L’empaquetage des huit segments du génome des virus influenza de type A est une des étapes clef du cycle viral. Il intervient également dans l’apparition de virus réassortants, les virus pandémiques par exemple, ce qui en fait un enjeu fondamental de la recherche actuelle.Nous avons étudié ce mécanisme au cours de deux études, la première portant sur les vaccins antigrippaux (réassortiment), la seconde visant à construire un virus vecteur (incorporation d’un segment hétérologue). Les semences vaccinales sont obtenues par co-infection d’oeufs de poule embryonnés avec deux souches virales une donneuse (souche circulante de référence) et une accepteuse (A/Puerto Rico/8/34 (H1N1) (PR8)). L'analyse de la composition génétique de treize semences vaccinales H3N2 montre que le segment PB1 de la souche donneuse est présent dans plus de 50 % des semences analysées et qu’une grande variété de réassortants,allant de 6:2 à 2:6 (PR8:H3N2), peut résulter de ces coinfections. Des expériences de compétition d'encapsidation de segments à l’aide de la génétique inverse révèlent que l'encapsidation sélective du segment PB1 dépend de son environnement génétique notamment l’origine virale des segments HA et NA. La seconde partie de mon travail de thèse a été consacrée à la construction d’un vecteur réplicatif sur la base d’un virus influenza H3 naturel sans segment NA. Aucune des constructions contenant le transgène gfp n’a été incorporée dans les particules virales, contrairement à ce qui a été décrit dans la littérature. Bien que les mécanismes moléculaires régissant l’incorporation des segments des virus influenza A demeurent très complexes, le fond génétique semble être déterminant pour ce processus. / The packaging of the eight segments corresponding to the influenza A viruses genomeis a key process of the viral replication as well as a stake of actual scientific researchesbecause it leads to reassortant viruses, e.g. pandemic viruses. We studied the two main facetsof influenza segment packaging: reassortment, during vaccine seeds production and foreignsegment incorporation for influenza vector construction. Vaccine seeds are produced bycoinfection of hens’ eggs with two viruses, a donor one (reference circulating strain) and anacceptor one (A/Puerto Rico/8/34 (H1N1) (PR8)). Analysis of internal genetic composition ofthirteen H3N2 vaccine seeds reveals that PB1 segment of H3N2 donor strain is incorporatedin more than fifty per cent of the cases. Moreover, coinfection events lead to an extremelywide range of reassortants from 6:2 to 2:6 (PR8:H3N2). Segment incorporation competitionassays performed using plasmid-based reverse genetics show that selective packaging of PB1segment is based on genetic environment, i.e. viral origin of HA and NA segments. Thesecond part of my PhD work has been devoted to replicative influenza vector based on H3virus isolated from patients without NA segment at the native stage. None of the gfptransgenic constructions containing reporter gene have been incorporated in viral particles,contrary to literature studies performed using H1N1 laboratory-adapted strains. Even ifmolecular mechanisms controlling influenza A viruses segments incorporation remain stillcomplex, genetic background seems to be an essential element which must be considered withinterest.

Papilomavírus (PVs) são vírus amplamente estudados, sendo enfatizada sua capacidade de infectar os tecidos epitelial e mucoso em diversos animais, incluindo humanos, causando lesões benignas que podem, ocasionalmente, resultar em câncer. Dentre os gêneros que infectam animais, os Deltapapillomavirus têm uma importância veterinária, ecológica e histórica, pois são capazes de infectar seu hospedeiro natural e outros animais. Por isso, esse trabalho contempla todos os tipos virais pertencentes aos Delta-PVs, incluindo sua história. Dentre as proteínas traduzidas pelos PVs, três são consideradas proteínas oncogênicas: E5, E6 e E7. Determinar a estrutura de uma proteína é crucial para a elucidação da sua função, possibilitando aplicações nas áreas de engenharia de proteínas, anotação genômica e desenho racional de fármacos. A estrutura tridimensional da proteína E6 de cada tipo viral pertencente ao gênero Deltapapillomavirus foi determinada por modelagem molecular por homologia. A história evolutiva dessas proteínas foi avaliada com base na geração de árvores filogenéticas e suas propriedades físico-químicas foram analisadas. Além disso, devido ao seu alto grau de conservação, a E6 demonstrou ser útil como um marcador molecular. Apesar de serem consideradas raras, foram observadas lesões papilomatosas em carneiros em uma fazenda do estado de São Paulo. Foi realizado o diagnóstico molecular dessas lesões. Os