The biological characteristics of the canine adenovirus type 1 from fox and the transcriptome analysis of the infected MDCK cell.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related research are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30,535 bp and had higher homology to the other reported CAdV-1 strains. After 0, 12, and 36 h of CAdV-1 infection, the difference gene of the 592 long noncoding RNA and 11,215 microRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, Wnt, Herpes simplex, hepatitis C, and Epstein-Barr virus infection pathway in Madin-Darby canine kidney cell line (MDCK). The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified.

Canine morbillivirus (canine distemper virus) with concomitant canine adenovirus, canine parvovirus-2, and Neospora caninum in puppies: a retrospective immunohistochemical study.

A retrospective immunohistochemical study was designed to investigate the frequency of concomitant traditional infectious disease pathogens in puppies that died suddenly and review the aspects of associated pathogenesis. Fifteen puppies were evaluated; the pathology reports and histopathologic slides of these animals were reviewed to determine the pattern of histopathologic lesions. The intralesional identification of antigens of canine (distemper) morbillivirus (CDV), canine adenovirus-1 and -2 (CAdV-1 and -2), canine parvovirus-2 (CPV-2), Toxoplasma gondii, and Neospora caninum was evaluated by IHC within the histopathologic patterns observed. All puppies contained CDV nucleic acid by molecular testing. The most frequent histopathologic patterns were intestinal crypt necrosis (n = 8), white matter cerebellar demyelination (n = 7), necrohaemorrhagic hepatitis (n = 7), interstitial pneumonia (n = 7), and gallbladder oedema (n = 5). All puppies contained intralesional antigens of CDV in multiple tissues resulting in singular (n = 3), and concomitant dual (n = 3), triple (n = 5) and quadruple (n = 4) infections by CAdV-1, and -2, CPV-2, and N. caninum; T. gondii was not identified. Concomitant infections by CDV was observed with N. caninum (100%; 1/1), CPV-2 (100%; 8/8), CAdV-1 (100%; 8/8), and CAdV-2 (100%; 8/8). Intralesional antigens of CDV and not CAdV-1 were identified in cases of gallbladder oedema. The "blue eye" phenomenon was histologically characterized by corneal oedema and degenerative lesions to the corneal epithelium, without inflammatory reactions.

A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons.

Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits.

Genetic dissection of the neuro-glio-vascular machinery in the adult brain.

The adult brain actively controls its metabolic homeostasis via the circulatory system at the blood brain barrier interface. The mechanisms underlying the functional coupling from neuron to vessel remain poorly understood. Here, we established a novel method to genetically isolate the individual components of this coupling machinery using a combination of viral vectors. We first discovered a surprising non-uniformity of the glio-vascular structure in different brain regions. We carried out a viral injection screen and found that intravenous Canine Adenovirus 2 (CAV2) preferentially targeted perivascular astrocytes throughout the adult brain, with sparing of the hippocampal hilus from infection. Using this new intravenous method to target astrocytes, we selectively ablated these cells and observed severe defects in hippocampus-dependent contextual memory and the metabolically regulated process of hippocampal neurogenesis. Combined with AAV9 targeting of neurons and endothelial cells, all components of the neuro-glio-vascular machinery can be simultaneously labeled for genetic manipulation. Together, we demonstrate a novel method, which we term CATNAP (CAV/AAV Targeting of Neurons and Astrocytes Perivascularly), to target and manipulate the neuro-glio-vascular machinery in the adult brain.

Recombinant canine adenovirus type 2 expressing rabbit hemorrhagic disease virus VP60 protein provided protection against RHD in rabbits.

