Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

No description available.

Zusammenfassung Zur Freisetzung ihres Genoms in das Innere der Wirtszelle müssen Hüllviren ihre Membran mit der Membran der Wirtszelle verschmelzen. Diese Fusion wird durch eine Konformations- umwandlung der Ektodomäne viraler Glykoproteine ausgelöst. Die Kenntnis der drei- dimensionalen Struktur der vollständigen Ektodomäne in der fusionsaktiven Konformation ist Voraussetzung für das Verständnis des Fusionsmechanismus. Die Fusion von Influenza mit entsprechenden Targetmembranen wird durch eine durch sauren pH ausgelöste Konformationsänderung des als Trimer vorliegenden Glykoproteins Hämagglutinin (HA) vermittelt. Bis jetzt war die dreidimensionale (Röntgenkristall-) Struktur einer enzymatisch abgespaltenen HA-Ektodomäne nur eines Influenzastammes bei neutralem pH-Wert bzw. von einigen Fragmenten der HA2-Untereinheit bei saurem pH-Wert bekannt. In der vorliegenden Arbeit wurden ein geeigneter Influenzastamm sowie geeignete Untersuchungsbedingungen ermittelt, um die 3D-Struktur des kompletten, nicht enzymatisch vorbehandelten HA sowohl in seiner nativen (bei neutralem pH-Wert vorliegenden) als auch in seiner fusionskompetenten (keinesfalls aber bereits inaktivierten) Struktur mittels Kryo- Elektronenmikroskopie und Bildverarbeitung aufzuklären. Es wurde erstmals die 3D-Struktur des kompletten HA eines anderen Influenzastammes (A/Japan) bei neutralem pH-Wert aufgeklärt und mit der bekannten 3D-Struktur von Influenza X-31 verglichen. Außerdem konnte eine fusionskompetente Form rekonstruiert werden, die im Vergleich zur nativen Konformation deutliche Veränderungen in der 3D-Struktur zeigt, ohne daß sich jedoch die Assoziation der Monomere aufhob. Die Befunde werden unter anderem in Hinblick auf die Bedeutung der HA1-Untereinheit für die Fusion diskutiert. Die vorgestellte Methode scheint geeignet, auch andere Membranproteine bzw. Membranfusion-vermittelnde Proteine in verschiedenen konformeren Zuständen aufzuklären (insbesondere jene, die der Röntgenkristallstrukturanalyse nicht zugänglich sind) und so einen diesen Fusionsprozessen eventuell zugrundeliegenden konservierten Funktionsmechanismus aufzuhellen. / Abstract Envelope viruses enter a host cell via fusion between the viral and endosomal membrane, thereby releasing the nucleocapsid into the cytoplasm. The fusion has been accompanied with an conformational change in the ectodomain of viral glycoproteins. To understand the mechanism leading to fusion the three-dimensional (3D) structure of the complete viral glycoprotein in its fusion competent conformation has to be determined. The Fusion of influenza virus which is triggered at low pH has been associated with an irreversible conformational change in the trimeric glycoprotein hemagglutinin (HA). The three-dimensional (crystal) structure is already known of the enzymatic-cleaved ectodomain of one influenza strain (X-31) at neutral pH or of some fragments of the HA2-subunit at low pH, respectively. In this work a suitable influenza strain as well as suitable experimental conditions for investigations of the 3D-structure of the complete and not enzymatically treated HA at neutral and acidic pH conditions have been determined. An cryo-microscopy/angular reconstitution approach has been employed for the 3D- reconstruction of the intact HA of an different influenza strain (A/Japan). This structure is in excellent agreement with the known X-ray crystallographic structure of the bromelain- cleaved ectodomain of HA from influenza X-31. Moreover, for the very first time the 3D structure of the intact HA of Influenza A/Japan in its fusion competent state (at acidic pH) has been calculated. The differences between the two structures are large compared to the marginal differences between the neutral pH structures by EM and by X-ray crystallography, respectively, although the monomers remain tightly connected. These findings will be discussed especially with regard to the role of the HA1 subunit in the fusion process. The procedure is in general applicable to pursue the 3D-structure of (fusion mediating) proteins in various conformational states (especially of those proteins which are not directly accessible by X-ray crystallography). This approach should offer to elucidate the (eventually conserved) mechanism of membrane fusion.

