Résumé
Objective To construct and rescue recombinant influenza virus strains expressing hu-man metapneumovirus ( hMPV) epitopes. -ethods B cell, CTL and Th epitopes predicted by bioinformat-ics software were coupled together in different combinations. These different array genes were inserted into the NS1 gene of influenza virus strain A/PR/8/34 ( PR8 ) , respectively. Recombinant PR8 influenza virus vectors expressing different hMPV antigenic epitopes were rescued by reverse genetics using eight-plasmid system. Sequencing analysis was conducted to verify whether the rescued viruses carried the chimeric hMPV epitopes. Hemagglutination ( HA) titers, half tissue culture infection dose ( TCID50 ) and growth curves were detected. Results Interval sequences GPGPG and KK were introduced into hMPV epitope combinations to construct multi-epitope antigens (MEA). These MEA were inserted into the PR8 NS gene, respectively. Using 8 plasmid system, three recombinant influenza virus strains were rescued successfully. After cultured for three passages in Madin-Darby canine kidney ( MDCK) cells and one in eggs, these three recombinant strains could proliferate steadily. Whole genome sequencing verified that the three recombinant strains car-ried the chimeric MEA sequences, named as rFLU/hMPV/B, rFLU/hMPV/CTL-Th and rFLU/hMPV/B-Th. HA titers of the recombinant strains were 128, 128 and 256 using turkey erythrocyte, respectively. Their TCID50 were 107. 0/ml, 106. 8/ml and 107. 0/ml, respectively. Growth curve tests also verified that the recombinant strains could proliferate steadily in MDCK cells. Conclusions Three recombinant influenza vi-rus vector strains carrying the B cell, CTL and Th epitopes of hMPV were rescued successfully. This study lays the foundation for further evaluation of the immune effects of these recombinant viruses and their poten-tial application value in vaccine development.
Résumé
Objective To construct an infectious full-length cDNA clone of enterovirus 71(EV71)and develop a technological platform for study on vaccine development as well as molecular virology of EV71.Methods According to the nucleotide sequence of EV71 strain 085 isolated in China,four pairs of primers were designed for amplification of four end to end overlapping subgenomic cDNA fragments,the cDNA fragments were directional cloned into pBluescript SK(+)vector,and the virus genome cDNA clone was obtained by ligation orderly.The rescued virus of parental strain 085 from RNA transfected host cells was identified by RT-PCR,IFA,titration as well as transmission electron microscope(TEM)after the transcription of the full-length cDNA clone in vitro.Results The full-length cDNA clone was constructed successfully,and the typical CPE was observed after its transcription into Vero cells.The rescued virus with 20-30 nm in diameter can not only be neutralized by EV71 special anti-serum but also react with anti-EV71 monoclonal antibody that virus infected cells stained with FITC can be detected by IFA.After amplification from the total RNA extraction of virus infected cells by RT-PCR with EV71 special primers,the 226 bp products can be detected.The growth curve showed that the rescued virus can propagate in Vero cells stably with a titer of 4.5 ～6.0 lgCCID50/ml during 8 passages.The plaque formed by rescued virus is identical as parental virus in morphology but smaller in size.Conclusion An infectious full-length clone of EV71 was developed successfully,which will be used for further study on pathogenesis and vaccine development of EV71.
Résumé
Objective To identify the helper plasmids from HEP-Flury strain rabies virus that could encapsidate the full-length genome of CTN strain. Methods Four overlapped fragments covering the full-length genome of rabies virus CTN strain were cloned into expression vector. A recombinant full-length genome plasmid (pCTN-GFP) contained the full-length genome of the CTN strain expect for ψ gene which was replaced by GFP gene was then constructed using restriction enzyme cleavage and ligation in vitro. In order to obtain the recombinant rabies virus CTN-GFP, the pCTN-GFP was transfected with helper plasmids carrying N, P, L gene of HEP-Flury strain. Results The four gene fragments of the genome were amplified and cloned into the expression vector. The recombinant genome cDNA plasmid pCTN-GFP was constructed and subjected to restriction endonuclease digestions. After sequenced to assure no absence and mutations compared with their parental viruses, it was ready for virus rescue. After the transfection of both pCTN-GFP and the helper plasmids from HEP-Flury strain into BHK-21 cells, the recombinant rabies virus CTN-GFP was rescued and confirmed by fluorescence analysis and RT-PCR, which demonstrated that the CTN-GFP was recovered from cloned cDNA. Conclusion The proteins of HEP-Flury strain rabies virus could encapsidate and transcribe the CTN strain rabies virus RNA genome.