RESUMO
The leader of the capsid (LC) protein is exclusive to the Vesivirus genus, and it is needed for successful feline calicivirus (FCV) replication, as well as an efficient apoptosis induction through the mitochondrial pathway. In this work, we aimed to determine if the LC protein from the FCV is a viroporin. Although lacking in a transmembrane domain or an amphipathic helix, the LC protein from the FCV is toxic when expressed in bacteria and it oligomerizes through disulfide bonds, which are both key characteristics of viroporins. An electron microscopy analysis of LC-expressing E. coli cells suggest that the protein induces osmotic stress. Moreover, we found that the previously studied C40A LC mutant, that fails to induce apoptosis and that hinders the replication cycle, also oligomerizes but it has a reduced toxicity and fails to induce osmotic stress in bacteria. We propose that the LC protein is a viroporin that acts as a disulfide bond-dependent antimicrobial peptide, similar to the Ebola virus delta peptide.
Assuntos
Infecções por Caliciviridae , Calicivirus Felino , Animais , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Gatos , Linhagem Celular , Dissulfetos , Escherichia coli/metabolismo , Proteínas ViroporinasRESUMO
Feline calicivirus is among the most common pathogenic microorganisms in upper respiratory tract disease (URTD) and oral lesions of cats. It leads to stomatitis, oral ulceration, ocular and nasal discharge, conjunctivitis, fever, lameness, anorexia, hypersalivation, pneumonia, respiratory distress, coughing, and depression in infected cats. This study aimed to determine the role of Feline calicivirus (FCV) in cats with the upper respiratory tract disease in the Diyarbakir region, Turkey, to provide treatment for infected cats and contribute to the disease prophylaxis. The study material consisted of 10 cats (control group) considered to be healthy according to the clinical examination and 20 cats with URTD that were not vaccinated against Feline calicivirus infection of different breeds, ages, and genders brought to Dicle University Veterinary Faculty Prof. Dr. Servet SEKIN Polyclinic with URTD. After routine clinical examinations of the animals, oral and conjunctival swabs and blood samples were taken. Hematological and biochemical analyzes of blood samples were performed. Swab samples were analyzed by the polymerase chain reaction (PCR) method for the diagnosis of the agent. Oral lesions, hypersalivation, ocular and nasal discharge, coughing, and breathing difficulties were seen in clinical examinations of cats with URTD. Feline calicivirus was detected in only one cat's conjunctival swab sample in PCR analyses. As a result, we found that Feline calicivirus infection was present in cats with URTD in the Diyarbakir region, and 5% positivity was found in cats with clinical symptoms according to PCR analysis.(AU)
O calicivírus felino está entre os microrganismos patogênicos mais comuns nas doenças do trato respiratório superior de gatos, determinando estomatites, ulcerações orais, descarga ocular e nasal, conjuntivite, febre, manqueira, anorexia, hipersalivação, pneumonia, distúrbios respiratórios, tosse e depressão. O presente trabalho foi delineado para determinar o papel do calicivírus felino (CVF) em gatos com doenças do trato respiratório superior na região de Diyarbakir, Turquia. Com o objetivo de orientar a prescrição do tratamento para os gatos infectados e contribuir com a profilaxia da doença. O material de estudo consistiu em 10 gatos saudáveis sem qualquer problema de saúde e 20 gatos acometidos por doenças do trato respiratório superior que não haviam sido vacinados contra a infecção pelo calicivírus felino. Os animais de diferentes raças, idades e gêneros foram encaminhados para a Universidade de Dicle, na Faculdade de Veterinária, na policlínica Professor Dr. Servet Sekin. Após o exame clínico de rotina dos animais, foram colhidos swabs orais e da conjuntiva e amostras de sangue. Análises hematológicas e bioquímicas das amostras de sangue foram realizadas e os swabs foram analisados pelo método da reação em cadeia pela polimerase (PCR) para diagnóstico do agente. Nos gatos infectados foram constatadas: lesões orais, hipersalivação, descargas oculares e nasais, tosse e dificuldade respiratória. O calicivírus felino foi detectado pela técnica de PCR no swab conjuntival de apenas um gato. A conclusão obtida foi que a infecção pelo calicivírus felino foi detectada pela técnica de PCR na região de Diyarbakir, Turquia, em gatos com doença do trato respiratório superior com a frequência de 5%.(AU)
Assuntos
