Canine Adenovirus Type Research Articles

Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R2) were >0.993 for all singleplex and multiplex real-time PCR assays with the exception of one that was 0.988; PCR amplification efficiencies (E) were between 92.1% and 107.8% for plasmid DNA, and 90.6–103.9% for RNA templates. In comparing singular and multiplex PCR assays, the three multiplex reactions generated similar R2 and E values to those by corresponding singular reactions, suggesting that multiplexing did not interfere with the detection sensitivities. The limit of detection (LOD) of the multiplex real-time PCR for DNA templates was 5, 2, 3, 1, 1, 1, 4, 24 and 10 copies per microliter for M. cynos, M. canis, B. brochiseptica, CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively; and 3, 2, 6, 17, 4 and 8 copies per microliter for CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively, when RNA templates were used for the four RNA viruses. No cross-detection was observed among the nine pathogens. For the 740 clinical samples tested, the newly designed PCR assay showed higher diagnostic sensitivity compared to an older panel assay; pathogen identities from selected samples positive by the new assay but undetected by the older assay were confirmed by Sanger sequencing. Our data showed that the new assay has higher diagnostic sensitivity while maintaining the assay's specificity, as compared to the older version of the panel assay.

Canine Adenovirus 1 Isolation Bioinformatics Analysis of the Fiber.

Canine adenovirus type 1 (CAdV-1) is a double-stranded DNA virus, which is the causative agent of fox encephalitis. The Fiber protein is one of the structural proteins in CAdV-1, which mediates virion binding to the coxsackievirus and adenovirus receptor on host cells. The suspected virus was cultured in the MDCK cells, and it was determined through the cytopathic effects, sequencing and electron microscopy. The informatics analysis of the Fiber was done using online bioinformatics servers. The CAdV-1-JL2021 strain was isolated successfully, and were most similar to the CAdV-1 strain circulating in Italy. The occurrence of negative selection and recombination were found in the CAdV-1-JL2021 and CAdV-2-AC_000020.1. Host cell membrane was its subcellular localization. The CAdV-1-JL2021 Fiber (ON164651) had 6 glycosylation sites and 107 phosphorylation sites, exerted adhesion receptor-mediated virion attachment to host cell, which was the same as CAdV-2-AC_000020.1 Fiber. The Fiber tertiary structure of the CAdV-1-JL2021 and CAdV-2-AC_000020.1 was different, but they had the same coxsackievirus and adenovirus receptor. “VATTSPTLTFAYPLIKNNNH” were predicted to be the potential CAdV-1 B cell linear epitope. The MHC-I binding peptide “KLGVKPTTY” were both presented in the CAdV-1-JL2021 and CAdV-2-AC_000020.1 Fiber and it is useful to design the canine adenovirus vaccine.

Establishment of Multiplex RT-PCR for Differentiating between the CD1901 and Lederle Strains of Canine Distemper Virus

Canine distemper virus (CDV) is a life-threatening pathogen in dogs. Clinical, pathological and molecular methods are required to diagnose CDV infection, and it is important to differentiate between the Korean CDV strain CD1901 and Lederle CDV vaccine strain. Therefore, in this study, we used multiplex reverse- transcription polymerase chain reaction (RT-PCR) to differentiate between the CD1901 and Lederle strains. A primer set was designed based on the CDV nucleoprotein gene and nucleotide sequence variation in the fusion (F) gene. First, 224-bp DNA bands were amplified from viral RNA of the CD1901 and Lederle strains. Then, 428- and 326-bp DNA bands were amplified in the CD1901 and Lederle strain, respectively. The multiplex RT-PCR detection limits were 2.53 and 0.8 median tissue culture infectious dose/reaction for the CD1901 and Lederle strains, respectively. No cross-reactions were detected in non-CDV reference viruses, including rabies virus, parvovirus, canine adenovirus types 1 and 2, and parainfluenza virus. The results indicate that our one-step multiplex RT-PCR is useful for differentiating between wildtype and vaccine CDV strains.

Co-circulation of five species of dog parvoviruses and canine adenovirus type 1 among gray wolves (Canis lupus) in northern Canada.

