1 Institutes of Molecular Biology, Friedrich-Loeffler Institutes, Federal Research Centre for Virus Diseases of Animals, D-17493 Greifswald-Insel Riems, Germany
2 Institutes of Diagnostic Virology, Friedrich-Loeffler Institutes, Federal Research Centre for Virus Diseases of Animals, D-17493 Greifswald-Insel Riems, Germany
3 Institutes of Infectology, 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
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ABSTRACT |
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Present address: Institute of Poultry Diseases, Department of Veterinary Medicine, FU Berlin, D-14195 Berlin, Germany.
Present address: Riemser Arzneimittel AG, D-17493 Greifswald-Insel Riems, Germany.
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INTRODUCTION |
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Recently, we demonstrated that in infected chicken cell cultures, seven of these ILTV-specific genes are indeed transcribed and translated into proteins (Veits et al., 2003b; Ziemann et al., 1998b
). The protein products of a cluster consisting of five ORFs (AE) located downstream of UL22 were detected predominantly in the host cell cytoplasm (Veits et al., 2003b
). In contrast, the products of the ILTV-specific UL0 and UL[-1] genes at the right end of the UL region (Fig. 1
) accumulate in the nuclei of infected cells (Ziemann et al., 1998b
). The common localization of the UL0 and UL[-1] proteins correlates with similar gene structures, since both ORFs contain small introns within their 5'-terminal parts, which are removed by mRNA splicing (Ziemann et al., 1998b
). Furthermore, UL0 and UL[-1] exhibit a significant amino acid sequence homology of 28 %, indicating that the two genes presumably originated from an ancient duplication event (Ziemann et al., 1998b
). By construction of genetically engineered virus recombinants, we demonstrated that the ILTV-specific ORF AE genes are dispensable for virus replication in cultured cells and speculated that these genes might be relevant only during infection of chickens (Veits et al., 2003b
).
In the present study, UL0 deletion mutants of ILTV were isolated and characterized in vitro and in vivo to evaluate their suitability as live virus vaccines. To date, the only ILT vaccines in use were generated by serial egg or cell culture passages of field virus isolates and these vaccines have not been genetically characterized (Bagust & Guy, 1997). These ILTV strains are suitable for mass application via eye drop, aerosol or drinking water, but, in numerous cases, turned out to be insufficiently attenuated or reverted to a virulent phenotype (Bagust & Guy, 1997
). ILTV recombinants with defined gene deletions might be safer vaccines, but, to date, only a few virus mutants of this type have been tested in vivo. Whereas deletion of the viral thymidine kinase gene UL23 led to attenuation of ILTV in chickens (Okamura et al., 1994
; Schnitzlein et al., 1995
), ILTV mutants lacking the UL50 gene, which encodes another enzyme of nucleotide metabolism, dUTPase, remained virulent (Fuchs et al., 2000
; Lüschow et al., 2001
).
Considering the easy mass application of ILTV live virus vaccines, ILTV recombinants might be useful vectors for expression of immunogenic proteins of other chicken pathogens. As demonstrated by recent epidemics in Hong Kong, Italy and The Netherlands (Abbott, 2003; Capua et al., 1999
; Capua & Marangon, 2000
; Shortridge et al., 1998
), fowl plague is one of the economically most important infectious diseases of chickens, with mortality rates of up to 100 %. Fowl plague is caused by highly pathogenic avian influenza A viruses (AIV) carrying the antigenically distinct haemagglutinin (HA) subtypes H5 or H7 (Alexander, 2000
). The HA of influenza viruses is an envelope glycoprotein that is required for attachment to and penetration of host cells and it represents a major target of the host immune response (Easterday et al., 1997
; Lamb & Krug, 2001
). Thus, not only inactivated AIV (Allan et al., 1971
; Swayne et al., 1999
, 2001
) but also subunit vaccines containing vaccinia virus- or baculovirus-expressed HA proteins (Chambers et al., 1988
; Crawford et al., 1999
), DNA vaccines containing the HA gene (Fynan et al., 1993
; Kodihalli et al., 2000
) or fowlpox virus recombinants expressing HA (Swayne et al., 2000
; Taylor et al., 1988
) have been shown to protect domestic fowl against infections with highly pathogenic AIV strains possessing the corresponding HA subtypes. HA subtype H5 has already been expressed in an UL50-negative ILTV recombinant and chickens vaccinated with this virus were protected against lethal AIV challenge (Lüschow et al., 2001
).
