ABSTRACT
Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Oligodeoxyribonucleotides, Antisense/analysis , Oligodeoxyribonucleotides, Antisense/chemistry , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , DNA Probes, HPV/analysis , DNA Probes, HPV/chemical synthesis , DNA Probes, HPV/chemistry , Flow Cytometry , Green Fluorescent Proteins , Humans , Luminescent Proteins/analysis , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Microscopy, Fluorescence/methods , Molecular Sequence Data , Oligodeoxyribonucleotides, Antisense/chemical synthesis , RNA, Messenger/chemistry , RNA, Viral/antagonists & inhibitors , RNA, Viral/chemistry , Recombinant Proteins/analysisABSTRACT
Fowl adenoviruses of the serotype 4 from Germany were characterised by restriction enzyme analysis in comparison to isolates from Asia, South America and the FAV4 reference strain KR5. Only strain Da60 which was isolated from a psittacine aviary was identical with the reference strain KR5. None of the isolates was identical with the highly pathogenic strains from India and Ecuador. One-day-old chicks were infected orally and intramuscularly with the reference strain KR5, the psittacine isolate Da60 and isolate K1013 from Ecuador. Whereas no mortality was seen with the two strains KR5 and Da60, the mortality with K1013 was 100%. The main pathological signs were a swollen liver with necrosis and a lymphocyte depletion with a loss of the follicle structure. To investigate a second subject of avian adenovirus epizootiology several FAVs were characterized serologically and with PCR which was combined with the digestion of the PCR products. Including the reference strains, both methods were compared. It was shown that the digestion of the PCR products allows a clear attribution to a specific serotype, which underlines the usefulness of this method for diagnostic purposes.
Subject(s)
Adenoviridae Infections/veterinary , Aviadenovirus , Bird Diseases/transmission , Adenoviridae Infections/pathology , Adenoviridae Infections/transmission , Animals , Asia , Aviadenovirus/classification , Aviadenovirus/genetics , Bird Diseases/pathology , Bird Diseases/virology , Chick Embryo , Germany , Liver/pathology , Necrosis , Psittaciformes , South AmericaABSTRACT
Three fowl adenovirus (FAV) isolates (341, 344, and 215) obtained during 1996-97 from field outbreaks of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) affecting broilers and broiler breeders in Chile were characterized by virus neutralization tests (VNTs) and restriction enzyme analysis of a DNA fragment. Furthermore, the pathologic characteristics of one of these FAV isolates (FAV 341) was studied in experimentally infected chickens. The VNTs conducted with isolates 341 and 344 against reference strains and antisera belonging to each of 12 FAV serotypes demonstrated a close antigenic relationship with strain KR5 of the FAV serotype 4. Polymerase chain reaction using the primers H3/H4 and subsequent HpaII digestion was used for serotype identification of isolates 341 and 215. The length of the PCR products and the restriction profiles of isolates 341, 215, and the reference strain KR5 (FAV4) were identical. The present results confirmed the classification of all three isolates as FAV4. The pathogenicity test with 1000 mean tissue infectious dose of isolate 341 inoculated intramuscularly in 20-day-old specific-pathogen-free chickens resulted in the death of 9% (two birds) six days postinoculation (PI). Both birds showed characteristic IBH/HPS gross and microscopic lesions; the remaining birds, sacrificed at day 10 PI, showed less severe lesions. On the basis of epidemiologic and experimental data of the virulence of Chilean FAV isolates, and the pathogenicity results with isolate 341, we speculate that Chilean FAV strains may require an association with other agents (immunosuppressive agents) to induce IBH/HPS outbreaks in the field.