Canine Adenovirus Type Research Articles

Booster effect of canine distemper, canine parvovirus infection and infectious canine hepatitis combination vaccine in domesticated adult dogs

Domesticated adult dogs with antibody titer classified as below 'high' to one or more of canine distemper virus (CDV), canine parvovirus type-2 (CPV-2) and canine adenovirus type-1 (CAdV-1) were then given an additional inoculation, and the effectiveness of this booster evaluated 2 months later. Consequently, CDV and CAdV-1 antibody titer experienced a significant increase, but the same effect was not observed in the antibody titer of CPV-2. These findings suggest that with additional inoculation, a booster effect may be expected in increasing antibody titers for CDV and CAdV-1, but it is unlikely to give an increase in CPV-2 antibody titer.

Bioprocess development for canine adenovirus type 2 vectors

Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors produced in MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ∼10(9) infectious particles (IP) ml(-1) and 2 × 10(3) IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.

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.

A Novel Recombinant Peste des Petits Ruminants-Canine Adenovirus Vaccine Elicits Long-Lasting Neutralizing Antibody Response against PPR in Goats

BackgroundPeste des petits ruminants (PPR) is a highly contagious infectious disease of goats, sheep and small wild ruminant species with high morbidity and mortality rates. The Peste des petits ruminants virus (PPRV) expresses a hemagglutinin (H) glycoprotein on its outer envelope that is crucial for viral attachment to host cells and represents a key antigen for inducing the host immune response.Methodology/Principal FindingsTo determine whether H can be exploited to generate an effective PPRV vaccine, a replication-competent recombinant canine adenovirus type-2 (CAV-2) expressing the H gene of PPRV (China/Tibet strain) was constructed by the in vitro ligation method. The H expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the BGH early mRNA polyadenylation signal, was inserted into the SspI site of the E3 region, which is not essential for proliferation of CAV-2. Infectious recombinant rCAV-2-PPRV-H virus was generated in transfected MDCK cells and used to immunize goats. All vaccinated animals produced antibodies upon primary injection that were effective in neutralizing PPRV in vitro. Higher antibody titer was obtained following booster inoculation, and the antibody was detectable in goats for at least seven months. No serious recombinant virus-related adverse effect was observed in immunized animals and no adenovirus could be isolated from the urine or feces of vaccinated animals. Results showed that the recombinant virus was safe and could stimulate a long-lasting immune response in goats.Conclusions/SignificanceThis strategy not only provides an effective PPR vaccine candidate for goats but may be a valuable mean by which to differentiate infected from vaccinated animals (the so-called DIVA approach).

DIAGNOSTIC BIOMARKERS OF INFECTIOUS CANINE HEPATITIS IN EXPERIMENTALLY INFECTED PUPPIES

Infectious canine hepatitis (ICH) is a systemic disease of Canidae and Ursidae caused by canine adenovirus type 1 (CAV-1) produces acute necrohemorrhagic hepatitis. The aim of the present study was to throw the light on the humoral and molecular diagnosis by polymerase chain reaction (PCR) and biochemical changes that occur in puppies infected with infectious canine hepatitis virus. Liver functions. tests, hematological profile, in addition to histopathological changes were performed. The study was carried out on fourteen native breed puppies of about three to four months old, apparently healthy, free from internal and external parasites. They were fed balanced diet and adequate water and housed under hygienic conditions in special isolated kennels. The animals were not vaccinated, and found to be free from canine hepatitis antibodies as screened by serum neutralization test. Puppies were divided into two groups, group 1, composed of 11 puppies were experimentally infected group with ICHV, and group 2, composed of 3 puppies were kept as non- infected control group. The results showed that, CAV-1 was detected in eight infected puppies by PCR showing amplicon of 508 bp sizes at the 1st and 2nd week post viral infection. The infected puppies showed an elevation of serum enzymes activities as ALT, AST, ALP and GGT throughout the experimental period, also serum total protein and serum albumin were decreased at the 3rd and 4th week post infection. Concerning hematological profile the infected puppies showed a decrease of HB, RBCs, platelets and haematocrit throughout the experimental period leading to anemia. The infected puppies showed a decrease of the total leukocytic count throughout the experimental period whereas neutrophils count increased significantly at the 1st week post viral infection then decline to the normal level at the 2nd week and finally decreased below the normal upper limits at the 3rd and 4th weeks. The infected puppies showed a decrease of lymphocytes count at 1st week post viral infection and then rapidly increased at the 3rd and 4th week post viral infection. Moreover, the mean values of monocytes and eosinophils count increased gradually throughout the experimental period. It was clearly observed from the present study that, PCR procedure is faster, sensitive and easier as diagnostic biomarkers for ICH especially at the acute stage. Moreover, liver functions tests as well as hematological indices were the best diagnostic biomarkers in the chronic stage of ICH.

Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells

Central nervous system (CNS) disorders remain a formidable challenge for the development of efficient therapies. Cell and gene therapy approaches are promising alternatives that can have a tremendous impact by treating the causes of the disease rather than the symptoms, providing specific targeting and prolonged duration of action. Hampering translation of gene-based therapeutic treatments of neurodegenerative diseases from experimental to clinical gene therapy is the lack of valid and reliable pre-clinical models that can contribute to evaluate feasibility and safety.Herein we describe a robust and reproducible methodology for the generation of 3D in vitro models of the human CNS following a systematic technological approach based on stirred culture systems. We took advantage of human midbrain-derived neural progenitor cells (hmNPCs) capability to differentiate into the various neural phenotypes and of their commitment to the dopaminergic lineage to generate differentiated neurospheres enriched in dopaminergic neurons. Furthermore, we describe a protocol for efficient gene transfer into differentiated neurospheres using CAV-2 viral vectors and stable expression of the transgene for at least 10days. CAV-2 vectors, derived from canine adenovirus type 2, are promising tools to understand and treat neurodegenerative diseases, in particular Parkinson’s disease. CAV-2 vectors preferentially transduce neurons and have an impressive level of axonal retrograde transport in vivo.Our model provides a practical and versatile in vitro approach to study the CNS in a 3D cellular context. With the successful differentiation and subsequent genetic modification of neurospheres we are increasing the collection of tools available for neuroscience research and contributing for the implementation and widespread utilization of 3D cellular CNS models. These can be applied to study neurodegenerative diseases such as Parkinson’s disease; to study the interaction of viral vectors of therapeutic potential within human neural cell populations, thus enabling the introduction of specific therapeutic genes for treatment of CNS pathologies; to study the fate and effect of delivered therapeutic genes; to study toxicological effects. Furthermore these methodologies may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells.

Atividade antiviral do extrato de própolis contra o calicivírus felino, adenovírus canino 2 e vírus da diarréia viral bovina

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. Neste artigo, descreve-se a atividade antiviral de dois extratos etanólicos de própolis (EP1 e EP2) frente aos vírus: calicivírus felino (FCV), adenovírus canino tipo 2 (CAV-2) e vírus da diarréia viral bovina (BVDV). Um dos extratos (EP1) foi obtido por extração etanólica de própolis obtida da região central do Estado do Rio Grande do Sul e o segundo (EP2), obtido comercialmente de uma empresa de Minas Gerais. A análise dos extratos de própolis através da cromatografia líquida de alta eficiência (CLAE) identificou a presença de flavonóides como: rutina, quercetina e ácido gálico. A atividade antiviral bem como a citotoxicidade dos extratos aos cultivos celulares foram avaliadas através do MTT [3- (4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo]. Ambos os extratos evidenciaram atividade antiviral frente ao BVDV e CAV-2 quando acrescidos ao cultivo celular anteriormente à inoculação viral. Os extratos foram menos efetivos contra o FCV em comparação aos resultados obtidos com os outros vírus, e a atividade antiviral neste caso foi observada apenas quando a própolis estava presente após a inoculação viral. O extrato obtido no laboratório (EP1) apresentou valores mais altos de índice de seletividade (IS=CC50/ CE50), quando comparado à outra amostra (EP2). Em resumo, a própolis apresentou atividade antiviral frente a três diferentes vírus, o que a torna alvo para o desenvolvimento de novos compostos naturais com atividade antiviral.

Pharmacovigilance in veterinary medicine in Chile: a pilot study

Iragüen, D., Urcelay, S., San Martín, B. Pharmacovigilance in veterinary medicine in Chile: a pilot study. J. vet. Pharmacol. Therap.34, 108-115. In Chile, there is no present government policy to survey and analyse adverse drug reactions (ADRs) in the field of veterinary medicine. The intent of this study is to assess, for the first time, ADR frequency in treated animals. To this purpose, a 6-month period pilot study based on WHO recommendations was conducted to monitor ADRs in cats and dogs for frequently used drugs and common labelled signs. Of a total of 149 detected ADRs, 29 (6 in cats and 23 in dogs) were notified by means of ADR report forms, while the rest was identified after reviewing patient clinical records, thus evidencing strong under-reporting problems. More than 70% of ADRs were related to antimicrobials, vaccines and tranquilizers. In dogs, there was a significant effect on ADRs' presentation when acepromazine, amoxicillin, carprofen, ivermectin, sextuple vaccine (polyvalent vaccine that confers immunity against canine distemper virus, canine parvovirus, Leptospira canicola, L.icterohemmoragiae, canine adenovirus type 2 and canine parainfluenza virus) and phytomenadione (subcutaneous injection) were administered. In the case of cats, a significant influence on ADRs was detected when acepromazine, amoxicillin or vitamin K was administered. Present results suggest the need for a pharmacovigilance programme in veterinary medicine for timely ADR-presenting drug detection and drug safety improvement.