Several viruses can infect wild carnivores but their impact on wildlife health is poorly understood. We investigated the presence, diversity and distribution of various DNA viruses in 303 wolves inhabiting a vast area of the Northwest Territories, Canada, over a period of 13 years. We found evidence for the presence of canine bufavirus (CBuV, 42.6%), canine parvovirus 2 (CPV-2, 34.0%), canine bocavirus 2 (CBoV-2, 5.0%), cachavirus (CachaV-1, 2.6%), canine adenovirus 1 (CAdV-1, 1%) and minute virus of canines (MVC, 0.3%). To our knowledge, this is the first detection of CBoV-2, MVC and CachV-1 in wild animals. We also demonstrate that CBuV and CachaV-1 were already circulating among wild animals at least 11 and 10 years, respectively, before their discoveries. Although CBuV prevalence was higher, CPV-2 was the most prevalent virus among juveniles, while CBuV infection was associated with poor nutrition conditions. Even if its prevalence was low, CachaV-1 had the highest multiple infection rate (87.5%). CadV-1 and MVC sequences were highly identical to reference strains, but we observed a high diversity among the other viruses and detected three new variants. One CPV-2 variant and one CBuV variant were endemic since the beginning of the 2000s in the entire investigated region, whereas one CBuV variant and two CBoV-2 variants were found in a more restricted area over multiple years and CachaV-1 was found only in one region. Two CPV-2 variants and one CachaV-1 variant were observed only once, indicating sporadic introductions or limited circulation. Different patterns of endemicity might indicate that viruses were introduced in the wolf population at different timepoints and that mixing between wolf packs may not be constant. Different epidemiological behaviors depend on viral factors like infectivity, transmission routes, pathogenicity and tissue-tropism, and on host factors like proximity to densely populated areas, carnivory and pack density and mixing.

Immunogenicity of an Inactivated Canine Adenovirus Type 1 Vaccine for Foxes.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis. As with most viral agents, the best method of prevention is vaccination. In this study, the CAdV-1 strain F1301 strain was used to construct a new type 1 canine adenovirus inactivated vaccine candidate, and its safety and immunogenicity were evaluated in silver foxes. Next, animals were challenged and survival rates of animals vaccinated with either the commercially available or the current candidate vaccine were examined. The results confirmed that the inactivated CAdV-1 vaccine prepared in this study can effectively protect against challenge with virulent CAdV-1 in silver foxes, and the safety profile was improved relative to that of the commercial vaccine. This study confirmed that the fox CAdV-1 F1301 strain can be used as a platform for an inactivated CAdV-1 vaccine.

Genetic analysis of Canine adenovirus type 2 strains circulating in Turkey from past to present

Canine adenoviruses (CAV) can infect both domestic and wild canid. Canine Adenovirus Type-1 (CAV-1) and Canine Adenovirus Type-2 (CAV-2) are the cause of infectious canine hepatitis and infectious canine laryngotracheitis, respectively. The epidemiology of the virus is not clear enough in the most territory. For this purpose, data on the virus in Turkey, which is a bridge between continents, were examined. Comparing the sequence data of strains circulated in the field over a ten-year period revealed a genetic heterogeneity. To make this comparision total of 32 fecal samples collected from shelter dogs with diarrhea in 2011 was used. Firstly, virus isolation was performed in MDCK cells and cytopathogenic effects were observed in 4/32 samples. Secondly, the supernatant of four isolates were found positive for the E3 gene of CAV-2 by PCR. Strains were placed on a different branch from other Turkish isolates under the same subgroup in the phylogenetic tree. According to partial E3 gene analysis, seven amino acid substitutions were detected between the strains. Due to Shannon entropy value and ConSurf analysis, all of them were indicated as important antigenic sites. This study pointed out the possible genetic heterogeneity among CAV-2 in Turkey.

Development of a Recombinase Polymerase Amplification Fluorescence Assay for the Detection of Canine Adenovirus 2.

