The N-Terminal Domain of Spike Protein Is Not the Enteric Tropism Determinant for Transmissible Gastroenteritis Virus in Piglets

Abstract

Transmissible gastroenteritis virus (TGEV) is the etiologic agent of transmissible gastroenteritis in pigs, and the N-terminal domain of TGEV spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. Here, we assembled a full-length infectious cDNA clone of TGEV in a bacterial artificial chromosome. Using a novel approach, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the N-terminal domain of the TGEV Spike gene (S_NTD224), which is analogous to the N-terminal domain of porcine respiratory coronavirus. S_NTD224 notably affected the TGEV growth kinetics in PK-15 cells but was not essential for recombinant virus survival. In animal experiments with 13 two-day-old piglets, the TGEV recombinant viruses with/without S_NTD224 deletion induced obvious clinical signs and mortality. Together, our results directly demonstrated that S_NTD224 of TGEV mildly influenced TGEV virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against TGEV. Importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research.

Minimum Determinants of Transmissible Gastroenteritis Virus Enteric Tropism Are Located in the N-Terminus of Spike Protein

“…Our results confirmed that the enteric tropism determinants are located in the N-terminus of TGEV S protein. Previously published data questioned the relevance of this domain in the enteric tropism [27]. It is worth noting that, in contrast to the data reported by other groups, we compared the tropism of engineered viruses containing a full-length S protein.…”

“…Then, the contribution of other S protein domains or other viral proteins to enteric tropism was for a long time an open question [26]. In fact, the relevance of S protein N-terminus in TGEV enteric tropism has even recently been questioned [27]. [16].…”

A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence

“…Although ␣-CoV nsp1 expression is known to suppress host gene expression, its biological functions in viral replication have been largely unexplored. To evaluate the role of TGEV nsp1 in inhibiting host gene expression during viral replication, we used our reverse-genetics system to construct a recombinant TGEV encoding a mutant nsp1 protein in which ORF3 was replaced by green fluorescent protein (GFP) (19). Based on the previously described results, we replaced the important motif (amino acids 91-95) in the nsp1-coding sequence.…”

“…with 10% fetal bovine serum. The pBAC-TGEV-GFP plasmid containing a full-length infectious cDNA clone, which was derived from the highly virulent TGEV strain WH-1 (GenBank TM accession number HQ462571), has been described previously (19). To acquire the TGEV recombinant virus, the plasmid was first transfected into HEK-293T cells, and then the virus was recovered to infect virus-susceptible cells.…”

“…To construct the recombinant virus containing mutant nsp1, a method previously reported by our laboratory was used (19). The sequences of the relevant primers are shown in Table 2.…”