Role of mutations in the phosphoprotein on RNA polymerase activity of canine distemper virus following cell culture adaptation

Abstract: The canine distemper virus A75/17 wild-type strain, which is unable to replicate in cell lines, was adapted to growth in Vero cells. Sequence comparison between the A75/17 and the Vero cell-adapted A75/17-V virus revealed 7 amino acid differences between the 2 viruses. Three of these were located in the matrix protein, three in the phosphoprotein also changing the V protein but not the C protein and one in the large protein. The phosphoprotein and the large protein constituted the viral RNA polymerase whose activity was studied by transfection experiments using a reverse genetic system with a plasmid encoding a minireplicon and expression plasmids encoding the nucleocapsid protein and the viral RNA polymerase subunits. Surprinsingly, the enzyme of A75/17 CDV was significantly more active in cell lines compared to the polymerase of A75/17-V CDV. The decrease in overall enzyme activity was found to be due to both decreased replication and transcription activity. This polymerase attenuation was confirmed in CHO cells infection stably expressing the dog SLAM receptor mainly found in dog's lymphoid organs and allowing both virus strains to enter these cells at the same efficiency. A75/17-V CDV replicated more slowly in CHODogSLAM cells than A75/17 CDV and syncytium formation was significantly decreased compared to A75/17 infected CHODogSLAM cells.. Cell culture adaptation lead to an attenuated virus strain both in vitro and in vivo with decreased polymerase activity and syncytium forming capability showing an important role of the polymerase in determining the phenoytpe of the virus. In addition, this reduced phenotype of A75/17-V CDV was shown to be due to the P mutations in the P protein only, showing an important function of the polycistronic P gene in the adaptation process. The role of the matrix protein was found not to have any effect on polymerase activity, however its participation in the adaptation process still needs to be elucidated. The accessory proteins V and C were shown to act on polymerase activity, but their functions in virus pathogenicity and in inhibiting the interferon system have not been studied in this thesis. The V proteins have an activating effect on the polymerase of both the A75/17 and the A75/17-V CDV strains. Although the C protein amino acid sequence was not changed during adaptation of wild-type canine distemper virus in Vero cells, the C protein was demonstrated to have opposite effects on polymerase activity of both virus strains suggesting a different interaction of the C protein with the proteins forming the polymerase complex, which could modulate polymeras activity. These effects were demonstrated by transfection experiments and studying recombinant viruses not expressing the C protein. Thus, the abrogation of the C protein decrease the activity of the wild-type polymerase. In contrast, the polymerase activity of the Vero cell- adapted virus is enhanced in the absence of the C protein and this has also been demonstrated with a recombinant virus, which grew faster in the first 48 hours of infection. Future studies will focus on the generation of recombinant wild-type viruses, which should be very helpful in understanding the molecular mechanisms underlying the adaptation process and the loss of pathogenicity.

Canine distemper virus detection in asymptomatic and non vaccinated dogs

A quantitative real time polymerase chain reaction (PCR) revealed canine distemper virus presence in peripheral blood samples from asymptomatic and non vaccinated dogs. Samples from eleven domestic dogs with no signs of canine distemper and not vaccinated at the month of collection were used. Canine distemper virus vaccine samples in VERO cells were used as positive controls. RNA was isolated with Trizol®, and treated with a TURBO DNA-free kit. Primers were designed for canine distemper virus nucleocapsid protein coding region fragment amplification (84 bp). Canine b-actin (93 bp) was utilized as the endogenous control for normalization. Quantitative results of real time PCR generated by ABI Prism 7000 SDS Software showed that 54.5% of dogs with asymptomatic canine distemper were positive for canine distemper virus. Dissociation curves confirmed the specificity of the real time PCR fragments. This technique could detect even a few copies of viral RNA and identificate subclinically infected dogs providing accurate diagnosis of this disease at an early stage.

Canine distemper virus infection in a lesser grison (Galictis cuja): first report and virus phylogeny

Infectious diseases in wild animals have been increasing as a result of their habitat alterations and closer contact with domestic animals. Canine distemper virus (CDV) has been reported in several species of wild carnivores, presenting a threat to wildlife conservation. We described the first case of canine distemper virus infection in lesser grison (Galictis cuja). A free-ranging individual, with no visible clinical sigs, presented sudden death after one day in captivity. Molecular diagnosis for CDV infection was performed using whole blood collected by postmortem intracardiac puncture, which resulted positive. The virus phylogeny indicated that domestic dogs were the probable source of infection.

Conjunctival effects of canine distemper virus-induced keratoconjunctivitis sicca

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Canine distemper virus infection in a lesser grison (Galictis cuja): first report and virus phylogeny

Doenças infecciosas em animais selvagens têm aumentado devido às alterações em seu habitat e ao maior contato com animais domésticos. A cinomose já foi descrita em diversas espécies de carnívoros selvagens, representando uma ameaça à conservação da vida selvagem. Nesse estudo é descrito o primeiro caso de infecção pelo vírus da cinomose em um furão (Galictis cuja). Um indivíduo de vida livre, sem sinais clínicos aparentes, apresentou morte súbita após um dia em cativeiro. Foi realizado o diagnóstico molecular para detecção do vírus da cinomose canina, sendo o resultado positivo. A filogenia do vírus indicou que cães domésticos foram a provável fonte de infecção.

