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1.
Braz. j. microbiol ; Braz. j. microbiol;45(4): 1555-1563, Oct.-Dec. 2014. ilus, graf, tab
Article de Anglais | LILACS | ID: lil-741314

RÉSUMÉ

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.


Sujet(s)
Animaux , Embryon de poulet , Recombinaison homologue , Virus de la bursite infectieuse/génétique , Génétique inverse/méthodes , Brésil , Cellules cultivées , Fibroblastes/virologie , Vecteurs génétiques , Instabilité du génome , Virus de la bursite infectieuse/isolement et purification , Virus de la bursite infectieuse/physiologie , Saccharomyces cerevisiae/génétique , Transfection , Culture virale , Réplication virale
2.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;39(7): 935-944, July 2006. ilus, tab
Article de Anglais | LILACS | ID: lil-431563

RÉSUMÉ

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98 percent) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.


Sujet(s)
Animaux , Bovins , Effet cytopathogène viral/génétique , Virus de la diarrhée virale bovine/génétique , Duplication de gène , Génome viral/génétique , Protéines virales non structurales/génétique , Séquence d'acides aminés , Virus de la diarrhée virale bovine/isolement et purification , Réarrangement des gènes , Données de séquences moléculaires , RT-PCR , ARN viral/génétique
3.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;39(3): 335-343, Mar. 2006. tab, graf
Article de Anglais | LILACS | ID: lil-421366

RÉSUMÉ

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.


Sujet(s)
Animaux , Bovins , Femelle , Mâle , Lapins , Encéphale/virologie , Encéphalite virale/virologie , Infections à Herpesviridae/virologie , /effets des médicaments et des substances chimiques , Méningoencéphalite/virologie , Activation virale/effets des médicaments et des substances chimiques , Maladie aigüe , Lignée cellulaire , Modèles animaux de maladie humaine , Dexaméthasone/pharmacologie , Glucocorticoïdes/pharmacologie , /isolement et purification , /physiologie , Latence virale/effets des médicaments et des substances chimiques
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