Animais , Gatos , Infecções Respiratórias , Gatos/anatomia & histologia , Infecções por Caliciviridae/diagnóstico , Reação em Cadeia da Polimerase , Calicivirus FelinoRESUMO
Feline calicivirus is among the most common pathogenic microorganisms in upper respiratory tract disease (URTD) and oral lesions of cats. It leads to stomatitis, oral ulceration, ocular and nasal discharge, conjunctivitis, fever, lameness, anorexia, hypersalivation, pneumonia, respiratory distress, coughing, and depression in infected cats. This study aimed to determine the role of Feline calicivirus (FCV) in cats with the upper respiratory tract disease in the Diyarbakir region, Turkey, to provide treatment for infected cats and contribute to the disease prophylaxis. The study material consisted of 10 cats (control group) considered to be healthy according to the clinical examination and 20 cats with URTD that were not vaccinated against Feline calicivirus infection of different breeds, ages, and genders brought to Dicle University Veterinary Faculty Prof. Dr. Servet SEKIN Polyclinic with URTD. After routine clinical examinations of the animals, oral and conjunctival swabs and blood samples were taken. Hematological and biochemical analyzes of blood samples were performed. Swab samples were analyzed by the polymerase chain reaction (PCR) method for the diagnosis of the agent. Oral lesions, hypersalivation, ocular and nasal discharge, coughing, and breathing difficulties were seen in clinical examinations of cats with URTD. Feline calicivirus was detected in only one cat's conjunctival swab sample in PCR analyses. As a result, we found that Feline calicivirus infection was present in cats with URTD in the Diyarbakir region, and 5% positivity was found in cats with clinical symptoms according to PCR analysis.(AU)
O calicivírus felino está entre os microrganismos patogênicos mais comuns nas doenças do trato respiratório superior de gatos, determinando estomatites, ulcerações orais, descarga ocular e nasal, conjuntivite, febre, manqueira, anorexia, hipersalivação, pneumonia, distúrbios respiratórios, tosse e depressão. O presente trabalho foi delineado para determinar o papel do calicivírus felino (CVF) em gatos com doenças do trato respiratório superior na região de Diyarbakir, Turquia. Com o objetivo de orientar a prescrição do tratamento para os gatos infectados e contribuir com a profilaxia da doença. O material de estudo consistiu em 10 gatos saudáveis sem qualquer problema de saúde e 20 gatos acometidos por doenças do trato respiratório superior que não haviam sido vacinados contra a infecção pelo calicivírus felino. Os animais de diferentes raças, idades e gêneros foram encaminhados para a Universidade de Dicle, na Faculdade de Veterinária, na policlínica Professor Dr. Servet Sekin. Após o exame clínico de rotina dos animais, foram colhidos swabs orais e da conjuntiva e amostras de sangue. Análises hematológicas e bioquímicas das amostras de sangue foram realizadas e os swabs foram analisados pelo método da reação em cadeia pela polimerase (PCR) para diagnóstico do agente. Nos gatos infectados foram constatadas: lesões orais, hipersalivação, descargas oculares e nasais, tosse e dificuldade respiratória. O calicivírus felino foi detectado pela técnica de PCR no swab conjuntival de apenas um gato. A conclusão obtida foi que a infecção pelo calicivírus felino foi detectada pela técnica de PCR na região de Diyarbakir, Turquia, em gatos com doença do trato respiratório superior com a frequência de 5%.(AU)
Assuntos
Animais , Gatos , Infecções Respiratórias , Gatos/anatomia & histologia , Infecções por Caliciviridae/diagnóstico , Reação em Cadeia da Polimerase , Calicivirus FelinoRESUMO
p53 is implicated in several cellular pathways such as induction of cell-cycle arrest, differentiation, senescence, and apoptosis. p53 is activated by a broad range of stress signals, including viral infections. While some viruses activate p53, others induce its inactivation, and occasionally p53 is differentially modulated during the replicative cycle. During calicivirus infections, apoptosis is required for virus exit and spread into the host; yet, the role of p53 during infection is unknown. By confocal microscopy, we found that p53 associates with FCV VP1, the protease-polymerase NS6/7, and the dsRNA. This interaction was further confirmed by proximity ligation assays, suggesting that p53 participates in the FCV replication. Knocked-down of p53 expression in CrFK cells before infection, resulted in a strong reduction of the non-structural protein levels and a decrease of the viral progeny production. These results indicate that p53 is associated with the viral replication complex and is required for an efficient FCV replication.
