A multiplex PCR method for the simultaneous detection of three viruses associated with canine viral enteric infections.

The aim of this study was to establish a multiplex PCR (mPCR) method that can simultaneously detect canine parvovirus (CPV-2), canine coronavirus (CCoV) and canine adenovirus (CAV), thereby eliminating the need to detect these pathogens individually. Based on conserved regions in the genomes of these three viruses, the VP2 gene of CPV-2, the endoribonuclease nsp15 gene of CCoV, and the 52K gene of CAV were selected for primer design. The specificity of the mPCR results showed no amplification of canine distemper virus (CDV), canine parainfluenza virus (CPIV), or pseudorabies virus (PRV), indicating that the method had good specificity. A sensitivity test showed that the detection limit of the mPCR method was 1 × 104 viral copies. A total of 63 rectal swabs from dogs with diarrheal symptoms were evaluated using mPCR and routine PCR. The ratio of positive samples to total samples for CPV-2, CCoV, and CAV was 55.6% (35/63) for mPCR and 55.6% (35/63) for routine PCR. Thirty-five positive samples were detected by both methods, for a coincidence ratio of 100%. This mPCR method can simultaneously detect CCoV (CCoV-II), CAV (CAV-1, CAV-2) and CPV-2 (CPV-2a, CPV-2b, CPV-2c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FK(CAV)), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FK(CAV) entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry. IMPORTANCE: Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family.

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

Production and purification of non replicative canine adenovirus type 2 derived vectors.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: a case study of three animal viruses.

Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID50 is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID50, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48 h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID50 at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID50 ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID50 indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48 h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.

Osteosarcoma cells as carriers to allow antitumor activity of canine oncolytic adenovirus in the presence of neutralizing antibodies.

Osteosarcoma (OSA) is the most common bone tumor affecting the dog. The veterinary options for therapeutic management of OSA are limited and prognosis for such patients is poor. Oncolytic adenoviruses are attractive tools for experimental therapeutics as they can replicate and spread within tumors to directly induce tumor destruction. However, a major impediment to systemic oncolytic adenoviruses injection is the presence of pre-existing neutralizing antibodies (Nabs). In this study, we investigated the effect of a replication-selective canine adenovirus (OCCAV) to treat OSA in the presence of Nabs and the use of canine OSA cells as carrier vehicles for evading Nabs. Our systemic biodistribution data indicated that canine tumor cells could successfully reach the tumor site and deliver OCCAV to tumor cells in an immunized mice model. Furthermore, the use of carrier cells also reduced adenovirus uptake by the liver. Importantly, OCCAV alone was not effective to control tumor growth in a pre-immunized xenograft mouse model. On the contrary, systemic antitumoral activity of carrier-cell OCCAV was evident even in the presence of circulating antibodies, which is a relevant result from a clinical point of view. These findings are of direct translational relevance for the future design of canine clinical trials.

Expression of the Foot-and-Mouth Disease Virus VP1 protein using a replication-competent recombinant canine adenovirus type 2 elicits a humoral antibody response in a porcine model.

To develop a new type vaccine for Foot-and-Mouth Disease (FMD) prevention by using canine adenovirus as vector, the VP1 cDNA of Foot-and-Mouth Disease Virus (FMDV) type O strain China 99 was amplified by RT-PCR and cloned into pEGFP-C1 by replacing the GFP gene with the VP1 cDNA, resulting in an expression plasmid pVP1-C1. The expression cassette of VP1 composed of the CMV promoter, the VP1 gene and the SV40 early mRNA polyadenylation signal was recovered by Nsi I / Mlu I digestion of pVP1-C1 and cloned into the Canine adenovirus type-2 (CAV-2) genome in which E3 region was partly deleted by removing the Ssp I- Ssp I fragment. The recombinant virus (CAV-2-VP1) was obtained by transfecting the recombinant CAV-2-VP1 genome into MDCK cells with Lipofectamine 2000. Immunization trial in pigs with the recombinant virus, CAV-2-VP1, showed that CAV-2-VP1 could stimulate a specific immune response to both FMDV and the vector virus. Immune response to the VP1 and FMDV after VP1 expression was confirmed by ELISA, western blotting analysis and neutralization test. It was indicated that CAV-2 may serve as a vector for FMD vaccine development in pigs.

[Construction and experimental immunity of recombinant replication-competent canine adenovirus type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus].

H5N1 highly pathogenic avian influenza virus was highly pathogenic and sometimes even fatal for tigers and cats. To develop a new type of vaccine for Felidae influenza prevention, recombinant replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003 (HSN1) HA gene was cloned into PVAX1. The HA expression cassette which included CMV and HA and PolyA was ligated into the E3 deletion region of pVAXdeltaE. The recombinant plasmid was named pdeltaEHA. The pdelta EHA and the pPoly2-CAV2 were digested with Nru I /Sal I, respectively. The purified Nru I/Sal I DNA fragment containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the recombinant plasmid pCAV-2/HA. The recombinant genome was released from pCAV-2/HA, and was transfected into MDCK cells by Lipofectamine. The recombinant virus named CAV2/HA was gained. Anti-H5N1 influenza virus HI antibody (1:8 - 1:16) was detected in the cat immunized with CAV-2/HA.