resultados mostraram pela primeira vez no mundo que, apesar de serem ovinos, o agente causador da papilomatose era um papilomavírus bovino, o BPV2, um Delta-PV. Logo, além de discutir os Delta-PVs, esta tese demonstra na prática a habilidade desse gênero em romper a barreira espécie-específica. / Papillomaviruses (PVs) are widely studied viruses, emphasizing their ability to infect the epithelial and mucosal tissues in several animals, including humans, causing benign lesions that may occasionally result in cancer. Among the genera that infect animals, Deltapapillomaviruses have a veterinary, ecological and historical importance because they are capable of infecting their natural host and other animals. Therefore, this work contemplates all viral types belonging to the Delta-PVs, including their history. Among the proteins translated by the PVs, three of them are considered oncogenic proteins: E5, E6 and E7. Determining the structure of a protein is crucial to the elucidation of its function, allowing applications in the areas of protein engineering, genomic annotation and rational design of drugs. The three-dimensional structure of the E6 protein of each viral type belonging to the genus Deltapapillomavirus was determined by molecular modeling by homology. The evolutionary history of these proteins was evaluated based on the generation of phylogenetic trees and their physicochemical properties were analyzed. In addition, due to its high degree of conservation, E6 has been shown to be useful as a molecular marker. Despite being considered rare, papillomas lesions were observed in sheep on a farm in the state of São Paulo. The molecular diagnosis of these lesions was performed. The results showed for the first time in the world that, despite being ovines, the causative agent of papillomatosis was a bovine papillomavirus, BPV2, a Delta-PV. Thus, in addition to discussing Delta-PVs, this thesis demonstrates in practice the ability of this genre to break the species-specific barrier.

<div>The family Flaviviridae includes important genera such as flavivirus and hepacivirus which comprise significant human pathogens that affect hundreds of millions annually. The understanding of these viruses, the viral life cycle, and pathogenicity is vital when it comes to developing therapeutics. Flavivirus virions undergo major conformational rearrangements during the life cycle, including the assembly and maturation steps. In order to create a reagent to investigate these processes, luminescent reporter viruses have been constructed. Luminescent reporter tags have yet to be incorporated into the structural proteins of dengue virus (DENV) without significantly affecting replication or infectivity and successful tagging would allow for targeted studies examining access to specific structural epitopes. Engineering tags in DENV structural proteins is particularly difficult because most reporter tags involve large insertions which may create steric hindrance and inhibit proper protein folding. However, the reporter system described here, developed by Promega, is much smaller than a full-size luciferase protein. It involves an eleven amino acid subunit (HiBiT) tagged to a viral protein that creates measurable luminescence when incubated with the larger subunit (LgBiT). Using the structure of the virion as a guide, the HiBiT reporter tag was incorporated into the structural region of the DENV genome including sites in capsid (C) as well as the glycoproteins membrane (M) and envelope (E). Resulting recombinant viruses were characterized and tag sites within the C protein membrane anchor as well as the transmembrane domain of M protein were found to tolerate HiBiT insertion and produce infectious particles. The recombinant virus possessing HiBiT in C protein was found to be stable over three rounds of serial passaging while virus containing the M protein tag site was found to be unstable. HiBiT activity of the capsid tagged virus was also found to directly correlate with purified infectious particles, suggesting the capsid membrane anchor may remain associated with the virus even after polyprotein processing. Additionally, insert composition was found to be a key determinant for the production of infectious virus. The lessons learned from engineering HiBiT in the DENV system were then applied to hepatitis C virus (HCV). </div><div>The highly lipophilic and pleiomorphic nature of HCV has made structural studies particularly difficult. However, by constructing multi-tagged reporter viruses containing both HiBiT and various purification tags, researchers will save time and resources in preparation for structural studies which are vital for vaccine development. In this study, HiBiT