Abstract
Transmissible gastroenteritis virus (TGEV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide, that possesses both enteric and respiratory tropism. The ability to replicate in the enteric tract directly correlates with virulence, as TGEVs with an exclusive respiratory tropism are attenuated. The tissue tropism is determined by spike (S) protein, although the molecular bases for enteric tropism remain to be fully characterized. Both pAPN and sialic acid binding domains (aa 506–655 and 145–155, respectively) are necessary but not sufficient for enteric tract infection. Using a TGEV infectious cDNA and enteric (TGEV-SC11) or respiratory (TGEV-SPTV) isolates, encoding a full-length S protein, a set of chimeric recombinant viruses, with a sequential modification in S protein amino terminus, was engineered. In vivo tropism, either enteric, respiratory or both, was studied by inoculating three-day-old piglets and analyzing viral titers in lung and gut. The data indicated that U655>G change in S gene (S219A in S protein) was required to confer enteric tropism to a respiratory virus that already contains the pAPN and sialic acid binding domains in its S protein. Moreover, an engineered virus containing U655>G and a 6 nt insertion at position 1124 (Y374-T375insND in S protein) was genetically stable after passage in cell cultures, and increased virus titers in gut by 1000-fold. We postulated that the effect of these residues in enteric tropism may be mediated by the modification of both glycosaminoglycan binding and S protein structure.
Highlights
Porcine enteric coronaviruses (CoVs) are one of the main threats for porcine industry worldwide, as acute infectious diarrhea is a major cause of high morbidity and mortality in piglets [1]
We postulated that the effect of these residues in enteric tropism may be mediated by the modification of both glycosaminoglycan binding and S protein structure
When the S genes (4350 nt) from an enteric (SC11) and a respiratory (SPTV) Transmissible gastroenteritis virus (TGEV) virus were compared, 17 nucleotide substitutions were identified and 15 of them leading to amino acid changes (Figure 2A)
Summary
Porcine enteric coronaviruses (CoVs) are one of the main threats for porcine industry worldwide, as acute infectious diarrhea is a major cause of high morbidity and mortality in piglets [1]. TGEV has both enteric and respiratory tropism, in contrast to other porcine enteric CoVs, making this virus a good model to analyze the molecular determinants of coronavirus tissue tropism. Using the same TGEV genetic background, the tropism of the recombinant virus rescued from the infectious cDNA was manipulated by substituting the S gene from an enteric virus (rTGEV-SC11) by that from a respiratory isolate (rTGEV-SPTV) [30], with no additional changes in other locations of the viral genome. It is worth noting that both rTGEV-SC11 and rTGEV-SPTV encode full-length S proteins (Figure 1), confirming that the determinants for TGEV enteric tropism rely on S protein and not in other viral proteins. The respiratory TGEV virus already contained the domains binding to pAPN (aa 506–655) and sialic acid (aa 145–155), which were necessary but not sufficient for enteric tract infection. A 6 nt insertion at position 1124 (leading to Y374-T375insND in S protein) led to a virus highly stable in cell cultures and increased by 1000-fold the virus titers in the enteric tract
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