RESUMEN
Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.
Asunto(s)
Enfermedades de los Perros/genética , Ehrlichia canis/genética , Ehrlichiosis/genética , Proteínas del Núcleo Viral/genética , Animales , Brasil , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/microbiología , Perros/microbiología , Ehrlichia canis/aislamiento & purificación , Ehrlichia canis/patogenicidad , Ehrlichiosis/diagnóstico , Ehrlichiosis/microbiología , Ehrlichiosis/veterinaria , Técnicas de Amplificación de Ácido Nucleico/métodos , Proteínas del Núcleo Viral/aislamiento & purificaciónRESUMEN
Rotavirus genome synthesis is a two-step process; the synthesis of the eleven viral mRNAs and the production of the complementary strand (minus strand synthesis) yielding the double stranded genomic RNA (dsRNA). Purified rotavirus open cores are a helpful tool to study this replication event. These open cores can use viral dsRNA and mRNAs as template for RNA synthesis. In the present communication it was demonstrated that open cores prefers to use viral mRNA as template instead of the genomic dsRNA. The use by the open cores of both templates can be blocked through two independent strategies. In the presence of ssRNAs molecules, the use of the viral dsRNA template is completely blocked without affecting the minus strand synthesis. Nevertheless, an antisense oligonucleotide strategy previously reported to block the minus strand synthesis of the target gene do not affect the use of the dsRNA template. These results suggest the recognition of different signals in each template by the open core protein complex.
Asunto(s)
ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Rotavirus/fisiología , Moldes Genéticos , Replicación Viral , Conformación de Ácido Nucleico , ARN Complementario/metabolismo , ARN Bicatenario/química , ARN Viral/biosíntesis , ARN Viral/química , Rotavirus/genética , Transcripción Genética , Proteínas del Núcleo Viral/aislamiento & purificación , Proteínas del Núcleo Viral/metabolismo , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismoRESUMEN
Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.