An improved system to generate recombinant canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. METHODS: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. RESULTS: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. CONCLUSION: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

Recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) designed for rapid detection of canine distemper virus.

In the present study, recombinase polymerase amplification (RPA) was combined with the colloidal gold lateral flow dipstick (LFD) method to establish a new, stable, and efficient assay for the detection of canine distemper virus (CDV). We designed a set of specific primers labeled with biotin and a specific probe labeled with dSpacer and C3 spacer, according to the conserved region in the N-terminal gene sequence of CDV. The reaction conditions and systems were then optimized, and the sensitivity and specificity were analyzed for potential clinical application. The results showed that the RPA-LFD assay for CDV detection was successfully established. We also found that the temperature in a closed fist (35°C) is optimal for the RPA reaction. The optimal ratio of primer to probe was 2:1. The minimum detection limit of the RPA-LFD assay was 1 × 101 the median tissue culture infective dose (TCID50)/mL. Using this assay with samples from experimentally infected dogs, CDV was detected in nasal secretions, eye secretions, and blood on the fourth day post infection. In summary, this novel RPA-LFD assay for CDV detection is simple to use, and preliminary findings indicate its high specificity and sensitivity.

Immunogenicity and protective efficacy of a novel bacterium-like particle-based vaccine displaying canine distemper virus antigens in mice and dogs.

Canine distemper virus (CDV) poses a severe threat to both domesticated and wild animals, including multiple carnivores. With the continued expansion of its host range, there is an urgent need for the development of a safer and more effective vaccine. In this study, we developed subunit vaccines based on a bacterium-like particle (BLP) delivery platform containing BLPs-F and BLPs-H, which display the CDV F and H glycoprotein antigens, respectively, using the antigen-protein anchor fusions produced by a recombinant baculovirus insect cell expression system. The combination of BLPs-F and BLPs-H (CDV-BLPs), formulated with colloidal manganese salt [Mn jelly (MnJ)] adjuvant, triggered robust CDV-specific antibody responses and a substantial increase in the number of interferon gamma (IFN-γ)-secreting CD4+ and CD8+ T cells in mice. Dogs immunized intramuscularly with this vaccine not only produced CDV-specific IgG but also displayed elevated concentrations of IFN-γ and interleukin 6 in their serum, along with an increase of the CD3+CD4+ and CD3+CD8+ T cell subsets. Consequently, this heightened immune response provided effective protection against disease development and reduced viral shedding levels following challenge with a virulent strain. These findings suggest that this BLP-based subunit vaccine has the potential to become a novel canine distemper vaccine. IMPORTANCE: Many sensitive species require a safe and effective distemper vaccine. Non-replicating vaccines are preferred. We constructed subunit particles displaying canine distemper virus (CDV) antigens based on a bacterium-like particle (BLP) delivery platform. The CDV-BLPs formulated with theMn jelly adjuvant induced robust humoral and cell-mediated immune responses to CDV in mice and dogs, thereby providing effective protection against a virulent virus challenge. This work is an important step in developing a CDV subunit vaccine.

Canine distemper virus infection of vaccinal origin in a 14-week-old puppy.

The body of a 14-wk-old puppy (Canis familiaris) was submitted to the Animal Health Laboratory, University of Guelph, Ontario for postmortem examination following a history of intermittent anorexia and lethargy progressing to pyrexia, pruritic skin rash, mucoid nasal discharge, decreased mentation, dysphagia, muscle twitches, and focal seizures. Gross examination revealed rhinitis and pulmonary edema. Histologically, there was fibrinonecrotizing bronchopneumonia, tracheitis, and neutrophilic and lymphohistiocytic rhinitis; rarely within the cortical gray and white matter of the brain were small clusters of glial cells, with rare individual neutrophils in the choroid plexus. Although canine distemper was suspected, none of the usual supportive histologic lesions of distinct syncytial cells, viral inclusion bodies, or demyelinating leukoencephalitis were observed. Lung and brain tissues were PCR-positive for canine distemper virus (CDV), and CDV was detected immunohistochemically in the brain. The agent from the PCR-positive sample from the brain was genotyped and was a 99.9% match to the CDV Rockborn strain, indicating that the disease agent in our case was vaccinal in origin. Our unusual case highlights the possibility of reversion to virulence in a modified-live virus vaccine, and the occurrence of a disease in the absence of a full complement of the usual and compatible histologic lesions.

