Canine Distemper Virus Nucleoprotein Research Articles

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis.

Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

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

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

Molecular Detection of Canine Distemper Virus in Dogs in Baghdad Province, Iraq

Canine distemper (CD) is an infectious disease that affects dogs and is extremely contagious and lethal, with a high mortality and morbidity rates. It infects a broad variety of animals, including primates, cetaceans, and carnivores causing a multi-systemic pathological condition. This study aimed to detect canine distemper virus (CDV) in blood samples of dogs clinically suspected with distemper at the Baghdad Veterinary Hospital, Baghdad, Iraq. CDV nucleoprotein gene (N) was detected in the whole blood of 46 dogs using reverse transcription-PCR (RT-PCR). The partially amplified (591 bp) fragment of the N gene was detected in 12 of 46 (26%) blood samples of dogs examined. Based on the partial sequencing data of the N gene, three local isolates might be similar to the NCBI-BLAST reference CDV virus isolates FJ977579.1 China, AF378705.1 USA, and AF305419.1 UK, while other strains EU072200.1 Hungary, AF164967.1 Switzerland, KU578257.1 Germany, and AB474397.1 Japan were found to be rather distinct. The isolates displayed a higher level of similarity with the Snyder Hill CDV strain and Onderstepoort CDV strain. There was less homology with the CDV strain A75/17 of Switzerland and 007Lm CDV strain of Japan. In conclusion, this study confirmed that CDV infection is present in domestic dogs in Iraq. This may indicate a risk of the disease spreading to parts of the country that may be disease-free.

The Investigation of Canine Distemper Virus in Different Diagnosis Materials of Dogs using Molecular and Pathological Methods, Northeastern Turkey

Background: Canine distemper virus (CDV) is highly contagious disease that affects dogs despite several control measures. This study was aimed at investigating the presence of CDV nucleic acid in different clinical and tissue materials, from naturally infected dogs, by reverse transcriptase-polymerase chain reaction (RT-PCR) and to molecularly characterize distemper strains according to the partial Nucleoprotein (NP) gene sequence. Furthermore, tissue samples under went histopathological examination for distemper infection. Methods: A total of 202 different diagnosis materials were collected from dogs (n=60) in the Kars region in northeastern Turkey. The samples were tested for CDV using RT-PCR with primers designed for the CDV NP gene. Samples determined as positive for CDV (n=7) were sequenced. Tissue samples underwent histopathological examination. Result: Most of the cases were in animals aged 0-6 months. The most common clinical finding was severe respiratory system infection. This finding was accompanied by gastrointestinal and nervous system infections. CDV nucleic acid was detected in 112 of 202 materials by RT-PCR. According to RT-PCR results, positivity rates of 88.2% (30/34), 72.2% (13/18), 60% (3/5), 55.5% (10/18), 55.5% (10/18), 51.6% (16/31), 45.5% (5/11), 37.8% (14/37) and 36.7% (11/30) were detected in nasal swab, lung, footpad, kidney, spleen, rectal swab, cerebrospinal fluids (CSF), leuokocyte and cerebellum samples, respectively. Viral nucleic acids were detected at higher rates in nasal swabs. The phylogenetic assessment of the amplicon sequences revealed a 97.7%-100% similarity among the Turkish CDV strains, which are independent from vaccine strains, were found to be more closely related to the European lineage. Intranuclear and intracytoplasmic inclusion bodies were detected by histopathology. This is the first study to investigate CDV in naturally infected dogs from northeastern Turkey and to provide novel and updated epidemiological information.

Identification of a novel linear B-cell epitope using a monoclonal antibody against the carboxy terminus of the canine distemper virus nucleoprotein and sequence analysis of the identified epitope in different CDV isolates

BackgroundThe Nucleoprotein (NP) is the most abundant and highly immunogenic protein in canine distemper virus (CDV), playing an important role in CDV viral replication and assembly.ResultsIn this study, a specific monoclonal antibody, named C8, was produced against the NP protein C terminal (amino acids 401–523). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis.The results indicated that 444GDKYPIHFNDER455 was the minimal linear epitope that could be recognized by mAb C8. Sequence alignments demonstrated that this linear epitope is less conserved among three CDV genotypes. We next analyzed the level of conservation of the defined epitope in19 Chinese CDV clinical isolates, and it has one site variation in amino acid among these CDV isolations. 2 isolates have the amino acid mutations F451L, while one has P448Ssubstitution.Phylogenetic analysis showed the two isolates with F451Lsubstitution had a closer relationship in a virulent strain ZJ-7, so the epitope may be a significant tag associated with virus virulence.ConclusionThis collection of mAb along with defined linear epitope may provide useful reagents for investigations of NP protein function and the development of CDV specific diagnostics.

The recombinant EHV-1 vector producing CDV hemagglutinin as potential vaccine against canine distemper

Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper.

Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions.

