PD1 CD44 antiviral peptide as an inhibitor of the protein-protein interaction in dengue virus invasion.

Dengue virus (DENV) infection is mediated by the interaction between the virus envelope protein and cellular receptors of the host cells. In this study, we designed peptides to inhibit protein-protein interaction between dengue virus and CD44 receptor, which is one of the receptors used by DENV for entry. In silico model complexes were designed between domain III of the viral envelope protein of dengue virus 2 and the domain of human CD44 receptor using ClusPro 2.0, (https://cluspro.bu.edu/login.php), and inhibition peptides were designed with Rosetta Online-Server(http://rosie.rosettacommons.org/peptiderive). We identified one linear antiviral peptide of 18 amino acids derived from the human CD44 receptor, PD1 CD44. It did not show hemolysis or toxicity in HepG2 or BHK cell lines, nor did it stimulate the release of IL-1ß, IL-6, TNF-α, and IFN-γ, below 100 µM. It had an IC50 of 13.8 µM and maximum effective dose of 54.9 µM evaluated in BHK cells. The decrease in plaque-forming units/mL for DENV1, DENV2, DENV3, and DENV4 was 99.60%, 99.40%, 97.80%, and 70.50%, respectively, and similar results were obtained by RT-qPCR. Non-structural protein 1 release was decreased in pre- and co-treatment but not in post-treatment. Competition assays between the DN59 peptide, envelope protein, and the fragment of domain III "MDKLQLKGMSYSMCTGKF" of the viral envelope of DENV2 and PD1 CD44 showed that our peptide lost its antiviral activity. We demonstrated that our peptide decreased endosome formation, and we propose that it binds to the envelope protein of DENV, inhibiting viral invasion/fusion.

In Vitro Inhibition of Replication of Dengue Virus Serotypes 1-4 by siRNAs Bound to Non-Toxic Liposomes.

Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1-4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.

K48-linked polyubiquitination of dengue virus NS1 protein inhibits its interaction with the viral partner NS4B.

Dengue virus (DENV) is a member of the Flaviviridae family, which is transmitted to mammalian species through arthropods, and causes dengue fever or severe dengue fever in humans. The DENV genome encodes for multiple nonstructural (NS) proteins including NS1. NS1 plays an essential role in replication by interacting with other viral proteins including NS4B, however how these interactions are regulated during virus infection is not known. By using bioinformatics, mass spectrometry analysis, and co-immunoprecipitation assays, here we show that DENV-NS1 is ubiquitinated on multiples lysine residues during DENV infection, including K189, a lysine residue previously shown to be important for efficient DENV replication. Data from in vitro and cell culture experiments indicate that dengue NS1 undergoes modification with K48-linked polyubiquitin chains, which usually target proteins to the proteasome for degradation. Furthermore, ubiquitinated NS1 was detected in lysates as well as in supernatants of human and mosquito infected cells. Ubiquitin deconjugation of NS1 using the deubiquitinase OTU resulted in increased interaction with the viral protein NS4B suggesting that ubiquitinated NS1 has reduced affinity for NS4B. In support of these data, a K189R mutation on NS1, which abrogates ubiquitination on amino acid residue 189 of NS1, also increased NS1-NS4B interactions. Our work describes a new mechanism of regulation of NS1-NS4B interactions and suggests that ubiquitination of NS1 may affect DENV replication.

Prevalencia de Fasciola hepatica en humanos y bovinos en el departamento del Quindío-Colombia 2012-2013 / Prevalence of Fasciola hepatica in humans and cattle in the departmentof Quindio-Colombia 2012-2013

Introducción: La fasciolosis es una parasitosis causada por Fasciola hepatica (F. hepatica). En el departamento del Quindío se desconoce su prevalencia tanto en humanos como en bovinos. Objetivo: Determinar la prevalencia de F. hepatica en heces de trabajadores del sector ganadero y bovinos en el departamento del Quindío entre los meses de septiembre de 2012 y marzo de 2013. Materiales y métodos: Se realizó un estudio descriptivo de corte transversal, mediante análisis parasitológico en heces de empleados del sector ganadero y en bovinos en los 12 municipios del departamento del Quindío, usando la técnica directa de Lugol, concentración de Kato-Katz y Ritchie; se realizó la determinación de antígenos de F. hepatica en heces mediante la prueba inmunológica Fascidig®. Se realizó una encuesta epidemiológica a los empleados y propietarios, en la que se consignaron la presencia de sintomatología y los factores de riesgo implicados en la adquisición de esta parasitosis. Resultados: La prevalencia de F. hepatica en bovinos fue 3,74%, por microscopia óptica y 3,01% mediante Fascidig®, y 0% en humanos. Los animales recibieron antiparasitarios en los meses previos a la toma de las muestras, sin embargo, se determinó presencia de huevos de Fasciola en las heces de los bovinos. Los municipios donde se encontraron resultados positivos fueron: Salento, Génova, Quimbaya, Montenegro y Circasia. Conclusión: Demostramos la presencia del parásito F. hepatica en los bovinos en pie de 4 municipios del departamento del Quindío. © 2014 ACIN. Publicado por Elsevier España, S.L.U. Todos los derechos reservados.

