Fluorescently tagged canine adenovirus via modification with protein IX–enhanced green fluorescent protein

Long P. Le1, Jing Li1, Vladimir V. Ternovoi1, Gene P. Siegal1,2 and David T. Curiel1

1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, 901 19th Street South, BMR2-502, Birmingham, AL 35294, USA
2 Departments of Pathology, Cell Biology and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294-2172, USA

Correspondence
David T. Curiel
curiel{at}uab.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX–enhanced green fluorescent protein (pIX–EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX–EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Adenoviral vectors have been widely implemented to deliver genes for the purpose of therapy (Curiel & Douglas, 2002). The attractiveness of this vector system includes the well-documented knowledge encompassing its biology and pathology (Shenk, 1996), relative safety associated with the virus and ease in upscaling vector production. More recently, manipulation of the viral genome by recombinant techniques has yielded cell-specific vectors founded on various concepts, including transductional (Wickham, 2003), transcriptional (Miller & Whelan, 1997; Nettelbeck et al., 2000) and translational (Ahmed et al., 2003) targeting. Furthermore, enhanced safety, long-term transgene expression and greater cloning capacity have resulted from the ability to generate helper-dependent adenoviral vectors that are completely devoid of native viral genes (Kochanek et al., 2001). Despite these advances in adenoviral vector engineering, two key unresolved issues remain. First, pre-existing humoral and cellular immunity against human adenoviruses, especially serotypes 2 and 5 (Ad2 and Ad5) on which most current adenoviral vectors are based, can have profound effects on initial vector administration and, increasingly, on repeat applications (Crystal et al., 1995; Zabner et al., 1994). Second, replication-competent adenovirus contamination of recombinant viral stocks poses a health risk, even in the case of gutless vectors (Lochmuller et al., 1994).

Vector systems based on other, less pathogenic human adenovirus serotypes and various xenotypes have been proposed to overcome these concerns (Both, 2004; Gao et al., 2003; Rasmussen et al., 1999; Reddy et al., 1999). Among the diverse approaches, a canine adenovirus type 2 (CAV2) vector demonstrated promising features, including high titres, undetectable replication-competent revertants, inability to replicate in various human cell lines tested, transduction efficiency comparable to that of human adenovirus serotype 5 (Ad5) vectors, minimal neutralization by human sera (Kremer et al., 2000) and neurotropism (Peltékian et al., 2002). CAV2 vectors have also gained importance in the field of adenoviral virotherapy for the treatment of cancer. Its disease presentation and prevalence in the dog population parallel similar properties of Ad5 found in the human population. These considerations establish the rationale for studying the effects of the immune system on a syngeneic CAV2 oncolytic adenovirus in the setting of a spontaneous osteosarcoma dog model (Hemminki et al., 2003).

The interest in CAV2 as a gene-delivery vector and as a model oncolytic adenovirus for immunological evaluation motivated us to consider genetic labelling of CAV2 via the fusion protein IX–enhanced green fluorescent protein (pIX–EGFP) as a means for vector detection. By using this system, we and others have previously generated fluorescent Ad5 particles with minimal effect on viral function, whilst demonstrating utility for vector detection in tracking, binding and biodistribution assays (Le et al., 2004; Meulenbroek et al., 2004). Likewise, another group was able to incorporate enhanced yellow fluorescent protein into a bovine adenovirus type 3 vector via pIX (Zakhartchouk et al., 2004). Based on studies involving human adenovirus serotypes 2 and 5, pIX is a small polypeptide of 140 aa (14·7 kDa) that acts as a cement protein to stabilize hexon–hexon interaction and therefore the capsid structure itself (Parks, 2005); however, pIX is structurally dispensable during virion formation (Colby & Shenk, 1981). Data suggest that four trimers of pIX interact with a group of nine hexons in each facet of the icosahedron (Stewart et al., 1991), resulting in 240 copies of the protein per virion (Lehmberg et al., 1999; van Oostrum & Burnett, 1985). During infection, pIX reorganizes the structure of the nucleus by sequestering the promyelocytic leukaemia protein (Rosa-Calatrava et al., 2003). In addition, pIX also serves as a transcriptional activator of several viral and cellular TATA-containing promoters, including adenoviral E1A, E4 and major late promoters (Lutz et al., 1997), although its transcriptional role during normal infection is not significant (Sargent et al., 2004). Taking advantage of its surface localization (Akalu et al., 1999), pIX was initially exploited as a locale for incorporation of a heterologous polylysine peptide onto its carboxy terminus for transduction purposes (Dmitriev et al., 2002). More recently, the fusion of spacers (Vellinga et al., 2004) and a biotin-acceptor peptide (Campos et al., 2004) onto Ad5 pIX have further expanded its utility for targeting.

