Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17493 Greifswald Insel Riems, Germany
Correspondence
Walter Fuchs
Walter.Fuchs{at}rie.bfav.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To confirm these results, additional Northern blot analyses of RNA from ILTV-infected cells with gene- and strand-specific probes for each of the five ORFs were performed in the present study. Furthermore, ORFs A to E were expressed as bacterial fusion proteins which were used for preparation of monospecific antisera after rabbit immunization. The antisera permitted identification of the protein products of the five ORFs in ILTV-infected cells. To investigate possible functions of ORFs A to E, respective gene deletion mutants of ILTV had to be generated. Such approaches are hampered by the narrow in vitro host range of ILTV, which replicates only in primary chicken cells and, much less efficiently, in a chicken hepatoma cell line (Bagust & Guy, 1997; Schnitzlein et al., 1994
). Since infectious full-length clones of the ILTV genome are also not available, only few virus recombinants, possessing deletions in the nonessential thymidine kinase (UL23), dUTPase (UL50) and UL10 genes, have been described previously (Fuchs & Mettenleiter, 1999
; Fuchs et al., 2000
; Lüschow et al., 2001
; Okamura et al., 1994
; Schnitzlein et al., 1995
). In the present study, we isolated five novel ILTV recombinants exhibiting deletions of ORF A, B, C, D or E, and expressing enhanced green fluorescent protein (EGFP). The in vitro growth properties of these ILTV mutants were compared to those of the parental wild-type virus strain.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro transcription and translation, and constitutive protein expression in eukaryotic cells.
ORFs A to E were amplified individually from genomic ILTV DNA by PCR with Pfx DNA-polymerase (Invitrogen), and custom-made primers which were deduced from the published DNA sequence (GenBank accession no. Y14300; Ziemann et al., 1998a). The PCR products representing nucleotides 71038337 (ORF A), 82679377 (ORF B), 935910385 (ORF C), 1047011603 (ORF D) or 1167012921 (ORF E) of the DNA sequence contained artificial restriction sites at their ends to facilitate directed insertion into the multiple cloning site of pcDNA3 (Invitrogen), which permits in vitro transcription of sense and antisense RNAs from the flanking T7 and SP6 promoters, as well as constitutive protein expression in eukaryotic cells under control of the human cytomegalovirus (HCMV) immediate early gene promoter. Insert fragments were sequenced using T7 and SP6 promoter primers (New England Biolabs), and the plasmids were used for calcium phosphate-mediated transfection (Graham & van der Eb, 1973
) of LMH cells, for in vitro transcription of 32P-labelled cRNA (T7/SP6 transcription kit, Roche), and for in vitro translation (TNT coupled reticulocyte lysate system, Promega) in the presence of [35S]methionine. Radiolabelled in vitro translation products were separated by discontinuous SDS-PAGE in Mini-Protean II cells (Bio-Rad). The gels were treated with Amplify (Amersham), dried and exposed to X-ray films at -70 °C.
RNA analyses.
CEK cells were infected with ILTV at an m.o.i. of 5 p.f.u. per cell and incubated in the presence of 100 µg cycloheximide ml-1 for 6 h (), in the presence of 250 µg phosphonoacetic acid ml-1 for 16 h (
), or in the absence of any drugs for 6 h (
1) and 16 h (
2) at 37 °C. Total RNA of infected and uninfected cells was prepared (Chomczynski & Sacchi, 1987
), separated in denaturing agarose gels and hybridized with radiolabelled cRNAs (see above) as described previously (Fuchs & Mettenleiter, 1996
).
Expression of fusion proteins in bacteria and antiserum preparation.
