Antibody Response and Virus Shedding of Chickens Inoculated with Left End Deleted Fowl Adenovirus 9-Based Recombinant Viruses Author(s) :Juan Carlos Corredor and Éva Nagy Source: Avian Diseases, 55(3):443-446. 2011. Published By: American Association of Avian Pathologists DOI: URL: http://www.bioone.org/doi/full/10.1637/9710-031311-Reg.1
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AVIAN DISEASES 55:443–446, 2011
Antibody Response and Virus Shedding of Chickens Inoculated with Left End Deleted Fowl Adenovirus 9-Based Recombinant Viruses Juan Carlos Corredor and E´va NagyA Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 15 March 2011; Accepted and published ahead of print 10 May 2011 SUMMARY. The nonpathogenic fowl adenoviruses (FAdVs) are suitable recombinant virus vectors. Two different replicationcompetent FAdV-9–based recombinant viruses carrying the enhanced green fluorescent protein (EGFP) gene within a nonessential DNA sequence at the left end genomic region were tested in chickens to study the antibody response by enzyme-linked immunosorbent assay to both the foreign proteins, EGFP and FAdV-9, and virus shedding through the feces. All inoculations were done intramuscularly: groups 1 and 2 with the recombinant viruses and group 3 with the wild-type FAdV-9 virus. Group 4 was mock inoculated. Sentinel birds also were included in groups 1–3 to study virus transmission. Boosting inoculations were done in all groups at 2, 3, and 4 wk after the first inoculation. Antibodies to EGFP were detected at 3–7 wk postinoculation in groups 1 and 2 only. Antibody response to FAdV-9 in groups 1–3 did not differ significantly (P . 0.06). Virus was not detected in the feces of chickens in groups 1 and 2, including the sentinel birds, but virus was present in the feces of chickens in group 3, including the sentinel birds. These results further supported our previous findings regarding the suitability of the nonessential region at the left end of the viral genome as an insertion site for foreign genes and its importance in in vivo replication. In this work, we demonstrated the potential of FAdV-9–based recombinant viruses as vaccines for poultry. RESUMEN. Respuesta de anticuerpos y diseminacio´n viral de pollos inoculados con una vacuna recombinante basada en un adenovirus aviar serotipo 9 con delecio´n en el extremo izquierdo. Los adenovirus aviares no patoge´nicos (FAdVs) resultan adecuados como virus vectores recombinantes. Dos diferentes adenovirus aviares del serotipo 9 recombinantes y competentes para la replicacio´n, que contenı´an el gene de la proteı´na verde fluorescente aumentada (EGFP), dentro de una secuencia de ADN no esencial en el extremo izquierdo de la regio´n geno´mica fueron probados en pollos para estudiar la respuesta de anticuerpos mediante un ensayo inmunoabsorcio´n con enzimas ligadas dirigido contra ambas proteı´nas externas; EGFP y del adenovirus serotipo 9, y tambie´n se analizo´ la diseminacio´n del virus a trave´s de las heces. Todas las inoculaciones se realizaron por vı´a intramuscular: los grupos uno y dos recibieron el virus recombinante y el grupo tres el virus de campo FAdV-9. El grupo cuatro fue el control negativo. Se incluyeron tambie´n aves centinelas en los grupos uno al tres para estudiar la transmisio´n del virus. Se realizaron inoculaciones de refuerzo en todos los grupos a las dos, tres y cuatro semanas despue´s de la primera inoculacio´n. Los anticuerpos contra EGFP se detectaron entre las semanas tres a siete posteriores a la inoculacio´n solamente en los grupos uno y dos. La respuesta de anticuerpos contra el FAdV-9 en los grupos de uno al tres no fue significativamente diferente (P . 