Published reports suggest that various human adenoviruses possess the IX gene (Chroboczek et al., 1992; Dijkema et al., 1981; Engler, 1981; Mei et al., 2003). Although ovine and avian adenoviruses do not appear to express pIX (Hess et al., 1997; Ojkic & Nagy, 2000; Vrati et al., 1996), analyses of some canine, bovine and porcine adenovirus genomes have shown the presence of the IX gene (Aggarwal & Mittal, 2000; Salmon & Haj-Ahmad, 1994; Shibata et al., 1989; Zheng et al., 1999). Of note, our CAV2 vector of interest does express pIX. We accomplished genetic labelling of CAV2 by generating a recombinant virus that expresses the fusion pIX–EGFP. The results indicate that pIX labelling was achieved, resulting in fluorescent viral particles that could be detected in vitro during viral binding and nuclear localization, and in situ after systemic administration. Furthermore, pIX–EGFP expression could be exploited to visualize virus replication and spread. Genetic labelling of CAV2 via pIX–EGFP provides a means for vector detection in gene-therapy applications and has potential utility for studying virotherapy in a canine osteosarcoma model.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cell culture.
Dog kidney DK and MDCK, dog osteosarcoma D22 and human lung adenocarcinoma A549 cells (ATCC, Manassas, VA, USA) were maintained according to the supplier's protocols. Cells were incubated at 37 °C and 5 % CO2 under humidified conditions.

CAV2-wt-IX-EGFP construction.
Similar to our previous report of pIX–EGFP-labelled Ad5 (Le et al., 2004), we generated replication-competent CAV2-wt-IX-EGFP with a carboxy-terminal fusion of EGFP onto pIX. Construction involved the cloning of an E1 shuttle vector produced by self-ligation of the 7·6 kb NdeI fragment liberated from pTG5412 (Transgene SA), which contains the CAV2 genome (strain Toronto A 26/61). Briefly, the EGFP gene from pEGFP-N1 (Clontech) was inserted into the Klenow large fragment-blunted AseI site between the last codon of the IX gene and the stop codon. The bovine growth hormone polyadenylation signal was placed after the EGFP gene. Finally, we inserted a kanamycin-resistance gene after the polyadenylation signal to facilitate double-selection recombination with the rescue backbone, which contains an ampicillin-resistance gene. The final shuttle plasmid was linearized with NdeI and NotI enzymes and recombined homologously with pTG5412 in BJ5183 cells. Colonies were selected on ampicillin/kanamycin double-selection plates to allow identification of recombinants containing the IX-EGFP modification with the kanamycin gene located in the CAV2 genome plasmid, which itself expresses ampicillin. This double-selection system was designed so that non-recombined original shuttle and genome-rescue plasmids would be eliminated, whilst preferentially selecting for recombinant plasmids that contain the IX-EGFP modification in the CAV2 genome. A correct clone was linearized with ClaI (which flanked the kanamycin gene) to remove the kanamycin gene and self-ligated to generate the final recombinant genome. The NotI-cut genome was transiently transfected into DK cells for virus generation and then amplification.