Es wird angenommen, dass das Influenzavirus-Glykoprotein Hämagglutinin (HA) für seine Funktion sowohl bei der Virusfreisetzung als auch bei der Fusion von viraler und zellulärer Membran mit Cholesterin- und Sphingolipidreichen Domänen, sogenannten Membran-Rafts, assoziiert sein muss. Aus diesem Grund sollte in dieser Arbeit die Membran-Raft-Affinität von HA in lebenden Zellen mittels FLIM-FRET gemessen werden. Dabei wurde mit Hilfe der Fluroreszenz-Lebenszeit-Messung (FLIM) der Förster-Resonanz-Energie-Transfer (FRET) von fluoreszenzmarkiertem HA auf einen etablierten Raft-Marker bestimmt. Diese Messungen zeigten, dass beide Proteine in gemeinsamen Klustern in der Plasmamembran vorkommen. Durch Cholesterinentzug und durch den Einsatz von Cytochalasin D, welches die Mikrofilamente zerstört, konnte diese Klusterbildung reduziert werden. Demnach tragen sowohl die Membran-Rafts als auch das Aktinnetzwerk zu dieser Klusterbildung bei. Mittels FLIM-FRET konnte zusätzlich bestätigt werden, dass die Signale für die Detergenslöslichkeit von HA in Triton-Extraktionsexperimenten, die Palmitylierung und die stark hydrophoben Aminosäuren zu Beginn der Transmembrandomäne (TMD), auch im lebenden System eine wichtige Rolle spielen. Zusätzlich konnten biochemische Experimente zeigen, dass die hydrophoben Aminosäuren zu Beginn der HA-TMD den intrazellulären Transport, nach der Trimerbildung, entscheidend verzögern. Diese Verzögerung ist vermutlich auf einer erschwerten Integration dieser Proteine in die Membran-Rafts begründet. Die virale Fusion mit der Wirtszellmembran wird durch eine pH5-Behandlung vermittelte Konformationsänderung von HA ausgelöst. FLIM-FRET-Messungen zeigten für die pH5-Konformation von HA eine verglichen mit der pH7-Konformation verringerte Klusterbildung mit dem Raft-Marker. Somit ist offensichtlich, dass die Membranfusion-vermittelnde HA-Konformation eine verringerte Raft-Affinität besitzt. Diese verringerte Raft-Affinität könnte eine wichtige Rolle bei der Störung der Lipide an der Fusionsstelle spielen und somit die Bildung und/oder Vergrößerung der Fusionspore erleichtern. / It has been supposed that the hemagglutinin (HA) of influenza virus is recruited to cholesterol- and sphingolipid-enriched domains, also named membrane-rafts, to accomplish its function in virus budding and membrane fusion. This study aimed at verifying the affinity of HA for membrane-rafts in living cells using fluorescence-lifetime imaging microscopy to measure Förster’s resonance energy transfer (FLIM-FRET). FLIM-FRET revealed strong clustering between a fluorescence-tagged HA-protein and a well-established raft-marker in CHO cells. Clustering was significantly reduced when rafts were disintegrated by cholesterol depletion and when microfilaments were disrupted with cytochalasin D. Thus, membrane-rafts as well as the actin meshwork contribute synergistically to clustering. Clustering was also reduced by the removal of the known signals for the association of HA with detergent-resistant-membranes, the palmitoylation and the first amino acids in the transmembrane region (TMR). Since these mutations are obviously important for the raft-association of HA their function during the transport through the ER and the Golgi-complex was studied. These investigations showed that the exchange of the first three amino acids of the HA-TMR led to a decelerated transport after trimer-formation of the protein, probably due to retarded integration of these proteins into membrane-raft domains. Mediating viral fusion with the host cell membrane requires an irreversible conformational change of HA. FLIM-FRET studies of this low pH conformation unveiled that the clustering with the raft-marker is decisively reduced compared to the pre-fusion conformation of the protein. It might be assumed that the fusion-mediating conformation of HA reduces the proteins affinity for membrane-rafts. Therefore it is likely that this reduced affinity for rafts after the conformational change is relevant to cause perturbation of lipids at the fusion site and thereby facilitating the formation and/or enlargement of the fusion pore.

