILTV DNA became available ists in tandem arrays of 1 to 13 copies in in the late 1980s [31]. In the mean- the IR- and TR sequences (Fig. 4) [95]. time, a complete sequence of the whole Like many other alphaherpesviruses ILTV genome has been assembled from over- possesses three origins of viral DNA repli- lapping genome fragments of different cation, one (OriL) located within the UL- virus strains analyzed in numerous labo- genome region, and two copies of another ratories [23–25, 32–34, 46, 48, 49, 52, 55– (OriS) within the IR and TR sequences, re- 57, 70, 87, 95, 101, 102]1,2. The result- spectively (Fig. 4)4 [101]. In ILTV, these ing molecule encompasses 148687 base elements contain exceptionally long palin- pairs (bp), and contains ca. 48.2% G and dromic regions, which hamper cloning and C residues. For several genome regions DNA sequencing. This might explain why more than one sequence is available, which OriS has not been detected in earlier stud- in some cases exhibit considerable differ- ies, and has not been included in the pub- ences. Most strikingly, close to the left lished genome sequence of ILTV [87]. genome end the Australian vaccine strain The gene content of ILTV clearly con- SA-2 contains an insertion of 3575 bp (nu- firms its previous classification as an alpha- cleotides 2173 to 5747 of the published herpesvirus [19]. The ILTV genome con- genome sequence), which is absent from tains 76 open reading frames (ORF) which the genome of the virulent ILTV strain have been predicted or demonstrated to A4893 [49]. Another feature contributing encode proteins [87]. Sixty-three of these to size variation of the ILTV genome is a ORF exhibit homologies to genes of the direct repeat element of 856 bp which ex- prototypic alphaherpesvirus HSV-1 [64], at least with respect to genome position 1 Johnson M.A., 1999, GenBank # AF168792. and overall structure of the deduced trans- 2 Kehu Y., Manfu Z., Yiping L., 2001, GenBank lation products. Eleven of the conserved # AY033143. 3 Fuchs W. et al., unpublished results. 4 Fuchs W., 2006, GenBank # AM238250. 268 W. Fuchs et al.

ORF encode homologues of the HSV-1 since it also possesses homologues of sev- glycoproteins gB, gC, gD, gE, gG, gH, eral open reading frames which have so gI, gJ, gK, gL and gM (Fig. 4). For clar- far been considered as unique for ILTV. ity, the commonly used HSV-1 gene des- Besides several poorly conserved genes ignations UL1 to UL54, US2 to US8, within the US region and adjoining parts and US10 were also adopted for ILTV of the IR- and TR sequences [95] a clus- (Fig. 4). Between the UL2 and UL3 ho- ter of five ILTV- and PsHV-1-specific ORF mologues the ILTV genome contains an (ORF A to ORF E) is localized in the UL additional ORF (UL3.5) which is not genome region [87, 101]. Whereas ORF present in herpes simplex virus genomes, A to ORF E share no significant homol- but conserved in most other mammalian ogy with any other known viral or cellular and avian alphaherpesviruses [23]. More- genes, marked similarities were detected over, the ILTV genome exhibits several between the deduced amino acid sequences unique characteristics, as e.g. the absence of another two ILTV-specific genes desig- of an UL16 homologue [24]. This pro- nated as UL0 and UL(-1), suggesting that tein is otherwise conserved throughout all these genes presumably result from an an- herpesvirus subfamilies, although its func- cient duplication event [102]. Interestingly, tion remains largely enigmatic [74]. The the genome of PsHV-1 apparently contains gene encoding a major tegument protein, only one of these genes, UL(-1) [87]. UL47, is usually localized within the UL The presence of several genes specific genome region of alphaherpesviruses [64], for ILTV and PsHV-1 already indicated but absent at the corresponding position that these viruses might represent a sepa- of ILTV DNA [101]. However, a signifi- rate branch within the phylogenetic tree of cantly conserved homologue of UL47 was herpesviruses. This was also confirmed by found to be inserted between the US3 comparative analyses based on the amino and US4 genes within the US genome re- acid sequences of conserved gene prod- gion of ILTV [95]. Besides this translo- ucts [47, 65]. These studies revealed that cation, compared to most other alphaher- the ancestors of ILTV probably separated pesviruses the ILTV genome also exhibits from those of the mammalian alphaher- a large internal inversion of a conserved pesviruses even earlier than the ancestors gene cluster within the UL region (Fig. 4) of Marek’s disease virus (MDV) and her- [101]. This inversion includes the UL22 to pesvirus of turkeys. Therefore, ILTV has UL44 genes, and, thus, is similar to a pre- been assigned as the hitherto only member viously described inversion of the UL27 to the genus Iltovirus of the Alphaher- to UL44 gene region within the genome pesvirinae subfamily of the of the porcine alphaherpesvirus pseudora- [19]. However, considering the recently bies virus (PrV) [7]. However, it is unclear published sequence of the PsHV-1 genome whether these findings indicate a special [87], this virus should also be grouped into phylogenetic relationship between ILTV the same genus. and PrV, or whether they reflect two in- dependent events. Recently, an inversion within the UL genome region identical to 4. IDENTIFICATION OF VIRAL that in ILTV, as well as a translocation of PROTEINS AND DIAGNOSTIC UL47, have also been found in the genome TOOLS of psittacid herpesvirus 1 (PsHV-1), the causative agent of Pacheco’s disease of Monoclonal antibodies (MAb) against parrots [87]. This virus represents the clos- ILTV particles have been prepared in sev- est cognate of ILTV identified up to now, eral laboratories [2,90,99]. However, since Molecular biology of ILTV 269

Table I. Identified gene products of ILTV.

