Establishment of a Simple and Efficient Reverse Genetics System for Canine Adenoviruses Using Bacterial Artificial Chromosomes.

Canine adenoviruses (CAdVs) are divided into pathotypes CAdV1 and CAdV2, which cause infectious hepatitis and laryngotracheitis in canid animals, respectively. They can be the backbones of viral vectors that could be applied in recombinant vaccines or for gene transfer in dogs and in serologically naïve humans. Although conventional plasmid-based reverse genetics systems can be used to construct CAdV vectors, their large genome size creates technical difficulties in gene cloning and manipulation. In this study, we established an improved reverse genetics system for CAdVs using bacterial artificial chromosomes (BACs), in which genetic modifications can be efficiently and simply made through BAC recombineering. To validate the utility of this system, we used it to generate CAdV2 with the early region 1 gene deleted. This mutant was robustly generated and attenuated in cell culture. The results suggest that our established BAC-based reverse genetics system for CAdVs would be a useful and powerful tool for basic and advanced practical studies with these viruses.

Infectious Canine Hepatitis in a Brown Bear ( Ursus arctos horribilis) from Alaska, USA.

We diagnosed infectious canine hepatitis in a free-ranging brown bear ( Ursus arctos horribilis) cub from Alaska, US, found dead in October 2015. Intranuclear inclusion bodies were present in hepatocytes, and immunohistochemistry showed reactivity to adenoviral antigens. Sequencing of the hexon protein of adenovirus showed 100% identity to canine adenovirus 1.

Novel sequence variants of viral hexon and fibre genes in two dogs with canine adenovirus type 1-associated disease.

There is little information on sequence variation of canine adenovirus type 1 (CAdV-1), the aetiological agent of infectious canine hepatitis (ICH). This study reports hexon and fibre gene sequence variants of CAdV-1 in a dog with systemic ICH and a dog with the ocular form of the disease ('blue eye') in Northern Italy in 2013. One of the sequence variants matched a CAdV-1 fox sequence previously detected in Italy.

A Novel Recombinant Canine Adenovirus Type 1 Detected from Acute Lethal Cases of Infectious Canine Hepatitis.

In this study, canine adenoviruses (CAdVs) from two acute fatal cases of infectious canine hepatitis (ICH) were analyzed using molecular detection and sequencing of the pVIII, E3, and fiber protein genes. Pathological findings in affected dogs were typical for CAdV-1 associated disease, characterized by severe centrilobular to panlobular necrohemorrhagic hepatitis and the development of disseminated intravascular coagulation in the terminal stages of disease. Comparison of partial genome sequences revealed that although these newly detected viruses mainly had CAdV-1 genome characteristics, their pVIII gene was more similar to that of CAdV-2. This likely suggests that a recombination has occurred between CAdV-1 and CAdV-2, which possibly explains the cause of vaccine failure or increased virulence of the virus in the observed ICH cases.

A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2.

Canine adenoviruses are a major cause of disease in dogs, coyotes, red foxes and wolves, as well as in other carnivores and marine mammals. Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB), respectively. In this study, a duplex real-time PCR assay for simultaneous detection and characterisation of CAdV-1 and CAdV-2 was developed by using a single primer pair and virus-specific probes. The assay was validated testing standard DNAs produced on purpose and clinical samples of various matrices known to be positive for CAdV-1, CAdV-2 or both viruses. Precise calculation of DNA loads in samples containing a wide range of viral amounts was allowed by generating a standard curve for absolute quantification. The assay was proven to be highly specific, since no cross-reactions with the different CAdV type was observed, and sensitive, being able to detect less than 10 copies of CAdV-1/CAdV-2 DNA. The low intra-assay and interassay coefficient of variations demonstrated a high repeatability, thus confirming the potential use of this assay for quantitative detection of CAdV-1 and CAdV-2 for rapid diagnosis and epidemiological investigations.

Assessing demographic and epidemiologic parameters of rural dog populations in India during mass vaccination campaigns.

Mass vaccination of dogs is a mainstay for efforts to control rabies and other viral pathogens. The success of such programs is a function of the ability to vaccinate sufficient proportions of animals to develop herd immunity. However, fully assessing success in reaching target vaccination-levels and in understanding the outcome of mass vaccination efforts is hindered if insufficient information is available on the demographics of dog populations and the prevalence of the targeted pathogens. While such information can sometimes be gained from questionnaire surveys, greater precision requires direct assessment of the dog populations. Here we show how such information can be gained from surveys of dogs conducted in association with mass-vaccination programs. We conducted surveys of dogs in six villages in rural Maharashtra, India, between February and July 2011 as part of an effort to reduce the risk of human rabies and virus transmission from dogs to wildlife. Mass vaccination efforts were conducted in each village, and paired with blood sample collection and photographic mark-recapture approaches to gain epidemiologic and demographic data. This data in turn facilitated estimates of dog abundance, population density and structure, vaccination coverage, and seroprevalence of antibodies against canine adenovirus (CAV), canine parvovirus (CPV), and canine distemper virus (CDV). The median dog population size for the six villages was 134 (range 90-188), the median dog population density was 719 dogs per km(2) (range 526-969), and the median human:dog ratio for these six villages was 34 (range 30-47). The median household:dog ratio for the six villages was 6 (range 5-8). Following vaccination efforts, the median vaccination coverage achieved was 34% (range 24-42%). The dog populations consisted mostly of adult dogs (67-86%) and the median sex ratio for the study area was male biased (1.55 males per female; range 0.9-2.5). The seroprevalence of antibodies against CAV, CPV and CDV was 68, 88 and 73%, respectively. Mass vaccination campaigns provide an opportunity to obtain vital epidemiological and demographic data, and develop a clearer understanding of the threats and impacts of diseases and disease control measures.