Parts of ORFs A to E (Fig. 1b) were inserted into plasmid vectors of the pGex-4T (Amersham) or pET-23 (Novagen) series, which permit expression of fusion proteins with glutathione S-transferase (GST), or with a T7-tag peptide in E. coli. Protein expression was induced as recommended by the manufacturers of the vectors. The viral DNA-fragments were recloned from plasmids pILT-S3.1 or pILT-S3.2, which contain a genomic 13 862 bp SalI-fragment of ILTV inserted in opposite orientations into pUC19 (Fig. 1a
; Ziemann et al., 1998a
). Prior to ligation, non-compatible restriction fragment overhangs were blunt-ended by treatment with Klenow polymerase. After cloning of a 1453 bp HpaI/XhoI-fragment representing codons 69377 of ORF A into the SmaI/XhoI double-digested vector pGex-4T1, a 60 kDa GST-fusion protein could be isolated. Codons 4163 of ORF B were expressed as a 20 kDa T7-fusion protein which was obtained after insertion of a 479 bp SphI/XhoI-fragment of ILTV DNA into the EcoRI/XhoI-cleaved plasmid pET-23b. A 1597 bp NcoI/XhoI-fragment containing the entire ORF C of ILTV was inserted into the EcoRI/XhoI-digested vector pET-23a to obtain a 40 kDa T7-fusion protein. An 18 kDa T7-fusion protein containing the 5'-terminal part of ORF D (codons 1126), preceded by 30 bp of originally noncoding DNA was expressed after insertion of a 407 bp MluI/HincII-fragment into the EcoRI/NotI-cleaved vector pET23c. Finally, a 1446 bp NruI/XhoI-fragment including codons 20411 of ORF E was inserted into the SalI/XhoI double-digested vector pET-23b. The 42 kDa T7-fusion protein obtained, as well as the other expression products, were purified after SDS-PAGE of bacterial cell lysates, and used for rabbit immunization as described previously (Fuchs et al., 2002
). Sera collected before and after immunization were analysed.
Western blot analyses and immunofluorescence (IF) tests.
CEK cells were infected with ILTV at an m.o.i. of 2 and incubated at 37 °C for 3, 6, 9, 12 or 24 h. LMH cells were harvested 48 h after calcium phosphate transfection with pcDNA3 expression plasmids. Lysates of infected or transfected, and of uninfected control cells, were separated by SDS-PAGE (ca. 104 cells per lane), and transferred to nitrocellulose filters (Trans-Blot SD cell, Bio-Rad). Blots were incubated with monospecific rabbit antisera, or a mouse monoclonal antibody (MAb) against glycoprotein C (Veits et al., 2003), at dilutions of 1 : 10001 : 5000, and binding of peroxidase-conjugated species-specific secondary antibodies (Dianova) was detected by chemiluminescence as described previously (Fuchs & Mettenleiter, 1999
). For indirect IF tests LMH or CEK cells were fixed with methanol and acetone (1 : 1) 2 or 3 days after either plasmid-transfection or ILTV-infection at low m.o.i., and subsequently incubated with antisera or the MAb (dilutions 1 : 100), and fluorescein-conjugated secondary antibodies as described earlier (Ziemann et al., 1998b
). After chromatin counterstaining with propidium iodide, samples were analysed either by conventional fluorescence microscopy (Diaphot 300, Nikon), or by confocal laser-scan microscopy (LSM 510, Zeiss).
Construction of ILTV recombinants.
For introduction of single gene deletions and concomitant reporter gene insertions within ORFs A to E of ILTV (Fig. 1c) the plasmids pILT-S3.1 or pILT-S3.2 (Ziemann et al., 1998a
) and pBl-GFP (Fuchs & Mettenleiter, 1999
) were used. The latter construct contains an EGFP-expression cassette (Clontech) inserted at the SmaI site within the polylinker of pBluescript SK(-) (Stratagene). To generate a transfer plasmid for deletion of codons 99172 of ORF A (Fig. 1c
), a 2333 bp BamHI/BssHII-fragment, and a 1056 bp PstI-fragment of the ILTV genome were isolated from pILT-S3.1 and subsequently inserted into pBl-GFP, which had been digested with BamHI and AflII, or PstI, respectively. As in the following experiments, non-compatible restriction fragment ends were blunt-ended by Klenow polymerase. For deletion of codons 2298 of ORF B (Fig. 1c
) a 1328 bp fragment generated by digestion of cloned ILTV DNA with EcoRI and partial cleavage with NsiI was inserted into the EcoRI/PstI double-digested vector pBl-GFP. Subsequently, a 4715 bp SstI-fragment of pILT-S3.1 was inserted downstream of the reporter gene cassette. To obtain unique restriction sites for deletion of ORF C, the insert of pILT-S3.2 was truncated by cleavage with XbaI followed by religation. Thereafter, codons 15278 of ORF C were removed by double-digestion with EagI and Eco47III, and replaced by the EGFP expression cassette contained in a 1623 bp EcoRV/XbaI-fragment of pBl-GFP (Fig. 1c
). A transfer plasmid for deletion of codons 126343 of ORF D (Fig. 1c
) was prepared by consecutive insertion of 1100 bp BamHI/NaeI- and 1934 bp KpnI/HincII-fragments of ILTV DNA into pBl-GFP, which had been cleaved with BamHI and AflII, or KpnI and EcoRV, respectively. In a similar manner, 1019 bp SalI/NruI- and 2654 SstI/FspI-fragments of the ILTV genome were inserted into pBl-GFP which had been doubly-digested with SalI and EcoRV, or BamHI and SstI. In the resulting plasmid codons 19357 of ORF E were replaced by the EGFP expression cassette (Fig. 1c
). GFP-expressing virus recombinants were obtained after calcium phosphate-mediated transfection (Graham & van der Eb, 1973
) of LMH cells with the above described transfer plasmids, virion DNA of ILTV A489, and expression constructs of the ILTV homologues of VP16 and ICP4 which have been shown to increase the infectivity of viral DNA (Fuchs et al., 2000
). After plaque purification of the ILTV mutants, genomic DNA was prepared (Fuchs & Mettenleiter, 1996
), cleaved with EcoRI, and analysed by Southern blot hybridization with the 32P-labelled (RediPrime kit, Amersham) plasmid pILT-S3.1 according to described protocols (Fuchs & Mettenleiter, 1999
).
