LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus.

Wild-type canine distemper virus (CDV) is an important pathogen of dogs as well as wildlife that can infect immune and epithelial cells through two known receptors: the signaling lymphocytic activation molecule (SLAM) and nectin-4, respectively. Conversely, the ferret and egg-adapted CDV-Onderstepoort strain (CDV-OP) is employed as an effective vaccine for dogs. CDV-OP also exhibits promising oncolytic properties, such as its abilities to infect and kill multiple cancer cells in vitro. Interestingly, several cancer cells do not express SLAM or nectin-4, suggesting the presence of a yet unknown entry factor for CDV-OP. By conducting a genome-wide CRISPR/Cas9 knockout (KO) screen in CDV-OP-susceptible canine mammary carcinoma P114 cells, which neither express SLAM nor nectin-4, we identified low-density lipoprotein receptor-related protein 6 (LRP6) as a host factor that promotes CDV-OP infectivity. Whereas the genetic ablation of LRP6 rendered cells resistant to infection, ectopic expression in resistant LRP6KO cells restored susceptibility. Furthermore, multiple functional studies revealed that (i) the overexpression of LRP6 leads to increased cell-cell fusion, (ii) a soluble construct of the viral receptor-binding protein (solHOP) interacts with a soluble form of LRP6 (solLRP6), (iii) an H-OP point mutant that prevents interaction with solLRP6 abrogates cell entry in multiple cell lines once transferred into recombinant viral particles, and (iv) vesicular stomatitis virus (VSV) pseudotyped with CDV-OP envelope glycoproteins loses its infectivity in LRP6KO cells. Collectively, our study identified LRP6 as the long sought-after cell entry receptor of CDV-OP in multiple cell lines, which set the molecular bases to refine our understanding of viral-cell adaptation and to further investigate its oncolytic properties. IMPORTANCE Oncolytic viruses (OV) have gathered increasing interest in recent years as an alternative option to treat cancers. The Onderstepoort strain of canine distemper virus (CDV-OP), an enveloped RNA virus belonging to the genus Morbillivirus, is employed as a safe and efficient vaccine for dogs against distemper disease. Importantly, although CDV-OP can infect and kill multiple cancer cell lines, the basic mechanisms of entry remain to be elucidated, as most of those transformed cells do not express natural receptors (i.e., SLAM and nectin-4). In this study, using a genome-wide CRISPR/Cas9 knockout screen, we describe the discovery of LRP6 as a novel functional entry receptor for CDV-OP in various cancer cell lines and thereby uncover a basic mechanism of cell culture adaptation. Since LRP6 is upregulated in various cancer types, our data provide important insights in order to further investigate the oncolytic properties of CDV-OP.

Disease outbreaks select for mate choice and coat color in wolves.

We know much about pathogen evolution and the emergence of new disease strains, but less about host resistance and how it is signaled to other individuals and subsequently maintained. The cline in frequency of black-coated wolves (Canis lupus) across North America is hypothesized to result from a relationship with canine distemper virus (CDV) outbreaks. We tested this hypothesis using cross-sectional data from wolf populations across North America that vary in the prevalence of CDV and the allele that makes coats black, longitudinal data from Yellowstone National Park, and modeling. We found that the frequency of CDV outbreaks generates fluctuating selection that results in heterozygote advantage that in turn affects the frequency of the black allele, optimal mating behavior, and black wolf cline across the continent.

Transcriptome Analysis Reveals the Alternative Splicing Changes in the Immune-Related Genes of the Giant Panda (Ailuropoda melanoleuca), in Response to the Canine Distemper Vaccine.

