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.
Highlights
Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses closely related to animal and human health [1,2,3]
Were generated by reverse transcriptase polymerase chain reaction (PCR) (RT-PCR) using total RNA extracted from PK-15 cells infected with Transmissible gastroenteritis virus (TGEV) WH-1 (Figure 1A,B)
We found that S_NTD224 not tropism determinant for TGEV
Summary
Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses closely related to animal and human health [1,2,3]. As the largest RNA genome viruses, CoVs have at least six typical overlapping open reading frames. TGEV is widespread in the pork industry, causes high mortality in neonatal pigs, and is generally thought to share a common ancestor with porcine respiratory coronavirus (PRCV) [13]. Both TGEV and PRCV have a common cell receptor, aminopeptidase N (APN), but a coreceptor (Neu5Gc) of TGEV is recognized to confer enteric tropism to TGEV [14,15,16,17].
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