0.06). No se detecto´ virus en las heces de los pollos en los grupos uno y dos, incluyendo las aves centinelas, pero el virus estaba presente en las heces de los pollos en el grupo tres, incluyendo las aves centinela. Estos resultados apoyan los resultados previos relacionados con la aplicabilidad de la regio´n no esencial en el extremo izquierdo del genoma viral como un sitio de la insercio´n de genes externos y su importancia en la replicacio´n in vivo. En este trabajo, se ha demostrado el potencial de los virus recombinantes FAdV-9 como vacunas para aves comerciales. Key words: fowl adenovirus 9, recombinant viruses, gene expression, virus vectors, recombinant vaccine, antibody response, virus shedding Abbreviations: Ab 5 antibody; d.p.i. 5 days postinoculation; EGFP 5 enhanced green fluorescent protein; ELISA 5 enzymelinked immunosorbent assay; FAdV-9 5 fowl adenovirus 9; GFP 5 green fluorescent protein; ORF 5 open reading frame; pfu 5 plaque-forming unit; p.i. 5 postinoculation; r 5 recombinant; w.p.i. 5 weeks postinfection; wt 5 wild type
Fowl adenoviruses (FAdVs) have a worldwide distribution, and some are associated with diseases such as inclusion body hepatitis (IBH) or hydropericardium syndrome (HPS) (1). The primary role of FAdVs in IBH/HPS has been shown previously (8,11,14), although other reports suggest the participation of immunosuppressive agents such as chicken anemia virus or infectious bursal disease virus (IBDV) (9,20,21). Nonpathogenic FAdVs have been shown to be suitable recombinant vaccines and gene delivery vehicles in avian and mammalian systems (6,10,12,18). FAdV-based recombinant viruses have been engineered by replacing nonessential early regions at the right and left end regions and used as recombinant vaccines (7,10,12,17,18). For example, chickens inoculated with FAdV-1 (CELO virus) and FAdV-10 (strain CFA20) expressing the VP2 protein of IBDV (10,18) and FAdV-8 (strain CFA40) expressing the S1 protein of infectious bronchitis virus (12) were protected against challenge. A
Corresponding author. E-mail:
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Despite recent progress, FAdVs are not well understood at the molecular level. As for all adenovirus genomes, the early regions of the FAdV genome are located mainly at the left and right ends. The open reading frame (ORF) organization of these regions for certain serotypes has been described previously (4,5), and the function of some viral genes at only the right end has been determined (13). The importance of the left end ORFs in virus replication in vivo has been reported previously (6), although their exact roles have yet to be determined. We have generated two replication-competent FAdV-9 recombinants carrying the enhanced green fluorescent protein (EGFP) cassette replacing the nonessential DNA sequences at nucleotides 1195–2342 and 491–2782 for FAdV-9D1-EGFP and FAdV-9D4-EGFP, respectively. A third recombinant virus, FAdV-9inEGFP, was generated through insertion of the EGFP expression cassette within the 10th codon of ORF1B (7). The EGFP expression cassette consists of the EGFP coding region under the control of the cytomegalovirus immediate early promoter and two simian virus-40 polyadenylation sites. In cell culture, FAdV-9D1-EGFP and FAdV-9D4-EGFP
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Fig. 1. Features of the recombinant viral genomes (as described by Corredor and Nagy [7]). In brief, FAdV-9inEGFP was generated by insertion of the EGFP expression cassette into ORF1B. FAdV-9D4-EGFP was generated by replacement of the deleted 2.3-kilobase nonessential DNA sequences in the parental FAdV-9 with the EGFP cassette.
replicate at wild-type (wt) levels, whereas FAdV-9inEGFP yields a somewhat (half-log) lower titer (7). Avian and mammalian cells infected with the recombinant viruses express EGFP, demonstrating the potential of these viruses as recombinant vectors in poultry vaccines or gene delivery vehicles for not only avian but also mammalian systems. In this work, we studied the antibody (Ab) response to EGFP and FAdV proteins and the virus shedding through the feces of chickens inoculated with FAdV-9inEGFP and FAdV-9D4-EGFP.