Canine distemper virus (CDV) is a highly fatal virus to the giant panda (Ailuropoda melanoleuca). Although vaccination is a key preventative measure in captive giant pandas, the immune response of giant pandas after vaccination remains unclear. Therefore, this study focuses on differential alternative splicing (DAS) events of giant pandas before and after vaccination to investigate the role of alternative splicing in the immune response of giant pandas after CDV vaccination. In this study, we identified 1113 DAS genes, which had 1288 DAS events. The KEGG functional enrichment analysis of DAS genes showed enrichment of some DNA damage repair and immune-related pathways. In the combined analysis of DAS and differentially expressed genes (from our previous research), we identified 66 differentially expressed genes with a DAS event, and found that some important immune-related genes, such as IL15, IL18, IL18RAP, CHUK, IFI44, CD40, and CD46 underwent DAS events and were involved in the immune response of giant pandas after CDV vaccination. We describe here the alternative splicing events of giant pandas after CDV vaccination for the first time and show that the results indicated that alternative splicing has an important role in regulating the immune response of giant pandas after vaccination.

Investigation of TLR1-9 genes and miR-155 expression in dogs infected with canine distemper.

This study aimed to determine the relationship of toll-like receptor (TLR) 1-9 genes and microRNA (miR) -155 expression levels with hematologic parameters in dogs diagnosed with canine distemper. In the study, two groups were used pre-treatment and post-treatment. Infected dogs were diagnosed with canine distemper with the help of a rapid test kit and Real Time-Polymerase Chain Reaction (RT-PCR). Based on the correlation coefficients between the expression levels of the genes examined within the scope of the study and hematologic values, a positive correlation was found between the TLR2 gene and the monocyte (MON) value and between the TLR4 gene and the platelet (PLT) value in the pre-treatment group. A strong positive correlation was identified between TLR3 and TLR9 genes and erythrocyte (RBC) and hemoglobin (HGB) values; between TLR5 gene and RBC, HGB and hematocrit (HCT) values and between TLR9 gene and RBC and HGB values in the post-treatment group, on the other hand, a positive correlation was found between TLR1 gene and MON and neutrophil (GRAN) values; between TLR3 gene and HCT value and between TLR9 gene and MON and HCT values. The study concluded that miR-155 and TLR8 gene were upregulated at a statistically significant level (P < 0.05) Post-treatment in dogs infected with canine distemper and there was a positive correlation between the upregulation of miR-155 and the upregulation of TLR8 in the same period. This result suggests that the upregulated miR-155 expression post-treatment increased TLR8 gene expression. In the light of these findings, it miR-155 may have the potential to be used in clinical practice in the treatment or prognosis of dogs infected with canine distemper.

A highly virulent canine distemper virus strain isolated from vaccinated mink in China.

An outbreak of canine distemper in 2017 in mink breeding farms (Shandong province, China) caused severe pneumonia, hardened footpads, and death in more than 5000 vaccinated animals. Sequencing of the hemagglutinin and fusion protein genes from the WH2 canine distemper virus (CDV) strain we isolated from the infected minks were clustered into the recently isolated CDV Asia-1 genotype group. The WH2 strain was distinct from the current vaccine strains, containing a novel potential N-glycosylation site in its hemagglutinin protein. It also contained amino acid mutations in the fusion protein gene (I87N, T110P and L386I), and the T110P mutation results in N-glycosylation site silencing. WH2 was highly virulent in both unvaccinated and vaccinated animals in our pathogenesis experiments. Immunohistochemistry results revealed positive staining of different organs in unvaccinated and vaccinated animals. The serum in vitro neutralizing antibody titers for the vaccinated mink group and a dog were higher for the WH2 strain than those of the HNly150520B strain (isolated from a dog). These findings indicate that the current commercial vaccines provide incomplete protection against WH2 challenge infections. Thus, a new vaccine strain is urgently needed to protect against variant CDV strains.

Computational Analysis Reveals a Critical Point Mutation in the N-Terminal Region of the Signaling Lymphocytic Activation Molecule Responsible for the Cross-Species Infection with Canine Distemper Virus.