An Outbreak of Canine Adenovirus Type 1 Infection at a Pet Shop in Japan

An Outbreak of Canine Adenovirus Type 1 Infection at a Pet Shop in Japan

Antibodies to parvovirus, distemper virus and adenovirus conferred to household dogs using commercial combination vaccines containing Leptospira bacterin

To examine how the inclusion (+) or exclusion (–) of inactivated Leptospira antigens in a vaccine for canine parvovirus type 2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type...

Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep

Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4+ and CD8+ lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.

An Update on Canine Adenovirus Type 2 and Its Vectors

Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors.

Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1

A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 101 and 1.20×101 DNA copies per 10μl−1 of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs.

Comparison of the diagnostic methods on the canine adenovirus type 2 infection.

ABSTRACT Background and aims: Canine adenovirus type 2 (CAV‐2) infection is typically diagnosed histopathologically since intranuclear inclusion bodies (IN/IBs) are demonstrable in the infected lung. However, it is sometimes difficult to identify IN/IBs, particularly in autolyzed tissues or samples from both early and late stages of infection, and other methods were presently developed. Methods: Stray dog samples were evaluated by histopathology, polymerase chain reaction (PCR), and immunohistochemistry (IHC) to investigate the status of the CAV‐2 infection on the stray dogs in Korea. Histologic tests were performed, and dogs with pneumonic lungs were further evaluated by IHC and PCR. Results: Pathognomonic IN/IBs were identified in 3 of 213 lungs; CAV‐2 PCR was positive for 27 of 213 pneumonic lungs. A total of 7 of 27 CAV‐2 PCR‐positive lungs were IHC‐positive. No PCR‐negative lung was IHC‐positive. Positive results were primarily detected in the IN/IBs of the bronchial epithelial cells, macrophages, and very rarely in the cytoplasm of bronchial epithelial cells. Conclusions: IHC was a more reliable diagnostic method than conventional pathologic methods in the present study, and suggests that IHC should be routinely used in the diagnosis of CAV‐2 infection. Further, PCR alone may not be adequate for CAV‐2 diagnosis.

570. Development of a MDCK Cell Line for the Manufacturing of Canine Adenovirus Type 2 (CAV-2) Vectors

570. Development of a MDCK Cell Line for the Manufacturing of Canine Adenovirus Type 2 (CAV-2) Vectors

SEROSURVEY FOR CANINE DISTEMPER VIRUS, CANINE ADENOVIRUS, LEPTOSPIRA INTERROGANS, AND TOXOPLASMA GONDII IN FREE-RANGING CANIDS IN SCANDINAVIA AND SVALBARD

Prevalence of antibodies reactive to canine distemper virus (CDV), canine adenovirus type 1 (CAV-1), Leptospira interrogans serovars Canicola and Icterohaemorrhagiae, and Toxoplasma gondii were examined in free-ranging Scandinavian canids. Sampling included 275 red foxes (Vulpes vulpes) from mainland Norway, 60 arctic foxes (Vulpes lagopus) from the high-arctic islands of Svalbard, and 98 wolves (Canis lupus) from the joint Swedish-Norwegian population. Methods used included virus neutralization tests for CDV and CAV-1, a microscopic agglutination test for L. interrogans, and a direct agglutination test for T. gondii. High prevalence of antibody to CAV-1 was identified in red foxes (59.6%), wolves (67.7%), and arctic foxes (37.8%). The prevalence of antibody to CDV varied between 9.6% and 12.3% in the three species. Antibodies to L. interrogans serovar Icterohaemorrhagiae were found in 9.9% of the red foxes and 8.4% of the wolves sampled, whereas no antibody-positive arctic foxes were found. All animals were antibody-negative for L. interrogans serovar Canicola. Antibodies to T. gondii were found in 66.9, 51.7, and 18.6% of red foxes, arctic foxes and wolves, respectively. Significantly more adults than juveniles were antibody-positive for CDV in red foxes and arctic foxes, for CAV-1 in wolves, and for T. gondii in red foxes and wolves. There was a general tendency for adult female red foxes to have a higher prevalence of antibodies for CDV than adult males; this difference was statistically significant. The results indicate that CDV and CAV-1 are endemic in red foxes and wolves on the Scandinavian mainland and in arctic foxes on Svalbard. Although infection with L. interrogans serovar Icterohaemorrhagiae was relatively common in wild canids on mainland Norway, it was not found on Svalbard, where the maintenance host (Rattus norvegicus) is absent. All three species are commonly exposed to T. gondii through predation on infected intermediate hosts.