Gene Protein Amino acids Expected masses (kDa) Apparent masses (kDa) UL0 ILTV-specific, nuclear protein 506 57.0 63 UL(-1) Iltovirus-specific, nuclear protein 501 57.6 73 ORF A Iltovirus-specific protein 376 41.3 40 ORF B Iltovirus-specific protein 340 38.1 34 ORF C Iltovirus-specific protein 354 37.4 38, 30 ORF D Iltovirus-specific protein 374 41.5 41 ORF E Iltovirus-specific protein 411 45.1 44 UL10 gM homologue, envelope protein 393 43.1 36, 31 UL22 gH, envelope glycoprotein 804 89.4 92 UL27 gB, envelope glycoprotein 883 100.2 110, 100, 58 UL31 nuclear protein 314 35.4 33 UL37 tegument protein 1022 113.0 112 UL44 gC, envelope glycoprotein 414 46.4 60 UL46 tegument protein 557 62.7 64 UL47 tegument protein 623 70.0 66 UL48 tegument protein 396 44.6 45 UL49 tegument protein 266 30.4 34 UL49.5 gN, envelope glycoprotein 117 12.7 17 US4 gG, secreted glycoprotein 292 31.7 52 US5 gJ, envelope glycoprotein 985, 611 106.5, 66.8 200, 160, 115, 85 Protein sizes and molecular masses of primary translation products were deduced from GenBank DNA sequences NC_006623, AM083948 (UL31), and AM083949 (UL37). Apparent masses of viral proteins were determined by Western blot analyses3 [24, 26, 27, 41, 71, 90, 91, 102]. cloned virus DNA and sequence data were In the latter assays the N-glycosylated limited in the past, identification of the vi- envelope protein gC of ILTV exhibited an ral target proteins was difficult. The genes apparent molecular mass of 60 kDa [52, of two viral envelope glycoproteins, des- 90]. The protein is conserved throughout ignated as gB and gp60, were detected by the alphaherpesvirus subfamily, and usu- screening of lambda-phage expression li- ally plays an important role during initial braries of cloned ILTV DNA with MAb virus attachment by binding to heparan sul- [55, 57]. However, a more detailed charac- fate chains of proteoglycans at the host terization of the respective viral gene prod- cell membrane [74]. However, obviously ucts revealed considerably different molec- neither gC, nor other envelope proteins of ular masses than described initially [71, ILTV bind to heparin, indicating that this 90]. Thus, up to now, only two ILTV pro- particular virus adsorbs to cells by mech- teins, gC and gJ, could be unambiguously anisms different from those used by most identified as targets of MAb by transient other herpesviruses [53]. expression of the respective UL44 and US5 At least with respect to antibody re- genes in eukaryotic cells [90]. The MAb sponses in mice, the most immunogenic against these two proteins were suitable surface protein of ILT virions seems to for indirect immunofluorescence tests, im- be the product of the ORF5 gene within munohistochemistry, immunoelectron mi- the US genome region [95], which was croscopy, radioimmunoprecipitations and detected by numerous MAb from differ- Western blot analyses (Fig. 2). ent laboratories. This protein was initially 270 W. Fuchs et al. designated as gp60 [57], but later renamed virus particles but secreted from infected gJ like the glycoprotein encoded at the cells, and, therefore, might possess im- corresponding genome position of HSV-1 munomodulating functions [56]. Further- [90]. This gene is missing in most other more, the UL22 gene product gH, the ma- alphaherpesvirus genomes, but in equine jor tegument proteins encoded by UL37, herpesvirus 1 a positional homologous UL46, UL47, UL48, and UL49, and the ORF also encodes an immunodominanten- non-structural UL31 protein of ILTV were velope glycoprotein (gp2) [85, 86] which also identified using monospecific antisera exhibits significant amino acid sequence (Tab. I) [41]3. homology to ILTV gJ. The precise function As mentioned above, detailed investiga- of ILTV gJ is still unknown, but it dis- tion of ILTV demonstrated the large bio- plays interesting characteristics, e.g. multi- logical variability within the herpesviruses. ple protein forms with apparent masses of Thus, the identification and localization 85, 115, 160 and 200 kDa. Two different of ILTV- or Iltovirus-specific gene prod- primary translation products are formed ucts with monospecific antisera could shed from non-spliced and spliced mRNA, mod- more light on specific characteristics of ified by N- and O-linked glycosylation, this genus. It has already been shown that and presumably further processed by pro- ORF A to ORF E, as well as the UL0 and teolytic cleavage [26]. UL(-1) genes of ILTV are translated into While the target proteins of other ILTV- proteins, which were detectable by West- specific MAb remain to be