Virus propagation and purification.
CAV2-wt-IX-EGFP was propagated in DK cells and purified by double caesium chloride (CsCl) ultracentrifugation and dialysis against PBS with 0·9 M Mg2+, 0·5 M Ca2+ and 10 % (v/v) glycerol. Final aliquots of virus were analysed for viral-particle titre (A260) and cytopathic effect unit titre (CPEU). CPEU was determined by infecting DK cells in 96-well plates with 1/10 serial dilutions of the virus and assayed on day 10 post-infection with an MTS viability assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega] to determine the viral dilution that causes 50 % cytopathic effect (n=6). Based on the cell number seeded (15 000), CPEU was calculated where one CPEU unit is defined as the amount of virus that causes cytopathic effect in one DK cell in 10 days. A similar protocol has been reported previously (O'Carroll et al., 2000). The viral particle (vp) : CPEU ratio of CAV2-wt-IX-EGFP was 49, whilst that of a control CAV2-wt was 21. All viruses were stored at –80 °C until use.

Fluorescence microscopy.
Fluorescence microscopy was performed with an inverted IX-70 microscope (Olympus) equipped with a Magnifire digital CCD camera (Optronics). Glass slides and coverslips were used to mount samples (Fisher Scientific). Images were acquired with a 100x objective by using oil immersion and deconvoluted digitally with IRIS version 4.15a (http://www.astrosurf.com/buil/) by applying the Richardson–Lucy algorithm with 15 iterations. An image of a single, fluorescent virus particle with strong signal-to-noise ratio was used to estimate the point-spread function as suggested by the software documentation. Green fluorescence, red autofluorescence and blue Hoechst-stain images were finally merged by using Adobe Photoshop 7.0. For pIX–EGFP expression and localization analysis, DK and MDCK cells seeded on glass coverslips were infected with CAV2-wt-IX-EGFP (100 vp per cell) and processed for imaging at 24 and 48 h post-infection.

Characterization of virus-gradient fractions.
CAV2-wt-IX-EGFP was propagated in ten 150 mm dishes and then purified by CsCl ultracentrifugation where the top and bottom viral bands were retained after two centrifugation steps, yielding one gradient from the ten dishes. After the second spin, fractions (approx. 100 µl) were collected dropwise through a perforation at the bottom of the tube into a 96-well white opaque plate. Plates with the viral fractions were measured with a microplate fluorometer (Fluostar Optima; BMG Labtechnologies) by using 490/10 nm excitation and 510/10 nm emission filters. To determine viral DNA content, a sample of each fraction (10 µl) was diluted in 90 µl 0·5 % SDS/PBS and incubated at room temperature for 10 min to release the viral genomes. A260 was then measured for each sample (MBA 2000; Perkin Elmer).

Western blot.
Fractionated samples (4 µl) were ethanol-precipitated, pelleted and resuspended in 20 µl RIPA buffer. Samples (5 µl) were resolved with SDS-PAGE and then transferred to a PVDF membrane (Bio-Rad). Blotting was performed with a primary monoclonal GFP antibody (1 : 1000 dilution; BD Biosciences Clontech) followed by a secondary horseradish peroxidase-linked anti-mouse antibody (1 : 5000 dilution; Amersham Biosciences). Bands were detected with a chemiluminescent ECL kit (Amersham Biosciences).

Tracking of CAV2-wt-IX-EGFP infection.
The day prior to infection, DK and MDCK cells (2·5x105) were seeded on glass coverslips that were placed in six-well plates with growth medium lacking phenol red (5 % fetal calf serum/Dulbecco's modified Eagle's medium). The cells were incubated for 1 h at 4 °C with CAV2-wt-IX-EGFP (10 000 vp per cell) in 1 ml phenol red-free growth medium containing 25 mM HEPES buffer. The viruses were allowed to bind to the cells at 4 °C for 1 h (cell binding), after which the cells were washed three times with PBS and then fixed to the coverslip with 3 % formalin (Tousimis) for 10 min. After another three washes, Hoescht (33342) staining was performed for 5 min, followed by three final washes with PBS before mounting and sealing. Another set of coverslips with the cells and virus were transferred to 37 °C for 1·5 h after incubation at 4 °C for 1 h (nuclear localization). Likewise, these samples were prepared for fluorescence imaging.