Assuntos
Calicivirus Felino/genética , Proteínas do Capsídeo/genética , Peptídeo Hidrolases/genética , RNA Viral/genética , Proteína Supressora de Tumor p53/genética , Replicação Viral/genética , Animais , Calicivirus Felino/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Gatos , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Rim/metabolismo , Rim/virologia , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Vírion/genética , Vírion/metabolismoRESUMO
Felid alphaherpesvirus 1 (FeHV-1) and feline calicivirus (FCV) affect cats worldwide. The aim of this study was to evaluate the frequency of occurrence of FeHV-1 and FCV in cats with clinical signs of respiratory, oral and/or ocular disease. Samples were collected from cats cared for in veterinary ambulatory and clinics and submitted to molecular detection and viral isolation. Of the 49 cats evaluated, 45 (92%) were positive for at least one of the viruses; 82% (40/49) were positive for FeHV-1 and 41% (20/49) for FCV. Of these, 31% (15/49) were coinfection cases. For FeHV-1, 45% (18/40) of the cats tested were positive from the collection of eye swab, and the same percentage (9/20) was obtained for the FCV by the oral swab. FeHV-1 and/or FCV were isolated in 35% (17/49) of the samples. The main clinical sign observed was ocular secretion in 71% (35/49) of cats, characterized as mild serous, purulent or serosanguineous, and in some cases associated with ocular injury and marked chemosis. Our findings demonstrate the high occurrence of FeHV-1 and FCV in domestic cats in southern Brazil and indicate that measures should be implemented to improve the diagnostic, prevention and management against of these important diseases.(AU)
Alphaherpesvírus felídeo 1 (FeHV-1) e calicivírus felino (FCV) afetam gatos mundialmente. O objetivo deste estudo foi identificar a frequência de ocorrência de FeHV-1 e FCV em gatos com sinais clínicos de doença respiratória, oral e/ou ocular. Amostras foram coletadas de gatos atendidos em ambulatório e clínicas veterinárias e submetidas à detecção molecular e isolamento viral. Dos 49 gatos avaliados, 45 (92%) foram positivos para ao menos um dos vírus; 82% (40/49) foram positivos para o FeHV-1 e 41% (20/49) para o FCV. Destes, 31% (15/49) foram casos de coinfecção. Para o FeHV-1, 45% (18/40) dos gatos foram positivos na coleta do swab ocular, e o mesmo percentual (9/20) foi obtido para o FCV a partir do swab oral. FeHV-1 e/ou FCV foram isolados em 35% (17/49) das amostras. O principal sinal clínico observado foi secreção ocular em 71% (35/49) dos gatos, caracterizada como serosa, purulenta ou serossanguinolenta e, em alguns casos, associada à lesão e quemose. Nossos resultados demonstram a alta ocorrência de FeHV-1 e FCV em gatos domésticos na região Sul do Brasil e indicam que devem ser implementadas medidas para melhorar o diagnóstico, a prevenção e o manejo contra essas importantes doenças.(AU)
Assuntos
Animais , Doenças do Gato/epidemiologia , Calicivirus Felino/isolamento & purificação , Alphaherpesvirinae/isolamento & purificação , Infecções por Caliciviridae/epidemiologia , Infecções por Herpesviridae/epidemiologia , Gatos , Infecções por Caliciviridae/veterinária , Infecções por Herpesviridae/veterináriaRESUMO
Felid alphaherpesvirus 1 (FeHV-1) and feline calicivirus (FCV) affect cats worldwide. The aim of this study was to evaluate the frequency of occurrence of FeHV-1 and FCV in cats with clinical signs of respiratory, oral and/or ocular disease. Samples were collected from cats cared for in veterinary ambulatory and clinics and submitted to molecular detection and viral isolation. Of the 49 cats evaluated, 45 (92%) were positive for at least one of the viruses; 82% (40/49) were positive for FeHV-1 and 41% (20/49) for FCV. Of these, 31% (15/49) were coinfection cases. For FeHV-1, 45% (18/40) of the cats tested were positive from the collection of eye swab, and the same percentage (9/20) was obtained for the FCV by the oral swab. FeHV-1 and/or FCV were isolated in 35% (17/49) of the samples. The main clinical sign observed was ocular secretion in 71% (35/49) of cats, characterized as mild serous, purulent or serosanguineous, and in some cases associated with ocular injury and marked chemosis. Our findings demonstrate the high occurrence of FeHV-1 and FCV in domestic cats in southern Brazil and indicate that measures should be implemented to improve the diagnostic, prevention and management against of these important diseases.(AU)