Canine distemper virus in a crab-eating fox (Cerdocyon thous) in Brazil: case report and phylogenetic analyses

Although canine distemper is enzootic worldwide and has a wide host range, there are no reports of canine distemper virus there are no reports of canine distemper virus provide information on virus phylogeny and histopathologic lesions. The objective of this study is report and describe canine distemper in a crab-eating fox (C. thous), with a focus oil the phylogeny of the virus strain and the histopathologic lesions in the animal.

Canine distemper virus and Toxoplasma gondii co-infection in dogs with neurological signs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

First Identification of Canine Distemper Virus in Hoary Fox (Lycalopex vetulus): Pathologic Aspects and Virus Phylogeny

Canine distemper virus (CDV) has been reported in several wild animal species, but there have been no reports of CDV in hoary fox (Lycalopex vetulus). This paper characterizes the first case of natural CDV infection in hoary fox, including the clinical and pathologic aspects of the disease as well as the viral strain phylogeny.

Toxoplasma gondii genotyping in a dog co-infected with distemper virus and ehrlichiosis rickettsia

This paper reports a toxoplasmosis, erhlichiosis and distemper co-infection in a dog with an exuberant neuropathological clinical picture. Primary involvement was discussed based on information collected in the analysis of the clinical case, such as neurological impairment, epidemiological data, poor immunoprophylactic scheme of the dog affected and the role of these diseases on immunosuppression. Canine distemper and ehrlichiosis were diagnosed based on epidemiologic data, clinical signs, hematological and cytological evaluation. Toxoplasma gondii was isolated and genetically characterized as Type I using restriction analysis (RFLP) with SAG-2 genes. Immunosuppression features of both dogs and human beings are discussed, as well as implications on animal and public health. This is the first report on toxoplasmosis, ehrlichiosis and distemper co-infection in a dog in Brazil, associated with genotyping determination of the T. gondii strain involved.

Expression of pro-and-anti-apoptotic antigens in the cerebellum of dogs naturally infected with canine distemper virus

Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The present work investigated apoptosis in white and gray matter (granular layer) in the cerebellum of naturally infected dogs by the analysis of the expression of the pro-apoptotic antigens caspase - 2 and - 3, b(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL-staining) positivity, annexin-V immunodetection, and the presence of the anti-apoptotic antigens, BCl-2 and p53. Cerebellum specimens were obtained from the Laboratory of Animal Pathology, from 1995 to 2009, and the 5-μm thick fragments were stained both with hematoxylin-eosin and Shorr. All samples were diagnosed as positive for CDV genome by reverse transcriptase polymerase chain reaction targeting the nucleocapsid gene. The anti-apoptotic pathways evidenced in this study were BCl-2 and p53 proteins that were intensively detected in cerebellum of CDV positive slides (40-80% of labeled cells/mm2). In addition, the apoptosis markers annexin-V and TUNEL are directly correlated among the same samples (80 and 40% of labeled cells, respectively). This is the first description of p53 and annexin-V expression, characterized as anti-apoptotic and apoptotic proteins, involvement in canine natural cases of CDV infections.

A immunosensor for the diagnosis of canine distemper virus infection using SPR and EIS

Canine distemper virus (CDV) is a viral disease that affects dogs and many other carnivores. Clinical diagnosis of CDV is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Laboratory analysis is required to diagnose suspected cases. In this study, surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) methodologies were developed for the detection of canine distemper virus simultaneously. The assay exhibited high specificity, as all the negative controls were not mistakenly detected. The CDV concentration was determined from successive injections into the apparatus, with a linear range from 1.1 to 116.0 ng mL-1. The system exhibited good reproducibility with 4.5% variation between runs after regeneration of the coated surface with a solution of 0.1 M glycine-HCL (pH 3.0). The capacitance and resistance values of the modified interface were calculated from EIS data using an equivalent circuit. It was possible to measure CDV in highly concentrated viruses with good specificity and reproducibility. © 2013 The Royal Society of Chemistry.

A fast and highly sensitive method for the detection of canine distemper virus by the naked eye

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Protein A in Detection of Canine Distemper Virus through immunosensor construction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Canine distemper virus and Toxoplasma gondii co-infection in dogs with neurological signs

O presente estudo relata a ocorrência de co-infecção entre o vírus da cinomose canina (CDV) e Toxoplama gondii em cães com sinais neurológicos. Amostras de soro e tecido nervoso (pos-mortem) de 21 cães, suspeitos de cinomose canina foram analisadas pela Reação de Imunofluorecência indireta (RIFI) para pesquisa de anticorpos contra T. gondii e N. caninum e por RT-PCR para CDV. Dezessete (80,9%) cães foram positivos para o CDV pela RT-PCR e 8 (38,1%) foram positivos para anticorpos contra T. gondii. Sete cães (41,1%) apresentaram-se positivos para ambos agentes, caracterizando processo de co-infecção. Somente 1 (4,7%) cão foi soropositivo para N. caninum (RIFI=100), entretanto este mesmo animal foi positivo para T. gondii (RIFI=4096) e para CDV (RT-PCR).