In the present study, the UL0 gene locus of ILTV was used for insertion of a H7 subtype HA gene, which had been reverse-transcribed and cloned from an AIV field isolate of the recent fowl plague epidemic in Italy (Capua & Marangon, 2000). The ILTV recombinant obtained was tested for HA expression by immunofluorescence and Western blot analyses. In vitro growth properties of the HA-expressing ILTV mutant, an UL0 deletion mutant without foreign gene insertion and an UL0 rescue mutant were also analysed. Furthermore, animal experiments were performed to determine whether the UL0 deletion mutants are attenuated and whether they confer protective immunity against ILT and fowl plague.
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METHODS |
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Construction of UL0 deletion plasmids and ILTV recombinants.
Plasmid pILT-E43, which contains an 11·2 kbp EcoRI fragment of the ILTV genome (Ziemann et al., 1998b), was shortened by consecutive HindIII/BstXI and EcoRI/XhoI double digestions and re-ligations. As in subsequent experiments, non-compatible, single-stranded overhangs of digested DNA were polished by treatment with Klenow polymerase prior to ligation. The 3997 bp insert of the resulting plasmid pILT-E43BX included the entire UL0 gene of ILTV (Fig. 1
). To obtain p
UL0, 1137 bp of the UL0 ORF were removed by double digestion of pILT-E43BX with BssHII/XbaI and subsequent re-ligation (Fig. 1
). A second deletion plasmid, p
UL0-G (Fig. 1
), was generated by substitution of a 984 bp ClaIBsrBI fragment by a 1615 bp ClaIAflII fragment of pBl-GFP (Fuchs & Mettenleiter, 1999
), which contains an expression cassette for EGFP under the control of the major immediateearly promoter of human cytomegalovirus (PHCMV-IE). Plasmid p
UL0-G and genomic DNA of ILTV A489 were used for calcium phosphate-mediated co-transfection of LMH cells (Fuchs et al., 2000
). Eukaryotic expression constructs encoding the trans-activating ICP4 and UL48 proteins of ILTV were also added to the transfection mixture, as they have been shown to enhance virus replication (Fuchs et al., 2000
). EGFP-expressing virus recombinants were identified by fluorescence microscopy and isolated by limiting dilutions of the transfection progeny on CEK cells grown in microtitre plates. Genomic DNA of the isolated virus recombinant ILTV
UL0-G (Fig. 1
) was prepared (Fuchs & Mettenleiter, 1996
) and used for subsequent co-transfection experiments with plasmids pILT-E43BX and p
UL0. By screening transfection progenies for non-fluorescent plaques, the UL0 rescue mutant ILTV UL0R and the deletion mutant ILTV
UL0, which contain no reporter gene insertion, were isolated.
Cloning and expression of influenza virus HA.
Influenza virus particles were sedimented from allantoic fluid harvested 4 days after inoculation of embryonated chickens' eggs with AIV A/chicken/Italy/445/99 by centrifugation at 50 000 g for 30 min, and genomic viral RNA was prepared (Chomczynski & Sacchi, 1987). Consensus primers for amplification of genome segment 4 were deduced from published nucleotide sequences of H7 subtype HA genes of other influenza viruses. The custom-made (Invitrogen) primers AI-H7F (5'-GGGATACAAAATGAAC/TACTC-3') and AI-H7R (5'-CCAAACTTATATACAAATAGTGC-3') were used for reverse transcription and subsequent PCR amplification, as described (Lüschow et al., 2001
). The 1711 bp amplification products were inserted into the SmaI-digested plasmid vector pUC19. Using 32P-labelled vector- and insert-specific primers, the cloned products of two independent PCR reactions were sequenced (Fuchs & Mettenleiter, 1999
). The resulting insert DNA sequence (GenBank accession no. AJ580353) of the two pUC-H7 plasmids was identical and encoded a HA protein of 564 aa. For HA expression, PHCMV-IE was excised from pcDNA3 (Invitrogen) as a 685 bp NruIHindIII fragment and inserted upstream of the cloned HA ORF after digestion of pUC-H7 with XbaI/SalI. From the plasmid obtained, the HA expression cassette was re-cloned as a 2431 bp HindIIIKpnI fragment into p
UL0-G, from which the GFP insertion had been removed by digestion with HindIII/XbaI. The resulting plasmid, p
UL0-H7 (Fig. 1
), together with genomic DNA of ILTV
UL0-G, was used for co-transfection of LMH cells. Recombinant ILTV
UL0-H7 was isolated from non-fluorescent progeny virus plaques.