Alphaherpesvírus felídeo 1 (FeHV-1) e calicivírus felino (FCV) afetam gatos mundialmente. O objetivo deste estudo foi identificar a frequência de ocorrência de FeHV-1 e FCV em gatos com sinais clínicos de doença respiratória, oral e/ou ocular. Amostras foram coletadas de gatos atendidos em ambulatório e clínicas veterinárias e submetidas à detecção molecular e isolamento viral. Dos 49 gatos avaliados, 45 (92%) foram positivos para ao menos um dos vírus; 82% (40/49) foram positivos para o FeHV-1 e 41% (20/49) para o FCV. Destes, 31% (15/49) foram casos de coinfecção. Para o FeHV-1, 45% (18/40) dos gatos foram positivos na coleta do swab ocular, e o mesmo percentual (9/20) foi obtido para o FCV a partir do swab oral. FeHV-1 e/ou FCV foram isolados em 35% (17/49) das amostras. O principal sinal clínico observado foi secreção ocular em 71% (35/49) dos gatos, caracterizada como serosa, purulenta ou serossanguinolenta e, em alguns casos, associada à lesão e quemose. Nossos resultados demonstram a alta ocorrência de FeHV-1 e FCV em gatos domésticos na região Sul do Brasil e indicam que devem ser implementadas medidas para melhorar o diagnóstico, a prevenção e o manejo contra essas importantes doenças.(AU)
Assuntos
Animais , Doenças do Gato/epidemiologia , Calicivirus Felino/isolamento & purificação , Alphaherpesvirinae/isolamento & purificação , Infecções por Caliciviridae/epidemiologia , Infecções por Herpesviridae/epidemiologia , Gatos , Infecções por Caliciviridae/veterinária , Infecções por Herpesviridae/veterináriaRESUMO
It is known that levels of the anti-apoptotic protein survivin are reduced during Murine norovirus MNV-1 and Feline calicivirus (FCV) infection as part of the apoptosis establishment required for virus release and propagation in the host. Recently, our group has reported that overexpression of survivin causes a reduction of FCV protein synthesis and viral progeny production, suggesting that survivin may affect early steps of the replicative cycle. Using immunofluorescence assays, we observed that overexpression of survivin, resulted in the reduction of FCV infection not only in transfected but also in the neighboring nontransfected CrFK cells, thus suggesting autocrine and paracrine protective effects. Cells treated with the supernatants collected from CrFK cells overexpressing survivin showed a reduction in FCV but not MNV-1 protein production and viral yield, suggesting that FCV binding and/or entry were specifically altered. The reduced ability of FCV to bind to the surface of the cells overexpressing survivin, or treated with the supernatants collected from these cells, correlate with the reduction in the cell surface of the FCV receptor, the feline junctional adhesion molecule (fJAM) 1, while no effect was observed in the cells transfected with the pAm-Cyan vector or in cells treated with the corresponding supernatants. Moreover, the overexpression of survivin affects neither Vaccinia virus (VACV) production in CrFK cells nor MNV-1 virus production in RAW 267.4 cells, indicating that the effect is specific for FCV. All of these results taken together indicate that cells that overexpress survivin, or cell treatment with the conditioned medium from these cells, results in the reduction of the fJAM-1 molecule and, therefore, a specific reduction in FCV entry and infection.