Molecular epidemiology of canine adenovirus type 1 and type 2 in free-ranging red foxes (Vulpes vulpes) in Italy.

To date, no studies exist regarding the presence of canine adenovirus (CAdV) infection in foxes in Italy. Furthermore, the majority of worldwide investigations regarding the presence of CAdV in foxes have been carried out using common serological assays which are unable to differentiate between CAdV type 1 and CAdV type 2. To assess the presence of viral infection in Italian red foxes (Vulpes vulpes), thirty-two subjects shot during the regular hunting season in the province of Pisa (Tuscany, Italy) were sampled and tested using a polymerase chain reaction (PCR) assay capable of distinguishing between CAdV type 1 and type 2. Two subjects were positive for CAdV-1 infection and one other for CAdV-2 infection. Sequence analysis of the two CAdV-1 viruses showed complete identity between them and a high genetic similarity with all reference strains sequenced in dogs in the last twenty years, indicating the presence of genetically stable CAdV-1 in red foxes in Italy which could easily be transmitted from the wild animal population to domestic dogs. Therefore, this is the first reliable identification of CAdV-2 in foxes, and cloning of the virus detected has revealed a possible coinfection involving two different CAdV-2 strains, raising new questions about the pathogenic role of CAdV-2 in wildlife. The presence of CAdV-1 and CAdV-2 infection in foxes could represent a problem for both wild animals and domestic dogs, and emphasises the central role of red foxes in maintaining these viruses in the territory.

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.

Characterization of a canine homolog of hepatitis C virus.

An estimated 3% of the world's population is chronically infected with hepatitis C virus (HCV). Although HCV was discovered more than 20 y ago, its origin remains obscure largely because no closely related animal virus homolog has been identified; furthermore, efforts to understand HCV pathogenesis have been hampered by the absence of animal models other than chimpanzees for human disease. Here we report the identification in domestic dogs of a nonprimate hepacivirus. Comparative phylogenetic analysis of the canine hepacivirus (CHV) confirmed it to be the most genetically similar animal virus homolog of HCV. Bayesian Markov chains Monte Carlo and associated time to most recent common ancestor analyses suggest a mean recent divergence time of CHV and HCV clades within the past 500-1,000 y, well after the domestication of canines. The discovery of CHV may provide new insights into the origin and evolution of HCV and a tractable model system with which to probe the pathogenesis, prevention, and treatment of diseases caused by hepacivirus infection.

Canine adenoviruses and herpesvirus.

Canine adenoviruses (CAVs) and canine herpesvirus (CHV) are pathogens of dogs that have been known for several decades. The two distinct types of CAVs, type 1 and type 2, are responsible for infectious canine hepatitis and infectious tracheobronchitis, respectively. In the present article, the currently available literature on CAVs and CHV is reviewed, providing a meaningful update on the epidemiologic, pathogenetic, clinical, diagnostic, and prophylactic aspects of the infections caused by these important pathogens.

Detection and differentiation of CAV-1 and CAV-2 by polymerase chain reaction.

Canine adenovirus type 1 (CAV-1) and type 2 (CAV-2) can be categorized in the laboratory by haemagglutination and neutralization tests, but they are difficult to differentiate from each other in specimens, especially when infection occurs in the digestive tract. The object of this study was to develop a simple method of detecting and differentiating them. One pair of common primers was designed and synthesized according to the sequences of the E3 and flanking regions and a polymerase chain reaction (PCR) assay was established using these two primers to amplify the virus-specific DNA fragment from clinical specimens as well as from cell cultures. After elecctrophoresis, under the same amplification conditions, 508 bp and 1030 bp PCR products were observed for CAV-1 and CAV-2, respectively. These were further shown to be adenovirus specific by dot hybridization and sequencing. As only one pair of primers was involved in the PCR procedure, it was faster and easier to perform than any of the other assays used for detecting canine adenovirus, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses.

Focal mesangial-sclerosing glomerulonephritis and acute-spontaneous infectious canine hepatitis: structural, immunohistochemical and subcellular studies.

The glomerular alterations observed in a dog with acute spontaneous infectious canine hepatitis (ICH) are described. Histologic changes of the glomeruli were enlargement of the mesangium with presence of intranuclear inclusion bodies and without proliferation of mesangial cells. Electron microscopy revealed adenovirus replication sites in glomerular mesangial cells and in endothelial cells of glomerular capillaries, as well as a focal mesangial-sclerosing glomerulonephritis associated with electron dense deposits which were closely related with extracellular ICH viral particles and immunohistochemically reactive for immunoglobulin (Ig) G, IgA, IgM and C3c complement components.