Canine adenovirus type 2 (CAdV-2) is often found in co-infections with other pathogens causing canine infectious respiratory disease (CIRD). Rapid, efficient, and convenient pathogen detection is the best approach for early confirmatory diagnosis. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) assay for detection of canine adenovirus 2 (CAV), which can detect CAV within 15 min at 39°C. The detection limit that assay was 214 copies/μl DNA molecules per reaction. The specificity was indicated by a lack of cross-reaction with canine distemper virus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV). Field and clinical applicability of this assay were evaluated using 86 field samples. The coincidence rate of the detection results for clinical samples between CAV-RPA and qPCR was 97.7%. In summary, the real-time CAV-RPA analysis provides an efficient, rapid and sensitive detection method for CAV.

In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9

AbstractGenetically 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 circovirus and Canine adenovirus type 1 and 2 in dogs with parvoviral enteritis.

Canine parvovirus type 2 (CPV-2) is one of the most relevant pathogens associated with enteritis in dogs and is frequently reported in association with the detection of other pathogens in faeces. In this study the concomitant presence of Canine circovirus (CanineCV) and Canine adenovirus (CAdV) DNA in faecal or intestine samples of 95 dogs with parvovirus enteritis sampled in Italy (1995–2017) was investigated and the viruses identified were genetically characterised. Potential correlations with the antigenic variant of CPV-2 and with signalment data and outcome were evaluated. Twenty-eight of 95 (29.5%) CPV-2 infected dogs tested positive to other viruses: 7/28 were also positive to CanineCV, 1/28 to CAdV-1, 18/28 to CAdV-2, 1/28 to CanineCV and CAdV-2, and 1/28 to CAdV-1 and CAdV-2. The frequency of CAdV DNA detection and coinfections was significantly higher in purebred dogs compared to mixed breed ones (P = 0.002 and 0.009, respectively). The presence of coinfection was not associated with any other relevant data available, including CPV-2 variant and final outcome. The detection of CanineCV in a dog sampled in 2009 allowed to backdating its circulation in dogs. The eight CanineCV completely sequenced were phylogenetically related to the CanineCV identified in dogs, wolves and a badger from Europe, USA, Argentina and China. Nine CAdV were partially sequenced and phylogenetic analysis showed a separate branch for the oldest CAdV-2 identified (1995). From the results obtained in this study population, CanineCV and CAdV coinfections in dogs with parvoviral enteritis did not result in more severe disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11259-021-09850-y.

Naturally Acquired Infectious Canine Hepatitis in Two Captive Maned Wolf (Chrysocyon brachyurus) Puppies

We report the clinicopathological manifestations of canine adenovirus type 1 (CAV 1) infection in captive-born naturally infected maned wolves (Chrysocyon brachyurus). Two 3-month-old maned wolves presented with lethargy, emesis, dehydration, pallor, hypothermia, leucocytosis, neutrophilia, lymphopaenia and thrombocytopaenia. One of the puppies died shortly after admission, with gross changes that included marked gastrointestinal petechiae, splenomegaly, hepatomegaly and pulmonary haemorrhage. Histologically, large eosinophilic intranuclear body inclusions were found in the liver and kidneys. The other wolf had elevated alkaline phosphatase, alanine aminotransferase and creatine kinase activities, and later developed anaemia, hypoalbuminaemia, bilirubinaemia, bilirubinuria, haematuria and proteinuria. Ultrasound demonstrated hepatomegaly, splenomegaly, inguinal lymphadenomegaly and lesions suggestive of gastritis and enteritis. Despite supportive treatment, the animal died. At necropsy, there was icterus, subcutaneous oedema in the inguinal region and hindlimbs, subchondral haemorrhage of articular cartilage of the femoral-tibial-patellar and tarsal joints of both hindlimbs, lymphadenomegaly, bronchopneumonia, hepatomegaly and petechiae in the gastrointestinal mucosa. Microscopically, there was a severe necrotizing hepatitis with intranuclear viral inclusions, fibrinous-necrotizing splenitis, non-suppurative meningoencephalitis and interstitial nephritis. A quantitative PCR test for CAV 1 using DNA extracted from peripheral blood was positive. The clinicopathological findings are similar to those of CAV 1 infection in dogs and other canids.

Isolation and genetic characterization of canine adenovirus type 2 from a domestic dog showing neurological symptoms.