Introduction: Fascioliasis is a parasitic disease caused by Fasciola hepatica (F. hepatica) . Theprevalence of this infection in the region of Quindío in humans and in cattle is unknown. Objectives: To determine the prevalence of F. hepatica in feces of cattle workers and cattle inthe region of Quindío from September 2012 to March 2013. Materials and methods: A descriptive, cross-sectional study was performed by parasitologicalanalysis of feces of cattle workers and cattle in 12 municipalities of department of Quindíousing the Lugol direct technique, Kato-Katz and Ritchie concentrations. The determination of Fasciola hepatic antigens in feces was performed by the Fascidig® immunological technique.In addition, an epidemiological survey concerning the symptomatology of the disease and therisk factors involved in the acquisition of this parasite was carried out. Results: The F. hepatica prevalence in cattle was 3,74%, by optical microscopy and 3,01% withFascidig® and 0% in humans. The animals received antiparasitics in the months prior to thetaking of samples; however the presence of F. hepatica eggs in cattle feces was determined.The cities where positive results were found include: Salento, Génova, Quimbaya, Montenegroy Circasia. Conclusion: We have demonstrated the presence of the parasite F. hepatica in cattle in 4 citiesin the region of Quindío.

Prevalencia de helmintos intestinales en gatos domésticos del departamento del Quindío, Colombia / Prevalence of intestinal helminths in cats in Quindío, Colombia

Introducción. Las enfermedades producidas por helmintos están ampliamente distribuidas en el mundo y muchas de ellas se consideran zoonosis. Los animales de compañía cumplen un papel trascendental en la transmisión a los humanos. Objetivo. Determinar la prevalencia de helmintos intestinales en gatos del departamento del Quindío. Materiales y métodos. Se estudiaron 121 gatos domésticos del departamento del Quindío, de los cuales se registraron los datos de sexo, edad y condición corporal. Se recolectaron heces y se procesaron mediante las técnicas de Ritchie modificada y de Kato-Katz. Resultados. De los 121 gatos, 42,14 % (IC95%: 33,35-50,94) y 45,45 % (IC95%: 36,58-54,32) resultaron parasitados con alguna especie de helminto adulto según la presencia de huevos en sus heces, mediante las técnicas de Ritchie y de Kato-Katz, respectivamente. Toxocara cati fue el parásito más prevalente(Ritchie: 37,2 %; Kato-Katz: 43 %), seguido por Ancylostoma spp. (Ritchie: 7,43 %; Kato-Katz: 5,78 %) y Aelurostrongylus abstrusus (Ritchie: 0,82 %). Sesenta y cinco (53,71 %) gatos eran hembras y 56 (46,28 %) eran machos; la prevalencia de infección fue similar en ambos sexos. Los felinos mayores de 4 años de edad presentaron mayor prevalencia de parásitos (81,8 %), seguidos por los de 1 a 4 años (48,8 %) y, por último, por los menores de un año (28,6 %). Se encontró una buena condición corporal en 77,68 % y este grupo presentó menor frecuencia de helmintos intestinales. Conclusión. La prevalencia de helmintos intestinales en gatos domésticos del departamento del Quindío fue de 43,8 %, lo que hace necesario establecer programas de vigilancia y prevención en la población humana y felina.

Introduction. Diseases caused by helminths are widely distributed in the world and many of them are considered zoonoses in which pets play a major role in transmission to humans. Objective. The prevalence of intestinal helminths was determined in cats in Quindío Province. Materials and methods. One hundred twenty-one cats were characterized --data recorded included sex, age and body condition. Fecal samples were collected and processed using the modified Ritchie and modified Kato-Katz techniques to determine the presence of intestinal helminths. Results. Of the 121 cats, 42.1%, (95% CI: 33.4-50.9) and 45.5% (95% CI: 36.6-54.3) were parasitized with at least one adult helminth species as evidenced by the presence of eggs in their fecal samples. Toxocara cati was the most prevalent parasite (Ritchie: 37.2%, Kato-Katz: 43%), followed by Ancylostoma spp. (Ritchie: 7.4%, Kato-Katz: 5.8%) and Aelurostrongylus abstrusus (Ritchie: 0.82%). Sixty-five cats (53.7%) were females and 56 (46.3%) males; the prevalence of infection was similar in both sexes. Cats older than 4 years had the highest prevalence (81.8%) followed by those aged 1 to 4 years (48.8%) and by those under 1 year (28.6%). The majority of cats, 77.7%, were found to be in good body condition and this group had the lowest frequency of intestinal helminths with both techniques. Conclusion. The prevalence of intestinal helminths in domestic cats in Quindío was 43.8%; it is necessary to establish surveillance and prevention programs in the human and feline populations.

Prevalencia de helmintos intestinales en caninos del departamento del Quindío / Prevalence of intestinal helminths in dogs from Quindío Province

Introducción. Los helmintos intestinales son agentes patógenos que afectan animales domésticos y que a través de ellos pueden infectar humanos. Objetivo. El objetivo del trabajo fue determinar la prevalencia de helmintos intestinales en perros con dueño del departamento del Quindío. Materiales y métodos. Estudio descriptivo prospectivo. Se aplicó una encuesta epidemiológica a los propietarios de los perros. Se recolectaron muestras de heces de los caninos registrados en la jornada de vacunación antirrábica del 2003 en el departamento del Quindío. Las muestras de materia fecal frescas fueron analizadas utilizando la técnica de diagnóstico de Ritchie. Resultados. Se analizaron 324 muestras de heces caninas; el 67,6 por ciento de los perros eran de razas puras y el 32,4 por ciento razas mestizas. Se encontró una prevalencia del 22,2 por ciento; Ancylostoma caninum fue el parásito más frecuente, 13,9 por ciento. También se observó Trichuris vulpis, 4,3 por ciento; Toxocara canis, 2,5 por ciento, y Strongyloides stercoralis, 4,0 por ciento. El 2,46 por ciento de las mascotas se encontraron multiparasitadas. Conclusión. La frecuencia de helmintos intestinales en el departamento del Quindío fue de 22,2 por ciento y la presencia de estos parásitos coincide con factores como la edad y la permanencia del canino en la calle, entre otros. Por esta razón, es necesario establecer programas de vigilancia y prevención en la población humana y canina