Plaque assays and one-step growth kinetics.
For determination of plaque sizes, LMH cell monolayers were infected with ILTV at a low m.o.i. (<0·001). After 2 h, the inoculum was replaced by medium containing 6 g methylcellulose l-1. After 3 days at 37 °C the diameters of 60 plaques of each GFP-expressing virus mutant were determined by fluorescence microscopy. Plaques of wild-type virus were visualized by indirect IF reactions of a MAb against glycoprotein C of ILTV. Average diameters and standard deviations were calculated. One-step growth analyses were performed essentially as described (Fuchs et al., 2000). Briefly, CEK cells were infected at an m.o.i. of 3, and after 1 h non-penetrated input virus was inactivated by treatment with citric acid (Mettenleiter, 1989
). At different times after infection, cells were scraped into the medium, lysed by freezing and thawing, and progeny virus titres were determined by plaque assays on LMH cells. The average results of two independent experiments were plotted.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Identification of the ORF A to ORF E proteins in plasmid-transfected and in ILTV-infected cells
For detection of the predicted viral gene products of ORF A, ORF B, ORF C, ORF D and ORF E parts of the coding sequences were expressed in bacteria, and monospecific antisera were prepared after immunization of rabbits with the isolated fusion proteins. All sera exhibited specific reactivity in Western blot analyses of LMH cells which had been transfected with the pcDNA3 expression plasmids of the individual ORFs (results not shown). All antisera also detected distinct protein bands in Western blot analyses of ILTV-infected CEK cell lysates (Fig. 3). These proteins were not found in uninfected cells (Fig. 3
) nor with the respective preimmune sera, and the specificity of the antisera could be further confirmed by competition experiments with the bacterial fusion proteins which had been used for immunization (results not shown). Detection of the ORF A to E proteins in ILTV-infected cells was in good agreement with the results of RNA analyses since the ORF C, D and E proteins were first recognized 6 h after infection, whereas the ORF A and ORF B proteins were only detectable after 12 h and 24 h (Table 1
).
|
The 34 kDa ORF B gene product of ILTV (Fig. 3) was significantly smaller than calculated and found after in vitro translation (Table 1
). In cells transfected with the pcDNA3 expression plasmid of ORF B both the 34 kDa protein and the expected 39 kDa gene product were detectable (results not shown). Since no 39 kDa ORF B protein was found at any time in ILTV-infected cells, translation of ORF B might generally initiate at the second in-frame ATG codon at position 27, which would result in a protein with a predicted molecular mass of 35·2 kDa.
The ORF C-specific antiserum reacted reproducibly with two proteins not only in plasmid-transfected cells, but also in cells which were harvested at late times after ILTV infection (Fig. 3). In contrast, only the expected 38 kDa ORF C protein was found in cells harvested 6 or 9 h after ILTV-infection (results not shown), indicating that the primary translation product might be partially cleaved by proteases.