MATERIALS AND METHODS Viruses and cells. The generation and characterization of the recombinant viruses FAdV-9inEGFP and FAdV-9D4-EGFP are described by Corredor and Nagy (7), and the features are shown in Fig. 1. Propagation and titration of recombinant fowl adenoviruses (rFAdV-9s) and wt FAdV-9 strain A-2A were carried out in chicken hepatoma cells (CH-SAH cell line) as described previously (2). Animal experiments. Birds were housed at the University Isolation Unit, University of Guelph, Guelph, Ontario, Canada, in cages according to The Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. They received feed (21% unmedicated starter ration) and water ad libitum. One-week-old barred rock chicks were wing-tagged, bled, and distributed into four groups of 20 each. Subsequently, birds were inoculated with 2 3 106 plaque-forming units (pfu) of the virus intramuscularly. Intramuscular boosting inoculations were done with the same virus doses at 2, 3, and 4 wk after
the first inoculation. Groups 1, 2, and 3 were inoculated with FAdV-9D4EGFP, FAdV-9inEGFP, and wt FAdV-9, respectively, and group 4 was mock inoculated with phosphate-buffered saline. Five sentinel birds were placed in contact with inoculated chickens in groups 1–3. Cloacal swabs were collected on 0 (before inoculation), 3, 5, 7, 10, 14, 21, and 28 days postinoculation (d.p.i.). The samples were prepared, and their virus titer was determined by the plaque assay as described previously (6). Chickens were bled weekly at 0–4 weeks postinfection (w.p.i.) and at 7 w.p.i. The Ab response to FAdV-9 was analyzed by enzyme-linked immunosorbent assay (ELISA) as described previously (16). The Ab response to GFP also was determined by ELISA, where plates were coated with 100 ng of GFP antigen (Clontech, Mountain View, CA) as described previously (3). In brief, 10 ml of GFP (1 mg/ml) was mixed with 10 ml of coating buffer (0.05 M bicarbonate buffer), and ELISA plates were coated with 100 ml of GFP antigen. For GFP ELISA, chicken sera were diluted 1/100 and assayed in duplicates. Chicken anti-GFP Ab (1 mg/ml; Millipore Bioscience Research Reagents, Temecula, CA) was diluted 1/300 and used as a positive control, whereas sera from groups 3 and 4 were the negative controls. The criteria to establish the cut-off values for GFP and FAdV-9 ELISA were the same as reported previously (16). The significance of the Ab response among the inoculated groups was determined by a t-test.
RESULTS AND DISCUSSION
We previously generated three FAdV-9–based recombinant viruses (FAdV-9inEGFP, FAdV-9D1-EGFP, and FAdV-9D4-EGFP) carrying
FAdV-9 based recombinant viruses as poultry vaccines
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Fig. 2. Ab response to FAdV-9 proteins in chickens inoculated with FAdV-9D4-EGFP (dark gray bars), FAdV-9inEGFP (light gray bars), and wt FAdV-9 (white bars), and mock-infected chickens (black bars) as measured by sample-to-positive ratios. The error bars correspond to 95% confidence intervals. The arrows indicate the boosting inoculations.
the EGFP expression cassette in a nonessential region at the left end of the viral genome, and we demonstrated their suitability as gene delivery vectors in vitro (7). This work aimed to demonstrate the ability of two of these recombinant viruses, FAdV-9inEGFP and FAdV-9D4-EGFP, to induce Ab response to the expressed foreign protein EGFP and thus the potential of FAdV-9–based recombinant viruses as vaccines. Ab to FAdV-9, measured by ELISA, was not detected in any of the groups before inoculation and in group 4 throughout the experiment. Abs to FAdV-9 appeared at 3 w.p.i. (1 wk after the first boosting inoculation). Although chickens in group 3 (wt FAdV-9) had the highest Ab response to FAdV-9 (Fig. 2), this response did not differ significantly from chickens in groups 1 and 2 (P . 0.06). No Abs to EGFP were present in any of the groups before inoculation, and in groups 3 (wt FAdV-9 group) and 4 (mock-infected control) throughout the experiment. In addition, no clinical disease associated with IBH was observed in birds inoculated with either recombinant virus or wt FAdV-9. Detectable Ab response to EGFP first appeared at week 2 in group 1 (inoculated with FAdV-9D4-EGFP) and at week 3 in group 2 (inoculated with FAdV-9inEGFP), and the levels for both continued to increase up to the end of the experiment (7 w.p.i.; Fig. 3). Chickens inoculated with FAdV-9D4-EGFP had higher Ab response to EGFP than those with FAdV-9inEGFP at 3 and 7 w.p.i., but the differences were not significant (P . 