In situ detection of CAV2-wt-IX-EGFP.
All methods were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham, AL, USA, and performed according to their guidelines. C57/BL6 mice (Charles River Laboratories) were anaesthetized with 2 % isoflurane at about 0·5 l min–1 for an open laparotomy procedure and injected via the inferior vena cava with 1011 vp CAV2-wt-IX-EGFP in 200 µl PBS. Twenty minutes after virus injection, the mice were sacrificed and the liver was frozen until sectioning (Minotome PLUS; Triangle Biomedical Sciences). Frozen sections (5 µm thick) of the liver were fixed onto glass slides and stained with Hoechst 33342 (Molecular Probes) for nuclear DNA. Glass coverslips were mounted on the slides with mounting medium (Biomeda) prior to fluorescence microscopy as described above.

In vitro visualization of CAV2-wt-IX-EGFP replication and spread.
Dog osteosarcoma D22 and human lung adenocarcinoma A549 cells were seeded on glass-bottomed 3 cm diameter dishes (200 000 cells per dish; Willco Wells) using phenol red-free medium. The next day, the cells were infected with 100 vp CAV2-wt-IX-EGFP per cell. Fluorescence microscopy was performed to detect pIX–EGFP expression in the same field of view over the course of 8 days.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
CAV2 pIX–EGFP expression and localization
CAV2-wt-IX-EGFP was constructed with the same strategy used to construct Ad5-IX-EGFP (Le et al., 2004), in that EGFP was fused to the carboxy terminus of protein IX (Fig. 1a). No other modifications were made in the CAV2 genome, including the position of the IX gene and its promoter. Therefore, expression of pIX–EGFP would be under the control of the endogenous pIX promoter. To demonstrate pIX–EGFP expression from the virus genome, DK and MDCK dog kidney cells were infected with purified CAV2-wt-IX-EGFP. Twenty-four hours post-infection, pIX–EGFP showed heterogeneous intracellular localization in both DK and MDCK cells. Representative data are shown for MDCK cells (Fig. 1b). In some cells, weak, diffuse green fluorescence could be visualized in the nucleus (white arrow, 200x magnification). A number of cells showed nuclear inclusions, which were either few in number and localized strongly in intense, round bodies (red arrows, 200x and 1000x magnification) or small and speckled in appearance, but abundant in number (white arrow, 1000x magnification). This particular localization pattern corresponds with results obtained for our previous Ad5 pIX–EGFP version (Le et al., 2004) and agree with reported pIX immunofluorescence data (Rosa-Calatrava et al., 2003). In other cells, the inclusions were numerous, irregular in shape, intense and concentrated throughout the nucleus and around the nuclear periphery (orange arrow, 1000x magnification). Certain cells also showed strong cytoplasmic accumulations of pIX–EGFP (blue arrow, 1000x magnification). Forty-eight hours post-infection, pIX–EGFP appeared even more speckled throughout the nucleus, with some cells showing overwhelming amounts of the fusion protein (data not shown). Cells transduced with the control vector CAVGFP showed diffuse GFP localization throughout the cytoplasm and nucleus, without any nuclear inclusion bodies or cytoplasmic aggregates as observed with CAV2-wt-IX-EGFP (data not shown).



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Fig. 1. (a) Schematic of CAV2-wt-IX-EGFP. The genome layout is shown with the IX-EGFP fusion gene followed by a polyadenylation signal. Note that no other modifications were made to the CAV2 genome. (b) pIX–EGFP expression and localization from CAV2-wt-IX-EGFP. MDCK cells were infected with 100 vp CAV2-wt-IX-EGFP per cell. The cells were fixed and stained with Hoechst stain 24 h post-infection. Fluorescence microscopy was performed to image pIX–EGFP expression and localization (green) along with nuclear DNA (blue). Red arrows indicate a few round, intense nuclear inclusions (applies to both sets of images). White arrows indicate weak, diffuse nuclear green fluorescence (200x magnification); abundant, small, speckled inclusions (1000x magnification). Orange arrows indicate abundant, irregular and strongly intense inclusions in the nucleus and its periphery (applies to both sets of images). Blue arrow indicates strong cytoplasmic localization of pIX–EGFP (1000x magnification).