Western blot analyses.
CEK cells were inoculated with AIV A/chicken/Italy/445/99 (H7N1), wild-type ILTV A489 or ILTV mutants at an m.o.i. of 2 and incubated at 37 °C for 24 h. Lysates of ca. 104 infected or uninfected cells per lane were separated by SDS-PAGE in 10 % polyacrylamide gels and transferred to nitrocellulose filters, as described (Fuchs & Mettenleiter, 1999). Blots were incubated with an ILTV UL0-specific rabbit antiserum at a dilution of 1 : 10 000 (Ziemann et al., 1998b
), an ILTV glycoprotein C-specific monoclonal antibody (mAb) at a dilution of 1 : 500 (Veits et al., 2003a
) or with chicken antiserum, which was obtained after repeated immunization with the inactivated influenza virus isolate A/FPV/Dutch/27 (H7N7) and subsequent booster infection with the same virus (dilution 1 : 1000). Antibody binding was detected by luminescence reactions (SuperSignal System, Pierce) of peroxidase-conjugated secondary antibodies (Dianova) and recorded on X-ray films (Hyperfilm MP, Amersham).
Indirect immunofluorescence (IIF) assays.
CEK or LMH cells were fixed with a 1 : 1 mixture of methanol and acetone at 13 days after ILTV- or AIV-infection at low m.o.i. (<0·001) and incubated with an AIV-specific antiserum (see above), with chicken sera from the animal trials (see below) or with an ILTV glycoprotein J-specific mAb (Veits et al., 2003a) at dilutions of 1 : 100, as described (Veits et al., 2003a
). Binding of fluorescein-conjugated secondary antibodies (Dako and Dianova) was analysed by fluorescence microscopy (Diaphot 300, Nikon) after chromatin counterstaining with propidium iodide.
Plaque assays and one-step growth kinetics.
For determination of plaque sizes, LMH cell monolayers were infected in parallel with the respective ILTV mutants at low m.o.i. After 2 h, the inoculum was replaced with medium containing 6 g methylcellulose l-1 and incubation was continued for 3 days at 37 °C. The diameters of 50 plaques per virus mutant were determined by fluorescence microscopy either directly (ILTV UL0-G) or after IIF with a glycoprotein J-specific mAb. Average diameters and SD values were calculated and the statistical significance of differences was analysed using Student's t-test. One-step growth analyses of ILTV were performed essentially as described previously (Fuchs et al., 2000
). At different times post-infection (p.i.) (1, 6, 12, 18, 24 and 48 h) at an m.o.i. of 4, CEK cells were scraped into the medium, lysed by freezethaw incubation and progeny virus titres were determined by plaque assays on LMH cells. The average results of two independent experiments were plotted.
Animal experiments.
White Leghorn chickens were bred from specific-pathogen-free eggs (Lohmann Tierzucht). At 10 weeks of age, the animals were separated into four groups, and infected with either wild-type ILTV A489 or recombinants ILTV UL0,
UL0-H7 or UL0R. The viruses were diluted in a blue-coloured solvent (Intervet) and ca. 5x103 p.f.u. of virus per animal were administered via eye drop. Chickens were observed daily for clinical symptoms for a 2 week period and tracheal swabs were taken at 3, 4 and 5 days after infection (p.i.) for re-isolation of ILTV. Sera collected before infection and at day 15 p.i. were tested for ILTV- and AIV-specific antibodies by IIF or HAI tests (Lüschow et al., 2001
), respectively. At day 25 p.i., six animals from each group, as well as eight non-immunized chickens, were infected intratracheally with 2x105 p.f.u. of the virulent ILTV strain A489. Furthermore, 14 chickens that had been immunized with ILTV
UL0-H7, and three chickens that had been immunized with ILTV
UL0, were challenged by oculonasal inoculation with 0·2 ml allantoic fluid containing 6x107 mean embryo infectious doses (EID50) of the highly pathogenic AIV isolate A/chicken/Italy/445/99 (H7N1). Clinical signs were monitored and tracheal and cloacal swabs were taken to re-isolate AIV or ILTV. For re-isolation of ILTV, tracheal swabs taken at 3, 4 and 5 days after immunization or challenge infection were analysed by plaque assay on LMH cells, whereas AIV was identified in tracheal and cloacal swabs taken at 3, 6 and 10 days after infection by passage in embryonated chickens' eggs and subsequent testing of the allantoic fluids for haemagglutinating activity (Lüschow et al., 2001
). At 2 weeks after challenge infection, all surviving animals were necropsied and investigated for pathological alterations. Clinical scores were determined over the 2 week period after immunization as well as after challenge infection. To this end, the chickens were classified on a daily basis as healthy (0), ill (1), severely ill (2) or dead (3). For all animals of each group, mean values were calculated for each day (daily scores) and for the period of days 212 after ILTV, or days 110 after AIV, infection, respectively (total scores). To determine total scores, dead animals were considered until the end of the monitoring period but were no longer included in the daily scores after the day of death.