Rational attenuation of canine distemper virus (CDV) to develop a morbillivirus animal model that mimics measles in humans.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.

Study of Canine Distemper Virus Presence in Catalonia's Wild Carnivores through H Gene Amplification and Sequencing.

Canine distemper virus (CDV) is recognised worldwide as an important pathogen in both domestic and wild carnivores. Few data are available on its impact and spread on the wildlife/wildlife-domestic animal-environment interface. This study, aimed at developing a conservation-oriented control strategy, analysed 89 sick or deceased animals from 2019 to 2023 at the Wildlife Rehabilitation Centre in Torreferrussa. RT-PCR and sequencing of the partial H gene were used to detect and analyse CDV in tissues. The total positive percentage was 20.22% (18/89), comprising 13 red foxes (44.8%), 4 European badgers (28.6%), and 1 American mink (4.5%), while 24 Eurasian otters tested negative. Phylogenetic analysis indicated that all of the CDV strains belong to the European lineage. Geographically distant individuals and different species shared the same viral strain, suggesting a strong capacity of CDV for interspecies and long-distance transmission. This calls for further research, particularly focusing on potential impacts of CDV on endangered carnivores.

Ferret Pediatrics.

Ferrets are bred to be pets, utilized for hunting, and as laboratory models. Despite the fact that ferrets in some areas of the world are neutered by the breeder before entering the pet trade, the importance of pediatric management should not be overlooked. Pregnant, whelping, and lactating jills should be closely monitored and kept in a quiet, stress-free environment. Hand-rearing baby kits is very challenging due to their requirement for ferret milk. Minimizing maternal stress and disease can prevent the need to hand rear kits. Infectious diseases in juvenile ferrets include canine distemper virus, rotavirus, coccidiosis, feline panleukopenia virus (experimental only), and Toxoplasma-like disease. All juvenile ferrets should be vaccinated against canine distemper and rabies. Congenital diseases are reported to affect the auditory, ocular, cardiovascular, urogenital, central nervous, and musculoskeletal systems. Early detection of these diseases is important to prevent the progression of curable diseases.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology.

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4-15 (scFv 4-15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4-15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.

Trace detection of canine distemper virus based on Michelson-interferometer sensing probe.

A single-mode-fiber (SMF)-multimode-fiber (MMF)-tri-core-fiber (TCF) Michelson probe structure is proposed for trace detection of canine distemper virus (CDV). One end of the TCF is cut flat and fused with the multimode fiber, and the other end is coated with a silver film to enhance the reflection, and an optic-fiber sensing probe with SMF-MMF-TCF structure is obtained. The (PDDA/PSS)3 multilayer film is modified on the surface of the fiber by layer-by-layer self-assembly method as a polyelectrolyte binder to immobilize CDV antibodies to form a (PDDA/PSS)3 /CDV antibody composite membrane for specific detection of CDV antigens. The response-recovery test of the sensor is performed to verify its repeatability. The detection limit, the sensitivity, and the linear fitting degree for CDV antigen are 0.1236 pg/mL, 1.1776 dB/(pg/mL), and 0.9899, respectively. At the same time, the stability, selectivity, and clinical samples of the sensors were also verified.

Oncolytic Activity of Canine Distemper Virus in Human Ductal Breast Carcinoma Cells.

INTRODUCTION: Oncolytic virotherapy is a novel strategy for cancer treatment in humans and companion animals. Canine distemper virus (CDV) is known to induce apoptosis in tumor cells, thus serving as a potential candidate for oncolytic therapy. However, the mechanism of viral oncolytic activity is less studied and varies depending on the type of cancer and cell lines. METHODS: In the present study, the susceptibility of the MCF-7 cell line to CDV infection was assessed using the CDV strain, which was confirmed previously through sequence analysis in the Vero cell line. The impact of CDV infection on cell proliferation and apoptosis was studied by evaluating the expression of four target genes including the myeloid cell leukemia 1 (MCL-1), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), transcription factor (SP1), and DNA (cytosine-5)-methyltransferase 3A (DNMT3A). RESULTS: CDV replication in the cells induced cytopathic effect and decreased in the cell proliferation rates compared to the uninfected control. MCL-1, SP1, and PIK3R1 gene expression was down-regulated, while the expression of DNMT3A was up-regulated 3 days post-infection. The expression levels of the target genes suggest that CDV may be inducing the intrinsic apoptotic pathway in the cancer cell line. CONCLUSION: Overall, the results strongly propose CDV strain as a potential candidate for cancer therapy after detailed studies.