was incorporated into sites within HCV previously shown to tolerate tags of various sizes. Different insert compositions were engineered within the genome and the construct containing both FLAG and HiBiT tags within the N-terminus of E2 yielded highly infectious and quantifiable, luminescent virus. The recombinant HCV containing FLAG and HiBiT displayed similar peak titer as compared to WT while also demonstrating HiBiT activity. Furthermore, the FLAG peptide was found to be partially surface exposed and capable of being used for virus purification purposes. The multi-tagged reporter virus characterized in this study provides a robust platform for quantification and purification of HCV, two facets of research that are critical for the determination of viral structure via cryo-EM and other imaging techniques. The findings from both the DENV and HCV studies provide a robust foundation for future tagging of viruses within the family Flaviviridae and offer insight on the structural proteins that compose the virion.</div>

The objective of this study was to identify possible roles of the class II antigen-associated, electrophoretically invariant chain (Ii) in class II antigen functions through analysis of the kinetics of synthesis, processing, and turnover of Ii in various cellular systems of Ii hyperexpression. Using [35S]methionine metabolic labeling of microsomal membrane proteins and analysis in one-dimensional SDS polyacrylamide gradient gels and two-dimensional electrophoretic gels, enhanced expression of Ii was demonstrated in leukemic cells of a subset of patients with hairy cell leukemia (HCL), in normal peripheral blood cells freshly transformed with EBV, and in Burkitt's lymphoma cell lines treated with n-butyrate. As part of an initial effort to identify leukemic cell subset-defining, membrane-associated molecules which might then be tested as targets for, or regulators of, the anti-leukemic cell immune response, subsets of HCL patients were identified on the basis of leukemic cell enhanced expression of 35,000 and 15,000 dalton proteins (p35 and p15). A similar enhanced expression of the p35 molecule was demonstrated in peripheral blood or cord blood lymphocytes freshly transformed with Epstein-Barr virus (EBV), as compared to long-term cultured EBV cell lines. Further structural characterization of the HCL p35 by one-dimensional SDS-PAGE and two-dimensional gel electrophoresis of HCL total microsomal membrane proteins, anti-class II antigen immunoprecipitates, and anti-Ii immunoprecipitates showed that the HCL p35 molecule was the human class II antigen-associated Ii chain. In order to determine the functional significance of altered Ii expression on class II antigen function, the relative kinetics of Ii synthesis, processing, and turnover was examined in an in vitro model system of regulated Ii synthesis. Treatment of the Burkitt's lymphoma cell line, Jijoye, and its class II antigen-negative, mutant, daughter cell line, P3HR-1, with 4 mM n-butyrate for 48 hr, enhanced the rate of synthesis of the Ii chain. One-dimensional SDS-PAGE and two-dimensional gel, electrophoretic analysis of anti-Ii and anti-class II antigen immunoprecipitates isolated from pulse-/pulse-chase-/or continuously labeled, control and butyrate-treated P3HR-1 and Jijoye cells demonstrated enhanced levels of synthesis of the dominant, most basic form of Ii in the butyrate-treated samples (to a greater degree in Jijoye cells). The butyrate enhancement of Ii synthesis occurred in the presence or absence of detectable class II alpha and beta chains, as did the relative turnover, although the terminal processing of Ii to an electrophoretically slower and more acidic form was dependent upon its association with class II antigens. The level of the dominant, most basic form of Ii expressed in the hairy leukemic cells equaled that in butyrate-treated Jijoye cells. However, hairy leukemic cell, class II antigen-associated Ii was not terminally processed. The results of these experiments were consistent with the following. In lymphoblastoid cells, two pathways for Ii turnover might exist. One is through association with class II antigen complexes and progressive carbohydrate-chain terminal processing, and a second is not associated with class II antigens and such processing. Class II antigens may direct the processing of Ii towards some function (rather than vice versa). Butyrate treatment rather uniquely enhances the synthesis of Ii in some lymphoid cells in the presence or absence of class II antigens. Hairy leukemic cells demonstrate an inability to terminally process Ii which might be relevant to the function of Ii and/or class II antigens on those cells.

No description available.