characterized, ern blot analyses of infected cells (Tab. I) many viral gene products could be iden- [91, 102]. However, more detailed studies tified after expression in bacteria or other were hampered by the low expression lev- heterologous systems, and preparation of els of most of these proteins. Only the monospecific antisera (Tab. I). Using such related UL0 and UL(-1) proteins, which an antiserum, the envelope glycoprotein are both translated from spliced mRNA, gB of ILTV was shown to be processed by could be shown to accumulate in the nuclei addition of N-linked carbohydrate chains of ILTV-infected cells [102]. This localiza- and by proteolytic cleavage into two sub- tion might indicate functional similarities units, as it has also been described for to the transcription-regulating ICP0 pro- the gB polypeptides of most other her- teins encoded at corresponding genome pesviruses [71]. Whereas the functional positions of other alphaherpesviruses [74]. form of gB seems to be homodimeric, The available monoclonal and mono- the glycoproteins gM and gN of many specific antibodies against defined ILTV herpesviruses have been shown to form proteins may be helpful tools for in situ in- a heterodimeric complex. This has re- vestigations of the viral replication cycle cently been confirmed for the correspond- by immunofluorescence or immunoelec- ing UL10 and UL49.5 gene products of tron microscopy. On the other hand, these ILTV [27]. However, whereas the UL49.5 antibodies can also be used for diagnos- proteins of ILTV and several other viruses tic virus detection in tissue samples or are O-glycosylated (and, therefore, named tracheal swabs of ILTV infected chickens gN), the UL10 protein of ILTV, unlike [1, 38, 90, 98]. ILTV-specific MAb might all known gM homologues, is apparently also facilitate serological diagnosis by in- not modified by glycosylation [24]. An- direct or blocking ELISA tests, similar to other conserved glycoprotein identified in those that are already applied e.g. for infec- ILTV is the US4 gene product gG, which, tious bovine rhinotracheitis, or Newcastle like gG homologs of several other al- disease of chickens [18, 59]. In particular, phaherpesviruses, is not incorporated into the ILTV gJ- and gC-specific MAb might Molecular biology of ILTV 271 be suitable for this type of assay, since directed deletion of virulence-determining naturally or experimentally infected chick- genes. The successful construction of ens produce antibodies against these two ILTV recombinants could also provide glycoproteins, which are detectable by in- deeper insights into fundamental mech- direct immunofluorescence tests on cells anisms of virus replication, but is still expressing the cloned genes [26,90]. It was hampered by the lack of suitable cell further shown that bacterial fusion proteins lines for efficient in vitro propagation and expressed from the cloned gJ (gp60) or gE manipulation. Whereas many herpesvirus genes of ILTV can be used as antigens for genomes, including that of the chicken detection of ILTV-specific serum antibod- pathogen MDV, have in the meantime ies in direct ELISA tests [13]. been cloned as infectious bacterial artifi- cial chromosomes (BAC) and mutagenized in Escherichia coli [66, 82], this technique 5. GENETICALLY ENGINEERED has not yet been successfully applied to ILTV MUTANTS, AND OTHER ILTV. A possible reason for this failure is NOVEL APPROACHES FOR the presence of several long palindromic VACCINE DEVELOPMENT DNA sequences within the ILTV genome (see above), which are known to be unsta- Since many years, classically attenu- ble in E. coli [14]. Nevertheless, infectious ated live-virus vaccines are in use for the ILTV could be recovered after transfec- prevention of ILT in chickens [39]. How- tion of chicken hepatoma (LMH) cells with ever, many of them possess residual vir- virion DNA, and virus yields were fur- ulence, which can further increase during ther increased by simultaneous expression animal passages (see above). To overcome of the viral transcription activators ICP4 this problem, inactivated whole-virus vac- or UL48 of ILTV [25]. The infectivity cines and subunit preparations containing of genomic ILTV DNA allowed the gen- affinity-purified glycoproteins were suc- eration of virus mutants by homologous cessfully tested as alternatives [5, 100]. recombination with cotransfected transfer However, due to the high costs of pro- plasmids which carried the desired dele- duction and delivery these vaccines are tions or insertions. To facilitate the time- not suitable for