Generation of E3-deleted canine adenovirus type 2 expressing the Gc glycoprotein of Seoul virus by gene insertion or deletion of related terminal region sequences

Seoul virus (SEOV) is one of the four hantaviruses known to cause haemorrhagic fever with renal syndrome. The medium genome segment encodes the Gn/Gc glycoproteins of SEOV, which form the major structural part of the virus envelope. Gc and/or Gn are the candidate antigens of hantavirus for induction of a highly immunogenic response for hantavirus vaccine. In this study, the immune response induced by a replication-competent recombinant canine adenovirus type 2 expressing the Gc protein of SEOV was evaluated in BALB/c mice. Sera from immunized mice contained neutralizing antibodies that could specifically recognize SEOV and neutralize its infectivity in vitro. Moreover, the recombinant virus induced complete protection against an intensive infectious challenge with approximately 1000 50 % infective doses for SEOV strain CC-2. Protective-level neutralizing antibodies were maintained for at least 20 weeks. This recombinant virus is therefore a potential alternative to the inactivated vaccine.

Infectious canine hepatitis in red foxes (Vulpes vulpes) in the United Kingdom

The pathological findings are described in three cases of infectious canine hepatitis in free-ranging red foxes (Vulpes vulpes) in England. The foxes died after short periods of clinical illness. Mild...

Construction and Characterization of a Recombinant Canine Adenovirus Expressing GP5 and M proteins of Porcine Reproductive and Respiratory Syndrome Virus

The causative agent of porcine reproductive and respiratory syndrome (PRRS) is PRRS virus (PRRSV), which belongs to the family Arteriviridae. GP5/M protein complex of PRRSV binds to sialoadhesion expressed on the cells to infect the cells. In this study, we developed a canine adenovirus type 2 (CAV-2) recombinant, termed rCAV2-GP5/M, expressing GP5 and M proteins. To evaluate the immunogenicity of the recombinant virus, mice were inoculated subcutaneously with rCAV2-GP5/M, and specific antibodies against PRRSV in the sera were measured by enzyme-linked immunosorbent assay and the viral neutralization test. Two weeks post-immunization (w.p.i.), anti-PRRSV antibodies were detected in the sera, slightly increased by booster immunization at four w.p.i., and then gradually decreased. The viral neutralizing test showed that neutralizing antibodies were present in the sera collected at two w.p.i., increased by booster immunization, and reached the maximum titer at six w.p.i. Lymphocyte proliferation responding to PRRSV antigens was also observed from two w.p.i. Although further studies are needed to evaluate the usefulness of the recombinant virus to protect pigs from PPRSV, we succeeded in developing a candidate vaccine against PPRSV infection by using CAV-2 vector.

A real-time PCR assay for quantification of canine adenoviral vectors

The pre-existing humoral and cellular immunity found in the great majority of the population raises concerns about the clinical efficacy and safety of vectors derived from ubiquitous human adenovirus serotypes. To alleviate these concerns, canine adenovirus type 2 vectors (CAV-2) were developed. Owing to their extraordinary neuronal tropism and efficient axonal retrograde transport, CAV-2 vectors hold great promise for the treatment of neurodegenerative diseases. The development and validation of a SYBR Green qPCR assay for determination of CAV-2 titers is reported in the present study. This method uses specific primers designed to amplify a small genomic fragment of CAV-2 structural genes (pVI-hexon). The method was accurate and reproducible as determined by the low intra- and inter-assay variability (<15% R.S.E.). It is sensitive and useful over a 5-log range (1 × 10 3 to 1 × 10 7 genome copies/reaction). The assay can be used to quantify purified vector samples as well as crude viral lysates. The titers obtained by qPCR correlated well with both, those obtained by OD 260 and TCID 50 as indicated by the high coefficients of determination obtained by regression analysis ( r 2 > 0.83). The development of this simple and rapid CAV-2 quantitation method should be helpful for process development and monitoring.