๐ Generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome
Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E+ packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-ฮ3abฮE) was successfully rescued in these cell lines. rTGEV-ฮ3abฮE reached high titers (107 PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 x 105 PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-ฮ3abฮE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for > 10 passages in the E+ packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.
keywords
๐ cells expressing (60)
๐ gastroenteritis coronavirus (57)
๐ cell lines (125)
๐ cell line (211)
๐ electron microscopy (149)
๐ transmissible gastroenteritis (226)
year
โฐ 2002
journal
๐ Journal of Virology
issn
๐ 0022538X
volume
76
number
22
page
11518-11529
citedbycount
102
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