immunization of large consuming selection and isolation of ILTV chicken flocks. Thus, the development recombinants, reporter genes encoding β- of genetically engineered live-virus vac- galactosidase or green fluorescent protein cines against ILT appears more promising. (GFP) have frequently been inserted into In one approach, immunogenic envelope the virus genome [24, 69, 79]. However, proteins of ILTV were expressed in the at least in ILTV, these reporter gene in- avian virus vectors herpesvirus of turkeys sertions might be unstable, or impair viral or attenuated fowlpox virus, and the ob- replication [25], and, therefore, should be tained virus recombinants were shown to removed from the viral genome by a sec- protect experimentally immunized chick- ond recombination step. ens against subsequent challenge infec- To our knowledge, up to now four- tions with virulent ILTV [20,76,88]. How- teen genes were successfully deleted from ever, unlike conventional ILT live-virus the ILTV genome (Tab. II). All of them vaccines, these virus recombinants require are dispensable for virus replication in individual administration, and are not suit- cell culture, since construction of trans- able for mass application. complementing chicken cell lines that Therefore, it was also attempted to gen- would permit propagation of ILTV recom- erate stably attenuated ILTV mutants by binants lacking essential genes, appears to 272 W. Fuchs et al.

Table II. In vitro and in vivo replication properties of ILTV recombinants.

Deleted gene Protein function In vitro growth defects In vivo attenuation UL0 nuclear protein +++ ORF A unknown + n.t ORF B unknown + n.t. ORF C unknown + n.t. ORF D unknown + n.t. ORF E unknown + n.t. UL10 “gM”, envelope protein + n.t. UL21 unknown ++ n.t. UL23 thymidine kinase – + UL47 tegument protein ++ UL49.5 gN, envelope glycoprotein ++ n.t. UL50 dUTPase – + US4 gG, secreted glycoprotein – + US5 gJ, envelope glycoprotein ++ ++ The degrees of in vitro growth defects and in vivo attenuation observed after deletion of the indicated genes3,5 [24–27, 40, 41bis, 61, 79, 91, 92] were estimated as moderate (+), or pronounced (++), since standardized experiments would be required for precise evaluation. (–) No effect; (n.t.) not tested. be very difficult. Remarkably, deletion of and gN, as well as the tegument proteins the five Iltovirus-specific genes ORF A to encoded by UL21 and UL47 were also ORF E did not result in severe replication shown to be nonessential in different defects with respect to plaque formation or alphaherpesviruses including ILTV [24, maximum virus titers in cultured cells [91]. 26, 27, 41bis]3. In contrast, recent attempts Thus, these genes apparently do not repre- to delete glycoproteins gE and gI did not sent crucial adaptations to avian host cells, lead to viable recombinants, indicating although more important functions during that these proteins might be essential for infection of chickens cannot be excluded. ILTV replication [21]. Deletion of the ILTV-specific UL0 gene Several of the described gene deletion also caused only minor in vitro growth de- mutants of ILTV have already been tested fects, whereas isolation of ILTV mutants in animals for their suitability as live- lacking the adjacent UL(-1) gene was not virus vaccines (Tab. II). As in other her- possible [92]. This finding indicates that pesviruses, deletion of the TK gene led UL0 and UL(-1), although structurally re- to a marked attenuation, which was also lated, are not functionally equivalent. the case after deletion of gG, or UL47 The conserved herpesvirus genes [40, 41bis, 79]5.DeletionofgJorthe deleted from the ILTV genome include ILTV-specific UL0 gene rendered ILTV UL23 and UL50 encoding two functional almost completely apathogenic for chick- enzymes of nucleotide metabolism, thymi- ens (Fig. 1A) [26, 92]. In contrast, dUT- dine kinase (TK) [40,69,79], and dUTPase Pase negative ILTV was shown to re- [25], respectively. Since most host cells tain a considerable virulence after intratra- possess enzymes with similar activity, the cheal application of high virus doses [25]. two genes are dispensable for replication of ILTV and other herpesviruses analyzed 5 Wild M.A., Cochran M.D., 2001, US patent so far. The glycoproteins gG, gJ, gM No. 6,875,856. Molecular biology of ILTV 273