In the infection process, native poliovirus (160S) first converts to a cell-entry intermediate (135S) particle, which causes the externalization of capsid proteins VP4 and the N-terminus of VP1 (residues 1-53). The externalization of these entities is followed by release of the RNA genome, leaving an empty (80S) particle. Three antibodies were utilized to track the location of VP1 residues in different states of poliovirus by cryogenic electron microscopy (cryo-EM). "P1" antibody binds to N-terminal residues 24-40 of VP1. Three-dimensional reconstruction of 135S-P1 showed that P1 binds to a prominent capsid peak known as the "propeller tip". In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, ~60 Å distant, at the icosahedral twofold axes. Analysis of 80S-P1 reconstructions showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips; only at the twofold axes; or simultaneously at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions. "C3" antibody binds to 93-103 residues (BC loop) of VP1. The C3 epitope shifts outwards in radius by 4.5% and twists through 15° in the 160S-to-135S transition, but appears unchanged in the 135S-to-80S transition. In addition, binding of C3 to either 160S or 135S particles causes residues of the BC loop to move an estimated 5 (±2) Å, indicating flexibility. The flexibility of BC loop may play a role in cell-entry interactions. At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and the N-terminus of VP1 externalize reversibly. An antibody, binding to the residues 39-55 of VP1, was utilized to track the location of the N-terminus of VP1 in 160S particle in the "breathing" state. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered 135S particle, but the N-terminus of VP1 is located near the twofold axes, instead of the propeller tip as in 135S particles.

No description available.

Viruses have huge impact on controlling diseases and regulating many key ecosystem processes. As metagenomic data can contain many microbiomes including many viruses, by analyzing metagenomic data we can analyze many viruses at the same time. The first step towards analyzing metagenomic data is to identify and quantify viruses present in the data. In order to answer this question, we developed a computational pipeline, FastViromeExplorer. FastViromeExplorer leverages a pseudoalignment based approach, which is faster than the traditional alignment based approach to quickly align millions/billions of reads. Application of FastViromeExplorer on both human gut samples and environmental samples shows that our tool can successfully identify viruses and quantify the abundances of viruses quickly and accurately even for a large data set. As viruses are getting increased attention in recent times, most of the viruses are still unknown or uncategorized. To discover novel viruses from metagenomic data, we developed a computational pipeline named FVE-novel. FVE-novel leverages a hybrid of both reference based and de novo assembly approach to recover novel viruses from metagenomic data. By applying FVE-novel to an ocean metagenome sample, we successfully recovered two novel viruses and two different strains of known phages. Analysis of viral assemblies from metagenomic data reveals that viral assemblies often contain assembly errors like chimeric sequences which means more than one viral genomes are incorrectly assembled together. In order to identify and fix these types of assembly errors, we developed a computational tool called VirChecker. Our tool can identify and fix assembly errors due to chimeric assembly. VirChecker also extends the assembly as much as possible to complete it and then annotates the extended and improved assembly. Application of VirChecker to viral scaffolds collected from an ocean meatgenome sample shows that our tool successfully fixes the assembly errors and extends two novel virus genomes and two strains of known phage genomes. / Doctor of Philosophy / Virus, the most abundant micro-organism on earth has a profound impact on human health and environment. Analyzing metagenomic data for viruses has the beneFIt of analyzing many viruses at a time without the need of cultivating them in the lab environment. Here, in this dissertation, we addressed three research problems of analyzing viruses from metagenomic data. To analyze viruses in metagenomic data, the first question needs to answer is what viruses are there and at what quantity. To answer this question, we developed a computational pipeline, FastViromeExplorer. Our tool can identify viruses from metagenomic data and quantify the abundances of viruses present in the data quickly and accurately even for a large data set. To recover novel virus genomes from metagenomic data, we developed a computational pipeline named FVE-novel. By applying FVE-novel to an ocean metagenome sample, we successfully recovered two novel viruses and two strains of known phages. Examination of viral assemblies from metagenomic data reveals that due to the complex nature of metagenome data, viral assemblies often contain assembly errors and are incomplete. To solve this problem, we developed a computational pipeline, named VirChecker, to polish, extend and annotate viral assemblies. Application of VirChecker to virus genomes recovered from an ocean metagenome sample shows that our tool successfully extended and completed those virus genomes.