 
Characterization of purified CAV-wt-IX-EGFP CsCl gradient fractions
After visualizing expression and heterogeneous intracellular localization of the fusion pIX–EGFP, the CAV2-wt-IX-EGFP virus was purified by double caesium chloride gradient ultracentrifugation to analyse the virus gradient. Both the top (immature particles) and bottom (mature particles) bands were retained through two rounds of centrifugation. Fractions collected from the final gradient were analysed for EGFP fluorescence (Fluostar Optima; BMG Labtechnologies) and viral DNA content (A260; MBA 2000). Similar to our pIX–EGFP-labelled Ad5 vector, we detected fluorescent peaks for both the bottom and top bands of the CAV2-wt-IX-EGFP gradient. The bottom peak coincided with the viral DNA peak detected for the band containing mature viral particles (Fig. 2a). Western blot analysis of the same fractions further confirmed the association of the pIX–EGFP label with viral particles (Fig. 2a). Samples of representative top- and bottom-band fractions were diluted and visualized by fluorescence microscopy. Single, fluorescent adenoviral particles were clearly visible for both the bottom (Fig. 2b) and top (data not shown) bands, although the signal was more homogeneous for the mature virus sample than for the empty capsid sample.



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Fig. 2. (a) Analysis of CAV2-wt-IX-EGFP viral gradient. CAV2-wt-IX-EGFP was propagated in DK cells (10x15 cm dishes). The crude viral lysate was purified by CsCl gradient double ultracentrifugation, retaining both the bottom and top bands. Fractions were collected and quantified for fluorescence ({circ}) and viral DNA content ({blacksquare}). The same fractions were also subjected to SDS-PAGE for Western blotting with anti-GFP antibody. (b) Visualization of CAV2-wt-IX-EGFP. Purified CAV2-wt-IX-EGFP was visualized by fluorescence microscopy using a 100x oil-immersion lens (1000x magnification).

 
Detection of CAV2-wt-IX-EGFP cell attachment and nuclear localization
To demonstrate utility of the fluorescent CAV2-wt-IX-EGFP vector, MDCK cells were infected with the virus at 4 °C for 1 h to allow binding of the virus, but not internalization. Fluorescence microscopy revealed abundant green viral particles bound to the cell membrane of MDCK cells (white arrows, Fig. 3). In a separate experiment, infection with CAV2-wt-IX-EGFP was carried out at 37 °C for 1·5 h after pre-incubation of the cells with the virus at 4 °C for 1 h. As expected, the virus trafficked to the nuclear membrane after infection at 37 °C in MDCK cells (note the rings of green fluorescent particles surrounding the nuclei). Non-infected MDCK cells only showed green cytoplasmic and nuclear autofluorescence, similar to that indicated in the images of cells infected with virus (orange arrows). No green fluorescent particles or speckled patterns were detected under the non-infected conditions (Fig. 3). Similar results were obtained with DK cells (data not shown).



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Fig. 3. Tracking of CAV2-wt-IX-EGFP infection of MDCK cells. MDCK cells seeded on glass coverslips were infected with CAV2-wt-IX-EGFP (10 000 vp per cell) in duplicate for 1 h at 4 °C. One set of infected cells was fixed, DNA-stained (blue signal) and mounted for microscopy (cell binding). The second set was transferred to 37 °C for an additional 1·5 h incubation and then prepared like the first set (nuclear localization). Bound (cell binding) and internalized (nuclear localization) fluorescent virions were imaged by fluorescence microscopy (1000x magnification). Orange arrows indicate cellular background green autofluorescence; white arrows indicate green fluorescent viral particles.

 
Detection of CAV2-wt-IX-EGFP particles in situ after intravenous administration
To further show the versatility of CAV2-wt-IX-EGFP detection, two C57/BL6 mice were injected in the inferior vena cava during a laparotomy procedure. Twenty minutes after virus injection, the mice were sacrificed to harvest the livers. Frozen liver sections were stained for nuclear DNA with Hoechst stain and then mounted for imaging. CAV2-wt-IX-EGFP particles could be detected along the sinusoids in some liver sections (Fig. 4). A few conglomerates of fluorescent viral particles (white arrows) were observed, whilst some signal resembling single particles could be detected (orange arrows). The results indicate that CAV2-wt-IX-EGFP could be detected in liver-tissue sections in a manner similar to our previously reported Ad5-wt-IX-EGFP vector (Le et al., 2004). This detection capability could potentially be exploited to study the biodistribution of CAV2-wt-IX-EGFP particles in vivo for gene-therapy applications.



