RESUMEN
Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus causing Crimean-Congo haemorrhagic fever (CCHF), a disease reported to have a high fatality rate in numerous countries. The virus is geographically widespread due to its vector, and numerous wild and domestic animals can develop asymptomatic infection. Serological and limited molecular evidence of CCHFV has previously been reported in Camelus dromedarius (the dromedary, or one-humped camel) in the United Arab Emirates (UAE). In this study, 238 camel samples were screened for CCHFV RNA where 16 camel samples were positive for CCHFV by RT-PCR. Analysis of full-length CCHFV genome sequences revealed a novel lineage in camels from the UAE, and potential reassortment of the M segment of the genome.
Asunto(s)
Camelus/virología , Genoma Viral/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Fiebre Hemorrágica de Crimea/veterinaria , Animales , Virus de la Fiebre Hemorrágica de Crimea-Congo/aislamiento & purificación , Fiebre Hemorrágica de Crimea/sangre , Fiebre Hemorrágica de Crimea/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Emiratos Árabes UnidosRESUMEN
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5 × 10-4 and 2.0 × 10-5 transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wild-type (P < 0.001). OriC-plasmids described in this study may, therefore open the way for functional genomics in M. hyorhinis. Furthermore, this is a first study demonstrated the gene associated with a hemolytic phenotype in mycoplasmas.
Asunto(s)
Mycoplasma hyorhinis/genética , Plásmidos/genética , Origen de Réplica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Replicación del ADN , Regulación Bacteriana de la Expresión Génica , Orden Génico , Hemólisis , Recombinación Homóloga , Mutación , Mycoplasma hyorhinis/fisiología , Fenotipo , Recombinación Genética , Análisis de Secuencia de ADN , Transformación BacterianaRESUMEN
Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.
Asunto(s)
Escherichia coli/genética , Marcación de Gen/métodos , Recombinación Homóloga , Mycoplasma hyorhinis/genética , Rec A Recombinasas/genética , Animales , Proteínas Bacterianas/genética , ADN Bacteriano , Genes Bacterianos , Vectores Genéticos , Proteínas Hemolisinas/genética , Ratones , Mutación , Mycoplasma/genética , Infecciones por Mycoplasma/diagnóstico , Mycoplasma hyorhinis/patogenicidad , Fenotipo , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Recombinación Genética , Reparación del ADN por Recombinación , Porcinos , Resistencia a la Tetraciclina/genéticaRESUMEN
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.
RESUMEN
Mycoplasma hyopneumoniae (M. hyopneumoniae) infection affects the swine industry. Lithium chloride (LiCl), is a drug used to treat bipolar disorder and has also shown activity against bacterial and viral infections. Herein, we evaluated the antibacterial activity of LiCl on PK-15 cells infected with M. hyopneumoniae. Incubation of LiCl (40mM) with cells for 24h, did not significantly affect the cell viability. The qRT-PCR showed ~80% reduction in M. hyopneumoniae genome when LiCl added post-infection. A direct effect of LiCl on bacteria was also observed. However, treatment of cells with LiCl prior infection, does not protect against the infection. Anti-bacterial activity of LiCl was further confirmed by IFA, which demonstrated a reduction in the bacterial protein. With 40mM LiCI, the apoptotic cell death, production of nitric oxide and superoxide anion induced by M. hyopneumoniae, were prevented by ~80%, 60% and 58% respectively. Moreover, caspase-3 activity was also reduced (82%) in cells treated with 40mM LiCl. LiCl showed activity against various strains of M. hyopneumoniae examined in our study. Collectively, our data showed that LiCl inhibited the infection of M. hyopneumoniae through anti-apoptotic mechanism.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Cloruro de Litio/farmacología , Mycoplasma hyopneumoniae/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mycoplasma , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , PorcinosRESUMEN
Griffithsin (GRFT) is a broad-spectrum antiviral protein against several glycosylated viruses. In our previous publication, we have shown that GRFT exerted antiviral activity against Japanese encephalitis virus (JEV) infection. Herein, we further elucidated the mechanism by which GRFT inhibits JEV infection in BHK-21 cells. In vitro experiments using Pull-down assay and Co-immunoprecipitation (CO-IP) assay showed that GRFT binds to the JEV glycosylated viral proteins, specifically the enveloped (E) and premature (prM) glycoproteins. The binding of GRFT to the JEV was competitively inhibited by increasing concentrations of mannose; in turns abolished anti-JEV activity of GRFT. We suggested that, the binding of GRFT to the glycosylated viral proteins may contribute to its anti-JEV activity. Collectively, our data indicated a possible mechanism by which GRFT exerted its anti-JEV activity. This observation suggests GRFT's potentials in the development of therapeutics against JEV or other flavivirus infection.
Asunto(s)
Antivirales/metabolismo , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Lectinas de Plantas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Línea Celular , Centrifugación , Cricetinae , Inmunoprecipitación , Pruebas de Sensibilidad Microbiana , Unión ProteicaRESUMEN
Griffithsin (GRFT) is a broad-spectrum antiviral protein that is effective against several glycosylated viruses. Here, we have evaluated the in vitro and in vivo antiviral activities of GRFT against Japanese encephalitis virus (JEV) infection. In vitro experiments showed that treatment of JEV with GRFT before inoculation of BHK-21 cells inhibited infection in a dose-dependent manner, with 99 % inhibition at 100 µg/ml and a 50 % inhibitory concentration (IC(50)) of 265 ng/ml (20 nM). Binding assays suggested that binding of GRFT to JEV virions inhibited JEV infection. In vivo experiment showed that GRFT (5 mg/kg) administered intraperitoneally before virus infection could completely prevent mortality in mice challenged intraperitoneally with a lethal dose of JEV. Our study also suggested that GRFT prevents JEV infection at the entry phase by targeting the virus. Collectively, our data demonstrate that GRFT is an antiviral agent with potential application in the development of therapeutics against JEV or other flavivirus infections.
Asunto(s)
Antivirales/farmacología , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Encefalitis Japonesa/prevención & control , Lectinas de Plantas/farmacología , Internalización del Virus/efectos de los fármacos , Animales , Línea Celular , Cricetinae , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos BALB C , Análisis de SupervivenciaRESUMEN
Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase ribosomal-inactivating protein isolated from Phytolacca americana. The antiviral activity of PAP has been described in several viruses. This study was to investigate the antiviral activity of PAP against Japanese encephalitis virus (JEV) infection in vitro and in vivo. Antiviral activity of PAP against JEV infection was evaluated in vitro using plaque forming assay, qRT-PCR and Western blot analysis. In vitro results showed that PAP inhibited replication of JEV in a dose-dependent manner with 50% inhibitory concentration (IC(50)) of 300 ng/ml (23.1 nM). Depurination assay suggested that the antiviral activity of PAP against JEV infection might be partially due to depurination of JEV genomic RNA. In vivo studies showed that PAP (1.0mg/kg) administered intraperitoneally decreased infection in mice challenged with a lethal dose of JEV, presenting a survival of 87.5% or 85.7% when administered pre-infection or post-infection. Collectively, our studies demonstrated that PAP possesses antiviral activity against JEV infection in vitro and in vivo, providing evidences for further development of PAP as an antiviral agent against JEV infection.