Canine Distemper Virus Replication Research Articles

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.

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.

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.

Host E3 ligase Hrd1 ubiquitinates and degrades H protein of canine distemper virus to inhibit viral replication

Canine distemper (CD) is a highly contagious and an acutely febrile disease caused by canine distemper virus (CDV), which greatly threatens the dog and fur industry in many countries. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control system for the degradation of misfolded proteins in the ER. In this study, a proteomic approach was performed, and results found the E3 ubiquitin ligase 3-hydroxy-3-methylglutaryl reductase degradation protein 1 (Hrd1), which is involved in ERAD, as one of the CDV H-interacting proteins. The interaction of Hrd1 with CDV H protein was further identified by Co-IP assay and confocal microscopy. Hrd1 degraded the CDV H protein via the proteasome pathway dependent on its E3 ubiquitin ligase activity. Hrd1 catalyzed the K63-linked polyubiquitination of CDV H protein at lysine residue 115 (K115). Hrd1 also exhibited a significant inhibitory effect on CDV replication. Together, the data demonstrate that the E3 ligase Hrd1 mediates the ubiquitination of CDV H protein for degradation via the proteasome pathway and inhibits CDV replication. Thus, targeting Hrd1 may represent a novel prevention and control strategy for CDV infection.

Replication competence of canine distemper virus in cell lines expressing signaling lymphocyte activation molecule (SLAM) of goat, sheep and dog origin

Cellular receptors play an important role in entry and cell to cell spread of morbillivirus infections. The cells expressing SLAM and Nectin-4 have been used for successful and efficient isolation of canine distemper virus (CDV) in high titre. There are several methods for generation of cells expressing receptor molecules. Here, we have used a comparatively cheaper and easily available method, pcDNA 3.1 (+) for engineering Vero cells to express SLAM gene of goat, sheep and dog origin (Vero/Goat/SLAM (VGS), Vero/Sheep/SLAM (VSS) and Vero/Dog/SLAM (VDS), respectively). The generated cell lines were then compared to test their efficacy to support CDV replication. CDV could be grown in high titre in the cells expressing SLAM and a difference of log two could be recorded in virus titre between VDS and native Vero cells. Also, CDV could be grown in a higher titre in VDS as compared to VGS and VSS. The finding of this study supports the preferential use of SLAM expressing cells over the native Vero cells by CDV. Further, the higher titre of CDV in cells expressing dog-SLAM as compared to the cells expressing SLAM of non-CDV hosts (i.e. goat and sheep) points towards the preferential use of dog SLAM by the CDV and may be a plausible reason for differential susceptibility of small ruminants and Canines to CDV.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

Ribavirin and boceprevir are able to reduce Canine distemper virus growth in vitro

Canine distemper virus (CDV) is a major infectious disease of dogs. Although vaccines were successful to control CDV spread in canine population, the disease is still common and may pose a threat to unvaccinated dogs. In the attempt to develop specific anti-viral therapeutic tools, the efficacy of several molecules against CDV has been investigated in vitro. In this study the antiviral efficacy in vitro against CDV of ribavirin and boceprevir alone or in combination was evaluated.CDV growth in VERO cells was inhibited by ribavirin, by boceprevir and by a combination of the two molecules at non-cytotoxic concentrations, as evaluated by end-point viral titration in cell monolayers and by quantification of viral RNA using quantitative RT-PCR. By end-point titration, a statistically significant reduction in CDV replication was observed only using ribavirin and boceprevir in combination. By quantitative RT-PCR, a significant reduction of viral growth was observed either in cells treated with ribavirin or boceprevir or with both the two molecules. The association of ribavirin or boceprevir was able to decrease CDV growth by up to 3.4458 logs with respect to untreated infected cells, chiefly at the highest virus dilutions. The results obtained in this study may constitute an important basis for the development of CDV therapies.

The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells

Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3−CD5−CD21− NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

In vitro anti-canine distemper virus activity of fucoidan extracted from the brown alga Cladosiphon okamuranus.