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.

Molecular characterization of canine adenovirus type 2 in dogs from India.

A total of 26 nasal swab samples were collected from dogs with gastroenteritis and respiratory tract infections in and around Chennai, India during 2019–20. All the samples were subjected to PCR using common primers for rapid diagnosis and differentiation of CAV1 and CAV2. Only one sample produced an amplicon of 1030 bp indicating the presence of CAV2 which was confirmed by further sequencing. The analysis of the sequence revealed 100 per cent identity with other CAV type 2 isolates from Brazilian, Canadian and USA strains and 95.9 per cent identity with other Indian CAV2 strains. The phylogenetic analysis of E3 gene reveal two distinct clusters (Asian and America-Europe subgroup) in which our strain (ABT/MVC/CAV2/001) grouped with CAV2 of America-Europe subgroup instead of Asian continent subgroup.This study confirms a novel CAV2 strain using molecular techniques which are genetically distinct in nature from other Indian CAV2 strains that is currently circulating in India.

Optimized Parameters for Transducing the Locus Coeruleus Using Canine Adenovirus Type 2 (CAV2) Vector in Rats for Chemogenetic Modulation Research.

IntroductionThe locus coeruleus noradrenergic (LC-NA) system is studied for its role in various neurological and psychiatric disorders such as epilepsy and Major Depression Dissorder. Chemogenetics is a powerful technique for specific manipulation of the LC to investigate its functioning. Local injection of AAV2/7 viral vectors has limitations with regards to efficiency and specificity of the transduction, potentially due to low tropism of AAV2/7 for LC neurons. In this study we used a canine adenovirus type 2 (CAV2) vector with different volumes and viral particle numbers to achieve high and selective expression of hM3Dq, an excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD), for chemogenetic modulation of LC neurons.MethodsAdult male Sprague-Dawley rats were injected in the LC with different absolute numbers of CAV2-PRSx8-hM3Dq-mCherry physical particles (0.1E9, 1E9, 5E9,10E9, or 20E9 pp) using different volumes (LowV = 3 nl × 300 nl, MediumV = 3 × 600 nl, HighV = 3 × 1200 nl). Two weeks post-injection, double-labeling immunohistochemistry for dopamine β hydroxylase (DBH) and mCherry was performed to determine hM3Dq expression and its specificity for LC neurons. The size of the transduced LC was compared to the contralateral LC to identify signs of toxicity.ResultsAdministration of Medium volume (3 × 600 nl) and 1E9 particles resulted in high expression levels with 87.3 ± 9.8% of LC neurons expressing hM3Dq, but low specificity with 36.2 ± 17.3% of hM3Dq expression in non-LC neurons. The most diluted conditions (Low volume_0.1E pp and Medium Volume_0.1E pp) presented similar high transduction of LC neurons (70.9 ± 12.7 and 77.2 ± 9.8%) with lower aspecificity (5.5 ± 3.5 and 4.0 ± 1.9%, respectively). Signs of toxicity were observed in all undiluted conditions as evidenced by a decreased size of the transduced LC.ConclusionThis study identified optimal conditions (Low and Medium Volume with 0.1E9 particles of CAV2-PRSx8-hM3Dq-mCherry) for safe and specific transduction of LC neurons with excitatory DREADDs to study the role of the LC-NA system in health and disease.

Wolf (Canis lupus) as canine adenovirus type 1 (CAdV-1) sentinel for the endangered cantabrian brown bear (Ursus arctos arctos).

Canine adenovirus type 1 (CAdV-1) causes infectious canine hepatitis (ICH) and has recently been described as a cause of death among endangered populations of European brown bear (Ursus arctos arctos) in the Cantabrian mountain range in Asturias, Spain. Sympatric wild and domestic carnivores can act as reservoirs of the virus and likely spread it into the environment and subsequently transmit it to brown bears. The present work investigates the prevalence and geo-temporal distribution of CAdV-1 among free-ranging wolves (Canis lupus) in Asturias from 2009 to 2018, during which three fatal cases of ICH were reported among brown bears in the region. A total of 149 wolves were analysed in this study, of which 21 (14%) were found to have CAdV-1 DNA based on real-time polymerase chain reaction (RT-PCR) of spleen samples. Prevalence of the virus was similar between males and females. All but one of the 20 CAdV-1-positive animals of estimable age were younger than 2years, and only one of the 46 adult animals (>2years) tested positive. Prevalence was highest in the western area of Asturias and during 2010 and 2011. Our results confirm that CAdV-1 is circulating in Asturian free-ranging wolves, supporting their possible role as virus reservoirs and sentinels in the region of this emerging disease in brown bears.

