Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production.

Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin-Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC's MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells.

The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

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.

Sequence-independent VIDISCA-454 technique to discover new viruses in canine livers.

In many mammals, viruses cause hepatitis. Despite many efforts a specific virus responsible for canine idiopathic hepatitis has not been identified. The discovery of a viral etiology in canine hepatitis will promote the development of specific drugs and vaccines for the treatment of idiopathic hepatitis in dogs. The objective of this study was the application of the sequence-independent Virus Discovery cDNA-amplified fragment length polymorphism (VIDISCA) technique combined with high through-put sequencing on a Roche-454 sequencer to identify unknown viruses. Liver tissue of a dog with idiopathic acute hepatitis was cultured on a canine liver cell line and the cell culture medium was submitted to the VIDISCA-454 technique. Without prior knowledge of the viral species involved, this technique identified Canine adenovirus type 1 (CAV-1) as the infecting agent. This demonstrates the power of VIDISCA-454 to identify viruses, independent of preliminary information about the genomic sequence. Consequently, the strategy of propagation in this cell line followed by the VIDISCA-454 technique is valuable to identify the viral etiology of idiopathic hepatitis in dogs.

Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI 1640 medium without animal protein.

Madin Darby canine kidney (MDCK) cells were adapted to serum-free RPMI 1640 medium and used for cultivation of canine viruses. RPMI 1640 medium was supplemented with a soybean peptone, L-glutamine and antibiotics, so that the protein concentration was less than 5 microg/ml (RPMI/SP medium). The resulting adapted MDCK-SP cells showed steady growth after the twenty-eighth passage in RPMI/SP medium (MDCK-SP cell culture). Canine distemper virus, canine parvovirus, canine adenoviruses and canine parainfluenza virus, which are the principal components of canine combined virus vaccines, grew in the MDCK-SP cell culture as efficiently as the parental MDCK cells cultured in the conventional Eagle's MEM containing fetal bovine serum. Consequently, the use of MDCK-SP cell culture can make current canine vaccine products much safer, of higher quality and at lower cost.

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

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.

Survival of viruses in fermented edible waste material.

The survival of selected viruses in fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Seven viruses, including pseudorabies, Newcastle disease, infectious canine hepatitis, avian infectious bronchitis, measles, vesicular stomatitis, and a porcine picornavirus were inoculated into a mixture of ground food waste (collected from a school lung program) containing Lactobacillus acidophilus. Mixtures were incubated at 5 C, 10 C, 20 C, and 30 C for 96 hours. Temperature, pH, and redox potential were monitored. Samples for virus isolation were obtained daily. Newcastle disease virus and infectious canine hepatitis virus survived the entire test period. The porcine picornavirus was inactivated at 30 C after 74 hours, but survived for the entire test period at the other temperatures. Pseudorabies virus was inactivated at 20 C and 30 C within 24 hours, but survived for 48 hours at 10 C and 96 hours at 5 C. Avian infectious bronchitis virus was inactivated at 20 C and 30 C within 24 hours, but survived 72 hours at 5 C and 10 C. Measles and vesicular stomatitis viruses were rapidly inactivated at all 4 temperatures.

Hepatitis in skunks caused by the virus of infectious canine hepatitis.

Two cases of acute, fatal, hepatitis occurred in young, striped skunks (Mephitis mephitis) trapped in southern Ontario. Histologically, lesions in the liver were similar to infectious canine hepatitis. A virus was isolated which produced large intranuclear inclusions in dog kidney cell cultures. These inclusions were Feulgen-positive and fluoresced green with acridine orange stain. The skunk hepatitis isolate was identified as the virus of infectious canine hepatitis by virus neutralization tests.

Cytoplasmic inclusions in canine cells infected with infectious canine laryngotracheitis (ICL) adenovirus.

Spherical dark inclusions were observed in both the cytoplasm and nuclei of infectious canine laryngotracheitis (ICL) adenovirus infected MDCK (Madin-Darby Canine Kidney) cells. The distribution of these inclusions in the cells appeared to indicate that they were formed in both the cytoplasm and in the nucleus at about the same time and there did not appear to be movement of these inclusions between the cytoplasm and the nucleus during the early stages of infection. Morphological appearance, 3H-leucine autoradiography, and immunoferritin labelling showed that the cytoplasmic inclusions were similar in nature to the dark nuclear inclusions, and contained the adenovirus hexon antigen, but not the penton base and fiber antigens.

Identification of a canine adenovirus (infectious canine hepatitis virus) inhibitor in dog liver extracts as arginase.

Extracts of canine liver inhibited growth of infectious canine hepatitis (ICH) virus, a canine adenovirus. Purified extracts from mammalian, but not avian, liver tissue contained the inhibitor, and evidence is presented that the inhibitory factor is the enzyme arginase (arginine ureohydrolase). This study further emphasized the need for arginine in adenovirus growth and may explain some of the difficulties in isolating small amounts of ICH virus from suspensions of liver.

Continuous-flow ultracentrifugation of canine distemper virus and infectious canine hepatitis virus.

The use of a Spinco L-4 zonal centrifuge with the B-XVI continuous-flow rotor for the purification of canine distemper and infectious canine hepatitis viruses is described. Up to 68 liters of virus was processed at one time. Infectious canine hepatitis virus was found to band at 39% sucrose and canine distemper virus banded between 32 and 48% sucrose. The virus was concentrated 10-fold, and the purity of the virus, as measured by protein concentration, was increased by 99%.