0.1). One sentinel bird in group 1 was positive for FAdV-9 at 7 w.p.i. In group 2, none of the sentinel birds seroconverted. In group 3, one sentinel bird was positive for FAdV-9 at 7 w.p.i. All sentinel birds within the inoculated groups remained negative for EGFP Abs. Abs to EGFP and FAdV-9 reached their maximum levels at 7 w.p.i. after boosting inoculations at 2, 3, and 4 wk. Therefore, boosting inoculations with FAdV-9–based recombinant viruses in barred rock chickens, and perhaps in other chicken breeds as well, are necessary to achieve optimal Ab response to the foreign protein (EGFP). Although not significantly different, the Ab response to EGFP was higher in chickens inoculated with FAdV-9D4-EGFP than that of chickens with FAdV-9inEGFP. The foreign protein should be considered carefully when recombinant vaccines are generated, because not all proteins are good immunogens. Although not determined in chickens, it is known that the antigenicity of EGFP depends on the animal strain and, perhaps, species (19). The Ab response to FAdV-9 in all inoculated groups did not differ significantly (P . 0.06) in this study. These findings are inconsistent with a similar study conducted in white leghorn chickens, where the Ab response to the wt FAdV-9 was significantly higher than in birds
Fig. 3. Ab response to GFP in chickens inoculated with FAdV-9D4EGFP (dark gray bars), FAdV-9inEGFP (light gray bars), and wt FAdV9 (white bars), and mock-infected chickens (black bars) as measured by sample-to-positive ratios. The error bars correspond to 95% confidence intervals. The arrows indicate the boosting inoculations.
inoculated with FAdV-9D4 (6), the parental virus of FAdV-9D4-EGFP (7). Antibodies to FAdV-9 in white leghorn chickens were apparent from 2 w.p.i., with higher (5 3 106 pfu/bird) (16) or similar virus doses (2 3 106 pfu/bird) (6), as opposed to the current study in barred rock chickens, where Ab was shown to rise from 3 w.p.i. Moreover, white leghorns seem to mount a better Ab response to FAdV-9 than barred rocks, despite the boosting inoculations given to the latter birds (6,16). In our previous work, we demonstrated that Ab response to FAdV-9 in white leghorns is dose- and route-dependent (16), which is also probably the case for barred rocks, although in the current work we studied only the intramuscular route. Furthermore, in white leghorn chickens, there was a significant increase in Ab response to FAdV-9 at 2 w.p.i. with respect to 1 w.p.i. Moreover, in this study, the Ab response of barred rock chickens, inoculated with either of the recombinant viruses or wt FAdV-9, was delayed, and it did not differ significantly at weeks 1 and 2 p.i., but it increased from week 3. Therefore, it is plausible that the genetic background of these chickens influences the immune response to FAdV-9 and thus the number of boosting inoculations required to elicit a robust Ab response to both the foreign and viral proteins. FAdV-9D4-EGFP was generated through replacement of a nonessential region at nucleotides 491–2782, which is absent in the FAdV9D4 parental virus (6), with the EGFP expression cassette (7). Both FAdV-9D4 and FAdV-9D4-EGFP are devoid of the first 6 rightwardoriented ORFs (0, 1, 1A, 1B, 1C, and 2; Fig. 1), whose transcription has been demonstrated previously (15). These viruses replicate at wt levels in vitro (6,7). However, FAdV-9D4 does not replicate at wt levels in vivo (white leghorn chickens inoculated once at 2 wk of age) based on the significant low virus titers in the feces and Ab response to FAdV-9, and the general low viral genome copy number in analyzed tissues with respect to those of the wt FAdV-9–inoculated group (6). In this study, live virus in the feces from barred rock chickens inoculated with the recombinant viruses (groups 1 and 2) was not detected at any time p.i. Virus was detected in the feces from wt FAdV-9–inoculated and sentinel birds in group 3. Virus shedding in group 3 was observed at 5 d.p.i., and virus titer reached the highest level at 14 d.p.i. Virus shedding in sentinel birds was observed at 10 and 14 d.p.i. (Table 1). The virus shedding data are consistent with our previous findings (6), further supporting the importance of the first six rightward-oriented ORFs for in vivo replication at wt levels. In addition, the lack of detection of live virus in
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Table 1. Virus titers (pfu/ml) in swabs in the feces of chickens inoculated with FAdV-9D4-EGFP, FAdV-9inEGFP, and wt FAdV-9. Chickens shedding the virus (%) FAdV-9D4-EGFP (group 1) d.p.i.