Nevertheless, this virus may be usable for Furthermore, epidemiological control of mass vaccination with low amounts of avian influenza in the field by serology is virus. Irrespective of their different resid- more difficult after vaccination with com- ual virulence, which remains to be eval- plete AIV. In contrast, the use of vector uated in detail in parallel immunization based or subunit vaccines containing HA experiments with identical virus doses, as the only AIV component would al- all tested ILTV recombinants were able low differentiation of vaccinated from nat- to confer solid protection against chal- urally infected animals using serological lenge infections with virulent ILTV (see tests detecting antibodies against other in- e.g. Fig. 1B) and, therefore, should re- fluenza virus proteins like neuraminidase place the genetically ill-defined vaccines or nucleoprotein (NP) [11, 83, 93]. As currently in use. An additional advantage expected, NP-specific antibodies were not of ILTV recombinants with deletions of found in chickens immunized with HA- immunogenic glycoproteins like gJ, and expressing ILTV but became detectable possibly also gG, would be their suitability after challenge with HPAIV, although no as marker vaccines, which permit serolog- clinical signs were observed (Fig. 5B). ical differentiation of field-virus infected These results clearly demonstrate that from vaccinated animals (DIVA strategy) attenuated ILTV mutants are useful viral [89] on the basis of the presence or ab- vectors, which may also be used for ex- sence of antibodies against the respective pression of immunogenic proteins of other viral gene product [26]. chicken pathogens like infectious bronchi- tis virus, bursal disease virus or fowlpox virus, and also of bacteria or parasites. 6. ILTV AS A VECTOR FOR Further experiments are needed to clarify ANTIGENS OF OTHER CHICKEN whether these ILTV-based vector vaccines PATHOGENS are also suitable for mass administration via aerosol or drinking water, and whether Since ILTV vaccines are suitable for they are effective in the presence of mater- mass application by aerosol or drinking nally derived or vaccination-induced anti- water [39], they may also be useful vectors ILTV immunity. for immunogenic proteins of other chicken pathogens. Besides the genes encoding the fusion protein of Newcastle disease virus 7. CONCLUSION and gB of MDV3, the H5 and H7 hemag- glutinin (HA) genes of highly pathogenic ILTV is significantly less well ana- avian influenza viruses (HPAIV) were suc- lyzed than several other human and animal cessfully expressed in ILTV recombinants herpesviruses. Although its basic param- lacking the dUTPase gene or UL0 [28, 61, eters reflect its inclusion in the Alpha- 92]. Animal trials with the latter constructs herpesvirinae subfamily, there are specific induced ILTV- and HA-specific antibodies features in ILTV that require further anal- and revealed a good protection of immu- ysis. Comparative studies between alpha- nized chickens against challenge infections herpesviruses of the genera with virulent ILTV, as well as with HPAIV and , and the phylogeneti- of the respective serotypes (Fig. 5A). Pro- cally distant Iltoviruses could shed more tection against fowl plague was compa- light on the specific co-evolution of her- rable to that obtained with inactivated pesviruses with their respective hosts. Fur- AIV preparations which, however, require thermore, molecular characterization and subcutaneous or intramuscular application. genetic engineering of ILTV can contribute 274 W. Fuchs et al.

Figure 5. Protection of chickens against fowl plague by marker-vaccination with recombinant ILTV. Ten-week old chickens were immunized by eye-drop application of 105 PFU of an attenuated UL0- negative ILTV mutant expressing H7-hemagglutinin of HPAIV (ILTV-∆UL0H7). After four weeks, vaccinated and control animals (NI) were challenged with 108 egg infectious doses of H7N1 HPAIV. (A) Cumulative clinical scores and mortality rates are shown. (B) At days 22 after immunization (p.i.) and challenge (p.c.) the vaccinated animals were tested for influenza virus nucleoprotein- specific serum antibodies by an indirect ELISA [93]. The mean relative extinctions were plotted, and the proportion of positive (> 2-fold above negative control) sera indicates that hemagglutinin- expressing ILTV might be suitable as marker vaccine against fowl plague. For more details see references [28, 92]. to development of novel, safe and effica- 395/1) and by Intervet International. The expert cious vaccines not only against infectious technical help of G. Czerwinski, C. Ehrlich, M. laryngotracheitis, but also against other im- Jörn and P. Meyer is greatly appreciated. portant chicken diseases.

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