Three dog shelters in Rio Grande do Sul were investigated for associations between the occurrence of respiratory viruses and shelter environmental conditions. Nasal secretions randomly collected during the cold season were tested via PCR, and this data collection was followed by nucleotide sequencing of the amplicons. In shelter #1 (poor sanitary and nutritional conditions, high animal density and constant contact between dogs), 78% (58/74) of the nasal samples were positive, 35% (26/74) of which were in single infections and 44% (32/74) of which were in coinfections. Shelters #2 and #3 had satisfactory sanitary and nutritional conditions, outdoors exercise areas (#2) and animal clustering by groups (#3). In shelter #2, 9% (3/35) of the samples were positive for Canine parainfluenza virus (CPIV), and 6% (2/35) were positive for Canid herpesvirus 1 (CaHV-1). In shelter #3, 9% (7/77) of the samples were positive for Canine adenovirus type 2 (CAdV-2), and 1% (1/77) were positive for Canine distemper virus (CDV). The amplicon sequences (CPIV and CDV nucleoprotein gene; CAdV-2 E3 gene; CaHV-1 glycoprotein B gene) showed 94–100% nucleotide identity with GenBank sequences. Our results demonstrate that CPIV, CAdV-2 and CDV are common in dog shelters and that their frequencies appear to be related with environmental and nutritional conditions. These results indicate the need for control/prevention measures, including vaccination and environmental management, to minimize these infections and improve dog health.

Morbillivirus and Pilot Whale Deaths, Canary Islands, Spain, 2015

Morbillivirus and Pilot Whale Deaths, Canary Islands, Spain, 2015

Case report : Canine distemper virus infection in a wild Korean raccoon dog

Abstract: A female wild raccoon dog was referred with a history of generalized seizure. Mild leukocytosis was noted onlaboratory tests. Gross lesions included nasal hemorrhage, hemothorax, and hemorrhage in the urinary bladder withhematuria. Microscopically, interstitial and purulent bacterial pneumonia was observed in the lungs. In the cerebellum,characteristic eosinophilic intracytoplasmic and intranuclear inclusion bodies were found in Purkinje cells, and severedemyelination was observed in the cerebellar white matter. Canine distemper virus (CDV) infection was suspected andconfirmed after detection of CDV nucleoprotein RNA in the cerebrum and the lungs by nested reverse transcriptionpolymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Based onthe histopathological and molecular diagnostic findings, it was concluded that the raccoon dog was infected with CDV.

Human and Canine Codon Optimization of Measles and Canine Distemper Viral Nucleic Acid Sequences Increases Sequence Identity and Protein Expression Compared to Wild Type and Human Codon Sub‐Optimized Constructs

The role of codon usage bias (CUB) in the host adaptation and pathogenicity of human measles virus (HMV) and canine distemper virus (CDV) is being investigated and compared. Analysis of nucleic acid sequences for HMV and CDV nucleoprotein (N) revealed that human and canine CUB optimized constructs, as compared to wild type (WT) and human CUB sub‐optimized constructs, were characterized by increased nucleic acid and codon identities, and increased protein expression when transfected into human (293 HEK) cells. The purpose of this study was to characterize human and canine codon optimized nucleic acid sequences of the MHV and CDV F, M, H,L and P/C proteins. All human and canine CUB optimized HMV and CDV nucleic acid sequences had increased nucleic acid and codon identity as compared to WT sequences. Protein expression in human cells transfected with human and canine CUB optimized constructs of the F, M, H and P/C proteins was assessed using florescence, Cellomic and where possible western analysis. Higher protein expression was present in cells transfected with the human and canine codon optimized constructs compared to cells transfected with HMV and CDV WT or HMV suboptimized constructs. These results suggest that further adaptation of HMV and CDV to the mammalian CUB could enhance viral protein expression and pathogenesis.

Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies

Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits with CDV DNA vaccines. Virus neutralising (VN) antibody responses were induced in mink kits vaccinated with a plasmid encoding the haemaglutinin protein (H) of CDV (n=5, pCDV-H) or a combination of the H, fusion (F) and nucleoprotein (N) of CDV (n=5, pCDV-HFN). These DNA vaccinated kits were protected against virulent experimental infection with field strains of CDV. The pCDV-H was more efficient in inducing protective immunity in the presence of maternal antibodies compared to the pCDV-HFN. The results show that DNA vaccination with the pCDV-H or pCDV-HFN (n=4) only given once at 5 days of age induces virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life.

Aphylogenetic study on the NP gene of detected canine distemper virus in (2008-2011) Iran

BACKGROUND: Canine Distemper (CD) is a lethal systemicdisease affecting a wide variety of terrestrial carnivores.OBJECTIVES: This study was performed to survey epidemiologicaland molecular characteristics of the canine distemper virus(CDV) strains circulating in Iran. METHODS: In this study, 19CDV-suspected unvaccinated dogs from Northeast and center ofIran were analyzed for presence of CDV nucleoprotein (NP) geneusing Nested-PCR during 2008- 2011. Different biological samplesof 14 dogs were positive. RESULTS: The phylogenetic analysisbased on partial NP gene sequences indicated the presence of twomajor clusters that are clearly different from vaccine strains in Iran.One cluster belongs to the European group and the other one to theArctic group. CONCLUTIONS: Due to a lack of phylogeneticanalysis on CDV in countries bordering Iran, except Turkey, theancestor of Iranian sequences specially Iranian Arctic sequencescould not be definitely identified. This study is the first report onphylogenetic analysis of CDV from domestic dogs in Iran.

Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms “viral replication” and “humoral immune response” as well as down-regulated genes functionally related to “metabolite and energy generation”.

Epizootic of dolphin morbillivirus on the Catalonian Mediterranean coast in 2007

BETWEEN 1990 and 1992, thousands of striped dolphins ( Stenella coeruleoalba ) stranded along the Mediterranean coast due to a newly described virus, the dolphin morbillivirus (DMV) (Domingo and others...

Isolation and molecular characterization of canine distemper virus from India

Ocular swabs from canine distemper virus (CDV) suspected live or brain tissue from dead dogs were tested for the presence of CDV nucleoprotein (N) gene using reverse transcriptase polymerase chain reaction (RT-PCR). Partial "N" gene sequencing of the RT-PCR-positive samples and the local vaccine virus revealed that the Ind/Andaman 01/07 virus was highly divergent from the rest of the CDV isolates and from the vaccine strain. Quantitative real-time PCR (qRT-PCR) using SYBR Green I chemistry for CDV haemagglutinin "H" gene quantification showed C(t) values ranging from 29.76-30.67 in the RT-PCR-positive samples. Two of the positive samples, designated Ind/TN 01/07 and Ind/Andaman 01/07 were used for virus isolation in B95a cell line. Characteristic cytopathic changes such as rounding of cells, syncytia formation, and ballooning were seen from the first passage onwards. Specific cytoplasmic fluorescence was seen in infected cells with a commercial reference serum against CDV. To the best of our knowledge, this is the first report of CDV isolation from clinical cases in India.

Molecular Detection of Canine Distemper Virus and the Immunohistochemical Characterization of the Neurologic Lesions in Naturally Occurring Old Dog Encephalitis

The current article describes a spontaneous case of old dog encephalitis (ODE) in a 7-year-old, intact, female Miniature Schnauzer dog from Londrina, Paraná, southern Brazil. Unlike conventional distemper encephalomyelitis, ODE is a poorly understood and extremely rare manifestation of Canine distemper virus (CDV) infection. The dog was presented with progressive clinical manifestations consistent with cerebral dysfunction. Briefly, histopathologic lesions were restricted to the forebrain and included chronic multifocal lymphoplasmacytic encephalitis with extensive perivascular cuffing, astrocytosis, and intranuclear inclusions within astrocytes and giant cells, with both intracytoplasmic and intranuclear inclusions. Immunohistochemistry (IHC) was used to identify the antigens of the nucleoprotein (NP) of CDV and to detect cluster of differentiation (CD)3, CD79a, macrophage (MAC) 387, glial fibrillary acidic protein, and vimentin to characterize the neuroparenchymal lesions. By IHC, CDV NP was demonstrated predominantly within neurons and astrocytes. Cells that formed perivascular cuffs and some astrocyte-like cells reacted intensely to vimentin. Reverse transcription polymerase chain reaction assay from brain sections further confirmed a role for CDV in this disease by the amplification and partial sequence analysis of the NP gene. These findings confirmed simultaneous detection of CDV in ODE by IHC and molecular assays. In addition, results of the current study could contribute to the neuropathologic characterization of this rare manifestation of CDV.

Virus isolation and molecular characterization of canine distemper virus by RT-PCR from a mature dog with multifocal encephalomyelit

A case of multifocal distemper encephalomyelitis in a mature dog is described. In the presented case the ante mortem clinical diagnosis of canine distemper virus (CDV) infection could not be ideally performed due to the absence of typical signs of distemper, such as myoclonus and systemic signs accompanying the nervous signs. The definitive diagnosis of distemper encephalomyelitis was only carried out at post mortem through virus isolation in cell culture from fresh central nervous system (CNS) fragments and CDV nucleoprotein gene detection in the CNS by RT-PCR.

Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados

Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV) by a reverse transcription-polymerase chain reaction (RT-PCR) assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and neurological signs. In 66.5% (125/188) of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8% (76/125) of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2% (49/125) of the dogs just a type of clinical sample (urine: n=37; leucocytes: n=12) was positive. These results demonstrate that the different forms of clinical distemper disease can hinder the choice of only one type of clinical sample to carry out the ante mortem etiological diagnosis of CDV infection, and false-negative results can be generated.

Morphometric analysis of the thymus of puppies infected with the Snyder Hill Strain of canine distemper virus

The thymic morphometry analysis was used for determining apoptosis and atrophy of the thymus of eight puppies inoculated with canine distemper virus (CDV). Three healthy dogs were used as negative controls. Sections, 5µm thick, were stained by HE and Shorr, and the latter were evaluated by morphometry. CDV nucleoprotein was detected by immunohistochemistry. Morphometric results confirmed lymphoid hypotrophy in CDV inoculated dog thymuses, more stroma, less parenchyma and higher apoptotic index/field than negative control (not inoculated) puppies. Apoptosis plays a role in the mechanism of thymus atrophy that develops in canine distemper.

Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro

Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.