Aujourd’hui, le Virus de l'Hépatite C (VHC) infecte plus 70 millions de personnes dans le monde. L’Organisation mondiale de la santé prévoit l’élimination du virus VHC d’ici 2030, grâce aux récentes découvertes dans le milieu du développement médical. Ces derniers ont conduit à la production des antiviraux pangenotypiques à action directe (ADD). Le VHC est un virus enveloppé de l’ARN, avec une polarité positive. Il est constitué de nucléocapside entouré d’une membrane lipidique. La nucléocapside contient l’acide ribonucléique (ARN) et la protéine core. La membrane lipidique quant à elle contient à la surface les glycoprotéines E1 et E2. Ainsi ces protéines, sont les premières à rencontrer les hépatocytes, c’est donc grâce à elles que le virus parvient à entrer dans les cellules. Parmi les deux protéines, l’E2 a été la mieux caractérisée pour ses fonctions de liaisons aux récepteurs spécifiques. De plus les anticorps neutralisants ciblent majoritairement cette protéine. En se basant sur le fait que ce virus est membre de la famille des Flaviviridae, il a été suggéré par analogie, que le VHC contient des protéines de fusion de classe II et que la protéine E2 est la protéine de fusion. Cependant, les structures cristallines récentes d’E2 ont révélé qu'il lui manquait les caractéristiques structurelles des protéines de fusion de classe II. Ainsi, tous les regards se sont tournés sur la glycoprotéine E1, suggérant qu’elle est responsable de l’étape de fusion, seule ou à l’aide d’E2. En effet, la partie N-terminale de l'ectodomaine E1 a été récemment cristallisée. La caractérisation des résidus conservés dans cette région a démontré son importance pour l'infectivité du virus, pour l'interaction entre E1 et E2, ainsi que pour son implication dans l'interaction avec les récepteurs du VHC. En soutenant le rôle potentiel d'E1 dans le processus de fusion, différents segments de l'extrémité C-terminale de l'ectodomaine seraient impliqués dans les interactions avec les membranes modèles. Nous avons étudié en particulier deux régions d’intérêt. La première située dans la zone du peptide de fusion putatif (PFP) entre les acides aminés 270 et 291. Cette région se compose des séquences hydrophobes, soutenant son implication dans l'étape de fusion. La deuxième région englobant les acides aminés 314-342, d’une activité membranotrope située à proximité de la zone transmembranaire d’E1, a été démontrée par la cristallographie aux rayons X et les études de RMN comme comprenant deux hélices α (α2 et α3).Nous avons introduit 22 mutations dans la partie C-terminale de l'ectodomaine E1 dans le contexte d'un clone infectieux JFH1. Nous avons remplacé les résidus les plus conservés par de l'alanine, puis analysé l'effet des mutations sur le cycle de vie du virus. Vingt des vingt-deux mutants ont été atténué ou ont perdu leur pouvoir infectieux, ce qui indique leur importance dans le cycle viral. Nous avons observé différents phénotypes; certaines mutations ont modulé la dépendance du virus vis-à-vis des récepteurs CLDN1 et SRBI pour l’entrée cellulaire. Plusieurs mutations dans la région PFP, ont affecté la sécrétion et l'assemblage du virus, ainsi que l'hétérodimérisation E1E2. D’autres mutations, telles que les mutations de l'hélice α2 ont entraîné une atténuation grave ou une perte complète d'infectivité, sans affecter le repliement d’E1 et E2, ni la morphogenèse virale. Une caractérisation plus poussée de certains mutants au sein de la région hélice α2 a suggéré l'implication de cette région dans une étape tardive de l'entrée du VHC. Enfin, nos résultats montrent le rôle important joué par la glycoprotéine E1 dans l'hétérodimérisation de E1E2, la morphogenèse du virus, ainsi que son interaction avec les récepteurs du VHC et son implication potentielle dans l'étape de fusion. / Hepatitis C virus is currently estimated to infect around 71 million people around the world. However, recent advances in drug development led to the generation of pangenotypic direct acting antivirals (DAA), which may make it possible to eliminate HCV by 2030 as planned by the World health organization (WHO). HCV is a small RNA enveloped virus of positive sense. The RNA is encapsidated and surrounded by a lipid bilayer in which the E1 and E2 envelope glycoproteins are anchored on the surface. Thus, E1 and E2 are the first viral proteins to encounter the hepatocytes and mediate the entry step. HCV entry into hepatocytes is a sophisticated process that includes several steps ranging from interaction of glycoproteins with cellular host attachment factors and HCV specific-receptors, which is followed by internalization via clathrin-mediated endocytosis. Finally, viral and endosomal membranes merge at acidic pH leading to the release of viral RNA into the cytoplasm. Among the two glycoproteins, E2 has been the better characterized, as it is responsible for binding to cellular receptors and targeted by neutralizing antibodies. As a member of the Flaviviridae family, it