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Fig. 4. Detection of CAV2-wt-IX-EGFP in liver-tissue section. A C57/BL6 mouse was injected with CAV2-wt-IX-EGFP (1011 vp) into the inferior vena cava through laparotomy and then sacrificed at 20 min post-injection. Green fluorescent CAV2-wt-IX-EGFP particles were detected in a frozen liver section under fluorescence microscopy (1000x magnification). Nuclear DNA was stained with Hoescht (blue signal). White arrows indicate accumulations of fluorescent viral particles and orange arrows indicate signal resembling single particles.

 
Visualization of CAV2-wt-IX-EGFP replication and spread in vitro
Because pIX–EGFP is a structural protein that is incorporated into virions, detection of the fluorescent fusion protein would provide a representation of the viral mass and localization. The augmentation and spread of signal would also indicate active replication with lateralization. We exploited this property of the genetic-labelling system to monitor the replication and spread of CAV2-wt-IX-EGFP in a monolayer of dog osteosarcoma D22 cells. Examining the same field of view over the course of 8 days, pIX–EGFP expression could be detected in a few cells early on, which eventually involved the entire monolayer by day 8 post-infection (Fig. 5), resulting in extensive cytopathic effect. The increase in number of cells expressing pIX–EGFP clearly suggested active replication and spread of CAV2-wt-IX-EGFP in D22 cells. The same experiment was also performed on human lung adenocarcinoma A549 cells, which were employed as a substrate that is limited for productive CAV2 replication. Previous data indicate that CAV2 infectious progeny production was nearly two orders of magnitude lower in human A549 cells than in canine MDCK cells and that productive replication was only observed during the first passage (Rasmussen et al., 1999). Of note, we have recently demonstrated the reverse scenario to be true, i.e. productive replication of human Ad5 in various canine tumour cells (Ternovoi et al., 2005). Consistent with published results showing some level of CAV2 DNA replication and gene expression in A549 cells (Rasmussen et al., 1999), CAV2 pIX–EGFP expression was noted in a few A549 cells; however, no augmentation or spread of the pIX–EGFP signal was detected at the end of 8 days (Fig. 5). The pIX–EGFP signal detected appeared to be limited only to the initially infected A549 cells, suggesting absent or poorly productive replication of the CAV2-wt-IX-EGFP virus in human A549 cells. These results demonstrate that detection of pIX–EGFP expression could be exploited to monitor CAV2 replication and spread.



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Fig. 5. Visualization of CAV2-wt-IX-EGFP replication and spread in vitro. CAV2 pIX–EGFP expression in D22 cells was imaged by fluorescence microscopy on days 1, 2, 4, 6 and 8 post-infection with 100 vp per cell. Also shown is an image of A549 cells on day 8 post-infection with CAV2-wt-IX-EGFP.

 
We have shown herein that the technique of capsid labelling of adenovirus with pIX–EGFP is applicable across species for CAV2. Fluorescent labelling of CAV2 with pIX–EGFP provides a means to detect viral particles in the design of vectors for targeting and also to track virus infection of cells. In another body of work, we have demonstrated non-invasive fluorescence imaging of human adenovirus type 5 replication by using the pIX labelling system (L. Le, H. Le, I. Dmitriev, J. Davydova, T. Gavrikova, S. Yamamoto, D. T. Curiel & M. Yamamoto, unpublished data). We believe that the same methodology may be adapted to CAV2 for monitoring CAV2-based oncolytic adenoviruses in a syngeneic dog tumour model. Furthermore, our results support the fact that CAV2 pIX may serve as a platform for incorporation of other imaging reporters for vector detection, and large proteins or peptides for targeting purposes.


   ACKNOWLEDGEMENTS
 
This work was supported with grants from the National Institutes of Health (P30 AR41031, RO1 CA93796, RO1 CA940840 and P50 CA83591), the Department of Defense (W81XWH-04-1-0025), the Haley's Hope Memorial Support Fund for Osteosarcoma Research and the Medical Scientist Training Program at the University of Alabama at Birmingham.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 13 February 2005; accepted 9 September 2005.



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