Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain.

Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells.

Development of a hamster kidney cell line expressing stably T7 RNA polymerase using retroviral gene transfer technology for efficient rescue of infectious foot-and-mouth disease virus.

Reverse genetics systems, with the ability to manipulate viral genomes at the DNA molecular level, are an important platform for study of the assembly and function of viruses. Genome manipulation, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for increasing the efficiency of rescuing recombinant viruses.

[Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].

To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end.

[Construction and sequencing of full-length cDNA clone of swine vesicular disease virus strain HK'1/70].

By RACE, 2 overlapping cDNA fragments (3'PCR and 5'PCR fragments) covering the full genome of swine vesicular disease virus strain HK'1/70 were amplified from total RNA extracted from experimentally infected suckling mice. These fragments were cloned into pGEM-T Easy vector, respectively. 5'PCR fragment was digested by enzymes of Aat II and BssH II, and the Aat II-BssH II-digested 5'PCR fragment was obtained and cloned into the recombinant pGEM-T Easy vector containing 3'PCR fragment,the recombinant plasmid encoding full-length cDNA of SVDV HK'I/70 strain was then obtained and sequenced.

Infective viruses produced from full-length complementary DNA of swine vesicular disease viruses HK/70 strain.

The full-length cDNA clone of swine vesicular disease virus HK/70 strain named pSVOK was constructed in order to study the antigenicity, replication, maturation and pathogenicity of swine vesicular disease virus. transcription RNA from pSVOK transfected IBRS-2 cells and the recovered virus RNA were isolated and sequenced, then indirect hemagglutination test, indirect immunofluorescence assays, eleectron microscope test, 50% tissue culture infecting dose (TCID) assays and mouse virulence studies were performed to study the antigenicity and virulence of the recovered virus. The result showed that the infectious clones we obtained and the virus derived from pSVOK had the same biological properties as the parental strain HK/70.