Infection of hosts by morbilliviruses is facilitated by the interaction between viral hemagglutinin (H-protein) and the signaling lymphocytic activation molecule (SLAM). Recently, the functional importance of the n-terminal region of human SLAM as a measles virus receptor was demonstrated. However, the functional roles of this region in the infection process by other morbilliviruses and host range determination remain unknown, partly because this region is highly flexible, which has hampered accurate structure determination of this region by X-ray crystallography. In this study, we analyzed the interaction between the H-protein from canine distemper virus (CDV-H) and SLAMs by a computational chemistry approach. Molecular dynamics simulations and fragment molecular orbital analysis demonstrated that the unique His28 in the N-terminal region of SLAM from Macaca is a key determinant that enables the formation of a stable interaction with CDV-H, providing a basis for CDV infection in Macaca. The computational chemistry approach presented should enable the determination of molecular interactions involving regions of proteins that are difficult to predict from crystal structures because of their high flexibility.

Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus.

BACKGROUND: Canine morbilivirus (canine distemper virus, CDV) is a highly contagious pathogen associated with high morbidity and mortality in susceptible carnivores. Although there are CDV vaccines available, the disease poses a huge threat to dogs and wildlife hosts due to vaccine failures and lack of effective treatment. Thus, the development of therapeutics is an urgent need to achieve rapid outbreak control and reduce mortality in target species. Gene silencing by RNA interference has emerged as a promising therapeutic approach against different human and animal viruses. In this study, plasmid-based short hairpin RNAs (shRNAs) against three different regions in either CDV nucleoprotein (N), or large polymerase (L) genes and recombinant adenovirus-expressing N-specific multi-shRNAs were generated. Viral cytopathic effect, virus titration, plaque-forming unit reduction, and real-time quantitative RT-PCR analysis were used to check the efficiency of constructs against CDV. RESULTS: In CDV-infected VerodogSLAM cells, shRNA-expressing plasmids targeting the N gene markedly inhibited the CDV replication in a dose-dependent manner, with viral genomes and titers being decreased by over 99%. Transfection of plasmid-based shRNAs against the L gene displayed weaker inhibition of viral RNA level and virus yield as compared to CDV N shRNAs. A combination of shRNAs targeting three sites in the N gene considerably reduced CDV RNA and viral titers, but their effect was not synergistic. Recombinant adenovirus-expressing multiple shRNAs against CDV N gene achieved a highly efficient knockdown of CDV N mRNAs and successful inhibition of CDV replication. CONCLUSIONS: We found that this strategy had strong silencing effects on CDV replication in vitro. Our findings indicate that the delivery of shRNAs using plasmid or adenovirus vectors potently inhibits CDV replication and provides a basis for the development of therapeutic strategies for clinical trials.

RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus.

Virulent morbillivirus infections, including Meals Virus (MeV) and Canine Distemper Virus (CDV), caused severe immune suppression and leukopenia, while attenuated vaccine strains developed protective host immune responses. However, the detailed molecular foundations of host antiviral responses were poorly characterized. In order to better understand the interactions between attenuated vaccine and host antiviral responses, the global gene expression changes in CDV-11-infected DH82 cells, a macrophage-derived cell line from canine, were investigated by transcriptomic analysis, and portions of results were confirmed with quantitative RT-PCR. The results exhibited that 372 genes significantly up-regulated (p < .01) and 119 genes were significantly down-regulated (p < .01) in CDV-infected macrophages DH82 at 48 h p.i.. The enriched functions of the significantly up-regulated (p < .01) genes were closely associated with interferon stimulated genes (ISGs), chemokine genes and pro-inflammatory factor genes. Gene ontology and pathway analysis of differentially expressed genes (DEGs) revealed that the most significantly involved pathways in CDV-infected DH82 cells were NF-κB and TNF signaling pathway, cytokine-cytokine receptor interaction, and pathogen associated molecular patterns (PAMPs), such as Toll-like, RIG-I-like and NOD-like receptor signalings. Thus, the findings indicated that pattern recognition receptors (PRRs) possibly mediated host innate and protective antiviral immune responses in CDV-11 infected DH82 cells.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis.