Assuntos
Infecções por Caliciviridae/virologia , Calicivirus Felino/fisiologia , Survivina/metabolismo , Animais , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Calicivirus Felino/metabolismo , Gatos , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Moléculas de Adesão Juncional/metabolismo , Receptores Virais/metabolismo , Especificidade da Espécie , Survivina/genética , Proteínas Virais/biossíntese , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Calicivirus infection causes intrinsic apoptosis, leading to viral propagation in the host. During murine norovirus infection, a reduction in the anti-apoptotic protein survivin has been documented. Here we report that in feline calicivirus infection, a downregulation of the anti-apoptotic proteins survivin and XIAP occur, which correlates with the translocation of the pro-apoptotic protein Smac/DIABLO from the mitochondria to the cytoplasm and the activation of caspase-3. Inhibition of survivin degradation by lactacystin treatment caused a delay in apoptosis progression, reducing virus release, without affecting virus production. However, the overexpression of survivin caused a negative effect in viral progeny production. Overexpression of the leader of the capsid protein (LC), but not of the protease-polymerase NS6/7, results in the downregulation of survivin and XIAP, caspase activation and mitochondrial damage. These results indicate that LC is responsible for the induction of apoptosis in transfected cells and most probably in FCV infection.
Assuntos
Apoptose , Infecções por Caliciviridae/metabolismo , Calicivirus Felino/fisiologia , Proteínas do Capsídeo/metabolismo , Regulação para Baixo , Survivina/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Animais , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Gatos , Linhagem Celular , Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Survivina/metabolismo , Proteínas Virais/biossíntese , Replicação ViralRESUMO
Cellular proteins have been identified to participate in calicivirus replication in association with viral proteins and/or viral RNAs. By mass spectrometry from pull-down assays, we identified several cellular proteins bound to the feline calicivirus (FCV) genomic RNA; among them the lipid raft-associated scaffold protein Annexin (Anx) A2. AnxA2 colocalizes with FCV NS6/7 protein and with the dsRNA in infected cells; moreover, it was found associated with the viral RNA in the membrane fraction corresponding to the replication complexes (RCs), suggesting its role during FCV replication. AnxA2-knockdown from CrFK cells prior to infection with FCV caused a delay in the cytopathic effect, a strong reduction of viral non-structural proteins and dsRNA production, and a decrease of FCV yield in both cell-associated and supernatant fractions. Taken together, these results indicate that AnxA2 associates to the genomic RNA of FCV and is required for an efficient FCV replication.
Assuntos
Anexina A2/metabolismo , Calicivirus Felino/fisiologia , Interações Hospedeiro-Patógeno , RNA Viral/metabolismo , Replicação Viral , Animais , Calicivirus Felino/crescimento & desenvolvimento , Gatos , Linhagem Celular , Efeito Citopatogênico Viral , Espectrometria de Massas , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Carga Viral , Proteínas não Estruturais Virais/metabolismoRESUMO
ABSTRACT The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.