RESUMO
Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.
Assuntos
Adaptação Biológica , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Vírus da Diarreia Epidêmica Suína/genética , Proteínas do Envelope Viral/genética , Animais , Chlorocebus aethiops , Proteínas M de Coronavírus , Proteínas do Nucleocapsídeo de Coronavírus , Análise Mutacional de DNA , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/genética , Análise de Sequência de DNA , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas da Matriz Viral/genética , VirulênciaRESUMO
Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3 x 10(6)U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.