Explore the vast range of titles available.

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.

•The immune response to vaccination is moderately heritable in dogs.•Many SNPs of low to high effect contribute to vaccine-induced immune response.•Genes involving the spliceosome may play a role in vaccine-induced immune response. Both genetic and non-genetic factors contribute to individual variation in the immune response to vaccination. Understanding how genetic background influences variation in both magnitude and persistence of vaccine-induced immunity is vital for improving vaccine development and identifying possible causes of vaccine failure. Dogs provide a relevant biomedical model for investigating mammalian vaccine genetics; canine breed structure and long linkage disequilibrium simplify genetic studies in this species compared to humans. The objective of this study was to estimate the heritability of the antibody response to vaccination against viral and bacterial pathogens, and to identify genes driving variation of the immune response to vaccination in Beagles. Sixty puppies were immunized following a standard vaccination schedule with an attenuated combination vaccine containing antigens for canine adenovirus type 2, canine distemper virus, canine parainfluenza virus, canine parvovirus, and four strains of Leptospira bacteria. Serum antibody measurements for each viral and bacterial component were measured at multiple time points. Heritability estimations and GWAS were conducted using SNP genotypes at 279,902 markers together with serum antibody titer phenotypes. The heritability estimates were: (1) to Leptospira antigens, ranging from 0.178 to 0.628; and (2) to viral antigens, ranging from 0.199 to 0.588. There was not a significant difference between overall heritability of vaccine-induced immune response to Leptospira antigens compared to viral antigens. Genetic architecture indicates that SNPs of low to high effect contribute to immune response to vaccination. GWAS identified two genetic markers associated with vaccine-induced immune response phenotypes. Collectively, these findings indicate that genetic regulation of the immune response to vaccination is antigen-specific and influenced by multiple genes of small effect.

Canine adenoviruses (CAdVs) are divided into two serotypes, CAdV1 and CAdV2, whose members mainly cause infectious hepatitis and laryngotracheitis, respectively, in canids. To gain insight into the molecular basis of viral hemagglutination, we constructed chimeric viruses whose fiber proteins or their knob domains, which play a role in viral attachment to cells, were swapped among CAdV1, CAdV2, and bat adenovirus via reverse genetics. The results revealed that, in each case, viral hemagglutination was specifically mediated by the fiber protein or knob domain, providing direct evidence for fiber-protein-directed receptor-binding characteristics of CAdVs.

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 × 10 4 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.

Viral enteritis is a significant cause of death among dogs younger than 6 months. In this study, the presence of canine chaphamaparvovirus (CaChPV), canine bufavirus (CBuV), and canine adenovirus (CAdV) was investigated in 62 diarrheal dogs previously tested for other viral pathogens (canine parvovirus type 2, canine coronavirus, and canine circovirus). CBuV was detected in two dogs (3.22%) and CaChPV in one dog (1.61%). One dog tested positive for three parvoviruses (CPV-2b, CBuV, and CaChPV). All dogs tested negative to CAdV-1/CAdV-2. A long genome fragment of one of the two identified CBuVs and of the CaChPV was obtained and analyzed. New Turkish CBuVs had high identity rates (96%–98% nt; 97%–98% aa) with some Italian CBuV strains (CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA). The phylogenetic analysis powerfully demonstrated that these viruses belonged to a novel genotype (genotype 2). A part of the genome ChPV-TR-2021–19 revealed high identity rates (> 98% nt and > 99% aa) with some Canadian CaChPV strains (NWT-W88 and NWT-W171) and the Italian CaChPV strain Te/37OVUD/2019/IT. This study is the first report on the detection of CBuV-2 and the concomitant presence of three canine parvoviruses in Turkey. The obtained data will contribute to the molecular epidemiology and the role in the etiology of enteric disease of new parvoviruses.

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly contagious, multifactorial syndrome that primarily affects dogs in crowded environments such as shelters, kennels, and breeding facilities. Three major CIRDC-associated pathogens: Canine distemper virus (CDV), Canine adenovirus type 2 (CAV-2), and (Bb) have been reported in the canine population of India. In this study, a multiplex PCR (mPCR) assay was developed and optimized for the simultaneous detection of these three pathogens. The multiplex assay was designed targeting three genes H, E3 and of CDV, CAV-2 and Bb respectively. This multiplex assay was optimized in both singleplex and multiplex formats by adjusting key PCR parameters such as primer concentration, annealing temperature, and incubation time to achieve distinct and reproducible amplification of all three targets. The developed assay demonstrated high analytical sensitivity, detecting 1,060 copies/μL for CDV, 11,403 copies/μL for CAV-2, and 11,016 copies/μL for Bb, with 100% specificity and no cross-reactivity with non-target organisms. The assay was validated on 55 clinical samples of dogs suspected with CIRDC, the assay detected pathogens in 32.2% of cases, with CDV being the most prevalent (25%). Compared with previously published singleplex PCR methods, the mPCR showed excellent diagnostic performance, achieving 94.12% sensitivity, 94.74% specificity, and 94.55% overall accuracy. This study demonstrates a rapid, specific, and cost-effective diagnostic mPCR assay capable of efficiently identifying key CIRDC pathogens in a single reaction. The assay is highly suitable for molecular diagnosis as well as large-scale field surveillance.

Canine adenoviruses (CAVs) are of two types: canine adenovirus type 1 (CAV-1), which causes infectious canine hepatitis, and canine adenovirus type 2 (CAV-2), which is mainly associated with the respiratory type of disease in dogs. Due to the widespread use of modified live vaccines to control canine adenoviral infections and subsequently reduced disease incidence, CAVs are often neglected by clinicians. Although a number of studies are available about CAV-1 prevalence in India, only meagre information is available about CAV-2. This study reports the CAV-2 infection in a vaccinated dog with neurological and respiratory symptoms which was found negative for other canine pathogens like canine distemper virus and canine parvovirus. The virus was successfully isolated from rectal swab in MDCK cells and characterized by immunofluorescence assay and virus neutralization test. On phylogenetic analysis of partial E3 region, the Indian CAV-2 grouped in a separate clade different from established subgroups. An insertion of “G” nucleotide was reported at nucleotide (nt.) position 1077 in the E3 gene of Indian CAV-2 isolates which led to a frameshift in the coding region of E3 gene thereby imparting additional eleven amino acids to its C-terminal end in comparison to isolates from other parts of the world. This may have an implication on the functional role of E3 protein inside the cell. This study reinforces the unique signature insertion in the E3 gene of Indian CAV-2 and is the second study in the world to report the association of CAV-2 with neurological disease in dogs.

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM3D(Gq)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM3D(Gq)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM3D(Gq)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM3D(Gq)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.

Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.

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.