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RESULTS AND DISCUSSION |
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Protein synthesis of wild-type ILTV and UL0 mutants was compared by Western blot analyses of infected CEK cells (Fig. 2). Whereas similar amounts of glycoprotein C were detectable with a specific mAb (Veits et al., 2003a
) in cells infected with either virus, the reactions of an UL0-specific antiserum revealed that the 63 kDa gene product (Ziemann et al., 1998b
) was only expressed by ILTV A489 and ILTV UL0R (Fig. 2
). Furthermore, the analyses provided no evidence for expression of truncated UL0 gene products from the 3'-terminal part of the ORF which is retained in the deletion mutants.
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Apart from these effects of foreign gene insertions, our studies demonstrated that the UL0 gene plays only a minor role during in vitro replication of ILTV. Possibly, this can be explained by the presence of the adjacent UL[-1] gene (Fig. 1), which encodes a protein that shares significant sequence homology with the UL0 gene product (Ziemann et al., 1998b
) and therefore might possess related functions.
UL0 deletion mutants are attenuated in vivo and protect against ILTV challenge infection
Although UL0 is not required for ILTV replication in cultured chickens' cells, the situation in the animal host might be different. To test this possibility, 10-week-old chickens were infected by ocular administration of 5x103 p.f.u. of wild-type ILTV A489, ILTV UL0R or deletion mutants ILTV UL0 and
UL0-H7. From days 2 to 12 after immunization, clinical signs of ILT, such as respiratory disorder and conjunctivitis, were monitored and scored (Table 1
and Fig. 4
A). Only 3 of 29 chickens infected with the UL0 deletion mutants ILTV
UL0 or
UL0-H7 developed slight respiratory symptoms for a few days, whereas the other animals remained completely healthy (Table 1
). Morbidity rates and clinical scores were significantly higher in animals infected with wild-type ILTV A489 or with the rescue mutant virus ILTV UL0R (Table 1
and Fig. 4A
). Tracheal swabs were taken from all animals at days 3, 4 and 5 after infection to detect virus shedding. Whereas ILTV A489 and UL0R were isolated from most animals of the respective groups, ILTV
UL0 was re-isolated from only 2 of 9 animals (Table 1
). This finding indicates a correlation between the efficiency of in vivo replication and virulence. Attenuation of UL0 deletion mutants of ILTV has also been confirmed by safety tests performed by intratracheal administration of 103 p.f.u. of virus to 10-day-old chickens. In these studies, all animals tested survived infection with ILTV
UL0, whereas in chickens infected with virulent ILTV A489 the mortality rate was 49 % (J. Claessens & W. Fuchs, European patent no. EP1241177).
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Immunization with an ILTV UL0 deletion mutant expressing influenza virus HA (H7) protects chickens from fowl plague
To generate a vector vaccine against one group of fowl plague viruses, we have previously used the UL50 gene locus of ILTV for insertion and expression of an AIV HA gene, subtype H5 (Lüschow et al., 2001). Since fowl plague is also caused by influenza viruses possessing HA genes of subtype H7, a second ILTV recombinant containing the respective ORF of the highly pathogenic AIV isolate A/chicken/Italy/445/99 (H7N1) was constructed. However, considering the insufficient attenuation of ILTV UL50 mutants (Fuchs et al., 2000
; Lüschow et al., 2001
), the foreign gene was inserted at the UL0 gene locus. In ILTV
UL0-H7 (Fig. 1
), the HA gene is preceded by a strong heterologous promoter (PHCMV-IE), and the endogenous polyadenylation signal of the co-terminally transcribed UL0, UL1 and UL2 genes (Fig. 1
) (Fuchs & Mettenleiter, 1996
) permits processing of the 3' end of the HA mRNA.