Administration of a conditionally replicative oncolytic canine adenovirus in normal dogs.

Conditionally replicative adenoviruses (CRAds) are engineered to replicate only in the target tissue and destroy tumor through their cytopathic effect. Because of restricted in vivo replication, it is difficult to model behavior of human Ad5-based vectors in animal subjects. To circumvent this, we developed a "syngeneic" canine CRAd based on canine adenovirus type 2 (CAV2) transcriptionally targeted to canine osteosarcoma (OS) cells. Canine OS is an outstanding model of human OS and is the most common primary bone tumor of dogs. Because conventional therapies extend median survival by approximately 6-8 months, canine OS remains a serious therapeutic challenge shared by human OS patients. Prior to using any CRAd for clinical trials in dogs, we sought to examine the effects and safety of administration of OS-targeted CAV2 CRAd in normal dogs. Short-term physiologic indicators of stress and shock, as well as gross and histological changes in a variety of tissues, were examined, and no major signs of virus-associated toxicity were noted. In addition, short-term immunosuppression did not increase CRAd toxicity. This study marks the first administration of a CRAd in an outbred large animal model and is an important milestone in the application of this modality in human patients.

An adenovirus vector with a chimeric fiber derived from canine adenovirus type 2 displays novel tropism.

Many clinically relevant tissues are refractory to Ad5 transduction because of negligible levels of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of Ad vectors that display CAR-independent tropism could lead directly to therapeutic gain. The Toronto strain of canine adenovirus type 2 (CAV2) exhibits native tropism that is augmented by, but not fully dependent upon, CAR for cellular transduction. We hypothesized that an Ad5 vector containing the nonhuman CAV2 knob would provide expanded tropism and constructed Ad5Luc1-CK, an E1-deleted Ad5 vector encoding the fiber knob domain from CAV2. Ad5Luc1-CK gene delivery to CAR-deficient cells was augmented up to 30-fold versus the Ad5 control vector, and correlated with increased cell surface binding. Further, we confirmed the importance of cellular integrins to Ad5Luc1-CK transduction. Herein, we present the rationale, design, purification, and characterization of a novel tropism modified, infectivity-enhanced Ad vector.

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway.

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.

Nedocromil sodium inhibits canine adenovirus bronchiolitis in beagle puppies.

Nedocromil sodium is a nonsteroidal anti-inflammatory drug used to control asthmatic attacks. Our hypothesis is that nedocromil sodium inhibits virus-induced airway inflammation, a common trigger of asthma. We nebulized nedocromil sodium into beagle dogs (n = 10, mean +/- SEM ages: 149 +/- 13 days) before and after inoculation with canine adenovirus type 2 (CAV2). Control dogs (n = 10) received saline aerosols and were either infected with CAV2 (Sal/CAV2, n = 7, mean +/- SEM ages: 140 +/- 11 days) or were not infected (Sal/Sal, n = 3, ages: 143 +/- 0 days). All dogs were anesthetized with choralose (80 mg/kg i.v.), intubated, and mechanically ventilated. Pulmonary function tests and bronchoalveolar lavage (BAL) were performed using standard techniques. Pulmonary function tests revealed no significant change between the nedocromil sodium and non-nedocromil-treated groups. The percentage of infected bronchioles was quantitated as the number of inflamed airways of 40 bronchioles examined times 100 for each dog. Nedocromil-treated dogs had significantly (p < 0.05) less mucosal inflammation (mean +/- SEM, 39% +/- 5%), epithelial denudation (36% +/- 5%), and BAL neutrophilia (11 +/- 3) than did Sal/CAV2 dogs (51% +/- 6%, 57% +/- 4%, and 33% +/- 8%, respectively). We concluded that pretreatment with nedocromil sodium aerosols attenuated CAV2-induced airway inflammation in these beagle puppies.

Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.

Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.

A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.

Fractal analysis of lung alveoli during the acute phase vs. repair phase of an adenoviral infection in canines.

Acute viral respiratory infections are commonly associated with alterations in lung growth. Recently, fractal techniques have been demonstrated to show changes in alveolar perimeter after canine adenovirus type 2 (CAV2) infection in a beagle puppy model. In the present study, we investigated whether the fractal dimension (Df) of the alveolar perimeter was changed in the acute phase (2-3 weeks after inoculation, 131d CAV2 group) or during the recovery phase (approximately 22 weeks after inoculation, 235d CAV2 group) after a single bout of CAV2. There were sham CAV2 groups, 130d and 238d controls, corresponding to the CAV2 groups. The Df of alveolar perimeter length was significantly increased in the 235d CAV2 puppies compared to all of the other beagle puppy groups. On the other hand, the fractal dimensions for alveolar perimeter length for the other beagle puppy groups were very similar. In a related human study of patients (age range of 25 h to 19 y, N = 11), who died of non-respiratory causes, showed no consistent change in Df of alveolar perimeter length with normal lung growth and development. We conclude that fractal analysis of alveolar perimeter length can be used as an index of permanent lung injury after insult to the growing lungs.