Résumé: Chaque année, les épidémies saisonnières d’influenza causent de 3 à 5 millions de cas sévères de maladie, entraînant entre 250 000 et 500 000 décès mondialement. Seulement deux classes d’antiviraux sont actuellement commercialisées pour traiter cette infection respiratoire : les inhibiteurs de la neuraminidase, tels que l’oseltamivir (Tamiflu) et les inhibiteurs du canal ionique M2 (adamantanes). Toutefois, leur utilisation est limitée par l’apparition rapide de résistance virale. Il est donc d’un grand intérêt de développer de nouvelles stratégies thérapeutiques pour le traitement de l’influenza. Le virus influenza dépend de l’activation de sa protéine de surface hémagglutinine (HA) pour être infectieux. L’activation a lieu par clivage protéolytique au sein d’une séquence d’acides aminés conservée. Ce clivage doit être effectué par une enzyme de l’hôte, étant donné que le génome du virus ne code pour aucune protéase. Pour les virus infectant l’humain, plusieurs études ont montré le potentiel de protéases à sérine transmembranaires de type II (TTSP) à promouvoir la réplication virale : TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 et matriptase, identifiée récemment par notre équipe (Beaulieu, Gravel et al., 2013), activent l’HA des virus influenza A (principalement H1N1 et H3N2). Toutefois, il existe peu d’information sur le clivage de l’HA des virus influenza B, et seulement TMPRSS2 et HAT ont été identifiées comme étant capables d’activer ce type de virus. Les travaux de ce projet de maîtrise visaient à identifier d’autres TTSP pouvant activer l’HA de l’influenza B. L’efficacité de clivage par la matriptase, hepsine, HAT et Desc1 a été étudiée et comparée entre ces TTSP. Ces quatre protéases s’avèrent capables de cliver l’HA de l’influenza B in vitro. Cependant, seul le clivage par matriptase, hepsine et HAT promeut la réplication virale. De plus, ces TTSP peuvent aussi supporter la réplication de virus influenza A. Ainsi, l’utilisation d’un inhibiteur de TTSP, développé en collaboration avec notre laboratoire, permet de bloquer significativement la réplication virale dans les cellules épithéliales bronchiques humaines Calu-3. Cet inhibiteur se lie de façon covalente et lentement réversible au site actif de la TTSP par un mécanisme slow tight-binding. Puisque cet inhibiteur cible une composante de la cellule hôte, et non une protéine virale, il n’entraîne pas le développement de résistance après 15 passages des virus en présence de l’inhibiteur dans les cellules Calu-3. L’inhibition des TTSP activatrices d’HA dans le système respiratoire humain représente donc une nouvelle stratégie thérapeutique pouvant mener au développement d’antiviraux efficaces contre l’influenza. / Abstract: Seasonal influenza epidemics cause between 3 and 5 millions severe cases of disease, leading to 250 000 to 500 000 deaths worldwide. Only two classes of drugs are currently available to treat influenza infections: neuraminidase inhibitors, such as oseltamivir (Tamiflu) and M2 channel inhibitors (adamantanes). However, the use of these antivirals is restricted by rapid emergence of viral resistance. It is therefore of great interest to develop new therapeutic strategies for the treatment of influenza disease. The influenza virus requires activation of its surface protein hemagglutinin (HA) to become infectious. This activation is achieved by proteolytic cleavage in a highly conserved amino acid sequence of the protein. Host cell proteases are responsible for this cleavage since the viral genome doesn’t encode any protease. For viruses that infect humans, many studies have shown the potential of type II transmembrane serine proteases (TTSP) to promote viral replication: TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 and matriptase, recently identified by our team (Beaulieu, Gravel et al., 2013), activate HA of influenza A viruses (mainly H1N1 and H3N2). However, little is known about cleavage of influenza B virus HA, and only TMPRSS2 and HAT have been identified as being capable of activating this type of virus. This project aimed to identify other TTSPs able to activate influenza B HA. Cleavage efficacies of matriptase, hepsin, HAT and Desc1 were studied and compared. These four proteases were shown to be able to cleave influenza B HA using in vitro assays. However, only cleavage by matriptase, hepsin and HAT promoted viral replication. Moreover, these TTSPs also supported the replication of influenza A viruses. Thus, the use of a slow, tight-binding inhibitor (developed in collaboration with our laboratory) that binds to the TTSP active site, forming a covalent and reversible bond, significantly blocked viral replication in human bronchial epithelial Calu-3 cells. Since this inhibitor targets a host cell component, instead of a viral protein, viruses did not develop resistance after 15 passages in presence of the inhibitor in Calu-3 cells. Thus, inhibition of HA-activating TTSPs in the human respiratory tract represents a novel therapeutic strategy against influenza.