Abundant HA expression was shown by IIF and Western blot analyses of infected CEK cells (Figs 5 and 6). After incubation with an AIV (H7N7)-specific chicken antiserum, a pronounced cytoplasmic fluorescence was detectable in cells infected with either AIV A/chicken/Italy/445/99 (H7N1) or ILTV
UL0-H7 but not in cells infected with wild-type ILTV A489 (Fig. 5
, left panels). As expected, a mAb against glycoprotein J of ILTV (Veits et al., 2003a
) reacted with cells infected with ILTV A489 or
UL0-H7 but not with AIV-infected cells (Fig. 5
, right panels). In Western blot analyses of infected cell lysates, the AIV-specific chicken serum detected two proteins (ca. 48 and 26 kDa) of ILTV
UL0-H7, which were not expressed by ILTV A489 but which correspond to proteins found in AIV-infected cells (Fig. 6A
). Presumably, these proteins represent HA1 and HA2, which are generated by protease cleavage during the processing of influenza virus HA (Lamb & Krug, 2001
). Similar to the situation that occurs in other highly pathogenic AIV strains, the predicted cleavage site at position 343 of the protein investigated is preceded by multiple basic amino acids (KGSRVRR/GLF), which permit efficient processing by ubiquitous cellular proteases (Alexander, 2000
). Identical cleavage site sequences were also determined for other H7N1 influenza virus isolates of the fowl plague outbreak in Italy during 1999 and 2000 (Capua & Marangon, 2000
). Efficient cleavage might explain that no HA proteins exhibiting the apparent mass of the primary translation product (62·7 kDa) or larger glycosylated precursor proteins were detectable in cells infected with ILTV
UL0-H7. A third AIV-specific protein of ILTV
UL0-H7 (ca. 42 kDa) (Fig. 6A
) does not resemble any of the authentic AIV gene products and might result from either incomplete glycosylation or degradation of HA1. A control blot was incubated with an ILTV-specific mAb (Veits et al., 2003a
) that detected comparable amounts of glycoprotein C in cells infected with ILTV
UL0-H7 or A489 (Fig. 6B
).
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Although it remains to be tested to what extent ILTV UL0-H7 is efficacious against heterologous fowl plague viruses of the H7 subtype, like that of the recent outbreak in The Netherlands (Abbott, 2003
), our studies demonstrate that a single live virus vaccination with this recombinant ILTV protected chickens from a lethal infection with homologous AIV and from severe symptoms of fowl plague. Virus shedding and the minor clinical signs observed after challenge infection might be reduced further by repeated immunization or by administration of higher doses of ILTV
UL0-H7. On the other hand, it should be mentioned that inactivated AIV, as well as other vector, subunit and DNA vaccines, did not completely prevent shedding of highly pathogenic AIV strains of the H5 or H7 subtypes (Crawford et al., 1999
; Kodihalli et al., 2000
; Swayne et al., 1999
, 2000
, 2001
). Apparently, the efficacy of inactivated AIV and of recombinant vaccines containing only the HA gene or protein is comparable (Easterday et al., 1997
). However, the use of recombinant HA vaccines would facilitate serological discrimination between immunized and field virus-infected animals by diagnostic tests for antibodies against other AIV proteins and might permit a relaxation of the present restrictions on vaccination against fowl plague. When compared to inactivated AIV, as well as to subunit, DNA or fowlpox vector vaccines, ILTV live virus vaccines have the additional advantage that they are suitable for mass application, not only by eye drop but also by aerosol or drinking water (Bagust & Guy, 1997
). The only disadvantage of the vector vaccine presented might be that the narrow host range of ILTV permits efficient replication only in chickens but not in turkeys and ducks, which are also infected frequently with AIV (Bagust & Guy, 1997
; Alexander, 2000
).