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.

Infectious Causation of Abnormal Host Behavior: Toxoplasma gondii and Its Potential Association With Dopey Fox Syndrome.

The apicomplexan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, can infect all warm-blooded animals. T. gondii can subtly alter host behaviors—either through manipulation to enhance transmission to the feline definitive host or as a side-effect, or “constraint,” of infection. In humans, T. gondii infection, either alone or in association with other co-infecting neurotropic agents, has been reliably associated with both subtle behavioral changes and, in some cases, severe neuropsychiatric disorders, including schizophrenia. Research on the potential impact of T. gondii on the behavior of other long-lived naturally infected hosts is lacking. Recent studies reported a large number of wild red foxes exhibiting a range of aberrant behavioral traits, subsequently classified as Dopey Fox Syndrome (DFS). Here we assessed the potential association between T. gondii and/or other neurotropic agents with DFS. Live, captive foxes within welfare centers were serologically tested for T. gondii and, if they died naturally, PCR-tested for vulpine circovirus (FoxCV). Post-mortem pseudo-control wild foxes, obtained from pest management companies, were PCR-tested for T. gondii, FoxCV, canine distemper virus (CDV), canine adenovirus type (CAV)-1 and CAV-2. We also assessed, using non-invasive assays, whether T. gondii–infected foxes showed subtle behavioral alterations as observed among infected rodent (and other) hosts, including altered activity, risk, and stress levels. All foxes tested negative for CAV, CDV, CHV, and DogCV. DFS was found to be associated with singular T. gondii infection (captives vs. pseudo-controls, 33.3% (3/9) vs. 6.8% (5/74)) and singular FoxCV infection (66.7% (6/9) vs. 11.1% (1/9)) and with T. gondii/FoxCV co-infection (33.3% (3/9) vs. 11.1% (1/9)). Overall, a higher proportion of captive foxes had signs of neuroinflammation compared to pseudo-controls (66.7% (4/6) vs. 11.1% (1/9)). Consistent with behavioral changes seen in infected rodents, T. gondii–infected foxes displayed increased attraction toward feline odor (n=6 foxes). These preliminary results suggest that wild foxes with DFS are infected with T. gondii and likely co-infected with FoxCV and/or another co-infecting neurotropic agent. Our findings using this novel system have important implications for our understanding of both the impact of parasites on mammalian host behavior in general and, potentially, of the infectious causation of certain neuropsychiatric disorders.

Novel Genotype Definition and the First Epidemiological Investigation of Canine Adenovirus Type 2 in Dogs in Central China.

Infections caused by canine adenovirus (CAdV) type 1 have been reported worldwide in the past two decades. However, only few studies have specifically reported the prevalence of CAdV type 2 (CAdV-2). The present study investigated the persistent circulation of CAdV-2 in dogs with diarrhea in the Henan, Hubei, and Jiangsu provinces in central China from 2017 to 2019. We conducted polymerase chain reaction for detecting CAdV-2 and other related pathogens in 224 rectal swabs of pet dogs and the co-infection of canine diseases was also analyzed. In addition, the structural protein genes—Fiber, Hexon, and Penton—of the isolated CAdV-2 strains were sequenced and analyzed. The similarity between Hexon and Penton among the 19 strains was 97.4%, as revealed by sequence alignment. Multiple sequence alignment results showed that the Fiber gene sequences of these CAdV-2 strains shared 97.4–99.8% nucleotide and 94.1–99.3% amino acid identity with reference sequences and shared only 79.0–80.5% nucleotide and 77.3–80.5% amino acid identity with the vaccine strain CLL, indicating that Fiber harbored most of the variant sites. Furthermore, pairwise sequence comparisons of Hexon of CH-JS-1901 and CH-HN-1801 with that of India2006 revealed a novel genotype. Furthermore, protein model prediction showed that the amino acid mutation of fiber protein in 19 strains was located in the head region, that may cause structural changes on the surface of the fiber protein. These findings are of significance for monitoring the epidemiology of CAdV-2 infection and developing a novel vaccine which contribute to understanding genetic evolution of CAdV-2 in China.