has been suggested by analogy that HCV encodes class II fusion proteins and that E2 is the fusion protein. Nevertheless, the recent crystal structures of E2 revealed that it lacks structural features of class II fusion proteins. Thus, E1 glycoprotein became under the spotlight with the assumption that it is responsible for the fusion step whether alone or with the help of E2. Indeed, the N-terminal part of E1 ectodomain was recently crystallized, and the characterization of conserved residues within this region demonstrated its importance for virus infectivity, E1E2 interaction as well as its involvement in the interplay with HCV receptors. Supporting the potential role of E1 in the fusion process, different segments in the C-terminal of the ectodomain have been reported to be involved in interactions with model membranes. In particular, we investigated two regions of interest. The first one located in the putative fusion peptide (PFP) region between amino acid 270 and 291, containing hydrophobic sequences, supporting its involvement in the fusion step. The second region spanning amino acids 314-342, a membranotropic region located proximal to the transmembrane region of E1 and has been shown by X-ray crystallography and NMR-studies to comprise two α-helices (α2 and α3). We introduced 22 mutations in the C-terminal part of E1 ectodomain in the context of a JFH1 infectious clone. We replaced the most conserved residues with alanine and analyzed the effect of the mutations on the viral life cycle. Twenty out of the 22 mutants were either attenuated or lost their infectivity, indicating their importance for the viral life cycle. We observed different phenotypes; some mutations modulated the dependence of the virus on CLDN1 and SRBI receptors for cellular entry. Most mutations in the PFP region affected virus secretion and assembly as well as E1E2 heterodimerization. Nevertheless, the majority of mutations in the α2-helix (aa 315-324) led to severe attenuation or complete loss of infectivity without affecting E1E2 folding or viral morphogenesis. Further characterization of some mutants within this region suggested the involvement of the α2-helix in a late step of HCV entry. Finally, our results show the important role of E1 played in E1E2 heterodimerization, virus morphogenesis, interaction with HCV receptors and its potential involvement in the fusion step.

O vírus da cinomose canina (VCC) é um patógeno viral, altamente, contagioso que pode causar doença sistémica letal, em cães e outros carnívoros em toda parte do mundo. Os cães afetados podem apresentar sintomas gastrentéricos, respitatórios e nervosos. As manifestações clínicas da doença inclue depressão, diarréia, vómito, desidratação, hiperqueratose dos coxins e focinho e espasmos musculares ou paresia de membros pélvicos, a qual pode persistir por longos períodos. Cães infectados, com sintomas clínicos de VCC, foram estudados para detecção do RNA viral pela técnica de PCR e Nested-PCR. Neste estudo, amplificou-se o gene da nucleoproteína (NP) em células mononucleares do sangue periférico (linfócitos), urina e saliva, de cães infectados com VCC, para detectar o genoma do mesmo, por RT-PCR, em diferentes amostras clínicas. A identificação do RNA viral foi concluída com sucesso, pelo método de RT-PCR, utilizando 2 pares de \"primers\" específicos do gene da nucleoproteína (NP). A técnica de RT-PCR, descrita neste etudo, pode ser um sistema de ensaio útil para determinar se cães suspeitos de infecção, por VCC, tenha níveis detectáveis de genes. Os resultados demonstram que a técnica de RT-PCR é exequível para o diagnóstico laboratorial de cinomose canina. / Canine distemper vírus (CDV) is a highly contagious Viral pathogen which may cause lethal systemic in dogs and other carnivores throughout the world. Affected dogs show gastrointestinal and respiratory clinical slgns, and frequently develop clinical signs in the central nervous system (CNS). Clinical manifestations of the disease include depression, progressive loss of weight, dehydration, hyperkeratosis of the foot pads and nose, nervous symptoms and muscular spasms or posterior paralysis which may perslst for long periods. Infected dogs with clinical symptoms for CDV, were by detection of viral RNA by Polymerase Chaln Reaction (PCR) and Nested PCR. In this study,w e determinebdy the RT-PCRth e presenceo f nucleoprotein (NP) gene in peripheral blood mononuclear cells, urine and saliva from dogs infected with CDV. The goals of this study was to detect CDV renome by RT-PCR in different clinical samples. In this study, Identificatlon of NP mRNA was successfully achieved by using the RT-PCR method with two sets of NP gene specific primers. The RT-PCR technique described in thls study, may provide a useful assay system to determine whether the dogs suspected of CDV infection have detectable leveis of CDV genes. The results demonstrate that RT-PCR technique is rapid, sensitivity and specificity for vírus diagnosis.