Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence

Targeted recombination within the S (spike) gene of transmissible gastroent eritis coronavirus (TGEV) was promoted by passage of helper respiratory vir us isolates in cells transfected with a TGEV-derived defective minigenome c arrying the S gene from an enteric isolate. The minigenome was efficiently replicated in trans and packaged by the helper virus, leading to the format ion of true recombinant and pseudorecombinant viruses containing the S prot eins of both enteric and respiratory TGEV strains in their envelopes. The r ecombinants acquired an enteric tropism, and their analysis showed that the y were generated by homologous recombination that implied a double crossove r in the S gene resulting in replacement of most of the respiratory, attenu ated strain S gene (nucleotides 96 to 3700) by the S gene of the enteric, v irulent isolate. The recombinant virus was virulent and rapidly evolved in swine testis cells by the introduction of point mutations and in-phase codo n deletions in a domain of the S gene (nucleotides 217 to 665) previously i mplicated in the tropism of TGEV. The helper virus, with an original respir atory tropism, was also found in the enteric tract, probably because pseudo recombinant viruses carrying the spike proteins from the respiratory strain and the enteric virus in their envelopes were formed. These results demons trated that a change in the tropism and virulence of TGEV can be engineered by sequence changes in the S gene.