Targeting Catecholaminergic Systems in Transgenic Rats With a CAV-2 Vector Harboring a Cre-Dependent DREADD Cassette

Techniques that allow the manipulation of specific neural circuits have greatly increased in the past few years. DREADDs (Designer receptors exclusively activated by designer drugs) provide an elegant way to manipulate individual brain structures and/or neural circuits, including neuromodulatory pathways. Considerable efforts have been made to increase cell-type specificity of DREADD expression while decreasing possible limitations due to multiple viral vectors injections. In line with this, a retrograde canine adenovirus type 2 (CAV-2) vector carrying a Cre-dependent DREADD cassette has been recently developed. In combination with Cre-driver transgenic animals, the vector allows one to target neuromodulatory pathways with cell-type specificity. In the present study, we specifically targeted catecholaminergic pathways by injecting the vector in knock-in rat line containing Cre recombinase cassette under the control of the tyrosine hydroxylase promoter. We assessed the efficacy of infection of the nigrostriatal pathway and the catecholaminergic pathways ascending to the orbitofrontal cortex (OFC) and found cell-type-specific DREADD expression.

Location of the Cell Adhesion Molecule "Coxsackievirus and Adenovirus Receptor" in the Adult Mouse Brain.

The coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane cell adhesion molecule (CAM). CAR is expressed in numerous mammalian tissues including the brain, heart, lung, and testes. In epithelial cells, CAR functions are typical of the quintessential roles of numerous CAMs. However, in the brain the multiple roles of CAR are poorly understood. To better understand the physiological role of CAR in the adult brain, characterizing its location is a primordial step to advance our knowledge of its functions. In addition, CAR is responsible for the attachment, internalization, and retrograde transport of canine adenovirus type 2 (CAV-2) vectors, which have found a niche in the mapping of neuronal circuits and gene transfer to treat and model neurodegenerative diseases. In this study, we used immunohistochemistry and immunofluorescence to document the global location of CAR in the healthy, young adult mouse brain. Globally, we found that CAR is expressed by maturing and mature neurons in the brain parenchyma and located on the soma and on projections. While CAR occasionally colocalizes with glial fibrillary acidic protein, this overlap was restricted to areas that are associated with adult neurogenesis.

Targeted Transgene Expression in Cholinergic Interneurons in the Monkey Striatum Using Canine Adenovirus Serotype 2 Vectors.

The striatum, the main input structure of the basal ganglia, is critical for action selection and adaptive motor control. To understand the neuronal mechanisms underlying these functions, an analysis of microcircuits that compose the striatum is necessary. Among these, cholinergic interneurons (ChIs) provide intrinsic striatal innervation whose dysfunction is implicated in neuropsychiatric diseases, such as Parkinson’s disease and Tourette syndrome. The ability to experimentally manipulate the activity of ChIs is critical to gain insights into their contribution to the normal function of the striatum and the emergence of behavioral abnormalities in pathological states. In this study, we generated and tested CAV-pChAT-GFP, a replication-defective canine adenovirus type 2 (CAV-2) vector carrying the green fluorescent protein (GFP) sequence under the control of the human choline acetyltransferase (ChAT) promoter. We first tested the potential specificity of CAV-pChAT-GFP to label striatal ChIs in a rat before performing experiments on two macaque monkeys. In the vector-injected rat and monkey striatum, we found that GFP expression preferentially colocalized with ChAT-immunoreactivity throughout the striatum, including those from local circuit interneurons. CAV-2 vectors containing transgene driven by the ChAT promoter provide a powerful tool for investigating ChI contributions to circuit function and behavior in nonhuman primates.