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species.

Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion.

Construction of a mavs-inactivated MDCK cell line for facilitating the propagation of canine distemper virus (CDV).

Canine distemper is a highly contagious disease of wild and domestic carnivores. Obtaining of a suitable cell line for canine distemper virus (CDV) propagation is very important for field CDV isolation and vaccine antigen preparation. However, the cell line currently developed cell lines for CDV propagation are a marmoset lymphoid cell line (B95a), which could cause the virus to potentially infect human cells, and canine SLAM-expressing Vero cells, which may cause the virus to lose virulence. Therefore, a canine cell line constructed for efficient CDV propagation would be attractive. In the present study, a Madin-Darby Canine Kidney Epithelial (MDCK) cell line with mavs (mitochondrial antiviral signaling) inactivation was constructed by CRISPR/Cas9 technology. The interferon-I response induced by poly(I:C), an analogue of viral RNA, was significantly blocked in the constructed cell line, designated MDCK-KOmavs. Moreover, the propagation of a filed CDV strain was approximately 100 times higher in MDCK-KOmavs cells than in wild-type MDCK cells. Therefore, in the present study, a canine cell line facilitating CDV propagation was successfully constructed, and the results suggested that the constructed canine cell line was more efficient than the wild-type cell line for the isolation of field CDVs. In addition, the rapid propagation of CDVs to high titers in the constructed MDCK-KOmavs cell line indicated that this cell line could also be an alternative cell line for the preparation of vaccine antigens.

Molecular and serological surveys of canine distemper virus: A meta-analysis of cross-sectional studies.

BACKGROUND: Canine morbillivirus (canine distemper virus, CDV) persists as a serious threat to the health of domestic dogs and wildlife. Although studies have been conducted on the frequency and risk factors associated with CDV infection, there are no comprehensive data on the current epidemiological magnitude in the domestic dog population at regional and national levels. Therefore, we conducted a cross-sectional study and included our results in a meta-analysis to summarize and combine available data on the frequency and potential risk factors associated with CDV infection. METHODS: For the cross-sectional study, biological samples from dogs suspected to have canine distemper (CD) were collected and screened for viral RNA. Briefly, the PRISMA protocol was used for the meta-analysis, and data analyses were performed using STATA IC 13.1 software. RESULTS: CDV RNA was detected in 34% (48/141) of dogs suspected to have CD. Following our meta-analysis, 53 studies were selected for a total of 11,527 dogs. Overall, the pooled frequency of CDV positivity based on molecular and serological results were 33% (95% CI: 23-43) and 46% (95% CI: 36-57), respectively. The pooled subgroup analyses of clinical signs, types of biological samples, diagnostic methods and dog lifestyle had a wide range of CDV positivity (range 8-75%). Free-ranging dogs (OR: 1.44, 95% CI: 1.05-1.97), dogs >24 months old (OR: 1.83, 95% CI: 1.1-3) and unvaccinated dogs (OR: 2.92, 95% CI: 1.26-6.77) were found to be positively associated with CDV infection. In contrast, dogs <12 months old (OR: 0.36, 95% CI: 0.20-0.64) and dogs with a complete anti-CDV vaccination (OR: 0.18, 95% CI: 0.05-0.59) had a negative association. CONCLUSION: Considering the high frequency of CDV positivity associated with almost all the variables analyzed in dogs, it is necessary to immediately and continuously plan mitigation strategies to reduce the CDV prevalence, especially in determined endemic localities.

Interferon-Stimulated Genes-Mediators of the Innate Immune Response during Canine Distemper Virus Infection.

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.

Genetic and spatial characterization of the red fox (Vulpes vulpes) population in the area stretching between the Eastern and Dinaric Alps and its relationship with rabies and canine distemper dynamics.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

Canine Distemper Virus Spread and Transmission to Naive Ferrets: Selective Pressure on Signaling Lymphocyte Activation Molecule-Dependent Entry.