Canine distemper virus (CDV) is a morbillivirus related to measles virus that infects dogs and other carnivores. CDV has a significant global impact on animal health; however, there is no current antiviral treatment for CDV infection. In recent years, it has been demonstrated that sulfated polysaccharides exhibit antiviral properties both in vivo and in vitro, despite their low cytotoxicity to host cells. Fucoidan is a sulfated polysaccharide found in the cell wall matrix of brown algae. In this study, we evaluated in vitro anti-CDV activity of fucoidan, which was derived from Cladosiphon okamuranus. Fucoidan actively inhibited CDV replication in Vero cells at a 50% inhibitory concentration (IC50) of 0.1µg/ml. The derived selectivity index (SI50) was >20,000. This polysaccharide likely inhibits viral infection by interference in the early steps and by inhibiting CDV-mediated cell fusion. Fucoidan may be useful in development of pharmacological strategies to treat and control CDV infection.

The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor

Recent outbreaks in monkeys have proven that canine distemper virus (CDV) causes diseases in a wide range of mammals. CDV uses SLAM and nectin4 as receptors to replicate in susceptible animals. Here, we show that human nectin4, but not human SLAM, is fully functional as a CDV receptor. The CDV Ac96I strain hardly replicated in nectin4-expressing human epithelial NCI-H358 cells, but readily adapted to grow in them. Unsurprisingly, no amino acid change in the H protein was required for the adaptation. The original Ac96I strain possessed a truncated C protein, and a subpopulation possessing the intact C protein was selected after growth in NCI-H358 cells. Other CDV strains possessing the intact C protein showed significantly higher growth abilities in NCI-H358 cells than the Ac96I strain with the truncated C protein. These findings suggest that the C protein is functional in human epithelial cells and critical for CDV replication in them.

Antiviral efficacy of EICAR against canine distemper virus (CDV) in vitro

Canine distemper virus (CDV) is a highly contagious pathogen of carnivores. In dogs, the disease is characterized by high lethality rates and no specific antiviral therapy is available. The aim of this study was to verify the in vitro antiviral activity of the 5-ethynyl-1-β- d-ribofuranosylimidazole-4-carboxamide (EICAR) and to compare it with the 1-(β- d-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin, RBV). EICAR was more active than RBV against CDV replication, while both molecules exhibited low selectivity indexes. A reversal of their antiviral activity was observed after addition of guanosine, suggesting their involvement in the inhibition of the inosine monophosphate dehydrogenase enzyme (IMPDH). RBV and EICAR had a time- and concentration-dependent anti-CDV activity, mainly displayed during the first 10 h post-infection. The involvement of the inhibition of the viral RNA-dependent RNA polymerase (vRdRp) is discussed, as well as the role of CDV as a model to study more potent and selective antiviral molecules active against other Paramyxoviridae.

In vitro efficacy of ribavirin against canine distemper virus

Despite vaccination, canine distemper virus (CDV) remains one of the important pathogen of dogs with worldwide distribution. Ribavirin (RIB) inhibits replication of measles virus (MV), a morbillivirus closely related to CDV, both in vitro and in vivo. In this report the antiviral activity of RIB against CDV in cell cultures was assessed. Quantitative real-time RT-PCR was used to measure viral RNA in VERO cells infected by CDV and to evaluate the inhibitory effects of RIB. RIB caused a dose- and time-dependent decrease in accumulation of CDV RNA when added after virus adsorption. RIB was highly effective in preventing CDV replication at low concentrations with 50% virus-inhibitory concentrations ranging from 0.02 to 0.05 mM. Such low values were comparable to values displayed by highly susceptible strains of MV. In addition, CDV was passaged sequentially in VERO cell monolayers in the presence of RIB to trigger viral extinction. The virus was no longer detected after three passages, suggesting that error catastrophe is one of the modes of action of RIB against CDV. These findings suggest RIB as a promising tool for the therapy of CD in dogs.

Oligodendroglial degeneration in distemper: apoptosis or necrosis?

Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. It was previously shown that the initial demyelinating lesions are directly virus induced since a correlation between the occurrence of demyelination and CDV replication in white matter cells was observed. During the course of infection oligodendrocytes undergo distinct morphological alterations, partly due to a restricted CDV infection of these cells, and eventually disappear from the lesions. This phenomenon has been described in vivo as well as in vitro. However, the reason for the morphological alterations and the following oligodendroglial depletion remained unclear. Since virus infection can induce cell death, it was investigated whether apoptosis or necrosis plays a role in the pathogenesis of demyelination in canine distemper. In brain tissue sections from dogs with acute distemper apoptotic cells were not detected within the demyelinating lesions using morphological and biochemical cell death criteria. In chronic distemper, apoptotic cells - presumably inflammatory cells - were seen within the perivascular cuffs. These in vivo findings were correlated to the in vitro situation using CDV-infected primary dog brain cell cultures as well as Vero cells. Infection with culture-adapted CDV lead to massive necrosis but not to apoptosis. After infection with virulent CDV neither apoptosis nor necrosis was a predominant feature in either culture system. These findings suggest that virus-induced demyelination in canine distemper is not the direct consequence of apoptosis or necrosis. It is speculated that another mechanism must be responsible for the observed morphological alterations of oligodendrocytes, ultimately leading to demyelination.

Oligodendroglial pathology in canine distemper.

Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. The mechanism of acute demyelination in distemper is still poorly understood. The initial demyelinating lesion in distemper is directly virus induced, since there is a clear correlation between the occurrence of demyelination and CDV replication in the cells of the white matter. Yet, there is little evidence for oligodendroglial infection. Changes of these cells have been reported in vitro and in vivo. The in vitro studies showed that – in contrast to other cells such as astrocytes and macrophages – oligodendrocytes hardly express CDV protein. However, we could show that these cells underwent a restricted infection with transcription of CDV RNA and that this phenomenon correlated with down-regulation of myelin gene transcription. The extension of these in vitro findings in vivo was obscured by the lack of reliable oligodendrocyte labelling techniques in canine brain tissue sections. In this study we combined immunohistochemistry with in situ hybridization to examine oligodendrocytes in demyelinating lesions and to investigate the question of oligodendrocyte infection in vivo. We could demonstrate that CDV infection leads to massive down-regulation of myelin gene expression in demyelinating lesions and that this effect correlates in part with a restricted infection of oligodendrocytes.

Inhibition of Tyrosine Hydroxylase Expression within the Substantia Nigra of Mice Infected with Canine Distemper Virus

Experimental infection of mouse brain with a neuroadapted strain of canine distemper virus (CDV) leads to early acute encephalitis, followed by late neurological diseases such as motor pathologies (paralysis and turning behavior) or obesity syndrome. We have previously shown that, during the early stage of infection, CDV replicates transiently in selective structures of the brain including the substantia nigra, a structure known to play a critical role in motor control. In this study we demonstrate that CDV replication in the substantia nigra induces an early decrease in transcript level of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. The CDV infection of neuroblastoma cell culture, constitutively expressing TH, results in downregulation of TH transcription in the absence of cell death. In the few surviving mice with motor deficiencies, a pronounced decrease in TH expression is associated with a loss of dopaminergic cell bodies in the absence of any viral transcripts and proteins, suggesting that the initial CDV infection was sufficient to trigger irreversible neurodegenerative processes.

Phosphorylation of Canine Distemper Virus P Protein by Protein Kinase C-ζ and Casein Kinase II

Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-ζ) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-ζ has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-ζ has only minor activity. Selective inhibition of PKC-ζ activity within CV1 cells eliminated permissiveness to CDV replication, indicating anin vivorole for PKC-ζ in the virus replication cycle. The broad tissue expression of PKC-ζ parallels the pantropic nature of CDV infections, suggesting that PKC-ζ activity is a determinant of cellular permissiveness to CDV replication.

Selective induction of cytokines in mouse brain infected with canine distemper virus: structural, cellular and temporal expression

We have previously shown that, in experimentally inoculated mice, canine distemper virus (CDV), a neurotropic virus, selectively infects certain brain structures (hypothalamus, hippocampus, monoaminergic nuclei, etc). Here we demonstrate that tumor necrosis factor (TNF)-α, interleukin (IL)-1 β and IL-6 transcripts are selectively expressed in these CDV-targeted structures, except in the dentate gyrus, where cytokines are induced without prior CDV replication. The time-course of TNF-α expression vs. viral replication in the hypothalamus was different from that in hippocampus. In addition, we show that a substantial number of neurons express TNF-α and IL-6. These findings provide new insights into the possible participation of cytokines in the neurological disorders triggered by CDV infection.