Inoculated chickens
3 5 7 10 14 21
0 0 0 0 0 0
A
Sentinel birds
0 0 0 0 0 0
FAdV-9inEGFP (group 2) B
Inoculated chickens
Sentinel birds
0 0 0 0 0 0
0 0 0 0 0 0
wt FAdV-9 (group 3) Inoculated birds
1.8 3.7 5.5 8.2
3 3 3 3
0 103 103 103 103 0
(5) (5) (40) (35)
Sentinel birds
0 0 0 4.9 3 103 (100) 2.1 3 104 (80) 0
A B
Twenty chickens. Five chickens.
the feces from chickens inoculated with FAdV-9inEGFP suggests that the insertion of the EGFP cassette into the 10th codon of ORF1B may have disrupted the splicing of the left end ORF transcripts, thereby preventing their transport from the nucleus to the cytoplasm to be translated. It is also possible that disruption of ORF1B itself by insertion of the EGFP expression rendered FAdV-9inEGFP unable to replicate at wt levels in vivo, judged by its lack of detection in the feces. Our laboratory is currently investigating the role of each ORF within the left end viral genome on virus replication in vivo. In conclusion, we have demonstrated the potential of FAdV-9 as a vaccine vector based on replacement of a nonessential region at the left end of the viral genome with a foreign gene. The lack of detection of live virus in the feces in the inoculated groups with the recombinant viruses further support our previous studies about the importance of the first six rightward ORFs in virus replication in vivo at wt levels. Although we demonstrated that such recombinants could be developed as vaccines for poultry, the intramuscular route is not a viable inoculation for the industry; thus, we initiated experiments in the in ovo application. This work and our previous findings suggest that the genetic background of the chicken breed also may influence the immune response to FAdV-9 and probably all FAdVs and needs to be considered for such recombinant vaccines. REFERENCES 1. Adair, B. M., and S. D. Fitzgerald. Group 1 adenovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Wiley Blackwell, Ames, IA. pp. 260–286. 2008. 2. Alexander, H. S., P. Huber, J. Cao, P. J. Krell, and E´. Nagy. Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line. J. Virol. Methods 74:9–14. 1998. 3. Carter, E. W., and D. E. Kerr. Optimization of DNA-based vaccination in cows using green fluorescent protein and protein A as a prelude to immunization against staphylococcal mastitis. J. Dairy Sci. 86:1177–1186. 2003. 4. Corredor, J. C., A. Garceac, P. J. Krell, and E´. Nagy. Sequence comparison of the right end of fowl adenovirus genomes. Virus Genes 36:331–344. 2008. 5. Corredor, J. C., P. J. Krell, and E´. Nagy. Sequence analysis of the left end of fowl adenovirus genomes. Virus Genes 33:95–106. 2006. 6. Corredor, J. C., and E´. Nagy. A region at the left end of the fowl adenovirus 9 genome that is non-essential in vitro has consequences in vivo. J. Gen. Virol. 91:51–58. 2010. 7. Corredor, J. C., and E´. Nagy. The non-essential left end region of fowl adenovirus 9 genome is suitable for foreign gene insertion/replacement. Virus Res. 149:167–174. 2010. 8. Dahiya, S., R. N. Srivastava, M. Hess, and B. R. Gulati. Fowl adenovirus serotype 4 associated with outbreaks of infectious hydropericardium in Haryana, India. Avian Dis. 46:230–233. 2002.
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ACKNOWLEDGMENTS We thank Sheila Watson, Bryan Griffin, and Dan-Hui Yang for technical help and the Isolation Unit personnel for professional animal care. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Poultry Research Council (CPRC), Agriculture and Agri-Food Canada (AAFC), and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA).