Upon infection, morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) initially target immune cells via the signaling lymphocyte activation molecule (SLAM) before spreading to respiratory epithelia through the adherens junction protein nectin-4. However, the roles of these receptors in transmission from infected to naive hosts have not yet been formally tested. To experimentally addressing this question, we established a model of CDV contact transmission between ferrets. We show here that transmission of wild-type CDV sometimes precedes the onset of clinical disease. In contrast, transmission was not observed in most animals infected with SLAM- or nectin-4-blind CDVs, even though all animals infected with the nectin-4-blind virus developed sustained viremia. There was an unexpected case of transmission of a nectin-4-blind virus, possibly due to biting. Another unprecedented event was transient viremia in an infection with a SLAM-blind virus. We identified three compensatory mutations within or near the SLAM-binding surface of the attachment protein. A recombinant CDV expressing the mutated attachment protein regained the ability to infect ferret lymphocytes in vitro, but its replication was not as efficient as that of wild-type CDV. Ferrets infected with this virus developed transient viremia and fever, but there was no transmission to naive contacts. Our study supports the importance of epithelial cell infection and of sequential CDV H protein interactions first with SLAM and then nectin-4 receptors for transmission to naive hosts. It also highlights the in vivo selection pressure on the H protein interactions with SLAM.IMPORTANCE Morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) are highly contagious. Despite extensive knowledge of how morbilliviruses interact with their receptors, little is known about how those interactions influence viral transmission to naive hosts. In a ferret model of CDV contact transmission, we showed that sequential use of the signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors is essential for transmission. In one animal infected with a SLAM-blind CDV, we documented mild viremia due to the acquisition of three compensatory mutations within or near the SLAM-binding surface. The interaction, however, was not sufficient to cause disease or sustain transmission to naive contacts. This work confirms the sequential roles of SLAM and nectin-4 in morbillivirus transmission and highlights the selective pressure directed toward productive interactions with SLAM.

Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam.

Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China.

MYD88 and functionally related genes are associated with multiple infections in a model population of Kenyan village dogs.

The purpose of this study was to seek associations between immunity-related molecular markers and endemic infections in a model population of African village dogs from Northern Kenya with no veterinary care and no selective breeding. A population of village dogs from Northern Kenya composed of three sub-populations from three different areas (84, 50 and 55 dogs) was studied. Canine distemper virus (CDV), Hepatozoon canis, Microfilariae (Acantocheilonema dracunculoides, Acantocheilonema reconditum) and Neospora caninum were the pathogens studied. The presence of antibodies (CDV, Neospora), light microscopy (Hepatozoon) and diagnostic PCR (Microfilariae) were the methods used for diagnosing infection. Genes involved in innate immune mechanisms, NOS3, IL6, TLR1, TLR2, TLR4, TLR7, TLR9, LY96, MYD88, and three major histocompatibility genes class II genes were selected as candidates. Single nucleotide polymorphism (SNP) markers were detected by Sanger sequencing, next generation sequencing and PCR-RFLP. The Fisher´s exact test for additive and non-additive models was used for association analyses. Three SNPs within the MYD88 gene and one TLR4 SNP marker were associated with more than one infection. Combined genotypes and further markers identified by next generation sequencing confirmed associations observed for individual genes. The genes associated with infection and their combinations in specific genotypes match well our knowledge on their biological role and on the role of the relevant biological pathways, respectively. Associations with multiple infections observed between the MYD88 and TLR4 genes suggest their involvement in the mechanisms of anti-infectious defenses in dogs.

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis.