The 34 kDa ORF B protein, as well as the 38 and 30 kDa ORF C gene products, were also detectable by immunoprecipitations performed after metabolic labelling of ILTV-infected CEK cells with [35S]methionine (results not shown), whereas the anti-ORF A, anti-ORF D and anti-ORF E sera showed no specific reactions in this assay. Only the ORF C-specific antiserum was also suitable for indirect IF tests of ILTV-infected CEK cells. Confocal microscopy revealed an almost even distribution of the ORF C protein within the virus-induced syncytia (Fig. 4). Most of the punctate green fluorescent signals were localized in the cytoplasm, but the merged yellow fluorescence observed after chromatin counterstaining with propidium iodide indicated that a portion of the ORF C protein also enters the host-cell nucleus (Fig. 4
). In contrast, envelope glycoprotein gC of ILTV was completely excluded from the nuclei of infected cells, but accumulated in cytoplasmic vesicles and vacuoles which presumably represent the sites of virion maturation (Granzow et al., 2001
; Guo et al., 1993
). In indirect IF analyses of cells transfected with expression plasmids, not only the ORF C, but also ORF B, ORF D and ORF E gene products, exhibited a predominantly cytoplasmic localization (results not shown). However, in ILTV-infected cells expression of the latter proteins was too weak for IF detection, and the ORF A gene product could be localized neither in ILTV-infected nor in plasmid-transfected cells.
|
Western blot analyses of cells infected with the different ILTV recombinants were performed to confirm the absence of the deleted gene products, and to investigate whether expression of adjacent genes was affected (Fig. 5). Whereas similar amounts of glycoprotein C (gC) were found in all ILTV-infected CEK cells, the ORF A, ORF B, ORF C, ORF D or ORF E proteins were not detectable in cells infected with the respective deletion mutants (Fig. 5
). These results did not rule out the possibility that the remaining 5'-terminal parts of the deleted ORFs (Fig. 1c
) are still expressed, since the resulting proteins could be too small or not sufficiently antigenic for detection. However, it appears unlikely that the truncated gene products, if stable at all, are still functional. The undeleted adjacent ORFs of each of the five ILTV recombinants were still expressed, and the detected proteins exhibited similar sizes like the gene products of wild-type ILTV (Fig. 5
). In most cases, the protein expression levels were also comparable, except that the amount of ORF D was reduced in cells infected with ILTV
ORF E-G, whereas ORF A was overexpressed by ILTV
ORF B-G. These effects might indicate a functional interdependence of the different ORF proteins, but could also be caused by the deletion or insertion of transcription-regulating DNA sequences. In cells infected with some of the mutants and with wild-type ILTV, smaller than full-length forms of the ORF A and ORF B proteins were visible (Fig. 6
). However, these protein bands were not reproducibly detected (see Fig. 3
) and presumably represent degradation products.
|
|
Previous studies have shown that insertion of an EGFP reporter gene cassette might affect replication of ILTV irrespective of the deleted viral DNA sequences (Fuchs et al., 2000). Therefore, a recombinant LMH cell line carrying the ORF A, B, C, D and E genes of ILTV was generated and used for plaque-assays with the EGFP-expressing virus mutants. In these cells the plaque sizes of ILTV
ORF A-G,
ORF C-G,
ORF D-G or
ORF E-G were not restored to the wild-type level, although cellular expression of all five proteins could be demonstrated by Western blot analyses performed after infection with the respective ILTV deletion mutants (results not shown). At present it is not clear whether the amount of protein provided by the cells was insufficient, whether the ORF deletion mutants possess unexpected second site mutations, or whether the observed plaque formation defects are indeed caused by the reporter gene insertion.
Toxic effects of the strongly expressed reporter protein would also explain the rapid reappearance of non-fluorescent virus plaques during passage of some of the purified EGFP-expressing ILTV recombinants in non-complementing cells. Two such virus mutants derived from ILTV ORF A-G and
ORF C-G, respectively, were plaque-purified, and their genomes were analysed by Southern blot hybridization, PCR amplification and DNA sequencing, which confirmed that in both cases parts of the reporter gene cassette including the HCMV immediate early gene promoter and coding sequences of EGFP were deleted. Interestingly, these spontaneous mutations also affected flanking viral DNA sequences (Fig. 1c
). The virus mutant ILTV
ORF A-C exhibited a deletion including ORF A (codons 99377), ORF B (complete) and ORF C (codons 315335), whereas the deletion of ILTV
ORF C-E contained ORF C (codons 1278), ORF D (complete) and ORF E (codons 183411). Thus, not only single, but also triple gene deletion mutants of the ILTV-specific ORFs A to E are viable in cell culture. However, the triple mutants exhibited significant in vitro growth defects. Plaque diameters of both viruses on LMH cells were reduced to ca. 35 % of the wild-type size, and maximum titres on CEK cells were reduced ca. tenfold. These findings indicate that the replication deficiencies of the above described single gene deletion mutants are presumably not solely caused by expression of the EGFP reporter protein. Repeated attempts to generate an ILTV recombinant lacking all five unique ORFs were not successful. Therefore, it remains possible that ORFs A to E possess important but redundant functions for virus replication, like those discussed for several conserved tegument and envelope proteins involved in alphaherpesvirus egress (Mettenleiter, 2002
).
