Characterization and Identification of a Novel Torovirus Associated With Recombinant Bovine Torovirus From Tibetan Antelope in Qinghai-Tibet Plateau of China

Toroviruses (ToVs) are enteric pathogens and comprise three species, equine torovirus (EToV), bovine torovirus (BToV), and porcine torovirus (PToV). In this study, a novel torovirus (antelope torovirus, AToV) was discovered from fecal samples of Tibetan antelopes (Pantholops hodgsonii) with viral loads of 2.10×109 to 1.76×1010 copies/g. The genome of AToV is 28,438 nucleotides (nt) in length encoding six open reading frames (ORFs) with 11 conserved domains in pp1ab and a putative slippery sequence (14171UUUAAAC14177) in the overlapping region of ORF1a and ORF1b. Phylogenetic analysis illustrated strains of AToV form a unique clade within ToVs and comparative analysis showed AToV share relatively low sequence identity with other ToVs in six ORFs (68.2–91.6% nucleotide identity). These data suggested that AToV represents a novel and distinct species of ToVs. Based on the M genes, evolutionary analysis with BEAST of AToV and other ToVs led to a most recent common ancestor estimate of 366years ago. Remarkably, recombination analysis revealed AToV was the unknown parental ToV that once involving in the recombinant events of HE genes of two Dutch strains of BToV (B150 and B155), which indicated that AToV occurred cross-species transmission and existed both in the Netherlands and China. This study revealed a novel torovirus, a natural reservoir host (Tibetan antelope) of toroviruses for the first time, and appealed to further related studies to better understand the diversity of toroviruses.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease

ABSTRACT The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro. Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro. In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase’s substrate specificities and the rational development of the antinidovirus drugs. IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The “noncanonical” substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes

Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.