INTRODUCTION: CDV-DL (Canine distemper virus-induced demyelinating leukoencephalitis) represents a spontaneously occurring animal model for demyelinating disorders. Axonopathy represents a key pathomechanism in this disease; however, its underlying pathogenesis has not been addressed in detail so far. This study aimed at the characterization of axonal cytoskeletal, transport, and potential regenerative changes with a parallel focus upon Schwann cell remyelination. METHODS: Immunohistochemistry of canine cerebellar tissue as well as a comparative analysis of genes from an independent microarray study were performed. RESULTS: Increased axonal immunoreactivity for nonphosphorylated neurofilament was followed by loss of cytoskeletal and motor proteins. Interestingly, a subset of genes encoding for neurofilament subunits and motor proteins was up-regulated in the chronic stage compared to dogs with subacute CDV-DL. However, immunohistochemically, hints for axonal regeneration were restricted to up-regulated axonal positivity of hypoxia-inducible factor 1 alpha, while growth-associated protein 43, erythropoietin and its receptor were not or even down-regulated. Periaxin-positive structures, indicative of Schwann cell remyelination, were only detected within few advanced lesions. CONCLUSIONS: The present findings demonstrate a complex sequence of axonal cytoskeletal breakdown mechanisms. Moreover, though sparse, this is the first report of Schwann cell remyelination in CDV-DL. Facilitation of these very limited endogenous regenerative responses represents an important topic for future research.

Molecular detection and phylogenetic relationship of wild-type strains of canine distemper virus in symptomatic dogs from Uberlândia, Minas Gerais / Detecção molecular e relação filogenética de cepas selvagens do vírus da cinomose canina em cães sintomáticos oriundos de Uberlândia, Minas Gerais

This study investigated the occurrence of canine distemper virus (CDV) by evaluating the presence of viral RNA within urine samples of dogs from Uberlândia, MG, with clinical manifestations suggestive of infection by CDV by targeting the CDV N gene. Of the clinical samples collected ( n =33), CDV viruria was detected in 45.5%. Five dogs died spontaneously; all had characteristic CDV-associated histopathological alterations and demonstrated CDV viruria. Statistical analyses revealed that the age, gender, breed, or the organ system of the dog affected had no influence on the occurrence of canine distemper. Myoclonus and motor incoordination were the most significant neurological manifestations observed. A direct association was observed between keratoconjunctivitis and dogs with CDV viruria. These findings suggest that CDV viruria in symptomatic dogs might not be age related, and that symptomatic dogs can demonstrate clinical manifestations attributed to CDV without viruria identified by RT-PCR. Additionally, the results of the sequence identities analysed have suggested that all Brazilian wild-type strains of CDV currently identified are closely related and probably originated from the same lineage of CDV. Nevertheless, phylogenetic analyses suggest that there are different clusters of wild-type strains of CDV circulating within urban canine populations in Brazil.

A presença do ácido nucleico (RNA) do vírus da cinomose canina (CDV) foi avaliada por meio da amplificação parcial do gene N pela técnica RT-PCR realizada em urina de cães provenientes de Uberlândia, Minas Gerais, que apresentavam sinais clínicos sugestivos de cinomose. Das 33 amostras de urina avaliadas, o CDV foi identificado em 45,5%. Em cinco cães que morreram espontaneamente, além da excreção do CDV na urina, foram observadas alterações histopatológicas associadas à infecção por esse vírus. Análises estatísticas demonstraram que a idade, gênero, raça e o sistema orgânico comprometido dos cães avaliados não exerceram influência no diagnóstico da cinomose canina. Mioclonia e incoordenação motora foram as manifestações neurológicas que apresentaram frequência de ocorrência significativa (P<0,05). Uma associação direta foi observada entre a presença de ceratoconjuntivite e a identificação de virúria pelo CDV. Esses achados sugerem que a excreção do CDV pela urina em cães com sinais clínicos compatíveis com cinomose pode não ser relacionada com a idade do animal, e que animais sintomáticos podem apresentar manifestações clínicas atribuídas ao CDV, porém sem a caracterização de virúria por RT-PCR. Adicionalmente, análises filogenéticas sugerem que várias cepas de CDV podem estar circulando em populações caninas de áreas urbanas no Brasil.