Post-vaccinal distemper-like disease in two dog litters with confirmed infection of vaccine virus strain.

Modified live canine distemper virus (CDV) vaccines are widely used and considered both safe and effective. Although there are occasional literature reports of suspected vaccine-induced disease, there are none where the vaccine strain has been identified in affected tissues. Here we describe two such cases in different litters. In litter A, five of ten puppies presented with fever, anorexia, vomiting, and diarrhea a few days post-vaccination. Four puppies died or were euthanized, and autopsy revealed atypical necrosis of the lymphoid tissue. In litter B, two of five puppies developed typical neurological signs some months post-vaccination and autopsy revealed encephalitis. In all cases, affected organs tested positive for CDV on immunohistochemistry, and CDV RNA extracted from the lesions confirmed the presence of vaccine strain. Since multiple puppies from each litter were affected, it cannot be excluded without further studies that some undiagnosed inherited immunodeficiency disorder may have been involved.

Novel high-coverage primers for detection of canine morbillivirus by end-point and real-time RT-PCR assays.

Canine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100 % (194/194) and 99 % (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15 % (29/194) and 9 % (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.001 TCID50/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32 %) positive samples selected for comparison purposes. In addition, our assays did not amplify other canine viruses associated with respiratory and neurological diseases: canine adenovirus 2, canine parainfluenza virus 2, canine herpesvirus 1 and rabies virus. Overall, we describe a high-coverage primer set for CDV detection, which represents an attractive tool for laboratory diagnosis of canine distemper.

Serological Survey for Three Canine Viruses in Brazilian Wild Carnivores : Antibodies Against Canine Viruses in Wild Carnivores.

We evaluated the presence of antibodies against CaHV-1, CDV, and CPV-2 in serum samples from Brazilian wild carnivore species. Nine maned wolves and six crab-eating foxes were tested for CaHV-1 and CDV by virus neutralization test and CPV-2 by hemagglutination inhibition assay. Antibodies to CaHV-1, CDV, and CPV-2 were detected in serum samples of 1 (6.7%), 5 (33.3%), and 10 (66.7%) wild carnivores, respectively. Two maned wolves and one crab-eating fox were seropositive simultaneously for CDV and CPV-2. Antibodies against all viruses were detected in one crab-eating fox. This is the first report of CaHV-1 antibody detection in crab-eating foxes.

Antiviral activity of nitazoxanide against Morbillivirus infections.

The measles virus (MeV) and canine distemper virus (CDV) belong to the genus Morbillivirus of the Paramyxoviridae family. They are enveloped viruses harboring a non-segmented negative-sense RNA. Morbilliviruses are extremely contagious and transmitted through infectious aerosol droplets. Both MeV and CDV may cause respiratory infections and fatal encephalitis, although a high incidence of brain infections is unique to CDV. Despite the availability of a safe and effective vaccine against these viruses, in recent years we are witnessing a strong resurgence of Morbillivirus infection. Measles still kills more than 100,000 people each year, and CDV causes widespread outbreaks, especially among wild animals, including non-human primates. No drugs are currently approved for MeV and CDV. Therefore, the identification of effective antiviral agents represents an unmet medical need. Here, we have investigated the potential antiviral properties of nitazoxanide (NTZ) against MeV and CDV. Antiviral activity was explored with live virus and cell-based assays. NTZ is a thiazolide that is approved by the FDA as an antiprotozoal agent for the treatment of Giardia intestinalis and Cryptosporidium parvum. Further, nitazoxanide and its metabolite tizoxanide have recently emerged as broad-spectrum antiviral agents. We found that NTZ blocks the MeV and CDV replication, acting at the post-entry level. Moreover, we showed that NTZ affects the function of the viral fusion protein (F), impairing viral spread. Our results indicate that NTZ should be further explored as a therapeutic option in measles and canine distemper virus treatment.