Coronavírus bovino (BCoV) é o agente causador de doença, tanto entérica como respiratória em bovinos, mas até agora existem controvérsias sobre a relação genealógica entre as amostras de BCoV em diferentes tecidos. Neste estudo, amostras de fezes e secreções nasais de 14 vacas de um mesmo rebanho apresentando simultaneamente disenteria epizoótica e doença respiratória foram estudados quanto a presença de BCoV. As amostras virais detectadas tiveram tanto o gene de espícula (S) como o gene hemaglutinina-esterase (HE) parcialmente sequenciados. Para o gene HE, foram obtidas 12 sequências de secreções nasais e 12 de amostras de fezes e para o gene S, foram obtidas 14 sequências de secreções nasais e 12 de amostras de fezes, com 100% de identidade nucleotídica para cada gene para as amostras deste estudo. Estes resultados apresentam algumas divergências com estudos anteriores os quais relatam que linhagens diferentes de BCoV podem ser esperados em casos de disenteria e doença respiratória em vacas, pois linhagens com sequências idênticas dos genes S e HE podem não mostrar diferenças em relação tropismo pelos diferentes tecidos. Sequências completas de duas amostras brasileiras de BCoV mostram que o já descrito padrão filogeográfico baseado no sequenciamento do gene S parcial foi mantido, foram encontradas substituições de aminoácidos específicos. / Bovine coronavirus (BCoV) is the causative agent of both enteric and respiratory disease in cattle, but hitherto there were some controversy on the genealogic relationship amongst strains from these different tissues. In this study, samples of feces and nasal secretions of 14 cows from a same herd simultaneously presenting epizootic dysentery and respiratory disease were screened for BCoV and the strains detected had both the spike (S) and hemagglutinin-esterase (HE) genes partially sequenced. For HE gene, 12 sequences from nasal secretions and 12 from fecal samples were obtained and for S gene, 14 sequences from nasal secretions and 12 from fecal samples were obtained, with 100% nucleotide identities for each gene for the strains of this study. These results have some disagreements with previous reports which try to put forward that divergent BCoV strain should be expected in cases of dysentery and respiratory disease in cows, showing that strain with identical S and HE sequences might show no differences in tropisms. Complete S gene sequences of two Brazilian BCoV strains show that the already described phylogeographic pattern based on partial S gene is sustained, though specific amino acids subtitutions are found.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-10-18T12:42:59Z No. of bitstreams: 1 DISS_2014_Leticya Lerner Lopes.pdf: 2057200 bytes, checksum: 5e81c161d271f2cc49db59d9720875b7 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-11-07T15:49:22Z (GMT) No. of bitstreams: 1 DISS_2014_Leticya Lerner Lopes.pdf: 2057200 bytes, checksum: 5e81c161d271f2cc49db59d9720875b7 (MD5) / Made available in DSpace on 2017-11-07T15:49:22Z (GMT). No. of bitstreams: 1 DISS_2014_Leticya Lerner Lopes.pdf: 2057200 bytes, checksum: 5e81c161d271f2cc49db59d9720875b7 (MD5) Previous issue date: 2014-02-28 / CAPES / O vírus da cinomose canina (VCC) é um vírus RNA, que pertence ao gênero Morbillivírus e família Paramyxoviridae. A capacidade de resposta imune, assim como sua virulência são fatores críticos para a invasão viral dos tecidos epiteliais e do sistema nervoso central (SNC). O VCC é o maior responsável pelas encefalites em cães, acometendo diversas idades. O objetivo desse estudo foi detectar o VCC nos cães com sinais neurológicos encaminhados para necropsia no Laboratório de Patologia Veterinária da Universidade Federal de Mato Grosso (LPV-UFMT). Durante um período de um ano, 85% (68/80) dos cães necropsiados tinham lesões microscópicas compatíveis com encefalomielite causada pelo VCC. Desses, 67.6% (46/68) foram confirmadas positivas através da imuno-histoquímica (IHQ). Microscopicamente, as lesões do SNC foram classificadas em encefalite desmielinizante aguda em 15.2% (7/46) dos cães, em subaguda em 73.9% (34/46) e crônica em 10.8% (5/46) dos cães. O cerebelo foi principal órgão a apresentar marcação positiva na IHQ (97.8%). O VCC é responsável pelos sinais neurológicos em cães principalmente abaixo de um ano de idade. A cinomose demonstrou sua relevância dentro da população canina de Cuiabá, sendo necessário ao nosso entendimento, caracterizar a estirpe viral relacionada à região. / Canine distemper virus (CDV) is a RNA virus classified under the genus Morbillivirus within the family of Paramyxoviridae. The time of onset of the immune response and, likely, also the virulence of the virus are critical factors in the extent of viral invasion of epithelial tissues and of the central nervous system. The CDV is the most responsible of encephalitis in dogs from different ages. In this study, the aim was to detected CDV in dogs with neurologicals signs referred for necropsy at the Laboratory of Veterinary Pathology, Federal University of Mato Grosso (LPV-UFMT).Over a period of 1 year, 85% (68/80) of the dogs necropsied had microscopic lesions compatible encephalomyelitis by CDV. Which 67.6% (46/68) were confirmed by immunohistochemistry (IHC). Microscopically, the CNS lesions were classified demyelinating encephalitis in 15.2% (7/46) to acute, 73.9% (34/46) in subacute and 10.8% (5/46) to chronic. The cerebellum (97.8%) was the main target organ to verify positivity in the IHC. Canine distemper virus is a pathogen responsible for neurological clinical signs in dogs mainly under one year of age. Distemper demonstrated its relevance within the canine population of Cuiabá, being necessary to our understanding, characterizing the viral strain related to the region.