In vivo studies have to be performed to assay the influence of the gene deletions on virulence of ILTV. Such studies are of particular interest, since ORFs A to E, like other unique genes of individual herpesviruses, might only be relevant for replication in the natural host organism. For example, several cytomegalovirus-specific gene products were identified as functional homologues of host proteins, and were shown to mediate immune evasion by the virus (Hengel et al., 1998). In a similar manner, the expression of a viral homologue of cellular oncogenes by MDV was discussed as a possible reason for the induction of T-cell tumours in chickens (Jones et al., 1992
). ILTV does not induce tumours, and considering its rapid replication in the respiratory tract of chickens during acute infection (Bagust & Guy, 1997
), immune evasion strategies might be dispensable. However, like MDV and unlike most other alphaherpesviruses, ILTV exhibits a very narrow host range consisting only of chickens and their closest cognates (Bagust & Guy, 1997
). Therefore, it is possible that the species-specific genes of ILTV are adaptations that benefit replication in its avian host. In consequence, some of the generated deletion mutants might be sufficiently attenuated to be suitable as live-virus vaccines against infectious laryngotracheitis of chickens.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ben-Porat, T., Veach, R. A. & Ihara, S. (1983). Localization of the regions of homology between the genomes of herpes simplex virus type 1 and pseudorabies virus. Virology 127, 194204.[Medline]
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Anal Biochem 162, 156159.[CrossRef][Medline]
Davison, A. J. & Scott, J. E. (1986). The complete DNA sequence of varicella-zoster virus. J Gen Virol 67, 17591816.[Abstract]
Fuchs, W. & Mettenleiter, T. C. (1996). DNA sequence and transcriptional analysis of the UL1 to UL5 gene cluster of infectious laryngotracheitis virus. J Gen Virol 77, 22212229.[Abstract]
Fuchs, W. & Mettenleiter, T. C. (1999). DNA sequence of the UL6 to UL20 genes of infectious laryngotracheitis virus and characterization of the UL10 gene product as a nonglycosylated and nonessential virion protein. J Gen Virol 80, 21732182.
Fuchs, W., Ziemann, K., Teifke, J. P., Werner, O. & Mettenleiter, T. C. (2000). The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor. J Gen Virol 81, 627638.
Fuchs, W., Klupp, B. G., Granzow, H., Osterrieder, N. & Mettenleiter, T. C. (2002). The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J Virol 76, 364378.
Graham, F. L. & van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456467.[Medline]
Granzow, H., Klupp, B. G., Fuchs, W., Veits, J., Osterrieder, N. & Mettenleiter, T. C. (2001). Egress of alphaherpesviruses: comparative ultrastructural study. J Virol 75, 36753684.
