Expression of porcine interferon-gamma and its safe antiviral assay / 生物工程学报

In order to ensure the biosafety of the IFN-gamma antiviral activity assay, we used a replication-deficient VSV carrying GFP as an interferon sensitive indicator virus (VSVdeltaG*G). The antiviral activities of porcine IFN-gamma expressed in Escherichia coli and in baculovirus on MDBK cells were assessed. The results showed that the antiviral activity of porcine IFN-gamma expressed in baculovirus could reach 10(5) IU/mL, while the porcine IFN-gamma expressed in E. coli showed some antiviral activity (32 IU/mL) after refolding. The results of the VSVdeltaG*G-based antiviral assay were almost identical to that of the VSV*GFP-based assay, suggesting it is highly feasible to use VSVdeltaG*G as a substitute for VSV*GFP, making assays for IFN-gamma antiviral activity safer and more accurate.

Preparation of recombinant human metapneumovirus expressing green fluorescent protein / 中华微生物学和免疫学杂志

Objective To construct the recombinant human metapneumovirus(hMPV) (defined as rhMPV NL/1/00 GFP) in vitro by reverse genetics technique. Methods BSR-T7 cells were transfected using LipofectAMINE 2000 with the full-length cDNA plasmid, and four major protein expressing plasmids, pCITE-N, pCITE-P, pCITE-M2.1 and pCITE-L. After 3 d, cells were subjected to one -80℃ freeze-thaw cycle to prepare lysates. The supernatant of lysate was used to inoculate Vero-E6 cells. After 1-4 d, cells were found for the obvious development of cytopathic effects under light microscope and green fluoroscopic signals under fluorescence microscope, and were observed up to 10 d. The supernatant were collected to de-tect virus titer. Viral RNA was extracted from the supernatant and reverse transcriptase polymerase chain re-action (RT-PCR) was used to amplify N, F and G genes of rescued virus. Results Cytopathic effects and green fluoroscopic signals was readily and obviously observed after 1-4 d post-inoculation in Vero-E6 cells, then cytopathic effects got worse and green fluoroscopic signals became stronger gradually up to 10 d. The ti-ters of the 1st, 5th, 10th,15th and 20th generation virus ranged from 105.0 to 106.5 TCID50/ml. Amplicons with size of 910 bp (N), 450 bp (F) and 980 bp (G) by RT-PCR were accordant with expectant. Nucleotide sequence analysis of above cDNA fragments showed 100% similarity with reported sequence of hMPV NI/1/00 strain. The recombinant virus was genetically constant and GFP-labeled after 20 passages in Vero-E6 cells. Conclusion Recombined hMPV was successfully rescued by reverse genetics technique. This study lays ground for exploring pathogenesis of hMPV infection and development of hMPV attenuated vac-cines.