Rabbit hemorrhagic disease virus (RHDV) is responsible for rabbit hemorrhagic disease (RHD), which is an acute, lethal and highly contagious disease in both wild and domestic rabbits. Although current vaccines are highly effective for controlling RHD, they are derived from infected rabbit livers and their use is thus associated with safety and animal-welfare concerns. In this study, we generated a recombinant lentogenic canine adenovirus type 2 (CAV2) vector expressing the RHDV vp60 gene, named rCAV2-VP60. rCAV2-VP60 expressed VP60 protein in Madin-Darby canine kidney cells as demonstrated by western blot and immunofluorescence assay. Polymerase chain reaction confirmed that the vp60 gene was successfully inserted into rCAV2-VP60 and was still detectable after 20 passages, indicating its stable genetic character. We evaluated the feasibility of rCAV2-VP60 as a live-virus-vectored RHD vaccine in rabbits. rCAV2-VP60 significantly induced specific antibodies to RHDV and provided effective protection against RHDV lethal challenge. These results suggest that rCAV2 expressing RHDV VP60 could be a safe and efficient candidate vaccine against RHDV in rabbits.

Study of adenovirus and CAR axonal transport in primary neurons.

Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals. These abilities are due to the engagement of virions to the coxsackievirus and adenovirus receptor at the surface of neurons, which is linked to the endocytic and axonal transport machineries. The trafficking of CAV-2 and the coxsackievirus and adenovirus receptor (CAR) can be visualized ex vivo by incubating primary neurons (e.g., motoneurons and hippocampal neurons) with fluorescently labeled virions or recombinant viral proteins. Using this approach, we could recapitulate the mechanisms responsible for long-range transport of adenovirus in neurons.

An endocytic CARriage tale: Adenoviruses internalization and trafficking in neurons.

In immune-competent hosts, adenoviruses (Ads) are mild pathogens that cause mainly infections of the respiratory and ocular tracks. The advent of Ad-based gene transfer vectors made the understanding of their interaction with the host cellular machinery an intensive field of research over the last decade. As studies focused primarily on epithelial-like cells, the mechanism of neuronal uptake of Ads was still missing. Using a combination of biochemical and cell biology approaches, we characterized the axonal trafficking pathway used by the canine adenovirus serotype 2 (CAV-2) to reach the neuronal soma. We showed that CAV-2 and CAR (coxsackievirus and adenovirus receptor) are entering a vesicular pathway coupled to the axonal transport machinery. The lumen of the multivalent Rab7 (+) vesicles that transport CAV-2 and CAR is, surprisingly, pH neutral. Among other issues, our study opens numerous questions concerning the neuronal function of CAR.

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.

Functional organization of the major late transcriptional unit of canine adenovirus type 2.

Vectors derived from canine adenovirus type 2 (CAV-2) are attractive candidates for gene therapy and live recombinant vaccines. CAV-2 vectors described thus far have been generated by modifying the virus genome, most notably early regions 1 and 3 or the fiber gene. Modification of these genes was underpinned by previous descriptions of their mRNA and protein-coding sequences. Similarly, the construction of new CAV-2 vectors bearing changes in other genomic regions, in particular many of those expressed late in the viral cycle, will require prior characterization of the corresponding transcriptional units. In this study, we provide a detailed description of the late transcriptional organization of the CAV-2 genome. We examined the major late transcription unit (MLTU) and determined its six families of mRNAs controlled by the putative major late promoter (MLP). All mRNAs expressed from the MLTU had a common non-coding tripartite leader (224 nt) at their 5' end. In transient transfection assays, the predicted MLP sequence was able to direct luciferase gene expression and the TPL sequence yielded a higher amount of transgene product. Identification of viral transcriptional products following in vitro infection confirmed most of the predicted protein-coding regions that were deduced from computer analysis of the CAV-2 genome. These findings contribute to a better understanding of gene expression in CAV-2 and lay the foundation required for genetic modifications aimed at vector optimization.

Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus.

Seoul virus glycoprotein Gn is a major structural protein and candidate antigen of hantavirus that induces a highly immunogenic response for hantavirus vaccine. In this study, a replication-competent recombinant canine adenovirus type-2 expressing Gn was constructed by the in vitro ligation method. The Gn expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the SV40 early mRNA polyadenylation signal, was cloned into the SspI site of the E3 region which is not essential for proliferation of CAV-2. Expression of Gn was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

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.