Guo, P., Scholz, E., Turek, J., Nodgreen, R. & Maloney, B. (1993). Assembly pathway of avian infectious laryngotracheitis virus. Am J Vet Res 54, 20312039.[Medline]
Hengel, H., Brune, W. & Koszinowski, U. H. (1998). Immune evasion by cytomegalovirus survival strategies of a highly adapted opportunist. Trends Microbiol 6, 190197.[CrossRef][Medline]
Johnson, M. A. & Tyack, S. G. (1995). Molecular evolution of infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1): an ancient example of the Alphaherpesviridae? Vet Microbiol 46, 221231.[CrossRef][Medline]
Johnson, M. A., Tyack, S. G., Prideaux, C. T., Kongsuwan, K. & Sheppard, M. (1997). Nucleotide sequence of the left-terminus of infectious laryngotracheitis virus (gallid herpesvirus 1) SA-2 strain. Arch Virol 142, 19031910.[CrossRef][Medline]
Jones, D., Lee, L., Liu, J. L., Kung, H. J. & Tillotson, J. K. (1992). Marek disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblastoid tumors. Proc Natl Acad Sci U S A 89, 40424046.[Abstract]
Kawaguchi, T., Nomura, K., Hirayama, Y. & Kitagawa, T. (1987). Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res 47, 44604464.[Abstract]
Leib, D. A., Bradbury, J. M., Hart, C. A. & McCarthy, K. (1987). Genome isomerism in two alphaherpesviruses: herpesvirus saimiri-1 (herpesvirus tamarinus) and avian infectious laryngotracheitis virus. Arch Virol 93, 287294.[Medline]
Lüschow, D., Werner, O., Mettenleiter, T. C. & Fuchs, W. (2001). Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene. Vaccine 19, 42494259.[CrossRef][Medline]
McGeoch, D. J., Dalrymple, M. A., Davison, A. J., Dolan, A., Frame, M. C., McNab, D., Perry, L. J., Scott, J. E. & Taylor, P. (1988). The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol 69, 15311574.[Abstract]
McGeoch, D. J., Dolan, A. & Ralph, A. C. (2000). Toward a comprehensive phylogeny for mammalian and avian herpesviruses. J Virol 74, 1040110406.
Mettenleiter, T. C. (1989). Glycoprotein gIII deletion mutants of pseudorabies virus are impaired in virus entry. Virology 171, 623625.[Medline]
Mettenleiter, T. C. (2002). Herpesvirus assembly and egress. J Virol 76, 15371547.
Minson, A. C., Davison, A., Eberle, R., Desrosiers, R. C., Fleckenstein, B., McGeoch, D. J., Pellet, P. E., Roizman, B. & Studdert, D. M. J. (2000). Family Herpesviridae. In Virus Taxonomy, pp. 203225. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Okamura, H., Sakaguchi, M., Honda, T., Taneno, A., Matsuo, K. & Yamada, S. (1994). Construction of recombinant infectious laryngotracheitis virus expressing the lacZ gene of E. coli with thymidine kinase gene. J Vet Med Sci 56, 799801.[Medline]
Rauh, I. & Mettenleiter, T. C. (1991). Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration. J Virol 65, 53485356.[Medline]
Roizman, B. & Knipe, D. M. (2001). Herpes simplex viruses and their replication. In Fields Virology, 4th edn, pp. 23992459. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Roizman, B. & Pellett, P. E. (2001). The family Herpesviridae: a brief introduction. In Fields Virology, 4th edn, pp. 23812397. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Schnitzlein, W. M., Radzevicius, J. & Tripathy, D. N. (1994). Propagation of infectious laryngotracheitis virus in an avian liver cell line. Avian Dis 38, 211217.[Medline]
Schnitzlein, W. M., Winans, R., Ellsworth, S. & Tripathy, D. N. (1995). Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus. Virology 209, 304314.[CrossRef][Medline]
Telford, E. A. R., Watson, M. S., McBride, K. & Davison, A. J. (1992). The DNA sequence of equine herpesvirus-1. Virology 189, 304316.[Medline]
Tulman, E. R., Alfonso, C. L., Lu, Z., Zsak, L., Rock, D. L. & Kutish, G. F. (2000). The genome of a very virulent Marek's disease virus. J Virol 74, 79807988.
Veits, J., Köllner, B., Teifke, J. P., Granzow, H., Mettenleiter, T. C. & Fuchs, W. (2003). Isolation and characterization of monoclonal antibodies against structural proteins of infectious laryngotracheitis virus. Avian Dis (in press).
Wild, M. A., Cook, S. & Cochran, M. (1996). A genomic map of infectious laryngotracheitis virus and the sequence and organization of genes present in the unique short and flanking regions. Virus Genes 12, 107116.[Medline]
Ziemann, K., Mettenleiter, T. C. & Fuchs, W. (1998a). Gene arrangement within the unique long genome region of infectious laryngotracheitis virus is distinct from that of other alphaherpesviruses. J Virol 72, 847852.
Ziemann, K., Mettenleiter, T. C. & Fuchs, W. (1998b). Infectious laryngotracheitis herpesvirus expresses a related pair of unique nuclear proteins which are encoded by split genes located at the right end of the UL genome region. J Virol 72, 68676874.
Received 26 October 2002;
accepted 18 February 2003.