RESUMO
Twelve Newcastle disease virus (NDV) strains were isolated from chickens involved in outbreaks of Newcastle disease (ND) in western China (Shaanxi, Gansu, Xinjiang, Qinghai and Guangxi provinces) between 1979 and 1999. All strains were determined to be velogenic by plaque formation, the mean death time (MDT) of embryonated eggs, and the intracerebral pathogenicity index (ICPI). For preparation of virus RNA, the acid guanidinium-thiocyanate method was used. A 908bp fragment of nucleotide was amplified by RT-PCR starting from the N terminal of the F gene and the PCR segments were cloned into the PGEM-T vector and sequenced. The similarities of the nucleotide sequences (1-519bp) and predicted amino acid sequences of the F gene (1-125) were analyzed by comparing the 12 NDV isolates with the NDV vaccine strains Lasota, B1, H1 and V4, with classical NDV strains and recent epizootic strains. Phylogenetic analysis demonstrated that all strains were of two novel genotypes; the NDV strains that caused the outbreak of ND in western China during 1998-1999 was of the genotype VIIa, whereas the strains from the Qinghai province (1979-1985) were of genotype VIII, which has been found predominately in southern Africa.
Assuntos
Galinhas , Surtos de Doenças/veterinária , Doença de Newcastle/epidemiologia , Vírus da Doença de Newcastle/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , China/epidemiologia , DNA Complementar/química , DNA Complementar/genética , Dados de Sequência Molecular , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/isolamento & purificação , Proteínas do Nucleocapsídeo , Nucleoproteínas/química , Nucleoproteínas/genética , Filogenia , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de Sequência de Aminoácidos , Organismos Livres de Patógenos Específicos , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
To overcome the drawbacks of encapsulating plasmid DNA (pDNA) in poly (D,L-lactic-co-glycolic acid) (PLGA) by water-in-oil-in-water double-emulsion solvent-evaporation method, we have developed a novel procedure for encapsulating pDNA in PLGA microparticles called DNA organic phase self-emulsification (DOPSM). This method was based on both the extraction plasmid DNA from aqueous phase into organic phase and the spontaneous emulsification DNA in organic phase by solvent diffusion method. The efficiency of extraction plasmid DNA into organic phase is 99% and the concentration of pDNA in organic phase is up to 2.4 mg/ml. The efficiency of microencapsulation of plasmid DNA in PLGA is up to 76% and can be enhanced by lowering the pH of aqueous solution of emulsion. The microparticles size of PLGA of pDNA is in a narrow range of 1-2 microm. This procedure does not involve the high mechanical energy to emulsify which may damage the integrity of pDNA. This method can be applied to encapsulate the pDNA into microparticles of other biocompatible polymers with high efficiency.