However, since chickens are among the most economically important domestic animals, we started to generate further ILTV recombinants containing foreign gene insertions at the UL0 gene locus, which apparently confers a better attenuation than the UL50 gene locus used previously (Fuchs et al., 2000; Lüschow et al., 2001
). These insertions include not only the HA gene of highly pathogenic AIV of the H5 subtype but also genes encoding immunogenic proteins of other chicken pathogens, such as Newcastle disease virus and Marek's disease virus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alexander, D. J. (2000). A review of avian influenza in different bird species. Vet Microbiol 74, 313.[CrossRef][Medline]
Allan, W. H., Madeley, C. R. & Kendal, A. P. (1971). Studies with avian influenza A viruses: cross protection experiments in chickens. J Gen Virol 12, 7984.[Medline]
Bagust, T. J. & Guy, J. S. (1997). Laryngotracheitis. In Diseases of Poultry, 10th edn, pp. 527539. Edited by B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald & Y. M. Saif. Ames: Iowa State University Press.
Capua, I. & Marangon, S. (2000). Avian influenza in Italy (19992000): a review. Avian Pathol 29, 289294.[CrossRef]
Capua, I., Marangon, S., Selli, L., Alexander, D. J., Swayne, D. E., Dalla Pozza, M., Parenti, E. & Cancellotti, F. M. (1999). Outbreaks of highly pathogenic avian influenza (H5N2) in Italy during October 1997 to January 1998. Avian Pathol 28, 455460.[CrossRef]
Chambers, T. M., Kawaoka, Y. & Webster, R. G. (1988). Protection of chickens from lethal influenza infection by vaccinia-expressed hemagglutinin. Virology 167, 414421.[Medline]
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Anal Biochem 162, 156159.[CrossRef][Medline]
Crawford, J., Wilkinson, B., Vosnesensky, A., Smith, G., Garcia, M., Stone, H. & Perdue, M. L. (1999). Baculovirus-derived hemagglutinin vaccines protect against lethal influenza virus infections by avian H5 and H7 subtypes. Vaccine 17, 22652274.[CrossRef][Medline]
Easterday, B. C., Hinshaw, V. S. & Halvorson, D. A. (1997). Influenza. In Diseases of Poultry, 10th edn, pp. 583605. Edited by B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald & Y. M. Saif. Ames: Iowa State University Press.
Everett, R. D., Barlow, P., Milner, A., Luisi, B., Orr, A., Hope, R. & Lyon, D. (1993). A novel arrangement of zinc-binding residues and secondary structure in the C3HC4 motif of an alpha herpes virus protein family. J Mol Biol 234, 10381047.[CrossRef][Medline]
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.
Fynan, E. F., Robinson, H. L. & Webster, R. G. (1993). Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. DNA Cell Biol 12, 785789.[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., Prideaux, C. T., Kongsuwan, K., Sheppard, M. & Fahey, K. J. (1991). Gallid herpesvirus 1 (infectious laryngotracheitis virus): cloning and physical maps of the SA-2 strain. Arch Virol 119, 181198.[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]
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]
Kodihalli, S., Kobasa, D. L. & Webster, R. G. (2000). Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines. Vaccine 18, 25922599.[CrossRef][Medline]
Lamb, R. A. & Krug, R. M. (2001). Orthomyxoviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 14871531. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
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.
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. Seventh Report of the International Committee on Taxonomy of Viruses, 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]
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]
Shortridge, K. F., Zhou, N. N., Guan, Y. & 12 other authors (1998). Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology 252, 331342.[CrossRef][Medline]
Swayne, D. E., Beck, J. R., Garcia, M. & Stone, H. D. (1999). Influence of virus strain and antigen mass on efficacy of H5 avian influenza virus inactivated vaccines. Avian Pathol 28, 245255.[CrossRef]
Swayne, D. E., Garcia, M., Beck, J. R., Kinney, N. & Suarez, D. L. (2000). Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 18, 10881095.[CrossRef][Medline]
Swayne, D. E., Beck, J. R., Perdue, M. L. & Beard, C. W. (2001). Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza. Avian Dis 45, 355365.[Medline]
Taylor, J., Weinberg, R., Kawaoka, Y., Webster, R. G. & Paoletti, E. (1988). Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine 6, 504508.[CrossRef][Medline]
Veits, J., Köllner, B., Teifke, J. P., Granzow, H., Mettenleiter, T. C. & Fuchs, W. (2003a). Isolation and characterization of monoclonal antibodies against structural proteins of infectious laryngotracheitis virus. Avian Dis 47, 330342.[Medline]
Veits, J., Mettenleiter, T. C. & Fuchs, W. (2003b). Five unique open reading frames of infectious laryngotracheitis virus are expressed during infection but are dispensable for virus replication in cell culture. J Gen Virol 84, 14151425.
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 12 August 2003;
accepted 22 September 2003.