Phenotypic Characterization of Encephalitis in the BRAINS of Badgers Naturally Infected with Canine Distemper Virus.

Canine distemper virus (CDV) affects a huge diversity of domestic and wild carnivores, with increasing numbers of mortality events worldwide. The local cell-mediated immune response elicited against a natural infection is an important factor in determining the outcome of CDV infection. Therefore, the purposes of this study were to describe the local immune response within the central nervous systems (CNSs) of seven badgers naturally infected with CDV in Asturias (Atlantic Spain) and to determine the phenotype and distribution of microglial cells, T and B lymphocytes, and astrocytes in the foci of gliosis located in the thalamus and cerebellum using immunohistochemistry. The immunohistochemical assessment demonstrated the presence of Iba1-positive microglia and GFAP-positive astrocytes in the foci of gliosis, whereas T (CD3-negative) or B (CD20-negative) lymphocytes in those same lesions were absent. Our results also revealed that the badgers with natural CDV encephalitis presented lesions mostly located in the white matter of the thalamus and cerebellum, suggesting a CDV-specific tropism for the white matter of badger brains in those locations. The knowledge gained in the field of the immunopathogenesis of distemper disease affecting the CNSs of badgers could help to clarify CDV disease patterns in this species.

Establishment and evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type canine distemper virus from vaccine strains.

This study sought to establish a real-time reverse transcription (RT)-PCR method to differentially detect canine distemper virus (CDV) wild-type and vaccine strains. To this end, a pair of CDV universal primers and two specific minor groove binder (MGB) probes, harboring a T/C substitution in the hemagglutinin (H) gene, were designed. Using a recombinant plasmid expressing the H gene of the CDV wild-type or vaccine strain as standards, a sensitive and specific multiplex real-time RT-PCR was established for quantitative and differential detection of CDV wild-type and vaccine strains. The limit of detection for this multiplex assay was 22.5 copies/µL and 2.98 copies/µL of viral RNA for wild-type and vaccine strains, respectively. Importantly, the wild-type and vaccine MGB probes specifically hybridized different genotypes of wild-type CDV circulating in China as well as globally administered vaccine viruses, respectively, with no cross-reactivity observed with non-CDV viruses. Moreover, this method was successfully applied for the quantitative detection of CDV RNA in tissue samples of experimentally infected breeding foxes, raccoon dogs, and minks. Additionally, the multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples as early as 3 days post-infection, 3 to 4 days prior to the onset of clinical signs in these CDV infection animals. Hence, the established multiplex real-time RT-PCR method is useful for differentiating wild-type CDV and vaccine strains in China, and for conducting canine distemper early diagnosis as well as dynamic mechanism of CDV replication studies in vivo.

DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret (Mustela putorius furo)-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 105.0 TCID50. Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

Death comes for us all: relating movement-integrated habitat selection and social behavior to human-associated and disease-related mortality among gray wolves.

Avoiding death affects biological processes, including behavior. Habitat selection, movement, and sociality are highly flexible behaviors that influence the mortality risks and subsequent fitness of individuals. In the Anthropocene, animals are experiencing increased risks from direct human causes and increased spread of infectious diseases. Using integrated step selection analysis, we tested how the habitat selection, movement, and social behaviors of gray wolves vary in the two months prior to death due to humans (being shot or trapped) or canine distemper virus (CDV). We further tested how those behaviors vary as a prelude to death. We studied populations of wolves that occurred under two different management schemes: a national park managed for conservation and a provincially managed multi-use area. Behaviors that changed prior to death were strongly related to how an animal eventually died. Wolves killed by humans moved slower than wolves that survived and selected to be nearer roads closer in time to their death. Wolves that died due to CDV moved progressively slower as they neared death and reduced their avoidance of wet habitats. All animals, regardless of dying or living, maintained selection to be near packmates across time, which seemingly contributed to disease dynamics in the packs infected with CDV. There were no noticeable differences in behavior between the two management areas. Overall, habitat selection, movement, and sociality interact to put individuals and groups at greater risks, influencing their cause-specific mortality.

Functional Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Delivered by Canine Histiocytic Sarcoma Cells Persistently Infected with Engineered Attenuated Canine Distemper Virus.

The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte-macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ondneon-GM-CSF). The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.